VI KONGRES DRUŠTVA ZA NEURONAUKE SRBIJE
sa meñunarodnim učešćem
6th CONGRESS OF THE SERBIAN NEUROSCIENCE SOCIETY
APSTRAKTI / ABSTRACTS
BEOGRAD
14.novembar – 16.novembar 2013. godine
Sava Centar
Izdavač:
Društvo za neuronauke Srbije
Ministarstvo prosvete, nauke i tehnološkog razvoja
Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu
Za izdavača:
Selma Kanazir
Tomislav Jovanović
Pavle Pavlović
Urednici:
Selma Kanazir
Danijela Savić
Aleksandra Isaković
Tehnički urednici:
Danijela Savić
Maja Jovanović
Jelena Popić
Nataša Lončarević-Vasiljković
Smilja Todorović
Marjan Popović
Dizajn:
Katarina Lung
Tehnička priprema:
Aleksandar Mandić
Autorska prava © 2013 od strane Društva za neuronauke Srbije i drugih saradnika. Sva prava zadržana. Nijedan deo
ove publikacije ne sme biti reprodukovan u bilo kom obliku ili bilo kojim sredstvom, bez pismene dozvole izdavača.
ISBN: 978-86-917255-0-1
ORGANIZATORI
Društvo za neuronauke Srbije
Ministarstvo prosvete, nauke i tehnološkog razvoja Republike Srbije
Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu
Medicinski fakultet, Univerzitet u Beogradu
Organizacioni odbor (Organizing Committee)
Selma Kanazir- predsednik
Danijela Savić, Ivanka Marković, Milka Perović, Aleksandra Mladenović Đorñević, Irena Lavrnja, Vesna Pešić,
Jelena Popić, Kosara Smiljanić, Nataša Lončarević-Vasiljković, Desanka Milanović, Smilja Todorović, Vesna Tešić,
Divna Lazić, Marjana Brkić, Iva Božić, Maja Jovanović, Marjan Popović
Naučni odbor (Scientific Committee)
Akademik Ljubiša Rakić- predsednik
Sabera Ruždijić, Selma Kanazir, Mirjana Stojiljković, Ivanka Marković, Sanja Peković, Nadežda Nedeljković,
Nikola Tanić, Anica Horvat, Nataša Petronijević, Tihomir Ilić, Aleksandra Isaković, Ivan Milenković, Zoran Redžić,
Sanja Ivković, Igor Jakovčevski
Počasni naučni odbor (Honorary Scientific Committee)
Akad. Vladimir Bumbaširević- rektor Univerziteta u Beogradu; Akad. Vladimir Kostić - predsednik Društva
neurologa Srbije; Pavle Pavlović - direktor IBISS-a; Tomislav Jovanović - ministar za prosvetu, nauku i tehnološki
razvoj; Ivica Radović - pomoćnik ministra za prosvetu, nauku i tehnološki razvoj; Jovan Bazić - pomoćnik ministra
za prosvetu, nauku i tehnološki razvoj; Stanko Stojilković, Markus Kipp, Dieter Lütjohann
PROGRAM
14. NOVEMBAR
19:00
Svečano otvaranje Kongresa, Amfiteatar Sava centar
Predavanje laureata nagrade “Dr Laza Lazarević” Akademika Vladimira S. Kostića:
“Parkinsonova bolest – od transmitera do proteina”
Koktel dobrodošlice
15. NOVEMBAR
09:00-09:10
Uvodna reč: Akademik Ljubiša Rakić, predsednik Društva za neuronauke Srbije
09:10-09:40
Plenarno predavanje: Dieter Lütjohann (Institut za kliničku hemiju i farmakologiju,
Univerzitetska klinika Bon, Nemačka)
Oksisteroli-endogeni markeri u neurodegenerativnim oboljenjima
Ćelijski i molekularni mehanizmi neurodegenerativnih procesa
Koordinatori: Dieter Lütjohann, Aleksandra Isaković, Selma Kanazir
09:40-09:55
Igor Jakovčevski (ZMNH, Universitetska klinika Hamburg-Eppendorf, Nemačka)
Vezivanje adhezivnog molekula L1 za amiloid beta poboljšava kliničku sliku u modelu
Alchajmerove bolesti kod miša
09:55-10:10
Ivana Novaković (Medicinski Fakutet, Univerzitet u Beogradu)
Mutacije gena za glukocerebrozidazu (GBA) u Parkinsonovoj bolesti: naša iskustva
10:10-10:25
Ivanka Marković (Medicinski Fakutet, Univerzitet u Beogradu)
Uloga AMP-om aktivisane kinaze u neurotoksičnom delovanju alfa-sinukleina in vitro
10:25-10:40
Dušanka Savić-Pavićević (Biološki Fakutet, Univerzitet u Beogradu)
Asocijacija i epistaze mikro RNK 137 i enzima za editovanje adenozina u inozin u molekulima
RNK kod šizofrenije i bipolarnog poremećaja
10:40-10:55
Miroslav Adžić (Institut za nuklearne nauke Vinča, Univerzitet u Beogradu)
Ispitivanje kliničkog potencijala fosforilisanog glukokortikoidnog receptora kao markera
depresije
11:00-11:15
Kafe pauza
Razviće, plastičnost i ćelijska smrt
Koordinatori: Sabera Ruždijić, Nadežda Nedeljković, Igor Jakovčevski
11:15-11:30
Zoran Redžić (Institut za fiziologiju, Medicinski fakultet, Univerziteta u Kuvajtu)
Uloga eritropoetina i neurovaskularne jedinice u ishemijskom moždanom udaru
11:30-11:45
Vladimir Trajković (Medicinski Fakutet, Univerzitet u Beogradu)
Uloga indukcije autofagije u neurotoksičnim efektima atipičnog antipsihotika olanzapina
11:45-12:00
Sanja Ivković (Institut za molekularnu medicinu, Lisabon)
Produžena ekspresija EGFR utiče na sudbinu neuralnih progenitora
12:00-12:15
Aleksandra Mladenović-Đorñević (Institut za biološka istraživanja „Siniša Stanković“,
Univerzitet u Beogradu)
Homeostaza holesterola u mozgu: efekat starenja i dijetalne restrikcije
12:15-12:30
Vesna Pešić (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
Efekti opštih anestetika na nervni sistem u razvoju
12:30-14:00
Poster sekcija
Ručak
14:00-14:30
Plenarno predavanje: Markus Kipp (Institut za neuroanatomiju, Univerzitet u Ahenu)
Selektivna osetljivost oligodendrocita u ćelijskom odgvoru na neuvijene proteine (UPR) tokom
nastanka demijelinizacionih lezija u multiploj sklerozi
Ćelijski i molekularni mehanizmi neuroinflamatornih procesa
Koordinatori: Sanja Peković, Zoran Redžić, Ivanka Marković
14:30-14:45
Nadežda Nedeljković (Biološki Fakutet, Univerzitet u Beogradu)
Uloga ektonukleotidaza u interakciji nervnog i imunskog sistema
14:45-15:00
Đorñe Miljković (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
Redoks procesi u inicijaciji multiple skleroze
15:00-15:15
Emina Savić (Medicinski Fakutet, Univerzitet u Beogradu)
Ekspresija gena Th1 i Th17 osovine kod bolesnika sa multiplom sklerozom lečenih interferonomβ: trogodišnje praćenje
15:15-15:30
Irena Lavrnja (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
BV-2 mikroglijska ćelijska linija kao model izučavanja neuroinflamacije - efekat benfotiamina
15:30–16:00
Kafe pauza
Poremećaj homeostaze i bolesti centralnog nervnog sistema
Koordinatori: Mirjana Stojiljković, Nataša Petronijević, Jasna Šaponjić
16:00-16:15
Predrag Vujović (Biološki Fakutet, Univerzitet u Beogradu)
Efekat gladovanja na ekspresiju i unutarćelijsku lokalizaciju Fto u neuronima mozga pacova
16:15-16:30
Milka Perović (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
Uticaj dijetalne restrikcije na glijski odgovor nakon traumatske povrede mozga
16:30-16:45
Nataša Nestorović (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
Dugotrajni efekti somatostatina na somatotropne i gonadotropne ćelije hipofize
16:45-17:00
Nikola Tanić (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
Identifikacija novih genetičkih alteracija u humanim malignim gliomima
17:00-17:15
Milica Pešić (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
Nove strategije u prevazilaženju višestruke rezistencije na hemioterapiju kod glioblastoma
17:15-18:30
Panel diskusija: Advocacy in Neuroscience
20.30-23:00
Svečana večera –restoran “Top of the Hub”, Poslovni centar “Ušće”
16. NOVEMBAR:
09:00-09:30
Plenarno predavanje: Stanko Stojilković (Američki nacionalni institut zdravlja, Betesda, SAD)
Polno-specifičan efekat gonadotropnog oslobañajućeg hormona hipotalamusa u indukciji
ekspresije Dmp1 u hipofizi
Neurofiziologija i ponašanje
Koordinatori: Stanko Stojilković, Ivan Milenković, Markus Kipp
09:30-09:45
Olivera Stanojlović (Medicinski Fakutet, Univerzitet u Beogradu)
Hipermetioninska dijeta i hiperekscitabilnost: električne i bihejvioralne promene kod pacova
09:45-10:00
Jasna Šaponjić (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
EEG arhitektura budnosti i spavanja u centralnoj holinergičkoj neurodegeneraciji
10:00-10:15
Branka Janać (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
Uticaj magnetnih polja na ponašanje različitih vrsta
10:15-10:30
Tihomir Ilić (Vojno medicinska akademija, Beograd)
Neinvazivna stimulacija mozga – translacioni principi
10:30-10:45
Kafe pauza
10:45-11:00
Nataša Petronijević (Medicinski Fakutet, Univerzitet u Beogradu)
Ekspresija NADPH oksidaze i mitohondrijalna disfunkcija kao pokretači oksidativnog stresa u
mozgu džerbila trovanih aluminijumom
11:00-11:15
Miroslav Savić (Farmaceutski Fakultet, Univerzitet u Beogradu)
Uticaj modulacije GABA-ergičke neurotransmisije na ponašanje
11:15-11:30
Nataša Todorović (Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu)
Jonski kanali aktivirani kiselim pH (ASIC): nove mete za terapiju u neurologiji...ali čemu služe?
11:30-11:45
Pavle Anñus (Biološki Fakutet, Univerzitet u Beogradu)
Uloga tenascina C u strukturnoj plastičnosti CNS
11:45-12:00
Vandenbroucke Roosmarijn (Odeljenje za molekulana biomedicinska istraživanja, Univerzitet
u Gentu, Belgija)
Identifikacija novih terapeutskih meta u prevenciji oštećenja krvno-likvorne barijere u
sistemskim inflamatornim poremećajima i tokom starenja
12:00-12:15
Nevena Radonjić, (University of Connecticut Health Center, SAD)
Sonic hedgehog i specifikacija humanih kortikalnih interneurona
12:15-14:00
Poster sekcija
Ručak
14:00-16:00
Okrugli sto: usmene prezentacije odabranih postera
Koordinatori: Mirjana Stojiljković, Aleksandra Isaković, Ivan Milenković
16:00-16:15
Dodela nagrada za najbolji poster
16:15-16:30
Kafe pauza
16:30
Skupština DNS
PROGRAMME
NOVEMBER 14th
19:00
Opening ceremony
Coctail reception wording
Award “Dr Laza Lazarevic“
NOVEMBER 15th
09:00-9:10
Welcome address: Ljubisa Rakic, President of the Serbian Neuroscience Society
09:10-9:40
Plenary lecture: Dieter Lütjohann (Institute of Clinical Chemistry and Clinical
Pharmacology, University Clinics of Bonn)
Oxysterols-endogenous marker in neurodegenerative diseeases
Cellular and molecular mechanisms of neurodegenerative processes
Chairs: Dieter Lutjohann, Aleksandra Isakovic, Selma Kanazir
09:40-09:55
09:55-10:10
10:10-10:25
Igor Jakovcevski (Faculty of Medicine, University of Belgrade)
Adhesion molecule L1 binds to amyloid beta and reduces Alzheimer’s disease pathology
in mice
Ivana Novakovic (Faculty of Medicine, University of Belgrade)
Glucocerobrosidase (GBA) gene mutations in Parkinson disease: our experience
Ivanka Markovic (Faculty of Medicine, University of Belgrade)
The protective role of AMP-activated protein kinase in alpha-synuclein neurotoxicity in
vitro
10:25-10:40
Dusanka Savic-Pavicevic (Faculty of Biology, University of Belgrade)
Association and epistasis of miR-137 and adenosine to inosine RNA editing enzymes in
schizophrenia and bipolar disorder
10:40-10:55
Miroslav Adzic (Instutute for nuclear sciences, Vinca, University of Belgrade)
Exploring the clinical potential of phosphorylated glucocorticoid receptor as biomarker
of depression
11:00-11:15
Coffee break
Development, plasticity and cell death
Chairs: Sabera Ruzdijic, Nadezda Nedeljkovic, Igor Jakovcevski
11:15-11:30
Zoran Redzic (Department of Physiology, Faculty of Medicine, Kuwait University)
Erythropoietin and neurovascular unit in the settings of ischemic stroke
11:30-11:45
Vladimir Trajkovic (Faculty of Medicine, University of Belgrade)
The role of autophagy induction in the neurotoxicity of atypical antipsychotic
olanzapine
11:45-12:00
Sanja Ivkovic (Instituto de Medecina Molecular, Lisbon, Portugal)
Prolonged EGFR expression alter the fate of neural progenitors
12:00-12:15
Aleksandra Mladenovic Djordjevic (Institute for Biological Research „Sinisa
Stankovic“, University of Belgrade)
Cholesterol homeostasis in the brain: the effect of aging and dietary restriction
12:15:12:30
Vesna Pesic (Institute for Biological Research „Sinisa Stankovic“, University of
Belgrade)
The effects of general anesthetics on developing nervous system
12:30-14:00
Poster presentation
Buffet lunch
14:00-14:30
Plenary lecture: Markus Kipp (Institute for Neuroanatomy, University Aachen)
Implications of the unfolded protein response for the selective vulnerability of
oligodendrocytes during multiple sclerosis lesion development
Cellular and molecular mechanisms of neuroinflammation
Chairs: Sanja Pekovic, Zoran Redzic, Ivanka Markovic
14:30-14:45
Nadezda Nedeljkovic (Faculty of Biology, University of Belgrade)
The role of ectonucleotidases in the interplay between the brain and the immune system
14:45-15:00
Djordje Miljkovic (Institute for Biological Research „Sinisa Stankovic“, University of
Belgrade)
Redox processes in multiple sclerosis initiation
15:00-15:15
Emina Savic (Faculty of Medicine, University of Belgrade)
Тh1/Th17 axes gene expression in multiple sclerosis patients: 3-year follow-up
15:15-15:30
Irena Lavrnja (Institute for Biological Research „Sinisa Stankovic“, University of
Belgrade)
BV2 cells as a model of studying neuroinflammation – effect of benfotiamine
15:30-16:00
Coffee break
Dysregulation of homeostasis and CNS disorders
Chairs: Mirjana Stojiljkovic, Natasa Petronijevic, Jasna Saponjic
15:45-16:00
Predrag Vujovic (Faculty of Biology, University of Belgrade)
The effect of fasting on the neuronal Fto expression and intracellular localization in rat
brain
16:00-16:15
Milka Perovic (Institute for Biological Research „Sinisa Stankovic“, University of
Belgrade)
Astrogliosis following traumatic brain injury – the impact of dietary restriction
16:15-16:30
Natasa Nestorovic (Institute for Biological Research „Sinisa Stankovic“, University of
Belgrade)
Long-term effects of somatostatin on pituitary somatotropic and gonadotropic cells
16:30-16:45
Nikola Tanic (Institute for Biological Research „Sinisa Stankovic“, University of
Belgrade)
Identification of novel genetic alterations in human malignant glioma.
16:45-17:00
Milica Pesic (Institute for Biological Research „Sinisa Stankovic“, University of
Belgrade)
New strategies for overcoming multi-drug resistance in glioblastomas
17:00-18:30
Panel discussion: Advocacy in Neuroscience
20:30
Dinner - Top of the hub, Usce Bussiness Center, 25th floor
NOVEMBER 16th
09:00-09:30
Plenary lecture: Stanko Stojilkovic (National Institute of Health, Bethesda, USA)
Sex –specific induction of pituitary Dmp1 by hypothalamic GonadotropinReleasingHormone
Neurophysiology and behaviour
Chairs: Stanko Stojilkovic, Ivan Milenkovic, Markus Kipp
09:30- 09:45
Olivera Stanojlovic, (Faculty of Medicine, University of Belgrade)
Hypermethionine diet and hyperexcitability: electrical and behavioral changes in rats
09:45-10:00
Jasna Saponjic, (Institute for Biological Research „„Sinisa Stankovic“, University of
Belgrade)
Sleep/wake states EEG architecture in the central cholinergic neurodegeneration
10:00-10:15
Branka Janac (Institute for Biological Research „Sinisa Stankovic“, University of
Belgrade)
The influence of magnetic fields on behaviour of different species
10:15-10:30
Tihomir Ilic (Military Medical Academy, Belgrade)
Non-invasive brain stimulation – translational principles
10.30-10.45
Coffee break
10:45-11:00
Natasa Petronijevic (Faculty of Medicine, University of Belgrade)
NADPH oxidase expression and mitochondrial dysfunction as initiators of oxidative
stress in the brain of gerbils treated with aluminum
11:00-11:15
Miroslav Savic (Faculty of Pharmacy, University of Belgrade)
The influence of modulation of GABAergic neurotransmission on behavior
11:15-11:30
Natasa Todorovic (Institute for Biological Research „Sinisa Stankovic“, University of
Belgrade)
Acid-sensing ion channels: novel therapeutic targets in neurology...but what are they
for?
11:30-11:45
Pavle Andjus (Faculty of Biology, University of Belgrade)
Role of tenascin C in structural brain plasticity
11:45-12:00
Vandenbroucke Roosmarijn (Department for Molecular Biomedical Research, Gent
University)
Identification of new therapeutic targets to prevent epithelial blood-CSF barrier
disruption in systemic inflammatory disorders and aging
12:00-12:15
Nevena Radonjić, (University of Connecticut Health Center, SAD)
Sonic hedgehog and specification of human cortical interneurons
12:15-14:00
Poster presentation
Buffet lunch
14:00-16:00
Selected posters oral presentations
Chairs: Mirjana Stojiljkovic, Aleksandra Isakovic, Ivan Milenkovic
16:00-16:15
Best poster award ceremony
16:15-16:30
Coffe break
16:30
SNS Assembly meeting
PLENARNA PREDAVANJA / PLENARY LECTURES
PLENARNA PREDAVANJA / PLENARY LECTURES
1
PLENARNA PREDAVANJA / PLENARY LECTURES
OXYSTEROLS – ENDOGENOUS MARKER IN
NEURODEGENERATIVE DISEASES
Dieter Lütjohann
Institute for Clinical Chemistry and Clinical
Pharmacology; University Clinics of Bonn; Germany
[email protected]
Since the blood-brain barrier efficiently
prevents cholesterol uptake from the circulation, de
novo synthesis is responsible for almost all cholesterol
present there. Neurons downregulate their cholesterol
synthesis and rely on delivery from ApoE lipoproteins
secreted by astrocytes. ApoE transcription is regulated
by 24S-hydroxycholesterol (24OHC) released by
neurons, via LXR. In order to maintain homeostasis,
excess of cholesterol is converted into 24OHC by the
neuronal
specific
cholesterol
24-hydroxylase
(CYP46A1). In neurodegenerative disorders such as
Multiple Sclerosis, Alzheimer and Huntington disease,
plasma 24OHC was found reduced proportionally to
the degree of brain atrophy as measured by MRI. Less
than 1% of the total excretion of 24OHC occurs via
the cerebrospinal fluid (CSF). In CSF from patients
affected by neurodegenerative diseases, increased
levels of 24OHC were found. In case of MCI and AD,
the CSF concentration of 24OHC was correlated with
ApoE, cholesterol and Tau. CSF tau is considered
related to the neurodegenerative process in AD, ApoE
and cholesterol are involved in the β-amyloid
deposition. 27-Hydroxycholesterol (27OHC), formed
outside the brain, crosses the blood-brain barrier
proportionally to the barrier dysfunction. There is a
positive correlation between levels of cholesterol and
27OHC in the circulation. This oxysterol antagonizes
the preventive effect of 24OHC on generation of βamyloid.
The analysis of oxysterols in plasma and CSF
seems to contribute to investigate the role of
cholesterol
metabolism
in
pathogenesis
of
neurodegeneration.
IMPLICATIONS OF THE UNFOLDED
PROTEIN RESPONSE FOR THE SELECTIVE
VULNERABILITY OF OLIGODENDROCYTES
DURING MULTIPLE SCLEROSIS LESION
DEVELOPMENT
Markus Kipp, Tim Clarner, Johannes Goldberg,
Marion Victor, Cordian Beyer
Institute for Neuroanatomy; Aachen University;
Aachen; Germany
[email protected]
Insults that impair endoplasmic reticulum
function, such as an impaired energy homeostasis,
trigger an adaptive programme called the unfolded
protein stress response (UPR). The physiology of
oligodendrocytes combines a vast amount of (lipo-)
protein synthesis with a high requirement of ATP.
Hence, the specific vulnerability of oligodendrocytes
during Multiple Sclerosis (MS) lesion formation and
progression might be due to an excessive UPR ones
tissue homeostasis is disturbed.
We investigated the UPR signature and
functional relevance in a well defined MS animal
model, where mitochondrial dysfunction leads to
selective oligodendrocyte death and finally
demyelination. Gene array experiments revealed a
dramatic reduction in myelin-associated mRNA
expression levels which was paralleled by the
induction of UPR-related marker proteins such as
CHOP, activating transcription factor-3 (ATF3) or
growth arrest and DNA damage-inducible protein-34
(GADD34). Double-labelling displayed that this
cellular stress response is exclusively activated in
oligodendrocytes.
Furthermore,
western-blotting
revealed an activation of UPR-associated protein
degradation. Attenuation of UPR negative feedbackloops by salubrinal, a drug which acts as a specific
inhibitor of eIF2α phosphatase enzymes, led to a
significant attenuation of oligodendrocyte apoptosis.
The same protection was observed in CHOP-deficient
animals. Inhibition of the respiratory chain in cultured
oligodendrocytes induced the activation of an UPR
and cell death, which was counteracted by salubrinal.
This is the first study which links the selective
vulnerability of oligodendrocytes during disturbed
cellular homeostasis with their high (lipo) -protein
synthesis rate. Further studies have to reveal the
significance of an UPR in oligodendrocytes for MS
lesion development and progression.
2
PLENARNA PREDAVANJA / PLENARY LECTURES
SEX-SPECIFIC INDUCTION OF PITUITARY
DMP1 BY HYPOTHALAMIC
GONADOTROPIN-RELEASING HORMONE
Stanko Stojilkovic
National Institute of Health; Bethesda; USA
[email protected]
Hypothalamic
gonadotropin-releasing
hormone (GnRH) is the primary regulator of
reproduction in vertebrates, acting via the G proteincoupled GnRH receptor (GnRHR) in pituitary
gonadotrophs to control synthesis and release of
gonadotropins. To identify the GnRHR-coupled gene
network, GnRH was applied in a pulsatile manner to
perifused pituitary cells from cycling females for 6
hours, mRNA was extracted and RNA sequencing
analysis was performed. This analysis revealed 83
candidate-regulated genes, including a large number
of those coding for secreted proteins. Most notably,
GnRH induces a >600-fold increase in expression of
dentin matrix protein-1 (Dmp1), one of five members
of the small integrin-binding ligand N-linked
glycoprotein gene family. The Dmp1 response is
mediated by the GnRHR, not elicited by other
hypothalamic releasing factors, and is ~20-fold
smaller in adult male pituitary cells. The in vitro sexspecific Dmp1 response is established during
peripubertal period and attenuated during pregnancy.
In vivo, pituitary Dmp1 is elevated during late
proestrus, suggesting that GnRH-induced ovulation
and the increase in Dmp1 expression are
synchronized. In contrast to the sexual dimorphism in
the Dmp1, Gnrhr expression is similar in both sexes
throughout development. Cell signaling studies
revealed that GnRH-induction of Dmp1 is mediated
by the protein kinase C signaling pathway and reflects
opposing roles of ERK1/2 and p38 MAPK; in
addition, the response is facilitated by progesterone.
These results establish that DMP1 production in
female rat gonadotrophs is controlled by the
hypothalamus through the GnRHR signaling pathway
and raises intriguing questions about its intrapituitary
and downstream effects.
3
PREDAVANJA / LECTURES
PREDAVANJA / LECTURES
4
PREDAVANJA / LECTURES
Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / Cellular and molecular mechanisms of neurodegenerative processes
ADHESION MOLECULE L1 BINDS TO AMYLOID
BETA AND REDUCES ALZHEIMER’S DISEASE
PATHOLOGY IN MICE
I. Jakovčevski*, N. Djogo, M. Schachner
Center for Molecular Neurobiology; University Hospital
Hamburg-Eppendorf; Hamburg; Germany
[email protected]
Introduction: Alzheimer’s disease (AD) is a
devastating neurodegenerative disorder and the most
common cause of elderly dementia.
Aim: In an effort to contribute investigate
molecular approaches to reduce degenerative processes in
AD we tested if the neural adhesion molecule L1
ameliorates some characteristic structural and molecular
parameters of neurodegeneration in a mouse model of AD.
Materials
and
methods:
Three-month-old
mice
overexpressing mutated forms of amyloid precursor protein
and presenilin-1 under the control of a neuron-specific
promoter received an injection of adeno-associated virus
encoding the full-length L1 (AAV-L1) or green fluorescent
protein (AAV-GFP) into the hippocampus and occipital
cortex. Four months after virus injection, the mice were
analyzed for histological and biochemical parameters of
AD.
Results:AAV-L1 injection decreased the Aβ
plaque load, levels of Aβ42, Aβ42/40 ratio and astrogliosis
compared with AAV-GFP controls. AAV-L1 injected mice
also had increased densities of inhibitory synaptic terminals
on pyramidal cells in the hippocampus. To probe for a
molecular mechanism that may underlie these effects, we
analyzed whether L1 interacts with Aβ in a label-free
binding assay.Concentration dependent binding of the
extracellular domain of L1 to Aβ40 and Aβ42 was seen,
with the fibronectin type III homologous repeats 1-3 of L1
mediating this effect. Aggregation of Aβ42 in vitro was
reduced in the presence of the extracellular domain of L1.
Conclusion: The combined observations indicate
that L1 is a candidate molecule to ameliorate the pathology
of AD, when applied in therapeutically viable treatment
schemes.
5
PREDAVANJA / LECTURES
Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / Cellular and molecular mechanisms of neurodegenerative processes
MUTACIJE GBA GENA U PARKINSONOVOJ
BOLESTI: NAŠA ISKUSTVA
GBA GENE MUTATIONS IN PARKINSON DISEASE:
OUR EXPERIENCE
I. Novaković
Klinika za neurologiju; Medicinski fakultet Univerziteta u
Beogradu; Beograd; Srbija
[email protected]
I. Novakovic
Neurology Clinic; Faculty of Medicine; University of
Belgrade; Belgrade; Serbia
[email protected]
Beta-glukocerebrozidaza (GBA) je enzim koji
raskida beta-glikozidne veze u glukocerebrozidu,
intermedijarnom proizvodu metabolizma glikolipida. U
humanom genomu GBA gen je lociran na hromozomu br. 1,
12kb ushodno od svog pseudogena. Dobro je poznato da
mutacije GBA gena uzrokuju Gošeovu bolest, autozomo
recesivan lizozomski poremećaj. U poslednjih desetak
godina u različitim populacijama je zapažena asocijacija
heterozigotnih promena u GBA genu i Parkinsonove bolesti
(PB). Meta analize su potvrdile GBA mutacije kao faktor
predispozicije za PB, ali sa različitom snagom zavisno od
etničkog porekla ispitanika. U grupi od 300 bolesnika sa PB
iz Srbije sproveli smo ciljanu DNK analizu radi detekcije
najčešćih GBA mutacija u našoj sredini. Direktnim
sekvenciranjem po Sangeru analizirani su egzoni 8, 9 i 11
GBA gena. Prevalenca GBA mutacija u našoj grupi
bolesnika bila je značajno veća nego u zdravoj populaciji
(OR=5,5). U drugoj fazi urañena je transkranijalna
sonografija moždanog parenhima (TKS) kod GBApozitivnih bolesnika sa PB, kao i kod asimptomatskih
nosilaca GBA mutacije, kod bolesnika sa Gošeovom
bolešću bez PB, sporadičnih PB bolesnika i zdravih
ispitanika. Pokazano je da se nalaz TKS kod GBApozitivnih bolesnika sa PB značajno razlikuje u odnosu na
zdrave ispitanike, ali po svemu odgovara nalazu tipičnom za
PB. Naši rezultati potvrdili su GBA mutacije kao značajan
faktor predispozicije za PB kod stanovništva Srbije, ali
posebne fenotipske odlike GBA-pozitivnih bolesnika sa PB
tek treba da budu utvrñene.
Beta-glucocerebrosidase (GBA) is an enzyme that
cleaves the beta-glucosidic linkage of the glucocerebroside,
an intermediate in glycolipid metabolism. Human GBA
gene is located on chromosome 1, 12kb upstream of its
related pseudogene. It is well known that mutations in the
GBA gene cause Gaucher's disease, an autosomal recessive
lysosomal storage disease. Recently, associations between
heterozygous GBA gene changes and Parkinson disease
(PD) have been observed in different populations. Meta
analyses confirmed GBA mutation as PD susceptibility
factor, but with various strength depend on ethnical origin.
In cohort of 300 Serbian PD patients we have performed
targeted DNA analysis in order to detect most frequent
GBA mutations in our population. Direct sequencing by
Sanger method of exons 8, 9 and 11of GBA gene was
conducted. The prevalence of GBA mutations in our PD
patients was significantly higher than in healthy population
(OR=5.5). In addition, we have performed transcranial brain
sonography-TCS in GBA-related PD patients in comparison
to asymptomatic carriers of heterozygous GBA mutations,
Gaucher disease patients without PD, sporadic PD (sPD)
patients and healthy controls. TCS findings in GBA-related
PD are significantly different from healthy subjects, but
quite consistent to those of patients with sPD. Our results
confirmed GBA mutations as important PD susceptibility
factor in Serbian PD cohort, but phenotypic characteristics
of GBA-related PD cases remain to be elucidated.
6
PREDAVANJA / LECTURES
Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / Cellular and molecular mechanisms of neurodegenerative processes
ULOGA AMP-OM AKTIVISANE KINAZE U
NEUROTOKSIČNOM DELOVANJU ALFASINUKLEINA IN VITRO
THE PROTECTIVE ROLE OF AMP-ACTIVATED
PROTEIN KINASE IN ALPHA-SYNUCLEIN
NEUROTOXICITY IN VITRO
I. Marković1, M. Dulović1, M. Jovanović1, Lj. HarhajiTrajković2, V. Kostić3, V. Trajković4
1
Institut za medicinsku i kliničku biohemiju; Medicinski
fakultet; Univerzitet u Beogradu; Srbija;
2
Institut za biološka istraživanja „Siniša Stanković“;
Univerzitet u Beogradu; Srbija;
3
Klinika za neurologiju; KCS; Univerzitet u Beogradu;
Srbija;
4
Institut za mikrobiologiju i imunologiju; Medicinski
fakultet; Univerzitet u Beogradu; Srbija
[email protected]
I. Markovic1, M. Dulovic1, M. Jovanovic1, Lj. HarhajiTrajkovic2, V. Kostic3, V. Trajkovic4
1
Institute of Medical and Clinical Biochemistry; School of
Medicine; University of Belgrade, Serbia;
2
Institute for Biological Research ”Sinisa Stankovic“;
University of Belgrade, Serbia;
3
Clinic for Neurology; Clinical Center of Serbia; University
of Belgrade, Serbia;
4
Institute of Microbiology and Immunology; School of
Medicine; University of Belgrade, Serbia
[email protected]
Alfa-sinuklein (ASYN) ima vrlo značajnu ulogu u
patogenezi Parkinsonove bolesti. Prekomerna ekspresija
(ASYN) u ćeliji dovodi do poremećaja funkcije
mitohondrija, što može uzrokovati smanjen kapacitet za
sintezu ATP-a, uz narušavanje odnosa ATP/AMP. Usled
toga, može doći do aktivacije najvažnijeg unutarćelijskog
energetskog senzora, AMP-om aktivisane kinaze.
Cilj ovog istraživanja je bio ispitivanje uloga
AMPK u preživljavanju diferentovanih SH-SY5Y ćelija
humanog neuroblastoma koje prekomerno eksprimiraju
normalni (wt) ASYN.
U uslovima diferencijacije retinoičnom kiselinom,
ASYN je indukovao smanjenje vijabiliteta SH-SY5Y ćelija,
uz smanjenje akrivacije AMPK i njoj nishodne kinaze
Raptor. Primena farmakoloških aktivatora AMPK,
metformina i AICAR-a, dovela je do smanjenja
neurotoksičnog efekta uzrokovanog ASYN, pri čemu ovaj
efekat nije bio bio praćen aktivacijom serin-treonin kinaze
Akt/PKB. Uprkos smanjenoj aktivaciji AMPK u uslovima
prekomerne ekspresije ASYN, došlo je do indukcije
autofagije, Činjenica da aktivatori AMPK nisu doveli do
dodatne indukcije autofagije, ukazuje da uočena autofagija
ne zavisi od aktivacije AMPK, i ne doprinosi
neuroprotektivnom dejstvu aktivacije AMPK. Primena
medijuma koji sadrži sekretovani ASYN, koji potiče od
ćelija sa prekomernom ekspresijom ASYN, uzrokovala je
smanjeno preživljavanje diferentovanih SH-SY5Y ćelija,
što je bilo praćeno smanjenjem aktivacije AMPK. Primena
aktivatora AMPK je i u ovom slučaju imala
neuroprotektivni efekat, dok je smanjenje ekspresije AMPK
dovelo do povećane osetljivosti SH-SY5Y ćelija na
neurotoksično delovanje vanćelijskog ASYN.
Dobijeni rezultati ukazuju da bi modulacija aktivnosti
AMPK mogla biti neuroprotektivna u uslovima delovanja
unutarćelijskog i vanćelijskog ASYN, što može biti
značajno u razmatranju novih terapijskih strategija u
Parkinsonovoj bolesti.
Alpha-synuclein (ASYN) is regarded as essential
in Parkinson’s disease pathogenesis. It has been
demonstrated that ASYN-overexpressing cells exhibit
multiple markers of mitochondrial dysfunction. As a result,
capacity for ATP generation may be decreased, as well as
ATP/AMP ratio, leading to the activation of main cellular
energy sensor, AMP-activated protein kinase (AMPK). In
the present study, we investigated the role of the main
intracellular energy sensor, AMP-activated protein kinase
(AMPK), in survival of retinoic acid-differentiated SHSY5Y human neuroblastoma cells inducibly expressing
wild type (wt) ASYN. The loss of viability in retinoic aciddifferentiated ASYN-overexpressing cells was associated
with a decrease in activation of AMPK and its downstream
target Raptor. AMPK activation by metformin or AICAR
reduced the neurotoxicity associated with wt ASYN
overexpression, acting independently of the prosurvival
kinase Akt. Despite AMPK downregulation in ASYNoverexpressing cells, autophagy induction, confirmed by an
increase in autophagic flux, was present and remained
unaltered by AMPK activators, implicating that the
observed autophagy was AMPK-independent and not
responsible for the neuroprotective effect. The conditioned
medium from ASYN-overexpressing cells, containing
secreted ASYN, inhibited AMPK activation in RAdifferentiated SH-SY5Y cells and reduced their viability,
but not in the presence of metformin or AICAR. Finally, the
RNA interference-mediated knockdown of AMPK
increased the sensitivity of SH-SY5Y cells to the harmful
effects of extracellular ASYN. These data confirm the
protective role of AMPK against toxicity of both
intracellular and extracellular wt ASYN, suggesting that
modulation of AMPK activity may be a promising
therapeutic strategy in Parkinson’s disease.
7
PREDAVANJA / LECTURES
Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / Cellular and molecular mechanisms of neurodegenerative processes
ASOCIJACIJA I EPISTAZE MIKRO RNK 137 I
ENZIMA ZA EDITOVANJE ADENOZINA U INOZIN
U MOLEKULIMA RNK KOD ŠIZOFRENIJE I
BIPOLARNOG POREMEĆAJA
ASSOCIATION AND EPISTASIS OF MIR-137 AND
ADENOSINE TO INOSINE RNA EDITING
ENZYMES IN SCHIZOPHRENIA AND BIPOLAR
DISORDER
S. Đurica1, S. Šviković1, M. Pantović2, J. Karanović1, G.
Brajušković1, S. Romac1, M. Ivković2, D. Savić Pavićević1
1
Centar za humanu molekularnu genetiku; Biološki fakultet
Univerziteta u Beogradu; Beograd; Srbija;
2
Klinika za psihijatriju; Klinički centar Srbije; Beograd;
Srbija
[email protected]
Uvod: Genetička predispozicija za razvoj
šizofrenije (SCZD) i bipolarnog poremećaja (BD) delimično
se preklapa. MIR137 rs1625579 pokazao je jednu od
najjačih asocijacija sa SCZD u studiji asocijacije čitavog
genoma. Dodatno, pokazana je asocijacija targeta
mikroRNK137 (miR137) sa SCZD i BD. Dezaminaze
adenozina koje deluju na RNK (ADAR enzimi) mogu
modifikovati i regulisati funkciju mikroRNK. Iako su
pokazane promene u obrascu editovanja nekoliko
transkripata kod psihijatrijskih poremećaja, regulatorni
uticaj ADAR enzima na mikroRNK kod ovih poremećaja
još nije ispitana.
Cilj: Ispitivanje asocijacije i epistaza izmeñu MIR137,
ADAR i ADARB1 gena sa SCZD i BD.
Materijal i metode: Populaciona studija slučajeva i
kontrola obuhvatila je 128 SCZD i 77 BD pacijenata,
dijagnostikovanih prema DSM-IV kriterijumu, kao i 163
zdrave kontrole. Dvanaest tagSNP-ova koji pokrivaju ceo
ADAR gen i jedanaest tagSNP-ova iz dezaminaznog
domena i 3-UTR-a ADARB1 gena odabrani su korišćenjem
HapMap baze podataka i HaploView softvera. Svi odabrani
SNP-ovi genotipizirani su pomoću TaqMan® eseja za
genotipizaciju SNP-ova. Podaci su analizirani korišćenjem
PLINK i MDR softvera.
Rezultati: Pokazali smo asocijaciju MIR137
rs1625579 sa združenim SCZD i BD entitetima (p=0.03,
OR=1.531) i ADARB1 rs1051385 sa SCZD (p=0.04,
OR=1,701). MDR analiza pokazala je sinergističku epistazu
izmeñu MIR137 i ADAR (rs6699729) i redundantnu
epistazu izmeñu MIR137 i ADARB1 (rs7277143 and
rs4818766) kod združenih SCZD i BD pacijenata.
Zaključak: Naši rezultati upućuju da miR137 i
ADAR enzimi, kao i njihove interakcije, doprinose
patogenezi SCZD i BD, podržavajući stanovište da su ovi
poremećaji suprotni krajevi kontinuuma psihoze. Oba
sistema mogla bi imati poligenski efekat i doprinos
heterogenosti fenotipa.
S. Đurica1, S. Šviković1, M. Pantović2, J. Karanović1, G.
Brajušković1, S. Romac1, M. Ivković2, D. Savić Pavićević1
1
Center for Human Molecular Genetics; Faculty of Biology;
University of Belgrade; Belgrade, Serbia;
2
Clinic of Psychiatry; Clinical Centre of Serbia; Belgrade,
Serbia
[email protected]
Introduction:
Genetic
vulnerability
to
schizophrenia (SCZD) and bipolar disorder (BD) is partially
overlapped. MIR137 rs1625579 showed one of the strongest
associations with SCZD identified by genome-wide
association study and microRNA137 (miR137) target loci
were associated with SCZD and BD. Adenosine deaminases
acting on RNA (ADARs) can modify and regulate
micoRNA function. Although changes in the editing pattern
of a few transcripts have been reported in psychiatric
patients regulatory impact of ADARs on microRNA in
psychiatric disorders has not been studied yet.
Aim: To examine association and epistasis
between MIR137 and ADARs genes with major psychosis.
Material and methods: A population-based casecontrol study included 128 SCZD individuals, 77 BD
individuals, all diagnosed according to DSM-IV, and 163
healthy controls. Twelve tagSNPs covering entire ADAR
gene and eleven tagSNPs covering deaminase coding region
and 3’-UTR of ADARB1 gene were selected using HapMap
database and HaploView software. All selected SNPs were
genotyped using TaqMan® SNP Genotyping Assays. Data
were analyzed by PLINK and MDR software.
Results: We showed association of MIR137
rs1625579 with grouped SCZD and BD entities (p=0.03,
OR=1.531) and ADARB1 rs1051385 with SCZD (p=0.04,
OR=1,701). MDR analysis showed synergistic epistasis
between MIR137 and ADAR (rs6699729) and redundant
epistasis between MIR137 and ADARB1 (rs7277143 and
rs4818766) in grouped SCZD and BD patients.
Conclusion: Our findings highlight miR137 and
ADARs and their interactions as potential contributors to
the pathogenesis SCZD and BD, supporting the idea of
them being the opposite ends of psychosis continuum. They
could both have polygenic effect and contribute to
phenotypic heterogeneity.
8
PREDAVANJA / LECTURES
Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / Cellular and molecular mechanisms of neurodegenerative processes
ISPITIVANJE KLINIČKOG POTENCIJALA
FOSFORILISANOG GLUKOKORTIOKOIDNOG
RECEPTORA KAO MARKERA DEPRESIJE
EXPLORING THE CLINICAL POTENTIAL OF
PHOSPHORYLATED GLUCOCORTICOID
RECEPTOR AS BIOMARKER OF DEPRESSION
M. Adžić1, I. Lukić1, M. Mitić1, J. Đorñević1, M.
Mihaljević2, M, Jovičić3, S. Andrić3, Z. Pavlović2, I.
Soldatović3, D. Savić4, N. Marić2,3, M. Krstić-Demonacos5
M. Radojčić1
1
Laboratorija za molekularnu biologiju i endokrinologiju;
Institut za nuklearne nauke “Vinča”; Univerzitet u
Beogradu; Beograd; Srbija
2
Klinika za psihijatriju; Klinički centar Srbije; Beograd;
Srbija
3
Medicinski fakultet; Univerzitet u Beogradu; Beograd;
Srbija
4
Laboratorija za teoretsku fiziku; Institut za nuklearne
nauke “Vinča”; Univerzitet u Beogradu; Beograd; Srbija
5
Univerzitet Salford; Mančester; Velika Britanija
[email protected]
M. Adžić1, I. Lukić1, M. Mitić1, J. Djordjević1, M.
Mihaljević2, M, Jovičić3, S. Andrić3, Z. Pavlović2, I.
Soldatović3, D. Savić4, N. Marić2,3, M. Krstić-Demonacos5,
M. Radojčić1
1
Laboratory of molecular biology; Institute of nuclear
sciences “Vinča”; University of Belgrade; Belgrade; Serbia;
2
Clinic for Psychiatry; Clinical Center of Serbia; Belgrade;
Serbia;
3
Faculty of Medicine; University of Belgrade; Belgrade;
Serbia;
4
Laboratory of theoretical physics; Institute of nuclear
sciences “Vinča”; University of Belgrade; Belgrade; Serbia
5
School of environment and life sciences; University of
Salford; Manchester; United Kingdom
[email protected]
Narušena signalizacija preko gluko-kortikoidnog
receptora (GR) je dobro poznat nalaz u istraživanjima
vezanim za depresivni poremećaj. Takoñe, zna se da
fosforilacija GR-a bitno utiče na njegovu funkciju.
S obzirom na gore pomenuto, ciljevi ovog
istraživanja su bili da utvrdimo da li postoji: 1) povezanost
izmeñu nivoa fosforilacije GR-a i trenutnih negativnih
emotivnih stanja kod zdravih ljudi, 2) razlike u fosforilaciji
GR-a izmeñu pacijenata sa akutnom epizodom depresije u
odnosu na kontrole i 3) da li dolazi do promene u
fosforilaciji GR-a sa poboljšanjem depresivnog stanja
tokom terapije repetitivnom transkranijalnom magnetnom
stimulacijom (rTMS) kao adjuvantnom metodom.
Fosforilacija GR-a na serinu 211 (pGR-S211) i 226
(pGR-S226) analizirana je Western blot tehnikom, u
jedarnom ekstraktu limfocita periferne krvi. Težina
depresije kod pacijenata je merena Hamiltonovom skalom
depresivnosti, a nivoi depresije, anksioznosti i stresa kod
zdravih ljudi korišćenjem Depression Anxiaty Stress Scale.
Kod zdravih žena postojala je značajna pozitivna
korekacija izmeñu pGR-S226 i nivoa depresije,
anksioznosti i stresa, kao i negativna korelacija izmeñu
odnosa pGR-S211/pGR-S226 i simptoma depresije.
Depresivni pacijenti su imali značajno više nivoe pGR-S226
kao i niži odnos pGR-S211/pGR-S226, što je ukazivalo na
smanjenu funkciju GR-a u depresiji. Poboljšanje
depresivnih simptoma kod pacijenata praćenih nakon rTMS
terapije, bilo je praćeno povećanjem odnosa pGRS211/pGR-S226.
Naši rezultati nude prve dokaze o vrednosti
istraživanja fosforilacije GR-a kao funkcionalnog
biomarkera za identifikavanje osoba sa rizikom za
oboljevanje od depresivnog poremćaja (barem kod žena),
osoba sa akutnom depresivnom epizodom, kao i za praćenje
poboljšanja depresivnih simptoma tokom lečenja.
Impairment in glucocorticoid-mediated negative
feedback is a well-established finding in depression. This
negative feedback involved the glucocorticoid receptor
(GR) whose function is reduced in depressive patients. The
GR function is also regulated via posttranslational
modification, phosphorylation.
Therefore, we sought to determine 1) a possible
relations of GR phosphorylation with current reports of
negative affective states in healty subjects, 2) the diffrences
in GR phosphorylation between currently depressed patients
and controls, and 3) changes in GR phosphorylation during
follow-up study of patients treated with repetitive
transcranial magnetic stimulation (rTMS) as adjuvant
method.
The GR phosphorylation at serine 211 (pGR-S211)
and 226 (pGR-S226) were measured using Western blot, in
nuclear extracts of peripheral lymphocites. The severity of
depression in patients was evaluated by Hamilton
Depression Scale, while the levels of depression, anxiaty
and stress in healty subjects were assessed by Depression
Anxiaty Stress Scale (DASS).
In healthy women, there was a positive correlation
between pGR-S226 and DASS scores, while pGRS211/pGR-S226 ratio was negatively correlated with reports
of depression. Depressed patients had significantly higher
levels of pGR-S226 but lower pGR-S211/pGR-S226 ratio
compared to the healthy, implying reduced GR
transcriptional activity. In follow-up study of rTMS treated
patients, improvement of depressive symptoms was
accompanied by elevation of pGR-S211/pGR-S226 ratio.
Our results offer the first evidences for the value of
GR phosphorylation-related research as potential biomarker
for indentifying individuals at risk (at least females),
patients with current depressive episode, as well as the
follow-up of improvement of depressive symptoms during
treatment.
9
PREDAVANJA / LECTURES
Razviće, plastičnost i ćelijska smrt / Development, plasticity and cell death
ERYTHROPOIETIN AND NEUROVASCULAR UNIT
IN THE SETTINGS OF ISCHEMIC STROKE
Z. Redzic
Department of Physiology; Faculty of Medicine; Kuwait
University; Kuwait
[email protected]
Hypoxia/ anoxia that occur during ischemic stroke
induces the activation of hypoxia-inducible factors (HIFs)
that modulate expression of over one hundred genes, which
control erythropoiesis, vasculogenesis and anaerobic
metabolism. HIFs also control expression of erythropoietin
(EPO). It has been revealed that cells of the neurovascular
unit (NVU), mainly astrocytes, but also neurons and
pericytes express EPO and release it during hypoxia/anoxia
in vitro and during middle cerebral artery occlusion
(MCAO) in vivo. EPOR is expressed by all cells of the
NVU and its expression increases several folds during
hypoxia/anoxia and during MCAO. Expression of EPOR in
BECs and pericytes appears to be complexly regulated,
since the observed increase in expression of EPOR during
hypoxia/anoxia is affected by paracrine signaling from
astrocytes and pericytes and from astrocytes, respectively.
EPO exerts clear protective effects on cells of the NVU
during hypoxia / anoxia. Presence of EPO significantly
reduces number of BECs in primary culture in early and late
apoptosis, but does not affect number of necrotic cells after
24h near anoxia. EPO also reduces number of pericytes in
primary culture in late apoptosis, but does not change
number of pericytes in early apoptosis after 24h near
anoxia. EPO also exerts clear protective effects for neurons
during near anoxia and oxygen glucose deprivation in vitro
as well as during MCAO in vivo. These data indicate that
EPO is involved in complex adaptive mechanism in the
NVU that is activated during cerebral ischemia.
10
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
INHIBICIJA AUTOFAGIJE DOVODI DO
ISPOLJAVANJA NEUROTOKSIČNOSTI
OLANZAPINA
Lj.Vučićević,1,2 V. Paunović,2 M. Misirkić,1,2 N. Marić3,
T.Martinović,4 D. Ćirić,4 S. Petričević,5 Lj. Harhaji-Trajković,1
V.Bumbaširević,4 V. Trajković2
1
Institut za biološka istraživanja; Univerzitet u Beogradu; Beograd;
Srbija;
2
Institut za mikrobiologiju i imunologiju; Medicinski fakultet u
Beogradu, Dr. Subotića 1, 11000 Beograd; Srbija;
3
Klinika za psihijatriju Kliničkog centra i Medicinskog fakulteta u
Beogradu; Beograd; Srbija;
4
Institut za histologiju i embriologiju Medicinskog fakulteta u
Beogradu; Beograd, Srbija;
5
Institut za biomedicinska istraživanja; Galenika a.d., Beograd;
Srbija
[email protected]
Uvod: Dugogodišnja terapija antipsihoticima je
povezana sa gubitkom moždane mase pacijenata sa
shizofrenijom. Autofagija može da spreči ili da doprinese
procesu ćelijske smrti neurona. Pokazano je da neki
antipsihotici indukuju autofagiju, ali njena uloga u
shizofreniji do sada nije istraživana.
Cilj: Ispitivanje uloge autofagije u preživljavanju neurona
izloženih atipičnom antipsihotiku olanzapinu.
Metode:Vijabilitet SH-SY5Y ćelija (ćelijska linija humanih
neurona) je odreñivan kristal violet i LDH testovima.
Oksidativni stres, apoptoza, depolarizacija mitohondrija i
unutarćelijska acidifikacija su analizirane protočnom
citofluorimetrijom. Indukcija autofagije i aktivacija
signalinih puteva je odreñivana metodoma imunoblota i/ili
real time PCR-om. Morfologija ćelija, autofagni fluks, kao i
ko-lokalizacija LC3 proteina i mitohondrija su
vizuelizovane
fluorescentnom,
elektronskom
i/ili
konfokalnom mikroskopijom.
Rezultati: Indukcija autofagije olanzapinom u SHSY5Y ćelijama je pokazana povećanjem unutarćelijske
acidifikacije, prisustvom autofagnog fluksa i povećanom
ekspresijom gena regulatora autofagije Atg4b, Atg5, Atg7 i
Atg12. Autofagija je indukovana proizvodnjom reaktivih
kiseoničnih vrsta i depolarizacijom mitohondrija bez učešća
AMPK/Akt/mTOR signalnog puta. Ko-lokalizacija
mitohondrija sa p62 i LC3, kao i elektronska mikroskopija
su potvrdile uklanjanje oštećenih mitohondrija autofagijom.
Ovim rezultatima je pokazano da sam olanzapin nije
toksičan za ćelije, meñutim blokiranje autofagije
farmakološkim inhibitorima, RNK interferencom i NO
donorima izaziva ćelijsku smrt. Čertnaestodnevni tretman
miševa sa olanzapinom povećava ekspresiju LC3-II proteína
kao i iRNK za p62, Atg4b, Atg7 i Atg12 u mozgu, dok
istovremeni tretman olanzapina sa inhibitorom autofagije
hlorokinom povećava ekspresiji proapoptotskih gena p53,
Noxa, Bax, Bim and p27.
