Potentiation of Gamma Aminobutyric Acid Receptors (GABAAR) by Ethanol: How Are Inhibitory Receptors Affected?

In recent years there has been an increase in the understanding of ethanol actions on the type A -aminobutyric acid chloride channel (GABAAR), a member of the pentameric ligand gated ion channels (pLGICs). However, the mechanism by which ethanol potentiates the complex is still not fully understood and a number of publications have shown contradictory results. Thus many questions still remain unresolved requiring further studies for a better comprehension of this effect. The present review concentrates on the involvement of GABAAR in the acute actions of ethanol and specifically focuses on the immediate, direct or indirect, synaptic and extra-synaptic modulatory effects. To elaborate on the immediate, direct modulation of GABAAR by acute ethanol exposure, electrophysiological studies investigating the importance of different subunits, and data from receptor mutants will be examined. We will also discuss the nature of the putative binding sites for ethanol based on structural data obtained from other members of the pLGICs family. Finally, we will briefly highlight the glycine gated chloride channel (GlyR), another member of the pLGIC family, as a suitable target for the development of new pharmacological tools

Presence of Inhibitory Glycinergic Transmission in Medium Spiny Neurons in the Nucleus Accumbens

It is believed that the rewarding actions of drugs are mediated by dysregulation of the mesolimbic dopaminergic system leading to increased levels of dopamine in the nucleus accumbens (nAc). It is widely recognized that GABAergic transmission is critical for neuronal inhibition within nAc. However, it is currently unknown if medium spiny neurons (MSNs) also receive inhibition by means of glycinergic synaptic inputs. We used a combination of proteomic and electrophysiology studies to characterize the presence of glycinergic input into MSNs from nAc demonstrating the presence of glycine transmission into nAc. In D1 MSNs, we found low frequency glycinergic miniature inhibitory postsynaptic currents (mIPSCs) which were blocked by 1 μM strychnine (STN), insensitive to low (10, 50 mM) and high (100 mM) ethanol (EtOH) concentrations, but sensitive to 30 μM propofol. Optogenetic experiments confirmed the existence of STN-sensitive glycinergic IPSCs and suggest a contribution of GABA and glycine neurotransmitters to the IPSCs in nAc. The study reveals the presence of glycinergic transmission in a non-spinal region and opens the possibility of a novel mechanism for the regulation of the reward pathway

A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy

C-to-U RNA editing of glycine receptors (GlyR) can play an important role in disease progression of temporal lobe epilepsy (TLE) as it may contribute in a neuron type-specific way to neuropsychiatric symptoms of the disease. It is therefore necessary to develop tools that allow identification of neuron types that express RNA-edited GlyR protein. In this study, we identify NH4 as agonist of C-to-U RNA edited GlyRs. Furthermore, we generated a new molecular C-to-U RNA editing sensor tool that detects Apobec-1- dependent RNA editing in HEPG2 cells and rat primary hippocampal neurons. Using this sensor combined with NH4 application, we were able to identify C-to-U RNA editing-competent neurons and expression of C-to-U RNA-edited GlyR protein in neurons. Bioinformatic analysis of 1,000 Genome Project Phase 3 allele frequencies coding for human Apobec-1 80M and 80I variants showed differences between populations, and the results revealed a preference of the 80I variant to generate RNA-edited GlyR protein. Finally, we established a new PCR-based restriction fragment length polymorphism (RFLP) approach to profile mRNA expression with regard to the genetic APOBEC1 dimorphism of patients with intractable temporal lobe epilepsy (iTLE) and found that the patients fall into two groups. Patients with expression of the Apobec-1 80I variant mostly suffered from simple or complex partial seizures, whereas patients with 80M expression exhibited secondarily generalized seizure activity. Thus, our method allows the characterization of Apobec-1 80M and 80l variants in the brain and provides a new way to epidemiologically and semiologically classify iTLE according to the two different APOBEC1 alleles. Together, these results demonstrate Apobec-1-dependent expression of RNA-edited GlyR protein in neurons and identify the APOBEC1 80I/M-coding alleles as new genetic risk factors for iTLE patients

K-shell X-ray spectroscopy of laser produced aluminum plasma

Optimization of a laser produced plasma (LPP) X-ray source has been performed by analyzing K-shell emission spectra of Al plasma at a laser intensity of 1013-1014 W/cm2. The effect of varying the laser intensity on the emissivity of the K-shell resonance lines is studied and found to follow a power law, E α I α with α=2.2, 2.3, 2.4 for Heβ, Heγ, Heδ respectively. The emission of these resonance lines has been found to be heavily anisotropic. A Python language based code has been developed to generate an intensity profile of K-shell spectral lines from the raw data. In theoretical calculations, the temperature is estimated by taking the ratio of the Li-like satellite (1s2 2p-1s2p3p) and the Heβ (1s2 -1s3p) resonance line and the ratio of the He-like satellite (1s2p-2p2 ) and the Lyα (1s-2p) resonance line. To determine the plasma density, stark broadening of the Lyβ spectral line is used. Simulation was carried out using the FLYCHK code to generate a synthetic emission spectrum. The results obtained by FLYCHK are Te=160 eV, Th=1 keV, f=0.008, ne=5 x 1020 cm-3 and the analytical model resulted Te=260-419 eV and ne=3x1020 cm-3

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)

Glycine receptors caught between genome and proteome - functional implications of RNA editing and splicing

Information processing in the brain requires a delicate balance between excitation and inhibition. Glycine receptors (GlyR) are involved in inhibitory mechanisms mainly at a synaptic level, but potential novel roles for these receptors recently emerged due to the discovery of posttranscriptional processing. GLR transcripts are edited through enzymatic modification of a single nucleotide leading to amino acid substitution within the neurotransmitter binding domain. RNA editing produces gain-of-function receptors well suited for generation and maintenance of tonic inhibition of neuronal excitability. As neuronal activity deprivation in early stages of development or in epileptic tissue is detrimental to neurons and because RNA editing of GlyR is up-regulated in temporal lobe epilepsy patients with a severe course of disease a pathophysiological role of these receptors emerges. This review contains a state-of-the-art discussion of (patho)physiological implications of GlyR RNA editing

Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development

Extracellular matrix (ECM) molecules are pivotal for central nervous system development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axonal guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during central nervous system development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn towards the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons