The epilepsy gene TBC1D24 encodes a novel synaptic protein that is required for the maintenance of excitatory synapses in hippocampal neuron

Abstract

Multiple inherited neurological disorders are resulted from mutations that disrupt synapse development and function. Dozens of mutations on the human TBC1D24 gene are strongly associated with epilepsy and intellectual disability. However, the function of TBC1D24 in brain is not well understood. In particular, there is a lack of study using genetic mouse model that mimics loss-of-function TBC1D24. Here we report that TBC1D24 is required to maintain dendritic spines and excitatory synapses through inhibition of the small GTPase ARF6. Using intrabodies and super-resolution SIM microscopy, we found that TBC1D24 colocalized with PSD95 and Gephryin, a postsynaptic marker of excitatory and inhibitory synapses respectively. The knockdown of TBC1D24 by short hairpin RNA (shRNA) in cultured hippocampal neurons resulted in loss of dendritic spines and excitatory synapses, which could be rescued by inhibition of ARF6 activity. Moreover, the shRNA-mediated depletion of TBC1D24 in neurons led to impairment of both excitatory and inhibitory neurotransmission. Interestingly, we found that among those mutations that are linked to intellectual disability, the missense mutation F251L drastically reduced TBC1D24 protein expression. To better understand the functional role of TBC1D24 in vivo, we generated F251L knock-in mice by CRISPR/Cas9 system, which exhibited an increase in number but a decrease in size of Homer1 puncta in the hippocampus, enhanced susceptibility to seizure and impaired memory formation. To further investigate the function of TBC1D24 in human neurons, we differentiated TBC1D24 knockout human induced pluripotent stem cells (hiPSCs) into excitatory neurons. The TBC1D24 knockout hiPSCs-derived neurons showed a remarkable reduction in the number of synapses. Our findings therefore reveal a previously uncharacterized postsynaptic function of TBC1D24, and suggest that the impairment of synapse maintenance contributes to the pathophysiology of neurological diseases caused by TBC1D24 gene mutations. The F251L knock-in mice likely represent a useful animal model for investigation of mechanisms on TBC1D24-related neurodevelopmental disorders.