Zaključak: Rezultati ove studije pokazuju da
olanzapin izaziva autofagiju koja uklanja oštećene
mitohondrije i da njena inhibicija dovodi do ispoljavanja
neurotoksičnih efekata olanzapina.
AUTOPHAGY INHIBITION UNCOVERS THE
NEUROTOXIC ACTION OF OLANZAPINE
Lj. Vucicevic,1,2 V. Paunovic,2 M. Misirkic,1,2 N.Maric,3
T.Martinovic,4 D. Ciric,4 S. Petricevic,5 Lj. Harhaji-Trajkovic,1
V.Bumbasirevic,4 V. Trajkovic2
1
Institute for Biological Research; University of Belgrade;
Belgrade; Serbia;
2
Institute of Microbiology and Immunology; School of Medicine;
University of Belgrade,;
Dr. Subotica 1, 11000 Belgrade; Serbia;
3
Clinic of Psychiatry; Clinical Centre of Serbia and School of
Medicine; University of Belgrade;
Belgrade; Serbia;
4
Institute of Histology and Embryology; School of Medicine;
University of Belgrade; Belgrade; Serbia;
5
Institute of Biomedical Research; Galenika a.d; Belgrade; Serbia
[email protected]
Introduction: Antipsychotic drugs may contribute
to brain tissue loss in schizophrenia. Autophagy can prevent
or enhance cell death, but its role in the side-effects of
antypsychotics is unclear.
Purpose: Investigating the role of autophagy in survival of
neurons exposed to atypical antipsychotic olanzapine.
Methods: The viability of human SH-SY5Y
neuronal cell line was measured by crystal violet and LDH
assay. Oxidative stress, mitochondrial depolarization and
intracellular acidification were analyzed by flow cytometry.
Induction of autophagy and autophagy-related signaling was
monitored by immunoblotting and/or real-time RT-PCR.
Cell morphology, autophagosome-lysosome fusion and colocalization of LC3 and mitochondria were assessed by
electron/fluorescence microscopy.
Results: Olanzapine induced autophagy in SHSY5Y cells, as it increased intracellular acidification,
autophagic flux, the presence of autophagosomes and their
fusion with lysosomes, and the expression of autophagyrelated genes Atg4b, Atg5, Atg7 and Atg12. Oxidative
stress and mitochondrial depolarization, but not modulation
of AMP/Akt/mTOR signaling, were responsible for
olanzapine-triggered autophagy. Mitochondrial association
with LC3 and p62, as well as electron microscopy,
confirmed the autophagic clearance of dysfunctional
mitochondria. While olanzapine was not toxic to SH-SY5Y
cells, their death was initiated upon pharmacological,
RNAi-mediated or nitric oxide-mediated inhibition of
autophagy. The treatment of mice with olanzapine for 14
days increased brain levels of LC3-II, p62, Atg4b, Atg7 and
Atg12, while the concomitant inhibition of autophagy
increased the expression of proapoptotic genes p53, Noxa,
Bax, Bim and p27.
Conclusions: Olanzapine-triggered autophagy
protects neurons from otherwise fatal mitochondrial damage
and the inhibition of autophagy unmasks the neurotoxic
action of the drug.
57
PREDAVANJA / LECTURES
Razviće, plastičnost i ćelijska smrt / Development, plasticity and cell death
KONSTITUTIVNA EKSPRESIJA RECEPTORA
EPIDERMALNOG FAKTORA RASTA (EGFR)
MENJA SUDBINU I KARAKTERISTIKE
NEURALNIH PROGENITORA
CONSTITUTIVE EXPRESSION OF EPIDERMAL
GROWTH FACTOR
RECEPTOR (EGFR) ALTERS THE FATE AND
PROPERTIES OF NEURAL PROGENITORS
S. Ivković
Institut za molekularnu medicinu; Lisabon; Portugal
[email protected]
S. Ivkovic
Instituto de Medecina Molecular; Lisbon; Portugal
[email protected]
Razviće zahteva pravilnu orkestraciju migracije,
diferencijacije i proliferacije progenitora. Signalizacija
preko receptora epidermalnog faktora rasta (EGFR) je
uključena u regulaciju ovih procesa u različitim ćelijama,
uključujući i neuralne progenitore. Ispitivali smo dve vrste
modela neuralne diferencijacije – gliogenezu u postnatalnoj
beloj masi kod pacova i postnatalno razviće retine kod miša.
Metodom infekcije retrovirusima
indukovali smo
konstitutivnu ekspresiju EGFR-GFP proteina u postnatalnoj
beloj masi i u retinalnim progenitorima.
EGFR-GFP+ ćelije u beloj masi nastavljaju da
proliferišu i migriraju i da na taj način postepeno
popunjavaju moždani prostor ipsi i kontra-lateralno od
mesta inekcije. Ove ćelije se, pri tome, nikada ne
diferencirale u zrelu gliju. Akumulacija ovih ćelija duplira
totalnu ćelijsku gustinu u beloj masi i povećava
desetostruko broj glijalnih progenitora, dovodeći na taj
način do patoloskog stanja poznatog kao ‘hiperplazija’
progenitora.
EGFR-GFP+ celije u retinalnim eksplantim se
takodje razlikuju od kontrolnih ćelija. Dok se kontrolne
ćelije diferenciraju u fotoreceptore, amakrine, horizontalne
ili bipolarne ćelije, EGFR-GFP+ ćelije kasne sa
diferencijacijom a zatim prevashodno postaju amakrine ili
horizontalne ćelije. Stimulacija eksplanata sa EGF-om i/ili
inhibitorom Notch signalizacije (LY) dodatno menja EGFRGFP+ ćelije forsirajući ih da se diferenciraju u
fotoreceptore.
Ovi rezultati sugerišu da konstitutivna ekspresija
EGFR-a dovodi do toga da inficirane progenitorske celije
reaguju drugačije na signale iz okoloćelijske sredine i na taj
način ili postaju permanentno nediferencirane ili prihvataju
alternativne ćelijske sudbine.
Proper development requires the careful
orchestration of migration, differentiation, and proliferation
of progenitors. Signaling through the epidermal growth
factor receptor (EGFR) has been implicated in regulating
these processes in a variety of cell types, including neural
progenitors. We have explored two different models of
neural differentiation – gliogenesis in rat postnatal white
matter and retinal postnatal development in mice. By
retroviral infection, we constitutively expressed an EGFRGFP fusion protein in both postnatal white matter and in
retinal progenitors.
EGFR-GFP+ cells in white matter remained
proliferative and migratory, gradually populating the brains
ipsilateral and contralateral to the side of viral infection, but
never differentiated into mature glia. The accumulation of
these cells doubled the total cell density in white matter and
led to a 10-fold increase in the abundance of glial
progenitors, giving rise to a progenitor "hyperplasia."
The marker profile of infected cells indicated a close
resemblance to oligodendrocyte progenitors.
Similarly, EGFR-GFP+ cells differ from controlGFP infected cells in retinal explants. While Control-GFP +
cells differentiated into photoreceptors, amacrine, horizontal
or bipolar cells EGFR-GFP+ cells exhibited delayed
differentiation and preferentially acquired the fate of
horizontal or amacrine cells. Addition of EGF and/or Notch
signaling inhibitors (LY) further changes the fate of EGFRGFP+ cells driving them to differentiate into
photoreceptors.
These data strongly suggest that the constitutive
EGF signaling in progenitor cells renders the cells
unresponsive to differentiating cues from the surrounding
tissue thus keeping them undifferentiated or forcing them to
adopt the alternative fate choices.
11
PREDAVANJA / LECTURES
Razviće, plastičnost i ćelijska smrt / Development, plasticity and cell death
HOMEOSTAZA HOLESTEROLA U MOZGU:
EFEKAT STARENJA I DIJETALNE RESTRIKCIJE
CHOLESTEROL HOMEOSTASIS IN THE BRAIN:
THE EFFECTS OF AGING AND DIETARY
RESTRICTION
A. Mladenović Đorñević 1, K. Smiljanić1, M. Perović1, V.
Tešić1, N. Lončarević-Vasiljković1, S. Todorović1, Lj.
Rakić2, S. Ruždijić1, S. Kanazir1
1
Institut za biološka istraživanja “Siniša Stanković”,
Univerzitet u Beogradu, 11060 Beograd, Srbija
2
Srpska akademija nauka i umetnosti, 11000 Beograd,
Srbija
[email protected]
A. Mladenovic Djordjevic1, K. Smiljanic1, M. Perovic1, V.
Tesic1, N. Loncarevic-Vasiljkovic1, S. Todorovic1, Lj.
Rakic2, S. Ruzdijic1, S. Kanazir1
1
Institute for Biological Research “Sinisa Stankovic”,
University of Belgrade, 11060 Belgrade, Serbia
2
Serbian Academy of Sciences and Arts, 11000 Belgrade,
Serbia
[email protected]
Holesterol je najzastupljeniji lipid u ćelijama
sisara. Više od 25% totalnog holesterola u ljudskom telu se
nalazi u CNS-u, a skoro čitav moždani holesterol se
sintetiše in situ. Neophodan je za formiranje membrana,
reguliše njihovu fluidnost, propustljivost i rigidnost.
Učestvuje u formiranju aksona i sinapsi, biogenezi
sinaptičkih vezikula, procesima učenja i pamćenja, pa se
zbog svojih mnogobrojnih i kompleksnih uloga u CNS-u
smatra važnim faktorom nervne plastičnosti. Održavanje
homeostaze holesterola u mozgu se postiže strogo
koordinisanom regulacijom sinteze, transporta i eliminacije
viška holesterola iz mozga, a narušena homeostaza
holesterola se nalazi u osnovi brojnih neurodegenerativnih
bolesti.
Da bi ispitali efekte starenja i različitih režima
ishrane (DR) na homeostazu holesterola pratili smo
promene u količini holesterola, njegovih prekursora i
metabolita, u mozgu i serumu, kao i promene
u ekspresiji ključnih proteina uključenih u metabolizam
holesterola (HMGCR, Cyp46, ApoE) u različitim
strukturama mozga.
Eksperimenti su rañeni na mužjacima pacova
Wistar soja različite starosti i podvrgnutim različitim
režimima ishrane. Promene u ekspresiji iRNK su praćene
Real-Time PCR tehnikom, a promene na nivou proteina
Western blot analizom. Količina holesterola, njegovih
prekursora i metabolita, kao i biljnih sterola je odreñena
gasnom
hromatografijom/masenom
spektrometrijom
(GC/MS). Dobijeni rezultati su pokazali da starenje i DR
utiču na na prekursore holesterola i ekspresiju proteina
uključenih u metabolizam holesterola na regionalnospecifičan način, dok je nivo holesterola i njegovih
metabolita ostao nepromenjen.
Ova studija ukazuje na još jedan od mehanizama
pozitivnog delovanja dijetalne restrikcije na modulaciju
sinaptičke plastičnosti kroz održavanje stabilnog nivoa
holesterola u korteksu i hipokampusu tokom starenja.
Cholesterol is an important factor in neural
plasticity. It is required for the membrane and synapses
formation, synaptic vesicle biogenesis, learning and
memory. More than 25% of the total cholesterol in the
human body located in the CNS, and almost the whole brain
cholesterol is synthesized in situ. Cholesterol homeostasis in
the brain is achieved by strictly coordinated regulation of its
synthesis, transport and elimination, while disturbed
cholesterol homeostasis underlies many neurodegenerative
diseases.
To investigate the effects of aging and various
dietary regimen (DR) on cholesterol homeostasis, we
monitored changes in the amount of cholesterol and its
precursors and metabolites in the brain and serum, as well
as changes in the expression of key proteins involved in
cholesterol metabolism (HMGCR, Cyp46, ApoE) in
different brain structures.
Experiments were performed on male Wistar rats
of different ages subjected to different feeding regiments.
mRNA expression were followed by real-time PCR
technique while protein levels were determined by Western
blot analysis. Cholesterol, its precursors and metabolites
were measured by gas chromatography/mass spectrometry.
The results showed that aging and DR influence on
cholesterol precursors and the expression of proteins
involved in cholesterol metabolism in regionally-specific
manner, whereas the level of cholesterol and its metabolites
remained unchanged.
In conclusion, this study points to additional
beneficial mechanism of dietary restriction on the
modulation of synaptic plasticity by maintaining a stable
cholesterol level in the cortex and hippocampus during
aging.
12
PREDAVANJA / LECTURES
Razviće, plastičnost i ćelijska smrt / Development, plasticity and cell death
KRATKOTRAJNA ANESTEZIJA INDUKOVANA
PROPOFOLOM UTIČE NA NEURALNU
AKTIVNOST I SINAPTICKU PLASTIČNOST U
NEONATALNOM PERIODU I IZAZIVA
DUGOTRAJNE PROMENE U PONAŠANJU
SHORT-TERM PROPOFOL ANESTHESIA
INFLUENCES NEURAL ACTIVITY AND SYNAPTIC
PLASTICITY IN NEONATES AND INDUCES
LASTING BEHAVIORAL CHANGES
V. Pešić1, J. Popić1, D. Milanović1, 3, Lj. Rakic2, S. Kanazir1,
V. Jevtović-Todorović3, S. Ruždijić1
1
Institut za biološka istraživanja Siniša Stanković;
Univerzitet u Beogradu; 11060 Beograd; Srbija;
2
Srpska akademija nauka i umetnosti, Beograd; Srbija;
3
Department of Anesthesiology; University of Virginia
Health System; Charlottesville; VA; USA
[email protected]
V. Pesic1, J. Popic1, D. Milanovic1, 3, Lj. Rakić2, S. Kanazir1,
V. Jevtovic-Todorovic3, S. Ruzdijic1
1
Institute for Biological Research Sinisa Stankovic;
University of Belgrade; 11060 Belgrade; Serbia;
2
Serbian Academy of Science and Arts; Belgrade; Serbia;
3
Department of Anesthesiology; University of Virginia
Health System; Charlottesville; VA; USA
[email protected]
Propofolska anestezija je široko korišćena u
pedijatrijskoj medicini, mada eksperimentalni rezultati
ukazuju da tokom neonatalnog perioda ona može da
indukuje neuroapoptozu i dugotrajne promene u morfologiji
dendrita. Kao i većina opštih anestetika propofol snažno
utiče na neuronsku aktivnost talamokortikalnog sistema.
Shodno tome naša istraživanja su rañena sa ciljem da se
ispita kako kratkotrajna propofolska anestezija primenjena u
periodu intenzivne sinaptogeneze kod pacova (postnatalni
dan 7 – PND7) utiče na ekspresiju odreñenih gena i proteina
koji su regulisani upravo aktivnošću neurona, i da li ima
dugotrajnih posledica na ponašanje.
Tehnikama RT-PCR i Western blot analize
praćene su promene u ekspresiji BDNF-a i c-Fos-a u
korteksu i talamusu PND7 pacova. Takoñe su ispitivane
promene u zastupljenosti sinaptičkih proteina (GAP-43,
sinaptofizin, drebrin). D-amfetaminom izazvana motorika je
praćena na PND35 pacovima koji su bili izloženi propofolu
u PND7.
Rezultati su pokazali da kod PND7 pacova
kratkotrajna propofolska anestezija smanjuje ekspresiju
iRNK za BDNF u korteksu i talamusu, naročito za egzon IV
i egzon VI transkripte. Detektovane su i promene u nivou
BDNF-a, c-Fos-a kao i svih ispitivanih sinaptičkih proteina.
Nakon primene d-amfetamina kod PND35 pacova (koji su
dobiili propofol u PND7) detektovano je značajno
povećanje totalne lokomotorne aktivnosti u drugom i
značajno smanjenje stereotipiji-sličnoj aktivnosti u prvom
satu.
Naša studija ukazuje da kratkotrajna propofolska
anestezija primenjena u periodu intenzivne sinaptogeneze
značajno utiče na procese koji su zavisni od neuralne
aktivnosti i moždanu plastičnost, što ima trajne posledice na
integritet kortiko-strijato-talamo-kortikalnih petlji. Ovim je
otvoreno pitanje funkcionalnih posledica primene
kratkotrajne anestezije u periodu intenzivne sinaptogeneze.
Propofol anesthesia is widely used in pediatric
medicine, although experimental data indicate that in
neonates it can induce neuroapoptosis and persistent
changes in dendritic spine density. As most general
anesthetics, propofol strongly depresses neuronal activity in
the thalamocortical system. We examined whether shortterm propofol anesthesia applied to 7-day-old rats (PND7;
the period of rapid synaptogenesis), perturbs the expression
of activity-regulated genes, the respective mRNAs and
synaptic proteins. A functional outcome of neonatal
exposure to anesthesia was expected.
We investigated by RT-PCR and Western blot
analysis how propofol anesthesia changes BDNF and c-Fos
expression in the cortex and thalamus of PND7 rats.
Synaptic proteins GAP-43, synaptophysin and drebrin were
also examined. The behavioral consequences of neonatal
propofol anesthesia were estimated through analysis of
spontaneous- and d-amphetamine-induced changes in the
motor activity of PND35 animals.
Short-term exposure of PND7 rats to propofol in a
time-dependent manner decreased BDNF mRNA
expression in the cortex and thalamus, especially exon IVand exon VI-containing transcripts. We detected time- and
region-specific alterations in BDNF, c-Fos, GAP-43,
synaptophysin and drebrin expression. After d-amphetamine
injection, significant increases in the total distance traveled
during the second hour, and a significant decrease of total
stereotypy-like activity during the first hour were detected
in PND35 rats exposed to propofol at PND7.
Our study revealed that in PND7 rats, short-term
propofol anesthesia influenced activity-dependent processes
and brain plasticity, permanently changing the integrity of
the cortico-striato-thalamo-cortical loops. This opens an
important question as to the functional consequences of
exposure to short-term anesthesia during intensive
synaptogenesis.
13
PREDAVANJA / LECTURES
Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / Cellular and molecular mechanisms of neuroinflammation
ULOGA EKTONUKLEOTIDAZA U INTERAKCIJI
NERVNOG I IMUNSKOG SISTEMA
THE ROLE OF ECTONUCLEOTIDASES IN THE
INTERPLAY BETWEEN THE BRAIN AND THE
IMMUNE SYSTEM
N. Nedeljkovića, D. Briševaca, A. Parabuckib, M. Adžića, D.
Savićb, I. Lavrnjab, D. Laketaa, I. Božićb, S. Pekovićb, M.
Stojiljkovićb, I. Bjelobabab
a
Institut za fiziologiju i biohemiju; Biološki fakultet
Univerziteta u Beogradu; Srbija
b
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; Srbija
[email protected]
N. Nedeljkovića, D. Briševaca, A. Parabuckib, M. Adžića, D.
Savićb, I. Lavrnjab, D. Laketaa, I. Božićb, S. Pekovićb, M.
Stojiljkovićb, I. Bjelobabab
a
Institute for Physiology and Biochemistry, Faculty of
Biology; University Belgrade; Belgrade; Serbia;
b
Institute for Biological Research “SInisa Stanković”;
University of Belgrade; Serbia
[email protected]
Adenozin trifosfat (ATP), osim uloge glavnog
energetskog molekula unutar ćelije, u vanćelijskom prostoru
deluje kao signal povezan sa oštećenjem tkiva (engl.
danger-associated molecular pattern), odnosno, kao signalni
molekul koji u centralnom nervnom sistemu (CNS) inicira
reaktivni odgovor mikroglije i astrocita. Signalna uloga
ATP direktno je povezana sa signalnom ulogom adenozina,
koji nastaje enzimskom razgradnjom ATP. Adenozin,
suprotno ATP, ostvaruje snažne anti-inflamatorne efekte u
CNS. Vanćelijske koncentracije ATP i adenozina pod
direktnom su kontrolom ektonukleotidaza, enzima koji
katalizuju postupnu hidrolizu ATP do Ado kao krajnjeg
proizvoda. Glavni predstavnici porodice ektonukleotidaza
su nukleozid-trifosfat difosfohidrolaza1 (NTPD-aza1), koja
razgrañuje ATP i ADP i ekto-5’-nukleotidaza (eN), koja
katalizuje završnu fazu hidrolize AMP do adenozina.
Prethodni rezultati naše grupe pokazuju snažnu ushodnu
regulaciju NTPD-aze1 i eN na reaktivnim astrocitima i
mikrogliji u nekoliko eksperimentalnih modela neuroloških
bolesti (traumatska mozga, autoimunski encefalomijelitis i
amiotrofna lateralna skleroza). Rezultati dobijeni u in vitro
sistemu astrocita, dodatno su potvrdili da je ekspresija
NTPDaze1 and eN pod transkripcionom kontrolom
nekoliko ključnih inflamatornih citokina, kao što su TNF-α i
INF-γ. Oba enzima, NTPD-aza1 i eN, pripadaju porodici
ćelijskih adehezionih molekula (engl. cluster of
differention) i nose oznake CD39, odnosno, CD73. Na
osnovu naših rezultata i rezultata drugih grupa, postavili
smo hipotezu da NTPD-aza1/CD39 i eN/CD73, regulišući
odnos vanćelijskog ATP i adenozina i delujući kao ćelijski
adhezioni molekuli, presudno utiču na razvoj inflamatornog
fenotipa astrocita i mikroglije in vivo. Biće predstavljeni i
diskutovani najnoviji rezultati naše grupe koji idu u prilog
navedenoj hipotezi.
Besides well-know intracellular roles, adenosine
triphosphate (ATP) in the extracellular space acts as
damage-associated molecular pattern, i.e. signaling
molecule that initiates immune responses of microglia and
astrocytes in the CNS. Signaling role of ATP is closely
related to its final breakdown product, the nucleoside
adenosine, which in contrast to ATP, exhibits antiinflammatory actions. Extracellular ATP and adenosine
levels are under the control of ectonucleotidase enzymes,
which are involved in the sequential conversion of ATP to
adenosine. The principal enzymes of the family are
nucleoside triphoshate diphosphohydrolase1 (NTPDase1),
which degrades ATP and ADP, and ecto-5’-nucleotidase
(eN), which catalyzes final degradation of AMP to
adenosine. Previous results of our group consistently
demonstrate a link between up-regulation of NTPDase1 and
eN and reactive astrogliosis in several models of human
brain pathologies. We were also able to show that
expression of NTPDase1 and eN are under modulatory
control by several pro-inflammatory factors, including
master proinflammatory cytokines TNF-α and INF-γ. It is
worth noting that both NTPDase1 and eN belong to the
cluster of differentiation family with a designation CD39
and CD73, respectively. Based on our work and work of
other groups, we hipothesized that NTPDase1/CD39 and
eN/CD73, by controling the levels of pro-inflammatory
ATP and anti-inflammatory adenosine and by acting as cell
adhesion molecules, crucially determine inflammatory
phenotype of astrocytes and microglia in vivo. Most recent
results of our group that support this view will be presented
and discussed.
14
PREDAVANJA / LECTURES
Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / Cellular and molecular mechanisms of neuroinflammation
REDOKS PROCESI U INICIJACIJI MULTIPLE
SKLEROZE
REDOX PROCESSES IN MULTIPLE SCLEROSIS
INITIATION
Đ. Miljković1, I. Spasojević2
1
Odeljenje za imunologiju; Institut za biološka istraživanja
“Siniša Stanković”; Univerzitet u Beogradu; Beograd;
Srbija;
2
Odsek za nauku o živim sistemima; Institut za
multidisciplinarna istraživanja; Univerzitet u Beogradu;
Beograd; Srbija
[email protected]
Đ. Miljković1, I. Spasojević2
1
Department of Immunology; Institute for Biological
Research “Siniša Stanković”; University of Belgrade;
Belgrade; Serbia;
2
Life Sciences Department, Institute for Multidisciplinary
Research; University of Belgrade; Belgrade; Serbia
[email protected]
Multipla skleroza (MS) je inflamatorno,
demijelinizirajuće i neurodegenerativno oboljenje CNS.
Klinička ekspresija ove bolesti je umnogome posledica
autoimunskog odgovora usmerenog protiv antigena i
struktura CNS. Postoje dve osnovne hipoteze inicijacije
MS. Po jednoj, promene i oštećenja u CNS prethode
autoimunskoj reakciji, takozvana “inside-out” hipoteza, dok
su po drugoj autoreaktivne ćelije imunskog sistema te koje
dovode do primarnih promena u MS, takozvana “outsidein” hipoteza. Mi predlažemo novi concept inicijacije MS
koji uključuje nekoliko bitnih komponenti patogeneze ove
bolesti,
uključujući
vaskularnu,
redoks,
inflamatornu/autoimunsku i neurodegenerativnu. Smatramo
da bi redoks procesi mogli imati centralnu ulogu u inicijaciji
neuroinflamacije, demijenizacije i neurodegeneracije u MS.
Multiple
sclerosis
is
an
inflammatory,
demyelinating and neurodegenerative disease of the CNS.
Autoimmune response directed against antigens and
structures of the CNS largely contributes to the clinical
expression of MS. Yet, there is a disagreement within the
MS research community regarding the chain of events that
initiate clinically determined MS. There are those who favor
the “inside-out” hypothesis, i.e. damage to the CNS comes
first followed by an autoimmune reaction, and those who
support “outside-in” hypothesis, i.e. CNS-specific
autoreactive lymphocytes are the initial pathogenic entities.
We propose a novel concept of the disease initiation that
integrates several important components, including
vascular,
redox,
inflammatory/autoimmune
and
neurodegenerative. We believe that redox processes might
have a central role in the initiation of neuroinflammation,
demyelination and neurodegeneration in some forms of MS.
15
PREDAVANJA / LECTURES
Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / Cellular and molecular mechanisms of neuroinflammation
EKSPRESIJA GENA POVEZANIH SA
ODGOVOROM POMOĆNIČKIH T-ĆELIJAMA TIP 1
I 17 U TERAPIJI PACIJENATA SA RELAPSNOREMITENRNOM MULTIPLOM SKLEROZOM
TOKOM TERAPIJE INTERFERONONOM-Β TROGODIŠNJE PRAĆENJE
EXPRESSION OF T HELPER CELLS 1/17-RELATED
GENES IN INTERFERON-Β-TREATED
RELAPSING-REMITTING MULTIPLE SCLEROSIS
PATIENTS - THREE YEAR FOLLOW-UP
E. Savić1, M. Marković1, J. Drulović2, I. Dujmović
Bašuorski2, S. Mesaroš2, V. Pravica1, D. Popadić1, M.
Mostarica Stojković1
1
Institut za mikrobiologiju i imunologiju; Medicinski
fakultet; Unverzitet u Beogradu; Beograd; Srbija;
2
Institute za neurologiju; Klinički centar Srbije; Medicinski
fakultet; Unverzitet u Beogradu; Beograd; Srbija
[email protected]
E. Savic1, M. Markovic1, J. Drulovic2, I. Dujmovic
Basuorski2, S. Mesaros2, V. Pravica1, D. Popadic1, M.
Mostarica Stojkovic1
1
Institute of Microbiology and Immunology; School of
Medicine; University of Belgrade; Belgrade; Serbia;
2
Institute of Neurology; Clinical Centre of Serbia; School of
Medicine; University of Belgrade; Belgrade; Serbia
[email protected]
Uvod: U multiploj sklerozi (MS), pomoćnički Tlimfociti (Th) 1 i Th17 se smatraju orkestratorima
uništavanja mijelina. Mehanizam povoljnog dejstva terapije
interferonom (IFN)-β kod dela pacijenata sa relapsnoremitentnom (RR) MS, i njegov neuspeh kod ostalih nije
objašnjen.
Cilj: Ispivali smo profile genske ekspresije
molekula Th1/17 osovine tokom 36 meseci kod pacijenata
sa RRMS na terapiji IFN-β.
Material i metode: Smatrali smo da RRMS
pacijenti (n=45) odgovaraju na IFN-β1b terapiju (R, n=20),
ako nisu imali relapse ni povećanje onesposobljenosti
tokom 3 godine, a da ostali ne odgovaraju (NR, n=25).
Relativna genska ekspresija je odreñena qPCR-om za:
marker biološkog odgovora na IFN-β (MX1), Th1/17
polarišuće citokine (IL-12/23p40, IL-12p35, IL-23p19),
njihove receptore (IL-12Rβ1, IL-12Rβ2, IL-23R),
transcripcione faktore (Tbet, RoRγt) i efektorske citokine
(IFN-γ, IL-17A, IL-17F, GM-CSF) u mononuklearnim
ćelijanma periferne krvi pre početka i nakon 6, 12, 24 i 36
meseci terapije IFN-β.
Rezultati: R i NR su imali slične MX1 nivoe
iRNK. U obe grupe, IL-12/23p40, IL-12p35, IL-23p19 i
GM-CSF iRNK ekspresija je snižena, a IL-23R, RoRγt i
IFN-γ statistički značajno povećala nakon 6 meseci. Nivoi
svih iRNK su se vratili na nivoe pre terapije nakon 24
meseca. Nakon 36 meseci, IL-12/23p40, IL-23p19 i Tbet
iRNK nivoi su se snizili, dok su nivoi drugih gena, osim
IFN-γ i IL-12Rβ1, statistički značajno porasli, i kod R i NR.
Zaključak: R i NR se nisu razlikovali u biološkom
odgovoru na IFN-β, prema nivoima iRNK za MX1. Nivoi
ekspresije gena Th1/17 osovina nisu razlikovali R and NR.
Ipak, efekat terapije IFN-β na promene iRNK u različitim
vremenskim trenucima zahteva dalje ispitivanje.
Introduction: In multiple sclerosis (MS), T helper
cells (Th) 1 and Th17 are considered the orchestrators of the
myelin destruction. However, mechanism of favorable
action of interferon (IFN)-β treatment in proportion of
relapsing-remitting (RR) MS patients, and its flaw in the
rest remains elusive.
Aim: We investigated 36 months expression
profiles of molecules of Th1/17 axes in IFN-β-treated
RRMS patients.
Material and methods: RRMS patients (n=45) on
IFN-β1b therapy were considered responders (R, n=20), if
without relapses or progression on EDSS during 3 year
follow-up and the rest as non-responders (NR, n=25).
Relative gene expression was determined by qPCR for
marker of IFN-β biological response (MX1), Th1/17
polarizing cytokines (IL-12/23p40, IL-12p35, IL-23p19),
respective receptors (IL-12Rβ1, IL-12Rβ2, IL-23R),
transcription factors (Tbet, RoRγt) and effector cytokines
(IFN-γ, IL-17A, IL-17F, GM-CSF) in peripheral blood
mononuclear cells before start and at 6, 12, 24 and 36
months of IFN-β therapy.
Results: R and NR had similar MX1 mRNA levels.
In both groups, IL-12/23p40, IL-12p35, IL-23p19 and GMCSF mRNA expression decreased, while IL-23R, RoRγt
and IFN-γ statistically significantly increased at 6 months.
All mRNA levels reverted to baseline at 24 months. At 36
months, IL-12/23p40, IL-23p19 and Tbet mRNA levels
decreased, while other for other genes, beside IFN-γ and IL12Rβ1, they increased statistically significantly, both in R
and NR.
Conclusion: R and NR did not differ in IFN-β
biological response, according to MX1 mRNA levels.
Expression levels of Th1/17 axes’ genes did not distinguish
R and NR. However, the effect of IFN-β treatment on
timepoint-to-timepoint changes in mRNA warrants further
investigation.
16
PREDAVANJA / LECTURES
Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / Cellular and molecular mechanisms of neuroinflammation
BV-2 MIKROGLIJSKA ĆELIJSKA LINIJA KAO
MODEL IZUČAVANJA NEUROINFLAMACIJE EFEKAT BENFOTIAMINA
BV-2 MICROGLIAL CELL LINE AS A MODEL FOR
THE STUDY OF NEUROINFLAMMATION-EFFECT
OF BENFOTIAMINE
I. Lavrnja1, I. Božić1, D. Savic1, N. Nedeljković2,
S.Peković1, M. Stojiljković1
1
Odeljenje za neurobiologiju; Institut za biološka
istraživanja "Siniša Stanković"; Univerzitet u Beogradu;
Beograd; Srbija;
2
Institut za fiziologiju i biohemiju; Biološki fakultet;
Univerzitet u Beogradu; Beograd; Srbija
[email protected]
I. Lavrnja1, I. Bozic1, D. Savic1, N.Nedeljkovic2, S.
Pekovic1, M. Stojiljkovic1
1
Department of Neurobiology; Institute for Biological
Research “Sinisa Stankovic”; University of Belgrade;
Belgrade; Serbia;
2
Institute of Physiology and Biochemistry; Faculty of
Biology; University of Belgrade; Belgrade; Serbia
[email protected]
Mikroglijske ćelije igraju važnu ulogu u
patogenezi neurodegenerativnih bolesti. Ispitivanje uloge
mikroglije
u
neurodegeneraciji,
toksikologiji
i
farmaceutskim studijama zahteva veliki broj životinja za
rad. Upotrebom ćelijskih linija omogućava se dobijanje
veće količine ćelijskog materijala uz ograničeno korišćenje
životinja; a lakše su i jeftinije za rad i održavanje. BV-2
ćelijska linija je dobijena retrovirusnom transformacijom
mikroglijskih ćelija neonatalnih miševa (C57BL/6) na v-raf
i v-myc onkogenu.
Imajući u vidu da se agensi koji doprinose
smanjenju inflamacije mogu koristiti u tretiranju različitih
neurodegenerativnih bolesti, cilj ove studije je bio da se
ispita uticaj benfotiamina (S-benzoiltiamin O-monofosfata),
koji predstavlja liposolubilnu formu vitamina B1, na
aktiviranu mikrogliju u in vitro uslovima.
BV-2 ćelije su aktivirane lipopolisaharidom (LPS),
za koga je pokazano da је moćni aktivator imunskog
sistema. LPS izaziva promene različitih pokazatelja
neuroinflamacije. Ove promene su merene ELISOM,
Western
blotom,
RT-PCRom,
FACSom
i
imunofluorescencom i uporedjivane sa BV-2 aktiviranim
ćelijama koje su tretirane benfotiaminom.
BV-2 ćelije koje su aktivirane LPSom produkuju
znatno veću količinu azot-oksida i proinflamatornih
citokina, kao što je faktor nekroze tumora -α u odnosu na
nestimulisane ćelije. Pretretman S-benzoiltiamin Omonofosfatom značajno smanjuje nivo koncentracije NO i
proinflamatornih citokina kod BV-2 ćelija aktiviranih
LPSom. Benfotiamin smanjuje apoptozu aktiviranih
mikroglijskih ćelija. Takoñe, benfotiamin učestvuje u
regulaciji signalnih puteva, kao što su nuklearni faktor B
(NF-κB) i mitogenom aktivirane proteinske kinaze
(MAPK).
Predstavljeni rezultati pokazuju da benfotiamin
može da moduliše mikroglijski inflamatorni odgovor. Stoga,
benfotiamin može imati primenu u tretiranju različitih
neuroinflamatornih bolesti.
Microglial cells play an important role in the
pathogenesis of neurodegenerative diseases. Investigation of
the role of microglia in neurodegeneration, toxicology and
pharmaceutical studies require large numbers of animals.
Use of a microglia cell line would facilitate getting larger
amounts of cellular material with limited use of animals;
they are easier and less expensive to work and maintain.
BV-2 cell line has been generated by retroviral
transformation of microglial cells of neonatal mice
(C57BL/6) with v-raf/v-myc carrying retrovirus.
Having in mind that drugs involved in attenuating
inflammation can be used in treating various
neurodegenerative diseases, the aim of this study was to
explore the effect of benfotiamine, (S-benzoiltiamin Omonofosfata) a lipid-soluble analogue of vitamin B1, on
activated microglial cells in vitro.
BV-2 cells were activated with lipopolysaccharide
(LPS), a powerful activator of the immune system. LPS
causes changes in various indicators of neuroinflammation
that were measured by ELISA, Western blot, RT-PCR,
FACS and compared with activated BV-2 cells treated with
benfotiamine.
LPS-activated BV-2 cells produce a significantly
greater amount of nitric oxide and proinflammatory
cytokines such as tumor necrosis factor - α compared to
unstimulated cells. Pretreatment with benfotiamine reduces
the concentration of NO and pro-inflammatory cytokines in
LPS-stimulated BV-2 cells. Benfotiamine prevents
apoptosis of activated BV-2 cells. Also, benfotiamine is
involved in the regulation of nuclear factor kappa B and
mitogen-activated protein kinases signaling pathways.
In conclusion, our results suggest that
benfotiamine modulate the microglial inflammatory
response. Therefore, benfotiamine may have application in
the treatment of various neuroinflammatory diseases.
17
PREDAVANJA / LECTURES
Poremećaj homeostaze i bolesti CNS / Dysregulation of homeostasis and CNS disorders
UTICAJ GLADOVANJA NA EKSPRESIJU I
UNUTARĆELIJSKU LOKALIZACIJU FTO ENZIMA
U NEURORONIMA RAZLIČITIH REGIONA
MOZGA PACOVA
THE EFFECT OF FASTING ON THE NEURONAL
FTO EXPRESSION AND INTRACELLULAR
LOCALISATION IN RAT BRAIN
P. Vujović1, S. Stamenković2, N. Jasnić1, I. Lakić1, S.
Đurašević1, G. Cvijić1, J. Đorñević1
1
Katedra za uporednu fiziologiju i ekofiziologiju; Instittut
za fiziologiju i biohemiju; Biološki fakultet Univerziteta u
Beogradu; Srbija;
2
Centar za lasersku mikroskopiju; Instittut za fiziologiju i
biohemiju; Biološki fakultet Univerziteta u Beogradu;
Srbija
[email protected]
P. Vujović1, S. Stamenković2, N. Jasnić1, I. Lakić1, S.
Djurasević1, G. Cvijić1, J. Djordjević1
1
Department
for
Comparative
Physiology
and
Ecophysiology; Institute for Physiology and Biochemistry;
Faculty of Biology; University of Belgrade; Serbia;
2
Center for Laser Miscroscopy; Institute for Physiology and
Biochemistry; Faculty of Biology; University of Belgrade;
Serbia
[email protected]
Fto (eng. fat mass and obesity associated protein)
je demetilaza nukleinskih kiselina, koja po novijim
saznanjima pokazuje povišen afinitet prema timinu i uracilu.
Ova činjenica ukazuje da je jednolančana RNK, pre nego
DNK, primarni supstrat pomenutog enzima. Fto je
eksprimiran u brojnim regionima mozga, a naročito u
delovima hipotalamusa uključenim u regulaciju energetske
homeostaze.
Imajući u vidu podatke koji ukazuju na vezu
izemedju eskpresije Fto i ponašanja u vezi sa ishranom, cilj
eksperimenta bio je da se ispita uticaj dvodnevnog
gladovanja (48 h) na ekspresiju ovog gena u različitim
regionima mozga pacova.
Dvodnevno gladovanje uzrokovalo je povećanje
eskpresije Fto gena, kao i količine samog proteina u
hipotalamusu. Takoñe, uočena je i promena unutarćelijske
lokalizacije ovog proteina. Naime, u nekim neuronima
paraventrikularnog i ventromedijalnog jedra, kao i
lateralnog hipotalamusa, većina enzima bila je locirana
izvan ćelijskog jedra. Pomenute promene u ekspresiji i
unutarćelijskoj distribuciji izostale su u neuronima
arkuatičnog jedra, hipokampusa i kore prednjeg mozga.
Naši rezultati ukazuju da gladovanje ne utiče na
isti način na ekspresiju i unutarćelijsku lokalizaciju Fto
enzima u neuronima različitih regiona mozga pacova.
Sličnost u gladovanjem izazvanim promenama ekspresije
gena koji kodiraju Fto i katehol-O-metil transferazu ukazuje
na moguću vezu izmedju aktivnosti Fto i metabolizma
monoamina u stanju narušene energetske homeostaze.
Fat mass and obesity associated protein (Fto) is a
nucleic acid demethylase, which according to the recent
structural data exhibits preference for thymine and uracil.
This suggests that methylated single-stranded RNA, rather
than DNA, may be primary Fto substrate. Fto is abundantly
expressed in all hypothalamic sites governing feeding
behaviour.
Considering that selective modulation of Fto levels
in the hypothalamus can influence food intake, we
examined the effect of 48 h fasting on the Fto expression in
various brain regions.
We have demonstrated that 48 h fasting causes not
only an increase in the overall hypothalamic levels of both
Fto mRNA and protein, but also alters Fto intracellular
distribution. This switch happens in some neurons of
paraventricular and ventromedial nucleus, as well as lateral
hypothalamic area, resulting in the majority of the enzyme
being localized outside the cell nuclei. Interestingly, the
change in the Fto expression and intracellular localization
was not observed in neurons of arcuate nucleus, cortex and
hippocampus.
Our findings suggest that fasting does not
universally affect Fto in all the examined brain regions.
Both Fto mRNA and catechol-O-methyltransferaze mRNA
were upregulated in the identical time-dependent manner in
the hypothalamus of fasting animals. This fact, combined
with the knowledge of the Fto substrate preference, may
provide further insight into monoamine metabolism in the
state of disturbed energy homeostasis.
18
PREDAVANJA / LECTURES
Poremećaj homeostaze i bolesti CNS / Dysregulation of homeostasis and CNS disorders
UTICAJ RESTRIKCIJE HRANE NA ASTROGLIJSKI
ODGOVOR NAKON TRAUMATSKE POVREDE
MOZGA
1
1
1
M. Perović , N. Lončarević-Vasiljković , D. Lazić , M.
Brkić1, K. Smiljanić1, A. Mladenović Đorñević1, Lj.
Rakić2, S. Ruždijić1, S. Kanazir1
1
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; 11060 Beograd; Srbija;
2
Srpska akademija nauka i umetnosti; 11000 Beograd;
Srbija
[email protected]
Reaktivna astroglioza predstavlja proces aktivacije
astrocita nakon povrede mozga koji karakteriše promena
morfologije, kao i prekomerna proliferacija astrocita.
Aktivirane astrocite karakteriše pojačana ekspresija
glijalnog fibrilarnog kiselog proteina (engl. Glial Fibrilary
Acidic Protein – GFAP), intermedijernog filamenta koji ima
niz važnih funkcija u ćeliji. Opisano je 9 izoformi ovog
proteina, a za izoforme označene kao delta i kapa je
pokazano i da ometaju formiranje GFAP alfa filamenata,
kao i da time utiču na pokretljivost astrocita.
Cilj ovog rada je bio da se utvrdi efekat restrikcije
hrane na reaktivnu astrogliozu nakon traumatske povrede
mozga. Za potrebe eksperimenta, korišćeni su mužjaci
pacova soja Wistar stari 3 meseca hranjeni ad libitum ili sa
50% dnevnog unosa hrane. Nakon dostizanja 6 meseci
starosti, na životinjama je izvedena ubodna lezija u regionu
senzorimotorne kore prednjeg mozga, a zatim je praćen
vremenski profil ekspresije GFAP-a RQ-PCR, Western blot
i imunohistohemijskom analizom do 28. dana nakon lezije.
Vremenski profil ekspresije mRNK je bio isti za
sve ispitivane izoforme Gfap-a. Efekat dijete je bio
najuočljiviji 7. dana nakon lezije, kada je detektovan i
znatno manji broj GFAP imunoobeleženih ćelija u regionu
povrede. U istoj vremenskoj tački oporavka uočava se i
izražen porast kapa izoforme GFAP-a.
Rezultati ukazuju da dijeta značajno smanjuje
reakciju astrocita nakon traumatske povrede mozga.
Povećanje sinteze kapa izoforme GFAP-a može biti razlog
smanjene pokretljivosti astrocita i veličine glijalnog ožiljka
kod životinja na restrikciji hrane.
ASTROGLIOSIS FOLLOWING TRAUMATIC BRAIN
INJURY – THE IMPACT OF FOOD RESTRICTION
M. Perovic1, N. Loncarevic-Vasiljkovic1, D. Lazic1, M.
Brkic1, K. Smiljanic1, A. Mladenovic Djordjevic1, Lj.
Rakic2, S. Ruzdijic1, S. Kanazir1
1
Institute for Biological Research “Sinisa Stankovic”;
University of Belgrade; 11060 Belgrade; Serbia;
2
Serbian Academy of Sciences and Arts; 11000 Belgrade;
Serbia
[email protected]
Reactive astrogliosis represents activation of
astrocytes following brain injury, characterized by their
excessive proliferation and changes in morphology. Glial
fibrillary acidic protein (GFAP) is the major intermediate
filament protein in astrocytes. GFAP is a subject of intense
research due to growing evidence of its involvement in
numerous cellular functions. Nine splicing isoforms of
GFAP were recently described. For isoforms designated as
delta and kappa a negative effect on GFAP alpha filament
formation and thus on astrocyte motility was demonstrated.
Our previous results demonstrated beneficial effect
of food restriction (FR) prior to cortical stab injury (CSI) on
the activation of microglia. The goal of this study was to
determine the influence of FR on morphological and
molecular changes in astrocytes.
A CSI was performed on male Wistar rats fed ad
libitum or with 50% of mean daily food intake. The
expression profile of GFAP alfa, kappa and delta, was
assessed in cortical tissues collected 2, 7, 14 and 28 days
after the injury by RQ-PCR and Western blot.
Morphological
analysis
was
performed
by
immunohistochemistry.
Time course of mRNA expression was the same
for all Gfap isoforms examined. The prominent FR effect
was observed on the 7th day of the recovery when an intense
decrease in immunostaining around the site of injury was
also observed. At the protein level FR induced an increase
of kappa isoform.
These results indicate that FR strongly attenuates
astroglial reaction following TBI, throughout an increase in
the expression of specific GFAP isoform-kappa.
19
PREDAVANJA / LECTURES
Poremećaj homeostaze i bolesti CNS / Dysregulation of homeostasis and CNS disorders
DUGOROČAN UTICAJ SOMATOSTATINA NA
GONADOTROPNE I SOMATOTROPNE ĆELIJE
HIPOFIZE
LONG-TERM EFFECT OF SOMATOSTATIN ON
PITUITARY SOMATOTROPIC AND
GONADOTROPIC CELLS
N. Nestorović, M. Manojlović-Stojanoski, N. Ristić, I.
Medigović, B. Filipović, D. Petrović-Kosanović, V.
Milošević
Odeljenje za citologiju; Institut za biološka istraživanja
“Siniša Stanković”; Univerzitet u Beogradu; Beograd;
Republika Srbija
[email protected]
N. Nestorović, M. Manojlović-Stojanoski, N. Ristić, I.
Medigović, B. Filipović, D. Petrović-Kosanović, V.
Milošević
Department of Cytology; Institute for Biological Research
“Siniša Stanković”; University of Belgrade; Belgrade;
Republic of Serbia
[email protected]
Ispitivani su dugoročni efekti somatostatina 14
(SRIH-14) na imunohistohemijske i morfometrijske
karakteristike gonadotropnih i somatotropnih ćelija
hipofize.
Ženke Wistar pacova su pet dana (od 11. do 15.
dana života) subkutano tretirane sa po 20 µg/100g dva puta
dnevno. Žrtvovane su u infantilnom (16. dan),
peripubertalnom (38. dan) i u adultnom (80. dan) periodu
života. Svaka od uzrastnih gupa imala je odgovarajuću
kontrolnu grupu. Promene volumena, volumenske gustine i
broja po jedinici površine (mm2) folikulostimulirajućih
(FSH), luteinizirajućih (LH) i somatotropnih (GH)
imunohistohemijski obeleženih ćelija, praćene su pomoću
stereoloških i morfometrijskih metoda. RIA metodom
odreñene su koncentracije FSH i LH u serumu.
Nakon tretmana sa SRIH-14, FSH, LH i GH ćelije
su bile manje u odnosu na kontrolne, izmenjenog oblika i
imunohistohemijskih karakteristika u svim ispitivanim
periodima života. SRIH-14 je izazvao smanjenje volumena
FSH i LH ćelija i koncentracije FSH i LH u serumu u
infantilnom i peripubertalnom periodu života, dok su
volumenska gustina i broj ćelija po jedinici površine ostali
neizmenjeni. U adultnom periodu života, vrednosti svih
parametara su bile bliske kontrolnim. Smanjenje volumena i
volumenske gustine GH ćelija, nastalo kao rezultat tretmana
u infantinom periodu, uočeno je i u adultnom dobu.
Dobijeni rezultati ukazuju da SRIH-14 ima
značajan kratkoročan i dugoročan inhibitoran uticaj na
gonadotropne i somatostropne ćelije hipofize.
Long-term effect of somatostatin 14 (SRIH-14) on
immunohistochemical and morphometric characteristics of
pituitary gonadotropic and somatotropic cells was
examined.
Female Wistar rats received subcutaneously two
daily 20 µg/100g b.w. doses for five consecutive days in
infantile period of life (from 11 to 15 days of age). The
effect of SRIH-14 was examined in infantile (16th day),
peripubertal (38th day) and adult period of life (80th day).
Every treated group had a corresponding control group.
Changes in cell volume, volume density and number per
unit area (mm2) of follicle-stimulating (FSH), luteinizing
(LH) and somatotropic (GH) immunolabeled cells were
evaluated by stereology and morphometry. Serum FSH and
LH concentrations were determined by RIA.
After the SRIH-14 treatment, gonadotropic and
GH cells appeared smaller when compared to the controls
with changed shapes and immunohistochemical features at
all examined periods of life. SRIH-14 treatment reduced
FSH and LH cell volume and serum concentrations of
gonadotropins in infantile and peripubertal animals, while
volume density of both gonadotrops and their number per
unit area were unaltered. In adult females all measured
parameters were at control levels. Decrease of volume and
volume density of GH cells, caused by SRIH-14 treatment
in infantile period of life, persisted till adulthood. Number
of GH cells per unit area was unaltered.
These findings suggest that infantile SRIH-14
treatment exerts a significant short- and long-term
inhibitory effect on pituitary gonadotropic and somatotropic
cells.
20
PREDAVANJA / LECTURES
Poremećaj homeostaze i bolesti CNS / Dysregulation of homeostasis and CNS disorders
IDENTIFIKACIJA GENETIČKIH ALTERACIJA
KOD PACIJENATA OBOLELIH OD MALIGNIH
GLIOMA
V. Milinković1, J. Banković1, M. Rakić2, T. Stanković1, M.
Skender-Gazibara3, S. Ruždijić1, N. Tanić1
1
Instutu za biološka istraživanja „Siniša Stanković“;
Univerzitet u Beogradu; Odeljenje za neurobiologiju;
Beograd; Republika Srbija;
2
Klinički centar Srbije; Klinika za neurohirurgiju; Beograd;
Republika Srbija;
3
Medicinski fakultet Beograd; Univerzitet u Beogradu;
Institu za patologiju; Beograd; Republika Srbija
[email protected]
Uvod. Glioblastomi su najčešći i najmaligniji
humani tumori mozga. Uprkos sličnoj histologiji, primarni i
sekundarni glioblastomi predstavljaju različite entitete koje
karakterišu različite genetičke promene. Zajednička
karakteristika ovih različitih entiteta je veoma visok nivo
genomske nestabilnosti što implicira da se tokom
patogeneze glioma akumulira veliki broj genetičkih
alteracija.
Metode. Analizirali smo genomsku nestabilnost u
uzorcima 30 pacienata obolelih od glioma uporednim
poreñenjem DNK profila tumorskog i normalnog tkiva
metodom AP-PCR DNK profilisanja.Izabrane trake
izmenjene pokretljivosti su izolovane, klonirane i
sekvencirane. Pored toga, testirali smo mutacioni status
najčešće mutiranih tumor supresor gena (p53, PTEN i p16)
metodama PCR SSCP i sekvenciranja i onkogeni status
EGFR gena metodom diferencijalnog PCR-a i meñusobno
korelisali dobijene rezultate.
Rezultati. Identifikovali smo 11 gena izmenjene
primarne strukture (LHFPL3, SGCG, HTR4, ITGB1, CPS1,
PROS1, GP2, KCNG2, PDE4D, KIR3DL3 i INPP5A) koji
ranije nisu povezivani sa promocijom i progresijom glioma.
Većina njih (osam) je po našem mišljenju uključeno u
procese nastanka i progresije glioma, a promene u preostala
tri gena (GP2, KCNG2 i KIR3DL3) predstavljaju posledicu
„mutator“ fenotipa i povećene stope mutacija u tumorskim
ćelijama. Neki od identifikovanih gena pokazuju značajnu
povezanost sa alteracijama u p53, p16 i EGFR genima ali ne
i sa gubitkom PTEN tumor supresor genom.
