LOSS OF TBC1D24 RESULTS IN IMPAIRED SYNAPTIC VESICLE CYCLING AT EXCITATORY BOUTONS AND DISRUPTED NETWORK STABILITY​​​​​

TBC1D24 is mutated across a wide spectrum of neurodevelopmental disorders, ranging from mild epilepsy to severe early‑onset epileptic encephalopathy. Although TBC1D24 has been implicated in neuronal development and synaptic vesicle (SV) trafficking, its role in the regulation of excitation inhibition balance and neuronal network excitability has remained unclear. To dissect its synaptic functions, we performed live-imaging experiments of mouse hippocampal neurons using the Synaptophysin‑pHluorin probe coupled to V-GAT live labeling to monitor SV acidification and recycling dynamics at excitatory and inhibitory terminals. Tbc1d24 knockout neurons revealed a pronounced defect in vesicle re-acidification and endocytic recovery. These impairments were selectively observed at excitatory boutons, while inhibitory terminals showed minimal alterations. Consistent with this functional specificity, Tbc1d24 protein was found to be predominantly enriched at excitatory presynaptic sites. These results indicate that TBC1D24 is required for efficient vesicle acidification and proper SV recycling at excitatory synapses. To translate these findings to a human context, we generated NGN2-induced excitatory neurons (iNeurons) from patients carrying TBC1D24 mutations and respective controls. Multi-electrode array recordings uncovered a dysregulated electrophysiological phenotype, characterized by prolonged network bursts and disrupted temporal organization, reflecting impaired excitatory synaptic homeostasis. Collectively, our data identify a synapse-type-specific vulnerability to TBC1D24 loss, linking impaired vesicle recycling to excitatory synaptic dysfunction and network instability. Ongoing synaptosome-based proteomic and lipidomic analyses will delineate the molecular signatures associated with TBC1D24 loss. This integrated approach will help to support the dissection of the pathophysiology of TBC1D24 diseases and the development of targeted therapeutic interventions.