SignificanceMutations in genes regulating neurotransmission in the brain are implicated in neurological disorders and neurodegeneration. Synapsin is a crucial regulator of neurotransmission and allows synapses to maintain a large reserve pool of synaptic vesicles. Human mutations in synapsin genes are linked to epilepsy and autism. How synapsin function is regulated to allow replenishment of synaptic vesicles and sustain neurotransmission is largely unknown. Here we identify a function for the endocytic scaffold protein intersectin, a protein overexpressed in patients with Down syndrome, as a regulator of synapsin nanoscale distribution and function that is controlled by a phosphorylation-dependent autoinhibitory switch. Our results unravel a hitherto unknown molecular connection between the machineries for synaptic vesicle reserve pool organization and endocytosis.

Highlights d Reagents that bind to the IDR of synapsin cause dispersal of synaptic vesicle clusters d Inhibition of SH3 domain interactions with synapsin do not cause vesicle dispersal d Synaptic vesicle clustering in vivo can be explained by phase separation

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