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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. |
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