Monday, April 28, 2025
4/28/2025
Mechanisms of intracellular transport and processing of neuropeptides - Project Summary/Abstract A major form of communication between cells, including the nervous system, is the release of small, chemical factors, like neuropeptides (NPs) and hormones. These molecules are used for communication both on a local level and at a systemic level, where they regulate and mediate developmental, physiological, and behavioral events. Molecules like neuropeptides are packaged in specialized cellular compartments called Dense Core Vesicles (DCVs). Despite decades of research, little is known about the in vivo biology of DCVs, from the mechanism governing their sorting into appropriate cargo, to how they are properly processed into mature bioactive peptides, to how they undergo release from cells. There are hundreds of different NPs in the human brain, most with completely unique cellular and physiological impacts. Some of society’s most severe and debilitating diseases and conditions including neurodegenerative and neurological disorders result from abnormalities in the packaging or release of these molecules. The long-term objective of this research seeks to understand the molecular machinery regulating the synthesis, sorting, trafficking, and release of NPs from DCVs. To initially investigate this process, we created 14 different transgenically tagged NP Drosophila fly lines to directly visualize the intracellular localization, sorting, trafficking, and release of neuropeptides from neurons in an in vivo system. We initially uncovered evidence that individual NPs are sorted on an mRNA level to regulate the identity of each DCV. In Aim 1 we propose to solidify this result by creating several new transgenic fly lines using the most novel and modern cloning approaches like tissue specific CRISPR and newly created fluorescent tags like mScarlett3. This approach will tag endogenous NPs within the genome and facilitate a real-time examination of their sorting, trafficking, and release mechanisms when combined with out high-resolution-ultra fast fluorescence microscopy. Aims 1 and 2 use genetic, biomolecular, cloning, electrophysiological and pharmacological approaches combined with confocal and live imaging in an in vivo system to define the proteins and processes involved in NP sorting in the trans-Golgi network. We predict that the complex of proteins found on each DCV is unique to the NP within, and this NP-dependent protein complex defines the machinery used for downstream trafficking and release. We have identified a subset of proteins known to regulate trafficking and release (e.x. synaptotagmin) and will genetically alter the structure of these proteins to identify their roles. Mutations in synaptotagmins in humans lead to severe neurodegenerative and neurological disorders like Alzheimer’s. These studies will greatly advance our comprehension of NP sorting and illuminate the proteins and pathways controlling their trafficking and release from neurons.
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