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.
| Original language | English |
|---|---|
| Article number | 107321 |
| Pages (from-to) | 1-13 |
| Number of pages | 13 |
| Journal | Journal of Biological Chemistry |
| Volume | 300 |
| Issue number | 6 |
| Early online date | 25 Apr 2024 |
| DOIs | |
| Publication status | Published - Jun 2024 |
Bibliographical note
Publisher Copyright:© 2024 The Authors
Funding
The authors thank Joke Wortel for animal breeding, Robbert Zalm for cloning and producing viral particles, Lisa Laan for astrocyte culture and cell culture assistance, and Ingrid Saarloos for assistance in protein chemistry. Desiree Schut, Judith Huijgen, and Solange Lopez Cardozo for iPSC culture and CRISPR-Cas9 engineering. Prof. Michiel Pegtel for gifting CD63-Nanoluc and sec-Nanoluc constructs. U. B. R. F. T. and M. V. writing-review and editing; U. B. and G. B. investigation; U. B. writing-original draft; U. B. visualization; U. B. validation; U. B. methodology; U. B. formal analysis; U. B. data curation; U. B. R. F. T. and M. V. conceptualization; R. F. T. and M. V. funding acquisition; R. F. T. and M. V. supervision. This work was supported by a European Research Council (ERC) Advanced grant (322966) of the European Union (to M. V.), COSYN (Comorbidity and Synapse Biology in Clinically Overlapping Psychiatric Disorders, Horizon 2020 Program of the European Union under RIA grant agreement 667301, to M. V.), and the JPND Neuron Cofund ERA-Net SNAREopathy (to R. F. T.). This work was supported by a European Research Council (ERC) Advanced grant (322966) of the European Union (to MV), COSYN (Comorbidity and Synapse Biology in Clinically Overlapping Psychiatric Disorders, Horizon 2020 Program of the European Union under RIA grant agreement 667301, to MV) and the JPND Neuron Cofund ERA-Net SNAREopathy (to RFT).
| Funders | Funder number |
|---|---|
| EU Joint Programme – Neurodegenerative Disease Research | |
| European Commission | |
| European Research Council | 322966 |
| European Research Council | |
| South Carolina Rural Infrastructure Authority | 667301 |
| South Carolina Rural Infrastructure Authority |
Keywords
- DCV exocytosis
- high-throughput assay
- Nanoluc
- neuropeptides