Zaključak. Većina identifikovanih gena ima
značajnu ulogu u prenosu signala i u regulaciji ćelijske
adhezije, izuzetno važnim procesima za nastanak i
progresiju kancera. Prema našim rezultatima LHFPL3 bi
mogao biti karakteristika primarnih glioblastoma, promene
u SGCG, HTR4, ITGB1, CPS1, PROS1 i INPP5A genima su
detektovane pretežno u anaplastičnim astrocitomima što
sugeriše da igraju važnu ulogu u progresiji sekundarnih
glioblastoma dok promene u PDE4D genu imaju ulogu u
progresiji oba entiteta. Na kraju, identifikovali smo
promene u genima koji ranije nisu povezivani sa
promocijom i progresijom glioma a koji bi mogli biti dobri
molekularni markeri različitih podtipova glioblastoma.
IDENTIFICATION OF NOVEL GENETIC
ALTERATIONS IN PATIENTS WITH MALIGNANT
GLIOMA
V. Milinkovic1, J. Bankovic1, M. Rakic2, T. Stankovic1, M.
Skender-Gazibara3, S. Ruzdijic1, N. Tanic1
1
Institute for Biological Research „Sinisa Stankovic“;
University of Belgrade; Department of Neurobiology;
Belgrade; Republic of Serbia;
2
Clinical Center of Serbia; Clinic for Neurosurgery;
Belgrade; Republic of Serbia;
3
University of Belgrade; School of Medicine; Institute of
Pathology; Belgrade; Republic of Serbia
[email protected]
Background: Glioblastoma is the most frequent
and the most malignant human brain tumor. Despite a
similar histological appearance, primary and secondary
glioblastomas are distinct tumor entities with different
genetic alterations but none being specific enough to
distinguish them. High level of genomic instability detected
in glioma cells implies that numerous genetic alterations
accumulate during glioma pathogenesis.
Methods: We investigated genomic instability in
30 glioma patients by comparing DNA fingerprints of
paired tumor and normal tissues using AP-PCR DNA
fingerprinting method. Selected bands with altered mobility
were isolated, cloned and sequenced. In addition, we tested
mutational status of most frequently altered tumor
suppressors (p53, PTEN and p16) by PCR SSCP and
sequencing and EGFR oncogene by differential PCR and
correlated obtained results.
Results: We have demonstrated changes in 11
novel genes previously not associated with gliomas:
LHFPL3, SGCG, HTR4, ITGB1, CPS1, PROS1, GP2,
KCNG2, PDE4D, KIR3DL3, and INPP5A. According to
their biological function, 8 of them might play important
role in pathogenesis of glial tumors, while detected changes
in GP2, KCNG2 and KIR3DL3 might be considered as
passenger mutations, consequence of high level of genomic
instability. Some of the identified genes showed significant
association with p53, p16, and EGFR, but there was no
significant correlation with loss of PTEN.
Conclusion: Most of identified genes have a
significant role in signal transduction or cell adhesion,
which are important processes for cancer development and
progression. According to our results, LHFPL3 might be
characteristic of primary glioblastoma, SGCG, HTR4,
ITGB1, CPS1, PROS1 and INPP5A were detected
predominantly in anaplastic astrocytoma, suggesting their
role in progression of secondary glioblastoma, while
alterations of PDE4D seem to have important role in
development of both glioblastoma subtypes. In conclusion
our study revealed genetic alterations that were not
previously associated with glioma pathogenesis and could
be potentially used as molecular markers of different
glioblastoma subtypes.
21
PREDAVANJA / LECTURES
Poremećaj homeostaze i bolesti CNS / Dysregulation of homeostasis and CNS disorders
NOVE STRATEGIJE U PREVAZILAŽENJU
VIŠESTRUKE REZISTENCIJE NA
HEMIOTERAPIJU KOD GLIOBLASTOMA
NEW STRATEGIES FOR OVERCOMING MULTIDRUG RESISTANCE IN GLIOBLASTOMAS
A. Podolski-Renić, J. Banković, T. Stanković, J. Dinić, S.
Stojković, Z. Milošević, N. Tanić, M. Pešić
Institut za biološka istraživanja “Siniša Stanković“;
Univerzitet u Beogradu; Beograd; Srbija
[email protected]
A. Podolski-Renić, J. Banković, T. Stanković, J. Dinić, S.
Stojković, Z. Milošević, N. Tanić, M. Pešić
Institute for Biological Research “Siniša Stanković”;
University of Belgrade; Belgrade; Serbia
[email protected]
Višestruka
rezistencija
(MDR)je
glavni
ograničavajući faktor za postizanje uspešne terapije
kancera. MDR fenotip je često povezan sa prekomernom
ekspresijom
P-glikoproteina
(P-gp),
membranskog
transportera koji efikasno izbacuje citotoksične droge iz
kancer ćelija i menja njihovu farmakokinetiku. Pored toga,
P-gp je prisutan u fiziološkoj CNS barijeri, gde sprečava
prodor sistemski dopremljenih molekula i ograničava
tretman moždanih maligniteta. Stoga, P-gp se smatra
terapeutskom metom za prevazilaženje MDR kod
glioblastoma.
In vitro modeli kao što su rezistentne tumorske
ćelijske linije su potrebne i neophodne za translacione
studije ćelijskih karakteristika nastalih nakon tretmana
citotoksičnim lekovima. Njihov doprinos u razvoju antikancer lekova je, takoñe, veoma važan. Stoga smo
uspostavili novu MDR ćelijsku liniju glioblastoma – U87TxR. Indukcija rezistencije dovela je do značajnih
molekulskih i citogenetskih promena kod U87-TxR ćelija:
indukcija P-gp ekspresije i aktivnosti, inaktivacija p53 i
redukcija poliploidije.
Pronalaženje novih jedinjenja koja mogu izbeći ili
prevazići MDR ubrzano raste u protekle dve decenije i još
uvek traje. Testirali smo neka od njih na MDR modelu
glioblastoma: sulfinozin (SF), relativno slabo proučen
nukleozidni analog; prirodni proizvodi – jatrofanski
diterpenoidi koji efikasno inhibiraju P-gp aktivnost i
podstiču spajanje tubulina, kao Akt i Ras inhibitori.
Pokazali smo da ovi novi agensi mogu biti korisni
u tretmanu rezistentnih glioblastoma. Stoga, predlažemo
njihov razvoj kao lekova koji mogu pomoći pacijentima sa
progresivnom bolešću, koja se ne može lečiti standardnim
terapeutskim pristupima.
Multi-drug resistance (MDR) is the main limitation
for the accomplishment of successful cancer treatment.
MDR phenotype often relates to the over-expression of Pglycoprotein (P-gp), a membrane transporter that effectively
extrudes the cytotoxic drugs from cancer cells and changes
their pharmacokinetics. In addition, P-gp is expressed in
physiological CNS barrier where it prevents penetration of
systemically delivered molecules and limits treatment of
brain malignancies. Therefore, P-gp is considered as
therapeutic target for overcoming MDR in glioblastomas.
In vitro models such as resistant cancer cell lines
are required and necessary for translational studies of
cellular properties impaired after cytotoxic drug treatment.
Their contribution to anti-cancer drug development is also
very important. Therefore, we established new MDR
glioblastoma cell line – U87-TxR. The induction of
resistance led to the significant molecular and cytogenetic
changes in U87-TxR cells: induction of P-gp expression and
activity, inactivation of p53 and polyploidy reduction.
The discovery of new lead compounds that could
abrogate or overcome the MDR grew rapidly in the last two
decades and still emerges. We have tested some of them in
MDR glioblastoma model: sulfinosine (SF), a quite
unexplored nucleoside analog; natural products - jatrophane
diterpenoids that efficiently inhibit P-gp activity and
promote tubulin-assembly as well as Akt and Ras inhibitors.
We have shown that these new agents may be
advantageous in treatment of resistant glioblastomas.
Therefore, we propose their development as drugs that
could help the patients with the progressive disease
refractory to standard therapeutic regiments.
22
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
HIPERMETIONINSKA DIJETA I
HIPEREKSCITABILNOST: ELEKTRIČNE I
BIHEJVIORALNE PROMENE KOD PACOVA
HYPERMETHIONINE DIET AND
HYPEREXCITABILITY: ELECTRICAL AND
BEHAVIORAL ALTERATIONS IN RATS
O. Stanojlović1, D. Hrnčić1, A. Rašić - Marković1, Đ.
Macut2, V. Šušić3, D. Đurić1
1
Laboratorija za neurofiziologiju; Institut za medicinsku
fiziologiju ”Rihard Burijan”; Medicinski fakultet;
Univerzitet u Beogradu; Beograd; Srbija;
2
Institut za endokrinologiju, dijabetes i bolesti metabolizma;
KCS; Medicinski fakultet u Beogradu; Univerzitet u
Beogradu; Beograd; Srbija;
3
SANU; Beograd; Srbija
[email protected]
O. Stanojlović1, D. Hrnčić1, A. Rašić - Marković1, Đ.
Macut2, V. Šušić3, D. Djurić1
1
Laboratory of Neurophysiology; Institute of Medical
Physiology ”Richard Burian”; Faculty of Medicine;
University of Belgrade; Belgrade; Serbia;
2
Institute of Endocrinology; Diabetes and Metabolic
Disease; CCS; Faculty of Medicine; University of Belgrade;
Belgrade; Serbia;
3
Serbian Academy of Sciences and Arts; Belgrade; Serbia
[email protected]
Brojne studije su dokazale postojanje kompleksne
povezanosti hiperhomocisteinemije i različitih neuroloških
oboljenja
uključujući
demencije
različitog
tipa,
Alchajmerovu
bolest,
Parkinsonovu
bolest
i
cerebrovaskularne poremećaje. Visoke koncentracije
homocisteina ili njegovih derivata, u prvom redu
homocistein tiolaktona, mogu izmeniti neurotransmiterske
sisteme i prouzrokovati hiperekscitabilnost, što smo
pokazali uspostavljanjem eksperimentalnog modela dva tipa
napada nakon akutne administracije homocistein tiolaktona.
Homocistein se sintetiše iz metionina, i njegov metabolizam
je regulsisan balansom remetilacionih i transsulfuracionih
puteva. Ishrana obogaćena metioninom kod pacova
prouzrokova je pojavu umerene hiperhomocisteinemije sa
posledičnim povećanjem ekscitabilnosti. To je bilo
evidentno u obrazcu kako konvulzivnog ponašanja, tako i
iktalne bioelektrične aktivnosti. Ove promene bilo je
moguće pripisati izmenenjenoj aktivnosti jonskih pumpi i
drugim nedavno prepoznatim mehanizmima uključenim u
epileptogenezu. Štaviše, hipermetioninska dijeta, prema
našim rezultatima, je izmenila ponašanje životinja u testu
otvorenog polja, ukazujući na izmenu ponašanja povezanog
sa anksioznšću u smislu povećane anksioznosti, što sugeriše
na vezu izmeñu hiperhomocisteinemije i anksioznosti.
Numerous studies provided evidences for a
complex relation between hypehomocysteinemia and
different CNS disorder including cognitive decline, vascular
dementia and Alzheimer’s disease, Parkinson disease and
stroke. High brain concentrations of either homocysteine or
its derivatives, like homocysteine thiolactone, might alter
neurotransmission and produce hyperexcitability as a
consequence, what we have shown by establishing
experimental model of two types of seizures upon acute
homocysteine thiolactone administration. Homocysteine is
synthesized from methionine and its metabolism is
regulated in order to achieve a balance between the
remethylation and transsulfuration pathways. Methionine
nutritional
overload
in
rats
induced
mild
hyperhomocysteinemia with consecutive increase of
hyperexcitability. It was evident in convulsive behavior, as
well as in ictal electrical profile. These changes could be
attributed to alterations in sodium/potassium ion pump
activity and other recently-recognized mechanisms of
epileptogenesis. Moreover, methionine-enriched diet,
according to our results, altered rat behavior in open field
test, indicating increased anxiety-related behavior in these
rats. Therefore, a link between hyperhomocysteinemia and
anxiety could also be supposed.
23
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
EEG ARHITEKTURABUDNOSTIISPAVANJA U
CENTRALNOJHOLINERGIČKOJNEURODEGENER
ACIJI
SLEEP/WAKE STATES EEG ARCHITECTURE IN
THE CENTRAL CHOLINERGIC
NEURODEGENERATION
J. Šaponjić
Univerzitet u Beogradu; Odeljenje za neurobiologiju;
Institut za biološka istraživanja “Siniša Stanković”; 11 060
Beograd; Srbija
[email protected]
J. Šaponjić
University of Belgrade; Department of Neurobiology;
Institute for Biological Research “Sinisa Stankovic”; 11 060
Belgrade; Serbia
[email protected]
Neurodegenerativne bolesti, kao sto su Alchajmerova (AB) i Parkinson-ova bolest (PB), podrazumevaju
selektivni gubitak specifičnih neuronskih populacija mozga.
U odnosu na holinergičke neurone, dok AB podrazumeva
selektivni gubitak holinergičkih neurona prednjeg bazalnog
mozga (NB), PB podrazumeva selektivni gubitak pontinskih
holinergičkih neurona (PPT). Skorašnje humane “imidžing”
i neuropatološke studije su dokazale holinergičku
denervaciju talamusa ili gubitak oko 50% PPT holinergičkih
neuronau PB nasuprot AD, i ova neurodegeneracija je
povezana sa pojavom poremećaja ponašanja u toku REM
faze spavanja (RBD), poremećajima hoda, ravnoteže,i
učestalim padovima u PB.
Sa ciljem da identifikujemo moguće EEG markere
u toku budnosti i spavanja, koji su znak početka i progresije
funkcionalno različitih holinergičkih poremećaja inervacije
mozga, mi smo koristili animalne modele ekscitotoksične
NBi PPT lezije. Efekte poremećaja holinergičkih inervacija
mozga smo pratili na izmenama ciklusa budnost/spavanje,
njihovim EEG karakteristikama, kao i na izmenama u
prelaznim stanjima u toku spavanja, u toku 5 nedelja nakon
lezija.
U našim animalnim modelima neuropatologije
humanih neurodegenerativnih bolesti dokazali smo po prvi
put da gubitak NB holinergičkih neurona tronedeljno
pojačava samo REM teta amplitude, posebno u
senzomotornoj kori. Gubitak holinergičkih PPT neurona
prouzrokuje petonedeljni poremećaj structure prelaznih
stanja spavanja, pojačava beta i gama amplitude budnosti,
REM i NeREM faze spavanja, i istovremeno smanjuje delta
amplitude budnosti i NeREM faze spavanja.
Različita ekspresija poremećaja budnost/spavanje
EEG arhitekture kao i structure prelaznih stanja, razlikuje
funkcionalno različite holinergičke denervacija mozga sa
specifičnom topografskom ekspresijom u senzomotornoj
nasuprot motornoj kori velikog mozga, posebno u toku
NeREM i REM faze spavanja.
Neurodegenerative
diseases,
such
as
Alzheimer’s(AD) and Parkinson (PD) diseases, involve the
selective loss of specific neuronal populations within the
brain. Regarding to the brain cholinergic neurons, while AD
involves the selective loss of basal forebrain (NB),PD
involves the selective loss of pontine cholinergic system
(PPT). Recent imaging and neuropathological studies in
humans demonstrated the thalamic cholinergic denervation
or degeneration of about 50% of the PPT cholinergic
neurons in the PDs in contrast to AD, and related it to rapideye-movement sleep behavioral disorder (RBD),gait and
balance impairment, including falls in PD.
In order to identify the possible sleep/wake state
related EEG markers for the onset and progression of the
functionally distinct cholinergic innervation disorders, we
used the animal models of the NB and PPT excitotoxic
lesions, and followed the effects on sleep/wake states
distribution, sleep/wake state related EEG microstructure
and transitions structure during the 5 post-lesions weeks.
In our animal models of the human neurodegenerative
diseases pathology we proved for the first time that NB
cholinergic neuronal loss sustainably augmentedonly REM
theta amplitude for 3 weeks, particularly in sensorimotor
cortex.PPT cholinergic neuronal loss sustainably augmented
Wake, NREM and REM beta and gamma amplitude, while
attenuated Wake and NREM delta amplitude, and disturbed
sleep/wake transitions structure for 5 weeks.
Distinct sleep/wake state-related EEG architecture
and transitions structure differentiated the functionally
distinct cholinergic innervation disorders in rat, with
specific topographical expression within the sensorimotor
versus motor cortex, particularly during NREM and REM
sleep.
24
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
UTICAJ MAGNETNIH POLJA NA PONAŠANJE
RAZLIČITIH VRSTA
THE INFLUENCE OF MAGNETIC FIELDS ON
BEHAVIOUR OF DIFFERENT SPECIES
B. Janać, D. Todorović, S. Rauš Balind, T. Savić, Lj.
Nikolić, Z. Prolić
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; Beograd; Srbija
[email protected]
B. Janać, D. Todorović, S. Rauš Balind, T. Savić, Lj.
Nikolić, Z. Prolić
Institute for Biological Research “Siniša Stanković”;
University of Belgrade; Belgrade; Serbia
[email protected]
Živa bića su svakodnevno izložena prirodnim i
veštačkim magnetnim poljima poreklom od različitih
izvora. Stoga je vrlo važno ispitati efekte magnetnih polja
kod različitih vrsta u cilju sagledavanja mogućih rizika
takvog izlaganja po zdravlje ljudi. Studije ponašanja su
veoma korisne za detekciju promena kod životinja
izazvanih magnetnim poljima. Eksperimenti u kojima je
praćena orijentacija i navigacija životinja su ukazali na
sposobnost živih bića da detektuju i odgovore na magnetna
polja i pokrenuli su dalja istraživanja mogućih mehanizama
koji su u osnovi uočenih efekata magnetnih polja. Takoñe je
ispitivan i uticaj magnetnih polja, kao ekološkog faktora, na
motornu aktivnost, učenje i pamćenje različitih vrsta.
Uočeni efekti bi mogli biti pripisani strukturnim i
funkcionalnim promenama u strukturama mozga
odgovornim za kontrolu ispitivanih ponašanja, koje nastaju
kao posledica delovanja magnetnog polja. Odgovori su
uglavnom zavisni od osobina primenjenog magnetnog polja
(tip, frekvencija, magnetna indukcija, dužina izlaganja,...) i
ispitivane vrste (funkcionalno stanje, pol, starost,...).
Poslednjih godina, magnetna polja se razmatraju i kao
potencijalno medicinsko sredstvo s obzirom na pokazane
povoljne efekte u terapiji nekih psihijatrijskih i neuroloških
oboljenja (shizofrenija, depresija, moždani udar,
Parkinsonova bolest,...) povezanih sa promenama u
ponašanju.
Living beings are everyday exposed to natural and
artificial magnetic fields derived from different sources.
Therefore, it is very important to investigate magnetic field
effects on different species in order to evaluate the possible
risks of such exposure on human health. Behavioural
studies are very useful tool for the detection of magnetic
field-induced changes in animals. Experiments dealing with
animal orientation and navigation revealed the ability of
living beings to detect and respond to magnetic fields
initiating further investigation of possible mechanism(s)
underlying observed magnetic field effects. The influence of
magnetic fields, as an ecological factor, on motor activity,
learning and memory of different species was also
investigated. The observed effects could be attributed to
magnetic field-induced morphological and functional
changes in the brain structures responsible for the control of
examined behaviours. Mostly, the effects are dependent on
the properties of the applied magnetic field (type,
frequency, magnetic induction, exposure duration,…) and
the examined species (functional state, sex, age,…). Resent
research revealed that magnetic fields can be a promising
tool in medicine concerning their beneficial effects in the
therapy of some psychiatric and neurological disorders
(schizophrenia, depression, stroke, Parkinson’s disease,…)
associated with behavioural changes.
25
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
NEINVAZIVNA STIMULACIJA MOZGA –
TRANSLACIONI PRINCIPI
NONINVASIVE BRAIN STIMULATION –
TRANSLATIONAL PRINCIPLES
T.V.Ilić
Klinika za neurologiju; Vojnomedicinska akademija;
Medicinski fakultet VMA; Univerzitet odbrane; Beograd;
Srbija
[email protected]
T.V.Ilić
Clinic of neurology; Military Medical Academy; Medical
Faculty of MMA; University of defence; Belgrade; Serbia
[email protected]
Neinvazivna stimulacija mozga (NIMS) kod ljudi u
budnom stanju obuhvata tehnike repetitivne transkranijalne
magnetne stimulacije (rTMS) i transkranijalne stimulacije
istosmernom strujom (transcranial direct current
stimulation - tDCS), koje tokom poslednje dve decenije
privlače veliku pažnju u eksperimentalnoj fiziologiji. Ove
metode razvijene su na translacionim principima prema
analogiji iz klasičnih modela dugoročne potencijacije i
dugoročne depresije sinaptičke transmisije opisanih u in
vivo i in vitro studijama, ali za razliku od njih prouzrokuju
masivnu pre- i post-sinaptičku aktivaciju unutar mreža
kortikalnih neurona. Brojne studije tokom protekle decenije
su pokazale da različiti protokoli ponavljane ili kontinuirane
stimulacije dovode do promene nadražljivosti motorne kore,
koja se održava izvesno vreme i nakon prestanka
stimulacije,
sugerišući
i
mogućnosti
kortikalne
reorganizacije pod dejstvom NIMS. Uporedo sa razvojem
protokola fokalne rTMS, uvedena je i tehnika tDCS,
relativno difuzne stimulacije korteksa istosmernom strujom
niskog intenziteta (1-2 mA). Za razliku od TMS motorne
kore koja izaziva motorne odgovore u ciljnom mišiću, tDCS
dovodi do promena membranskog potencijala neurona u
mirovanju (smer pomaka membranskog potencijala zavisan
je od polariteta); anodnom tDCS se uvećava kortikalna
ekscitabilnost putem pomaka membranskog potencijala u
pravcu depolarizacije (olakšano spontano okidanje
neurona), suprotno od katodne polarizacije koja izaziva
sniženje kortikalne ekscitabilnosti pomakom u pravcu
hiperpolarizacije membranskog potencijala. Ovakva
biološko dejstvo vodi ka reverzibilnim promenama spontane
aktivnosti neurona i procesiranja aferentnih signala, što se
već primenjuje u pokušajima tretmana oboljenja centralnog
nervnog sistema. Uprkos značajnim naprecima u modulaciji
kortikalne ekscitabilnosti, ostaje potreba da se
neurohemijski i fiziološki efekti ovih metoda detaljnije
upoznaju kroz animalne modele.
Non-invasive brain stimulation (NIBS) in humans awake
includes techniques of repetitive transcranial magnetic
stimulation (rTMS) and transcranial direct current
stimulation (tDCS), which during the last two decades
become very attractive in field of experimental physiology.
These methods have been developed on translational
principles by analogy from classical model long-term
potentiation and long-term depression of synaptic
transmission described in the in vivo and in vitro studies,
but unlike them, causing massive pre-and post-synaptic
activation within a network of cortical neurons. Numerous
studies in humans over the past decade have shown that
different protocols of repeated or continuous stimulation
leads to changes in cortex excitability, which takes place
some time after the cessation of stimulation, suggesting
possibilities and cortical reorganization under the influence
of NIBS. Along with the development of more focal rTMS
protocols, introduced the technique tDCS, relatively diffuse
cortical stimulation downdraft current of low intensity (1-2
mA). Unlike rTMS of motor cortex that can causes motor
responses in the target muscle, tDCS lead exclucively to
changes in membrane potential of neurons at rest (direction
of of membrane potential shifts is dependent on the
polarity) anodal tDCS increases cortical excitability via
depolarization of membrane potential (facilitated
spontaneous firing of neurons), the opposite of cathodic
polarization that causes decreased cortical excitability
shifting towards membrane potential hyperpolarization.
Such biological effects leading to reversible changes in
spontaneous activity of neurons and afferent signal
processing, which is already applied in attempts to treat
diseases of the central nervous system. Despite significant
advances in the modulation of cortical excitability, there
remains a need to explore neurochemical and physiological
effects of these methods on animal models in much more
detailed way.
.
.
26
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
EKSPRESIJA NADPH OKSIDAZE I
MITOHONDRIJALNA DISFUNKCIJA KAO
POKRETAČI OKSIDATIVNOG STRESA U MOZGU
DŽERBILA TROVANIH ALUMINIJUMOM
NADPH OXIDASE EXPRESSION AND
MITOCHONDRIAL DYSFUNCTION AS
INITIATORS OF OXIDATIVE STRESS IN THE
BRAIN OF GERBILS TREATED WITH ALUMINUM
N. Petronijević1, S. Vučetić-Arsić2, G. Jevtić1, M.
Jovanović3, V. Selaković3, T. Nikolić1, M. Velimirović1, T.
Stojković1, N. Radonjić1
1
Institut za kliničku i medicinsku biohemiju; Medicinski
fakultet; Univerzitet u Beogradu; Beograd; Srbija;
²Specjalna bolnica za bolesti zavisnosti; Beograd; Srbija;
³Vojnomedicinska Akademija; Beograd; Srbija
[email protected]
N. Petronijević1, Vučetić-Arsić2, G. Jevtić1, S. M.
Jovanović3, V. Selaković3, T. Nikolić1, M. Velimirović1, T.
Stojković1, N. Radonjić1
1
Institute of Clinical and Medical Biochemistry; School of
Medicine; University of Belgrade; Pasterova 2, Belgrade;
Serbia;
²Special Hospital for Addictions; Belgrade; Serbia;
³Military Medical Academy; Belgrade; Serbia
[email protected]
Uvod: Aluminijum (Al) je prepoznat kao kofaktor
u etiologiji nekih mentalnih poremećaja uključujući i
Alchajmerovu bolest (AB). Tačan mehanizam kojim Al
indukuje promene u mozgu nije poznat.
Cilj: Cilj ovog rada je bio da se ispitaju efekti
ingestije Al na dinamiku promena u ekspresiji NADPH
oksidaze (NOX2), aktivnosti citohrom C oksidaze (COX) i
parametara oksidativnog stresa u mozgu Mongolskih
džerbila.
Materijal i metode: Odrasli džerbili (n=30) su bili
akutno tretirani aluminijum hloridom (LD25), gavažom, i
žrtvovani posle 2, 6 ili 24 sata. Deset džerbila je subakutno
tretirano (LD10) i žrtvovano posle 21 dan. Kontrolne
životinje su dobijale fiziološki rastvor. Aktivnost COX,
superoksid dizmutaze (SOD), katalaze (CAT) i nivo lipidnih
peroksida (MDA) su odreñivani spektrofotometrijski a
ekspresija membranskih (gp91phox, p22phox) i citosolnih
(p40phox, p47phox, p67phox) subjedinica NOX2 je
ispitivana tehnikom Western blot u pojedinim strukturama
mozga.
Rezultati: Oksidativni stres, izražen kroz povećanje
nivoa MDA i promene u aktivnosti SOD i CAT, je zapažen
već 2 sata posle trovanja. Sniženje aktivnosti COX i
smanjenje ekspresije membranskih i citosolnih subjedinica
NOX2 su takoñe bili meñu najranijim efektima tretmana sa
Al. Posle subakutnog tretmana oksidativni stress je bio još
izraženiji. Smanjenje ekspresije gp91phox i povećanje
ekspresije p67phox je zapaženo u korteksu, dok je u
hipokampusu ekspresija p67phox bila smanjena.
Zaključak: Mitohondrijalna disfunkcija bi mogla
biti inicijator oskidativnog stresa posle akutnog trovanja sa
Al. Promene u ekspresiji NOX2 subjedinica koje su
zapažene posle subakutnog davanja Al odgovaraju
promenama koje se zapažaju u obolelih od AB.
Introduction: Aluminum (Al) was recognized as a
cofactor in the etiology of some mental impairments
including Alzheimer’s disease (AD). The exact mechanism
responsible for changes induced by Al is not known.
Aim: The aim was to analyze the effects of
ingested Al on the dynamic of changes in NADPH oxidase
(NOX2) expression, cytochrome C oxidase (COX) activity
and oxidative stress parameters in Mongolian gerbil’s brain.
Material and methods: Adult gerbils (n=30) were
acutely exposed to aluminum chloride (LD25), by gavage,
and sacrificed 2, 6 or 24 hours later. Ten gerbils were
subacutely treated (LD10) and sacrificed after 21 days.
Control animals received saline. The activities of COX,
superoxide dismutase (SOD), catalase (CAT), and lipid
peroxide levels (MDA) were measured in specific brain
structures. The expression of membrane-bound (gp91phox,
p22phox) and cytosolic (p40phox, p47phox, p67phox)
NOX2 subunits was determined by Western blot.
Results: Oxidative stress, expressed as increase in
MDA levels and changes in the activity of SOD and CAT
was seen 2 hours after posioning. The activity of COX and
the expression of membrane-bound and cytosolic NOX2
subunits were also decreased as the earliest effect of Al
treatment. After subacute Al ingestion the oxidative stress
was pronounced. Decreased gp91phox and increased
p67phox expressions were seen in cortex while in the
hippocampus the decrease of p67phox was noticed.
Conclusion: Mitochondrial failure can be the
initiator of oxidative stress after Al poisoning. The changes
in NOX2 subunit expression seen after subacute poisoning
correspond to the changes seen in AD patients.
27
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
UTICAJ MODULACIJE GABA-ERGIČKE
NEUROTRANSMISIJE NA PONAŠANJE
M. Savić
Katedra za farmakologiju; Univerzitet u Beogradu Farmaceutski fakultet; Vojvode Stepe 450, 11221 Beograd;
Srbija
[email protected]
Uvod: Generalno je prihvaćena snažna uključenost
GABA-ergičke neurotransmisije u regulaciji pažnje,
anksioznosti, memorijskih funkcija, mišićnog tonusa i
epileptogene aktivnosti. Veliki deo relevantnog istraživanja
potiče iz studija uloge GABAA receptora i njihove
modulacije.
Cilj: Prikazani rezultati su imali za cilj postizanje
daljeg uvida u posledice finog podešavanja neuronske
inhibicije posredovane GABAA receptorima na ponašanje.
Materijal i metode: Bihejvioralni efekti seta
novosintetisanih pozitivnih alosternih modulatora, sa
kodnim nazivima YT-III-31, SH–I–048A, WYS8, SH–053–
2’N i SH–053–R–CH3–2’F, ispitani su u testovima
spontane lokomotorne aktivnosti, uzignutog plus lavirinta,
Morris-ovog vodenog lavirinta, rotirajućeg vretena, jačine
stiska i izlaganja pentilentetrazolu, koji prevashodno
procenjuju, redom, sedativne, anksiolitičke, amnezijske,
ataksične, miorelaksantne i antikonvulzivne efekte.Ovi
ligandi poseduju odreñene kombinacije afiniteta i
efikasnosti na četiri populacije GABAA receptora, koje na
meñupovršini izmeñu gama2 i alfa 1/2/3 ili 5 podjedinice
sadrže benzodiazepinsko mesto vezivanja.
Rezultati: Parametri ponašanja kod pacova bili su
menjani na različite načine, u zavisnosti od in vitro profila
ispitivanog liganda. Dok su relativno male razlike u
afinitetima i/ili efikasnostima često bile praćene jasnim
razlikama u profilima ponašanja, suprotno je takoñe
ponekad bilo istinito.
Zaključak: U smislu ishoda na ponašanje, čini se
da je korektna ravnoteža na različitim populacijama
podjedinica najmanje onoliko važna koliko su i individualni
modulatorni uticaji na pojedinim podtipovima GABAA
receptora.
THE INFLUENCE OF MODULATION OF
GABAERGIC NEUROTRANSMISSION ON
BEHAVIOR
M. Savić
Department of Pharmacology; University of Belgrade Faculty of Pharmacy; Vojvode Stepe 450, 11221 Belgrade;
Serbia
[email protected]
Background: It is generally accepted that
GABAergic neurotransmission is profoundly involved in
regulation of vigilance, anxiety, memory functions, muscle
tone and epileptogenic activity. The major part of the
relevant research stems from studies on the role of GABAA
receptors and their modulation.
Objective: The presented results are aimed to
provide further insight into the behavioral consequences of
fine tuning of GABAA-mediated neuronal inhibition.
Material and Methods: Behavioral effects in rats of
a set of newly-synthesized positive allosteric modulators of
GABAA receptors, with code names YT-III-31, SH–I–
048A, WYS8, SH–053–2’N and SH–053–R–CH3–2’F,
were assessed in spontaneous locomotor activity, elevated
plus–maze, Morris water maze, rotarod, grip strength, and
pentylenetetrazole tests, designed to assess the sedative,
anxiolytic, amnesic, ataxic, muscle relaxant, and
anticonvulsant effects, respectively. These ligands possess
distinct combinations of affinities and efficacies at four
populations of GABAA receptor, which at the interface
between gamma2 and alpha1/2/3 or 5 subunits contain the
benzodiazepine binding site.
Results: Behavioral parameters in rats were
affected in different ways in dependence on in vitro profile
of the examined ligand. While relatively small differences
in affinities and/or efficacies were often accompanied by
distinct differences in behavioral profiles, the opposite was
also true sometimes.
Conclusion: In regard to the behavioral output, it
seems that a correct balance at various receptor populations
is at least as important as individual modulatory influences
at distinct GABAA receptor subtypes.
28
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
JONSKI KANALI AKTIVIRANI KISELIM PH
(ASIC): NOVE METE ZA TERAPIJU U
NEUROLOGIJI...ALI ČEMU SLUŽE?
N. Todorović
Institut za biološka istraživanja
Univerzitet u Beogradu; Srbija
[email protected]
“Siniša stanković”;
Jonski kanali aktivirani kiselim pH (ASIC) su pH
senzori sposobni da reaguju na širok opseg promena u
fiziološkim procesima i patološkim stanjima. ASIC kanali
su voltažno nezavisni katjonski kanali aktivirani protonima
sa širokom rasprostranjenošću u centralnom i perifernom
nervnom sistemu.
Kod ljudi je pronadjeno 4 ASIC gena, označenih 1-4, koji
alternativnom obradom daju devet jedinstvenih proteina.
Aktivacija ovih kanala protonima izgleda da igra važnu
ulogu u osećanju bola, zaustavljanju epileptičnog napada i
oštećivanju neurona izazvanog acidozom.
ASIC kanali su modulisani cinkom, laktatom,
serotoninom i kanabinoidnim receptorom tip 1.
Najviše se istražuje uloga ASIC kanala u percepciji bola, s
obzirom na njihovo izraženo prisustvo na nociceptivnim
neuronima,
direktnu
inhibiciju
nesteroidnim
antiinflamatornim lekovima i, kako je skoro pokazano,
nekim lokalnim anesteticima. Zanimljivi ligandi ASIC
kanala medju peptidnim toksinima izazivaju osećaj bola
(toksin iz otrova teksaške koralne zmije) aktivacijom
perifernih ASIC kanala i uklanjaju bol (toksin iz otrova
crne mambe) inhibirajući ASIC kanale.
Regulacija ASIC kanala je nedovoljno istražena.
Iako je prisustvo ASIC kanala u mozgu i kičmenoj moždini
kao i povećanje aktivnosti u patološkim stanjima jasno
utvrdjeno, podaci dobijeni na životinjama sa uklonjenom
funkcijom ASIC kanala nisu pomogli da se odgovori na
pitanje o njihovoj ulozi u fiziologiji mozga.
Potrebna su dalja istraživanja farmakoloških i
biofizičkih osobina ASIC kanala kao i njihovih regulatornih
mehanizama da bi se omogućio razvoj novih terapeutskih
pristupa u tretmanu bola. Druge moguće primene
farmakološke inhibicije ASIC kanala su u tretmanu
ishemične povrede mozga, migrene i Parkinsonove i
Hantingtonove bolesti.
ACID-SENSING ION CHANNELS: NOVEL
THERAPEUTIC TARGETS IN NEUROLOGY...BUT
WHAT ARE THEY FOR?
N. Todorović
Institute for biological research “Siniša Stanković”;
Belgrade University; Serbia
[email protected]
Acid-sensing ion channels (ASICs) function as pH
sensors able to detect a broad range of pH fluctuations
during normal physiological processes and pathological
conditions. ASICs are voltage independent, proton-gated
cation channels widely expressed throughout the central and
peripheral nervous system. Four ASIC genes have been
identified in humans, designated 1–4, with splice variants
encoding a total of nine proteins. Activation of these
channels by protons seems to play an important role in
nociception, seizure termination and acidosis-mediated
neuronal injury.
ASICs can be modulated by zinc, lactate, serotonin
and G protein coupled receptors like cannabinoid receptor
type 1. The role of ASICs in perception of pain is the most
worked on, since they are abundantly expressed in
nociceptive neurons, directly inhibited by ibuprofen and, as
recently demonstrated, by some local anesthetics.
Interesting ASIC ligands among peptide toxins (from Texas
coral snake) evoke pain by activating peripheral ASICs and
abolish pain (from black mamba) through inhibition of
ASICs.
However, the regulation of ASICs remains poorly
understood. While the wide expression of ASICs in central
neurons and their upregulation in pathological conditions is
firmly established, the inconclusive data from knockout
animals leaves the question of the role of ASICs in normal
brain physiology unanswered.
Further investigation into the pharmacological and
biophysical properties of ASICs and their regulatory
mechanisms is needed for development of novel therapeutic
strategies for the treatment of pain. The other prospective
applications of pharmacological ASIC inhibition are
treatments of ischemic neuronal injury, migraine and
Parkinson's and Huntington’s disease.
29
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
ULOGA TENASCINA C U STRUKTURNOJ
PLASTIČNOSTI MOZGA
ROLE OF TENASCIN C IN STRUCTURAL BRAIN
PLASTICITY
V. Stamenković1, S. Stamenković1, M. Gawlak2, T.
Jaworski2 , M. Jovanović1, G. Wilczynski2, M. Schachner3,
L. Radenović1, P. R. Anñus1
1
Centar za lasersku mikroskopiju; Biološki fakultet
Univerziteta u Beogradu; Srbija;
2
Nencki Institut eksperimentalne biologije; Poljska
akademija nauka; Varšava; Poljska;
3
Centar za molekularnu neurobiologiju; Univerzitet u
Hamburgu; Hamburg; Nemačka
[email protected]
V. Stamenković1, S. Stamenković1, M. Gawlak2, T.
Jaworski 2 , M. Jovanović1, G. Wilczynski2, M. Schachner3,
L. Radenović1, P. R. Andjus1
1
Center for Laser Microscopy; Department of Physiology
and Biochemistry; Faculty of Biology; University of
Belgrade; Serbia;
2
Nencki Institute of Experimental Biology; Laboratory of
Neuromorphology; Polish Academy of Science; Warsaw,
Poland;
3
Zentrum für Molekulare Neurobiologie; Universitaet
Hamburg; Hamburg; Germany
[email protected]
Tenascin C (TN-C) je glikoprotein ekstraćelijskog
matriksa sa važnom ulogom u morforegulaciji tokom
razvića, dok u oboljenjima reguliše adhezivna i signalna
svojstva neurona i glije. U adultnom CNS njegova
ekspresija je suprimirana, ali ostaje zapažena u zonama
neuroanlne plastičnosti.
Istraživana je uloga TN-C u uslovima gajenja u
obogaćenoj sredini (OS) imunohistohemijskim praćenjem
dsitribucije perineuronalnih mreža (PNM) u TNC-/- miševa
u poredjenju sa srodnim divljim sojem (WT) u raznim
strukturama CNS. Takodje nas je interesovao doprinos
matriks metaloproteinaza (MMP) mogućoj reorganizaciji
PNM što je proveravano in situ i gel zimografijom.
Rezultati pokazuju da se PNM značajno razlažu u
dubokim jedrima cerebeluma i hipokampusu WT miševa
nakon OS, dok je u TNC-/- miševa efekat bio obrnut sa
porastom ekspresije PNM nakon OS. Medjutim, u
retrospleničnoj zoni korteksa nisu detektovane slične
promene. In situ zimografijom je otkrivena najveća
aktivnost MMP u svim ispitivanim regionima u WT miševa
gajenih 8 nedelja u standardnoj sredini poredjenjem sa OS i
u TNC-/- miševima u oba uslova gajenja, bez posebne
razlike medju njima. Medjutim, gel zimografija otkrila je
značajan porast MMP-9 aktivnosti nakon 4 nedelje u OS za
oba genotipa. Takodje, konfokalnom mikroskopijom je
otrkrivena kolokalizacija MMP aktivnosti sa PNM.
Ovi rezultati otkrivaju izraziti tkivno-zavisni
doprinos TN-C strukturnom plasticitetu indukovanom u OS
i ukazuju na ulogu MMP u ovom efektu.
Tenascin C (TN-C) is an extracellular matrix
glycoprotein playing an important morphoregulatory role
during development, tissue remodeling, and in disease by
regulating the cell adhesive and signaling properties of
neural and non-neural cells. In the adult CNS its expression
is downregulated, but remains detectable in areas that
exhibit neuronal plasticity.
We aimed to examine the role of TN-C under
conditions of enriched environment (EE) in the organization
of perineuronal nets (PNNs). Thus, diverse CNS structures
in TNC-/- mice and their WT littermates were studied after
rearing under EE and standard conditions (SC). In addition,
we wanted to examine the activity of ECM degrading
matrix metalloproteinases (MMPs) and to check their
possible involvement in PNN reorganization after EE using
in situ and gel zymography.
Immunohistochemistry
of
PNNs
showed
significant reduction of nets in deep cerebellar nuclei and
the hippocampus of WT animals after EE, while in TNC-/animals the effect was opposite with increased expression of
PNNs after EE. However, in the retrosplenial area no
changes were detected. In situ zymography revealed in all
investigated regions the highest activity of MMPs in WT
mice reared 8 weeks in SC compared to EE and TNC-/mice in SC and EE, with no apparent differences. However,
gel zymography revealed a significant increase of MMP-9
activity after 4 weeks of EE in both genotypes. In addition,
confocal microscopy revealed co-localization of MMP
activity and PNNs.
These results reveal a striking tissue-specific
contribution of TN-C in structural plasticity induced by EE,
and suggest an involvement of MMPs in this effect.
30
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
IDENTIFICATION OF NEW THERAPEUTIC
TARGETS TO PREVENT EPITHELIAL BLOOD-CSF
BARRIER DISRUPTION IN SYSTEMIC
INFLAMMATORY DISORDERS AND
(INFLAMM)AGING
Vandenbroucke REa,b, Balusu Sa,b, Demeestere Da,b, Van
Wonterghem Ea,b, Van Hooren Va,b, De Rycke Ra,b, and
Libert Ca,b
a
Department for Molecular Biomedical Research, VIB,
Technologiepark 927, 9052 Ghent, Belgium
b
Department of Biomedical Molecular Biology, University
Ghent, Technologiepark 927, 9052 Ghent, Belgium
The blood-cerebrospinal fluid barrier (BCSFB) is
one of the CNS barriers that separates the CNS from the
periphery. Inflammatory conditions, such as systemic acute
inflammation and aging, are known to severely affect CNS
barrier functionality, and this has important consequences
on the homeostasis of their neural microenvironment.
Systemic acute inflammation occurs during sepsis, a highly
mortal, inflammatory disease initiated by an infection and
associated with systemic inflammation and organ
dysfunction. The prevalence is high and current treatments
are ineffective and mainly supportive. Aging is a
progressive degenerative process accompanied with chronic
low-grade inflammation, often called ‘inflammaging’. The
latter implies similar underlying mechanisms during aging
and acute inflammation.
We identified the BCSFB as the first CNS barrier
disrupted during systemic acute inflammation and we found
that this barrier is also severely affected during healthy
aging. This disruption results in deregulation of brain
homeostasis and consequent neurological complications.
We identified an important role for the cytokine tumor
necrosis factor (TNF) and matrix metalloproteinases
(MMPs) MMP3 and MMP8, which all seem to play a
detrimental role in the induction of BCSFB leakage. This
disruption of the BCSFB results in increased cytokine levels
in the CSF and consequent brain inflammation.
In conclusion, we found that BCSFB disruption
plays a detrimental role in systemic inflammation and aging
and we hope that unraveling the complex mechanism
behind BCSFB dysfunction will guide us towards more
effective therapies to treat systemic inflammatory disorders.
31
PREDAVANJA / LECTURES
Neurofiziologija i ponašanje / Neurophysiology and behaviour
SONIC HEDGEHOG I SPECIFIKACIJA HUMANIH
KORTIKALNIH INTERNEURONA
SONIC HEDGEHOG AND SPECIFICATION OF
HUMAN CORTICAL INTERNEURONS
N.V. Radonjić1, 2, N. Zecevic1
1
Department of Neuroscience; University of Connecticut
Health Center; Farmington; CT, 06030 USA;
2
Institut za medicinsku i kliničku biohemiju; Medicinski
fakultet; Univerzitet u Beogradu; Pasterova 2, 11000
Beograd; Srbija
[email protected]
N.V. Radonjić1, 2, N. Zecevic1
Department of Neuroscience; University of Connecticut
Health Center; Farmington; CT, 06030; USA;
2
Institute of Medical and Clinical Biochemistry; School of
Medicine; University of Belgrade; Pasterova 2, 11000
Belgrade; Serbia
[email protected]
Uvod: Kortikalni interneuroni predstavljaju
sastavnu komponentu kortikalnog kruga, gde vrše regulaciju
ekscitacije piramidalnih neurona i uspostavljaju normalnu
integrativnu funkciju moždanog korteksa. Kod primata,
kortikalniinterneuroni imaju dvojno poreklo, dorzalnu
kortikalnu
ventrikularnu/subventrikularnu
zonu
i
ventralnoganglionarno uzvišenje. Sonic hedgehog (Shh)
predstavlja jedan od osnovnih ventralnih razvojnih
morfogena koji indukuje brojne unutarćelijske odgovore i
utiče na specifikaciju i diferencijaciju kortikalnih neurona
kod glodara. Iako je Shh u toku razvoja eksprimiran i u
dorzalnom telencefalonu, funkcije Shh u ovoj regiji nisu u
potpunosti razjašnjene.
Cilj: Ispitati uticaj Shh na stvaranje i specifikaciju
dorzalnih humanih kortikalnih interneurona.
Materijal i metode: Izolovali smo ćelije radijalne
glije (RG) iz disociranih mešovitih ćelijskih kultura
uspostavljenih od dorzalne i ventralne proliferativne zone
fetalnog telencefalona starih 14-19 gestacionih nedelja. U
ovim ćelijskim kulturama smo farmakološki manipulisali
Shh signalizaciju i proučavali efekte Shh na stvaranje i
specifikaciju ćelija obeleženih interneuronskim markerima.
Rezultati: Pokazali smo da ćelije RG nezavisno od
regije imaju neophodne komponente za Shh signalni put,
uključujući Shh receptore, Patched1 i Smoothen kao i
članove Gli transkripcione familije. Dodavanje egzogenog
Shh-a ili njegovog analoga purmorfamina, dovodi do
povećanja broja Nkx2.1 progenitora, dok inhibicija
endogenog Shh signalnog puta ciklopaminom ili antiShhantitelom ne utiče na Nkx2.1 ekspresiju, ukazujući na
prisustvo Shh-nezavisnog puta za stvaranje Nkx2.1 celija in
vitro. Takoñe smo pokazali da Shh signalizacija vrsi
supresiju stvaranja kalretinin-pozitivnih celija, dok
inhibicija indukuje njihovo stvaranje.
Zaključak: Ovi podaci ukazuju da Shh signalizacija
u humanom fetalnom mozgu ima uticaj na nastanak i
specifikaciju dorzalnih kortikalnih interneurona od RG.
Cortical interneurons regulate the excitation of
pyramidal neurons, and establish normal integrative
function of the cerebral cortex. In primates, cortical
interneurons
have
dual
origin,
dorsal
cortical
ventricular/subventricular zone and ventral ganglionic
eminence (GE). Sonic hedgehog (Shh), an essential
morphogen, induces various intracellular responses
andinfluences specification and differentiation of cortical
interneuronsin rodents. In mice, parvalbumin- and
somatostatin-positive interneurons originate from the
Nkx2.1+ progenitors in the medial GE, whereas calretininpositive cells originate mainly from caudal GE. Although
Shh is also expressed in the dorsal human telencephalon
during development, its functions at this location remain
unclear.
We isolated radial glia cells (RG), multipotent
progenitors, from dorsal (cortical) proliferative zone and
ventral (GE) human fetal forebrain at 14 and 17 gestational
week. In these cultures we demonstrated that RG cells
differentiated into Nkx2.1+ progenitors and cells labeled
with interneuron markers, GABA and calretinin. We then
pharmacologically manipulated Shh signaling and studied
its effect on generation and specification of cells labeled
with interneuronal markers.
We demonstrated that human RG cells regardless
of the region of origin have necessary components for Shh
signaling pathway, including Shh receptors, Smoothen and
Patched-1, and members of Gli transcription factor family.
Adding Shh or its agonist purmorphamin, increased the
number of Nkx2.1+ progenitors. On the other hand,
inhibition of Shh signaling with its inhibitor cyclopamine or
anti-Shh antibody, did not affect Nkx2.1 expression.
Combined these results imply the existence of Shhindependent pathway for generation of Nkx2.1+progenitors.
Furthermore, we revealed that Shh signaling suppresses
production of calretinin+ cells, while its inhibition induces
their generation.
These data suggest that Shh signaling in human
fetal brain influences generation of cortical interneurons
from RG cells as well as their specification.
1
32
POSTER SESIJE / POSTER SESSIONS
POSTER SESIJE / POSTER SESSIONS
33
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
UTICAJ RESTRIKTIVNOG REŽIMA ISHRANE NA
EKSPRESIJU ANTIAPOPTOTSKIH PROTEINA U
ANIMALNOM MODELU TRAUMATSKE POVREDE
MOZGA
INFLUENCE OF FOOD RESTRICTION ON
EXPRESSION OF ANTI-APOPTOTIC PROTEINS IN
AN ANIMAL MODEL OF TRAUMATIC BRAIN
INJURY
M. Brkić1, N. Lončarević-Vasiljković1, V. Pešić1, V. Tešić1,
M. Perović1, Lj. Rakić2, S. Ruždijić1, S. Kanazir1
1
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; 11060 Beograd; Srbija;
2
Srpska akademija nauka i umetnosti; 11000 Beograd;
Srbija
[email protected]
M. Brkic1, N. Loncarevic-Vasiljkovic1, V. Pesic1, V. Tesic1,
M. Perovic1, Lj. Rakic2, S. Ruzdijic1, S. Kanazir1
1
Institute for Biological Research “Sinisa Stankovic”;
University of Belgrade; 11060 Belgrade; Serbia;
2
Serbian Academy of Sciences and Arts; 11000 Belgrade;
Serbia
[email protected]
Traumatska povreda mozga (TPM) je vodeći
uzročnik smrti i invaliditeta, prevashodno u populaciji
mlañeg uzrasta. Nakon primarne povrede mozga, sledi
sekundarna povreda pokrenuta od strane brojnih
inflamatornih procesa, koja uglavnom vodi ćelijskoj smrti,
prepoznatoj kao apoptoza. Pretpostavlja se da smanjenje
obima apoptotskih procesa može dovesti do boljeg
oporavka nakon povrede. U literaturi postoje podaci da više
mesečni restriktivni režim ishrane pre akutne TPM ima
neuroprotektivna svojstva.
Cilj ovog istraživanja je bio ispitivanje efekata
restriktivnog režima ishrane na ekspresiju dva proteina
odgovornih za preživljavanje ćelija, Bcl-2 I Bcl-XL.
Eksperimenti su radjeni na mužjacima pacova soja
Wistar starim tri meseca, koji su bili podvrgnuti ili ad
libitum (AL), ili restriktivnom režimu od 50% normalnog
dnevnog unosa hrane. Kod svih životinja je u šestom
mesecu života izvršena ubodna lezija u senzomotornom
delu kore velikog mozga. Koristeći PCR i Western blot
tehnike praćeni su nivoi ekspresije iRNK i proteina u
korteksu.
Dobijeni rezultati pokazuju da je nivo iRNK u
slučaju oba antiapoptotska proteina, Bcl-2 i Bcl-XL, bio viši
u korteksu povredjenih pacova prethodno podvrgnutih
restriktivnom režimu ishrane, sa najvišim porastom u
četrnaestom i sedmom danu, respektivno. Uprkos tome,
proteinska ekspresija Bcl-2 molekula je bila slična kod obe
grupe životinja, dok je ekspresija Bcl-XL proteina bila
značajno viša kod AL životinja.
Neočekivani porast Bcl-XL proteina u AL grupi
mogao bi da ukazuje na pokušaj endogenog odbrambenog
mehanizma nestabilnog i povreñenog sistema da se zaštiti
od dalje štete. U kontekstu manjeg povećanja Bcl-XL u
grupi podvrgnutoj restriktivnom režimu ishrane, možemo
špekulisati da se radi o dobrom preduslovljavanju sistema,
koje može pomoći u lečenju TPM.
Traumatic brain injury (TBI) is a leading cause of
death and disability worldwide, especially amongst
youngsters. After primary brain injury, secondary follows,
initiated by inflammation, that mainly leads to cell death,
recognized as apoptosis. Thus, attenuation of apoptosisrelated processes could lead to better general outcome. It
has been shown that several-months-long food restriction
(FR) prior to an acute TBI, exhibits neuroprotective
properties.
The goal of the present study was to examine the
effects of FR on expression of two survival promoting
proteins, Bcl-2 and Bcl-XL.
Experiments were performed on 3-months-old
male Wistar rats either fed ad libitum (AL) or exposed to
50% of the normal daily food intake (DR) until 6 months of
age, when stab lesion to the sensorimotor cortex was done.
Using PCR and Western blot, expression levels of mRNA
and proteins were investigated.
Our results have shown that the levels of mRNA
for both anti-apoptotic proteins, Bcl-2 and Bcl-XL, were
higher in injured cortex of FR compared to AL group, with
the peak at 14th and 7th day, respectively. However, unlike
protein expression of Bcl-2, being equally raised in FR and
AL animals, Bcl-XL was significantly higher in AL
animals.
Unexpectedly high levels of Bcl-XL protein in AL
group could indicate the attempt of endogenous defense
mechanism of an unstable injured system to protect itself
from deleterious damage. In this contex less increase of
Bcl-XL in FR group could signify good preconditioning
capabilities of such intervention for treatment of TBI.
34
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
DA LI POLIMORFIZAM TAQ1A DRD2 RECEPTOR
GENA DOPRINOSI LEVODOPA INDUKOVANIH
HALUCINACIJA KOD OBOLELIH OD
PARKINSONOVE BOLESTI?
DOES POLYMORPHISM TAQ1A DOPAMINERGIC
RECEPTOR GENE CONTRIBUTE TO LEVODOPA
INDUCED HALLUCINATIONS IN PATIENTS WITH
PARKINSON’S DISEASE?
I. Buzadžić1, D. Drakulić2, M. Stanojlović2, T. V Ilić3
1
Odeljenje za humanu genetiku i prenatalnu dijagnostiku;
Kliničko bolnički centar Zvezdara; Univerzitet Univerzitet u
Beogradu; Beograd; Srbija
2
Laboratorija za molekularnu biologiju i endokrinologiju;
Institut za nuklearne nauke “Vinča”; Univerzitet u
Beogradu; Beograd; Srbija
3
Klinika za neurologiju, Vojnomedicinska akademija,
Medicinski fakultet VMA, Univerzitet odbrane, Beograd,
Srbija
[email protected]
I. Buzadžić1, D. Drakulić2, M. Stanojlović2, T. V Ilić3
Department of Human Genetics and Prenatal Diagnostics,
University Medical Hospital Zvezdara, University of
Belgrade, Belgrade, Serbia
2
Laboratory of Molecular Biology and Endocrinology,
VINČA Institute of Nuclear Sciences, University of
Belgrade, Belgrade, Serbia
3
Department of Neurology, Military Medical Academy,
Belgrade, Serbia
[email protected]
Uvod. Parkinsonova bolest je progresiva bolest i
lekovi koji se koriste vremenom gube efikasnost dovode
dovodeći do ispoljavanja neželjenih efekata. Kod 40%
obolelih od Parkinonove bolesti efikasnost levodope koja se
obično koristi u kombinaciji sa inhibitorima dopa
dekarboksilaze ograničena je vremenom zbog pojave
halucinacija, diskinezije i drugih motornih komplikacija.
Istraživanja na polju farmakogenetoke su pokazala da je
genetička varijabilnost svakog pojedinca u velikoj meri
odgovorna za terapijski odgovor. S obzirom da
dopaminergični receptori regulišu nivo dopamina i time
doprinose održavanju vitalnih funkcija, cilj istraživanja je
ispitati ulogu Taq1A polimorfizma u pojavi halucinacija i
povećane osetljivosti.
Metod. Taq1A polimorfizam je odreñen tehnikom
lančane reakcije polimeraze sa odgovarajućim restrikcionim
enzimima
Rezultat. Uporeñujući pacijente sa i bez
halucinacija, najveći broj onih koji su imali halucinacije su
bili heterozigoti A1/A2, i time bi se prisustvo A1 alela
moglo dovesti u vezu sa pojavom ovog vida komplikacija.
Diskusija. Prisustvo jednog ili dva alela A1 može
se dovesti u vezu sa smanjenim vezivanjem levodope za
receptore u strijatumu, smanjena ekspresija DRD2 receptora
bi mogla dovesti do povećane osetljivosti, pojavu
halucinacija i drugih neželjenih efekata.
1
Introduction: As Parkinson's disease (PD) is
progressive disease, the drugs used in its treatment
gradually loose efficiency and lead to appearance of side
effects. For instance, in 40% PD patients, the long-term
efficiency of the drug levodopa that is usually used in
combination with dopa decarboxylase inhibitors is limited
in the course of time by severe hallucinations, motor
complications and drug-induced dyskinesia. Research in
pharmacogenetics has shown that the genetic variability of
each individual is largely responsible for therapeutic
response. Thus, given that dopaminergic (DRD2) receptors
regulate the activity levels of dopamine and thereby
contribute to the maintenance of vital functions, the aim of
current study was to examine the polymorphisms in genes
for DRD2 receptors that could be associated with increased
sensitivity and hallucinations.
Method: TaqIA genotype was determined by
polymerase chain reaction-restriction fragment length
polymorphism.
Results: Comparing patients with and without
hallucinations, most of those who had hallucinations were
heterozygous A1/A2, indicating that presence of A1 allele
may be associated with appearance of this type of
complication.
Discussion: The presence of one or two A1 alleles
could be associated with reduced binding of levodopa to
DRD2 receptors in striatum and, thus the reduced
expression of DRD2 receptors could contribute to increased
susceptibility to dopaminergic drugs, occurrence of
hallucinations and other side effects.
35
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
ULOGA ADENOZIN-MONOFOSFATOM
AKTIVISANE KINAZE (AMPK) U
NEUROTOKSIČNOM DELOVANJU
UNUTARĆELIJSKOG I VANĆELIJSKOG ALFASINUKLEINA IN VITRO
ADENOSINE MONOPHOSPHATE-ACTIVATED
PROTEIN KINASE (AMPK) AS A POTENTIAL
PLAYER IN NEUROTOXICITY CAUSED BY
INTRACELLULAR AND EXTRACELLULAR
ALPHA-SYNUCLEIN ACCUMULATION IN VITRO
M. Dulović1, M. Jovanović1, Lj. Harhaji-Trajković2, V.
Kostić3, I. Marković1, V. Trajković4
1
Institut za medicinsku i kliničku biohemiju; Medicinski
fakultet; Univerzitet u Beogradu; Srbija;
2
Institut za biološka istraživanja „Siniša Stanković“;
Univerzitet u Beogradu; Srbija;
3
Klinika za neurologiju; KCS; Univerzitet u Beogradu,
Srbija;
4
Institut za mikrobiologiju i imunologiju; Medicinski
fakultet; Univerzitet u Beogradu; Srbija
[email protected]
M. Dulovic1, M. Jovanovic1, Lj. Harhaji-Trajkovic2, V.
Kostic3, I. Markovic1, V. Trajkovic4
1
Institute of Medical and Clinical Biochemistry; School of
Medicine; University of Belgrade; Serbia; 2Institute for
Biological Research „Sinisa Stankovic“; University of
Belgrade; Serbia;
3
Clinic for Neurology; Clinical Center of Serbia;
4
Institute of Microbiology and Immunology; School of
Medicine; University of Belgrade; Serbia
[email protected]
Uvod: Prekomerna akumulacija alfa-sinukleina
(ASYN) smatra se značajnim patogenetskim činiocem u
nastanku Parkinsonove bolesti. Pokazano je da protein
ASYN iako uglavnom unutarćelijski, može biti prisutan i u
vanćelijskom okruženju. Imajući u vidu da su neuroni
veoma osetljivi na promenu energetskog statusa u ćeliji, kao
i da ASYN može uzrokovati oštećenje funkcije
mitohondrija, cilj našeg istraživanja je bio ispitivanje uloge
AMPK, ključnog unutarćelijskog energetskog senzora, u
oštećenju
neurona
prouzrokovanom
prekomernim
nakupljanjem ASYN.
Metode: Uloga AMPK signalnog puta ispitivana je
u retinoičnom kiselinom-diferentovanoj ćelijskoj liniji
humanog neuroblastoma SH-SY5Y koja prekomerno
eksprimira normalni (wt) ASYN (unutarćelijski ASYN),
kao i u kontrolnim SH-SY5Y ćelijama koje su gajene u
medijumu sa sekretovanim ASYN, sakupljanim od ćelija
koje prekomerno eksprimiraju ASYN (vanćelijski ASYN).
Rezultati: Diferencijacija retinoičnom kiselinom
dovela je do povećane citotoksičnosti ASYN u ćelijama
koje prekomerno produkuju ASYN, što je bilo praćeno
smanjenjem aktivacije AMPK i nishodne kinaze Raptor.
Farmakološki aktivatori AMPK, AICAR i metformin,
doveli su do smanjenja neurotoksičnog efekta ASYN,
nezavisno od aktivacije Akt-signalnog puta. Ćelije
neuroblastoma, gajene u medijumu sa sekretovanim ASYN,
pokazale su značajno smanjenje ćelijskog vijabiliteta, kao i
smanjenu aktivaciju AMPK, dok su aktivatori AMPK
(AICAR i metformin) poboljšali preživljavanje ovih ćelija.
Protektivna uloga AMPK je potvrñena je time što su ćelije
neuroblastoma sa smanjenom ekspresijom AMPK gena
(delovanjem shRNA) bole osetljivije na toksično delovanje
sekretovanog ASYN.
Zaključak: Dobijeni rezultati ukazuju na
protektivnu ulogu AMPK u neurotoksičnom delovanju kako
unutarćelijskog, tako i vanćelijskog ASYN, što ukazuje na
mogućnost da bi modulacija aktivnosti AMPK mogla biti
neuroprotektivna kod Parkinsonove bolesti.
Introduction: The accumulation of wild-type (wt)
alpha-synuclein (ASYN) in susceptible neurons is regarded
as essential in Parkinson’s disease pathogenesis. ASYN,
although mostly regarded as purely intracellular protein,
was recently also detected extracellularly. Having in mind
that ASYN can affect mitochondrial function, as well as
high sensitivity of neurons to changes in cellular energy
level; we investigated if modulation of AMPK, a key
intracellular energy sensor, might play a role in neurotoxic
effect of ASYN.
Materials and Methods: The role of AMPK was
investigated
in
retinoic-acid-differentiated
human
neuroblastoma SH-SY5Y cells stably expressing wtASYN
(intracellular ASYN), as well as in SH-SY5Y cells exposed
to secreted ASYN, present in conditioned medium (CM),
collected from ASYN-overexpressing cells (extracellular
ASYN).
Results: Differentiation with retinoic acidhad
pronounced cytotoxic effect on ASYN-overexpressing cells,
associated with decrease in activation of AMPK and its
downstream target Raptor. AMPK activators, AICAR and
metformin, reduced the neurotoxicity associated with
ASYN overexpression, in Akt-independent manner.
Application of CM containing secreted ASYN caused cell
death of recipient SH-SY5Y cells, as well as a decrease in
activation of AMPK, whereas application of AICAR and
metformin abolished those neurotoxic effects. The
protective role of AMPK was additionally confirmed, as
shRNA-mediated downregulation of AMPK significantly
affected cell survival of differentiated SH-SY5Y cells
cultivated in CM.
Conclusion: These results confirm the protective
role of AMPK against toxicity of both intracellular and
extracellular wtASYN, and indicate that modulation of
AMPK activity may be an intriguing neuro-protective
strategy in Parkinson’s disease.
36
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
POLNO-SPECIFIČNA MODULACIJA EKSPRESIJE
EKTO-5'-NUKLEOTIDAZE U HIPOKAMPUSU
PACOVA NAKON SUBHRONIČNOG TRETMANA
STEROIDNIM HORMONIMA
GENDER-SPECIFIC MODULATION OF ECTO-5'NUCLEOTIDASE EXPRESSION RAT
HIPPOCAMPUS FOLLOWING SUBCHRONIC
STEROID HORMONE TREATMENT
I. Guševac, D. Drakulić, M. Stanojlović, N. Mitrović, I.
Grković, A. Horvat
Laboratorija za molekularnu biologiju i endokrinologiju;
Vinča Institut za nuklearne nauke; Univerzitet u Beogradu;
P.O.Box 522, 11001 Beograd; Srbija
[email protected]
I. Guševac, D. Drakulić, M. Stanojlović, N. Mitrović, I.
Grković, A. Horvat
Laboratory of Molecular Biology and Endocrinology; Vinča
Institute of Nuclear Sciences; University of Belgrade;
P.O.Box 522, 11001 Belgrade; Serbia
[email protected]
Uvod: Adenozin je važan neuromodulator i
regulator homeostaze, uključen u veliki broj funkcija
centralnog nervnog sistema u fiziološkim i patofiziološkim
uslovima. Ekto-5’-nukleotidaza (5’NT) kontroliše njegov
nivo u sinaptičkoj pukotini. 5’NT, ektoenzim lokalizovan na
spoljašnjoj
membrani
ćelije
pomoću
glikozil
fosfatidilinozitol (GPI) repića, je ključni enzim u hidrolizi
AMP-a do adenozina u sinaptičkoj pukotini.
Cilj: S obzirom da progesteron i estradiol imaju
ključnu ulogu u funkcionisanju mozga i da se koriste u
terapiji različitih bolesti, u ovoj studiji je praćeno da li su
ženski gonadalni steroidni hormoni u mogućnosti da
menjaju proteinsku ekspresiju 5’NT.
Materijali i metode: Mužjaci i ženke pacova Wistar
soja starosti 3 meseca su tretirani 7 dana fiziološkim
dozama progesterona i estradiola apliciranim jednom
dnevno u obliku subkutanih injekcija. Ekspresija 5’NT u
sinaptozomalnim frakcijama hipokampusa je praćena
Western blot metodom.
Rezultati: U zadatim eksperimentalnim uslovima, kod
jedinki oba pola progesteron je doveo do statistički
značajnog povećanja ekspresije 5’NT u sinaptičkim
pukotinama. Estradiol nije uticao na ekpresiju 5’NT kod
mužjaka, dok je kod ženki uzrokovao porast njegove
ekspresije.
Zaključak: Ženski gonadalni hormoni mogu
menjati ekspresiju enzima ključnih za nastanak adenozina u
hipokampusu pacova na polno-specifičan način. Naši
rezultati su bitni zbog substitucionih hormonskih terapija,
jer je odnos izmeñu ovih hormona i adenozina još uvek
nerazjašnjen.
Introduction: Adenosine is an important
neuromodulator and homeostatic regulator, involved in a
broad array of functions in the central nervous system in
physiological and pathophysiological conditions. Level of
this molecule in synaptic cleft is controlled by ecto-5'nucleotidase (5’NT). Namely, 5’NT, an ectoenzyme
anchored to the extracellular surface of cell membrane
through a glycosyl phosphatidylinositol (GPI) linkage, is the
key enzyme responsible for the hydrolysis of synaptic AMP
to adenosine.
Aim: Given that progesterone and estradiol exert a
pivotal role in the brain functioning and also have been
widely used in various therapeutic purposes, the current
study investigated whether these female gonadal steroid
hormones are able to modulate the protein expression of
5’NT.
Materials and methods: Three-months-old male
and female Wistar rats were treated for seven days with
daily physiological subcutaneous injections of progesterone
or estradiol. The expression level of 5’NT in hippocampal
crude synaptosomal fraction was determined by Western
blot analysis.
Results: Progesterone significantly increased 5’NT
protein expression on rat hippocampal synaptic membranes
of both genders. Estradiol did not influence 5’NT protein
level in males while in females it induced a significant
enhancement.
Conclusion: Female gonadal hormones are capable
to modulate expression of the adenosine producing enzyme
in rat hippocampus in gender specific manner. These results
are essential for hormone replacement therapy, while the
relationship between these hormones and adenosine is still
matter of controversy.
37
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
ECTO-NUCLEOSIDE TRIPHOSPHATE
DIPHOSPHOHYDROLASE2 (NTPDASE2) IS
DOWNREGULATED IN OLIGODENDROCYTES IN
RESPONSE TO OXIDATIVE STRESS
M. Jovanović1, 2, J. Goldberg1, M. Victor1, N. Nedeljković2,
M. Kipp1
1
Institute of Neuroanatomy, Faculty of Medicine, University
Hospital Aachen, Germany
2
Institute of Physiology and Biochemistry, Faculty of
Biology, University of Belgrade, Serbia
[email protected]
Ectonucleoside triphosphate diphosphohydrolase 2
(NTPDase2) is an ecto-enzyme involved in the extracellular
hydrolysis of adenosine 5’ triphosphate (ATP) to adenosine,
as the final product. While adenosine exerts mainly antiinflammatory effects, ATP activates inflammatory signaling
cascades. With respect to demyelinating disorders it has
been demonstrated that ATP is cytotoxic for
oligodendrocytes. In the current project we follow the idea
that
oligodendrocytes
actively
participate
in
neuroinflammatory processes by regulating ATP
metabolism via the function of ectonuleotidases.
Male C57BL6 animals were treated for up to 5
weeks with the mitochondrial respiratory chain inhibitor
cuprizone
(0.25%),
transcardially
perfused
and
subsequently embedded for immunohistochemistry. In
parallel experiments, different brain regions were dissected
and analyzed for gene expression levels. Genome wide
Affymetrix array analysis were performed to study
genomics of purinergic signaling in this demyelination
model in a broader context. Additionally, two well
characterized oligodendrocyte cell lines, namely OLN93
and OLIneu, were exposed for 24 hours to specific
inhibitors of the respiratory chain (i.e. Sodiumazid,
Rotenon, Antimycin-A).
Affymetrix array analysis revealed a complex
regulation of purinergic signaling molecules, among
NTPDase2 which was dramatically reduced early after
initiation of the cuprizone diet. This reduction in NTPDase2
expression levels were paralleled by selective
oligodendrocyte apoptosis and microglia activation. In line
with these findings, cultured oligodendrocytes showed
reduced NTPDase2 expression levels once exposed to
respiratory chain inhibitors. During later disease stages,
NTPDase2 expression levels recovered once astrocytosis
became evident.
Our results suggest that oligodendrocytes can
regulate
extracellular
ATP
concentrations
under
neuroinflammatory paradigms. Further studies using
appropriate knock-down approaches have to reveal the
relevance of this regulation for oligodendrocyte viability,
demyelination and concomitant axonal damage.
38
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
PROMENA FOSFORILACIJE
GLUKOKORTIKOIDNOG RECEPTORA U
LIMFOCITIMA TOKOM PRAĆENJA
TARAPEUTSKOG EFEKTA REPETITIVNE
TRANSKRANIJALNE MAGNETNE STIMULACIJE
KOD PACIJENATA SA DEPRESIVNIM
POREMEĆAJEM (PILOT STUDIJA)
1
1
2
1
I. Lukić , N. Božović , M. Jovičić , M. Mitić , I.
Soldatović3, N. Marić2, 3, M. Adžić1
1
Laboratorija za molekularnu biologiju i endokrinologiju;
Institut za nuklearne nauke “Vinča”; Univerzitet u
Beogradu; Beograd; Srbija;
2
Klinika za psihijatriju; Klinički centar Srbije; Beograd;
Srbija ;
3
Medicinski fakultet; Univerzitet u Beogradu; Beograd;
Srbija
[email protected]
Odavno je poznato da narušena funkcija
glukokortikoidnog receptora (GR) igra važnu ulogu u
patogenezi depresivnog poremećaja. U našoj laboratoriji je
pokazano da je fosforilacija GR-a izmenjena kod pacijenata
sa akutnom depresijom i da pretstavlja još jedan od bitnih
aspekata funkcije GR-a koju treba dodati postojećim
saznanjima glukokortikoidne teorije depresije.
Cilj ove pilot studije je bio da pratimo promenu
fosforilacije GR-a u jedarnoj frakciji limfocita periferne
krvi, na serinima 211 (pGR-S211) i 226 (pGR-S226)
važnim za funkciju GR-a, kod pacijenata sa depresivnim
poremećajem, lečenih repetitivnom transkranijalnom
magnetnom stimulacijom (rTMS) kao adjuvantnom
terapijom, i utvrdimo da li postoji korelacija izmeñu
promena u fosforilaciji GR-a i poboljšanja depresivnog
stanja tokom vremena.
Testiranje pacijenata (težina depresivnih simptoma
merena je Hamiltonovom skalom depresivnosti) i uzimanje
uzorka periferne krvi vršeno je u tri tačke: 1) pre početka
tretmana rTMS-om, kada su pacijenti bili u akutnoj
depresivnoj epizodi, 2) posle 3 nedelje - nakon završetka
tretmana rTMS-om, i 3) 12 nedelja nakon prvog
uzorkovanja. Nivoi fosforilacije GR-a odreñivani su
Western blot tehnikom. Rezultati su obrañeni statističkom
metodom Generalised Estimated Equations.
Poboljšanje depresivnog stanja najbolje je bilo
praćeno povećanjem odnosa fosfoformi pGR-S211/pGRS226, koji se može smatrati markerom nivoa transkripcione
aktivnosti GR-a. Promene u pojedinim nivoima pGR-S226,
pGR-S211 i ukupnog GR-a nisu bile statistički značajne.
Iako je uzorak bio mali (4 pacijenta), rezultati ovog
istraživanja dopunjavaju naše prethodne nalaze i ukazuju da
praćenje odnosa pGR-S211/pGR-S226 bi mogao nadalje
biti israživan kao dobar pokazatelj ne samo akutnog
depresivnog stanja već i poboljšanja mentalnog stanja i
glukokortikoidne signalizacije tokom lečenja depresije.
ALTERATIONS IN LYMPHOCITE
GLUCOCORTICOID RECEPTOR
PHOSPHORYLATION DURING FOLLOW-UP OF
DEPRESSED PATIENTS TREATED WITH
REPETITIVE TRANSCRANIAL MAGNETIC
STIMULATION (A PILOT STUDY)
I. Lukić1, N. Božović1, M. Jovičić2, M. Mitić1, I.
Soldatović3, N. Marić2, 3, M. Adžić1
1
Laboratory of molecular biology and endocrinology;
Institute of nuclear sciences “Vinča”; University of
Belgrade; Belgrade; Serbia;
2
Clinic for Psychiatry; Clinical Center of Serbia; Belgrade;
Serbia;
3
Faculty of Medicine; University of Belgrade; Belgrade;
Serbia
[email protected]
The impared glucocorticoid signalization has for
long been recognised in depressed patients. Recently, we
showed that phosphorylation of limphocyte glucocorticoid
receptor (GR) is also atered in patients with acute
depressive episode and contributs to impared GR function
in depression.
The aim of this pilot study was to extand our
previous reserch, particularly, to assess changes in
limphocyte nuclear GR phosphorilation at serine 211 (pGRS211) and 226 (pGR-S226), in patients with major
depressive disorder treated with repetitive transcranial
magnetic stimulation (rTMS) as adjuvant method, and to
examine if there are correlations between alternations in the
GR phosphorylation and improvement of their depressive
state during time.
The measurements of patients’ depressive states
(assessed by Hamilton Depression Scale) and blood
samplings occurred three times: 1) before beginning of
rTMS treatment, when patient were acutely depressed, 2) 3
weeks afterwards – after the last rTMS treatment, and 3) 12
weeks after the first assessment. The levels of GR
phosphoisoforms were measured by Western blot. The data
were analyzed by statistical method Generalized Estimated
Equations.
The best indicator of improvement of depressive
state was the pGR-S211/pGR-S226 ratio, known as good
marker of GR transcriptional activity. The alterations of
pGR-S211, pGR-S226 and total GR levels were not
significantly correlated with alterations in depressive state.
This pilot study, although conducted with only 4
patients, complements our previous results and proposes
that pGR-S211/pGR-S226 ratio could be further explored as
biomarker not only for depressive state, but also for followup of depressive symptoms and improvement of GR
signalization during treatment.
39
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
KONSTITUTIVNO POVEĆANA EKSPRESIJA SOX2
GENA USPORAVA PROCES NEURALNE
DIFERENCIJACIJE NT2/D1 ĆELIJA
CONSTITUTIVE SOX2 OVEREXPRESSION
DELAYS THE PROCESS OF NEURAL
DIFFERENTIATION OF NT2/D1 CELLS
A. Klajn,1 D. Drakulić,1 Ž. Pavković,2 J. Marjanović,1 M.
Tošić,1 J. Tošić,1 M. Stevanović,1
1
Institut za molekularnu genetiku i genetičko inženjerstvo;
Univerzitet u Beogradu; Vojvode Stepe 444a, 11010
Beograd; Srbija;
2
Institut za Biološka Istraživanja "Siniša Stanković";
Univerzitet u Beogradu; Bulevar despota Stefana 142,
11060 Beograd; Srbija
[email protected]
A.Klajn,1 D. Drakulić,1 Ž. Pavković,2 J. Marjanović,1 M.
Tošić,1 J. Tošić,1 M. Stevanović,1
1
Institute of Molecular Genetics and Genetic Engineering,
University of Belgrade; Vojvode Stepe 444a, 11010;
Belgrade; Serbia;
2
Institute for Biological Research “Siniša Stanković”;
University of Belgrade; Bulevar despota Stefana 142,
11060; Belgrade; Serbia
[email protected]
Uvod: SOX2 transkripcioni faktor predstavlja
univerzalni marker pluripotentnih matičnih ćelija koji
reguliše održavanje pluripotentnosti i diferencijaciju ovih
ćelija. Ovaj transkripcioni faktor ima i ulogu regulatora
neuralne diferencijacije. Podaci iz literature sugerišu da
pravilno neuralno razviće zavisi od fine kontrole nivoa
ekspresije ovog faktora.
Cilj: Analiza efekata konstitutivno povećane
ekspresije SOX2 gena na neuralnu diferencijaciju
pluripotentnih NT2/D1 ćelija.
Materijal i metode: Kao model sistem korišćene su
NT2/D1 ćelije i njihov ćelijski klon sa konstitutivno
povećanom ekspresijom SOX2 gena (G3). Neuralna
diferencijacija ćelija je indukovana retinoičnom kiselinom
(RK). Primenjene tehnike: Western blot, semikvantitativni
RT-PCR, imunocitohemija, esej za praćenje ćelijske
proliferacije.
Rezultati:Nakon četiri nedelje tretmana RK kod G3
ćelijskog klona detektovan je manji broj neurona u
poreñenju sa brojem neurona kod NT2/D1 ćelija. Tretman
sa RK dovodi do gubitka ekspresije markera
pluripotentnosti OCT4 kod NT2/D1 i G3 ćelija. U toku
diferencijacije nivo ekspresije SOX1 proteina, markera
ranih prekursora, ne opada kod G3 ćelijskog klona, za
razliku od parentalne linije gde je detektovano smanjene
ekspresije. Takoñe, ekspresija NeuroD1 gena, markera
ćelija u inermedijarnoj fazi neuronalne diferencijacije,
povećana je kod G3 ćelijskog klona u svim analiziranim
fazama neuralne diferencijacije. Pored toga, u toku neuralne
diferencijacije stopa rasta G3 ćelijskog klona je povećana u
poreñenju sa stopom rasta NT2/D1 ćelija.
Zaključak:U uslovima konstitutivno povećane
ekspresije SOX2 gena proces neuralne diferencijacije
NT2/D1 nije blokiran, ali je usporen. Većina ćelija
G3ćelijskog
klona ostaje zaustavljena
u ranim i
intermedijarnim fazama neuralne diferencijacije, a samo
manji deo ćelija se diferencira u zrele neurone.
Introduction: SOX2 transcription factor represents
a universal marker of pluripotent stem cells regulating
maintenance of pluripotency and differentiation of these
cells. It has a role in regulation of neural differentiation and
literature data indicate that SOX2 expression level must be
tightly controlled for proper neural development.
Aim: Analysis of effects of constitutive SOX2
overexpression on neural differentiation of pluripotent cells.
Material and methods: NT2\D1 cells and SOX2overexpressing NT2/D1 cell clone (G3) were used as model
systems. Neural differentiation of the cells was induced by
retinoic acid (RA). Applied techniques: Western blot, semiquantitative
RT-PCR,
immunocytohemestry,
cell
proliferation essay.
Results: Upon four week of RA treatment the number of
mature neurons was decreased in G3 cell clone comparing
to NT2/D1 cells. Upon RA treatment the expression of
pluripotency marker OCT4 was undetectable in NT2/D1
and G3 cells. The level of early precursor marker SOX1 in
NT2/D1 cells was decreasing with longer exposure to RA,
whereas in G3 cells the level of SOX1 remained the same.
The expression of NeuroD1, a neuronal marker of cells in
intermediate stages of differentiation, was increased in G3
cell clone in all analyzed phases of differentiation
comparing to NT2/D1 cells. Additionally, during neural
differentiation the proliferation rate of G3 cell clone was
higher comparing to NT2/D1.
Conclusion: Constitutive SOX2 overexpression did
not block, but induced delay of the neural differentiation of
NT2/D1 cells. The majority of G3 cells are accumulated in
early and intermediate stages of neuronal differentiation,
while only minority of them differentiates into mature
neurons.
40
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
CREB PROTEIN JE POZITIVNI TRANSKRIPCIONI
REGULATOR EKSPRESIJE SOX3 GENA U NT2/D1
ĆELIJAMA
CREB (CYCLIC AMP RESPONSE ELEMENT
BINDING) PROTEIN ACTS AS A POSITIVE
REGULATOR OF SOX3 GENE EXPRESSION IN
NT2/D1 CELLS
N. Kovačević-Grujičić, M. Mojsin, J. Popović, I. Petrović,
V. Topalović, M. Milivojević, M. Stevanović
Institut za molekularnu genetiku i genetičko inženjerstvo;
Univerzitet u Beogradu; Beograd; Srbija
[email protected]
N. Kovačević-Grujičić, M. Mojsin, J. Popović, I. Petrović,
V. Topalović, M. Milivojević, M. Stevanović
Institute of Molecular Genetics and Genetic Engineering;
University of Belgrade; Belgrade; Serbia
[email protected]
Uvod: SOX3 i CREB imaju važnu ulogu u
neurogenezi kod kičmenjaka. Sox3 se tranzijentno
eksprimira u neuralnim progenitorima neurogenih zona
neonatalnog i adultnog prednjeg mozga miša sugerišući
ulogu SOX3 u regulaciji proliferacije i opstanka neuralnih
progenitora tokom celog života. Transkripcioni faktor
CREB je konstitutivno aktiviran u nezrelim, proliferišućim
neuralnim ćelijama neurogenih zona embrionalnog i
adultnog mozga kod kičmenjaka.
Cilj: Cilj ove studije je funkcionalna analiza uloge
CREB proteina u transkripcionoj regulaciji ekspresije SOX3
gena u NT2/D1 ćelijama.
Materijal i metode: Vezivanje CREB proteina za
CRE polumesto u okviru SOX3 promotorskog regiona je
ispitivano metodom usporene elektroforetske pokretljivosti
u
gelu,
“supershift”
esejem
i
hromatinskom
imunoprecipitacijom. Efekti CREB proteina i njegovog
dominantno negativnog inhibitora (A-CREB) na
promotorsku aktivnost i ekspresiju SOX3 su ispitivani
funkcionalnim CAT esejima i Western blotom. Ekspresija
SOX3, pCREB i CREB proteina tokom indukcije NT2/D1
ćelija retinoičnom kiselinom, praćena je Western blotom.
Rezultati: CREB protein se specifično vezuje za
CRE polumesto u okviru SOX3 promotora kako in vitro
tako i in vivo. Povećana ekspresija CREB dovodi do
povećane ekspresije SOX3 proteina kao i njegove
promotorske aktivnosti u NT2/D1 ćelijama. Takoñe,
povećana ekspresija A-CREB inhibitora, kao i mutacija u
okviru CRE polumesta dovode do pada promotorske
aktivnosti SOX3 gena. CREB ne dovodi do značajnog
porasta inducibilnosti SOX3 promotora pod dejstvom
retinoične kiseline.
Zaključak: CREB ima ulogu pozitivnog
transkripcionog regulatora ekspresije SOX3 gena u
neindukovanim NT2/D1 ćelijama, dok njegov doprinos
aktivaciji SOX3 promotora retinoičnom kiselinom nije
značajan.
Background: SOX3 and CREB play important
roles in vertebrate neurogenesis. Sox3 is expressed
transiently by proliferating and differentiating neural
progenitors in the neurogenic regions of the neonatal and
adult mouse forebrain suggesting that SOX3 continues to
regulate proliferation and survival of neural progenitor cells
throughout life. Several studies show that CREB is
constitutively activated in dividing immature neural cells in
neurogenic regions of both embryonic and adult vertebrate
brains.
Objective: The aim of this study was to functionally analyze
the role of CREB in transcriptional regulation of SOX3
gene in NT2/D1 cell line.
Material and methods: Binding of CREB to CRE
half-site within SOX3 promoter was assessed by
electrophoretic mobility shift, supershift and chromatin
immunoprecipitation assays. Effects of CREB and its
dominant-negative inhibitor A-CREB on SOX3 promoter
activity and expression were examined by functional CAT
assays and Western blot. Expression of SOX3, pCREB and
CREB during retinoic acid (RA) induction of NT2/D1 cells
was assessed by Western blot.
Results: CREB binds to CRE half-site within the
human SOX3 promoter both in vitro and in vivo. CREB
overexpression upregulates both SOX3 expression and
promoter activity in NT2/D1 cells. In addition,
overexpression of A-CREB as well as mutation of CRE
half-site lead to reduction in SOX3 promoter activity. RA
inducibility of the SOX3 promoter in the presence of CREB
is not considerably increased.
Conclusion: CREB acts as a positive regulator of
SOX3 gene expression in uninduced NT2/D1 cells, while its
contribution to RA induction of SOX3 promoter is not
prominent.
41
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
POLNO-SPECIFIČAN EFEKAT FLUOKSETINA NA
PONAŠANJE I FOSFORILACIJU
GLUKOKORTIKOIDNOG RECEPTORA U
HIPOKAMPUSU HRONIČNO STRESIRANIH
ŽIVOTINJA
GENDER-SPECIFIC EFFECTS OF FLUOXETINE
ON HIPPOCAMPAL GLUCOCORTICOID
RECEPTOR PHOSPHORYLATION AND BEHAVIOR
IN CHRONICALLY STRESSED RATS
M. Mitić, I. Simić, J. Đorñević, M.B. Radojčić, M. Adžić
Institut za nuklearne nauke “Vinča”; Univerzitet u
Beogradu; Beograd; Srbija
[email protected]
M. Mitic, I. Simic, J. Djordjevic, M.B. Radojcic, M. Adzic
VINCA Institute of Nuclear Sciences; University of
Belgrade; Serbia
[email protected]
Hroničnim stresorom izazvano ponašanje nalik
depresivnom
moduliše
funkciju
glukokortikoidnog
receptora (GR) kroz promene fosforilacionog statusa GR-a i
njegovih ushodnih kinaza.
Cilj ove studije je da ispita postojanje potencijalnih
polno-specifičnih razlika na terapiju antidepresivom
fluoksetinom na nivou ponašanja i signalizacije GR-a u
hipokampusu stresiranih životinja. Ženke i muzjaci su
tretirani fluoksetinom 21 dan i mereni su serumski
kortikosteron i ponašanje nalik depresivnom. Takoñe,
mereni su nivoi GR proteina, njegovih fosfo-izoformi na
serinu 232 (pGR232) i 246 (pGR246) i ushodnih kinaza,
ciklin-zavisne kinaze 5 (Cdk5) i c-Jun N-terminalnih kinaza
(JNK), metodom Western blota, dok je ekspresija gena za
GR i neurotroficki faktor u mozgu (BDNF) merena
metodom qRT-PCR.
Stres je promenio ponašanje životinja dovodeći do
povećane imobilnosti kod mužjaka za razliku od smanjene
imobilnosti kod ženki, i narušio signalni put, pGR232Cdk5, kao i signalizaciju JNK-ova u jedru na polnospecifičan način. Nasuprot tome, stres je povećao nivoe GRa i pGR246 u jedru, dok je koncentracija kortikosterona kao
i genska ekspresija GR-a i BDNF-a bila smanjena. Tretman
fluoksetinom je normalizovao ponašanje i izmenio pGR232Cdk5 signalizaciju na polno-specifičan način, dok je uticaj
leka na kortikosteron i nivoe GR-a i pGR246 u jedru bio
polno-nespecifičan. Fluoksetin je povećao nivo ekspresije
BDNF gena samo kod ženki, dok su nivoi ekspresije GR-a
ostali nepromenjeni u oba pola.
Nañene promene na nivou pGR232-Cdk5
signalizacije i ekspresije BDNF-a u hipokampusu ženki i
mužjaka kao i ponašanju pri tretmanu fluoksetinom
naglašavaju važnost ovog signalnog puta u polnospecifičnoj terapiji depresivnih poremećaja antidepresivima.
Depressive-like behavior induced by chronic stress
modulates glucocorticoid receptor (GR) functioning through
alteration in GR phosphorylation and its upstream kinases
signaling.
The current study investigated potential gender
specificities regarding the effect of chronic antidepressant
fluoxetine treatment on rat behavior and GR signaling in
hippocampus of stressed animals.
Fluoxetine was administrated to female and male
naïve or stressed animals for 21 days and serum
corticosterone and depressive-like behavior were analysed
in parallel with molecular parameters: GR protein, its
phosphorylation and GR upstream kinases using Western
blot and GR and brain-derived neurotrophic factor (BDNF)
mRNA using real time-PCR
Stress increased immobility in males versus
hyperactivity in females and disrupted nuclear phosphoGR232-Cycline-dependent kinase 5 (Cdk5) pathway and cJun N-terminal kinase (JNK) signalling in a gender-specific
way. In contrast, stress increased nuclear GR and phosphoGR246 but decreased corticosterone and mRNA of GR and
BDNF. Fluoxetine normalized the behavior and altered the
nuclear pGR232-Cdk5 signaling in a gender-specific
manner. In both genders, FLU reversed the nuclear GR and
pGR246 without affecting CORT. In contrast, Fluoxetine
exhibited gender-specific effect on BDNF mRNA in
stressed animals, by increasing it only in females, while GR
mRNA remained unchanged in both genders.
The gender-specific alterations of phosphoGR232-Cdk5 signaling and BDNF gene expression in
hippocampus and normalization of depressive-like behavior
upon fluoxetine treatment distinguishes these signaling
pathways as potential future antidepressant targets for
gender-specific therapy of stress-related mood disorders.
42
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
EFEKAT ESTRADIOLA NA HIDROLIZU
ADENINSKIH NUKLEOTIDA U KORI PREDNJEG
MOZGA ŽENKI PACOVA NAKON HRONIČNE
CEREBRALNE HIPOPERFUZIJE
ESTRADIOL AFFECTS ADENINE NUCLEOTIDES
HYDROLYSIS IN FEMALE RAT CEREBRAL
CORTEX AFTER CHRONIC CEREBRAL
HYPOPERFUSION
N. Mitrović, D. Drakulić, M. Stanojlović, I.Guševac,
I.Grković, A.Horvat
Institut za nuklearne nauke “Vinča”; Univerzitet u
Beogradu; Beograd; Srbija
[email protected]
N. Mitrović, D. Drakulić, M. Stanojlović, I.Guševac,
I.Grković, A.Horvat
Laboratory of Molecular Biology and Endocrinology;
VINCA Institute of Nuclear Sciences; University of
Belgrade; Belgrade; Serbia
[email protected]
Hronična
cerebralna
hipoperfuzija
CCH
predstavlja patofiziološki proces koji se javlja u stanjima
kao što su arterio-venske malformacije, arterioskleroza,
Alchajmerova bolest i vaskularna demencija. CCH
započinje padom energetskog stanja neurona koje se ogleda
naglim smanjenjem nivoa adenozin 3 fosfata (ATP) u ćeliji,
dramatičnim oslobañanjem solubilnih adeninskih nukleotida
što dovodi do ozbiljnog deficita ćelijske funkcije i ishemije.
Jedan od mnogobrojnih neuroprotektivnih agenasa je i
estradiol koji se koristi u terapiji neurodegenerativnih
poremećaja, a naročito u terapiji ishemične povrede mozga.
CCH je kod adultnih ovarijektomisanih (OVX) i
ne-OVX ženki pacova soja Wistar izazvana bilateralnom
okluzijom zajedničkih karotidnih arterija (2VO). Pošto su
sinapse u velikoj meri podložne ishemičnom tipu povrede
mozga, u kortikalnoj sinaptozomalnoj frakciji ženki pacova
odreñivan je efekta 7 dana duge CCH i hroničnog tretmana
estradiolom na nivoe hidrolize adeninskih nukleotida
malahit zeleno enzimskim esejom.
Ovarijektomija nije značajno promenila hidrolizu
ATP, ADP i AMP-a. Rezultati pokazuju da 2VO značajno
(P<0.05) smanjuje hidrolizu ATP-a (77%) i ADP-a (57%) u
poreñenju sa 2VO-sham grupom. Tretman estradiolom
poništava efekat 2VO, odnosno nema značajne razlike u
poreñenju sa sham ovarijektomisanim životinjama.
Hidroliza AMP-a se nije promenila nakon 2VO dok je
tretman estradiolom povećao hidrolizu AMP-a (122%,
124%) u poreñenju sa 2VO i OVX životinjama.
Ovi rezultati se mogu smatrati bitnim za
substitucionu hormonsku terapiju, pošto postoji dosta
kontraverznih podataka u ovoj oblasti.
Chronic cerebral hypoperfusion (CCH) represents
a common pathophysiological process that usually occurs in
conditions such as arteriovenous malformations or
artherosclerosis, Alzheimer‘s disease and vascular
dementia. The chain of events in CCH begins with neuronal
energy failure which is reflected in the rapid depletion of
ATP, the dramatic release of soluble adenine nucleotides
resulting in profound deficiencies in cellular function and
ischemia. Estradiol is one of the numerous neuroprotective
agents used in treatment of neurodegenerative disorders,
especially as therapy of ischemic brain damage.
CCH in adult ovariectomized (OVX) and nonOVX female Wistar rats was induced by bilateral common
carotid arteries occlusion (2VO) procedure. Given that
synapses are greatly affected by ischemia, malachite green
enzyme assay was used to determine effects of 7 days long
CCH, and chronic estradiol treatment on levels of adenine
nucleotide hydrolysis in cortical crude synaptosomal
fraction of female rats.
Obtained results indicate that OVX did not
significantly alter ATP, ADP and AMP hydrolysis. Also,
our findings demonstrated that 2VO significantly (P<0.05)
decreased ATP (77%) and ADP (57%) hydrolysis compared
to 2VO-sham operated group. Estradiol treatment reversed
this effect, and there is no significant difference compared
to non OVX animals. AMP hydrolysis did not change after
2VO treatment compared to 2VO-sham operated group,
while Estradiol treatment increased AMP hydrolysis (122%,
124%) compared to 2VO treated animals and non OVX
animals, respectively.
These results should be considered relevant for
hormone replacement therapy, since much controversy
exists surrounding this area.
43
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
UTICAJ MODULACIJA WNT SIGNALNOG PUTA
NA EKSPRESIJU GENA SOXB1 GRUPE U NT2/D1
ĆELIJAMA HUMANOG EMBRIONALNOG
KARCINOMA
MODULATIONS OF WNT SIGNALLING AFFECTS
SOXB1 GENE EXPRESSION IN HUMAN
EMBRYONAL CARCINOMA NT2/D1 CELLS
M. Mojsin, V. Topalović, J. Marjanović, D. Stanisavljević,
M. Stevanović
Institut za molekularnu genetiku i genetičko inženjerstvo;
Univerzitet u Beogradu; Beograd; Srbija
[email protected]
M. Mojsin, V. Topalović, J. Marjanović, D. Stanisavljević,
M. Stevanović
Institute of Molecular Genetics and Genetic Engineering;
University of Belgrade; Belgrade; Serbia
[email protected]
Uvod: Indukcija NT2/D1 ćelija retinoičnom
kiselinom predstavlja odličan in vitro model sistem za
proučavanje humane neuralne diferencijacije. Mogućnost
modulacije WNT signalnog puta u ovim ćelijama
omogućila bi bolje razumevanje mehanizama uključenih u
humanu neuralnu diferencijaciju.
Ciljevi: Cilj ovog rada bio je da se optimiziju
uslovi za tretman NT2/D1 ćelija kvercetinom i LiCl u cilju
modulacije aktivnosti WNT signalnog puta u ovim ćelijama
i da se ispita uticaj ovih modulacija na ekspresiju gena
SOXB1 grupe.
Materijal i metode: Za odreñivanje optimalne doze
kvercetina i LiCl za tretmane NT2/D1 ćelija korišćen je
MTT test. Stepen modulacije aktivnosti signalnog puta
praćen je Western blot analizom ekspresije c-myc, ciljnog
gena WNT signalnog puta. Distribucija beta-katenina u
ćeliji nakon tretmana kvercetinom i LiCl praćena je
imunocitohemijskom analizom. Promene ekspresije gena
SOXB1 grupe praćene su metodama RT-PCR-a, Western
blot-a i imunocitohemije.
Rezultati: Optimizovani su uslovi za modulaciju
WNT signalnog puta u NT2/D1 ćelijama i pokazano je da je
citotoksični efekat kvercetina i LiCl posredovan p53
proteinom. Praćenjem ekspresije c-myc gena, utvrñen je
stepen modifikacije aktivnosti ovog signalnog puta. Takoñe,
pokazano je da modulacije WNT signalnog puta u NT2/D1
ćelijama značajno menjaju ekspresiju gena SOXB1 grupe.
Zaključak: Dobijeni rezultati pokazuju da postoji
funkcionalna veza izmeñu promene aktivnosti WNT
signalnog puta i ekspresije gena SOXB1 grupe u NT2/D1
ćelijama.
Background: Since retinoic acid induction of
NT2/D1 cells are good in vitro model system for human
neural differentiation, studying WNT signalling modulation
in NT2/D1 would contribute to better understanding of
mechanisms involved in neural stem cells maintainance and
human neural development.
Objectives: In this study we used quercetin and
lithium chloride to investigate modulations of WNT
signalling pathway in human pluripotent embryonal
carcinoma NT2/D1 cell line. We also examined the effects
of canonical WNT signaling modulations on human SOXB1
genes expression in NT2/D1 cells.
Material and Methods: NT2/D1 cells were treated
with quercetin and lithium chloride for 24h. Cytotoxic
effects on NT2/D1 cells viability and proliferation rate were
assessed using MTT test. Cellular redistribution of betacatenin was examined using immunocytochemistry and
Western blot. Expression of p53 and c-myc proteins was
assessed by Western blot. SOXB1 proteins expression was
examined
using
RT-PCR,
Western
blot
and
immunocytochemistry.
Results: We optimized conditions for NT2/D1 cells
treatments with quercetin and lithium chloride. Our results
have shown that induction of cell death by quercetin and
LiCl is p53-dependent in NT2/D1 cells. We examined the
degree of WNT signalling modulations by analyzing the
expression of c-myc, well known WNT signalling target
gene. We also shown that WNT signalling modulations
have prominent effect on SOXB1 genes expression in
NT2/D1 cells.
Conclusions: Our results are of particular interest
since both canonical WNT signaling and human SOXB1
genes are recognized as important developmental
regulators.
44
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
DINAMIČKA EKSPRESIJA SOXB1
TRANSKRIPCIONIH FAKTORA TOKOM IN VITRO
INDUKOVANE NEURALNE DIFERENCIJACIJE
HUMANE EMBRIONALNE KARCINOMSKE
ĆELIJSKE LINIJE NT2/D1
DYNAMIC EXPRESSION OF SOXB1
TRANSCRIPTION FACTORS DURING IN VITROINDUCED NEURAL DIFFERENTIATION OF
HUMAN EMBRYONIC CARCINOMA (EC) CELL
LINE, NT2/D1
M.Schwirtlich, D.Stanisavljević, S.Davidović, D.Drakulić,
A.Klajn, M.Stevanović
Institut za molekularnu genetiku i genetičko inženjerstvo;
Univerzitet u Beogradu; Beograd; Srbija
[email protected]
M.Schwirtlich, D.Stanisavljevic, S.Davidovic, D.Drakulic,
A.Klajn, M.Stevanovic
Institute of Molecular Genetics and Genetic Engineering;
University of Belgrade; Belgrade; Serbia
[email protected]
Uvod: Iako se “de novo“ stvaranje neurona dešava
u diskretnim regijama u adultnom mozgu, njihov gubitak je
trajna posledica mnogih neuroloških oboljenja i trauma. Da
bi mogli da manipulišemo neuralnim prekursorima radi
povećanja broja neurona ili glijalnih ćelija u terapeutske
svrhe, neophodno je steći znanje o molekularnim
mehanizmima koji regulišu neurogenezu. I pored brojnih
istraživanja koja su pokazala da su SOX1, SOX2 i SOX3,
članovi SOXB1 familije transkripcionih faktora, uključeni u
regulaciju različitih procesa u nervnom sistemu tokom
razvića njihova pojedinačna uloga nije u potpunosti
razjašnjena.
Cilj: Karakterisali smo ekspresiju SOXB1
transkripcionih faktora u nediferenciranim embrionalnim
karcinomskim ćelijama i tokom njihove neuralne
diferencijacije retinoičnom kiselinom.
Metode: Neuralna diferencijacija je indukovana u suspenziji
ili adherentnim ćelijskim kulturama. Ekspresija specifičnih
gena je detektovana RT-PCR-om, a ekspresija proteina
Western blotom i imunocitohemijom.
Rezultati: Naši rezultati su pokazali da se sva tri
člana SOXB1familije eksprimiraju u nediferenciranim
NT2/D1 ćelijama ali da imaju različite profile ekspresije
tokom neuralne diferencijacije. Dok je nivo SOX2
ekspresije opadao u ćelijama paralelno sa njihovom
diferencijacijom i potpuno izostao u zrelim neuronima i
astrocitima, nivo SOX3 se naglo povećao u ćelijama 24 sata
nakon dodavanja retinoične kiseline u medijum. U kasnijim
fazama diferencijacije SOX3 je detektovan u pojedinačnim
neuronalnim progenitorima. Pad nivoa ekspresije SOX1 u
NT2/D1 ćelijama detektovan je tek nakon prve nedelje
indukcije.
Zaključak:
Dinamička
ekspresija
SOXB1
transkripcionih faktora tokom in vitro neuralne
differencijacije ukazuje na njihovu moguću različitu
funkciju u procesima tranzicije pluripotentnih ćelija u zrele
neuronalne i glijane ćelije.
Background: De novo neurogenesis occurs in
discrete regions of the adult brain, while, neuronal loss is a
common feature of many neurological disorders and
traumatic brain injury. To be able to influence neurogenesis
and increase number of neural progenitors towards
therapeutic neuronal or glial repopulation, a better
knowledge of the cellular and molecular signalling
underlying neural differentiation and neurogenesis is
needed. The members of SOXB1 family of transcription
factors, SOX1, SOX2 and SOX3 have been implicated in
regulation of numerous developmental processes in the
brain but their distinct functions have been difficult to
resolve.
Objectives: We characterized the expression of
SOXB1 transcription factors in undifferentiated human EC
cells and in the course of retinoic acid-induced (RA)
neuronal differentiation.
Methods: Neuronal differentiation was induced in
either adherent or suspension cell culture conditions.
Specific gene and protein expression was detected by semiquantitative
RT-PCR,
Western
blot
and
immunocytochemistry.
Results: We found that all three members of
SOXB1 family were already present in EC cells but had
distinct expression profiles during differentiation. While
SOX2
expression
declined
during
RA-induced
differentiation of NT2/D1 and disappeared in terminally
differentiated neurons and astrocytes, SOX3 expression was
significantly increased upon RA treatment and remained in
some differentiated neuronal populations. SOX1 was
expressed in cells at constantly high level during first week
of induction and then gradually decreased in parallel with
differentiation.
Conclusion: Overlapping expression patterns of
human SOXB1 genes reveal novel aspects underlying
distinct functions of these transcription factors during
transition from uncommitted cells to differentiated neurons
and glia.
