Posttranskriptionale Modifikation von Gephyrin und Glycinrezeptor messenger RNA in Temporallappenepilepsie

1\. Introduction 1.1 About the dichotomous nature of chloride signalling in the brain – The chloride equilibrium potential 10 1.2 GABA & Glycine 11 1.2.1 Inhibition 12 1.2.2 Glycine Receptor (GlyR) 13 1.2.3 GABA(A) Receptors (GABA(A)R) 15 1.3 Gephyrin 18 1.3.1 Gephyrin structure and aggregation 18 1.3.2 GlyR and GABA(A)R binding of gephyrin 20 1.3.3 Gephyrin trafficking and synaptogenesis 20 1.3.4 Molybdenum co-factor synthesis of gephyrin 24 1.4 Glia cells 24 1.5 The hippocampus 26 1.5.1 Hippocampal anatomy 26 1.5.2 The main hippocampal circuit 26 1.6 Epilepsy 28 1.6.1 Temporal Lobe Epilepsy 30 1.6.1.1 Network and Cellular Mechanisms of TLE 32 1.7 Aim of the PhD work 34 2\. Material and methods 2.1 Preface 35 2.1.1 Chemicals 35 2.1.2 Enzymes 37 2.1.2.1 Enzymes for cell culture 37 2.1.2.2 Polymerases 37 2.1.2.3 Restriction enzymes 37 2.1.3 Kits 37 2.1.4 Media and solutions 37 2.1.4.1 Primary hippocampal cell culture 38 2.1.4.2 Media and solutions for HEK293 cells 39 2.1.4.3 Substances for pharmacoligical manipulations 39 2.1.4.4 RHC whole-cell patch-clamp recording 39 2.1.4.5 HEK293 whole-cell patch-clamp recording 39 2.1.4.6 HEK293 outside-out patch-clamp recording 40 2.1.4.7 Solutions for immunofluorescence 40 2.1.4.8 Media and solutions for bacterial cultures 40 2.1.4.9 Solutions for molecular biology 40 2.1.5 Antibodies 41 2.1.6 Plasmids 41 2.1.7 Oligonucleotides 42 2.1.8 Human control RNA, Bacteria and Animals 43 2.1.9 Equipment and software 44 2.1.9.1 Epifluorescence microscopy 44 2.1.9.2 Confocal microscopy 44 2.1.9.3 Microtome 44 2.1.9.4 Molecular- and microbiology 44 2.1.9.5 Cell culture 45 2.1.9.6 Electrophysiology 45 2.1.9.7 Computer software 45 2.2 Legal information 46 2.3 Tissue preparation 46 2.3.1 Human patient hippocampectomies 46 2.3.2 Mouse brain tissue 48 2.3.3 Cryosections 48 2.4 Cell culture 49 2.4.1 Primary rat hippocampal neuron cell culture 49 2.4.1.1 Preparation 49 2.4.1.2 Effectene based cell transfection 50 2.4.1.3 Experimental cellular stress procedures 51 2.4.2 Human embryonic kidney cell culture and transfection 52 2.4.2.1 HEK293 cell cultivation 52 2.4.2.2 Calcium phosphate based transfection 52 2.5 Molecular biology 53 2.5.1 Gel extraction 53 2.5.2 TA vector 53 2.5.3 RNA isolation 53 2.5.4 Synthesis of complementary DNA 54 2.5.5 Polymerase chain reaction 55 2.5.5.1 Semi- quantitative PCR 56 2.5.5.2 Isolation of irregularly spliced GPHN transcripts 56 2.5.5.3 Detection of GlyR alpha2A and GlyR alpha2B splice variants 57 2.5.5.4 Detection of GlyR alpha3L and GlyR alpha3K splice variants 57 2.5.5.5 Detection of GABA(A)R subunits 58 2.5.6 DNA extraction and sequencing of genomic regions 58 2.5.7 Expression constructs 59 2.5.7.1 Gephyrin expression constructs 59 2.5.7.2 Gephyrin RNA splice reporter constructs 60 2.5.7.3 Epitope tagged and high affinity GlyR expression constructs 62 2.5.8 Molybdenum cofactor synthesis assay 62 2.6 Immunochemistry 63 2.6.1 Western blot analysis 63 2.6.2 Immunohistochemistry 64 2.6.3 Immunocytochemistry 65 2.6.3.1 Surface staining 65 2.6.3.2 Methanol fixation and permeabilisation 66 2.6.3.3 Paraformaldehyde fixation and Triton-X permeabilisation 66 2.6.3.4 Intracellular staining 67 2.6.4 Microscopy and image analysis 67 2.6.4.1 Fluorescence microscopy 67 2.6.4.2 Confocal microscopy 68 2.6.4.3 Splice reporter assays 68 2.6.4.4 Co-localisation analysis 68 2.6.4.5 Receptor cluster analysis 69 2.7 Electrophysiology 70 2.7.1 Whole-cell patch-clamp 70 2.7.2 Outside-out patch-clamp 71 2.8 Statistical data analysis 73 3\. Manuscripts 3.1 Publication 1: Irregular RNA splicing curtails postsynaptic gephyrin in the cornu ammonis of patients with epilepsy. 