Following Neuropeptide Secretion: The role of SM-proteins in Dense Core Vesicle exocytosis

The general aim of this thesis was to reveal the role of SM-proteins in neuronal secretion, and in particular, neuropeptide secretion from DCVs. Neuropeptide signaling occurs throughout the brain, with each neuron having its own subset of neuropeptides and receptors. This signaling network is important for many brain functions including memory, sleep, appetite and fear. Although the molecular determinants of neurotransmission are well described, including the essential role of SM-protein MUNC18-1, it has remained unclear which SM-protein is required for DCV exocytosis. We identified MUNC18-1 as the SM-protein for DCV exocytosis and showed that STXBP1 has the same role in human iNeurons. Additionally, we found evidence that reduced MUNC18-1/STXBP1 expression decreases DCV exocytosis, implicating neuropeptide secretion defects in STXBP1 syndrome. In search of a role of MUNC18-2 in neuronal secretion, we found that MUNC18-2 is not required for SV, DCV or GLUT4 exocytosis in mammalian neurons. Finally, because the upstream mechanisms required for DCV exocytosis are less well understood, we tested methods to investigate DCV biogenesis and maturation. We started with searching for the SM-protein required for mammalian DCV exocytosis. In Chapter 2, we identified MUNC18-1 as the essential SM-protein for neuropeptide secretion in primary mouse neurons, while MUNC18-2 and -3 did not support DCV exocytosis in absence of MUNC18-1. In addition, we showed that the reduced expression levels of MUNC18-1 in Munc18-1 HZ neurons led to a decrease in DCV exocytosis. This suggests that the neurodevelopmental, behavioral and cognitive deficits that Munc18-1 HZ mice display may be in part due to defects in neuropeptide secretion and that this provides a possible additional disease mechanism in STXBP1 syndrome. In Chapter 3, we tested DCV exocytosis in human iNeurons, which was readily visible as NPY-pHluorin dequenching events upon high-frequency train-stimulation, similar to previous experiments in mouse CNS neurons. We showed that STXBP1 in human iNeurons is essential for DCV exocytosis. In addition, we found that a STXBP1 syndrome patient mutation generated with Crispr-Cas9 reduced STXBP1 protein levels and hampered DCV exocytosis. Therefore, it is plausible that defective neuropeptide secretion caused by mutations in STXBP1 contributes to the neurodevelopmental, cognitive, psychiatric and neurological symptoms in STXBP1 syndrome. In Chapter 4, we tested the role of MUNC18-2 in neuronal secretion. We used conditional knock-out in primary mouse neurons to show that MUNC18-2 is not required for DCV exocytosis, or any property of DCV exocytosis including opening of the fusion pore and full collapse of the vesicle. Furthermore, we tested GLUT4 exocytosis marker GLUT4-pHluorin and observed GLUT4 exocytosis events during basal conditions and increased exocytosis upon high-frequency stimulation. A stimulation similar to natural burst-firing was most effective in triggering GLUT4 exocytosis. Next, we showed that MUNC18-2 is not required for basal or stimulated GLUT4 exocytosis. In search of upstream mechanisms important for DCV exocytosis, we explored tools to visualize DCV biogenesis and maturation in Chapter 5. Here, we tested DCV labeling with SNAP-Cell, which only sparsely labeled DCVs. In parallel, we tested a genetic pulse-chase strategy by doxycycline-induced expression of DCV cargo NPY-pHluorin. Induction of NPY-pHluorin led to a highly variable number of labeled DCVs in primary mouse neurons, which could not be explained by viral load and thus suggests that individual neurons possess different DCV biogenesis rates.