45
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
EFEKTI ASKORBINSKE KISELINE I VITAMINA D
NA VIJABILITET ASTROCITA U PRIMARNOJ
KULTURI U NORMALNIM USLOVIMA I
USLOVIMA OKSIDATIVNOG STRESA
INDUKOVANOG VODONIK PEROKSIDOM
EFFECTS OF ASCORBIC ACID AND VITAMIN D
ON VIABILITY OF PRIMARY RAT ASTROCYTES
CULTURED UNDER NORMAL CONDITIONS OR
SUBJECTED TO OXIDATIVE STRESS INDUCED BY
HYDROGEN PEROXIDE
M.Velimirović1, A. M Isaković1, S. Misirlić Denčić1, A.
Isaković1, N. Petronijević1
1
Institut za medicinsku i kliničku biohemiju; Medicinski
fakultet Univerziteta u Beogradu; Beograd; Srbija
[email protected]
M.Velimirović1, A. M Isaković1, S. Misirlić Denčić1, A.
Isaković1, N. Petronijević1
1
Institute of Medical and Clinical Biochemistry; Scool of
Medicin; University of Belgrade; Belgrade; Serbia
[email protected]
Uvod: Askorbinska kiselina (vitamin C), pored
svoje antioksidativne aktivnosti učestvuje i u proliferaciji,
diferencijaciji i preživljavanju ćelija nervnog sistema.
Poznata je uloga vitamina D u imunomodulaciji i
neuroprotekciji. Takoñe, vitamin D se svrstava u
membranske antioksidanse.
Cilj: Ispitati uticaj vitamina C i vitamina D na
vijabilitet primarne kulture astrocita pacova, kao i njihov
efekat na preživljavanje ovih ćelija u uslovima indukovanog
oksidativnog stresa.
Materijal i metode: Primarna kultura astrocita je
uspostavljena izolacijom mozga dva dana starih pacova soja
Wistar. Nakon potvrde astrocitne kulture imunohemijskim
bojenjem na glijalni fibrilarni kiseli protein ćelije su
korišćene za eksperimente. Vijabilitet ćelija je procenjivan
spektrofotometrijski, MTT metodom. Astrociti su tretirani
dvostruko opadajućim koncentracijama vitamina C (10mM40µM, 24h), i/ili vitamina D (100-0.2nM, 72h), a vijabilitet
ćelija procenjivan u uslovima sa (1mM vodonik peroksid,
30 minuta) ili bez indukcije oksidativnog stresa.
Rezultati: Vitamin C dozno-zavisno povećava
vijabilitet primarne kulture astrocita. Vitamin D nema
značajan uticaj na vijabilitet astrocita u odnosu na
netretirane ćelije. Primenjeni zajedno, u visokim dozama,
vitamini povećavaju preživljavanje ćelija, ali u manjoj meri
u odnosu na monotretman vitaminom C. U uslovima
oksidativnog stresa, vitamin C dozno-zavisno spašava
astrocite, dok vitamin D nema uticaja na njihovo
preživljavanje. Kombinovana primena vitamina C i D u
visokim dozama u potpunosti štite ćelije od smrti
uzrokovane oksidativnim stresom.
Zaključak: Očekivano, vitamin C pokazuje
antioksidativno dejstvo i utiče na proliferaciju primarnih
astrocita pacova. Vitamin D nema uticaj na vijabilitet ćelija
i ne pokazuje antioksidativno dejstvo. Primenjen u visokim
dozama deluje sinergistički sa vitaminom C u smislu
antioksidativne zaštite.
Introduction: Besides its well-documented
antioxidative properties, ascorbic acid has a role in
proliferation, differentiation and survival of nervous system
cells. Vitamin D affects both immunomodulation and
neuroprotection. Furthermore, vitamin D belongs to
membrane antioxidant family.
Aim: To determine effect of vitamins C and D on
primary rat astrocyte culture viability, as well as protection
of these cells in induced oxidative stress conditions.
Material and methods: Primary rat astrocyte
culture was established from 2-day old Wistar rats.
Astrocyte culture was confirmed by glial fibrillary acidic
protein staining, and cells used for experiments. Cell
viability was assessed using spectrophotometric MTT assay.
Astrocytes were treated with vitamin C (10mM-40µM, 24h)
and/or vitamin D (100-0.2nM, 72h), and viability
determined in both conditions with (1mM hydrogen
peroxide, 30 minutes) or without oxidative stress induction.
Results: Vitamin C induced dose-dependent
increase in astrocyte viability. Vitamin D showed no
significant influence on viability of treated cells. High
dosage of both vitamins increased cell number, however,
less efficiently than vitamin C monotreatment. Vitamin C
acted in dose-dependent manner against oxidative stress,
while vitamin D showed no protection. Combined high
dosage treatment with both vitamins prevented oxidative
stress-induced astrocyte cell death.
Conclusion: As expected, vitamin C exerts
antioxidative activity and influences primary rat astrocytes
proliferation. Vitamin D has no impact on cell viability and
shows no antioxidative capacity. Used in high
concentrations vitamin D acted synergistically with vitamin
C as far as antioxidative protection is concerned.
46
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
HRONIČNA SOCIJALNA IZOLACIJA POVEĆAVA
EKSPRESIJU CIKLOOKSIGENAZE-2 U
PREFRONTALNOM KORTEKSU ALI NE I
HIPOKAMPUSU MOZGA PACOVA
CHRONIC SOCIAL ISOLATION STRESS
UPREGULATES CYCLOOXYGENASE-2 PROTEIN
EXPRESSION IN THE PREFRONTAL CORTEX BUT
NOT THE HIPPOCAMPUS OF RAT BRAIN
J. Zlatković, N. Todorović, D. Filipović
Laboratorija za molekularnu biologiju i endokrinologiju;
Institut za nuklearne nauke Vinča; Univerzitet u Beogradu;
Srbija
[email protected]
J. Zlatković, N. Todorović, D. Filipović
Laboratory of Molecular Biology and Endocrinology;
Institute of Nuclear Sciences Vinča;
University of
Belgrade; Serbia
[email protected]
Uvod: Izlaganje hroničnom stresu može
prouzrokovati stanje oksidativnog stresa i pokretanje
inflamatornog odgovora u mozgu. Promena redoks
ravnoteže u smeru prooksidativnog stanja može dovesti do
aktiviranja nuklearnog transkripcionog faktora kapa beta
(NF-kB), koji ima značajnu ulogu u stresnom odgovoru
obzirom da njegova translokacija u jedro aktivira gene čija
je ekspresija regulisana od strane NF-kB, kao što je
ciklooksigenaza-2.
Cilj: Cilj studije bio je da se utvrdi da li hronična
socijalna izolacija (IZ) može prouzrokovati prooksidativno
stanje u ćeliji i time uticati na aktivaciju NF-kB i ekspresiju
proteina ciklooksigenaze-2 (COX-2), jednog od markera
inflamacije, u prefrontalnom korteksu (PFC) i hipokampusu
(HIP) mužjaka pacova Wistar soja.
Materijal i metode: Ispitivana je aktivnost ukupne
superoksid dismutaze (SOD) i nivo redukovanog glutationa
(GSH) u citosolu, preraspodela NF-kB izmeñu citosola i
jedra kao i ekspresija COX-2 proteina u citosolu pacova
izloženih 21-dnevnoj IZ, koji predstavlja animalni model
depresije. Nivo serumskog kortikosterona korišćen je kao
marker odgovora na stres.
Rezultati: IZ nije dovela do promene nivoa
kortikosterona, ali smanjena aktivnost ukupne SOD i nivoa
GSH u PFC ukazuju na ugroženu antioksidativnu odbranu i
poremećenu redoks ravnotežu. Ove promene najverovatnije
dovode do premeštanja NF-kB u jedro što je praćeno
povećanjem ekspresije COX-2 proteina, koji svojim
delovanjem
dodatno
može
doprineti
stvaranju
prooksidativnog stanja. U HIP, uprkos smanjenju nivoa
GSH, nisu uočene promene aktivnosti ukupne SOD, kao ni
ekspresije NF-kB i COX-2 proteina.
Zaključak: Rezultati ukazuju da izlaganje
hroničnoj IZ narušava redoks ravnotežu PFC ali ne i HIP,
što najverovatnije uzrokuje povećanje ekspresije COX-2
proteina i dovodi do neurotoksičnosti.
Introduction: Exposure to chronic stressors induces
oxidative stress and initiation of inflammatory response in
the brain. Stress-induced shift in the prooxidant-antioxidant
balance toward prooxidant state may activate nuclear
transcription factor kappa beta (NF-kB), which
translocation into the nucleus activates genes whose
expression is regulated by NF-kB, such as cyclooxygenase2, playing important role in stress responses.
Aim: Aim of our study was to determine whether
chronic social isolation (CSIS) stress may create prooxidant
state in the cell and mediate NF-kB activation and
concomitant expression of cyclooxygenase-2 (COX-2)
protein, one of the inflammatory markers, in the prefrontal
cortex (PFC) and hippocampus (HIPP) of male Wistar rats.
Material and methods: Cytosolic total superoxide
dismutase (SOD) activity and reduced glutathione (GSH)
level, cytosolic/nuclear NF-kB distributions and cytosolic
COX-2 protein expression in the PFC and HIPP of rats
exposed to 21-day CSIS, an animal model of depression,
were investigated. Serum corticosterone level was used as
marker of stress response.
Results: CSIS resulted in no change in
corticosterone level but decreased total SOD activity and
depleted GSH level in the PFC indicated compromised
detoxifying capacity. These changes in the cellular redox
equilibrium probably triggered NF-kB nuclear translocation
and concomitant COX-2 upregulation, which further may
intensify oxidative stress. Despite the fact that hippocampal
GSH was decreased, total SOD activity, NF-kB and COX-2
protein levels were not changed.
Conclusion: The data have shown that CSISinduced redox disbalance likely triggered COX-2
upregulation which may lead to neurotoxicity in the PFC,
but not in HIPP.
47
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
HIPERBARIČNA OKSIGENACIJA KAO
ADJUVANTAN TERAPIJA U REEDUKACIJI
PSIHOMOTORIKE KOD DECE SA BLAGOM
CEREBRALNOM PARALIZOM
1
3
3
1,2
P. Brkić , N. Jovanović Simić , I. Terzić , T. Jovanović
1
Institut za Medicinsku Fiziologiju; Medicinski fakultet;
Univerzitet u Beogradu; Beograd; Srbija;
2
Specijalna bolnica za hiperbaričnu medicinu; Beograd;
Srbija;
3
Fakultet za specijalnu edukaciju i rehabilitaciju;
Univerzitet u Beogradu; Beograd; Srbija
[email protected]
Uvod: Neadekvatna oksigenacije moždanog tkiva
prilikom
prevremenog
poroñaj
ili
poroñaj
sa
komplikacijama je jedan od najzanačajnijih uzročnika
poremećaja moždanih funkcija kod dece.
Cilj: U našoj studiji pratili smo efekte specifične procedure
restorativne reedukacije psihomotorike i hiperbarične
oksigenacije (HBO) kod dece sa dijagnozom blage
cerebralne paralize.
Metod: Deca uzrasta od 3 do 5 godina su
organizovana u dve grupe (n = 12, svaka). Prva grupa je
istovremeno sa reedukacionim tretmanom bila uključena u
hiperbaričnu oksigenaciju (protokol: 60 min, pritisak 1,7
ATA, 30 dana, u višemesnoj komori). Dece iz druge grupe
su bila uključena samo u reedukacioni tretman. Sledeći
parametri su praćeni: gruba i fina motorika, prostorna,
vremenska i telesna orijentacija, telesna šema, vokalizacija,
verbalizacija, sekvencijalno razmišljanje i lateralizacija.
Rezultati: Prva razlika u stepenu funkcionalnog
poboljšanja ja uočena nedelju dana po odpočinjanju HBO.
Na kraju tretmana značajan stepen poboljšanja je uočen kod
petoro dece iz prve grupe. Njihov funkcionalni status je
procenjen na 90% od za taj uzrast očekivanog fiziološkog
niva. Potrebno je istaći da je i sam intenzivni reedukativni
tretman doveo do poboljšanja praćenih funkcionalnih
parametara kod dece iz druge grupe. Meñutim, statistička
analiza rezultata jasno ukazuje da u znatnoj korelaciji HBO
i reedukativni tretman postižu značajno veći nivo
poboljšanja i smanjenja stepena disfunkcije.
Zaključak: Molekularni kiseonik primenjen pod
hiperbaričnim uslovima može doprineti poboljšanju
oporavka kognitivnih funkcija kod dece sa cerebralnim
disfunkcijama.
HYPERBARIC OXYGENATION AS AN ADJUVANT
THERAPY IN PSYCHOMOTOR RE–EDUCATION
OF CHILDREN WITH MILD CEREBRAL PALSY
P. Brkić1, N. Jovanović Simić3, I. Terzić3, T. Jovanović1,2
1
Institute of Medical Physiology; School of Medicine;
University of Belgrade; Belgrade; Serbia;
2
Centre for hyperbaric medicine; Belgrade; Serbia;
3
Faculty of special education and rehabilitation; University
of Belgrade; Belgrade; Serbia
[email protected]
Introduction: Inadequate oxygenation of the brain
tissue during premature or delivery with complication is one
of the most important causes of cerebral disturbances in
children.
Aim: In our study we have overseen the effects of specific
restorative procedure re–education of psychomotricity (RP)
and hyperbaric oxygenation (HBO) in children with the
diagnosis of mild cerebral palsy.
Methods: Children from the age of 3 to 5 years old
were divided in two groups (n = 12, each). The first group
was simultaneously with RP included in HBO protocol (60
min, pressure 1.7 ATA, during 30 days, in multiple
chamber). The second group was only included in RP. The
following rehabilitation parameters were monitored: gross
and fine motor skills, spatial, time and body orientation,
body scheme, vocalization, verbalization and sequential
thinking.
Results: First difference in the level of the
improvement was noticed 7 days after the beginning of
HBO. At the end of the treatment significant improvements
were noticed in 5 children from the first group, resulting in
re-establishing of the functional status at the 90% of the
physiological level. It is necessary to underline that
intensive RP has also improved the parameters in the
second group. However, statistical analyses of the results
have clearly shown that in a significantly strong correlation
HBO and RP induce the improvement and reduce the
dysfunctions.
Conclusion: The molecular oxygen applied under
the hyperbaric conditions may have beneficial effects and
can contribute to the restoration of the cognitive functions
in children.
48
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
POVEĆANA SINAPTIČKA PLASTIČNOST NAKON
TRETMANA B VITAMINIMA JE OSNOVA
OPORAVKA LOKOMOTORNE FUNKCIJE NAKON
KORTIKALNE ABLACIJE KOD ADULTNIH
PACOVA
S. Dacić1, I. Lavrnja2, D. Savić2, D. Laketa1, I. Božić, N.
Nedeljković1, Lj. Rakić, M. Stojiljković2, S. Peković2
1
Biološki fakultet; Institut za fiziologiju i biohemiju;
Univerzitet u Beogradu; Beograd; Srbija;
2
Institut za biološka istraživanja"Siniša Stanković;
Odeljenje za neurobiologiju; Univerzitet u Beogradu;
Beograd; Srbija
[email protected]
Poznato je da je kod odraslih oporavak izgubljene
funkcije nakon povrede mozga spor i nepotpun usled
uništenja velikog broja nervnih ćelija i nemogućnosti
remodelovanja postojećih i formiranja novih neuronskih
veza.
Cilj ove studije je bio da se ispita dejstvo
kompleksa B (B1, B2, B3, B6 i B12) vitamina na oporavak
motornih funkcija, posle unilateralne ablacije senzomotorne
kore (ASK) prednjeg mozga pacova.
Mužjaci Wistar pacova podvrgnuti su ASK po
sledećim koordinatama: 2 mm ispred, 4 mm iza bregme i 4
mm lateralno od središnje linije. Sa tretmanom se
započinjalo 15 min nakon ASK, a potom je tretman
ponavljan na svaka 24h tokom 14 dana. Kokteli B vitamina
davani su u sledećim dozama: 33 mg/kg/dan (B1 i B6); 7.5
mg/kg/dan (B2); 500 mg/kg/dan (B3); 0.5 µg/kg/dan (B12).
Testirane su sledeće kombinacije B vitamina: LK1 (B1, B6,
B12); LK2 (B2, B3); LK (B1, B2, B3, B6, B12). Oporavak
motornih funkcija praćen je testom prelaska preko grede.
Procesi plastičnosti praćeni su imunohistohemijski pomoću
specifičnih markera izrastanja aksona i dendrita i formiranja
sinapsi (GAP43, MAP2, sinaptofizin).
Pokazano je da primena B vitamina ubrzava i
poboljšava oporavak lokomotorne funkcije, ali sa različitom
efikasnošću: LK ≥ LK2 > LK1. Pored toga, njihova primena
je potpomogla izrastanje aksona i dendrita, grananje
dendrita i formiranje novih sinapsi.
Rezultati prikazani u ovoj studiji ukazuju da
povećana ekspresija markera sinaptičke plastičnosti, nakon
ponovljene administracije B vitamina, doprinosi
uspostavljanju novih neuronskih veza, što objašnjava
zapaženi brzi oporavak lokomotorne funkcije.
ENHANCED SYNAPTIC PLASTICITY AFTER
TREATMENT WITH B VITAMINS UNDERLIES
RECOVERY OF LOCOMOTOR FUNCTION
FOLLOWING CORTICAL ABLATION IN ADULT
RATS
S. Dacić1, I. Lavrnja2, D. Savić2, D. Laketa1, I Božić, N.
Nedeljković1, Lj. Rakić, M. Stojiljković2, S. Peković2
1
Department of Physiology and Biochemistry; Faculty of
Biology; University of Belgrade; Serbia
2
Department of Neurobiology; Institute for Biological
Research “Sinisa Stankovic; University of Belgrade;
Belgrade; Serbia
[email protected]
In adults, the recovery of lost function after brain
injury is slow and incomplete due to the destruction of a
large number of nerve cells and the inability to remodeling
existing and forming new neural connections.
The aim of this study was to investigate the effects
of vitamin B complex (B1,B2,B3,B6,B12) on the recovery
of motor function after unilateral sensorimotor cortex
ablation (SCA) of rat forebrain.
Male Wistar rats underwent SCA at the following
coordinates: 2mm before, 4mm behind the bregma and
4mm lateral to the midline. Treatment protocol started 15
min after the SCA and then continuously for 14 days, every
24h. The cocktail of B vitamins was given in following
doses: 33 mg/kg/day (B1 and B6); 7.5 mg/kg/day (B2); 500
mg/kg/day (B3); 0.5 µg/kg/day (B12). Different
combinations of B vitamins were tested: LC1 (B1,B6,B12);
LC2 (B2,B3); LC (B1,B2,B3,B6,B12). Beam walking test
was used to evaluate the recovery of motor functions.
Plasticity processes were monitored immunohistochemicaly
using specific markers for axon and dendrites outgrowth,
and synapses formation (GAP43, MAP2, synaptophysin).
It is shown that the use of B vitamins accelerates
and improves recovery of locomotor function, but with
different efficiency: LC ≥ LC2>LC1. Additionally, their use
induced sprouting of axons and dendrites, dendritic
branching and the formation of new synapses.
Results presented in this study revealed that
increased expression of synaptic plasticity markers after
repetitive administration of B vitamins contributed to the
establishment of new neural connections, which may
explain observed rapid recovery of locomotor function.
49
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
UTICAJ RESTRIKCIJE HRANE NA EKSPRESIJU
HONDROITIN SULFATNIH PROTEOGLIKANA U
MOZGU PACOVA NAKON TRAUMATSKE
POVREDE MOZGA
INFLUENCE OF FOOD RESTRICTION ON
EXPRESSION OF CHONDROITIN SULPHATE
PROTEOGLYCANS IN THE RAT BRAIN
FOLLOWING TRAUMATIC BRAIN INJURY
D. Lazić1, V. Tešić1, N. Lončarević-Vasiljković1, M. Brkić1,
M. Perović1, Lj. Rakić2, S. Ruždijić1, S. Kanazir1
1
Institut za biološka istraživanja “Siniša Stanković”,
Univerzitet u Beogradu, 11060 Beograd, Srbija
2
Srpska akademija nauka i umetnosti, 11000 Beograd,
Srbija
[email protected]
D. Lazic1, V. Tesic1, N. Loncarevic-Vasiljkovic1, M. Brkic1,
M. Perovic1, Lj. Rakic2, S. Ruzdijic1, S. Kanazir1
1
Institute for Biological Research “Sinisa Stankovic”,
University of Belgrade, 11060 Belgrade, Serbia
2
Serbian Academy of Sciences and Arts, 11000 Belgrade,
Serbia
[email protected]
Traumatska povreda mozga (eng. traumatic brain
injury, TBI) dovodi do serije procesa koji započinju
masovnim gubitkom neurona i aktivacijom astrocita.
Aktivirani astrociti, pored produkcije citokinskih molekula
u odgovoru na povredu sintetišu i brevikan i fosfakan,
članove familije hondroitin-sulfatnih proteoglikana. Ovi
proteoglikani modulišu rast aksona i na taj način utiču na
regeneraciju oštećenih neurona.
Cilj ovog istraživanja je bio da se utvrdi da li
restrikcija hrane (eng. food restriction, FR) utiče na
ekspresiju brevikana i fosfakana u ipsilateralnoj kori
velikog mozga pacova nakon TBI.
Eksperimenti su urañeni na mužjacima pacova
Wistar. Jedna grupa životinja je bila na FR (50% od
uobičajenog dnevnog unosa), dok je druga grupa imala
neograničen pristup hrani (lat. ad libitum, AL). Životinje su
bile na ovakvom režimu ishrane tri meseca, počev od tri
meseca starosti, nakon čega im je urañena ubodna lezija i
sakupljena kora velikog mozga oko lezije 2, 7, 14 i 28 dana
nakon povrede. Ekspresija brevikana i fosfakana je praćena
pomoću Western blot tehnike.
Rezultati su pokazali da je nivo brevikana i
fosfakana veći kod FR grupe u svim vremenskim tačkama u
poreñenju sa AL grupom životinja. Ta razlika je u sličaju
brevikana bila najuočljivija 7. dana, dok se za fosfakan
najveća promena uočava 2. dana nakon TBI. Restrikcija
hrane povećava ekspresiju brevikana i fosfakana u
ipsilateralnom korteksu nakon TBI. S obzirom da su ovi
proteoglikani ukjlučeni u sinaptičku reorganizaciju oko
povreñenog regiona, povećanje njihove ekspresije može
povoljno da utiče na oporavak nakon TBI.
One of the first hallmarks of traumatic brain injury
(TBI) is neuronal loss followed by activation of astrocytes
around damaged area. In addition to production of
cytokines, activated astrocytes produce brevican and
phosphacan, members of chondroitin sulphate proteoglycan
family, which can modulate axonal growth, thereby
influencing axon pathfinding in regeneration.
This research was aimed to examine whether food
restriction (FR) affects expression profiles of brevican and
phosphacan in rat ipsilateral cortex after TBI.
Experiments were done on male Wistar rats which
were divided in two groups. One group was subjected to FR
(lasting 3 months, starting at 3 months of age) fed with 50%
of mean daily food intake, while the other group was fed ad
libitum (AL) prior to the stab cortical injury. Cortex samples
were collected 2, 7, 14 and 28 days after the injury and
levels of brevican and phosphacan were examined using
Western blot analysis.
Our results have shown that protein levels of
brevican and phosphacan were higher in every time point
examined in the injured cortex of FR group comparing to
AL group. This difference in protein levels between groups
was most notable at 7th day for brevican and 2nd day after
the injury for phosphacan expression.
FR upregulates expression of brevican and
phosphacan in ipsilateral cortex after TBI. Knowing that
these proteoglycans are involved in synaptic reorganization
of injured area, upregulation of their expression could
represent an advantage for improvement of TBI outcome.
50
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
DUGOROČNI UTICAJ TERAPIJE
HALOPERIDOLOM I KLOZAPINOM NA
PROMENU KOŠTANE MASE U
FENCIKLIDINSKOM ANIMALNOM MODELU
LONG-TERM EFFECTS OF HALOPERIDOL AND
CLOZAPINE TREATMENT ON BONE MASS IN
PHENCYCLIDINE ANIMAL MODEL OF
SCHIZOPHRENIA
T. Nikolić1, M. Petronijević2, M. Velimirović1, T.
Stojković1, G. Jevtić1, N. Radonjić1, N. Petronijević1
1
Institut za medicinsku i kliničku biohemiju, Medicinski
fakultet, Univerzitet u Beogradu, Beograd, Srbija
2
Vojnomedicinska akademija, Klinika za reumatologiju,
Univerzitet u Beogradu, Beograd, Srbija
[email protected]
T. Nikolić1, M. Petronijević2, M. Velimirović1, T.
Stojković1, G. Jevtić1, N. Radonjić1, N. Petronijević1
1
Institute of Medical and Clinical Biochemistry, School of
Medicine, University of Belgrade, Belgrade, Serbia
2
Military Medical Academy-Department of Reumathology,
University of Belgrade, Belgrade, Serbia
[email protected]
Uvod: Shizofreni pacijenti na dugoročnoj terapiji
antipsihoticima imaju smanjenu koštanu mineralnu gustinu
(BMD) i povećani rizik od preloma. Perinatalna primena
fenciklidina (PCP) kod pacova predstavlja animalni model
shizofrenije.
Cilj: Cilj rada je bilo ispitivanje dugoročnog efekta
terapije haloperidolom i klozapinom na koštanu masu i
telesni sastav u PCP animalnom modelu shizofrenije.
Materijal i metode: Šest grupa životinja je
subkutano tretirano 2., 6., 9. i 12. postnatalnog (PN) dana,
PCP-om (10mg/kg) ili NaCl-om (0,9% fiziološki rastvor).
Od PN35, jedna NaCl i PCP grupa su počele da primaju
haloperidol (1mg/kg/dan; NaCl-H, PCP-H grupe), jedna
NaCl i PCP grupa su počele da primaju klozapin
(20mg/kg/dan; NaCl-C, PCP-C grupa), rastvorene u vodi za
piće. Preostala NaCl (kontrolna) i PCP grupa je dobila vodu
za piće. BMD, mineralni sadržaj kostiju (BMC) i količina
masnog tkiva su mereni in vivo dual rendgen
absorptiometrom (DXA) PN60 i PN98.
Rezultati: U PCP grupi je uočeno dugotrajno
smanjenje ukupne BMD u odnosu na kontrolu nakon
merenja PN60 i PN98. Ukupni BMC je smanjen PN60 u
PCP, PCP-H i PCP-C grupi. Prilikom merenja PN98, BMC
je bio smanjen u NaCl-H grupi i visoko statistički značajno
smanjen u PCP, PCP-H i PCP-C grupi. Došlo je do pada u
ukupnoj količini masnog tkiva u NaCl-H, PCP-H i PCP-C
grupi.
Zaljučak: Rezultati pokazuju da perinatalna
primena PCP-a dovodi do smanjenja koštane mase.
Promene u BMC uzrokovane PCP-om nisu normalizovane
nakon terapije lekovima, ali su lekovi uticali na sadržaj
masti.
Introduction: Schizophrenic patients, on long-term
anty-psychotic therapy, have decreased bone mineral
density (BMD) and increased fracture risk. Perinatal
phencyclidine administration to rodents represents an
animal model of schizophrenia.
Aim: The aim was to investigate long-term effects
of haloperidol and clozapine treatment on bone mass and
body composition in phencyclidine (PCP) animal model of
schizophrenia.
Material and methods: Six groups of animals were
subcutaneously treated on 2nd, 6th, 9th and 12th postnatal day
(PN), with either PCP (10 mg/kg) or vehicle (0.9% saline).
At PN35, one NaCl and PCP group have started to receive
haloperidol (1mg/kg/day; NaCl-H, PCP-H groups) and one
NaCl and PCP group have started to receive clozapine
(20mg/kg/day; NaCl-C, PCP-C groups) dissolved in
drinking water. The rest NaCl (controls) and PCP groups
have received drinking water. BMD, bone mineral content
(BMC) and fat mass were measured in vivo by dual X ray
absorptiometry (DXA) on PN60 and PN98.
Results: A long lasting reduction of total BMD was
observed on PN60 and PN98 in PCP group comparing to
control animals. Total BMC was decreased on PN60 in
PCP, PCP-H and PCP-C groups. On PN98, BMC was
decreased in NaCl-H and highly significantly decreased in
PCP, PCP-H and PCP-C groups. There was a decrease in
total fat in NaCl-H, PCP-H and PCP-C groups.
Conclusion: The results demonstrate that PCP
administrated perinatally reduces bone mass. The changes
in BMC caused by PCP were not normalized upon drugs
treatments, but drugs have influenced fat content.
51
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
ANTIGLIOMSKI EFEKAT INDOMETACINA
POSREDOVAN JE MODULACIJOM AMPK/MTOR
SIGNALNOG PUTA
ANTIGLIOMA EFFECT OF INDOMETHACIN IS
MEDIATED THROUGH MODULATION OF
AMPK/MTOR SIGNALING PATHWAY
A. Pantović 1,2, K. Arsikin 1, B. Ristić 1, M. Bošnjak 3, V.
Suzin-Živković 3, V. Trajković 1, LJ. Harhaji-Trajković 4
1
Institut za mikrobiologiju i imunologiju; Medicinski
fakultet; Univerzitet u Beogradu; Beograd; Srbija;
2
Klinika za neurologiju; Vojnomedicinska akademija;
Beograd; Srbija;
3
Institut za histologiju i embriologiju; Medicinski fakultet;
Univerzitet u Beogradu; Beograd; Srbija;
4
Institute za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; Beograd; Srbija
[email protected]
A. Pantović 1,2, K. Arsikin 1, B. Ristić 1, M. Bošnjak 3, V.
Suzin-Živković 3, V. Trajković 1, LJ. Harhaji-Trajković 4
1
Institute for Microbiology and Immunology; School of
Medicine; University of Belgrade; Belgrade; Serbia;
2
Neurology Clinic; Medical Military Academy; Belgrade;
Serbia;
3
Institute for Histology and Embriology; School of
Medicine; University of Belgrade; Belgrade; Serbia;
4
Institute for Biological Research "Siniša Stanković";
University of Belgrade; Belgrade; Serbia
[email protected]
Uvod: Nesteroidni antiinflamatorni lek i inhibitor
ciklooksigenaze indometacin ima antigliomski efekat.
Farmakološka aktivacija intracelularnog energetskog
senzora, AMP-aktivirane protein kinaze (AMPK), inhibira
rast tumorskih ćelija.
Cilj: Ispitivali smo efekat indometacina na
aktivaciju AMPK i mTOR (mammalian target of
rapamycin), glavni intracellularni inhibitor autofagije i
nishodnu metu AMPK, kao i njihovu ulogu u
antigliomskom dejstvu indometacina.
Metod: Vijabilitet U251 ćelija humanog glioma
odredjivan je kristal violet, MTT i LDH testom, dok je
proces apoptoze je analiziran protočnom citometrijom.
Aktivacija AMPK, Raptor-a, mTOR-a, p70S6K, kao i
ekspresija proteina koji regulišu autofagiju beklina-1 i LC3
analizirane su imunoblotom. RNK inteferencija je korišćene
za inhibiciju ekspresije AMPK i autofagije. Nivo ATP-a je
odredjivan HPLC tehnikom ili luminiscentnim esejem.
Rezultati: Antigliomski efekat indometacina je bio
posredovan oksidativnim stresom, aktivacijom kaspaza i
apoptozom. Indometacin je, nasuprot ostalim inhibitorima
ciklooksigenaze, aktivirao AMPK i nishodni molekul
Raptor, dok je inhibirao mTOR i njegov supstrat p70S6K.
Genska inaktivacija AMPK redukovala je indometacinom
stimulisanu inhibiciju mTOR-a i apoptozu gliomskih ćelija,
dok je aktivator mTOR-a leucin delimično smanjio
proapoptotsko dejstvo indometacina. Indometacin nije
indukovao ekspresiju beklina-1, konverziju LC3, niti
intracelularnu acidifikaciju. Inhibicija autofagije RNK
interferencijom ili farmakološkim inhibitorima nije imala
uticaj na antigliomsko dejstvo indometacina, što ukazuje na
dejstvo nezavisno od autofagije. Aktivacija AMPK nije
zavisila od porasta intracelularnog nivoa kalcijuma, ali je
korelisala sa deplecijom ATP.
Zaključak: Indometacin izaziva apoptozu ćelija
glioma smanjenjem nivoa intracelularnog ATP-a i
modulacijom AMPK/mTOR signalnog puta. Dejstvo
indometacina je nezavisno od inhibicije ciklooksigenaze i
autofagije. Dobijeni rezultati podržavaju dalja istraživanja
antigliomskog dejstva indometacina.
Introduction: Non-steroidal anti-inflammatory drug
and cyclooxigenase inhibitor indomethacin exerts
antiglioma effect. Activation of intracellular energy sensor
AMP-activated protein kinase (AMPK) by various drugs
inhibits growth of cancer cells.
Aim: We investigated the effect of indomethacin
on AMPK and its downstream mediator, the main
autophagy repressor mammalian target of rapamycin
(mTOR), as well as their role in indomethacin antiglioma
effect.
Methods: The viability of U251 human glioma
cells was determined by crystal violet, MTT and LDH tests,
while apoptosis parameters were assessed by flow
cytometry. Activation of AMPK, Raptor, mTOR, p70S6K
and expression of autophagy-related proteins beclin-1 and
LC3 were monitored by immunoblotting. RNA interference
was used for AMPK and autophagy inhibition. ATP levels
were determined by HPLC or luminescence assay.
Results: Indomethacin antiglioma effect was
mediated through oxidative stress, caspase activation and
apoptosis. Indomethacin, but not other cyclooxigenase
inhibitors, stimulated AMPK and its target Raptor, while
inhibited mTOR and its substrate p70S6K. AMPK
knockdown prevented indomethacin-mediated mTOR
inhibition and proapoptotic action, mimicking the effect of
the mTOR activator leucine. Indomethacin failed to
increase beclin-1 expression, LC3 conversion and
intracellular acidification, while autophagy inhibition by
RNA interference or pharmacological inhibitors had no
effect on its antiglioma activity, arguing against
involvement of autophagy in the observed effect. AMPK
activation was not mediated by the increase in intracellular
calcium, but correlated with indomethacin-induced ATP
depletion.
Conclusion: Indomethacin stimulates glioma cell
apoptosis
through
ATP
reduction-dependent,
cyclooxigenase inhibition- and autophagy-independent
modulation of AMPK/mTOR signaling. These results
support further exploration of indomethacin as potential
antiglioma drug.
52
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
BDNF MRNK IZOFORME U KORTEKSU I
HIPOKAMPUSU PACOVA TOKOM STARENJA I
UTICAJ RESTRIKCIJE HRANE
BDNF MRNA TRANSCRIPTS IN THE CORTEX AND
HIPPOCAMPUS OF RATS DURING AGING - THE
EFFECTS OF FOOD RESTRICTION
Ž. Pavković1, K. Smiljanić1, M. Perović1, V. Pešić1, A.
Mladenović Đorñević1, Lj. Rakić2, S. Ruždijić1, S. Kanazir1
1
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; 11060 Beograd; Srbija;
2
Srpska akademija nauka i umetnosti; 11000 Beograd;
Srbija
[email protected]
Z. Pavkovic1, K. Smiljanic1, M. Perovic1, V. Pesic1, A.
Mladenovic Djordjevic1, Lj. Rakic2, S. Ruzdijic1, S.
Kanazir1,
1
Institute for Biological Research “Sinisa Stankovic”;
University of Belgrade; 11060 Belgrade; Serbia;
2
Serbian Academy of Sciences and Arts; 11000 Belgrade;
Serbia
[email protected]
Restrikcija hrane (FR) je jedina poznata
manipulacija koja je u stanju da ublaži neurodegeneraciju
koja se javlja sa starenjem i poboljša sinaptičku plastičnost.
Neurotrofički faktor poreklom iz mozga (Bdnf) je član
familije neurotrofina i dobro poznati molekularni posrednik
neuronalne plastičnosti u adultnom mozgu. Njegovu
ekspresiju karakteriše precizna prostorno-vremenska kao i
stimulus-specifična regulacija
Cilj ovog rada je bio da se utvdi profil ekspresije
devet izoformi Bdnf-a (I-IXA) u korteksu i hipokampusu
pacova tokom starenja i pod dejstvom restrikcije hrane.
Eksperimenti su izvedeni na mužjacima pacova
soja Wistar. Životinje su podeljene u dve grupe, od kojih je
jedna imala slobodan pristup hrani (AL), a druga je hranjena
sa 100% prosečnog dnevnog unosa AL životinja, svaki
drugi dan počevši od tri meseca starosti (FR). Obe grupe su
bile na gajene do 12. i 24. meseca starosti, kada su izoforme
Bdnf -a u korteksu i hipokampusu analizirane RT-PCR-om.
Dobijeni rezultati su ukazali da su u korteksu i
hipokampusu najzastupljenije izoforme koje sadrže egzone
III, IV i VI, dok su transkripti sa egzonima I, II i IX slabo
eksprimirani. FR je značajno povećala ekspresiju
dominantnih transkripata u obe strukture kod životinja
starih 12 meseci, dok starenje nije dovelo do značajnih
promena.
Ekspresija
Bdnf-a
ispoljava
koordinisanu
regulaciju tokom starenja i pod uticajem FR. Izoforme
Bdnf-a koje sadrže egzone III, IV i VI se menjaju na isti
način tokom starenja u korteksu i hipokampusu, dok u
uslovima FR pokazuju starosno-zavisne promene.
Food restriction (FR) is an environmental
manipulation that attenuates age-related neurodegeneration
and promotes brain plasticity. Brain-derived neurotrophic
factor (BDNF), a member of the neurotrophin family, is
well established molecular mediator of neuronal plasticity
in the adult brain. Recently, its precise temporal, spatial,
and stimulus-specific regulation of has been described.
We assessed the expression profile of nine BDNF
transcripts (I-IXA) in the cortex and hippocampus of rats
during aging and compared them with the expressional level
in animals that were kept on FR.
Two groups of adult male Wistar rats were used:
(i) animals fed ad libitum (AL), (ii) animals fed with 100%
of the main daily intake of AL group, every other day
starting from 3 months. Both groups of rats were maintained
on this dietary regimen up to 12 and 24 months. Bdnf
transcripts level in cortex and hippocampus was analyzed
by RT-PCR.
Exon III-, IV-, VI-containing transcripts showed
dominant expression in the cortex and hippocampus, while
exon I-, II- and IX-containing transcripts showed very weak
expression. Aging per se did not induce significant changes
in any group, while FR induced significant elevation of
dominant transcripts in both structures in 12 months old
animals.
The results showed that in the cortex and
hippocampus exon III-, IV-, VI-containing BDNF
transcripts exhibited the same pattern of changes during the
aging. Age-specific effects of FR on Bdnf transcripts
expression were observed as age-specific.
53
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
UTICAJ PROPOFOLA NA EKSPRESIJU MARKERA
SINAPTIČKE PLASTIČNOSTI
U KORI I TALAMUSU PACOVA STARIH 14 DANA
PROPOFOL-INDUCED CHANGES IN SYNAPTIC
PLASTICITY MARKERS EXPRESSION
IN THE CORTEX AND THALAMUS OF 14-DAYOLD RATS
J. Popić1, V. Pešić1, D. Milanović1,3, Lj. Rakić2, S. Kanazir1,
V. Jevtović-Todorović3, S. Ruždijić1
1
Institut za biološka istraživanja Siniša Stanković,
Univerzitet u Beogradu, Beograd, Srbija;
2
Srpska akademija nauka i umetnosti, Beograd, Srbija;
3
Department of Anesthesiology, University of Virginia
Health System, Charlottesville, Va, USA
[email protected]
J. Popic1, V. Pesic1, D. Milanovic1, 3, Lj. Rakic2, S. Kanazir1,
V. Jevtovic-Todorovic3, S. Ruzdijic1
1
Institut za biološka istraživanja Siniša Stanković,
Univerzitet u Beogradu, Beograd, Srbija;
2
Srpska akademija nauka i umetnosti, Beograd, Srbija;
3
Department of Anesthesiology, University of Virginia
Health System, Charlottesville, Va, USA
[email protected]
Primena opštih
anestetika tokom
ranog
postnatalnog razvića može da utiče na morfologiju i
plastičnost neurona, dok stepen preživljavanja neurona ne
mora da bude izmenjen. Shodno tome, cilj ovog istraživanja
je da se utvrdi da li kratkotrajna anestezija indukovana
propofolom može da indukuje promene u ekspresiji markera
sinaptičke plastičnosti u prednjem mozgu postnatalnih
pacova.
Nakon jednokratne primene propofola (25 mg/kg
i.p.) na 14 dana starim pacovima (PND14) ispitane su
promene u ekspresiji proteina MAP-2 (microtubuleassociated proteins), drebrina (developmentally regulated
brain protein), GAP-43 (growth-associated protein-43),
sinaptofizina, sinukleina-1 i N-kadherina. Promene su
praćene primenom Western blot analize u kori i talamusu
kao primarnim moždanim strukturama na koje anestetici
deluju. Nakon tretmana životinje su dekapitovane u
različitim vremenskim tackama (0, 1, 2, 4, 8, 16 i 24 h).
Propofolski tretman je indukovao značajan pad u
ekspresiji MAP-2, GAP-43 proteina i N-kadherina u kori,
dok je značajan porast detektovan u talamusu. Suprotan
trend je detekovan u ekspresiji sinaptofizina, odnosno
pokazan je porast u kori i pad u talamusu. Porast u
ekspresiji sinukleina-1 je detektovan i u kori i talamusu.
Prolazni porasti u ekspresiji drebrina su detektovani u obe
ispitivane strukture.
Dobijeni rezultati ukazuju da propofolom
indukovana kratkotrajna anestezija ispoljava kompleksne
regionalno-specifične promene u zastupljenosti markera
sinaptičke plastičnosti u mozgu postnatalnih pacova
(PND14). Razumevanje molekulskih mehanizama koji se
pokreću nakon izlaganja mladih eksperimentalnih životinja
opštoj anesteziji može doprineti boljem razumevanju
starosno-zavisne komponente neuroplastičnosti koje izaziva
primena opštih anestetika u kliničkoj pedijatriji.
Several studies have revealed that during early
postnatal development general anesthetics could have an
impact on neuronal morphology and plasticity, without
changing neuronal survival rate. The goal of this study was
to explore whether short-term propofol anesthesia is able to
change the expression level of synaptic plasticity markers in
the forebrain of postnatal rats.
In this study we monitored by Western
immunoblot analysis the spatial and temporal changes in the
expression of synaptic plasticity markers MAP-2
(microtubule-associated proteins), drebrin (developmentally
regulated brain protein), GAP-43 (growth-associated
protein-43), synaptophysin, synuclein-1 and N-cadherin in
the cortex and thalamus of 14-day-old (PND14) Wistar rats.
The animals were decapitated at 0, 1, 2, 4, 8, 16 and 24 h
after the application of propofol anesthesia (25 mg/kg i.p).
The treatment induced significant decrease in the
expression of MAP-2, GAP-43 and N-cadherin in the
cortex, while significant increase in the expression of these
proteins was observed in the thalamus. Changes in
synaptophysin level showed opposite trend, to be exact
increase in the cortex and decrease in the thalamus. The
level of synuclein-1 was increased in the cortex and
thalamus. Drebrin level showed transient increase in both
examined structures.
The obtained results show that propofol exerts
complex region-specific effects on synaptic plasticity
markers expression in the postnatal rat brain. Potential
functional consequences of these changes remain to be
examined. Nevertheless, elucidation of propofol-induced
changes in young animals could contribute to better
understanding of age-dependent component of anesthesiainduced neuroplasticity, which is of clinical significance.
54
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
OKSIDATIVNI STRES INDUKOVAN
DONEPEZILOM POSREDUJE U APOPTOZI I
AUTOFAGIJI GLIOMA, IN VITRO
OXIDATIVE STRESS INDUCED BY DONEPEZIL
MEDIATES GLIOM APOPTOSIS AND
AUTOPHAGY IN VITRO
M. Popović1, J. Tošić1, Ž. Stanojević1, A. Isaković1, V.
Trajković2
1
Institut za Medicinsku i Klinicku Biohemiju; Medicinski
Fakultet Univerziteta u Beogradu; Srbija;
2
Institut za Mikrobiologiju i Imunologiju; Medicinski
Fakultet Univerziteta u Beogradu; Srbija
[email protected]
M. Popovic1, J. Tosic1, Z.Stanojevic1, A. Isakovic1, V.
Trajkovic2
1
Institute of Medical and Clinical Biochemistry; Faculty of
Medicine; University of Belgrade; Serbia;
2
Institute of Microbiology and Immunology; Faculty of
Medicine; University of Belgrade; Serbia
[email protected]
Uvod: Alchajmerova bolest se odlikuje gubitkom
neurona i sinapsi u cerebralnom korteksu i pojedinim
subkortikalnim regionima. Kao uzrok nastanka ove bolesti,
holinergička hipoteza pretpostavlja smanjenu sintezu
acetilholina. S toga je u terapiji obolelih mesto našao
donepezil kao reverzibilni inhibitor acetilholinesteraze.
Cilj: Ispitivanje potencijalnog citotoksičnog efekta
donepezila na C6 i U251 ćelijskim linijama pacovskog
odnosno humanog glioma.
Materijal i metode: Eksperimenti su izvedeni na
C6 i U251 ćelijskim linijama i na transfekovanim U251
ćelijama knockout-ima za AMPK i LC3II gene. Ćelijski
vijabilitet je odreñen kristal violet i MTT testom. Produkcija
slobodnih kiseoničnih radikala, pankaspazna aktivnost,
prisustvo
aneksin
pozitivnih
ćelija,
prisustvo
citoplazmatskih kiselih vezikula kao i ćelijski ciklus ispitani
su primenom odgovarajućih fluorohroma (dihidrorodaminDHR, pankaspazni inhibitor-ApoStat, aneksin/propidijum
jodid-Ann/Pi akridin oranž-AO i propidijum jodid)
metodom protočne citofluorimetrije.
Rezultati: Testovi vijabiliteta su dali približno iste
IC50 vrednosti (160µM za C6, i 250µM za U251 na 48h).
Donepezil povećava: stvaranje slobodnih kiseoničnih
radikala (80% C6, 200% U251), aktivnost kaspaza (60%
C6, 110% U251), broj dvostruko pozitivnih ćelija - Ann/Pi
(2.7 puta C6, 19.7 puta U251) kao i broj ćelija sa
hipodiploidnom
DNK.
Takoñe,
povećan
odnos
crvene/zelene fluorescence, FL3/FL1, ukazuje na prisustvo
autofagije. Sa druge strane, produkcija slobodnih
kiseoničnih radikala je bila smanjena na knockout ćelijama.
Zaključak: Donepezil indukuje apoptozu i
autofagiju i C6 i U251 ćelija koje delom zavise od
produkcije slobodnih kiseoničnih radikala.
Introduction: Alzheimer's disease is characterized
by loss of neurons and synapses in cerebral cortex and
certain subcortical regions. Cholinergic hypothesis assumes
decreased synthesis of acetylcholine as the cause of this
disease. Therefore donepezil found its place in the therapy,
as a reversible inhibitor of acetylcholinesterase.
Objective: Investigate potential cytotoxic effect of
donepezil on C6 and U251 cell lines.
Materials and methods: Experiments were
performed on C6 and U251 cells of mouse and human
gliom respectively, and on transfected U251 cells,
knockouts for AMPK and LC3II genes. Cell viability was
determined by crystal violet and MTT tests. Production of
reactive oxygen species, pan-caspase activity, presence of
anexin positive cells, presence of acidic vesicles and cell
cycle were investigated using appropriate fluorochromes
(dihydrorhodamine-DHR, pan-caspase inhibitor-ApoStat,
annexin-propidium iodide-Ann/Pi, acridine orange -AO and
propidium iodide-PI) by FACSCalibur flow cytometer.
Results: Viability tests gave fairly the same IC50
values (160µM for C6, and 250µM for U251 at 48h).
Donepezil increased production of reactive oxygen species
(80% C6, 200% U251), caspase activity (60% C6, 110%
U251), number of double positive cells Ann/Pi (2.7 times
C6, 19.7 times U251) and number of cells with hypodiploid
DNA. Increased ratio of red/green fluorescence (FL3/FL1)
indicated the presence of autophagy. On the other hand,
production of reactive oxygen species was decreased in
knockout cells.
Conclusion: Based on all evidence, we can
conclude that donepezil induces apoptosis and autophagy in
C6 and U251 cells that is partly dependent on reactive
oxygen species production.
55
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
POLNO ZAVISNA RAZLIKA U EKSPRESIJI
APOPTOTSKIH PROTEINA TOKOM HRONIČNE
CEREBRALNE HIPOPERFUZIJE
GENDER SPECIFIC EXPRESSION OF APOPTOSIS
RELATED PROTEINS IN RESPONSE TO CHRONIC
CEREBRAL HYPOPERFUSION
M. Stanojlović, D. Drakulić, I. Guševac, I. Grković, N.
Mitrović, A. Horvat
Institut za nuklearne nauke “Vinča”; Univerzitet u
Beogradu; Beograd; Srbija
milosmolbio@gmail.com
M. Stanojlovic, D. Drakulic, I. Gusevac, I. Grkovic, N.
Mitrovic, A. Horvat
“Vinca” Institute of Nuclear Sciences; University of
Belgrade; Serbia
milosmolbio@gmail.com
Hronična hipoperfuzija, umereno ali trajno
smanjenje protoka krvi kroz mozak, javlja se kao uzrok ali i
posledica mnogih neurodegenerativnih oboljenja kao što su
stenoza i okluzija arterija, arterioskleroza, vaskularna
demencija i Alchajmerova bolest.
S obzirom da brojna neurodegenerativna stanja
dovode do apoptotskog odgovora u sinaptičkom delu ćelije i
da je muški pol osetljiviji na oštećenja mozga koja nastaju
usled ishemije, cilj ove studije bio je da se utvrdi da li
cerebralna hipoperfuzija dovodi do polno specifične
aktivacije apoptotskog puta zavisnog od kaspaza u
sinaptozomalnoj frakciji hipokampusa mozga pacova.
U ovoj studiji korišćena je Western blot metoda
kako bi se odredile razlike u ekspresiji B-ćelijski limfom 2
proteina (Bcl-2), Bcl-2 spregnutog X proteina (Bax) i
proteina kaspaze 3 u sinaptozomima hipokampusa odraslih
pacova 7 dana nakon permanentne okluzije zajedničkih
karotidnih arterija.
Hronična cerebralna hipoperfuzija u trajanju od 7
dana dovela je do porasta ekspresije Bax proteina i
smanjenja ekspresije Bcl-2 proteina. Ove promene su bile
značajnije kod mužjaka. Porast u niva kaspaze 3 je,
meñutim, bio veći kod ženki.
Rezultati ove studije ukazuju da: hronična
cerebralna hipoperfuzija dovodi do pro-apoptotskih
promena u sinaptozomima hipokampusa pacova kod oba
pola; neusklañenost promena u ekspresiji Bcl-2 familije
proteina i kaspaze 3 može nalaziti u osnovi polno zavisnog
mehanizma uključenog u apoptotske procese tokom
hronične cerebralne ishemije.
Chronic cerebral hypoperfusion, a moderate but
persistent reduction in regional cerebral blood flow, occurs
in various pathophysiological conditions such as arterial
stenosis or occlusion, arteriosclerosis, vascular dementia,
and Alzheimer’s disease.
Given that number of different neurodegenerative
disorders induce apoptotic processes in synapses and that
male gender is more susceptible to ischemic brain damage,
the goal of current study was to establish whether chronic
cerebral hypoperfusion induces gender specific activation of
caspase dependent apoptotic pathway in synaptosomal
fraction in hippocampus of rat brain.
In this study, Western blot analysis was used to
determine the gender difference in protein expression of Bcell lymphoma 2 (Bcl-2), Bcl-2–associated X protein (Bax)
and caspase 3 in adult rats hippocampal synapses 7 days
after permanent bilateral occlusion of common carotid
arteries.
The obtained results indicated that chronic cerebral
hypoperfusion in duration of 7 days induced elevation of
Bax and decrease of Bcl-2 protein levels. These changes
were more prominent in males compared to females.
However, levels of caspase 3 were more elevated in
females.
Major findings of this study are: chronic cerebral
hypoperfusion
induces
pro-apoptotic changes
in
synaptosomes of rat hippocampus in both genders; the
observed discordance in expression of Bcl-2 protein family
and caspase 3 levels may point to the existence of gender
specific mechanism involved in apoptotic processes during
chronic cerebral ischemia.
