Epigenetic gene regulation in focal epilepsies [Elektronische Ressource] / vorgelegt von Katja Kobow

Kobowka

Sujets

• Biologie

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 31
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

„Epigenetic gene regulation in focal epilepsies“






Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität
Erlangen-Nürnberg



zur
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von



Katja Kobow
aus Berlin











Als Dissertation genehmigt
von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander Universität Erlangen-Nürnberg













Tag der mündlichen Prüfung: 28.10.2009

Vorsitzende/r der der Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter/in: Prof. Dr. Ingmar Blümcke
Zweitberichterstatter/in: Prof. Dr. Robert Slany















































Contents
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1. INTRODUCTION 1
1.1. EPILEPSY 2
1.1.1. EPILEPSY – MORE THAN SEIZURES
1.1.2. NEUROBIOLOGY OF EPILEPSY 3
1.1.3. TEMPORAL LOBE EPILEPSY 5
1.1.4. THE PILOCARPINE MODEL OF TEMPORAL LOBE EPILEPSY 8
1.2. REELIN 9
1.2.1. DEVELOPMENTAL ROLES OF REELIN
1.2.2. REELIN FUNCTION IN THE ADULT BRAIN 11
1.2.3. STRUCTURE AND REGULATORY PROPERTIES OF THE REELIN GENE 12
1.3. GLUTAMATE SIGNALING 14
1.3.1. GLUTAMATE – THE MAJOR EXCITATORY NEUROTRANSMITTER 14
1.3.2. MGLUR2 16
1.4. EPIGENETIC GENE REGULATION 17
1.4.1. THE HISTONE CODE
1.4.2. DNA METHYLATION 18
1.4.3. THE EPIGENOME IN HEALTH AND DISEASE 19
2. MATERIALS AND METHODS 21
2.1. MATERIALS 21
2.1.1. LIST OF SUPPLIERS
2.1.2. BUFFERS 22
2.1.3. KITS 23
2.1.4. ENZYMES 23
2.1.5. LIST OF ANTIBODIES 24
2.1.6. BACTERIAL STRAINS AND MEDIA 25
2.1.7. ANIMALS 26
2.1.8. HUMAN MATERIAL
2.1.9. LIST OF PRIMERS 27
2.2. METHODS 28
2.2.1. EPIGENETIC REGULATION OF HUMAN REELIN IN TLE SPECIMEN 28
2.2.2. EGENE REGULATION IN A RAT MODEL OF INDUCED CHRONIC EPILEPSY 32
3. RESULTS 35
3.1. REELIN PROMOTER METHYLATION IN MTS PATIENTS WITH ASSOCIATED GCD 35
3.1.1. PRESERVATION OF REELIN-SECRETING CAJAL-RETZIUS CELLS IN THE HIPPOCAMPUS OF
EPILEPSY PATIENTS WITH GCD 36
3.1.2. REELIN GENE EXPRESSION IS ALTERED IN EPILEPSY PATIENTS WITH GCD 37
3.1.3. REELIN PROMOTER METHYLATION IS INCREASED IN CHRONIC TLE 38
3.1.4. INCREASED REELIN PROMOTER METHYLATION ASSOCIATES WITH GCD 39
3.1.5. NO REELIN PROMOTER METHYLATION IN CHRONIC EPILEPTIC RATS 43
3.2. TIME DEPENDENT CHANGES OF GENE-SPECIFIC METHYLATION PATTERNS IN CHRONIC
EPILEPTIC RATS 45
3.2.1. IDENTIFICATION OF POTENTIAL TARGET GENES WITH SUSPECTED IMPACT ON
EPILEPTOGENESIS IN A MODEL OF TLE
3.2.2. REGION- AND TIME-DEPENDENT GENE EXPRESSION OF CANDIDATE GENES SUSCEPTIBLE
TO PROMOTER METHYLATION IN THE RODENT EPILEPTOGENIC HIPPOCAMPUS 46 Contents
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3.2.3. MGLUR2 GENE EXPRESSION IN DIFFERENT BRAIN REGIONS OF CHRONIC EPILEPTIC RATS
48
3.2.4. MGLUR2 PROMOTER METHYLATION IS INCREASED IN THE DG OF CHRONIC EPILEPTIC
RATS 50
3.2.5. GENE EXPRESSION OF DNMT1 AND MTHFR IS NOT INCREASED IN THE DG OF CHRONIC
EPILEPTIC RATS 52
4. DISCUSSION 54
4.1. INCREASED REELIN PROMOTER METHYLATION ASSOCIATES WITH GCD IN HUMAN TLE 54
4.1.1. CONTROL OF HIPPOCAMPAL ARCHITECTURE BY REELIN SECRETING CAJAL-RETZIUS
CELLS 54
4.1.2. EPIGENETIC REGULATION OF REELIN EXPRESSION IN CNS DISORDERS 55
4.1.3. EOF RPRESSION IN FOCAL EPILEPSIES 56
4.1.4. ANIMAL MODELS MIMIC THE CLINICO-PATHOLOGICAL FEATURES OF TLE 57
4.1.5. PARAMETERS INFLUENCING CLINICO-PATHOLOGICAL FEATURES OBSERVED IN
EXPERIMENTAL TLE 58
4.1.6. PYROSEQUENCING ALLOWS QUANTIFICATION OF DNA METHYLATION 60
4.2. EPIGENETIC REGULATION OF MGLUR2 IN CHRONIC EPILEPTIC RATS 62
4.2.1. GENE EXPRESSION PROFILING FROM TLE 62
4.2.2. TARGETING MGLUR2 SIGNALING IN TLE 63
4.2.3. INCREASED MGLUR2 PROMOTER METHYLATION IN EXPERIMENTAL EPILEPSY 63
5. OUTLOOK 66
6. SUMMARY 67
7. ZUSAMMENFASSUNG 70
8. REFERENCES 73
9. APPENDIX 82
LIST OF ABBREVIATIONS 82
BIBLIOGRAPHY 86
ACKNOWLEDGEMENTS 87
CURRICULUM VITAE 88 Introduction 1
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1. Introduction


