GABAergic mechanisms in epilepsy and contribution of the ClC-2 chloride channel to neuronal excitability

In this thesis, I aimed to investigate the role of anion conductances for neuronal hyperexcitability and inherited epilepsy. I examined the functional consequences of novel epilepsy-causing mutations identified in GABAA or GABAB receptors. Additionally, a ClC-2 knockout (KO) mouse model was used to investigate the ClC-2 channel contribution to neuronal excitability in the thalamus and hippocampus. In the first part, four novel mutations, identified by our collaborators in the alpha-3 subunit of the GABAA receptor (gene GABRA3 on the X chromosome), were functionally characterized. Two mutations, Q242L and T166M, were found in two families with epileptic encephalopathy (EE), intellectual disability (ID), and other symptoms, as nystagmus, cleft palate and micrognathia. The EE phenotype followed an X-chromosomal inheritance pattern, affecting more severely the males, whereas females were mildly affected. Furthermore, two non-co-segregating mutations were detected, T336M in one of two sisters affected with idiopathic/genetic generalized epilepsy (IGE/GGE), and G47R in one of two brothers with autism spectrum disorder, both inherited by their unaffected mothers. To assess the pathogenicity of these variants, we introduced them into the cDNA of the human GABRA3 gene and compared their properties to the wildtype (WT) using a Xenopus laevis expression system. The cRNAs coding for alpha-3 WT or mutant GABAA receptor subunits were co-injected with β2 and γ2 subunits and recordings were performed using automated two-electrode voltage-clamp recordings. A strong loss-of-function with >75% reduction in GABA-induced current amplitudes in comparison to the WT was found for both mutations associated with EE (Q242L and T166M), and the one associated with IGE (T336M). The reduction in GABA-induced current was much less pronounced (46.04 ± 9.46%) for G47R. The obtained results suggest that GABRA3 mutations with a severe loss-of-function can cause X-linked EE with dysmorphic features. The missing co-segregation and genotype-phenotype correlation for the IGE- and autism-associated mutations imply that further genetic factors are involved to cause the disease in these families. In the second part of my thesis, I have analyzed two de novo mutations found in GABBR2, encoding subunit 2 of the metabotropic GABAB receptor, in two unrelated patients with severe EE. Both patients present with profound ID and severe seizures. Functional analysis was also performed in Xenopus laevis oocytes. cRNAs of GABAB receptor subunits 1 and 2 were co-injected to form functional GABAB receptor, and Kir3.1/3.2 K+ channels were co-expressed as a reporter. Both mutations caused a gain of channel function manifesting at lower GABA concentrations. A significant increase in GABA sensitivity was found for I705N (EC50 of 0.48 ± 0.16 µM vs. 3.98 ± 0.68 µM for the WT) and S695I (EC50 of 0.31 ± 0.2 µM vs. 3.96 ± 0.85 µM for the WT). Additionally, a significantly slower deactivation time constant in comparison with the WT, indicating a gain of function effect was observed for the S695I mutation. The increased sensitivity of both mutations at nanomolar GABA concentration, suggest a common pathomechanism based on increased activity of GABABRs at extrasynaptic sites. Nevertheless, for a deeper understanding of how gain of function of the GABAB receptor could mediate the epileptic phenotype, further functional investigations using neuronal cells are necessary. In the third part of my thesis, I worked on the contribution of ClC-2 channels to neuronal excitability. To this end, I analyzed neuronal activity in thalamo-cortical brain slices and hippocampal primary cultures obtained from WT and ClC-2 KO mice. Recordings from brain slices revealed that the action potential firing rate was significantly reduced in inhibitory neurons of the nucleus reticularis thalami (NRT) in ClC-2 KO mice in comparison with those from WT animals, suggesting less inhibition in the thalamocortical system and possibly increased excitability of the whole network. To verify this hypothesis, I recorded network activity using extracellular synchronous recordings in NRT, ventrobasal nucleus (VB) and cortex. An increase in synchronized activity in the three areas was observed in ClC-2 KO mice when GABAA receptors were blocked by picrotoxin, suggesting that the lack of ClC-2 may present a susceptibility factor for increased neuronal excitability. In addition, microelectrode array (MEA) recordings of hippocampal primary cultured neurons derived from KO and WT mice revealed that the lack of ClC-2 increases neuronal excitability by increasing the duration of spontaneous burst activity. Furthermore, the network properties of neurons derived from ClC-2 KO mice were not further altered by the GABAA receptor antagonist bicuculline in contrast to those of WT mice, suggesting that the loss of ClC-2 alone modulates the action of GABAA receptors. These results suggest that the ClC-2 channel has complex and important physiological functions and does play a role in the regulation of neuronal excitability. Altogether, my thesis shows the importance of three players of neuronal excitability and inhibitory function of the nervous system, revealing different roles for epileptogenic mutations in GABAA and GABAB receptor subunits, and loss of the ClC-2 Cl- channel