56
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
PROTEIN KINAZA REGULISANA ADENOZIN
MONOFOSFATOM I KINAZA REGULISANA
VANĆELIJSKIM SIGNALIMA REGULIŠU
DIFERENCIJACIJU SH-SY5Y ĆELIJA
NEUROBLASTOMA IZAZVANU FORBOL
MIRISTAT ACETATOM PREKO MEHANIZAMA
ZAVISNIH I NEZAVISNIH OD AUTOFAGIJE
AMPK AND ERK REGULATE PHORBOL
MYRISTATE ACETATE-INDUCED
DIFFERENTIATION OF SH-SY5Y
NEUROBLASTOMA CELLS THROUGH
AUTOPHAGY-DEPENDENT AND –INDEPENDENT
MECHANISMS
N. Zogović1, G. Tovilović1, M. Misirkić-Marjanović1,2, K.
Janjetović1,2, Lj. Vučićević1,2, V. Trajković2
1
Institut za biološka istraživanja "Siniša Stanković";
Univerzitet u Beogradu; Beograd; Srbija;
2
Institut za mikrobiologiju i imunologiju; Medicinski
fakultet; Univerzitet u Beogradu; Beograd; Srbija
nevenar@ibiss.bg.ac.rs
N. Zogović1, G. Tovilović1, M. Misirkić-Marjanović1,2, K.
Janjetović1,2, Lj. Vučićević1,2, V. Trajković2
1
Institute for Biological Research "Siniša Stanković";
University of Belgrade; Belgrade; Serbia;
2
Institute for Microbiology and Immunology; School of
Medicine; University of Belgrade; Belgrade; Serbia
nevenar@ibiss.bg.ac.rs
Uvod: Diferencijacija neurona je proces
kontrolisan od strane nekoliko unutarćelijskih signala. U
toku diferencijacije dolazi do potpunog sazrevanja
progenitorskih ćelija u neurone.
Cilj: Cilj ovog rada je bio da se ispita uloga protein
kinaze regulisane adenozin monofosfatom (AMPK), kinaze
regulisane vanćelijskim signalima (ERK) i autofagije tokom
diferencijacije humanih neuroblastoma SH-SY5Y izazvane
forbol miristat acetatom (PMA).
Metode: Ekspresija neuronskih markera i
autofagija su praćene pomoću protočnog citofluorimetra
nakon bojenja ćelija sa odgovarajućim antitelima, odnosno
akridin oranžom. Aktivnosti AMPK, ERK, beklina, Atg7,
proteina lakog lanca 3 povezanog sa mikrotubulima (LC3),
p62, p70S6K i aktina su odreñene pomoću imunoblota.
Utišavanje AMPK i LC3 gena je izvedeno pomoću malih
interferirajućih RNK.
Rezultati:
Istovremeno
sa
započinjanjem
autofagije, PMA je aktivirao AMPK protein, glavni
energetski senzor u ćeliji, i ERK kinazu, zaduženu za
regulaciju diferencijacije i proliferacije. Farmakološka
inhibicija AMPK i utišavanje gena za AMPK usporili su
diferencijaciju SH-SY5Y ćelija, i inhibirali su aktivaciju
ERK kinaze i autofagiju izazvane pomoću PMA. Blokiranje
aktivnosti ERK proteina pomoću specifičnog inhibitora
značajno je usporilo diferencijaciju SH-SY5Y ćelija i
autofagiju izazvane posredstvom PMA, bez uticaja na
aktivnost AMPK. Sa druge strane, inhibicija autofagije
nishodno od AMPK/ERK (farmakološka ili utišavanjem
gena za LC3) je povećala ekspresiju neuronskih markera,
što ukazuje na činjenicu da autofagija izazvana pomoću
PMA najverovatnije suprimira diferencijaciju SH-SY5Y
ćelija.
Zaključak: Diferencijaciju SH-SY5Y ćelija
izazvanu posredstvom PMA pospešuju AMPK i ERK
proteini nezavisno od autofagnog procesa. Istovremeno oba
molekula inhibiraju diferencijaciju preko mehanizama
zavisnih od autofagije.
Introduction: Neural differentiation involves
intracellular signaling-controlled maturation of neural
progenitors into cells with fully developed neuronal
phenotype.
Aim: We explored the interplay between the
adenosine
monophosphate-activated
protein
kinase
(AMPK), extracellular signal-regulated kinase (ERK) and
autophagy in phorbol myristate acetate (PMA)-induced
differentiation of SH-SY5Y human neuroblastoma cells.
Methods: The levels of neuronal marker expression
and acidification of autophagic compartments were
determined by flow cytometry of cells labeled with
appropriate antibodies and acridine orange, respectively.
The activation (phosphorylation) of AMPK and ERK, as
well as the levels of autophagic proteins beclin-1, Atg7,
microtubule-associated protein light chain 3 (LC3) and p62,
were assessed using immunoblot. RNA interference was
used for AMPK and LC3 knockdown.
Results: PMA initiated autophagic response in SHSY5Y cells simultaneously with activation of AMPK, a
major intracellular energy sensor, and ERK, a kinase
involved in cell proliferation and differentiation.
Pharmacological inhibition of AMPK or AMPK gene
silencing attenuated differentiation of SH-SY5Y cells, as
well as PMA-induced ERK activation and autophagy. A
selective pharmacological blockade of ERK prevented
PMA-induced neuronal differentiation and autophagy
induction, without affecting AMPK phosphorylation. On the
other hand, the inhibition of autophagy downstream of
AMPK/ERK activation, either by pharmacological agents or
LC3 knockdown, actually promoted the expression of
neuronal markers, thus indicating a role for autophagy in
suppression of PMA-induced differentiation of SH-SY5Y
cells.
Conclusion: PMA-induced differentiation of SHSY5Y cells depends on a complex interplay between
AMPK, ERK and autophagy, where AMPK and ERK
promote differentiation independently of autophagy, while
simultaneously inhibiting differentiation process through
autophagy-dependent mechanisms.
58
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
VANĆELIJSKI ATP INDUKUJE REAKTIVNI
FENOTIP I POVEĆAVA EKSPRESIJU NUKLEOZID
TRIFOSFAT-DIFOSFOHIDROLAZE-1U
KORTIKALNIM ASTROCITIMA PACOVA IN
VITRO
EXTRACELLULAR ATP INDUCES ASTROGLIOTIC
PHENOTYPE AND UP-REGULATES EXPRESSION
OF NUCLEOSIDE TRIPHOSPHATE
DIPHOSPHOHYDROLASE-1 BYRAT CORTICAL
ASTROCYTES IN VITRO
M. Adžić1, D. Briševac1, D. Bijelić1, M. Milošević1, A.
Parabucki2, I. Bjelobaba2, M. Stojiljković2, N. Nedeljković1
1
Instut za fiziologiju i biohemiju; Biološki fakultet
Univerziteta u Beogradu;
2
Institut za biološka istraživanja “SinisaStanković”;
Univerziteta u Beogradu.
adzic_marija@yahoo.com
M. Adžić1, D. Briševac1, D. Bijelić1, M. Milošević1, A.
Parabucki2, I. Bjelobaba2, M. Stojiljkovic2, N. Nedeljković1
1
Institute for physiology and Biochemistry; Faculty of
Biology University of Belgrade;
2
Institute for Biological Research “SinisaStanković”;
University of Belgrade.
adzic_marija@yahoo.com
Koncentracija vanćelijskog ATP znatno se
povećava nakon mehaničke povrede ili metaboličkog
izazova mozga, što je važan faktor u neuroinflamatornoj
reakciji. ATP i proizvodi njegove razgradnje, ADP i AMP,
deluju na P2X i P2Y purinske receptore, ostvarujući tako
svoja proinflamatorna dejstva. ATP se razgrañuje
postupnom hidrolizom fosfatnih grupa, katalizovano
enzimima ekto-nukleozid trifosfat difosfohidrolazama
(NTPDaze).Ovako
NTPDaze
direktno
kontrolišu
koncentraciju vanćelijskog ATP, čijom razgradnjom nastaje
adenozin, koji ispoljava anti-inflamatorne efekte.Veoma
važnu ulogu u celokupnom sledu reakcija u akutnim ili
hroničnim dogañajima ima purinski P2X7 receptor.
U ovoj studiji izučavan je efekat ATP (0.01-1
mmol/l) u kulturi astrocita neonatalnih pacova. Aktivnost
NTPDaza odreñena je merenjem hidrolazne aktivnosti, dok
je ekspresija iRNK za NTPDazu1-3 odreñena primenom
qRT-PCR. Rezultati pokazuju da ATP dovodi do značajnog
povećanja hidrolize ATP i ADP, kao i do očekivanog
porasta ekspresije iRNK-NTPDaza1, što jasno ukazuje na
pozitivno regulisanu ekspresiju NTPDaze1 kod astrocita
tretiranih ATP. Ulogu P2X7 receptora u uočenom
povećanju ekspresije NTPDaze1 nakon stimulacije ATP,
proverili smo u eksperimentu gde smo astrocite tretirali
BenzATP ili oxATP. U kulturi tretiranoj BenzATP, koji je
agonist P2X7 receptora, aktivnost NTPDaze povećava se na
sličan način kao i kod delovanja ATP. Meñutim, ATP u
prisustvu oxATP, koji je antagonost P2X7 receptora, ne
dovodi do istog efekta kao kada se ATP primeni
samostalno.Takoñe smo pokazali da astrociti stimulisani
ATP povećavaju ekpresiju IL-1beta i TNF-alfa.
Uzimajući sve navedeno u obzir, može se zaključiti
da ATP deluje kao proinflamatorni faktor, koji indukuje
reaktivni fenotip astrocita in vitro i u isto vreme povećava
ekspresiju NTPDaze1, posredstvom P2X7 receptora.
Extracellular ATP, released from astrocytes
challenged by mechanical or metabolic stimuli, emerges as
an important player in neuroinflammation. ATP, together
with its metabolites, ADP and AMP, acts through P2X or
P2Ypurinoreceptors, exhibiting pro-inflammatory actions.
Sequential hydrolysis of ATP is catalyzed by ectonucleoside triphoshate diphosphohydrolase enzymes
(NTPDases). Thus, NTPDases directly control extracellular
ATP by shifting the signal from ATP to anti-inflammatory
acting adenosine. An important role in the general frame of
the events leading to response to acute and chronic harmful
events is also played by P2X7 receptor.
We have analyzed the effects of ATP (0.01-1
mmol/l) on cultured astrocytes obtained from 2-day old rat
pups. NTPDase activity was assayed by malachite green
phosphate kit, while NTPDase1-3 mRNA were determined
by qRT-PCR. Results show significant increase in ATP and
ADP hydrolysis and comparable increase in NTPDase1mRNA, which strongly indicates up-regulation of
NTPDase1 by astrocytes treated with ATP. To explore if the
effects were mediated through P2X7 receptor, we replicated
the same experiment, in the presence of BenzATP or
oxATP. We have found that BenzATP, being the agonist of
P2X7, induced the same effect on NTPDase activity as
ATP, while ATP, in the presence of P2X7 receptor
antagonist, failed to reproduce its previous effects. We were
also able to show that astrocytes stimulated by ATP
strongly upregulate IL-1beta and TNF-alfa expression.
Thus, taken together we conclude that ATP acts as
a proinflammatory signal, that activates reactive phenotype
of astrocytes in vitro and strongly up-regulates NTPDase1
expression, by acting through P2X7 receptor.
59
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
EKTO-5`-NUKLEOTIDAZA U AMIOTROFIČNOJ
LATERALNOJ SKLEROZI
ECTO-5´-NUCLEOTIDASE IN AMYOTROPHIC
LATERAL SCLEROSIS
D. Bijelić1, D. Briševac2, M. Adžić1, P.R. Anñus1, N.
Nedeljković2, M. Milošević1
1
Centar za lasersku mikroskopiju; Biološki fakultet;
Univerzitet u Beogradu; Beograd; Srbija;
2
Institut za Fiziologiju i Biohemiju; Biološki fakultet;
Univerzitet u Beogradu; Beograd, Srbija
dunja.bersha@gmail.com
D. Bijelić1, D. Briševac2, M. Adžić1, P.R. Andjus1, N.
Nedeljković2, M. Milošević1
1
Center for Laser Microscopy; Faculty of Biology,
University of Belgrade; Belgrade; Serbia;
2
Institute for Physiology and Biochemistry; Faculty of
Biology; University of Belgrade; Belgrade; Serbia
dunja.bersha@gmail.com
Amiotrofična latelarna skleroza (ALS) je teška
neurodegenertivna bolest koju karakteriše selektivna
degeneracija motoneurona. Koristili smo transgeni soj
pacova kod koga se povećano eksprimira hSOD1G93A, što je
ujedno i najčešća humana genska mutacija povezana sa
ALS-om. Astrociti koji eksprimiraju hSOD1G93A pokazuju
tipičan neuroinflamatorni fenotip i doprinose smrti motornih
neurona in vivo. Ekto-5’-nukleotidaza (CD73) je
membranski enzim koji katalizuje poslednji i ograničavajući
korak u metabolizmu ekstraćelijskog ATP, pri čemu je
krajnji produkt adenozin. Smatra se da CD73 funkcioniše
kao fino-podešavajući mehanizam tokom akutne i hronične
neuroinflamacije, jer kontorliše ravnotežu izmeñu proinflamatornog ATP i anti-inflamatornog adenozina. U ovom
istraživanju smo ispitivali ekspresiju i funkciju CD73 u
ćelijskim kulturama kortikalnih astrocita pripremljenih iz
hSOD1G93A transgenih životinja.
Primarne kulture kortikalnih astrocita su izolovane
iz neontalanih transgenih hSOD1G93A pacova i netransgenih
životinja iz istog legla. Stopa AMP hidrolize od strane
CD73 je odreñena pomoću eseja sa malahit zelenim na
membranskim
preparatima
astrocita,
dok
je
rasprostranjenost CD73 procenjena imunofluorecentim
obeležavanjem.
Jasno je uočeno da astrocti sa pojačanom
ekpresijom hSOD1G93A imaju značajno povećanu aktivnost
CD73 kao i povećanu rasprostranjenost ovog enzima na
membrani u poreñenju sa netransgenim životinjama iz istog
legla. Uzevši u obzir ulogu CD73 u kontroli nivoa
ekstraćelijskog ATP i adenozina, ova studija opravdava
dalja istraživanja koja će rasvetliti funkcionalni i klinički
značaj ovih rezultata.
Amyotrophic lateral sclerosis (ALS) is a severe
neurodegenerative disorder characterized by selective
degeneration of motor neurons. We used a transgenic rat
strain overexpressing the hSOD1G93A, which is the most
common genetic mutation associated with ALS in humans.
Astrocytes expressing hSOD1G93A display typical
neuroinflammatory phenotype and are able to exacerbate
motor neuron death in vivo. Ecto-5´-nucleotidase (CD73) is
a membrane- bound ecto-enzyme catalyzing the final and
rate-limiting step in the extracellular metabolism of ATP,
with adenosine being the final product. It is believed that
CD73 functions as a fine-tuning mechanism during acute
and chronic neuroinflammation, since it controls the balance
between pro-inflammatory ATP and anti-inflammatory
adenosine. In the present work we explored expression and
function of CD73 in cultured cortical astrocytes obtained
from hSOD1G93A transgenic animals.
Primary cortical astrocytes were isolated from
neonatal transgenic hSOD1G93A rats and their nontransgenic
littermates. Rate of AMP hydrolysis by CD73 was
determined in astrocytic membrane preparations by
Malachite green phosphate assay, while CD73 membrane
abundance was evaluated by immunofluorescencent
labeling.
We were clearly able to demonstrate that
hSOD1G93A overexpressing astrocytes exhibit significantly
increased CD73 activity and enhanced CD73 membrane
abundance in comparison to their nontransgenic littermates.
Given the role of CD73 in controlling the extracellular
levels of ATP and adenosine, the functional and clinical
relevance of these findings should be further explored.
60
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
VISOKA EKSPRESIJA FAKTORA NEKROZE
TUMORA U CENTRALNOM NERVNOM SISTEMU
PACOVA REZISTENTNIH NA EKSPERIMENTALNI
AUTOIMUNSKI ENCEFALOMIJELITIS
ELEVATED TUMOR NECROSIS FACTOR IN THE
CENTRAL NERVOUS SYSTEM OF RATS
RESISTANT TO EXPERIMENTAL AUTOIMMUNE
ENCEPHALOMYELITIS
J. Blaževski 1, F. Petković 1, M. Momčilović1, Đ.
Miljković1, M. Mostarica Stojković2
1
Odeljenje za imunologiju; Institut za biološka istraživanja
“Sinaša Stanković”; Univerzitet u Beogradu; Srbija;
2
Institut za Mikrobiologiju i Imunologiju; Škola za
imunologiju; Univerzitet u Beogradu; Srbija
janablazevski@yahoo.com
J. Blazevski1, F. Petkovic1, M. Momcilovic1, Dj. Miljkovic1,
M. Mostarica Stojkovic2
1
Department of Immunology; Institute for Biological
Research “Sinisa Stankovic”; University of Belgrade;
Serbia;
2
Institute of Microbiology and Immunology; School of
Immunology; University of Belgrade; Serbia
janablazevski@yahoo.com
Uvod:
Eksperimentalni
autoimunski
encefalomijelitis (EAE) je životinjski model multiple
skleroze. EAE se kod Dark Aguti (DA) pacova karakteriše
intenzivnom infiltracijom imunskih ćelija u kičmenu
moždinu (KM) i jakom inflamacijom koja vodi do
neuroloških oštećenja, dok je kod Albino Oksford (AO)
pacova bolest blaga, često bez simptoma, sa ograničenom
infiltracijom imunskih ćelija u KM. Citokini produkovani u
limfnim tkivima i u centralnom nervnom sistemu (CNS) u
velikoj meri doprinose patogenezi EAE.
Cilj: Odreñivanje genske ekspresije i produkcije
faktora nekroze tumora (TNF) u CNS i limfnim čvorovima
DA i AO pacova nakon encefalitogene imunizacije.
Materijal i metode: Pacovi su imunizovani injektiranjem
mijelin baznog proteina ili homogenata KM i kompletnog
Frojndovog adjuvansa. KM su izolovane iz pacova u
različitim fazama bolesti, a homogenati i infiltrati KM
(IKM) su korišćeni za odreñivanje genske ekspresije
metodom RT-PCR i proteinske produkcije metodom
ELISA. Drenirajući limfni čvorovi (DLČ) su izolovani iz
pacova u induktivnoj fazi bolesti. CD4+ T-ćelije su
izolovane iz IKM i DLČ metodom magnetne separacije.
Rezultati:
Pokazali
smo povišenu gensku
ekspresiju TNF na piku EAE u homogenatima KM, IKM i
CD4+ T-ćelijama IKM kao i povišenu produkciju TNF u
IKM AO pacova u poreñenju sa DA pacovima. Produkcija
TNF u DLČ i CD4+ DLČ nije bila povišena kod AO pacova.
Zaključak: TNF je citokin sa brojnim
proinflamatornim i imunosupresivnim ulogama u
patogenezi neuroinflamatornih bolesti. Naši rezultati
ukazuju da povišen TNF u CNS može biti važan za
rezistenciju AO pacova na indukovanje EAE.
Introduction:
Experimental
autoimmune
encephalomyelitis (EAE) is an animal model of multiple
sclerosis. EAE in Dark Agouti (DA) rats is characterized by
intense infiltration of immune cells into the spinal cord (SC)
and strong inflammation leading to neurological
impairments, while in Albino Oxford (AO) rats it is mild,
often asymptomatic with limited infiltration of immune
cells into SC. Cytokines produced in lymphoid tissues and
within the CNS largely contribute to EAE pathogenesis.
Objective: Tumor Necrosis factor (TNF) gene
expression and production determination within the CNS
and lymph nodes of DA and AO rats after encephalitogenic
immunization.
Material and methods: Rats were immunized with
myelin basic protein or SC homogenate and complete
Freund`s adjuvant. SC were isolated from rats at different
stages of the disease and homogenates and SC immune cells
(SCIC). Cytokine gene expression and protein production
were determined by RT-PCR and ELISA, respectively.
Draining lymph node cells (DLNC) were isolated from rats
at the induction phase of EAE. CD4+ T cells were obtained
from SCIC and DLNC by magnetic separation.
Results:
Higher expression of TNF was
determined at the peak of EAE in SC homogenates, SCIC
and CD4+ SCIC and higher TNF production in SCIC of AO
rats comparing to DA rats. TNF production in DLNC and
CD4+ DLNC was not higher in AO rats.
Conclusion: TNF is a cytokine that exerts
proinflammatory and immunosuppressive functions in the
pathogenesis of neuroinflammation. Our results imply that
elevated TNF within the CNS might be important for
resistance of AO rats to EAE.
61
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
UTICAJ S-BENZOILTIAMIN O-MONOFOSFATA NA
AKTIVIRANU MIKROGLIJU IN VITRO
THE EFFECT OF S-BENZOYLTHIAMINE-OMONOPHOSPHATE ON ACTIVATED MICROGLIA
IN VITRO
I. Božić1, D. Savić1, N. Nedeljković2, S. Peković1, M.
Stojiljković1, I. Lavrnja1
1
Odeljenje za neurobiologiju; Institut za biološka
istraživanja "Siniša Stanković"; Univerzitet u Beogradu;
Beograd; Srbija;
2
Institut za fiziologiju i biohemiju; Biološki fakultet;
Univerzitet u Beogradu; Beograd; Srbija
ivabo1@yahoo.com
I. Božić1, D. Savić1, N. Nedeljković2, S. Peković1, M.
Stojiljković1, I. Lavrnja1
1
Department of Neurobiology; Institute for Biological
Research “Sinisa Stankovic”; University of Belgrade;
Belgrade; Serbia;
2
Institute of Physiology and Biochemistry; Faculty of
Biology; University of Belgrade; Belgrade; Serbia
ivabo1@yahoo.com
Mikroglijske ćelije su rezidentne imunske ćelije
centralnog nervnog sistema, čija je preterana aktivacija
obeležje mnogih neurodegenerativnih oboljenja. Aktivirane
mikroglijske ćelije odlikuje reaktivni fenotip, proliferacija,
hemotaksa, kao i oslobañanje proinflamatornih medijatora,
citokina i azot-monoksida (NO), koji dovode do oštećenja
neurona i njihove smrti. Stoga su agensi koji smanjuju
aktivaciju mikroglije i njima izazvanu neuroinflamaciju,
potencijalni terapeutici u lečenju neurodegenerativnih
oboljenja. S-benzoiltiamin-O-monofosfat (benfotiamin) je
sintetski S-acil derivat vitamina B1 koji ispoljava
antiinflamatorna svojstva u nekim tipovima neuropatija. Cilj
ovog istraživanja bio je da se ispita sposobnost
benfotiamina da smanji stepen aktivacije mikroglije in vitro.
Eksperimenti su izvedeni na BV-2 ćelijskoj liniji
dobijenoj transformacijom mikroglijskih ćelija izolovanih iz
C57BL/6 mišjeg soja. BV-2 ćelije su aktivirane
lipopolisaharidom (LPS) u prisustvu ili odsustvu
benfotiamina. Uticaj benfotiamina na vijabilnost ćelija
proveren je MTT testom, dok je produkcija NO odreñena
Grisovom reakcijom. Ekspresija inducibilne azot oksid
sintaze (iNOS) odredjena je metodama qRT - PCR i
Western blot. Ispitana je sposobnost benfotiamina da
moduliše MAP kinazni signalni put, kao i njegov uticaj na
apoptozu indukovanu aktivacijom LPS-om.
Rezultati pokazuju da benfotiamin ne utiče na
vijabilnost BV-2 ćelija, a da smanjuje produkciju NO,
smanjujući ekspresiju iNOS-a. Tretman benfotiaminom
suprimira fosforilaciju MAP kinaza i utiče na smanjenje
broja ćelija koje ulaze u apoptozu pod dejstvom LPS-a.
Na osnovu svega može se zaključiti da benfotiamin
umanjuje neke aspekte reaktivne mikroglioze i ispoljava
antiinflamatorno dejstvo, a budući da korišćenje
benfotiamina ne dovodi do neželjenih efekata, može se
razmotriti njegova upotreba i u neuorodegenerativnim
oboljenjima.
Microglial cells are the resident immune cells of the
central nervous system whose excessive activation is a
hallmark of many neurodegenerative disorders. Activated
microglial cells have a reactive phenotype, are highly
proliferating and produce proinflammatory mediators,
cytokines and nitric oxide (NO), which damage neurons and
can lead to their death. Drugs that supress microglial
activation and alleviate inflammation are potential
therapeutics in treating neurodegenerative disorders. Sbenzoylthiamine-O-monophosphate (benfotiamine) is a
synthetic S-acyl derivative of vitamin B1 that has antiinflammatory properties in some types of neuropathy. The
aim of this study was to investigate the effects of
benfotiamine on activated microglial cells in vitro.
The experiments were performed on BV-2 cell line
which was generated by transformation of microglial cells
of C57BL/6 neonatal mice. BV-2 cells were activated with
lipopolysacharide (LPS) in the presence or absence of
benfotiamine. Viability of treated cells was determined by
MTT assay and production of nitric oxide (NO) was
measured with the use of Griess reagent. Expression of
inducible nitric oxide synthase (iNOS) was evaluated using
qRT - PCR and Western blot. Benfotiamine's ability to
modulate MAP kinases signalling pathway and its influence
on LPS induced apoptosis were also examined.
Results show that benfotiamine does not affect
viability of BV-2 cells but lowers NO production, by
decreasing iNOS expression. Benfotiamine supresses
phosphorilation of MAP kinases and decreases the number
of cells that underwent apoptosis in LPS treatment.
In summary, benfotiamine lessened certain aspects of
reactive microgliosis and attenuated inflammation,
consequently it may be beneficial in treatment of
neurodegenerative disorders.
62
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
POVEĆANA EKSPRESIJA MIKRORNK-155 U
RESTIMULISANIM ENCEFALITOGENIM T
ĆELIJAMA
1
2
1
B. Jevtić , G. Timotijević , M. Momčilović , M. Mostarica
Stojković3, Đ. Miljković1
1
Institut za Biološka istraživanja „Siniša Stanković“;
Univerzitet u Beogradu; Srbija;
2
Institut za Molekularnu Genetiku i Genetičko Inženjerstvo;
Univerzitet u Beogradu; Srbija;
3
Medicinski fakultet; Univerzitet u Beogradu; Srbija
bojanbh@gmail.com
Uvod: MikroRNK (miRNK) predstavljaju male
nekodirajuće RNK koje imaju značajnu ulogu u regulaciji
imunskog odgovora. Pretpostavlja se da miRNA-155
značajno
doprinosi
patogenezi
autoimunskih
neuroinflamatornih bolesti, kao što je eksperimentalni
autoimunski encefalomijelitis (EAE).
Cilj: Ispitati da li je miRNK-155 uključena u
procese restimulacije encefalitogenih T-ćelija in vitro i in
vivo.
Materijal i metode: Pacovi su imunizovani
mijelinskim baznim proteinom (MBP) i kompletnim
Frojndovim adjuvansom. Ćelije su izolovane iz ćelija
limfnih čvorova koji dreniraju mesto imunizacije (DLČ) i
CNS i prečišćene magnetnom separacijom do CD4+ Tlimfocita. Ekspresija miRNK-155 u ćelijama DLČ i CNS je
merena metodom „Real-time“ PCR.
Rezultati: miRNK-155 se eksprimira u ćelijama
DLČ i njena ekspresija je MBP in vitro. Ekspresija miRNK155 je značajno niža kod ćelija izolovanih iz CNS u
poreñenju sa ćelijama iz DLČ-a. Ekspresija miRNK-155 je
značajno viša kod CD4+ T-ćelija izolovanih iz CNS u
poreñenju sa CD4+ T-ćelijama iz DLČ, kao i u odnosu na
neprečišćene ćelije CNS.
Zaključak: miRNK-155 se eksprimira u DLČ
pacova, a njena ekspresija je povećana nakon restimulacije
DLČ antigenom in vitro. In vivo restimulacija u CNS
dovodi do povećanja ekspresije miRNK-155 u CD4+ Tćelijama. Ovi rezultati ukazuju da bi miRNK-155 mogla da
ima važnu ulogu u reaktivaciji encefalitogenih T-ćelija u
neuroinflamaciji.
MICRORNA-155 IS UP-REGULATED IN
RESTIMULATED ENCEPHALITOGENIC T CELLS
B. Jevtić1, G. Timotijević2, M. Momčilović1, M. Mostarica
Stojković3, Đ. Miljković1
1
Institute for Biological Research “Siniša Stanković“;
University of Belgrade; Serbia;
2
Institute for Molecular Genetics and Genetic Engineering;
University of Belgrade; Serbia;
3
Institute for Immunology and Microbiology; School of
Medicine; University of Belgrade; Serbia
bojanbh@gmail.com
Introduction: MicroRNA (miRNA) are small noncoding RNAs that have a profound role in regulating
immune response. It has been assumed that miRNA-155
significantly contributes to the pathogenesis of autoimmune
neuroinflammation, such as experimental autoimmune
encephalomyelitis (EAE).
Aim: To determine whether miRNA-155 is
involved in re-stimulation of encephalitogenic cells in vitro
and in vivo.
Material and methods: Rats were immunized with
myelin basic protein (MBP) and complete Freund’s
adjuvant (CFA). Immune cells were isolated from lymph
nodes draining the site of immunization (DLN) or from the
CNS and CD4+ T cells were purified by magnetic
separation. miRNA-155 expression was measured by “realtime” PCR.
Results: MicroRNA-155 was expressed in DLN
cells and its expression was further stimulated with MBP
treatment in both rat strains, in vitro. miRNA-155
expression was significantly lower in cells isolated from the
CNS in comparison to the cells of DLN, while it was
significantly higher in purified CD4+ T cells from the CNS
in comparison to those purified from DLN, as well as in
comparison to unpurified cells from the CNS.
Conclusion: MicroRNA-155 is expressed in DLNs.
Its expression is further elevated by in vitro re-stimulation
of the DLN cells with antigen. In vivo restimulation within
the CNS leads to increase of miRNA-155 expression in
CD4+ T cells. These results indicate that miRNA-155 might
have an important role in the reactivation of
encephalitogenic T cells within the CNS.
63
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
POVREDA MOZGA IZAZIVA PROMENU
EKTONUKLEOTIDAZNIH AKTIVNOSTI
PERIFERNIH LIMFOCITA I SERUMA KOD
PACOVA
BRAIN INJURY ALTERS ECTONUCLEOTIDASE
ACTIVITIES OF PERYPHERAL LYMPHOCYTES
AND SERUM IN RAT
D. Laketa1 I. Bjelobaba2, D. Savić2, I. Lavrnja2, S. Dacić1,
A. Parabucki2, I. Božić2, M. Stojiljković,2 S. Peković2, N.
Nedeljković1
1
Institut za Fiziologiju i biohemiju; Biološki fakultet;
Univerzitet u Beogradu; 11001 Beograd; Republika Srbija;
2
Odeljenje za Neurobiologiju; Institut za biološka
istraživanja “Sinisa Stanković; Univerzitet u Beogradu;
Beograd; Srbija
danijela@bio.bg.ac.rs
D. Laketa1 I. Bjelobaba2, D. Savic2, I. Lavrnja2, S. Dacic1,
A. Parabucki2, I. Bozic2, M. Stojiljkovic2, S. Pekovic2, N.
Nedeljkovic1
1
Department for General Physiology and Biophysics,
Institute for Physiology and Biochemistry; Faculty of
Biology; University of Belgrade; 11001 Belgrade; Republic
of Serbia;
2
Department for Neurobiology; Institute for Biological
Research “Sinisa Stankovic”; University of Belgrade; 11000
Belgrade; Republic of Serbia
danijela@bio.bg.ac.rs
Ektonukleotidaze čini porodica enzima koji
katalizuju postupnu razgradnju ATP u vanćelijskom
prostoru. Ovi enzimi pričvršćeni su za spoljašnju stranu
membrane svih tipova ćelija ili su slobodni u vanćelijskom
prostoru. Povreda mozga dovodi do povećanja vanćelijske
koncentracije ATP, koji tada postaje „signal opasnosti”, jer
izaziva reaktivnu aktivaciju glije. U nekoliko različitih
modela
pokazano je da je povreda mozga praćena
povećanjem ekspresije ektonukleotidaza na glijskim
ćelijama na mestu povrede, a neke studije su pokazale da se
ekspresija ektonukleotidaza menja i na ćelijama krvi i na
vaskularnim endotelnim ćelijama.
Cilj ovog rada bio je ispitavanje uticaja moždane
povrede na aktivnost ektonukleotidaza limfocita periferne
krvi i solubilnih ektonukleotidaza u serumu.
Eksperimenti su izvedeni na odraslim pacovima
podvrgnutim ubodnoj povredi moždane kore (L) ili lažnoj
operaciji (S), koji su žrtvovani 4h, odnosno 1, 2 ili 7 dana
nakon operacije. Iz krvi životinja izdvojeni su limfociti i
serum i odreñene su aktivnosti ektonukleotidaza.
Hidroliza ATP i ADP u limfocitima značajno je
povećana 2 i 7 dana nakon operacije kod L i kod S grupe,
dok je hidroliza AMP bila povećana počev od 1. dana.
Promena aktivnosti ektonukleotidaza u serumu uočena je
samo 4 h nakon povrede, kada je izmereno značajno
povećanje hidrolize ATP i AMP u L grupi.
Rezultati ukazuju da povreda mozga izaziva
promenu aktivnosti ektonukleotidaza na limfocitima i u
serumu, koje posledično dovode do promena nivoa purina u
cirkulaciji. To ukazuje da povreda mozga posredstvom
promene nivoa purinske signalizacije, ostvaruje uticaj na
imunski sistem.
Ectonucleotidases belong to a family of enzymes
which catalyze sequential degradation of ATP in the
extracellular space. These enzymes are either attached to the
cell membrane of various cells or free in the extracellular
compartment. Brain injury induces increase of extracellular
ATP, which becomes “danger signal”, inducing activation
of glial cells. In several experimental models it was shown
that brain injury leads to an increase of ectonucleotidase
expression at glial cells at the injury site, while some other
studies demonstrated increase of ectonucleotidase
expression at blood cells and vascular endothelial cells.
The aim of this study was to explore if brain injury
induces changes of ectonucleotidase activities at perypheral
lymphocytes and in serum.
Adult male Wistar rats were subjected to cortical
stab injury (L) or sham surgery (S). Animals were sacrificed
at 4-h, 1-, 2- and 7-days after the injury. Lymphocytes and
serum were isolated from perypheral blood and
ectonucleotidase activities were determined.
In perypheral lymphocyte fraction, ATP- and
ADP-hydrolysis were significantly increased at 2- and 7days post-injury both in L- and S-group, whilst increase in
AMP-hydrolysis started from the 1st-day. In serum, changes
in ectonucleotidase activities were observed only at 4-h
post-injury in L-group, when significant increase in ATPand AMP-hydrolysis were recorded.
Obtained results suggest that brain injury induces
changes in ectonucleotidase activities at perypheral
lymphocytes and in serum, which consequently lead to
alterations of purine levels in circulation. This further
implies that brain injury exerts its effects at immune system
through modulation of purinergic signalling.
64
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
REAKTIVNE KISEONIČNE VRSTE I
NEUROINFLAMACIJA U HUMORALNOJ
PATOGENEZI AMIOTROFIČNE LATERALNE
SKLEROZE
REACTIVE OXYGEN SPECIES AND
NEUROINFLAMMATION IN HUMORAL
PATHOGENESIS OF AMYOTROPHIC LATERAL
SCLEROSIS
M. Milošević1, P. Abushik2, R. Giniatullin2, P. R. Anñus1,
1
Centar za lasersku mikroskopiju; Biološki fakultet;
Univerzitet u Beogradu; Beograd; Srbija;
2
A. I. Virtanen Institute for Molecular Sciences; UEF
Department of Neurobiology; Kuopio; Finland
milmi@bio.bg.ac.rs
M. Milošević1, P. Abushik2, R. Giniatullin2, P. R. Andjus1
Center for laser microscopy, Faculty of Biology;
University of Belgrade; Belgrade; Serbia;
2
A. I. Virtanen Institute for Molecular Sciences,;
Department of Neurobiology; Kuopio; Finland
milmi@bio.bg.ac.rs
Nedavno
smo
pokazali
da
prečišćeni
imunoglobulin G (IgG) izolovan iz krvi obolelih od
amiotrofične lateralne skleroze (ALS) utiče na kalcijumovu
homeostazu pacovskih kortikalnih astrocita u kulturi. Cilj
nam je bio da istražimo potencijalne akutne efekte ALS IgG
na kalcijumovu homeostazu i signaliranje reaktivnih
kiseoničnih vrsta (ROS) u drugim tipovima ćelija koje
imaju značajnu ulogu u mehanizmu patogeneze ALS
nezavisne od tipa ćelije.
Korišćena su dva tipa ćelijskih linija: BV-2
(mikroglijske) ćelije i motoneuronska linija NSL-34. Ćelije
su inkubirane sa Fluo-3 AM za oslikavanje kalcijuma ili sa
DCF za praćenje ROS. Deo BV-2 ćelija je trasfekovan
probom CyPher (fluorescentni agens senzitivan na
peroksid). Korišćeni su prečišćeni uzorci IgG (50µg/ml)
poreklom iz 5 ALS bolesnika i 2 bolesna kontrolna subjekta
(radikulopatija i Parkinsonova bolest). Responsivnost ćelija
je testirana pomoću 1 mM ATP, dok je senzitivnost CyPher
probe za detekciju ROS ispitana izazivanjem odgovora
ćelija na 50 µM H2O2.
Dobijeni rezultati pokazuju da ALS IgG pacijenta
sa ranom formom bolesti utiče na kalcijumovu homeostazu
mikroglijske ćelijske linije. Takoñe, ovi humoralni imunski
faktori spregnuti sa ALS mogu da izazovu ROS signaliranje
u citosolu mikroglijskih i motoneuronskih ćeliskih linija.
Ova zapažanja opravdavaju dalje istraživanje sekundarnih
glasnika ALS IgG- izazvanog ROS signaliranja, koje može
da rasvetli mehanizme patogeneze ALS nezavisne od tipa
ćelija.
We have recently shown that purified
immunoglobulin G (IgG) isolated from amyotrophic lateral
sclerosis (ALS) patients affects calcium homeostasis in
cultured rat astrocytes. Our goal was to investigate the
possible acute effects of ALS IgGs on calcium homeostasis
and reactive oxygen species (ROS) signaling in other cell
types, microglia and motor neurons, taking important parts
in the non-cell autonomous mechanism of ALS
pathogenesis.
Two types of cell lines were used: BV-2
(microglial) cells and the motoneuronal line NSL-34. Cells
were loaded with Fluo-3 AM for calcium imaging or DCF
for ROS monitoring. A batch of BV-2 cells was also
transfected with CyPher (peroxide-sensitive fluorescent
agent). Samples used were of purified IgGs (50µg/ml) from
5 ALS patients and 2 disease controls (radiculopathy,
Parkinson’s disease). Cells were tested for responsiveness
with 1 mM ATP, while the sensitivity of CyPher ROSdetection was done by challenging the cells with 50 µM
H2O2.
The obtained results indicate that ALS IgG from a
patient with the early stage of the disease affects Ca2+
homeostasis of microglial cell line. Furthermore, these
ALS-related humoral immune factors were able to induce
ROS signaling in the cytosol of microglial or motoneuronal
cell lines. These observations justify further research into
second messengers of ALS IgG-induced ROS signaling that
can shed new light on the non-cell autonomous mechanisms
in ALS.
1
65
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
POVEĆANA GENSKA EKSPRESIJA
METALOPROTEINAZA MATRIKSA-2,-9,-14 I
SMANJENA EKSPRESIJA TKIVNOG INHIBITORA
METALOPROTEINAZA-1 U KIČMENIM
MOŽDINAMA PACOVA KORELIŠU SA
REZISTENCIJOM PACOVA NA INDUKCIJU
EKSPERIMENTALNOG AUTOIMUNSKOG
ENCEFALOMIJELITISA
HIGH MATRIX METALLOPROTEINASE-2, -9 AND 14 AND LOW TISSUE INHIBITOR OF
METALOPROTEINASE-1 EXPRESSION IN THE
SPINAL CORD OF RATS CORRELATE TO
CENTRAL NERVOUS SYSTEM AUTOIMMUNITY
RESISTANCE
M. Momčilović , F. Petković , J. Blaževski , J. Živanović ,
V. Stamenković2, M. Mostarica Stojković3, Đ. Miljković1
1
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; Srbija;
2
Institut za fiziologiju i biohemiju; Biološki fakultet;
Univerzitet u Beogradu; Srbija;
3
Institut za imunologiju i mikrobiologiju, Medicinski
fakultet; Univerzitet u Beogradu; Srbija
mommilja@yahoo.com
M. Momčilović1, F. Petković1, J. Blaževski1, J. Živanović1,
V. Stamenković2, M. Mostarica Stojković3,
Đ. Miljković1
1
Institute of Biological Research “Siniša Stanković;
University of Belgrade; Serbia;
2
Institute for Physiology and Biochemistry; School of
Biology; University of Belgrade; Serbia;
3
Institute of Immunology and Microbiology; School of
Medicine; University of Belgrade; Serbia
mommilja@yahoo.com
Uvod:
Eksperimentalni
autoimunski
encefalomijelitis (EAE) je animalni model multiple skleroze
(MS). EAE se može indukovati kod Dark Agouti (DA)
pacova imunizacijom mešavine homogenata kičmene
moždine (HKM) i kompletnog Frojndovog adjuvansa
(KFA). Albino Oxford (AO) pacovi ne ispoljavaju klinički
manifestni EAE nakon imunizacije. Metaloproteinaze
matriksa (MMP) imaju značajnu ulogu u autoimunskom
odgovoru tokom MS-a i EAE.
Cilj: Ispitati gensku ekspresiju MMP-2, MMP-9,
MMP-14 i tkivnog inhibitora metaloproteinaza-1 (TIMP-1)
u homogenatima (HKM) i infiltratima kičmenih moždina
(IKM) imunizovanih AO i DA pacova.
Metode: EAE je indukovan kod DA i AO pacova
imunizacijom sa HKM+KFA. Inflamatorne ćelije kičmene
moždine iz KM pacova perfundovanih sterilnim PBS su
izolovane na gradijentu Perkola. Genska ekspresija MMP-2,
MMP-9, MMP-14 i TIMP-1 u HKM i IKM kod oba soja je
odreñivana metodom “Real time” PCR. Aktivnost MMP je
odreñivana in situ zimografijom na presecima KM.
Rezultati: Genska ekspresija MMP-2, MMP-9 i
MMP-14 je bila značajno povećana kod HKM i IKM EAErezistentnih AO pacova, ali ne i kod EAE-osetljivih DA
pacova, u poreñenju sa zdravim pacovima. TIMP-1 genska
ekspresija je bila povećana kod oba soja, ali je bila značajno
veća kod DA pacova u fazi pika i oporavka od EAE-a. Nije
uočena razlika u aktivnosti MMP izmeñu AO i DA pacova
u fazi pika EAE.
Zaključak: Otpornost AO pacova na indukciju
EAE koreliše sa povećanom ekspresijom TIMP-1 i
smanjenom ekspresijom pro-inflammatornih MMP-2,
MMP-9 i MMP-14.
Background:
Experimental
autoimmune
encephalomyelitis (EAE) is a model of multiple sclerosis
(MS). EAE can be induced in Dark Agouti (DA) rats by
injection of spinal cord homogenate (SCH) and an adjuvant,
complete Freund’s adjuvant (CFA). Albino Oxford (AO)
rats do not express clinically manifested EAE after the
immunization. Matrix metaloproteinases (MMP) play
significant role in the (auto)immune response in MS and
EAE.
Aim: To investigate gene expression of MMP-2,
MMP-9 and MMP-14 and a MMP inhibitor TIMP-1 in SCH
and infiltrates of immunized AO and DA rats.
Methods: EAE was induced in DA and AO rats by
immunization with SCH+CFA. Spinal cord inflammatory
cells (SCIC) were obtained from SC of rats perfused with
sterile PBS and separated on the Percoll gradient. Gene
expression of MMP-2, MMP-9, MMP-14 and TIMP-1 in
SCH and SCIC of both strains of rats was measured by Real
time RT-PCR. The activity of MMPs was determined by in
situ zymography on SC sections.
Results: MMP-2, MMP-9 and MMP14 gene
expression increased significantly in SCH and SCIC of
EAE-resistant AO rats, but not of EAE-prone DA rats, if
compared to healthy, non-immunized counterparts. TIMP-1
gene expression was up-regulated in both strains, but it was
significantly higher in DA rats at the peak and recovery of
EAE. There was no difference in MMPs activity between
AO and DA spinal cords at the peak of EAE.
Conclusion: Resistance of AO rats to EAE
correlated with low expression of TIMP-1 and high
expression of pro-inflammatory MMP2, MMP9 and
MMP14.
1
1
1
1
66
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
UTICAJ RIBAVIRINA I TIAZOFURINA NA
GENSKU I PROTEINSKU EKSPRESIJU
URAVNOTEŽUJUĆIH NUKLEOZIDNIH
TRANSPORTERA U KULTURI MIKROGLIJSKIH
ĆELIJA
1
1
1
1
D. Savić , I. Lavrnja , A. Parabucki , I. Bjelobaba , I. Božić,
S. Dacić2, D. Laketa, N. Nedeljković2, M. Stojiljković1, S.
Peković1
1
Odeljenje za neurobiologiju; Institut za biološka
istraživanja „Siniša Stanković“; Univerzitet u Beogradu;
Beograd; Srbija;
2
Institut za fiziologiju i biohemiju; Biološki fakultet;
Univerzitet u Beogradu; Beograd; Srbija
danisto@ibiss.bg.ac.rs
Uravnotežujući
nukleozidni
transporteri
(Equilibrative Nucleoside Transporter - ENT) obavljaju
olakšanu difuziju adenozina i drugih nukleozida niz
gradijent njihove koncentracije. Pokazano je da purinski
nukleozidni analozi, ribavirin (RBV) i tiazofurin (TR),
mogu da se transportuju u ćelije posredstvom članova ENT
familije (ENT1, ENT2).
Cilj ove studije bio je da se ispita genska i
proteinska ekspresija ENT1 i ENT2 na mikrogliji.
Nestimulisana i LPS-stimulisana primarna kultura
mikroglije izolovana iz Wistar pacova, inkubirana je sa
RBV, TR i njihovom kombinacijom. Ekspresija ENT1 i
ENT2 procenjena je na na nivou iRNK metodom
semikvantitativnog RT-PCR u realnom vremenu, kao i
imunocitohemijskim obeležavanjem.
U nestimulisanoj kulturi mikroglije, oba analoga,
pojedinačno i u kombinaciji, povećala su ekspresiju iRNK
za ENT1. Meñutim, primenjeni tretmani nisu uticali na nivo
iRNK za ENT2. LPS stimulacija uzrokovala je smanjenje
ekspresija iRNK za ENT1 i ENT2 na polovinu u odnosu na
nestimulisane ćelije. U istoj kulturi, samo je kombinovani
tretman izazvao povećanje ekspresije iRNK za ENT1 u
odnosu na LPS stimulisane ćelije. Imunocitohemijska
analiza pokazuje da se ENT1 i ENT2 u visokom stepenu
eksprimiraju na nestimulisanim i LPS-stimulisanim
ćelijama mikroglije, kotretiranim RBV-om, TR-om ili
njihovom kombinacijom.
Otpuštanje adenozina predstavlja endogeni
mehanizam imunosupresije tokom ćelijskog stresa i
inflamacije, pa registrovane kvantitativne promene u
ekspresiji ENT1 i ENT2 na mikrogliji izazvane LPS
stimulacijom, ukazuju na ulogu adenozina u procesu
mikroglijske aktivacije. Za potpuno razumevanje interakcije
ENT-ova sa RBV i TR, potrebno je funkcionalno
okarakterisati ove transportere.
EFFECT OF RIBAVIRIN AND TIAZOFURIN ON
GENE AND PROTEIN EXPRESSION OF
EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS
IN CULTURED MICROGLIA
D. Savić1, I. Lavrnja1, A. Parabucki1, I. Bjelobaba1, I. Božić,
S. Dacić2, D. Laketa, N. Nedeljković2, M. Stojiljković1, S.
Peković1
1
Department for Neurobiology; Institute for biological
research “Sinisa Stankovic”; University of Belgrade;
Belgrade; Serbia;
2
Institute for Physiology and Biochemistry; Faculty of
Biology; University of Belgrade; Belgrade; Serbia
danisto@ibiss.bg.ac.rs
Equilibrative Nucleoside Transporters (ENTs) are
passive, bidirectional transporters of adenosine and other
nucleosides across the cell membranes. It was shown that
transport of purine nucleoside analogues, ribavirin and
tiazofurin, into the cell is mediated by the ENT family
members, ENT1 and ENT2.
Since these transporters represent the possible way
of RBV and TR into the cells, in this study we investigated
expression of ENT1 and ENT2 in the microglia.
Non-stimulated and LPS-stimulated primary rat
microglia cultures were incubated with different
concentrations of RBV, TR and their combination.
Expression of ENT1 and ENT2 was estimated by real time
PCR and immunocytochemistry.
In non-stimulated microglia both analogues,
separately and in combination, increased ENT1 –mRNA,
while they did not affected level of ENT2-mRNA. LPS
stimulation induced down-regulation of ENT1 and ENT2
mRNAs compared to non-stimulated cells. In LPSstimulated cultures, only the combined treatment RBV+TR
caused the effect, by increasing ENT1-mRNA level.
Immunocytochemical analysis showed that non- and LPSstimulated microglia cells co-treated with RBV, TR and
RBV+TR were strongly immunopositive for both
nucleoside transporters.
Knowing that release of adenosine is an
endogenous mechanism of immunosuppression evoked by
cellular stress and inflammation, LPS-induced quantitative
changes of the ENT1/ENT2 expression pattern in microglia
point out to adenosine role in process of microglial
activation. However, for a full understanding of ENTs
interaction with RBV and TR, these transporters should be
functionally characterized.
67
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
UTICAJ NETIPIČNIH ANTIPSIHOTIKA NA
EKSPERIMENTALNI AUTOIMUNSKI
ENCEFALOMIJELITIS KOJI JE INDUKOVAN KOD
PACOVA DA SOJA
1
1
3
Ž. Stanojević, M. Popović , A.J. Isaković , S. Petrićević , J.
Tošić1, V. Trajković2
1
Institut za medicinsku i kliničku biohemiju; Medicinski
fakultet; Univerzitet u Beogradu; Beograd, Srbija;
2
Institut za imunologiju; Medicinski fakultet; Univerzitet u
Beogradu; Beograd;Srbija;
3
Centar za biomedicinska istrazivanja; R&D Institut;
Galenika a.d; Beograd; Srbija
zeljka.stanojevic@med.bg.ac.rs
Uvod: U terapiji autoimunskih bolesti centralnog
nervnog sistema (CNS), kao što je multipla skleroza (MS) i
njen animalni model eksperimentalni autouimunski
encefalomijelitis
(ЕАЕ),
se
primenjuju
brojni
imunosupresivni i imunomodulatorni lekovi. Meñutim,
danas nije poznato da li i na koji način atipični antipsihotici
ostvaruju antiinflamatorni efekat unutar CNS-a.
Cilj: Ispitati potencijalni antiinflamatorni uticaj
atipičnih antipsihotika primenom u EAE-u.
Materijal i metode: Pacovi DA soja starosti 6-8
nedelja, ženskog pola su imunizovani homogenatom
kičmene moždine kompletni Frojndov adjuvans. Atipični
antipsihotici su aplikovani intraperitonealno (i.p) u dozi 10
mg/kg telesne težine u različitim fazama bolesti i to: od
dana imunizacije do 7 dana bolesti, od 7. dana posle
imunizacije (d.p.i) do 21. dana bolesti i od dana imunizacije
do 21. dana bolesti. Tokom navedenog perioda praćen je
njihov uticaj na samu kliničku sliku EAE-a a korišćenjem
izolovanih mononuklearnih ćelija kičmene moždine
(MNĆKM) je analiziran efekat atipičnih antipsihotika na
ekspresiju proinflamatornih i antiinflamatornih citokina,
PCR metodom.