Temporal lobe epilepsy (TLE) is the most common epileptic syndrome in adult
humans. Patients frequently suffer from a progressive worsening of the disease,
insufficient treatment failing to achieve seizure control and, finally, development of
pharmacoresistance to common anti-epileptic drugs. Many studies have addressed
epileptogenicity underlying mechanisms. Therein, both associated histopathology and
gene expression patterns have been extensively analyzed. Aberrant region and cell
type specific gene expression has been suggested to be the basis for the chronification
of epilepsy and development of drug-resistance. However, in previous studies rarely
there has been provided information on mechanisms that regulate differential gene
expression in human and/or experimental TLE. The present study will address
epigenetic gene silencing in two particular genes (Reelin and mGluR2), explicitly
implicated in the histopathology and pathophysiology of TLE. DNA methylation of
susceptible promoters will be identified as potential new pathomechanism in focal
epilepsies. Furthermore, the hypothesis of DNA methylation acting as universal
regulatory element during the process of epileptogenesis offers an intriguing
perspective for new therapeutic strategies in TLE patients.
Introduction 2
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1.1. Epilepsy

1.1.1. Epilepsy – more than seizures

Epilepsy is one of the most common disorders of the brain. It is estimated that
one of every ten people will have at least one epileptic seizure during a normal lifespan
and about a third out of these will develop epilepsy [1].
An epileptic seizure is a transient occurrence of signs and/or symptoms due to
excessive synchronous and rhythmic neuronal activity in the brain [2]. It can be either
unprovoked or acute symptomatic, e.g. a response of the brain to stress, lack of sleep,
drug intoxication, inflammation or other events and, therefore, must not necessarily be
an indication for an epileptic disorder.
The fundamental characteristic of epilepsy is the recurrence of unprovoked
seizures. However, the term epilepsy does not refer to a specific disease or a single
syndrome. It rather summarizes several symptom complexes of an underlying disorder,
which may be genetic, infectious, traumatic, malignant, metabolic or pharmacologic [3].
The term epilepsy syndrome is used restrictively for a group of clinical entities that are
reliably identified concerning their characteristic localization, seizure types, and pattern
of seizure recurrence, age of onset, clinical signs, EEG findings, genetics and
prognosis [1].
Epilepsies are differentiated according to their anatomical origin in focal and
generalized epilepsies. Focal epilepsies are, thereby, consistent with the initial
activation of only a part of one cerebral hemisphere (e.g. the hippocampus), while
generalized epilepsies may arise from multiple foci and affect both cerebral
hemispheres. Classification by the presumptive first cause allows differentiation of
symptomatic, idiopathic or cryptogenic epilepsies [4]. Symptomatic epilepsies are
connected to specific lesions of the brain that can be either focal such as tumours,
infarctions, cortical malformations, or in case of a brain infection or inborn metabolic
disease may have widespread effects. Idiopathic epilepsies are of true genetic origin.
They, however, account only for the minority of epilepsy cases [5]. Finally, cryptogenic
epilepsies are of unknown origin. They may have a fundamental genetic defect at their
core or may as well be the consequence of a separate disorder or condition not yet
recognized. Further, epilepsies can be classified by seizure types or as part of discrete Introduction 3
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medical syndromes. Generally, recommendations by the International League Against
Epilepsy (ILAE) regarding terminology and concepts of classification for epilepsies and
epileptic seizures develop gradually with increasing knowledge.


1.1.2. Neurobiology of Epilepsy

Many attempts have been made to unravel the molecular pathogenesis of
epilepsy. This has been proven to be difficult due to the enormo