Abstracts

Rationale: Dravet syndrome (DS) is a rare epileptic encephalopathy mostly caused by mutations of SCN1A gene, encoding the a subunit of the voltage-gated sodium channel NaV1.1. Due to the genetics of this pathology, most studies focused on the functional and molecular  alterations of sodium channels and these efforts led to the formulation of the “interneuron hypothesis”. Indeed, many studies using mouse models of DS showed decreased GABA release from interneurons due to the preferential expression of NaV1.1 that show a “loss of function”, inducing a higher excitability of pyramidal neurons. Even if the relationship between this GABAergic dysfunction and sodium channel mutations may appear linear, the existence of other pathological events that underlie epileptogenesis in DS can not be excluded. Indeed, a GABAergic impairment has been previously described (Yu et al., 2006; Kurbatova et al., 2016; Stern et al., 2017) and alterations of GABAA receptor (GABAAR) function and subunit expression using human DS brain tissues have been shown (Ruffolo et al., 2018). Beyond the GABAergic system, the scenario could be more complex if we consider a putative role of an alteration of glutamatergic neurotransmission in DS as previously shown for other epileptic neurodevelopmental diseases (Ruffolo et al., 2016; Talos et al. 2012). Our main goal is to demonstrate that an altered neurotransmission, together with the loss of function of sodium channels, could explain not only the epileptic hyperexcitability of DS but also the cognitive impairment and the social behavior (i.e., autistic spectrum) of these patients that could stem from a sort of “brain dysmaturity”. In the light of this hypothesis, we delved deeper into the physiopathology of GABAergic neurotransmission in DS, extending the study also to glutamatergic transmission, in order to create new therapeutic opportunities for these patients, that almost invariably suffer of “drug-resistant” seizures. Methods: Here, for the first time, we transplanted in Xenopus oocytes cell membranes obtained  from autoptic brain tissue of Dravet patients, tuberous sclerosis complex (TSC) patients as pathological comparison, and age-matched controls. Additional experiments were performed on oocytes expressing human a1/a2b2g2 and a1/a2b2 GABAARs. GABAA currents were recorded using the two-microelectrodes voltage-clamp technique. Quantitative RT-PCR, immunohistochemistry and double-labelling techniques were carried out on the same tissue samples. Results: We found: (i) a decrease in GABA sensitivity in Dravet compared to controls, which was related to an increase in a4- relative to a1 or a2-containing GABAA receptors; (ii) a shift of the GABA reversal potential towards more depolarizing values in Dravet, and a parallel increase of the chloride transporters NKCC1/KCC2 expression ratio; (iii) an increase of GABAA currents induced by low doses of cannabidiol (CBD) both in Dravet and TSC comparable to that induced by flunitrazepam, and that still persists in g-less GABAA receptors; (iv) no difference in AMPA sensitivity nor in AMPA/GABA current ratio in Dravet patients compared to age-matched controls. Conclusions: Our study indicates that a dysfunction of the GABAergic but not glutamatergic AMPA-mediated system can contribute to a general reduction of inhibitory efficacy in Dravet brain, suggesting that GABAA receptors could be a target for new therapies. Funding: AICE-FIRE