A role in pH homeostasis regulation by genes related to Neurodevelopmental Disorders: TBC1D24 and ATP6V1A.

TBC1D24 is a gene mutated in a spectrum of neurodevelopmental disorders, from mild epilepsy to severe epileptic encephalopathy. TBC1D24 is involved in brain development, synaptic vesicle trafficking and synaptic function; yet the molecular mechanisms mediating these roles and their relationship to brain dysfunction are largely unknown. TBC1D24 is unique in containing conserved TBC and TLDc domains; importantly, TLDc proteins have been recently described as interactors of the essential complex of V-ATPase. V-ATPase is a multisubunit proton pump that acidifies intracellular organelles through the hydrolysis of ATP. In this thesis, we aimed to explore the interaction between Tbc1d24 and V-ATPase and its physio-pathological role in neuronal cells. We found that Tbc1d24 interacts with the V-ATPase V1 cytosolic domain subunits ATP6V1A and ATP6V1B2 in the brain. By employing a mouse model of chronic loss of Tbc1d24, we demonstrated that loss of Tbc1d24 led to a cytosolic shift of ATP6V1A and ATP6V1B2 subunits, suggesting an unproper assembly state of the complex. This phenotype was accompanied by the alteration of intracellular organelles acidification with increased pH and impairment of autophagic flux. Given the localization of Tbc1d24 at synaptic sites and the relevance of V-ATPase proton pumping activity in synaptic vesicles, we evaluated synaptic ultrastructure and synaptic vesicles acidification. In lack of Tbc1d24, presynaptic compartments showed fewer synaptic vesicles and the accumulation of aberrant endosomal-like structures that engulfed the pre-synapse. Moreover, we found an increased luminal pH of synaptic vesicles. Concurrently, in collaboration with the group of Professor Guerrini (Meyer Hospital, Florence) we identified de novo mutations in ATP6V1A in patients affected by epileptic encephalopathy with different severity. We characterized patients’ fibroblasts finding that pathogenic mutations can alter protein stability and the overall functionality of V-ATPase complex, leading to alterations in the endo-lysosomal pH. Ultrastructural analysis of patients’ fibroblasts and patients-derived iNeurons revealed the presence of aberrant lysosomes engulfed with different non-degraded materials, a typical hallmark of lysosomal diseases. All together these findings uncover a novel function for Tbc1d24 as regulator of V-ATPase activity and suggest pH dysregulation as key cellular mechanism that possibly underpin the pathogenesis in TBC1D24- and ATP6V1A-associated disorders.