# Uncovering the roles of ubiquitination and the ESCRT pathway in degradative sorting of SV proteins.

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2021 · $478,327

## Abstract

PROJECT SUMMARY
Synaptic vesicles (SVs) are highly specialized organelles that store and release neurotransmitters. The
accumulation of old or damaged proteins on SVs compromises neurotransmission and can lead to
dysfunctional neural circuits and networks. Indeed, recent studies have shown that mutations in genes that
regulate SV protein degradation are associated with neurological and neurodegenerative disorders,
demonstrating the critical importance of SV protein turnover for nervous system health. Yet the molecular
mechanisms responsible for SV turnover and degradation remain poorly understood. The overall goal of this
project is to elucidate these mechanisms, providing critical insights into the etiology of diseases that afflict
millions of Americans. Our recent work has shown that the ESCRT pathway mediates the activity-dependent
degradation of SV membrane proteins. The ESCRT pathway comprises a series of protein complexes that
sequentially recruit ubiquitinated cargo and catalyze the formation of multivesicular bodies (MVBs) for delivery
of these cargo to lysosomes. Intriguingly, we find that increased neuronal firing stimulates the activation of
de/ubiquitinating enzymes at the synapse, as well as the motility of axonal transport vesicles carrying initial
ESCRT protein Hrs, and their recruitment to SV pools. We hypothesize that these events are critical rate-
limiting steps for activity-dependent turnover of SV membrane proteins. We will test this hypothesis with three
aims. In Aim 1, we will evaluate the role of de/ubiquitination in the recycling of SV membrane proteins. Here,
we will use biochemical and fluorescence imaging assays to evaluate how ubiquitination regulates SV protein
recycling vs. degradation in hippocampal neurons. We will also investigate whether the deubiquitinating
enzyme UCHL1 is necessary for maintaining SV proteins on recycling SVs, counteracting their degradative
sorting. In Aim 2, we will characterize Hrs vesicles and the impact of Hrs on downstream ESCRT protein
recruitment to SV pools. We will use super-resolution fluorescence/electron microscopy and proximity
biotinylation to characterize the morphology and molecular composition of these vesicles, and Hrs gain- and
loss-of-function combined with live imaging to determine whether the recruitment of downstream ESCRT
proteins to SV pools requires Hrs. In Aim 3, we will investigate the mechanisms of activity-dependent Hrs
recruitment to SV pools. We will test the roles of specific kinesins in the axonal transport of Hrs, and test
whether its recruitment to SV pools requires the lipid PI(3)P, the presence of ubiquitinated proteins, and/or the
small GTPase Rab35. Together, these studies will uncover fundamental mechanisms underlying SV
proteostasis in neurons.

## Key facts

- **NIH application ID:** 10113675
- **Project number:** 5R01NS080967-08
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Clarissa Leigh Waites
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $478,327
- **Award type:** 5
- **Project period:** 2014-01-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10113675

## Citation

> US National Institutes of Health, RePORTER application 10113675, Uncovering the roles of ubiquitination and the ESCRT pathway in degradative sorting of SV proteins. (5R01NS080967-08). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10113675. Licensed CC0.

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