Investigating the effect of Depdc5 cKO: insights on synaptic transmission, plasticity and its role in mTOR-related epilepsy

Focal Epilepsy (FE) accounts for more than the half of the total cases of all epileptic syndrome, around 60%. It can be caused by either genetic or acquired factors, such as brain trauma, tumors or inflammation. However, it is widely recognized to have a mostly a genetic basis. Mutations at the level of the GATOR1 complex components, DEPDC5, NPRL2 and NPRL3, were found in patients with FE, confirming the key role of the GATOR1 complex in the pathogenesis of various FE syndromes. In particular, it has been discovered that mutations of DEPDC5 gene are implicated in the 5%–37% of a broad range of FEs, both lesional or non-lesional and are associated with mTOR hyperactivity. The specific mechanism underlying the epileptogenic phenotype following DEPDC5 loss is far from being clear. The aim of this thesis is to deepen the impact of Depdc5 loss-of-function at the cellular level, by focusing the attention on synaptic transmission and plasticity. In order to overcome the problem of embryonic lethality of Depdc5 full knockout in rodents, and the failure of Depdc5 constitutive heterozygous knockout mice to recapitulate the major epileptogenic traits, we used Depdc5-floxed mice and applied the Cre/LoxP technique to generate a model of Depdc5 cKO. We transfected primary cortical neurons obtained from Depdc5-floxed mice with lentiviruses expressing either active or inactive Cre recombinase and performed electrophysiological and biochemical experiments. We observed an increased in excitatory synaptic transmission and intrinsic excitability, while no difference occurred at the level of the inhibitory synaptic transmission. Given the involvement of mTOR in the mechanism of plasticity and the effect of its dysregulation on synaptic transmission, we assessed whether Depdc5 loss could also have an impact at the level of the short-term plasticity (STP). However, no effects were found at the level of either excitatory or inhibitory synaptic transmission. The data suggest that Depdc5 loss mostly induces a strengthening of excitatory transmission at the post-synaptic level and by increasing the number of connections, without directly affecting pre-synaptic mechanisms. Thus, the enhanced excitatory synaptic transmission and increased intrinsic excitability could generate a synergistic effect underlying the excitation/inhibition imbalance that triggers the epileptogenic process.