Final Report Summary - DCVFUSION (Telling the full story: how neurons send other signals than by classical synaptic transmission)
The regulated secretion of chemical signals in the brain occurs principally from two organelles, synaptic vesicles and dense core vesicles (DCVs). Synaptic vesicle secretion accounts for the well characterized local, fast signaling in synapses. DCVs contain a diverse collection of cargo, including many neuropeptides that trigger a multitude of modulatory effects with quite robust impact, for instance on memory, mood, pain, appetite or social behavior. Dysregulation of neuropeptide secretion is firmly associated with many diseases such as cognitive and mood disorders, obesity and diabetes. In addition, many other signals depend on DCVs, for instance trophic factors and proteolytic enzymes, but also signals that typically do not diffuse like guidance cues and pre-assembled active zones. DCVfusion has characterized the molecular principles that account for DCV delivery at release sites and their secretion. The project has addressed 3 fundamental questions: What are the molecular factors that drive DCV fusion in mammalian CNS neurons? How does Ca2+ trigger DCV fusion? and What are the requirements of DCV release sites and where do they occur? New cell biological and photonic approaches were developed that allow for the first time a quantitative assessment of DCV-trafficking and fusion of many cargo types, in living neurons with a single vesicle resolution, also in human neurons, re-programmed from human (patient) fibroblasts. Using this new methodology, the role of many candidate genes in DCV trafficking and fusion has now been characterized, such as Munc18-1/2, CAPS1/2, Munc13-1/2, SNAP-25, Vti1a/b, chromogranins, Synaptotagmin1, with several more underway (rab3, CaM-KII, Synaptotagmin7, Synaptobrevins/VAMPs, dynamin1/2/3, stxbp5/5l). The project also addressed pool sizes of DCVs in neurons and effective stimulation patterns. We conclude that DCVs have an exceptionally low release probability, much lower than observed for synaptic vesicles. Finally, we have characterized several calcium sensors involved in DCV trafficking and fusion, while other prime candidates were excluded, e.g. calmodulin and Ca2+/calmodulin kinase II. Together, these studies will produce the first systematic evaluation of the molecular identity of the core machinery that drives DCV fusion in mouse and human neurons, the Ca2+-affinity of DCV fusion and the characteristics of DCV release sites.