# High-throughput assay for regulated secretion of neuropeptides in mouse and human neurons

## TL;DR

This paper introduces a new high-throughput method to study how neuropeptides are released from neurons in mice and humans.

## Contribution

A novel, sensitive assay using a NPY-Nanoluc reporter for studying dense core vesicle exocytosis in mammalian neurons.

## Key findings

- NPY-Nanoluc accurately reports DCV exocytosis in rodent and human neurons.
- The assay is sensitive to known modulators of exocytosis and outperforms low-throughput methods.
- Nanoluc coupled to other markers can also detect constitutive secretion.

## Abstract

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 with little mislocalization to other cellular compartments. NPY-Nanoluc reported DCV exocytosis in both rodent and induced pluripotent stem cell-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 central nervous system disorders.