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High-throughput assay for regulated secretion of neuropeptides in mouse and human neurons.
Journal of Biological Chemistry 2024 April 26
Neuropeptides are the largest group of chemical signals in the brain. More than 100 different neuropeptides modulate various brain functions, and their dysregulation has been associated with neurological disorders. Neuropeptides are packed into dense core vesicles (DCVs), which fuse with the plasma membrane in a calcium-dependent manner. Here, we describe a novel high-throughput assay for DCV exocytosis using a chimera of Nanoluc luciferase and the DCV-cargo neuropeptide Y (NPY). The NPY-Nanoluc reporter colocalized with endogenous DCV markers in all neurons and little mis-localization to other cellular compartments. NPY-Nanoluc reported DCV exocytosis in both rodent and iPSC-derived human neurons, with similar depolarization-, Ca2+ -, RAB3- and STXBP1/MUNC18-dependence as low throughput assays. Moreover, NPY-Nanoluc accurately reported modulation of DCV exocytosis by known modulators diacylglycerol-analog and Ca2+ -channel blocker, and showed a higher assay sensitivity than a widely used single cell low throughput assay. Lastly, we showed that Nanoluc coupled to other secretory markers reports on constitutive secretion. In conclusion, the NPY-Nanoluc is a sensitive reporter of DCV exocytosis in mammalian neurons, suitable for pharmacological and genomic screening for DCV exocytosis genes and for mechanism-based treatments for CNS disorders.
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