# Localizing endogenous synaptic proteins in vivo

> **NIH NIH R21** · UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH · 2022 · $190,625

## Abstract

PROJECT SUMMARY
Determining the subcellular localization of a protein under different cellular states is a critical aspect of
assessing protein function and dysfunction. This is especially important in neuroscience as neurons are
complex, polarized cells with distinct functional compartments, including synapses. Synapse dysfunction
underlies many neural and psychiatric disorders. Interestingly, synapses connecting different neurons develop
unique structural and functional properties that differentially modulate circuit function. This structural and
functional diversity is mediated by molecular differences. However, our understanding of the proteins located at
different types of synapses is very limited. Suitable antibodies are simply not available for many proteins and
protein overexpression drives mis-localization. Therefore, methods to localize endogenous synaptic proteins in
brain tissue are urgently needed. Here, we developed a CRISPR gene editing strategy that, in one seamless
genetic modification, inserts an epitope tag onto a protein of interest and drives expression of a cell marker in
postmitotic neurons using a single AAV. Our innovative new method is the first to provide an integrated means
for selectively identifying only those neurons that correctly integrated the protein tag and provide a cell filling,
structural reference necessary for determining the synapse-specific localization of a protein. Our method is
highly flexible for a variety of proteins, tags, and cell markers. Preliminary data indicate that our method
correctly tags the synaptic protein N-cadherin in cultured neurons but it requires further optimization and
expansion in vitro (Aim 1) and in vivo (Aim 2). Successful completion of our proposal will yield new
technologies that allow the study of endogenously expressed, synapse-specific proteins in the brain. As an
example, we will test the hypothesis that different cadherins associated with distinct mental illnesses localize to
different types of synapses in vivo. Taken together, our results are expected to result in a new technology that
can be broadly applied to study synapse formation and function and provide new molecular insight to
mechanisms underlying synapse diversity.

## Key facts

- **NIH application ID:** 10308511
- **Project number:** 5R21MH122766-02
- **Recipient organization:** UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH
- **Principal Investigator:** Megan Elise Williams
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $190,625
- **Award type:** 5
- **Project period:** 2020-12-01 → 2023-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10308511

## Citation

> US National Institutes of Health, RePORTER application 10308511, Localizing endogenous synaptic proteins in vivo (5R21MH122766-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10308511. Licensed CC0.

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