Rezultati: Dobijeni rezultati su pokazali da atipični
antipsihotici ostvaruju povoljan terapijski efekat na kliničko
ispoljavanja EAE-a kada su aplikovani od dana imunizacije
i od 7. d.p.i. Analizom cDNK koja je dobijena reverznom
transkripcijom RNK iz MNĆKM primenom PCR metode je
pokazano da poboljšanje kliničke slike EAE-a nije praćeno
smanjenjem ekspresije gena kako za citokine IL-1, IL-12a,
IL-23, IL-6, IL-17α, TGF-β tako i za transkripcione
faktoreT-bet, RorγT i Foxp3 kod MNĆKM izolovanih iz
tretirane grupe životinja.
Zaključak: Povoljan terapijski efekat na kliničku
sliku EAE-a izostaje kada su aplikovani samo 7 dana od
dana imunizacije. Kako klinička slika nije praćena i
smanjenjem ekspresije gena za citokine koji imaju
dominatnu ulogu u oštećenju CNS-a predpostavlja se da
atipični antipsihotici svoje povoljno dejstvo ostaruju
nezavisno od pomenutih citokina.
EFFECT OF ATYPICAL ANTIPSYCHOTIC IN
EXPERIMENTAL AUTOIMMUNE
ENCEPHALOMYELITIS IN DA RAT
Ž. Stanojević, M. Popović1, A.J. Isaković1, S. Petrićević 3, J.
Tošić1, V. Trajković2
1
Institute of Medical and Clinical Biochemistry; Faculty of
Medicine; University of Belgrade; Belgrade; Serbia;
2
Institute of and Immunology, Faculty of Medicine;
University of Belgrade; Belgrade; Serbia;
3
Biomedical Research; R&D Institute; Galenika A.D.;
Belgrade; Serbia
zeljka.stanojevic@med.bg.ac.rs
Introduction: Series of different strategies
including usage of immunoregulatory and antiinflammatory drugs were tested for therapy of autoimmune
disease of the central nervous system like Multiple Sclerosis
or its corresponding animal model, Experimental
Autoimmune Encephalomyelitis (EAE). However, there is
not any data about the potential anti-inflammatory effects
that is generated within the CNS by atypical antipsychotics.
The aim: To examine the potential antiinflammatory effects of atypical antipsychotics in EAE.
Material and methods: EAE was induced in Dark
Agouti (DA) rats by immunization with rat spinal cord
homogenate (SCH) mixed with Complete Freund's
Adjuvant. Age (6-8 week) and gender matched animals
were used in the experiments. DA rats were injected daily
with atypical antipsychotics (10 mg/kg) in three different
settings: starting on the day of immunization; during first
seven days; starting from the 7. day post immunization.
Experimen lasted 21 day. The effects of atypical
antipsychotics were analysed using daily EAE clinical
course while spinal cord mononuclear cells (SCC) were
used for PCR analysis.
Results: Atypical antipsychotics generate favorable
therapeutic effect on clinical manifestation of EAE but only
when administered from the first untill 21. day and from the
7. untill 21. day post immunization. PCR analysis of SCC
shows that in vivo treatment with atypical antipsychotics
was not mediated through inhibition of gene expression for
cytokines: IL-1, IL-12a, IL-23, IL-6, IL-17a, TGF-β as well
as of transcription factors T-bet, RorγT and Foxp3.
Conclusion: Taken together, these results
demonstrate that amelioration of EAE by atypical
antipsychotics was absent when they were administered
during first seven days after immunization. Since the
clinical score was not followed by inhibition of cytokines
gene expression we can assume that the beneficial effect of
atypical antipsychotics is not mediated by cytokines within
CNS.
68
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
AKTIVACIJA GLIJALNIH ĆELIJA I OKSIDATIVNI
STRES U HIPOKAMPUSU I MOŽDANOM STABLU
SOD1 G93A PACOVA - MODELA AMIOTROFIČNE
LATERALNE SKLEROZE
ACTIVATION OF GLIAL CELLS AND OXIDATIVE
STRESS IN THE HIPPOCAMPUS AND BRAINSTEM
OF THE TRANSGENIC SOD1 G93A RAT MODEL
OF ALS
S. Stamenković1, V. Selaković2, L. Radenović1, P.R. Anñus1
1
Centar za lasersku mikroskopiju; Institut za fiziologiju i
biohemiju; Biološki fakultet; Univerzitet u Beogradu;
Srbija;
2
Instiutu za medicinska istraživanja; Vojnomedicinska
akademija; Beograd; Srbija
stefanstm@gmail.com
S. Stamenkovic1, V. Selakovic2, L. Radenovic1, P. R.
Andjus1
1
Center for Laser Microscopy; Institute for Physiology and
Biochemistry; Faculty of Biology; Univesity of Belgrade;
Serbia;
2
Insitute for Medical Research; Military Medical Academy;
Belgrade; Serbia.
stefanstm@gmail.com
Amiotrofična lateralna skleroza (ALS) je
progresivno neurodegenerativno oboljenje sa smrtnim
ishodom, koje se odlikuje odumiranjem motornih neurona u
kori velikog mozga, moždanom stablu i kičmenoj moždini.
Većina slučajeva ALS su sporadični, dok samo 10% ima
poznatu genetičku osnovu, a od kojih je 20% izazvano
mutacijama u Cu, Zn superoksid dismutazi (SOD1). Jedan
od najvažnijih i najbolje okarakterisanih patoloških
mehanizama u ALS je neuroinflamatorna reakcija u kojoj
učestvuju aktivirana mikroglija i astrociti. Predložena je
hipoteza da se mutirana SOD1 otpušta u vanćelijski prostor
gde može da izvrši aktivaciju ova dva tipa glijalnih ćelija i
pokrene neuroinflamatornu reakciju sa povećanom
proizvodnjom slobodnih radikala. Stoga smo rešili da
ispitamo markere aktivacije glijalnih ćelija i oksidativnog
stresa u moždanom stablu i hipokampusu transgenog
modela ALS pacova sa G93A mutacijom u SOD1.
Imunohistohemijska
bojenja
za
markere
mikroglije, astrocita i neurona su pokazala prisustvo jake
glijalne aktivacije u ispitivanim moždanim regionima i
postojanje izrazitih unutarćelijskih agregacija SOD1
(antitelima na SOD1) kako u neuronima, tako i u oba tipa
glijalnih ćelija. Ispitivanje parametara oksidativnog stresa
spektrofotometrijskim biohemijskim testovima je otkrilo
povećanu količinu kiseoničnih i azotnih radikalskih vrsta,
povišen indeks lipidne peroksidacije, smanjenu aktivnost
citosolne SOD1 i povišenu aktivnost motohondrijske Mn
superoksid dismutaze (SOD2), kako u hipokampusu tako i u
moždanom stablu ALS modela. Navedeni rezultati ukazuju
da je oksidativni stres ne samo posledica, već i mogući
uzrok neuroinflamacije koja se završava odumiranjem
neurona, čime se ističe važnost ovog fenomena kao jednog
od osnovih patogenih mehanizama u ALS.
Amyotrophic lateral sclerosis (ALS) is a
progressive and fatal neurodegenerative disorder
characterized by death of neurons in cerebral cortex, brain
stem and spinal cord. Most ALS cases are sporadic, while
only 10% occurs in familial forms, among which 20% are
caused by mutations in the Cu, Zn-superoxide dismutase
(SOD1). One of the most important and well characterized
pathological mechanisms in ALS is the neuroinflammatory
reaction consisting mainly of activated microglia and
astrocytes. It is proposed that the mutant SOD1 is released
to the extracellular space where it can activate these two
types of glial cells and lead to neuroinflammation and
increased reactive oxygen species production. Therefore, we
decided to check for markers of glial activation and
oxidative stress in the brain stem and hippocampus of the
rat transgenic SOD1 G93A model of ALS.
Immunohistochemistry with markers of microglia,
astrocytes and neurons showed strong glial activation in
these regions and revealed the existence of pronounced
intracellular aggregations of SOD1 (anti-SOD1 antibody) in
neurons and both types of glial cells as well. Examination of
oxidative stress parameters by spectrophotometric
biochemical assays detected increased presence of reactive
oxygen and nitrogen species, increased index of lipid
peroxidation, decreased cytosolic SOD1 and increased
mitochondrial SOD2 activity, in both hippocampus and
brainstem of the ALS model. These results point to
oxidative stress not just as a consequence, but also as a
likely cause of the neuroinflammation that ends in
neurodegeneration, thus emphasizing its importance as one
of the most essential pathogenic mechanisms in ALS.
69
POSTER SESIJA / POSTER SESSION
P3 - Ćelijski i molekularni mehanizmi neuroinflamatornih procesa / P3 - Cellular and molecular mechanisms of neuroinflammation
RESTRIKCIJA HRANE KAO POTENCIJALNI
PRETRETMAN U SUPRESIJI IMUNSKOG
ODGOVORA KOD PACOVA NAKON KORTIKALNE
LEZIJE
FOOD RESTRICTION AS A POTENTIAL
PRETREATMENT CAPABLE OF SUPPRESSING
INFLAMMATION FOLLOWING CORTICAL
INJURY IN RATS
S. Todorović1, N. Lončarević-Vasiljković1, A. Mladenović
Đorñević1, M. Perović1, Lj. Rakić2, S. Ruždijić1 ,S. Kanazir1
1
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; 11060 Beograd; Srbija;
2
Srpska akademija nauka i umetnosti; 11000 Beograd;
Srbija
smilja.pracer@ibiss.bg.ac.rs
S. Todorovic1, N. Loncarevic-Vasiljkovic1, A. Mladenovic
Djordjevic1, M. Perovic1, Lj. Rakic2, S. Ruzdijic1, S.
Kanazir1
1
Institute for Biological Research “Sinisa Stankovic”;
University of Belgrade; 11060 Belgrade; Serbia;
2
Serbian Academy of Sciences and Arts; 11000 Belgrade;
Serbia
smilja.pracer@ibiss.bg.ac.rs
Dobro je poznato da je smanjen unos hrane
neuroprotektivan, a novija istraživanja ukazuju i na
neuroprotektivno dejstvo restrikcije hrane nakon traumatske
povrede mozga (TPM). Molekulski procesi u osnovi ovog
dejstva još uvek nisu razjašnjeni. Glukokortikoidi, delujući
preko svojih receptora (GR), predstavljaju glavne
medijatore stresa. U prisustvu liganda, ovi receptori se
fosforilišu i aktiviraju. Nuklearni faktor kapa B (NF-kB) je
važan transkripcioni regulator inflamatornog odgovora.
Cilj ovog eksperimenta je da se utvrdi da li
restrikcija hrane dovodi do supresije imunskog odgovora
nakon ubodne kortikalne lezije (engl. Cortical Stab Injury,
CSI) posredstvom GR-a i NFkB signalnog puta.
Mužjaci Wistar pacova (tri meseca stari) su
podeljeni u dve grupe, životinje koje su dobijale 50% od
normalnog dnevnog unosa hrane (engl. Food Restricted,
FR) i životinje koje imaju slobodan, ad libitum (AL),
pristup hrani. Nakon navršenih 6 meseci, sve životinje su
podvrgnute CSI i žrtvovane 2., 7., 14. i 28. dana nakon
lezije. Nivo ekspresije GR-a, fosfo-GR-a i NF-kB je
odreñen Western Blot analizom.
Detektovan je povećan nivo fosfo-GR-a i NF-kB u
AL i FR grupi nakon povrede, u odnosu na odgovarajuću
kontrolu. Meñutim, poreñenjem AL i FR grupa, utvrñeno je
značajno povećanje fosfo-GR-a i NF-kB u 7. danu nakon
lezije kod FR pacova.
Rezultati našeg istraživanja daju novi uvid o
načinu dejstva FR u supresiji imunskog odgovora nakon
CSI i važnoj ulozi GR-a i NF-kB u tom procesu.
Food restriction (FR) has profound effects on brain
function and its vulnerability to injury or disease. It has
been suggested that FR is protective as a pretreatment to an
acute traumatic brain injury (TBI), but underlying signaling
pathways are still unclear. Glucocorticoids (GCs), acting
through their receptors (GR) are key mediators of stressrelated response and nuclear factor kappa B (NF-kB) plays
a key role in regulating the inflammation.
The goal of the present study was to examine
whether FR exhibits its neuroprotective role after cortical
stab injury (CSI) by modulating the inflammation and
whether it happens through GR and NF-kB signaling
pathway.
Three months old male Wistar rats were divided
into two groups, ad libitum (AL) and food restricted (FR).
FR animals were fed 50% of their normal daily food intake.
At 6 months of age, animals were subjected to the CSI and
sacrificed 2, 7, 14 and 28 days post injury. Expression of
glucocorticoid receptor (GR), phospho-GR (p-GR) and NFkB was detected by Western Blot analysis.
Our data revealed an increase in the levels of p-GR
and NF-kB in AL and FR groups after injury. On the 7^th
day following injury we found significant increase of p-GR
and NF-kB in FR group compared to AL group.
Present study further illuminates the mechanism by
which FR suppresses inflammation after CSI suggesting an
important role of NFkB and GR in this process.
70
POSTER SESIJA / POSTER SESSION
P4 - Poremećaj homeostaze i bolesti centralnog nervnog sistema / P4 - Dysregulation of homeostasis and CNS disorders
UTICAJ TERMOMINERALNE VODE NA
REGENERACIJU KOD ŽIVOTINJSKOG MODELA
LEZIJE KIČMENE MOŽDINE
THERMOMINERALWATER IMPROVES
REGENERATION IN AN ANIMAL MODEL OF
SPINAL CORD INJURY
D. Aleksić1, M. Aksić1, B. Marković1, N. Divac2, V.
Radonjić1, B. Filipović1, I. Jakovčevski3
1
Institut za anatomiju; Medicinski fakultet; Beograd; Srbija;
2
Institut
zafarmakologiju,
kliničkufarmakologiju
i
toksikologiju; Medicinskifakultet; Beograd; Srbija;
3
Centar za Molekularnu Neurobiologiju; Univerzitetska
Klinika Hamburg-Eppendorf; Hamburg; Nemačka
igor.jakovcevski@zmnh.uni-hamburg.de
D. Aleksić1, M. Aksić1, B. Marković1, N. Divac2, V.
Radonjić1, B. Filipović1, I. Jakovčevski3
1
InstituteforAnatomy, Medical Faculty, Belgrade, Serbia;
2
Institutefor Farmacology; Clinical Farmacology and
Toxicology; Medical Faculty; Belgrade; Serbia;
3
Center for Molecular Neurobiology; UniversityHospital
Hamburg-Eppendorf; Hamburg; Germany
igor.jakovcevski@zmnh.uni-hamburg.de
Uvod: Centralni nervni system odraslih sisara ima
mali kapacitet za regeneraciju posle povrede, te poboljšanje
regeneracije predstavlja važan zadatak neurobiologije.
Termomineralna voda Atomske Banje (GornjaTrepča, RS)
koristi se već 100 godina za terapiju neuroloških i
reumatoloških oboljenja. Ova voda sadrži brojne mikro- i
makroelemente, uključujući jone metala poput Li koji
stabilizuju membrane neurona i poboljšavaju njihovo
preživljavanje.
Cilj: Testirali smo uticaj oralnog unosa vode iz
Atomske Banje na regeneraciju kičmene moždine miševa
posle ozlede, kao klasičnog eksperimentalnog modela lezije
CNS-a.
Materijal i metode: Dve grupe od po 8 C57BL/6 ženki starih
3 meseca podvrgnuto je prignječenju kičmene moždine u
torakalnom delu i oporavak motorne funkcije praćen je
tokom 3 meseca. Miševi iz eksperimentalne grupe pili su
mineralnu vodu dok su kontrolni miševi pili
demineralizovanu vodu.Nakon 3 meseca miševi su
žrtvovani i kičmene moždine su pripremljene za
imunohistološku analizu grananja kateholaminergičkih
aksona, gustine astrocitnog ožiljka i mikroglijalne reakcije u
regionu lezije.
Rezultati: Miševi tretirani eksperimentalnom
vodom pokazali su bolji oporavak motorne funkcije u
odnosu na kontrolnu grupu, procenjen BMS indeksom 2 i 3
meseca posle ozlede. Grananje kateholaminergičkih aksona
u regionu lezije, takodje je bilo veće u eksperimentalnoj
grupi, dok nije bilo razlike u glijalnoj reakciji.
Zaključak: Termomineralna voda obogaćena
litijumom pomaže oporavak motiliteta i poboljšava
aksonalnu regeneraciju kod miševa posle povrede kičmene
moždine. Dalja istraživanja su potrebna da se ustanovi koji
joni iz vode ili njihov optimalni odnos su ključni za
primećene efekte.
Introduction: Adult mammalian CNSregenerates
poorly after injury, thus the improvement of regeneration is
an important goal of neurobiology. Thermomineral water
from AtomskaBanja (GornjaTrepča, RS) has been used for
more than 100 yearsfor therapy of neurological and
rheumatic disorders. This water contains several metal ions
such as lithium, which stabilizes neuronal membrane and
improves neuronal survival upon injury.
Aim: We tested how oral intake of water from
AtomskaBanja influences regeneration in a mouse spinal
cord injury, a commonly used experimental model for
injury of the CNS.
Materials and methods: Three-month-old female
C57BL/6 mice underwent lower thoracic spinal cord
compression with a 3 month follow-up of the motor
recovery. Experimental group drank mineral water, whereas
control group had demineralized water. After 3 months
mice were sacrificed and spinal cords were analyzed for
sprouting of catecholaminergic axons, density of astrocyte
scar, and microglia at the lesion site.
Results: Mice from the experimental group showed
better functional recovery than control group, at 2 and 3
months after injury, assessed by the BMS score. Sprouting
of catecholaminergic axons was better in mice that drank
experimental water at 3 months after injury, and there was
no difference in glia reaction to injury from control mice.
Conclusion: Thermomineral water enriched in
lithium
promotes
motor
recovery
and
axonal
regenerationafter spinal cord injury in mice. Further studies
should address which ions or their optimal ratio are causing
the effects of mineral water reported here.
71
POSTER SESIJA / POSTER SESSION
P4 - Poremećaj homeostaze i bolesti centralnog nervnog sistema / P4 - Dysregulation of homeostasis and CNS disorders
UTICAJ VITAMINA D NA EKSPRESIJU POJEDINIH
GLIJALNIH PROTEINSKIH MARKERA I
MARKERA NEURONA U MOZGU PUSTINJSKIH
MIŠEVA IZLOŽENIH PROLAZNOJ GLOBALNOJ
ISHEMIJI MOZGA
THE EFFECTS OF VITAMIN D ON THE
EXPRESSION OF GLIAL AND NEURONAL
MARKERS IN THE BRAIN OF GERBILS EXPOSED
TO TRANSIENT GLOBAL CEREBRAL ISCHEMIA
G. Jevtić, T. Stojković, M. Velimirović, N. Petronijević
Institut za medicinsku i kliničku biohemiju; Medicinski
fakultet;Univerzitet u Beogradu; Srbija
jevtic.gordana@yahoo.com
G. Jevtić, T. Stojković, M. Velimirović, N. Petronijević
Institute of Medical and Clinical Biochemistry; School of
Medicine; University of Belgrade; Serbia
jevtic.gordana@yahoo.com
Uvod: Podvezivanje karotidnih arterija kod
džerbila je opšte prihvaćeni animalni model prolazne
globalne ishemije mozga. Vitamin D ima neuroprotektivnu
ulogu u mozgu. Promene u neurogliji i nervnim ćelijama se
mogu pratiti preko ekspresije proteinskih markera: glijalnog
fibrilarnog
kiselog
proteina
(GFAP),
mijelin
oligodendrocitnog glikoproteina (MOG), jonizovanog
kalcijum-vezujućeg adapterskog molekula (Iba1) i neuron
specifičnog nuklearnog proteina (NeuN).
Cilj: Cilj rada je ispitivanje uticaja vitamina D na
ekspresiju GFAP, MOG, Iba1 i NeuN u mozgu džerbila
izloženih globalnoj ishemiji.
Materijal i metode: Mongolski džerbili su
razvrstani u tri grupe: Kontrolna grupa - podvrgnuta
operativnom zahvatu bez zatvaranja karotidnih arterija;
Grupa II - podvrgnuta ishemiji tokom 10 minuta i 24časovnoj reperfuziji; Grupa III - sedam dana pre operacije je
primala vitamin D3 (1µg/kg/dan, i.p.), potom podvrgnuta
desetominutnoj ishemiji i 24-časovnoj reperfuziji.
Ekspresija GFAP, MOG, Iba1 i NeuN odreñivana je
metodom Western blota.
Rezultati: Ekspresija GFAP u korteksu i
hipokampusu džerbila izloženih ishemiji je statistički
značajno smanjena u odnosu na kontrolnu grupu.
Pretretman vitaminom D je sprečio pojavu smanjenja
ekspresije GFAP u hipokampusu. Ekspresije MOG, Iba1
NeuN su nepromenjene u korteksu i hipokampusu u
eksperimentalnim grupama u odnosu na kontrolnu grupu.
Zaključak: Nakon desetominutne prolazne
globalne ishemije i 24-časovne reperfuzije mozga džerbila
dolazi do smanjenja ekspresije GFAP u korteksu i
hipokampusu, dok ova promena izostaje kada ishemiji
prethodi tretman vitaminom D. Ekspresija ostalih markera
glija ćelija i neurona je nepromenjena u ovoj fazi reperfuzije
bez obzira na primenu vitamina D. Neophodna su dalja
istraživanja sa različitim vremenima trajanja ishemije i
vremenima reperfuzije.
Introduction: Carotid artery occlusion in the
Mongolian gerbils is a widely used model of cerebral
ischemia. Vitamin D has protective effects in brain.
Changes in neuroglia and neurons can be monitored by the
expression of protein markers: glial fibrillary acidic protein
(GFAP), myelin oligodendrocyte glycoprotein (MOG),
ionized calcium-binding adapter molecule 1 (Iba1) and
neuron specific nuclear protein (NeuN).
Aim: The aim of the study was to examine the
effects of vitamin D on the expression of glial and neuronal
markers in gerbils subjected to cerebral ischemia.
Material and methods: Animals were divided into
three groups: Sham-operated group (control); Group IIsubjected to ischemia for 10 min and reperfused for 24h;
Group III-received injections of vitamin D (1µg/kg/day i.p.)
for 7 days, then subjected to ischemia for 10 min and
reperfused for 24h. Expression of GFAP, MOG, Iba1 and
NeuN was determined by Western blot.
Results: The expression of GFAP in cortex of
animals subjected to ischemia, and those treated with
vitamin D was significantly reduced, while in hippocampus
the decrease seen after ischemia/reperfusion was absent in
vitamin D pretreated group. The expressions of MOG, Iba1
and NeuN were unchanged in both brain structures in
experimental groups compared to control.
Conclusion: Reperfusion for 24h after ischemia
leads to change in the expression of GFAP in both
structures, while in hippocampus was noticed protective
effect of vitamin D. The expressions of other markers were
unchanged. Further experiments with different periods of
ischemia and reperfusion are needed to clarify the effects of
vitamin D.
72
POSTER SESIJA / POSTER SESSION
P4 - Poremećaj homeostaze i bolesti centralnog nervnog sistema / P4 - Dysregulation of homeostasis and CNS disorders
UTICAJ RESTRIKTIVNOG REŽIMA ISHRANE NA
EKSPRESIJU GFAP-ALFA I GFAP-KAPA
PROTEINA
FOOD RESTRICTION CHANGES GFAP-ALPHA
AND GFAP-KAPPA EXPRESSION FOLLOWING
CORTICAL INJURY IN RATS
N. Lončarević-Vasiljković1, M. Perović1, V. Tešić1, D.
Lazić1, M. Brkić1, Lj. Rakić2, S. Ruždijić1, S. Kanazir 1
1
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; 11060 Beograd; Srbija;
2
Srpska akademija nauka i umetnosti; 11000 Beograd;
Srbija
natasa.loncarevic@ibiss.bg.ac.rs
N. Loncarevic-Vasiljkovic1, M. Perovic1, V. Tesic1, D.
Lazic1, M. Brkic1, Lj. Rakic2, S. Ruzdijic1, S. Kanazir 1
1
Institute for Biological Research “Sinisa Stankovic”;
University of Belgrade; 11060 Belgrade; Serbia;
2
Serbian Academy of Sciences and Arts; 11000 Belgrade;
Serbia
natasa.loncarevic@ibiss.bg.ac.rs
Pokazano je da izlaganje životinja restriktivnom
režimu ishrane dovodi do redukcije astroglioze i modulacije
ekspresije glijalnog fibrilarnog kiselog proteina (GFAP)
nakon kortikalne lezije. Meñutim, ne postoje podaci o
uticaju restrikcije hrane na ekspresiju GFAP-alfa i GFAPkapa izoformi nakon lezije. Poznato je da povećani nivo
ekspresije GFAP-kapa izoforme dovodi do kolapsa u
formiranju intermedijernih filamenata sastavljenih od
GFAP-alfa izoforme. Takoñe, nivo ekspresije GFAP-kapa
može menjati svojstva intermedijernih filamenata i tako
uticati na pokretljivost astrocita.
Cilj ovog eksperimenta je da se pokaže da li
restrikcija hrane utiče na ekspresiju GFAP-alfa i GFAPkapa izoformi u povreñenom tkivu kore mozga.
Mužjaci Wistar pacova (stari tri meseca) su
podeljeni u dve grupe, životinje hranjene sa 50% od
normalnog dnevnog unosa hrane (dijetalna restrikcija, DR)
i životinje koje jedu neograničeno, ad libitum (AL). Nakon
navršenih 6 meseci, životinje su podvrgnute leziji, a tkivo je
sakupljeno 2., 7., 14. i 28. dana nakon lezije. Nivo
ekspresije GFAP-alfa i GFAP-kapa izoformi je odreñen
Western Blot analizom.
Rezultati ove studije su pokazali da povreda mozga
utiče na ekspresiju GFAP-alfa u obe grupe životinja (AL i
DR). Kod AL životinja, nivo GFAP-alfa bio je značajno
povećan u svim vremenskim tačkama, dok je restrikcija
hrane dovela do supresije porasta GFAP-alfa ekspresije.
Suprotno od toga, povreda je prouzrokovala blagi porast
GFAP-kapa ekspresije kod AL životinja, dok je porast
ekspresije ove izoforme bio značajno veći kod DR životinja.
Rezultati našeg istraživanja pokazuju da DR
značajno menja ekspresiju GFAP-alfa i GFAP-kapa
izoformi u povreñenom regionu mozga. Fiziološke
posledice ovih promena potrebno je dalje ispitati.
Exposing animals to food restriction (FR)
significantly reduces astrogliosis and modulates glial
fibrillary acidic protein (GFAP) expression in response to
cortical stab injury (CSI). However, there is no data
concerning the impact of FR to GFAP-alpha and GFAPkappa isoform expression following CSI. Increased GFAPkappa expression levels can cause a collapse of intermediate
filaments formed by GFAP-alpha thus influencing the
motility of astrocytes.
The goal of this study was to examine whether 3month long FR applied prior to CSI influences GFAP-alpha
and GFAP-kappa expression in the injured cortical tissue.
Three months old male Wistar rats were divided
into two groups, ad libitum (AL) and food restricted (FR).
FR animals were fed 50% of normal daily food intake. At 6
months of age, animals were subjected to the CSI and
cortical tissue was collected at 2, 7, 14 and 28 days
following injury. Expression of GFAP-alpha and GFAPkappa was detected by Western Blot analysis.
Present study reveals that brain injury affects
GFAP-alpha expression in both AL and FR groups. In AL
group, GFAP-alpha level was significantly increased at all
time points, while FR abolished GFAP-alpha increase
induced by CSI. In contrast, CSI caused a slight increase in
GFAP-kappa expression in AL animals, while a far greater
increase of this isoform expression was detected in FR
animals.
Our findings suggest that FR significantly changes
GFAP-alpha and GFAP-kappa expression in the injured
cortical tissue following CSI. The physiological
consequences of these changes remain to be elucidated.
73
POSTER SESIJA / POSTER SESSION
P4 - Poremećaj homeostaze i bolesti centralnog nervnog sistema / P4 - Dysregulation of homeostasis and CNS disorders
MORFOFUNKCIONALNE KARAKTERISTIKE
TIROTROPNIH ĆELIJA ADENOHIPOFIZE ŽENKI
PACOVA IZLOŽENIH KONSTANTNOM SVETLU
CONSTANT LIGHT ENVIRONMENT MODULATE
MORPHO-FUNCTIONAL FEATURES OF TSHBETAIMMUNOREACTIVE CELLS IN FEMALE RATS
M. Miler, N. Ristić, I. Medigović, J. Živanović, N.
Nestorović, V. Milošević, B. Šošić-Jurjević
Institut za biološka istraživanja „Siniša Stanković;
Univerzitet u Beogradu; Bulevar despota Stefana 142,
11060 Beograd; Srbija
marko.miler@ibiss.bg.ac.rs
M. Miler, N. Ristić, I. Medigović, J. Živanović, N.
Nestorović, V. Milošević, B. Šošić-Jurjević
Institute for Biological Research "Siniša Stanković";
University of Belgrade; 142 Despot Stefan Blvd, 11060
Belgrade; Serbia
marko.miler@ibiss.bg.ac.rs
Uvod: Tiroidni hormoni su neophodni za normalan
rast,
razviće
i
energetsku
homeostazu.
Tireostimulirajućihormon (TSH) regulišefunkcijuštitaste
žlezde u fiziološkim uslovimaiprilagoñava njenu aktivnost
promenama u spoljašnjoj sredini.
Cilj: Cilj ove studije je bio da se ispitana koji način
izlaganje ženki pacova konstantnom svetlu (CL) utiče na
morfofunkcionalne karakteristike TSHćelijaadenohipofize,
kao i na nivo TSH i tiroidnih hormona u serumu.
Materijal i metode:Ženke Wistar pacova su 95
dananeprekidno izlaganekonstantnom svetlu (600Lx),
počevši od30. postantalnog dana. Nakon dekapitacije
hipofize su izolovane i pripremljene za histološku analizu.
TSH
ćelije
su
vizualizovane
korišćenjem
imunohistohemijskih ili imunofluorescentnih metoda.
Kvantifikovan jerelativni volumen i relativna volumenska
gustina tirotropnih ćelija, kao i relativni intenzitet TSH beta
imunofluorescence (RIF) u njihovoj citoplazmi. Odreñen je
nivo TSH, ukupnih T4 i T3 hormona u serumu.
Rezultati:Tirotropne ćelije adenohipofize su
poligonalnog ili ovalnog oblika s ekscentrično postavljanim
jedrom. Granulisana TSHbeta imunoreaktivnost jako je
izražena u citoplazmi ovih ćelija. Relativni volumen i
volumenska gustina TSHćelijaje smanjena (p<0.05), dok je
RIF signal povećan (p<0.05) nakon izlaganja CL. Nije
uočena promena u nivou TSH u serumu, dok su ukupniT4i
T3sniženi (p<0.05).
Zaključak:
Akumulacija
TSH
beta
imunofluorescentnih granula u citoplazmi tirotropnih ćelija
adenohipofize ukazuje na povećanu sintezu TSH, koja
nijepraćenapovišenjemkoncentracijeTSH u serumu pacova
izloženih konstantnom svetlu. Smanjena sekretorna
aktivnost štitaste žlezde detektovana pod našim
eksperimentalnim uslovima može biti rezultat smanjene
bioaktivnosti TSH i/ili desenzitacije tiroidnog tkiva nakon
prolongiraneTSHstimulacije u uslovima dugotrajnog
izlaganja CL.
Background: Thyroid hormones are essential for
normal growth, development and energy balance. Pituitary
thyroid stimulating hormone (TSH) regulates and
adjuststhyroid functioning changes in the environment.
Objective: In this study, we tested whether and
how exposure of female rats to constant light(CL)
environment would affect themorpho-functional features of
pituitary TSH-betacells,as well as the level of thyroid
hormones in serum.
Material and Methods: Starting from the 30th
postnatal day, female Wistar rats were keptin CL
(600Lx)environment for the following95 days. After
decapitation,the pituitaries were excised and prepared for
further
histological
examination.TSH-betacells
wereanalyzed
using
immunohistochemicalor
immunofluorescentprocedures. The relativevolume and
volume density of thyrotroph cells, as well as therelative
intensity of fluorescent (RIF) TSH-beta signal within their
cytoplasm werequantified.Serum levels of TSH, total T4 and
T3were determined.
Results: Pituitary thyrotrophswere polygonal or
oval in shape with eccentrically located nuclei.Granular
TSH-beta immunoreactivity waspronounced in their
cytoplasm. CL treatmentdecreased (p<0.05) relative volume
and the volume density ofTSH-beta cells, while the RIF
signal increased (p<0.05). No change in serum TSH level
was determined, while total T4 and T3weredecreased
(p<0.05).
Conclusion:
Prolonged
exposure to
CL
environment induced changes in morphology of TSH cells
that indicate increased TSH synthesis and accumulation.
However, since no change in serum TSH was detected,
lower T4 and T3 levels in serum may bedue to decreased
bioactivity of TSH and/or desensitization of thyroid tissue
for TSH stimulation after prolonged exposure to CL.
74
POSTER SESIJA / POSTER SESSION
P4 - Poremećaj homeostaze i bolesti centralnog nervnog sistema / P4 - Dysregulation of homeostasis and CNS disorders
BIHEJVIORALNA,
IMUNOHISTOMORFOMETRIJSKA I
HORMONALNA STUDIJA ODGOVORA
KORTIKOTROPNIH ĆELIJA HIPOFIZE NA
POVREDU SENZOMOTORNOG KORTEKSA KOD
ODRASLIH PACOVA
BEHAVIORAL, IMMUNOHISTOMORPHOMETRIC
AND HORMONAL STUDY OF PITUITARY
CORTICOTROPES RESPONSE TO
SENSORIMOTOR CORTEX INJURY IN ADULT
RATS
S.Trifunović1, S. Peković2, I. Lavrnja2, V. Ajdžanović1, S.
Dacić3, N. Ristić1, M. Stojiljković2, V. Milošević1
1
Institut za biološka istraživanja"Siniša Stanković“;
Odeljenje za citologiju; Univerzitet u Beogradu; Beograd;
Srbija;
2
Institut za biološka istraživanja"Siniša Stanković“;
Odeljenje za neurobiologiju; Univerzitet u Beogradu;
Beograd; Srbija;
3
Biološki fakultet; Institut za fiziologiju i biohemiju;
Univerzitet u Beogradu; Beograd; Srbija
spekovic@ibiss.bg.ac.rs
S. Trifunovic1, S. Pekovic2, I. Lavrnja2, V. Ajdzanović1, S.
Dacic3, N. Ristic 1, M. Stojiljkovic2, V. Milosevic1
1
Department of Cytology; Institute for Biological Research
“Sinisa Stankovic”; University of Belgrade; Belgrade;
Serbia;
2
Department of Neurobiology; Institute for Biological
Research “Sinisa Stankovic”; University of Belgrade;
Belgrade; Serbia;
3
Department of Physiology and Biochemistry; Faculty of
Biology; University of Belgrade; Serbia
spekovic@ibiss.bg.ac.rs
Traumatska povreda mozga (TPM) predstavlja
ozbiljan dogañaj sa dugosežnim posledicama, koje
uključuju i poremećaj funkcije hipofize. Izgleda da TPM
utiče i na aktivnost pars distalis kortikotropnih (ACTH)
ćelija, koje predstavljaju aktivni modul hipotalamohipofizno-adrenokortikalne ose.
Imajući u vidu blisku povezanost poremećaja u
radu hipofize sa promenama u ponašanju, cilj ove studije
bio je da se ispita kako TPM utiče na oporavak motornih
funkcija, kao i na morfologiju i sekretornu aktivnost ACTH
ćelija u hipofizi adultnih pacova.
Mužjaci Wistar pacova kojima je urañena ablacija
senzomotornog korteksa (ASK) žrtvovani su 2, 7, 14 i 30
dana posle operacije (dpo). Oporavak motornih funkcija
praćen je pomoću testa prelaska preko grede. Hipofize i krv
su sakupljani za morfološke i hormonalne analize.
Tokom prve dve nedelje nakon povrede zapaža se
povećan oporavak lokomotornih funkcija, koji dostiže
kontrolne vrednosti 30-tog dpo. ASK dovodi do značajnog
povećanja težina hipofize u poreñenju sa odgovarajućim
kontrolama tog uzrasta. Volumen ACTH-imunopozitivnih
ćelija bio je smanjen u 7 dpo, dok je u 14 dpo njihov
volumen bio uvećan u odnosu na lažno operisane kontrole.
Povećanje volumenske gustine ACTH ćelija zapaža se samo
14-tog dpo, dok je 30-tog dpo to povećanje zanemarljivo.
Nivo ACTH u plazmi prolazno je povećan nakon povrede.
Najizraženije promene zapažaju se 7-og i 14-og dpo i
praćene su smanjenjem na nivo kontrolnih vrednosti u 30
dpo.
Na osnovu ovih rezultata može se zaključiti da su
vremenski-zavisne promene u hipotalamo-hipofiznoadrenalnoj osi, koje se zapažaju nakon traumatske povrede
mozga, u korelaciji sa procesom oporavka.
Traumatic brain injury (TBI) represents a serious
event with far reaching complications, including pituitary
dysfunction. Pars distalis corticotropes (ACTH cells), that
represents the active module of hipothalamo-pituitaryadrenocortical axis, seem to be affected as well.
Given the close association between TBI-induced
pituitary failure and neurobehavioral impairments, the aim
of this study was to evaluate the effects of TBI on recovery
of motor functions, and morphology and secretory activity
of ACTH cells in the pituitary of adult rats.
Wistar male rats, initially exposed to sensorimotor
cortex ablation (SCA), were sacrificed at the 2nd, 7th, 14th
and 30th day post-surgery (dps). Beam walking test was
used to evaluate the recovery of motor functions. Pituitary
glands and blood were collected for morphological and
hormonal analyses.
During the first two weeks post-injury increased
recovery of locomotor function was detected, reaching
almost control value at day 30. SCA induces significant
increase of pituitary weights compared to time-matched
controls. The volume of ACTH-immunopositive cells was
reduced at the 7th dps, while at the 14th dps their volume was
enlarged, related to corresponding sham controls. Volume
density of ACTH cells was increased only at 14th dps, while
at day 30 this increase was insignificant. Plasma level of
ACTH transiently increased after the injury. The most
pronounced changes were observed at the 7th and 14th dps,
and were followed by decrease toward control levels at the
30th dps.
Obtained data indicate that temporal changes in the
hypothalamic-pituitary-adrenal axis after traumatic brain
injury appear to correlate with the recovery process.
75
POSTER SESIJA / POSTER SESSION
P4 - Poremećaj homeostaze i bolesti centralnog nervnog sistema / P4 - Dysregulation of homeostasis and CNS disorders
EFEKAT DEKSAMETAZONA NA EKSPRESIJU
GENA UKLJUČENIH U METABOLIZAM
HOLESTEROLA U KORTEKSU I HIPOKAMPUSU
PACOVA TOKOM STARENJA I PRI
RESTRIKTIVNOM REŽIMU ISHRANE
THE EFFECTS OF AGING AND FOOD
RESTRICTION ON DEXAMETHASONE-INDUCED
CHANGES IN EXPRESSION OF GENES INVOLVED
IN REGULATION OF CHOLESTEROL
HOMEOSTASIS
V. Tešić1, M. Perović1, K. Smiljanić1, A. Mladenović
Đorñevic1, Lj. Rakić2, S. Ruždijić1, S. Kanazir1
1
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; 11060 Beograd; Srbija;
2
Srpska akademija nauka i umetnosti; 11000 Beograd;
Srbija
vesna.tesic@ibiss.bg.ac.rs
V. Tesic1, M. Perovic1, K. Smiljanic1, A. Mladenovic
Djordjevic1, Lj. Rakic2, S. Ruzdijic1, S. Kanazir1
1
Institute for Biological Research “Sinisa Stankovic”;
University of Belgrade; 11060 Belgrade; Serbia;
2
Serbian Academy of Sciences and Arts; 11000 Belgrade;
Serbia
vesna.tesic@ibiss.bg.ac.rs
Široka
upotreba
kortikosterteroida
kao
antiinflamatornih lekova uslovila je da ispitivanje brojnih
neželjenih efekta koji prate njihovu primenu postane veoma
značajno. Akutna primena kortikosteroida utiče i na
kognitivne funkcije, a mehanizmi u osnovi ovih promena
nisu dovoljno ispitani. Pravilan rad mozga zahteva očuvanje
homeostaze lipida, a glukokortikoidi izmeñu ostalog,
značajno menjaju lipidni metabolizam.
uključenih u metabolizam holesterola, 3-hidroksi3-metilglutaril-koenzime-A
reduktaze
(HMG-CoAR),
apolipoproteina E (ApoE) i holesterol 24-hidroksilaze
(CYP46A1), kao i da restrikcija hrane ( FR, od engl. Food
Restriction) ublažava ove starosno-zavisne promene. Cilj
ove studije je bio da se ispita da li restikcija hrane utice na
obrazac ekspresije ovih gena pod delovanjem sintetskog
glukokortikoida deksametazona tokom starenja.
Eksperiment je izveden na mužjacima pacova soja
Wistar. Životinje stare 6, 12 i 18 meseci, hranjene standarno
ili sa 50% od prosečnog dnevnog unosa hrane svaki drugi
dan (FR) su tretirane visokom dozom deksametazona (4
mg/kg, i.p.). Nivo ekspresije HMG-CoAR, ApoE i
CYP46A1 gena je utvrñen RQ-PCR-om.
Tretman deksametazonom doveo je do značajnog
smanjenja u ekspresiji ApoE gena kod starih životinja u
odnosu na životinje stare 6 meseci. Poboljšanja nije bilo ni
kod životinja koje su bile na restrikciji hrane, mada je kod
njih uočena povećana ekspresija HMG-CoAR i CYP46A1
gena.. Promene su bile izraženije u hipokampusu pacova.
FR
ublažava
deksametazonom-indukovane
promene u ekspresiji gena uključenih u metabolizam
holesterola tokom starenja na region-specifičan način kod
pacova.
Glucocorticoids are widely used anti-inflammatory
agents. Limited in vivo data are available to characterize the
mechanism underlying their cognitive side effects and
transient occurrence of steroid psychosis. Glucocorticoids
are known for their ability to alter lipid metabolism and this
may be of particular importance for the proper brain
functioning.
Our previous work has revealed that the agerelated increase in expression of genes involved in
cholesterol
metabolism
3-hydroxy-3-methylglutarylcoenzyme-A reductase (HMG-CoAR), apolipoprotein E
(ApoE) and cholesterol 24-hydroxylase (CYP46A1) was
counteracted by the long-term food restriction (FR), the
only intervention known to delay the aging process and
improve cognitive performance. Thus, we assessed the
influence of synthetic glucocorticoid dexamethasone (DEX)
on above mentioned cholesterol metabolism genes, during
aging and under long term FR.
The experiment was performed on male Wistar
rats. Animals 6, 12 and 18-month-old, fed ad libitum or
with 50% of the mean daily intake every other day (FR),
were treated with high dose of DEX (4 mg/kg, i.p.). The
expression of HMG-CoAR, ApoE and CYP46A1 mRNAs
was determined by RQ-PCR.
The most prominent change observed following
DEX treatment was the age-related ApoE mRNA decrease
that was not reversed by FR. In FR treated animals this
decrease was compensated by the increase in the expression
of HMG-CoAR and CYP46A1. Changes were more
prominent in the rat hippocampus, than in the cortex.
Food restriction improves the dexamethasoneinduced changes of cholesterol metabolism genes in aging
rats in a region-specific manner.
76
POSTER SESIJA / POSTER SESSION
P4 - Poremećaj homeostaze i bolesti centralnog nervnog sistema / P4 - Dysregulation of homeostasis and CNS disorders
EFEKAT SOKA ARONIAE MELANOCARPAE NA
NEUROHEMIJSKE PROMENE IZAZVANE
RAZLIČITIM TIPOVIMA STRESA
EFFECT OF ARONIA MELANOCARPA FRUIT
JUICE ON NEUROCHEMICAL CHANGES
INDUCED BY DIFFERENT STRESS MODELS
J. Tošić1, Ž. Stanojević1, M. Momčilović2, Đ. Miljković2,
M. Popović1, A. Isaković1, L. Radenović 3, D. Dekanski4
1
Institut za medicinsku i kliničku biohemiju; Medicinski
fakultet Univerziteta u Beogradu; Beograd; Srbija;
2
Institut za biološka istraživanja “Siniša Stanković”;
Univerzitet u Beogradu; Beograd; Srbija;
3
Institut za fiziologiju i biohemiju; Biološki fakultet
Univerziteta u Beogradu, Beograd, Srbija
4
Centar za biomedicinska istrazivanja; R&D Institut,
Galenika a.d.; Beograd; Srbija
toshka88@gmail.com
J. Tošić1, . Stanojević1, M. Momčilović2, Đ. Miljković2, M.
Popović1, A. Isaković1, L. Radenović3, D. Dekanski4
1
Institute of medical and clinical biochemistry; School of
Medicine; University of Belgrade; Belgrade; Serbia;
2
Department of Immunology; Institute for Biological
Research “Siniša Stanković”; University of Belgrade;
Belgrade; Serbia;
3
Institute for Physiology and Biochemistry, Faculty of
Biology; University Belgrade; Belgrade; Serbia
4
Biomedical Research; R&D Institute; Galenika a.d.;
Belgrade; Serbia
toshka88@gmail.com
Uvod: Sok Aroniae melanocarpae je bogat
polifenolima, koji imaju antioksidativni potencijal. Poznato
je da stres indukuje nastanak visokih nivoa reaktivnih oblika
kiseonika i proinflamatornih citokina.
Cilj: Ispitati potencijalno antioksidativno i
antiinflamatorno dejstvo soka A. melanocarpae u
hipokampusima pacova
na modelima
hroničnog
varijabilnog stresa (CVS) i imobilizacionog stresa ubrzanog
hladnoćom (ISUH).
Materijal i metode: U eksperimentima su korišćeni
pacovi soja Wistar, mužjaci (220±20g). Aktivnost ukupne
superoksid dismutaze (SOD), katalaze (CAT) i indeks
lipidne peroksidacije (ILP) su ispitivani metodama
spektrofotometrije, na modelu ISUH-a. Životinje su
podeljene u pet eksperimentalnih grupa od kojih su dve bile
negativna i pozitivna (5mg/kg kvercetin) kontrola a
preostalim grupama je aplikovan sok aronije u različitim
dozama (5ml/kg, 10ml/kg, jednokratno i 5ml/kg tokom
sedam dana pre izvoñenja stresa). Antiinflamatorni efekat
soka aronije je ispitivan na modelu CVS-a u trajanju od 40
dana. Pacovi su podeljeni u tri grupe. Kontrolna grupa nije
izlagana stresu, niti je konzumirala sok. Preostale dve su
izlagane stresu, pri čemu je jedna dobijala sok ad libitum
(5ml/kg) a druga nije. Ispitivanje nivoa proinflamatornih
citokina (IL-1beta, IL-6 i TNF-alfa) i aktivnosti acetilholinesteraze (AchE) je vršeno ELISA testom.
Rezultati: Jednokratni i sedmodnevni pretretman
sokom aronije je značajno snizio ILP i aktivnost SOD, dok
nije uticao na promenu aktivnosti CAT. CVS je doveo do
povećanje nivoa AchE i TNF-alfa a konzumiranje soka je
snizilo nivo istih. S druge strane, ni stres, kao ni
konzumacija soka nisu doveli do značajnih promena u nivou
IL-1beta i IL-6.
Zaključak: Dobijeni rezultati ukazuju na
antioksidativno dejstvo kao i imunomodulatorni potencijal
A. melanocarpae.
Introduction: Aronia melanocarpa fruit juice is a
rich source of polifenolic components, known by its
antioxidant potential. It is known that stress induces
generation of a high level of reactive oxigen species and
proinflammatory cytokines.
The aim of our study was to investigate antioxidant
and antiinflamatory potential of A. melanocarpa fruit juice
in rat hippocampuses on chronic variable stress (CVS) and
cold restraint stress (CRS) models.
Material and methods: Male Wistar rats (220±20g)
were used. In the CRS model, superoxide dismutase (SOD)
and catalase (CAT) activity and the lipid peroxidation index
(ILP) were determined spectrophotometrically. Two
experimental groups were negative and positive (5mg/kg
quercetin) control and three other were pretreated with juice
in different dosage (5ml/kg, 10ml/kg, as a single dose and
5ml/kg during seven days before CRS). Antiinflammatory
effect of Aronia juice was examined on CVS for fourty
days. Control group wasn’t stressed, nor pretreated with
juice. Other two groups were stressed and one of them had
long pretreatment with juice (5ml/kg). Level of
proinflammatory cytokines (IL-1beta, IL-6 and TNF-alpha)
and acetylcholine esterase activity (AchE) were measured
using ELISA test.
Results: Single dose and seven days pretreatment
with juice significantly reduced ILP and SOD activity,
while the activity of CAT wasn’t significantly changed, in
comparison to control. CVS increased level of TNF-alpha
and AchE activity in stressed group and consummation of
juice attenuated it. On the other hand, stress, as well as juice
cpnsumation didn’t have influence on IL-1beta and IL-6
levels.
Conclusion: A. melanocarpa showed antioxidative
effect as well as immunomodulatory potential on these two
models of stress.
77
POSTER SESIJA / POSTER SESSION
P5 - Neurofiziologija i ponašanje / P5 - Neurophysiology and behaviour
HIPERMETIONINSKA DIJETA POVEĆAVA
OSETLJIVOST PACOVA NA EPILEPTIČNU
AKTIVNOST: BIHEJVIORALNA I EEG STUDIJA
HYPERMETHIONINE DIET INCREASES
SUSCEPTIBILITY OF RATS TO EPILEPSY:
BEHAVIORAL AND EEG STUDY
D. Hrnčić 1, A. Rašić - Marković 1, Đ. Macut 2, V. Šušić 3,
D. Đurić 1, O. Stanojlović1
1
Laboratorija za neurofiziologiju; Institut za medicinsku
fiziologiju ”Rihard Burijan”; Medicinski fakultet;
Univerzitet u Beogradu; Beograd; Srbija;
2
Institut za endokrinologiju, dijabetes i bolesti metabolizma;
KCS; Medicinski fakultet u Beogradu; Univerzitet u
Beogradu; Beograd; Srbija;
3
SANU
drhrncic@yahoo.com
D. Hrnčić 1, A. Rašić - Marković 1, Đ. Macut 2, V. Šušić 3,
D. Djurić 1, O. Stanojlović1
1
Laboratory of Neurophysiology; Institute of Medical
Physiology ”Richard Burian”; Faculty of Medicine;
University of Belgrade; Belgrade; Serbia;
2
Institute of Endocrinology; Diabetes and Metabolic
Disease; CCS, Faculty of Medicine; University of Belgrade;
Belgrade; Serbia;
3
Serbian Academy of Sciences and Arts; Belgrade; Serbia
drhrncic@yahoo.com
Metabolizam metionina, esencijalne aminokiseline
koja sadrži sumpor, je u neposrednoj vezi sa metabolizmom
homocisteina.
Uloga
homocisteina
u
regulaciji
hiperekscitabilnosti CNS nije u potpunosti razjašnjenja. Cilj
ovog rada je bio da se ispitaju efekti ishrane obogaćene
metioninom na osetljivost pacova na epileptičnu aktivnost
izazvanu homocistein tiolaktonom koristeći bihejvioralni i
elekroencefalografski (EEG) epileptološki pristup.
Mužjaci Wistar pacova su nasumično rasporeñeni
u kontrolnu i eksperimentalnu grupu i dobijali su od 30 do
60. postnatalnog dana standardu hranu, odnosno
hipermetioninsku dijetu (sadržaj metionina duplo veći nego
u standardnoj hrani). Epileptična aktivnost izazvana je
pojedinačnom subkonvulzivnom dozom D,L homocistein
tiolaktona (HcT, 5,5 mmol/kg). Praćeni su sledeći parametri
konvulzivnog ponašanja tokom 90 min od administracije:
incidenca napada, latentno vreme do prve konvulzivne
epizode, broj konvulzivnih epizoda i nijhov intenzitet.
Uporedo je registrovana EEG aktivnost i kvantifikovana
pojava šiljak-talas pražnjenja (SWD).
