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Étude fonctionnelle de mutations des canaux sodiques potentiel-dépendants Nav1.1 et Nav1.2 : corrélation phénotype/génotype et mise en évidence d’un mécanisme spécifique pour les troubles du spectre de l’autisme

Abstract : The genes encoding for the voltage-gated sodium channels (Nav) expressed in the central nervous system are the target of numerous mutations leading to various phenotypes. The aim of my work is to understand why mutations in the same gene can lead to distinct pathologies in order to consider the development of new therapeutic approaches. The SCN1A gene encoding for the Nav1.1 channels, mainly expressed in GABAergic interneurons (GABA IN), is the target of mutations responsible for epileptic syndromes and familial hemiplegic migraine (FHM-3), a rare form of migraine with aura. The mutations responsible for epilepsy have been shown to cause a loss of function, which leads to hypoexcitability of GABA IN and subsequently to the network hyperexcitability. At the opposite, the mutations responsible for MHF-3 showed a gain of function and hyperexcitability of GABA IN which can lead to the cortical spreading depression, a pathological mechanism of migarine. In particular, the functional study of the L1649Q mutation showed that the mutation leads to an important decrease of the current density (loss of function). Analysis of the biophysical properties of the mutated channels after partial recovery of the current density showed that the overall effect of the mutation is a gain of function, consistent with an hyperexcitability of GABA IN (Cestele and al. 2013 PNAS). In order to identify if other FHM-3 mutations share the same mechanism (loss / gain of function), the first part of my thesis aimed to characterize a new mutation responsible for MHF-3, L1670W. This mutation leads to a defect in the Nav1.1 channels expression at the membrane but after partial recovery of the current density, the mutation induces a clear gain of function of Nav1.1 channels. These results showed that the L1670W mutation, like the L1649Q mutation, leads to a defect in the Nav1.1 channels expression at the membrane and a gain in function, thus reinforcing the hypothesis that this mechanism could be generalized to other mutations responsible for MHF-3. The SCN2A gene encodes for the α subunit of Nav1.2 channels mainly expressed in excitatory neurons. Mutations in the SCN2A gene are responsible for different pathologies such as benign epilepsies, epileptic encephalopathies and autism spectrum disorder (ASD). To date, the detailed mechanisms responsible for these different pathologies remain unclear. In order to elucidate the genotype/phenotype relationship, we studied the functional effects of 23 SCN2A mutants responsible for these different pathologies. Our results show that all the mutations responsible for ASD induce an important decrease (almost complete) of the current density while for the other pathologies the effects are heterogeneous. In order to reproduce the heterozygous conditions, we studied the co-expression of wild-type (WT) channels with each mutated channel. Our results showed a reduction in the WT channels current density only in the presence of channels carrying mutations responsible for ASD. Consequently, only the mutations responsible for ASD induce a negative dominance on WT channels. To determine whether this negative dominance mechanism is due to the interaction of α subunits described recently (Clatot et al., 2018 Nat Commun), we used different strategies to inhibit this interaction. The results obtained showed that the negative dominance effect of the mutants responsible for ASD is no longer observed when the interaction between the α subunits is inhibited. Therefore, our results allow us to describe for the first time that mutations in Na+ channels responsible for ASD act by a negative dominance mechanism, which is mediated by the interaction between WT and mutated channels.
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Submitted on : Tuesday, January 4, 2022 - 2:43:39 PM
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Sandra Dhifallah. Étude fonctionnelle de mutations des canaux sodiques potentiel-dépendants Nav1.1 et Nav1.2 : corrélation phénotype/génotype et mise en évidence d’un mécanisme spécifique pour les troubles du spectre de l’autisme. Biologie moléculaire. Université Côte d'Azur, 2020. Français. ⟨NNT : 2020COAZ6004⟩. ⟨tel-03510363⟩

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