74 3.2 Publication 1, Supplementary Data 92 3.3 Publication 2: Glycine receptors caught between genome and proteome - Functional implications of RNA editing and splicing. 109 3.4 Publication 3: Splice-specific roles of glycine receptor alpha3 in the hippocampus. 118 4\. Discussion 4.1 GABA(A)R, gephyrin & TLE 134 4.1.1 Gephyrin oligomerisation is essential for the post-synaptic enrichment & stabilisation of GABA(A)R 135 4.1.2 TLE patients express irregularly spliced TLE gephyrins 136 4.1.2.1 Dominant negative effects can be attributed to irregularly spliced TLE gephyrins 137 4.1.2.2 Cellular stress is sufficient to disrupt regular gephyrin RNA splicing 138 4.1.2.3 Consequences of impaired gephyrin clustering in TLE 140 4.2 GlyR 141 4.2.1 RNA-edited high affinity GlyR 141 4.2.1.1 Structural implications of GlyR RNA-editing 142 4.2.1.2 Functional relevance of extra-synaptic high affinity GlyR 143 4.2.2 GlyR alpha3 RNA splicing determines subcellular localisation 145 4.2.2.1 Neuronal phenotypic promiscuity of hippocampal GlyR depends on RNA splicing 146 4.3 Conclusion and Outlook 148 4.3.1 Finding avenues to combat the disruptive effects of cellular stress on GABAergic post-synaptic domains 149 5\. Appendix 5.1 Abbreviations 151 5.1.1 Units 154 5.2 Index of Figures and Tables 155 5.3 Declaration to the publications 157 5.4 Summary 159 5.5 Zusammenfassung 161 6\. References 163The main focus of this thesis is the involvement of the GABA(A) receptor alpha2 scaffolding protein gephyrin in hyperexcitability disorders. The recent observation of gradually declining hippocampal gephyrin immunoreactivity during epileptogenesis in an animal model of epilepsy (Fang et al. 2011) and a reduction of postsynaptic GABA(A) receptor alpha2 in the epileptic hippocampus (Bouilleret et al. 2000; Kneussel et al. 2001; Kumar and Buckmaster 2006) have linked gephyrin to temporal lobe epilepsy. Still, the mechanisms underlying reduced gephyrin immunoreactivity have remained enigmatic. Thus, aim of this PhD work was to identify and characterise cellular mechanisms responsible for loss of postsynaptic gephyrin in temporal lobe epilepsy. Immunohistochemical and western blot analyses unveiled aberrant gephyrin expression in the cornu ammonis of patients afflicted with temporal lobe epilepsy. Four abnormally spliced gephyrin variants lacking several exons in the G-domain were isolated from patient RNA and characterised in HEK293 cells and primary hippocampal neurons via EGFP tagged expression constructs. All 4 identified temporal lobe epilepsy gephyrins were found to be oligomerisation-deficient and interact with regularly spliced gephyrins in a dominant negative way, thereby curbing hippocampal postsynaptic gephyrin and GABA(A) receptor alpha2. While gephyrin gene mutations were not detected by sequencing of genomic DNA, cellular stress like hyperthermia or alkalosis proved suitable and sufficient to induce inhibition of regular gephyrin RNA splicing and subsequent expression of dominant negative temporal lobe epilepsy gephyrins, leading to curtailed postsynaptic gephyrin and GABA(A) receptor alpha2 scaffolds in primary hippocampal neurons of a wild type background. Thus, cellular stress, like rebound alkalosis occurring secondary to seizure activity, could facilitate the development of temporal lobe epilepsy by reducing GABA(A) receptor alpha2-mediated hippocampal synaptic transmission selectively in the cornu ammonis, and in turn reduce