Rare coding variants in genes encoding GABA_A receptors in genetic generalised epilepsies: an exome-based case-control study

BACKGROUND: Genetic generalised epilepsy is the most common type of inherited epilepsy. Despite a high concordance rate of 80% in monozygotic twins, the genetic background is still poorly understood. We aimed to investigate the burden of rare genetic variants in genetic generalised epilepsy. METHODS: For this exome-based case-control study, we used three different genetic generalised epilepsy case cohorts and three independent control cohorts, all of European descent. Cases included in the study were clinically evaluated for genetic generalised epilepsy. Whole-exome sequencing was done for the discovery case cohort, a validation case cohort, and two independent control cohorts. The replication case cohort underwent targeted next-generation sequencing of the 19 known genes encoding subunits of GABAA receptors and was compared to the respective GABAA receptor variants of a third independent control cohort. Functional investigations were done with automated two-microelectrode voltage clamping in Xenopus laevis oocytes. FINDINGS: Statistical comparison of 152 familial index cases with genetic generalised epilepsy in the discovery cohort to 549 ethnically matched controls suggested an enrichment of rare missense (Nonsyn) variants in the ensemble of 19 genes encoding GABAA receptors in cases (odds ratio [OR] 2·40 [95% CI 1·41-4·10]; pNonsyn=0·0014, adjusted pNonsyn=0·019). Enrichment for these genes was validated in a whole-exome sequencing cohort of 357 sporadic and familial genetic generalised epilepsy cases and 1485 independent controls (OR 1·46 [95% CI 1·05-2·03]; pNonsyn=0·0081, adjusted pNonsyn=0·016). Comparison of genes encoding GABAA receptors in the independent replication cohort of 583 familial and sporadic genetic generalised epilepsy index cases, based on candidate-gene panel sequencing, with a third independent control cohort of 635 controls confirmed the overall enrichment of rare missense variants for 15 GABAA receptor genes in cases compared with controls (OR 1·46 [95% CI 1·02-2·08]; pNonsyn=0·013, adjusted pNonsyn=0·027). Functional studies for two selected genes (GABRB2 and GABRA5) showed significant loss-of-function effects with reduced current amplitudes in four of seven tested variants compared with wild-type receptors. INTERPRETATION: Functionally relevant variants in genes encoding GABAA receptor subunits constitute a significant risk factor for genetic generalised epilepsy. Examination of the role of specific gene groups and pathways can disentangle the complex genetic architecture of genetic generalised epilepsy. FUNDING: EuroEPINOMICS (European Science Foundation through national funding organisations), Epicure and EpiPGX (Sixth Framework Programme and Seventh Framework Programme of the European Commission), Research Unit FOR2715 (German Research Foundation and Luxembourg National Research Fund)

Rare coding variants in genes encoding GABA(A) receptors in genetic generalised epilepsies : an exome-based case-control study

Background Genetic generalised epilepsy is the most common type of inherited epilepsy. Despite a high concordance rate of 80% in monozygotic twins, the genetic background is still poorly understood. We aimed to investigate the burden of rare genetic variants in genetic generalised epilepsy. Methods For this exome-based case-control study, we used three different genetic generalised epilepsy case cohorts and three independent control cohorts, all of European descent. Cases included in the study were clinically evaluated for genetic generalised epilepsy. Whole-exome sequencing was done for the discovery case cohort, a validation case cohort, and two independent control cohorts. The replication case cohort underwent targeted next-generation sequencing of the 19 known genes encoding subunits of GABA(A) receptors and was compared to the respective GABA(A) receptor variants of a third independent control cohort. Functional investigations were done with automated two-microelectrode voltage clamping in Xenopus laevis oocytes. Findings Statistical comparison of 152 familial index cases with genetic generalised epilepsy in the discovery cohort to 549 ethnically matched controls suggested an enrichment of rare missense (Nonsyn) variants in the ensemble of 19 genes encoding GABA(A) receptors in cases (odds ratio [OR] 2.40 [95% CI 1.41-4.10]; p(Nonsyn)=0.0014, adjusted p(Nonsyn)=0.019). Enrichment for these genes was validated in a whole-exome sequencing cohort of 357 sporadic and familial genetic generalised epilepsy cases and 1485 independent controls (OR 1.46 [95% CI 1.05-2.03]; p(Nonsyn)=0.0081, adjusted p(Nonsyn)=0.016). Comparison of genes encoding GABA(A) receptors in the independent replication cohort of 583 familial and sporadic genetic generalised epilepsy index cases, based on candidate-gene panel sequencing, with a third independent control cohort of 635 controls confirmed the overall enrichment of rare missense variants for 15 GABA(A) receptor genes in cases compared with controls (OR 1.46 [95% CI 1.02-2.08]; p(Nonsyn)=0.013, adjusted p(Nonsyn)=0.027). Functional studies for two selected genes (GABRB2 and GABRA5) showed significant loss-of-function effects with reduced current amplitudes in four of seven tested variants compared with wild-type receptors. Interpretation Functionally relevant variants in genes encoding GABA(A) receptor subunits constitute a significant risk factor for genetic generalised epilepsy. Examination of the role of specific gene groups and pathways can disentangle the complex genetic architecture of genetic generalised epilepsy. Copyright (C) 2018 The Author(s). Published by Elsevier Ltd.Peer reviewe

