Rabphilin-3A negatively regulates neuropeptide release, through its SNAP25 interaction

Neuropeptides and neurotrophins are stored in and released from dense-core vesicles (DCVs). While DCVs and synaptic vesicles (SVs) share fundamental SNARE/SM proteins for exocytosis, a detailed understanding of DCV exocytosis remains elusive. We recently identified the RAB3-RIM1 pathway to be essential for DCV-, but not SV exocytosis, highlighting a significant distinction between the SV– and DCV secretory pathways. Whether RIM1 is the only RAB3 effector that is essential for DCV release is currently unknown. In this study, we characterized the role of rabphilin-3A (RPH3A), a known downstream effector of RAB3A, in the DCV secretory pathway using RPH3A-deficient hippocampal mouse neurons. RPH3A did not travel with DCVs, but remained stationary at synapses. In the absence of RPH3A, the number of DCV exocytosis events was 3-fold higher than in wildtype (WT) neurons. RPH3A lost its punctate distribution when it was unable to bind RAB3A. However, this was not rate limiting, as expressing a mutant RPH3A that was unable to bind RAB3A restored exocytosis to WT levels, but not when RPH3A was unable to bind SNAP25. In addition, RPH3A null neurons had longer neurites, which was partly restored when ablating all regulated secretion with tetanus neurotoxin. Taken together, we conclude that RPH3A negatively regulates DCV exocytosis, potentially also affecting neuron size. Furthermore, RAB3A interaction is required for the synaptic enrichment of RPH3A, but not for limiting DCV exocytosis. Instead the interaction of RPH3A with SNAP25 is relevant for inhibiting DCV exocytosis. ### Competing Interest Statement The authors have declared no competing interest.