seizure threshold, making the network prone to further deregulation of gephyrin splicing and epileptogenesis in a self- propagating cycle. The novel RNA splice-reporter introduced in this work (see 3.2, Publication 1, Supplementary Methods and Figure 9) presents an invaluable molecular tool in drug screening for protective agents to brace neurons against cellular stress and the search for gene expression with compensatory properties for the design of causally-oriented therapies for the treatment of excitability diseases (Eichler and Meier 2008) as well, as other neuronal impairments, like affective mood disorders and stroke (Tyagarajan et al. 2011).Der Hauptschwerpunkt dieser Dissertation liegt auf der Beteiligung des Alpha2 GABA(A) Rezeptor (GABA(A)R) Verankerungsproteins Gephyrin an Temporallappenepilepsie (TLE). Unlängst publizierte Beobachtungen eines graduellen Verlusts hippocampaler Gephyrinimmunoreaktivität während der Epileptogenese in einem Tiermodell der Epilepsie (Fang et al. 2011) und der Minderung postsynaptischer Alpha2 GABA(A)R im epileptischen Hippocampus (Bouilleret et al. 2000; Kneussel et al. 2001; Kumar and Buckmaster 2006) bringen Gephyrin in Verbindung mit TLE. Die Mechanismen, welche der verringerten Gephyrinimmunoreaktivität zugrunde liegen, blieben bislang rätselhaft. Ziel dieser Doktorarbeit war es daher, die zellulären Mechanismen des Verlusts postsynaptischen Gephyrins in TLE zu identifizieren und zu charakterisieren. Immunohistochemische und Westernblotuntersuchungen enthüllten abnormale Gephyrinexpression in der Cornu Ammonis (CA) Region von TLE Patienten. Vier irregulär gespleißte Gephyrinvarianten, denen mehrere Exone in der G-Domäne fehlen, wurden aus Patienten-RNA isoliert und mittels EGFP-markierter Expressionskonstrukte in HEK293 Zellen und primären Hippocampusneuronen charakterisiert. Alle 4 fehlgespleißten Gephyrine wiesen Oligomerisationsdefizite auf und reduzierten mittels Interaktion mit regulärem Gephyrin in dominant-negativer Weise hippocampales postsynaptisches Gephyrin und Alpha2 GABA(A)R. Mutationen des Gephyringens bei der Sequenzierung genomischer DNA wurden nicht gefunden. Zellulärer Stress wie Hypertermie oder Alkalose, stellte sich hingegen als hinreichend heraus, reguläres Spleißen von Gephyrin RNA zu behindern und durch die resultierende Expression dominant- negativer Gephyrine postsynaptische Gephyrinaggregation und korrespondierende Alpha2 GABA(A)R-Verankerung in primären Hippocampusneuronen mit Wildtyphintergrund einzuschränken. Somit erweist sich zellulärer Stress, wie beispielsweise Alkalose infolge epileptischer Anfälle, als möglicher Verstärker in der Entwicklung von TLE, und zwar aufgrund einer selektiv in der CA Region eingeschränkten Verfügbarkeit postsynaptischer Alpha2 GABA(A)R. Die hieraus resultierende niedrigere Schwelle zur Auslösung epileptischer Aktivität kann das neuronale Netzwerk in einer selbstpropagierenden Schleife wiederum anfälliger für Deregulierung des Gephyrinspleißens machen. Der in dieser Arbeit vorgestellte neuartige Spleißreporter (3.2, Publication 1, Supplementary Data) stellt ein molekulares Werkzeug zur Identifkation potentiell protektiver Wirkstoffe gegen zellulären Stress dar und kann der Suche nach Genen mit kompensatorischen Eigenschaften gegenüber zellulärem Stress dienen, um Ansatzpunkte für die Entwickung neuer pharmakologischer Agenzien und ursachenorientierter Therapieformen nicht nur für die Behandlung von TLE (Eichler and Meier 2008) sondern auch von anderen neurodegenerativen Erkrankungen und affektiven Persönlichkeitsstörungen zu liefern (Tyagarajan et al. 2011)