The role of DEPDC5 in the pathogenesis of mTOR-dependent epilepsy and focal cortical dysplasia

DEP-domain containing 5 (DEPDC5) is part of the GATOR1 complex that functions as key inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1) in the absence of amino acids. Mutations in DEPDC5 have been identified as the most common cause of either lesional or non-lesional focal epilepsy and are associated with mTOR hyperactivity. Recently, it has been hypothesized that somatic “second-hit” mutations occur in the brain of patients with the more severe symptomatology, including focal cortical dysplasia type II, drug-resistant epilepsy and intellectual disability. However, the mechanisms underlying dysplastic and epileptic phenotype following DEPDC5 loss-of-function, especially at the cellular levels, are still largely unknown, particularly regarding the morpho-functional impact of DEPDC5 deficiency at level of synaptic connectivity and transmission. The scope of my PhD project is to investigate the pathological changes occurring with DEPDC5 loss-of-function, with particular emphasis on cellular and synaptic morphology and physiology, and to address the role of the loss of heterozygosity in DEPDC5-related pathogenesis. As the full knockout of Depdc5 is embryonically lethal in rodents, in this study I have first characterized a heterozygous knockout mouse (Depdc5+/-), which failed to recapitulate the major phenotypic tracts of the pathology, except for a reduced PTZ-induced epileptic threshold. Therefore, to uncover the phenotype induced by Depdc5 loss-of-function, I have compared the condition of the constitutive Depdc5+/- haploinsufficent mouse with the more effective acute neuronal knockdown of Depdc5 by RNA interference. While heterozygous Depdc5+/- neurons have a very mild phenotype with morpho-functional features that are not significantly different from wild type neurons, acutely knocked down neurons exhibit a much stronger phenotype characterized by mTOR hyperactivation, increased soma size and dendritic arborization, increased excitatory synaptic transmission and intrinsic excitability of excitatory neurons, leading to an excitation/inhibition imbalance. These results uncover a novel synaptic phenotype that is causally linked to acute Depdc5 knockdown and mTOR hyperactivity, highlighting the loss of heterozygosity as causal factor for the establishment of FCD-related neuronal phenotype, and suggesting an involvement of Depdc5 in the neurodevelopmental processes. The robust synaptic phenotype resulting from acute sh-mediated, but not constitutive, Depdc5 deficiency is reminiscent of the somatic second-hit mechanism in patients with focal cortical dysplasia and, together with the increased intrinsic excitability, can trigger the epileptogenic process.