Synaptotagmin 9 modulates spontaneous neurotransmitter release in striatal neurons by regulating substance P secretion

Synaptotagmin 9 (SYT9) is a tandem C2-domain Ca2+ sensor for exocytosis in neuroendocrine cells; its function in neurons remains unclear. Here, we show that endogenous SYT9 does not trigger rapid synaptic vesicle exocytosis in cultured cortical, hippocampal, or striatal neurons; rather, synaptotagmin 1 (SYT1) fulfills this function. SYT9 is able to regulate evoked synaptic vesicle exocytosis, but only when massively over-expressed. In striatal neurons, loss of SYT9 reduced the rate of spontaneous miniature neurotransmitter release events (minis). To delve into the underlying mechanism, we localized SYT9 within these neurons and found that it is targeted to dense core vesicles, where it regulates the release of substance P (SP), a neuropeptide known to modulate mini frequency. Exogenous SP, but not other striatal peptide hormones, completely rescued the Syt9 KO mini phenotype. Biochemical experiments revealed that Ca2+-binding to the C2A domain of SYT9 triggers membrane fusion in vitro, and mutations that disrupt this activity abolished the ability of SYT9 to regulate both SP release and mini frequency in striatal neurons. We conclude that SYT9 indirectly regulates synaptic transmission in striatal neurons by controlling SP release.