SUMMARY
Neuronal DCV exocytosis releases multiple signalling molecules, including neurotrophins and neuropeptides. These signalling molecules play a role in for instance neuronal development and plasticity. Currently, the secretory mechanisms that regulate fusion of neuronal DCVs with the plasma membrane are incompletely understood. In this thesis, we provide deeper understanding of the molecular mechanisms driving DCV exocytosis in neurons.
In Chapter 2 and 3, we show that DCV exocytosis increases 10-fold during neuronal in vitro development between DIV 4 and DIV 10. The release probability of neuronal DCVs reaches a plateau at DIV 6. Although VAMP2 levels are low in young neurons, it is not rate-limiting for DCV exocytosis. During in vitro development, the number of DCVs increases and their size remains constant. DCVs in immature neurons contain more often SCG2 and ChgA compared to ChgB and BDNF. In conclusion, the number of DCVs and the number of DCV fusion events increase during neuronal development.
In Chapter 3 and 4, we studied the role of the v-SNARE VAMP/synaptobrevin protein family in DCV exocytosis. We show that DCVs require multiple TeNT-sensitive VAMP/synaptobrevin proteins in their secretory pathway. VAMP1 and VAMP2 co-localize and co-traffic with DCVs. VAMP2 is highly enriched at the pre-synapse and its levels increase during neuronal development. VAMP1 is also present, but not enriched, at synapses. VAMP1 levels are relatively high in young neurons. In DIV 4 neurons, DCV exocytosis is mediated by TeNT-sensitive VAMPs and not by VAMP7. In conclusion, TeNT-sensitive VAMPs, most likely VAMP1 and VAMP2, are the v-SNARE proteins regulating DCV exocytosis in neurons.
In Chapter 5, we investigated the importance of several protein domains in the canonical vesicle priming protein CAPS-1. We identify that the C-terminus of CAPS-1, which contains the MHD1 and DCV domain, is essential for its enrichment at the pre-synapse. In addition, all the functional domains of CAPS-1 are required for its role in neuronal DCV exocytosis. Highly reduced DCV exocytosis in CAPS DKO neurons does not affect neurite outgrowth, synapse number, DCV biogenesis or DCV distribution. Hence, CAPS-1 requires its C2, PH, MHD1 and DCV domains for DCV exocytosis but CAPS-1 is not essential in neuronal development.
Our studies show differences in the molecular mechanisms for exocytosis of DCVs compared to SVs. First, in CAPS DKO neurons, DCV exocytosis is reduced by 96%, but SV exocytosis only with 39% using the same intense stimulation (Chapter 5). Second, TI-VAMP2 expression rescues SV exocytosis, but not DCV exocytosis, in TeNT treated neurons (Chapter 4). Third, maturation of the DCV fusion machinery occurs before SV fusion machinery maturation (Chapter 3).