In vitro neuronal network activity as a new functional diagnostic system to detect effects of Cerebrospinal fluid from autoimmune encephalitis patients

Abstract The intent of this study was to investigate if cerebrospinal fluid (CSF) from autoimmune encephalitis (AE) patients regulates in vitro neuronal network activity differentially to healthy human control CSF (hCSF). To this end, electrophysiological effects of CSF from AE patients or hCSF were measured by in vitro neuronal network activity (ivNNA) recorded with microelectrode arrays (MEA). CSF from patients with either N-methyl-D-aspartate-receptor-antibody (pCSFNMDAR, n = 7) or Leucine-rich-glioma-inactivated-1-Ab (pCSFLGI1, n = 6) associated AE suppressed global spiking activity of neuronal networks by a factor of 2.17 (p < 0.05) or 2.42 (p < 0.05) compared to hCSF. The former also suppressed synchronous network bursting by a factor of 1.93 (p < 0.05) in comparison to hCSF (n = 13). As a functional diagnostic test, this parameter reached a sensitivity of 86% for NMDAR-Ab- and 100% for LGI1-Ab-associated AE vs. hCSF at a specificity of 85%. To explore if modulation at the NMDAR influences effects of hCSF or pathological CSF, we applied the NMDAR-antagonist 2-Amino-5-phosphono-pentanoic acid (AP5). In CSF from NMDAR-Ab-associated AE patients, spike rate reduction by AP5 was more than 2-fold larger than in hCSF (p < 0.05), and network burst rate reduction more than 18-fold (p < 0.01). Recording ivNNA might help discriminating between functional effects of CSF from AE patients and hCSF, and thus could be used as a functional diagnostic test in AE. The pronounced suppression of ivNNA by CSF from NMDAR-Ab-associated AE patients and simultaneous antagonism at the NMDAR by AP5, particularly in burst activity, compared to hCSF plus AP5, confirms that the former contains additional ivNNA-suppressing factors

Additional file 1: Table S1. of SRF modulates seizure occurrence, activity induced gene transcription and hippocampal circuit reorganization in the mouse pilocarpine epilepsy model

Excel sheet including all microarray data. (XLSX 44537 kb

Characterization of the GABRB2-Associated Neurodevelopmental Disorders

Contains fulltext : 231652.pdf (Publisher’s version ) (Closed access)OBJECTIVE: We aimed to characterize the phenotypic spectrum and functional consequences associated with variants in the gene GABRB2, coding for the γ-aminobutyric acid type A (GABA(A) ) receptor subunit β2. METHODS: We recruited and systematically evaluated 25 individuals with variants in GABRB2, 17 of whom are newly described and 8 previously reported with additional clinical data. Functional analysis was performed using a Xenopus laevis oocyte model system. RESULTS: Our cohort of 25 individuals from 22 families with variants in GABRB2 demonstrated a range of epilepsy phenotypes from genetic generalized epilepsy to developmental and epileptic encephalopathy. Fifty-eight percent of individuals had pharmacoresistant epilepsy; response to medications targeting the GABAergic pathway was inconsistent. Developmental disability (present in 84%) ranged from mild intellectual disability to severe global disability; movement disorders (present in 44%) included choreoathetosis, dystonia, and ataxia. Disease-associated variants cluster in the extracellular N-terminus and transmembrane domains 1-3, with more severe phenotypes seen in association with variants in transmembrane domains 1 and 2 and the allosteric binding site between transmembrane domains 2 and 3. Functional analysis of 4 variants in transmembrane domains 1 or 2 (p.Ile246Thr, p.Pro252Leu, p.Ile288Ser, p.Val282Ala) revealed strongly reduced amplitudes of GABA-evoked anionic currents. INTERPRETATION: GABRB2-related epilepsy ranges broadly in severity from genetic generalized epilepsy to developmental and epileptic encephalopathies. Developmental disability and movement disorder are key features. The phenotypic spectrum is comparable to other GABA(A) receptor-encoding genes. Phenotypic severity varies by protein domain. Experimental evidence supports loss of GABAergic inhibition as the mechanism underlying GABRB2-associated neurodevelopmental disorders. ANN NEUROL 2021;89:573-586