Identification and functional characterization of extracellular signals affecting the expression of astroglial glutamate transporters [Elektronische Ressource] / Maciej Figiel

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Sujets

• Biologie

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2002
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Abteilung Anatomie und Zellbiologie
Universität Ulm
Leiter: Prof. Dr. Dr. h.c. Ch. Pilgrim
Identification and functional characterization of
extracellular signals affecting the expression of
astroglial glutamate transporters
Dissertation
zu Erlangung des Doktorgrades der Humanbiologie
an der Fakultät für Medizin
der Universität Ulm
Vorgelegt von
Diplom-Biotechnologe Maciej Figiel
aus Pozna , Polen
2001Amtierender Dekan: Prof. Dr. med. Reinhard Marre
1. Gutachter: Prof. Dr. Jürgen Engele
2. Gutachter: Prof. Dr. med. Albert C. Ludolph
Tag der Promotion: 19.10.2001Moim Rodzicom
For my ParentsTABLE OF CONTENTS
1. ABBREVIATIONS 3
2. INTRODUCTION 6
2.1. FUNCTION AND PHYSIOLOGY OF GLUTAMATE IN THE CNS....................................6
2.2. GLUTAMATE TRANSPORTERS AND THEIR PROPERTIES...........................................7
2.3. TRANSPORTERS IN ACUTE AND CHRONIC BRAIN DISEASES...................................9
2.4. REGULATION OF GLUTAMATE TRANSPORTERS........................................................10
2.5. PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDES (PACAPS).....11
2.6. PACAP FUNCTION IN THE CENTRAL NERVOUS SYSTEM.........................................13
2.7. FGF-2, EGF, TGF AND THEIR RECEPTORS IN THE CNS............................................13
2.8. AIMS OF THE WORK............................................................................................................14
3. MATERIALS AND METHODS 15
3.1. MATERIALS...........................................................................................................................15
3.1.1. Animals............................................................................................................................15
3.1.2. Reagents and media.........................................................................................................15
3.1.3. Plasticware.......................................................................................................................16
3.1.4. Solutions..........................................................................................................................16
3.1.5. Primers.............................................................................................................................20
3.1.6. Antisera............................................................................................................................20
3.1.7. Equipment........................................................................................................................21
3.2. METHODS..............................................................................................................................22
3.2.1. Animals............................................................................................................................22
3.2.2. Glial cultures...................................................................................................................22
3.2.3. Neuronal Cultures...........................................................................................................23
3.2.4. Treatment of Cultures......................................................................................................23
3.2.5. Total RNA isolation and RT-PCR..................................................................................24
3.2.6. Fos assay..........................................................................................................................25
3.2.7. Characterization of glial cultures....................................................................................25
3.2.8. Glutamate uptake.............................................................................................................25
3.2.9. Total protein isolation.....................................................................................................26
3.2.10. Neuronal membranes.....................................................................................................26
3.2.11. Protein determination....................................................................................................27
3.2.12. Neuron-conditioned medium........................................................................................27
3.2.13. Western blot analysis.....................................................................................................27
3.2.14. Statistics.........................................................................................................................28
4. RESULTS 29
4.1. ESTABLISHMENT OF ENRICHED CORTICAL ASTROGLIAL CULTURES................29
4.2. CELLULAR SOURCES AND TARGETS OF PACAP IN THE CNS..................................30
4.2.1. Neurons are the major source for PACAP in the neocortex...........................................30
1
a4.2.2. PACAP-38 acts on astroglia involved in glutamate turnover........................................31
4.2.3. Neuronal influences on glial glutamate uptake are mediated by PACAP.....................33
4.3. FACTORS REGULATING EXPRESSION OF GLIAL GLUTAMATE TRANSPORTERS
.........................................................................................................................................................34
4.3.1. PACAP promotes glutamate uptake in astroglia............................................................34
4.3.2. Increased glutamate uptake promoted by PACAP-38 is the result of increased
expression of Glt-1 and Glast....................................................................................................35
4.3.3. PACAP affects glial glutamate uptake through type-1 binding sites.............................36
4.3.4. PACAP promotes glutamate metabolism in astroglia....................................................37
4.3.5. EGF and TGF affect glutamate transporters expression..............................................38
4.4. REDUNDANT SIGNALING PATHWAYS REGULATE EXPRESSION OF GLT-1 AND
GLAST............................................................................................................................................40
4.5. THE IDENTIFIED EXTRACELLULAR FACTORS PROMOTE GLUTAMATE
TRANSPORTERS EXPRESSION INDEPENDENT OF THE MORPHOLOGICAL
DIFFERENTIATION OF CORTICAL GLIA ...............................................................................44
5. DISCUSSION 46
5.1. ASSAY FOR EFFECTS ON GLIAL GLUTAMATE TRANSPORT....................................46
5.2. IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF
EXTRACELLUALR SIGNALS AFFECTING THE EXPRESSION OF GLIAL GLUTAMATE
TRANSPORTERS..........................................................................................................................47
5.2.1. PACAP............................................................................................................................47
5.2.2. EGFR ligands and FGF-2................................................................................................49
5.2.3. Additional features of the regulatory influences of PACAP, EGF and TGF on glial
glutamate transport....................................................................................................................50
5.3. SIGNAL RECOGNITION AND TRANSDUCTION MECHANISMS UNDERLYING THE
STIMULATORY EFFECTS OF PACAP, EGF AND TGF ON GLIAL GLUTAMATE
TARNSPORTER EXPRESSION..................................................................................................52
5.3.1. Receptors.........................................................................................................................52
5.3.2. Signaling pathways..........................................................................................................53
5.4. THE STIMUALTORY EFFECTS OF PACAP, TGF AND EGF ON GLIAL
GLUTAMATE TARNSPORTER EXPRESSION OCCUR INDEPEDENTLY OF
MORPHOLOGICAL DIFFERENTIATION..................................................................................55
5.5. PHYSIOLOGICAL ROLE OF PACAP, EGF AND TGF IN THE REGULATION OF
GLUTAMATE TRANSPORTERS EXPRESSION......................................................................55
5.6. CONCLUSION........................................................................................................................57
6. SUMMARY 58
7. REFERENCES 60
2
aaaaaAbbreviations
1. ABBREVIATIONS
AA arachidonic acid
aa amino acid
dbcAMP dibutyryl cyclic adenine monophosphate
ABC avidin-biotin peroxidase complex
AC adenylate cyclase
ACh acetylcholine
ADS antibody diluting solution
AMPA -amino-3-hydroxy-5-methyl-isoxasole-4-
propionate
ALS amyothrophic lateral sclerosis
ASCT1 amino acid transporter 1
ASCT2 amino acid transporter 2
APS ammonium persulfate
BB bromophenol blue
BCA bicinchoninic acid
bFGF/FGF-2 basic fibroblast growth factor/fibroblast
growth factor-2
bp base pair
BSA bovine serum albumin
cAMP cyclic adenine monophosphate
CM conditioned medium
CRE cAMP response element
cDNA complementary deoxyribonucleic acid
CNS central nervous system
DAB 3,3’-diaminobenzindintetrahydrochloride
dihydrate
DMSO dimethyl sulfoxide
DTT dithiothreitol
E17 embryonic day 17
EAAC1 excitatory amino acid carrier 1
EAAT1/2/3/4/5 excitatory amino acid transporters 1/2/3/4/5
3
aAbbreviations
ECL enhanced chemiluminescence
EDTA