Latenca do prve konvulzivne epizode je bila
značajno kraća, a broj konvulzivnih epizoda povećan u
eksperimentalnoj grupi životinja koje su bile na režimu
hipermetioninske ishrane u odnosu na kontrolnu grupu
životinja. Incidenca napada je bila statistički značajno veća
u eksperimentalnoj nego u kontrolnoj grupi životinja nakon
administracije HcT, pri čemu nije bilo značajne razlike u
pogledu intenziteta napada. Broj SWD po pacovu, kao i
njihovo trajanje, po administraciji HcT, bili su značajno
veči u eksperimentalnoj u poreñenju sa kontrolnom grupom.
Ovi rezultati ukazuju da hipermetioninska dijeta
podiže nivo ekscitabilnosti CNS-a čineći ove pacove
podložnijim razvoju konvulzivne aktinvosti, što je bilo
evidentno na osnovu bihejvioralnih i EEG efekata.
Metabolism of methionine, sulfur-containing
essential amino acid, is closely related with those of
homocysteine. Homocysteine involvement in regulation of
hyperexcitability of the CNS is not completely understood.
The aim of this study was to examine the effects of
methionine nutritional overload on rat susceptibility to
homocysteine-thiolactone –induced epileptic activity in rats
using behavioral and electroencephalographic (EEG)
approach.
Male Wistar rats were randomly divided into
control and experimental group, fed from 30th to 60th
postnatal day with standard or hypermethionine diet (two
folded comparing to standard, 7.7 g/kg), respectively. A
single subconvulsive dose of D,L homocysteine –
thiolactone (HcT, 5.5 mmol/kg, i.p) was used to challenge
epileptic activity in these rats. Convulsive behavior was
assessed by seizure incidence, latency time to first seizure,
number and intensity of seizure episodes during 90 min post
injection. EEG activity was concomitantly recorded and
appearance of spike-and-wave discharges (SWD) was
quantified.
Seizure latency was significantly decreased, and
number of seizure episodes per rat significantly increased in
experimental group fed with hypermethionine diet
comparing to control group. Rats from experimental groups
developed seizures upon single subconvulsive dose of HcT
with significantly higher incidence comparing to those from
control group, while no significant difference in intensity of
seizures was observed. Number of SWD per rat, as well as
its duration, was significantly higher in experimental
comparing to control group upon HcT administration.
These results indicate that hypermethionine diet
increases excitability of the CNS making these rats more
susceptibility to seizure development, as evident on
behavioral and EEG effects.
78
POSTER SESIJA / POSTER SESSION
P5 - Neurofiziologija i ponašanje / P5 - Neurophysiology and behaviour
HIPERPOLARIZACIJA MEMBRANE
MITOHONDRIJA MIŠIJIH ASTROCITA U
KULTURI NAKON NORMOKSIJE / HIPOKSIJE U
USLOVIMA SMANJENE GLUKOZE
MITOCHONDRIAL MEMBRANE
HYPERPOLARIZATION FOLLOWING NORMOXIA
/ HYPOXIA IN GLUCOSE DEPRIVED MOUSE
ASTROCYTES IN CULTURE
A. Korenić1, M. Jaklin2, M. Peters2, J. Boltze2, P.R. Anñus1,
L. Radenović1
1
Centar za lasersku mikroskopiju; Institut za fiziologiju i
biohemiju; Biološki fakultet; Univerzitet u Beogradu;
Srbija;
2
Fraunhofer Institut za ćelijsku terapiju i imunologiju;
Lajpcig; Nemačka
agapije@gmail.com
A. Korenić1, M. Jaklin2, M. Peters2, J. Boltze2, P.R.
Andjus1, L. Radenović1
1
Centre for Laser Microscopy; Department of Physiology
and Biochemistry; Faculty of Biology; University of
Belgrade; Serbia;
2
Fraunhofer Institute for Cell Therapy and Immunology;
Leipzig; Germany
agapije@gmail.com
Poznato je da astrociti tolerišu duže periode
nedostatka kiseonika i glukoze (OGD) u poreñenju sa
neuronima koji su osetljiviji. Promene potencijala
membrane mitohondrija (pmm), kao pokazatelja redoks
stanja astrocita, posle duže izloženosti OGD-u još nisu
istražene. Primarne kulture mišijih astrocita izlagane su
hipoksiji različitog trajanja, odsustvu glukoze (GD) ili
OGD, kao i različitim kombinacijama navedenih tretmana.
Promene pmm su praćene u roku od jednog sata nakon
reperfuzije obeležavanjem sa JC-1, fluorescentnim
markerom koji prelazeći u unutrašnjost mitohondrija (u
zavisnosti od potencijala membrane), menja maksimu
fluorescencije od zelene ka crvenom delu spektra. Naši
rezultati ukazuju da je, za razliku od odsustva efekta
hipoksije, GD doprinela hiperpolarizaciji membrane
mitohondrija astrocita – negativni pmm. Kada su ta dva
tretmana kombinovana, hiperpolarizacioni nivo tokom
reperfuzije odgovarao je snižavanju glukoze u medijumu
nekoliko sati pre hipoksije. Iako efekat nije bio zabeležen
posle hipoksije u medijumu sa niskom glukozom, nakon
hemijske hipoksije izazvane natrijum azidom došlo je do
hiperpolarizacije tokom reperfuzije. Takoñe, kada je
produžena GD prethodila hipoksiji, ta dva tretmana su
pokazala značajnu interakciju u vidu povećane
hiperpolarizacije. Ovi rezultati povezuju dostupnost dva
glavna supstrata transportnog lanca elektrona (glukoze i
kiseonika) i njihov hiperpolarišući uticaj na pmm,
rasvetljavajući mehanizme otpornosti astrocita na duže
ishemijske epizode.
Astrocytes are known to tolerate long periods of oxygenglucose deprivation (OGD), however this time frame has
not been taken into account when compared to neuronal
injury. In fact, changes in mitochondrial membrane
potential (mmp), as the indicator of the cellular redox state,
after longer exposures to OGD have not been investigated
yet. Therefore, we have exposed primary mouse astrocyte
cultures to the effect of varying relatively longer durations
of hypoxia (oxygen deprivation), glucose deprivation (GD)
or OGD, and to various combinations of these treatments.
Changes of mmp were investigated within one hour of
reperfusion by applying JC-1, a fluorescent probe that
enters the mitochondrial matrix in a potential-dependent
manner, thus shifting its emission from green to red. Our
results indicated that, in contrast to ineffectiveness of
hypoxia, GD contributed to a more negative mmp. When
the two treatments were combined, the hyperpolarization
level during the reperfusion phase was related to lowering
of glucose in the culture medium several hours prior to
hypoxic treatment. When the effect was not initially
observed after hypoxia in low glucose, subsequent chemical
hypoxia induced by sodium-azide still caused
hyperpolarization during the reperfusion phase. However,
when GD preceded hypoxia, those two treatments showed
significant
interaction
towards
an
increased
hyperpolarization. These findings connect the availability of
the two main substrates of electron transport chain (glucose
and oxygen) and their hyperpolarizing effect on mmp, thus
shedding light on astrocyte resistance to prolonged ischemic
injury.
79
POSTER SESIJA / POSTER SESSION
P5 - Neurofiziologija i ponašanje / P5 - Neurophysiology and behaviour
RAZLIČITI NIVOI KETAMINSKE ANESTEZIJE
NAKON LEZIJE PEDUNKULOPONTINSKOG
TEGMENTALNOG JEDRA PACOVA
K. Lazić1, J. Petrović1, A. Kalauzi2, J. Šaponjić1
Univerzitet u Beogradu; Odeljenje za neurobiologiju;
Institut za biološka istraživanja “Siniša Stanković”; 11 060
Beograd; Srbija;
2
Univerzitet u Beogradu; Odeljenje prirodnih nauka;
Institut za multidisciplinarna istraživanja; 11 030 Beograd;
Srbija.
katarinalazic@ibiss.bg.ac.rs
1
Pratili smo uticaj deficit holinergičke inervacije iz
pedunkulopontinskog tegmentalnog jedra (PPT) na prelaze
iz budnog u “arteficijalno” spavanje i obrnuto, da bi
razotkrili
moguće
karakteristične
EEG
razlike
stepenovanog, farmakološki izazvanog nesvesnog stanja
(“arteficijalnog spavanja”) i razbuñivanja do svesnog stanja.
Odrasli Wista rkontrolni (n = 5) i PPT ledirani
pacovi (n = 4) su implantirani za registrovanje spavanja.
Bilateralne PPT lezije
su izvršene mikroinfuzijom
ibotenične kiseline. Nakon oporavka od 14 dana,
indukovano
je
farmakološko
spavanje
sa
ketamin/diazepam–om 100 mg/kg, i.p. EEG je registrovan
za vreme anestezije 60 min, počinjući 5 min nakon
administracije anestetika. EEG promene su analizirane u tri
20 min perioda: uvod u anesteziju, operativni nivo
anestezije, i razbuñivanje. Korišćenjem MATLAB 6.5,
izračunavane su grupne gustine verovatnoće distrubucija
(PDEs) relativnih amplitude svih konvencionalnih EEG
frekventnih opsega, a njihove srednje vrednosti na svakih
10 min za dalju primenu Mann-Whitney U dvosmernog
testa.
Mi smo pokazali da se uvod u ketaminsku
anesteziju ne razlikuje izmeñu kontrolnihi PPT lediranih
pacova, ali na operativnom nivou anestezije PPT ledirani
pacovi eksprimiraju veću delta i teta, a manju sigma i beta
amplitudu u odnosu na kontrolne pacove. U toku
razbuñivanja iz anestezije u PPT lediranih pacova je i dalje
pojačana delta, a smanjena beta amplituda u odnosu na
kontrolne pacove.
U pacovskom modelu holinergičke neuropatologije
Parkinsonove bolesti značajno su različiti operativni nivo
ketaminske anestezije i razbuñivanje iz anestezije nasuprot
kontrolnim pacovima.
DIFFERENT LEVELS OF KETAMINE
ANESTHESIAFOLLOWING THE LESION OF
PEDUNCULOPONTINE TEGMENTAL NUCLEUSIN
RAT
K. Lazić1, J. Petrović1, A. Kalauzi2, J. Šaponjić1
1
University of Belgrade; Department of Neurobiology;
Institute for Biological Research “Sinisa Stankovic”; 11 060
Belgrade; Serbia;
2
University of Belgrade; Department for Life Sciences;
Institute for Multidisciplinary Research; 11 030 Belgrade;
Serbia.
katarinalazic@ibiss.bg.ac.rs
We followed the impact of pedunculopontine
tegmental nucleus (PPT)cholinergic innervation deficit on
the transitions from wake to “artificial” sleep and vice
versa, to find out the possible differences of the
characteristic EEG markers for gradual pharmacological
induction of unconsciousness (“artificial sleep”), and
withdrawal to wake, conscious state.
Adult Wistar control (n = 5) and PPT lesioned (n =
4) rats were implanted for sleep recording.Bilateral PPT
lesions were performed by ibotenic acid microinfusion.
After recovery of 14 days, we induced the pharmacological
sleep by ketamine/diazepam 100 mg/kg, i.p. EEG was
recorded during 60 min of the anesthetized state,beginning
5 min after the anesthesia administration.We analyzed the
EEG changes during the 20 min periods of: anesthesia
introduction, operative level of anesthesia, and withdrawal
to wake state.We calculated the group probability density
distributions (PDEs)of the all conventional EEG frequency
bands relative amplitudes using MATLAB 6.5,and the
relative amplitude means per each 10 min for the MannWhitney U two-tailed tests.
We have shown that the anesthesia introduction
was not different between the controls and PPT lesioned
rats, but the PPT lesioned rats at operative level of
anesthesia expressed higher delta and theta, and lower
sigma and beta amplitudes. During withdrawal to wake state
the PPT lesioned rats were still with augmented delta and
attenuated beta amplitudes versus controls.
In the rat model of the cholinergic Parkinson`s
disease neuropathology the operative level of anesthesia and
withdrawal to wake state are significantly differently
expressed versus controls.
80
POSTER SESIJA / POSTER SESSION
P5 - Neurofiziologija i ponašanje / P5 - Neurophysiology and behaviour
EEG MIKROSTRUKTURA BUDNOSTI ISPAVANJA
RAZLIKUJE FUNKCIONALNO RAZLIČITE
HOLINERGIČKE DENERVACIJE U PACOVA
SLEEP/WAKE STATE RELATED EEG
MICROSTRUCTURE DIFFERENTIATES THE
FUNCTIONALLY DISTINCT CHOLINERGIC
DENERVATIONS IN RAT
J. Petrović1, K. Lazić1, A. Kalauzi2, J. Šaponjić1
Univerzitet u Beogradu; Odeljenje za neurobiologiju;
Institut za biološka istraživanja “Siniša Stanković; 11060
Beograd; Srbija;
2
Univerzitet u Beogradu; Odeljenje prirodnih nauka; Institut
za multidisciplinarna istraživanja; 11030 Beograd; Srbija.
jelenapetrovic@ibiss.bg.ac.rs
J. Petrović1, K. Lazić1, A. Kalauzi2, J. Šaponjić1
1
University of Belgrade; Department of Neurobiology;
Institute for Biological Research “Sinisa Stankovic”; 11 060
Belgrade; Serbia;
2
University of Belgrade; Department for Life Sciences;
Institute for Multidisciplinary Research; 11 030 Belgrade;
Serbia.
jelenapetrovic@ibiss.bg.ac.rs
1
U
cilju
identifikacije
EEG
markera
poremećajainervacije
dva
funkcionalno
različita
holinergička sistema, praćen je efekat bilateralne lezije
nucleus
basalis-a
(NB)
i
pedunkulopontinskog
tegmentalnog jedra (PPT) na arhitekturu spavanja i EEG
mikrostrukturu budnosti i spavanja. Tokom operativne
procedure hronične implantacije elektroda za registrovanje
spavanja, bilateralna NB ili PPT lezija je izvršena
mikroinfuzijom 0.1 M ibotenične kiseline i kasnije
identifikovana NADPH-diaforaza histohemijskim bojenjem.
Korišćenjem originalnog softvera razvijenog u MATLAB
6.5 svaka 10s epoha u 6 sati registrovanih signala je
diferencirana kao budnost, NeREM ili REM faza spavanja.
Za analizu EEG mikrostrukture budnosti i spavanja koristili
smo PDE rutinu u MATLAB 6.5.
Bilateralne NB i PPT lezije nisu promenile
arhitekturu spavanja, ali su promenile budnost/spavanje
EEG mikrostrukture. Dve nedelje nakon bilateralne PPT
lezije došlo je do smanjenja delta, a porasta beta i gama
amplitude budnosti u trajanju od 28 dana nakon lezije.
Bilateralna NB lezija je tek 28 dana nakon lezije
ekspirimirana istim poremećajem EEG mikrostrukture
budnosti kao i PPT lezija. NeREM beta amplituda je bila
povećana četiri nedelje samo nakon bilateralne PPT lezije.
Bilateralna NB lezija prouzrokovala je tronedeljni porast
REM teta amplitude, a bilateralna PPT lezija
četvoronedeljni porast beta i gama amplitude.
Naši rezultati pokazuju da se na osnovu EEG
mikrostrukture budnosti, NeREM i REM faze spavanja
mogu razlikovati dva funkcionalno različita poremećaja
holinergičke inervacije (bilateralna NB nasuprot bilateralne
PPT lezije), posebno tokom NeREM i REM faze spavanja.
To identify the EEG markers for the functionally
distinct cholinergic denervations in rat,we investigated the
effect of the bilateral nucleus basalis (NB) and
pedunculopontine tegmental nucleus (PPT) lesions on
sleep/wake states and sleep/wake state-related EEG
microstructure.During the operative procedure of chronic
electrodes implantation for sleep recording,the bilateral NB
or PPT lesions were performed by 0.1 M ibotenic acid
microinfusion, and later verified by NADPH-diaphorase
histochemistry. We applied Fourier analysis to signals
acquired throughout 6 h recording, and each 10 s epoch was
differentiated as Wake, NREM or REM state. To analyse
sleep/wake state-related EEG microstructurewe used PDE
routine supplied with MATLAB 6.5.
The bilateral NB and PPT lesions did not change
the sleep/wake architecture, but did change EEG
microstructure. Two weeks after the lesions Wake delta
amplitude decreased, while beta and gamma amplitude
increased only after bilateral PPT lesion. This delta and beta
Wake EEG microstructure disturbance maintained 28 days,
when the bilateral NB lesion was expressed equally as the
bilateral PPT lesion by delta amplitude decrease, and beta
amplitude increase.During NREM sleep there was
sustainable beta amplitude augmentation for four weeks
only after the bilateral PPT lesion. During REM the bilateral
NB lesion augmented theta amplitude for three weeks, and
returned to control values, while the bilateral PPT lesion
augmented beta and gamma amplitudefor overall four
weeks.
We have proved that the sleep/wake state-related
EEG microstructure differentiates the distinct cholinergic
denervations (bilateral NB versus bilateral PPT lesion),
particularly during NREM and REM sleep.
81
POSTER SESIJA / POSTER SESSION
P5 - Neurofiziologija i ponašanje / P5 - Neurophysiology and behaviour
ISPITIVANJE PONAŠANJA U TESTU OTVORENOG
POLJA MIŠEVA KOJI SU BILI TRETIRANI
PRENATALNO RAZLIČITIM DOZAMA
VALPROIČNE KISELINE
NEUROBEHAVIORAL PERFORMANCE IN OPEN
FIELD TEST IN MICE WHICH WERE EXPOSED
PRENATALLY TO DIFFERENT DOSES OF
VALPROIC ACID
J. Podgorac1, S. Sekulić2, Lj. Martać1, I. Čapo2, G.
Keković1, M. Dokić3
1
Institut za biološka istraživanja „Siniša Stanković;
Univerzitet u Beogradu; Srbija;
2
Medicinski fakultet; Univerzitet u Novom Sadu; Srbija;
3
Medicinski fakultet; Univerzitet u Beogradu; Srbija
jelenapodgorac@yahoo.com
J. Podgorac1, S. Sekulić2, Lj. Martać1, I. Čapo2, G.
Keković1, M. Dokić3
1
Institute for Biological Research „Siniša Stanković;
University of Belgrade; Serbia;
2
Medical Faculty; University of Novi Sad; Serbia;
3
Medical Faculty; University of Belgrade; Serbia
jelenapodgorac@yahoo.com
Uvod: Valproična kiselina (VK) predstavlja
antiepileptik širokog spektra dejastva, koji se koristi se za
lečenje različitih tipova epilepsija. Prvi lek izbora je kod
epilepsija tipa absansa, miokloničnih napada i LenoksGastoovog sindroma. Većina stručnjaka smatra da je lek
izbora u terapiji tonično-kloničnih epilepsija. Upotreba VK
tokom trudnoće u vezi je sa različitim kongenitalnim
malformacija kod potomaka. Pored defekta neuralne tube,
kod potomaka, registrovani su i kranio-facijalni
dizmorfizam, srčane, urogenitalne i malformacije koštanog
sistema.
Cilj: Cilj istraživanja bio je da se utvrdi, na
animalnom modelu, poremećaj u ponašanju i motorici
tokom razvoja mladih jedinki, bez telesnih malformacija,
čije majke su bile tretirane tokom cele gestacije
valproičnom kiselinom. Test otvorenog polja je korišćen za
procenu lokomotorne i eksplorativne aktivnosti i za
evaluaciju emocionalnog ponašanja.
Materijal i metod: Istraživanje se sastojalo iz tri
grupe, dve eksperimentalne i jedne kontrolne grupe. Odrasle
ženke miša, NMRI soja, tretirane su subkutano, injekcijom
valproične kiseline u dozi od 200mg/kg i 400mg/kg tokom
priploda i gestacije. Kontrolna grupa bila je tretirana
fiziološkim rastvorom, tokom istog perioda. U testu
otvorenog polja 40og dana, ispitivana je lokomotorna,
eksplorativna i emocionalna aktivnost. Analizirani su
sledeći parametri: preñena horizontalna distanca, vertikalno
uzdizanje, prostorna naklonost i timarenje.
Rezultati: Neurobihejvioralni parametri analizirani
su Kruskal-Wallis testom. Izmeñu eksperimentalnih grupa i
kontrolne grupe nañena je statistička značajnost.
Zaključak: Parametri korišćeni za procenu
lokomotorne, eksplorativne i emocionalne aktivnosti ukazali
su na slabiji kvalitet postnatalnog razvoja jedinki tretiranih
grupa od jedinki u kontrolnoj grupi.
Introduction: Valproic acid (VPA) as broadspectrum drug is very efficient antiepileptic for different
type of epilepsy and represent the drug of first choice in the
treatment of myoclonic and absence seizures and LennoxGastaut syndrome. Most experts regard valproic acid as first
line therapy for tonic-clonic epilepsy. When is used during
pregnancy it is associated with congenital malformations
(craniofacial dysmorphism, neural tube defect, urogenital,
hart and skeletal anomalies).
Aim: Our aim was to determine, in animal model,
neurobehavioral disorder of young, without body
malformations, which mothers were treated with valproic
acid during whole gestation. Open field test was used to
assess emotional behavior, general locomotor and
exploratory activity in mice.
Material and Method: Study was consisted of three
groups, two experimental and control one. Adult female
NMRI mice were treated with subcutaneous injection of
VPA in doses of 200 mg/kg and 400 mg/kg and control
group with saline, during breeding and whole gestation. We
investigated locomotor, exploratory and emotional activity
in open field test at 40th day of postnatal life, using
parameters such as horizontal distance, rearing, spatial
preferences and grooming activity .
Results: Neurobehavioral parameters were
analyzed using the Kruskal-Wallis-test. Between
experimental groups and control group was found
significant differences.
Conclusion: Test for neurobehavioral assessment
of offspring was conducted at 40th postnatal day, which
correspond
to
adolescent
period
of
humans.
Neurobehavioral parameters show less quality of postnatal
development in treated groups due to control.
82
POSTER SESIJA / POSTER SESSION
P5 - Neurofiziologija i ponašanje / P5 - Neurophysiology and behaviour
UTICAJ OBOGAĆENE SREDINE NA MODULACIJU
PERINEURONALNIH MREŽA I SINAPTIČKU
REORGANIZACIJU KOD MIŠEVA SA
NEDOSTATKOM TENASCINA-C
THE EFFECT OF ENRICHED ENVIRONMENT ON
THE MODULATION OF PERINEURONAL NETS
AND SYNAPTIC REMODELING OF TENASCIN-C
DEFICIENT MICE
V. Stamenković1, S. Stamenković1, T. Jaworski 2, M.
Gawlak3, M. Jovanović1, G.M. Wilczynski3, L. Kaczmarek2,
M. Schachner3, L. Radenović1, P.R. Anñus1
1
Centar za lasersku mikroskopiju; Institut za fiziologiju i
biohemiju; Biološki fakultet; Univerzitet u Beogradu;
Srbija;
2
Nencki Institute of Experimental Biology; Laboratory of
Neurobiology; Polish Academy of Science; Warsaw,
Poland;
3
Nencki Institute of Experimental Biology; Laboratory of
Neuromorphology; Polish Academy of Science; Warsaw;
Poland;
4
Zentrum für Molekulare Neurobiologie; Universitaet
Hamburg; Hamburg; Germany
sekeljicv@ikomline.net
V. Stamenkovic1, S. Stamenkovic1, T. Jaworski 2, M.
Gawlak3, M. Jovanovic1, G.M. Wilczynski3, L. Kaczmarek2,
M. Schachner3, L. Radenovic1, P.R. Andjus1
1
Center for Laser Microscopy; Department of Physiology
and Biochemistry; Faculty of Biology; University of
Belgrade; Serbia;
2
Nencki Institute of Experimental Biology; Laboratory of
Neurobiology; Polish Academy of Science; Warsaw;
Poland;
3
Nencki Institute of Experimental Biology; Laboratory of
Neuromorphology; Polish Academy of Science; Warsaw,
Poland;
4
Zentrum für Molekulare Neurobiologie; Universitaet
Hamburg; Hamburg; Germany;
sekeljicv@ikomline.net
Tenascin-C
(TnC)
je
glikoprotein
ekstraćelijskog matriksa sa važnom morforegulatornom
ulogom tokom razvića. U adultnom centralnom nervnom
sistemu ekspresija TnC ograničena je samo na oblasti koje
se odlikuju plastičnošću. S obzirom da gajenje životinja u
obogaćenoj sredini (OS) stimuliše neuronalnu i sinaptičku
plastičnost, cilj naše studije bio je da ispitamo: 1) uticaj OS
na prisustvo perineuronalnih mreža (PNM) i gustinu
inhibitornih i ekscitatornih sinapsi u dentatnom jedru malog
mozga miševa sa genskim nedostatkom TnC (TnC-/-) i
divljeg soja (TnC+/+) nakon 8 nedelja gajenja u OS i
standardnoj sredini počevši od 21. postnatalnog dana, i 2)
ulogu matriksnih metaloproteinaza (MMP) 2 i 9 u ovim
procesima, praćenjem njihove aktivnosti u različitim
vremenima (4 i 8 nedelja) pomoću tehnika gel i in situ
zimografije.
Ova studija je pokazala da dolazi do značajne
razgradnje PNM kod TnC+/+ miševa 8 nedelja nakon
gajenja u OS, dok kod TnC-/- miševa takav efekat izostaje.
Tehnike zimografije pokazale su povećanu aktivnost MMP9 nakon 4 nedelje gajenja u OS u oba genotipa, kao i da
nakon 8 nedelja dolazi do njenog smanjenja kod TnC+/+,
dok kod TnC-/- miševa ona ostaje generalno slaba bez
obzira na uslove gajenja. Ovim je ukazano na značajnu
ulogu MMP-9 (nasuprot MMP-2) u reorganizaciji PNM.
Takoñe, ova studija ukazuje i na važnu ulogu TnC u
sinaptičkoj plastičnosti pošto su imunohistohemijska
ispitivanja sinaptičke gustine pokazala povećan broj
inhibitornih sinapsi u TnC-/- u odnosu na TnC+/+ miševe
bez obzira na uslove gajenja, dok je gustina ekscitatornih
sinapsi povećana kod TnC+/+ nakon OS bez promena kod
TnC-/- miševa.
Tenascin-C (TnC) is an extracellular matrix
glycoprotein with an important morphoregulatory role
during development, while in adult CNS its expression is
limited to areas of neuronal plasticity. Since enriched
environment (EE) stimulates neuronal plasticity, we
examined the role of TnC by observing the distribution of
perineuronal nets (PNNs) in TnC deficient (TnC-/-) and
wild-type (TnC+/+) mice in deep cerebellar nuclei (DCN)
after rearing for 8 weeks in EE (vs. standard conditions)
starting from postnatal day 21. Furthermore, possible
involvement of matrix metalloproteinases, MMP-2 and
MMP-9, in PNN reorganization was examined by following
their activity at two distinct time points, 4 and 8 weeks,
using gel (GZ) and in situ zymography (ISZ), respectively.
A significant reduction of PNNs was shown in DCN of
TnC+/+ animals after 8 weeks in EE, while in TnC-/animals this effect was absent. In addition, GZ revealed a
significant increase of MMP-9 activity after 4 weeks of EE
in both genotypes, while MMP-2 activity remained
unchanged. Furthermore, ISZ showed a significant decrease
in MMPs activity after 8 weeks in TnC+/+ and a generally
weak ISZ signal in TnC-/- animals. Finally, investigation of
synaptic density in DCN revealed increased density of
inhibitory synapses in TnC-/- as compared to TnC+/+ mice
regardless of rearing conditions, while the density of
excitatory synapses was increased in TnC+/+ mice after EE
with no changes in TnC-/- mice. These results emphasize a
significant role of TnC in neuronal plasticity and synaptic
remodeling, and imply a possible involvement of MMP-9 in
this effect.
83
POSTER SESIJA / POSTER SESSION
P5 - Neurofiziologija i ponašanje / P5 - Neurophysiology and behaviour
UTICAJ RISPERIDONA NA PROMENE U
METABOLIZMU GLUTATIONA I ENZIMA
ANTIOKSIDATIVNE ZAŠTITE U MOZGU PACOVA
TRETIRANIH FENCIKLIDINOM
EFFECTS OF RISPERIDONE TREATMENT ON
PHENCYCLIDINE INDUCED ALTERATIONS IN
GLUTATHIONE METABOLISM AND
ANTIOXIDANT DEFENSE IN RAT BRAIN
T. Stojković, M. Velimirović, G. Jevtić, N. Radonjić, N.
Petronijević
Institut za medicinsku i kliničku biohemiju; Medicinski
fakultet; Univerzitet u Beogradu; Beograd; Srbija
stihomir85@yahoo.com
T. Stojković, M. Velimirović, G. Jevtić, N. Radonjić, N.
Petronijević
Institute of Medical and Clinical Biochemistry; School of
Medicine; University of Belgrade; Belgrade; Serbia
stihomir85@yahoo.com
Uvod:Perinatalna primena fenciklidina (PCP) kod
pacova predstavlja animalni model shizofrenije. Brojni
podaci ukazuju na redoks disregulaciju u ovom oboljenju.
Naše prethodno istraživanje pokazalo je da dugotrajni efekti
perinatalnog tretmana fenciklidinom kod pacova uzrokuju
promene u sadržaju redukovanog glutationa (GSH) i
aktivnosti antioksidativnih enzima koje su specifične za
odreñene moždane regione.
Cilj: Cilj rada je ispitivanje sposobnosti risperidona
(RSP) da izvrši reverziju redoks disbalansa uzrokovanog
fenciklidinom u mozgu pacova.
Materijal i metode:Wistar pacovi su perinatalno
tretirani fenciklidinom (10mg/kg PCP, dve grupe) ili
fiziološkim rastvorom (0,9% NaCl, dve grupe).
Postnatalnog (PN) 35. dana, u po jednoj NaCl i jednoj PCP
grupi započeta je terapija risperidonom (0,84mg/kg/dan)
rastvorenog u vodi za piće tokom devet nedelja (NaCl-RSP
i PCP-RSP grupe). Životinje su žrtvovane 100 PN dana i
odreñivani su nivoi GSH, aktivnosti gama-glutamat cistein
ligaze (GCL), glutation peroksidaze (GPx), glutation
reduktaze (GR) i superoksid dizmutaze (SOD), kao i
koncentracija lipidinih peroksida u različitim moždanim
strukturama.
Rezultati:Treatman risperidonom je doveo do
normalizacije sniženih nivoa GSH u korteksu i
hipokampusu PCP tretiranih životinja. Promene u aktivnosti
GCL, GPx i GR koje su zapažene u ispitivanim moždanim
strukturama nakon PCP tretmana su takoñe normalizovane
upotrebom risperidona. PCP je doveo do smanjenja SOD
aktivnosti, a tretman risperidonom je bio praćen daljim
smanjenjem u NaCl-RSP, kao i PCP-RSP grupi. Risperidon
je doveo do reverzije povišenog sadržaja lipidnih peroksida
u hipokampusu i talamusu PCP tretiranih životinja.
Zaljučak:Rezultati pokazuju da risperidon dovodi
do reverzije smanjenog nivoa GSH i promena u
antioksidativnoj zaštiti u mozgu pacova tretiranih
fenciklidinom.
Introduction: Phencyclidine (PCP) administration
to rat pups represents an animal model of schizophrenia.
Numerous data suggest redox dysregulation in this disease.
In our previous study, region-specific changes in the
reduced glutathione (GSH) content and the activity of
antioxidant enzymes were reported as the long-term effects
of perinatal PCP treatment in rats.
Aim: The aim was to elucidate whether the risperidone
(RSP) treatment can reverse PCP induced redox disbalance
in rat brain.
Material and methods: The Wistar rats were
perinatally treated with either PCP (10mg/kg PCP, two
groups) or saline (0.9%NaCl, two groups). At postnatal
(PN) day 35, one NaCl and one PCP group have started to
receive risperidone (0,84mg/kg/day) in drinking water for
nine weeks (NaCl-RSP and PCP-RSP groups). Animals
were sacrificed on PN100 and the GSH levels, the activities
of gama-glutamate cysteine ligase (GCL), glutathione
peroxidase (GPx), glutathione reductase (GR) and
superoxide dismutase (SOD), as well as the concentration of
lipid peroxides were determined in different brain regions.
Results: Risperidone treatment normalized
decreased GSH levels in cortex and hippocampus of PCP
treated animals. The changes in GCL, GPx, and GR
activities documented in investigated brain structures after
PCP treatment were also restored by RSP. PCP caused
decreased SOD activity and risperidone treatment was
followed by its further decrease in both NaCl-RSP and
PCP-RSP groups. Risperidone reversed increased levels of
lipid peroxides in hippocampus and thalamus of PCP treated
animals.
Conclusion: The results demonstrate that
risperidone reverses PCP induced decrease in GSH content
and antioxidant defense alterations in rat brain.
84
POSTER SESIJA / POSTER SESSION
P2 - Razviće, plastičnost i ćelijska smrt / P2 - Development, plasticity and cell death
PERINATALNA PRIMENA FENCIKLIDINA
UZROKUJE DEPOLARIZACIJU
MITOHONDRIJALNE MEMBRANE I BLOK U
PRVOJ KONTROLNOJ TAČKI (G1-S) ĆELIJSKOG
CIKLUSA ASTROCITA IZOLOVANIH IZ
MOZGOVA PACOVA STAROSTI 7 DANA
PERINATAL PHENCYCLIDINE ADMINISTRATION
CAUSES MITOCHONDRIAL MEMBRANE
DEPOLARISATION AND G1-S CELL CYCLE
ARREST OF ASTROCYTES ESTABLISHED FROM
BRAINS OF 7 DAYS OLD WISTAR RATS
S. Misirlić Denčić, A.M. Isaković, G. Jevtić, M.
Velimirović, I. Marković, N. Petronijević
Institut za medicinsku i kliničku biohemiju; Medicinski
fakultet; Univerzitet u Beogradu; Beograd; Srbija
sonja_dencic@med.bg.ac.rs
S. Misirlić Denčić, A.M. Isaković, G. Jevtić, M.
Velimirović, I. Marković, N. Petronijević
Institute of Medical and Clinical Biochemistry; Faculty of
Medicine; University of Belgrade; Belgrade; Serbia
sonja_dencic@med.bg.ac.rs
Uvod: Aktuelni animalni model shizofrenije (SCH)
počiva na perinatalnoj aplikaciji fenciklidina (PCP)
pacovima Wistar soja. Nije poznato do kakvih direktnih
funkcionalnih promena ćelija astroglije dolazi pod uticajem
PCP.
Cilj: Uspostaviti kulturu astrocita iz mozgova
pacova starosti 7 dana koji su tretirani fiziološkim
rastvorom (kontrolna grupa) odnosno PCP-om (PCP grupa).
Utvrditi zastupljenost oksidativnog stresa, kao i promene u
kiselosti citoplazme, aktivaciji kaspaza i ćelijskom ciklusu
ovih ćelija.
Materijal i metode: Pacovi Wistar soja su 2. i 6.
postatalnog dana (PD)
primali 0.9%NaCl
ili PCP
(10mg/kg). Sedmog postantalnog dana su žrtvovani i iz
mozgova (8 po grupi) su izolovani astrociti. Paralelno su
izolovani i kultivisani astrociti iz mozgova tek roñenih
pacova u cilju dobijanja kondicionog medijuma za
održavanje astrocitnih kultura dobijenih iz mozgova pacova
starosti 7PD. Nakon kultivacije koja je trajala 3 nedelje,
kulture astrocita su tripsinizirane a ćelije su nakon bojenja
odgovarujućim fluorohromom analizirane na protočnom
citometru.
Rezultati: Astrociti iz PCP grupe nisu pokazili
razliku u odnosu na astrocite iz kontrolne grupe u
intenzitetu produkcije superoksidnog anjona i aktivaciji
kaspaza, kao ni porast u količini kiselog sadržaja u
citoplazmi koji bi mogao da ukaže na prisustvo
oksidativnog stresa i apoptoze tj. autofagije u ovim
ćelijama. Meñutim, astrociti iz PCP grupe su imali
depolarizovanu mitohondrijalnu membranu i blok u prvoj
kontrolnoj tački ćelijskog ciklusa u odnosu na astrocite iz
kontrolne grupe.
Zaključak: S obzirom da signali izmeñu astrocita i
neurona u tripartitnim sinapsama imaju značajnu ulogu u
razvoju neuronskih krugova, zapažene promene bi mogle
biti u osnovi
poremećaja glijoneurotransmisije
pretpostavljenog u SCH.
Introduction: Perinatal phencyclidine (PCP)
administration to Wistar rats represents valid animal model
of schizophrenia (SCH). Still, immediate functional changes
of astroglial cells under PCP influence remain unknown.
Aim: Establishing the astrocytes cell culture from
brains of 7 days old Wistar rats treated with saline (control
group) or PCP (PCP group). Determine the presence of
oxidative stress, changes in the acidic cytoplasmatic
content, caspase activation and astrocytes cell cycle.
Materials and Methods: Wistar rats were treated on
the 2nd and 6th postnatal day (PD) with either phencyclidine
(10 mg/kg) or saline and sacrificed on 7PD. Astrocytes
cultures were established from the obtained brains
(8/group). At the same time astrocyte culture was
established from brains of newborn rats in order to obtain
conditioned medium. After 3 weeks, astrocytes were
detached by trypsinisation and analyzed by flow cytometry
after staining with appropriate fluorochromes.
Results: PCP group astrocytes produced the same
amount of superoxide as astrocytes from control group. No
change in caspase activation was observed together with the
decrease in red fluorescence originating from lysosomes
suggesting the absence of oxidative stress mediatied
apoptosis or autophagy in 7PD astrocytes cell culture. On
the other hand, PCP group astrocytes showed mitochondrial
membrane depolarization and G1-S cell cycle arrest in
comparison to control group astrocytes.
Conclusion: Since the signaling between astrocytes
and neurons at the tripartite synapse plays a significant role
during formation of neural circuits, the changes observed in
our investigation could be involved in the development of
glioneurotransmission imbalance suggested in SCH.
85
POSTER SESIJA / POSTER SESSION
P1- Ćelijski i molekularni mehanizmi neurodegenerativnih procesa / P1 - Cellular and molecular mechanisms of neurodegenerative processes
NEUROPROTEKTIVNA ULOGA ADENOZIN
MONOFOSFATOM AKTIVISANE KINAZE I
SERIN-TREONIN KINAZE AKT/PKB U 1METIL-4-FENILPIRIDINIJUM (MPP+) MODELU
PARKINSONOVE BOLESTI
M. Jovanović 1, M. Dulović 1, G. Tovilović 2, Lj. HarhajiTrajković 2,V. Kostić 3, I. Marković 1, V. Trajković 4
1
Institut za medicinsku i kliničku biohemiju, Medicinski
fakultet Univerziteta u Beogradu, Srbija; 2Institut za
biološka istarživanja „Siniša Stanković“, Univerzitet u
Beogradu, Srbija;
3
Klinika za neurologiju, Klinički Centar Srbije, Medicinski
fakultet, Univerzitet u Beogradu, Srbija;
4
Institut za mikrobiologiju i imunologiju, Medicinski
fakultet Univerziteta u Beogradu, Srbija
zmajce84@gmail.com
Uvod: Oštećenje ćelija neurotoksinom MPP+ je
jedan od najznačajnijih modela bolesti za analizu
mehanizama neurodegeneracije u Parkinsonovoj bolesti.
Cilj: Cilj ovog rada je ispitivanje neuroprotektivne
uloge AMP-om aktivisane protein kinaze (AMPK) i serintreonin
kinaze
Akt/PKB
u
MPP+-indukovanom
neurotoksičnom
modelu
na
ćelijama
humanog
neuroblastoma SH-SY5Y.
Materijali i metode: Ćelijski vijabilitet je odreñen
merenjem aktivnosti endogene kisele fosfataze i MTT
testom. Analiza prisustva slobodnih kiseoničnih radikala,
promene potencijala unutrašnje membrane mitohondrija i
fragmentacije DNK je izvršena protočnom citometrijom.
Aktivacija proteina uključenih u signalne puteve odreñena
je primenom imunoblota. Za smenjenje ekspresije gena za
AMPK koristili smo inhibiciju transkripcije korišćenjem
shRNA.
Rezultati: MPP+ je pokazao vremenski i doznozavisno toksično dejstvo na neuroblastomske ćelije, koje
karakteriše indukcija stvaranja superoksidnog anjona i
posledična depolarizacija mitohondrija, što dovodi do
povećanja fragmentacije DNK. Analiza ekspresije proteina
pokazala je da dolazi do vremenski zavisne aktivacije
kinaza Akt/PKB, AMPK i Raptor. Primena supstanci sa
antioksidativnom aktivnošću (N-acetil cistein i butilirani
hidroksianizol) su dovele dio smanjenja citotoksičnog
efekta MPP+, bez uticaja na fosforilaciju kinaza Akt/PKB i
AMPK. Smanjena ekspresija gena za AMPK je dovela do
povećanog stvaranja superoksida i smanjenja aktivacije
Akt/PKB, što je za posledicu imalo povećanu osetljivost na
MPP+. Primena farmakoloških inhibitora Akt/PKB (10DEBC hidrohorid i LY294002), dovela je do intenzivnijeg
neurotoksičnog efekta MPP+, bez uticaja na aktivnost
AMPK.
Zaključak: Dobijeni rezultati ukazuju da AMPKzavisna aktivacija kinaze Akt/PKB ima protektivno dejstvo
u neurotoksičnom oštećenju izazvanom MPP+-om, kao i da
modulacija
ovog
signalnog
puta
može
imati
neuroprotektivno dejstvo kod oštećenja uzrokovanih
oksidativnim stresom u Parkinsonovoj bolesti.
THE NEUROPROTECTIVE ROLE OF AMPKDEPENDENT AKT ACTIVATION IN MPP+ MODEL
OF PARKINSON’S DISEASE
M. Jovanović1, M. Dulović1, G. Tovilović2, Lj. HarhajiTrajković 2,V. Kostić3, I. Marković 1, V. Trajković4
1
Institute of Medical and Clinical Biochemistry, School of
Medicine, University of Belgrade, Serbia;
2
Institute for Biological Research 'Sinisa Stankovic',
University of Belgrade, Serbia;
3
Institute of Neurology, Clinical Center of Serbia, School of
Medicine, University of Belgrade, Serbia;
4
Institute of Microbiology and Immunovogy, School of
Medicine, University of Belgrade, Serbia
Background: Parkinson´s disease (PD) is the
second most common neurodegenerative disorder. The
neurotoxicity induced by MPP+ is one of the most valuable
models for analysing pathological aspects of PD.
Objective: In the present study we investigated the
neuroprotective role of AMP-activated protein kinase
(AMPK) and Akt in the MPP+-induced neurotoxic effect in
human neuroblastoma SH-SY5Y cell line.
Methods: The cell viability was determined by
MTT and acid phosphatase assay, whereas superoxide
production, mitochondrial membrane potential and DNA
fragmentation were quantified by flow cytometry after
application of appropriate fluorochromes (dihydroethidium,
JC-1 and propidium iodide, respectively). Activation of
proteins involved in signalling pathways was investigated
by immunoblotting. The transfection with short hairpin
RNA (shRNA) was used to inhibit AMPK activity.
Results: MPP+ showed time- and dose-dependent
neurototoxic effect, through induction of superoxide
production, followed by mitochondrial membrane
depolarization and subsequent DNA fragmentation. This
was associated with time-dependent activation of Akt,
AMPK, and Raptor. The reactive oxygen species (ROS)
scavengers (BHA and NAC) partly reversed the MPP+
cytotoxic effect, without decrease in phosphorylation of Akt
and AMPK. AMPK knockdown with shRNA resulted in
increase in superoxide production, accompanied by
decrease in Akt phosphorylation, leading to increased cell
death. Akt inhibitors (10-DEBC hydrochloride and
LY294002), further potentiated MPP+ neurotoxic effect,
with no reduction in AMPK phosphorylation.
Conclusion: Our results have shown that AMPKdependent Akt activation protects against MPP+-elicited
oxidative stress and cell damage, and that modulation of
AMPK/Akt action may provide a valid neuro-protective
approach in ameliorating the ROS-induced neurotoxic
effects in Parkinson's disease.
86
INDEKS AUTORA
INDEKS AUTORA
INDEKS AUTORA
Abushik P. 65
Adžić M. 9, 39, 42
Adžić M. 14, 59, 60
Ajdžanović V. 75
Aksić M. 71
Aleksić D. 71
Anñus P.R. 30, 60, 65, 69, 79, 83
Andrić S. 9
Arsikin K. 52
Balusu S. 31
Banković J. 21, 22
Beyer C. 2
Bijelić D. 59, 60
Bjelobaba I. 14, 59, 64, 67
Blaževski J. 61, 66
Boltze J. 79
Bošnjak M. 52
Božić I. 14, 17, 49, 62, 64, 67
Božović N. 39
Brajušković G. 8
Briševac D. 14, 59, 60
Brkić M. 19, 34, 50, 73
Brkić P. 48
Bumbaširević V. 57
Buzadžić I. 35
Čapo I. 82
Ćirić D. 57
Clarner T. 2
Cvijić G. 18
Dacić S. 49, 64, 67, 75
Davidović S. 45
De Rycke R. 31
Dekanski D. 77
Demeestere D. 31
Dinić J. 22
Divac N. 71
Đogo N. 5
Đordjević J. 18
Đorñević J. 9, 42
Đurašević S. 18
Đurić D. 23, 78
Dokić M. 82
Drakulić D. 40, 45
Drakulić D. 35, 37, 43, 56
Drulović J. 16
Dujmović Bašuorski I. 16
Dulović M. 7, 36, 86
Đurica S. 8
Filipović B. 20
Filipović B. 71
Filipović D. 47
Gawlak M. 30, 83
Giniatullin R. 65
Goldberg J. 2, 38
Grković I. 37, 43, 56
Guševac I. 37, 43, 56
Harhaji-Trajković Lj. 7, 36, 52, 57, 86
Horvat A. 37, 43, 56
Hrnčić D. 23, 78
Ilić T.V. 26, 35
Isaković A.M. 46, 85
Isaković A.J. 68
Isaković A. 46, 55, 77
Ivković M. 8
Ivković S. 11
Jaklin M. 79
Jakovčevski I. 5, 71
Janać B. 25
Janjetović K. 58
Jasnić N. 18
Jaworski T. 30, 83
Jevtić B. 63
Jevtić G. 27, 51, 72, 84, 85
Jevtović-Todorović V. 13, 54
Jovanović M. 27
Jovanović M. 38
Jovanović M. 30, 83
Jovanović M. 7, 36, 86
Jovanović Simić N. 48
Jovanović T. 48
Jovičić M. 9, 39
Kaczmarek L. 83
Kalauzi A. 80, 81
Kanazir S. 12, 13, 19, 34, 50, 53, 54, 70, 73, 76
Karanović J. 8
Keković G. 82
Kipp M. 2, 38
Klajn A. 40, 45
Korenić A. 79
Kostić V. 7, 36, 86
Kovačević-Grujičić N. 41
Krstić-Demonacos M. 9
Laketa D. 14, 49, 64, 67
Lakić I. 18
Lavrnja I. 14, 17, 49, 62, 64, 67, 75
Lazić D. 19, 50, 73
Lazić K. 80, 81
Libert C. 31
Lončarević-Vasiljković N. 12, 19, 34, 50, 70, 73
Lukić I. 9, 39
Lütjohann D. 2
Macut Đ. 23, 78
INDEKS AUTORA
Manojlović-Stojanoski M. 20
Marić N. 9, 39, 57
Marjanović J. 40, 44
Marković B. 71
Marković I. 7, 36, 85, 86
Marković M. 16
Martać Lj. 82
Martinović T. 57
Medigović I. 20, 74
Mesaroš S. 16
Mihaljević M. 9
Milanović D. 13, 54
Miler M. 74
Milinković V. 21
Milivojević M. 41
Miljković Đ. 15, 61, 63, 66, 77
Milošević M. 59, 60, 65
Milošević V. 20, 74, 75
Milošević Z. 22
Misirkić M. 57
Misirkić-Marjanović M. 58
Misirlić Denčić S. 46, 85
Mitić M. 9, 39, 42
Mitrović N. 37, 43, 56
Mladenović Đorñević A. 12, 19, 53, 70, 76
Mojsin M. 41, 44
Momčilović M. 61, 63, 66, 77
Mostarica Stojković M. 16, 61, 63, 66
Nedeljković N. 14, 17, 38, 49, 59, 60, 62, 64, 67
Nestorović N. 20, 74
Nikolić Lj. 25
Nikolić T. 27, 51
Novaković I. 6
Pantović A. 52
Pantović M. 8
Parabucki A. 14, 59, 64, 67
Paunović V. 57
Pavković Ž. 40, 53
Pavlović Z. 9
Peković S. 14, 17, 49, 62, 64, 67, 75
Perović M. 12, 19, 34, 50, 53, 70, 73, 76
Pešić M. 22
Pešić V. 13, 34, 53, 54
Peters M. 79
Petković F. 61, 66
Petričević S. 57, 68
Petronijević M. 51
Petronijević N. 27, 46, 51, 72, 84, 85
Petrović I. 41
Petrović J. 80, 81
Petrović-Kosanović D. 20
Podgorac J. 82
Podolski-Renić A. 22
Popadić D. 16
Popić J. 13, 54
Popović J. 41
Popović M. 55, 68, 77
Pravica V. 16
Prolić Z. 25
Radenović L. 30, 69, 77, 79, 83
Radojčić M. 9, 42
Radonjić N. 27, 32, 51, 84
Radonjić V. 71
Rakić Lj. 12, 13, 19, 34, 49, 50, 53, 54, 70, 73, 76
Rakić M. 21
Rašić - Marković A. 23, 78
Rauš Balind S. 25
Redzic Z. 10
Ristić B. 52
Ristić N. 20, 74, 75
Romac S. 8
Ruždijić S. 12, 13, 19, 21, 34, 50, 53, 54, 70, 73, 76
Šaponjić J. 24, 80, 81
Savić D. 9
Savić D. 14, 17, 49, 62, 64, 67
Savić E. 16
Savić M. 28
Savić Pavićević D. 8
Savić T. 25
Schachner M. 5, 30, 83
Schwirtlich M. 45
Sekulić S. 82
Selaković V. 27, 69
Simić I. 42
Skender-Gazibara M. 21
Smiljanić K. 12, 19, 53, 76
Soldatović I. 9, 39
Šošić-Jurjević B. 74
Spasojević I. 15
Stamenković S. 18, 30, 69, 83
Stamenković V. 30, 66, 83
Stanisavljević D. 44, 45
Stanković T. 21, 22
Stanojević Ž. 55, 68, 77
Stanojlović M. 35, 37, 43, 56
Stanojlović O. 23, 78
Stevanović M. 40, 41, 44, 45
Stojiljković M. 14, 17, 49, 59, 62, 64, 67, 75
Stojilkovic S. 3
Stojković S. 22
Stojković T. 27, 51, 72, 84
Šušić V. 23, 78
INDEKS AUTORA
Suzin-Živković V. 52
Šviković S. 8
Tanić N. 21, 22
Terzić I. 48
Tešić V. 12, 34, 50, 73, 76
Timotijević G. 63
Todorović D. 25
Todorović N. 29
Todorović N. 47
Todorović S. 12, 70
Topalović V. 41, 44
Tošić J. 40
Tošić J. 55, 68, 77
Tošić M. 40
Tovilović G. 58, 86
Trajković V. 7, 36, 52, 55, 57, 58, 68, 86
Trifunović S. 75
Van Hooren V. 31
Van Wonterghem E. 31
Vandenbroucke R.E. 31
Velimirović M. 27, 46, 51, 72, 84, 85
Victor M. 2, 38
Vučetić-Arsić S. 27
Vučićević Lj. 57, 58
Vujović P. 18
Wilczynski G.M. 30, 83
Zecevic N. 32
Živanović J. 66, 74
Zlatković J. 47
Zogović N. 58
CIP - Каталогизација у публикацији
Народна библиотека Србије, Београд
616.8(048)(0.034.2)
ДРУШТВО за неуронауке Србије (Београд). Конгрес (6 ; 2013 ; Београд)
Knjiga sažetaka [Elektronski izvor] = Book of Abstract / VI Kongres DNS
[Društvo za neuronauke Srbije], Beograd, 14-16, Novembar 2013. = 6th SNS
[Serbian Neuroscience Society] Congress ; [organizatori Društvo za
neuronauke Srbije ... [et al.] ; urednici Selma Kanazir, Danijela Savić,
Aleksandra Isaković]. - Beograd : Društvo za neuronauke
Srbije : Ministarstvo prosvete, nauke i tehnološkog razvoja : Institut za
biološka istraživanja "Siniša Stanković", 2013 (Beograd : Društvo za
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