# Establishing the cohort of early active zone proteins and their role in synaptic strength and maturation at the Drosophila neuromuscular junction.

> **NIH NIH F31** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2022 · $46,752

## Abstract

PROJECT SUMMARY/ABSTRACT
Presynaptic active zones (AZs) cluster synaptic vesicle (SV) fusion machinery across from postsynaptic receptor
fields, facilitating efficient neural signaling. Proper development of glutamatergic synapses and AZs is critical for
normal mammalian brain development. These synapses are involved in learning and memory and their
dysfunction causes neurodevelopmental disorders like intellectual disability and autism spectrum disorder.
However, the development and maturation of these AZs is poorly understood. Drosophila melanogaster larval
motor neurons form many AZs which serve as a genetically and experimentally tractable model for mammalian
glutamatergic synapses. Previous work at the Drosophila neuromuscular junction (NMJ) has established that AZ
material accumulates in two steps: early in AZ development, proteins such as Syd-1, Liprin-a, and Unc-13B form
an initial release scaffold and Brp, Cac (Drosophila voltage-gated calcium channel), RIM and Unc-13A arrive
hours later. AZ age and incorporation of the late components Brp and Cac correlate with maturation and synaptic
vesicle release probability (Pr) at individual AZs. The contribution of the early scaffolds to synaptic strength and
AZ maturation is an open question. In addition, the full cohort of early proteins that can contribute to AZ seeding
and maturation is unknown. In Aim 1, structural and functional maturity of individual AZs will be assessed
following depletion and overexpression of early AZ scaffolds. Accumulation of fluorescently tagged Glutamate
receptor (GluR) subunits will be measured throughout development using high resolution confocal imaging of
live animals. At mature AZs, GluRIIA and GluRIIB subunits segregate into distinct rings. Levels of presynaptic
Brp and Cac at individual AZs will also be quantified to assess structural maturity. Functional maturity of individual
AZs will be assessed by calculating Pr. Using a fluorescent calcium sensor attached to the postsynaptic
membrane, individual SV fusion events following electrical stimulation are visualized by calcium entry through
GluRs. In Aim 2, proteins which contribute to formation and maturation of the AZ will be identified using a
CRISPR-based screen in single neurons. Many currently identified AZ proteins have lipid binding domains which
may bind specific regions of synaptic membrane rich in individual lipid species. Lipid kinases and phosphatases
will be eliminated in single neurons with Cas9 in order to identify disruptions in AZ formation and maturation.
These experiments are made possible by experimental approaches only available in Drosophila, but will provide
insights relevant to human neurodevelopmental disease and glutamatergic synapse development. All of the work
and prerequisite training to accomplish these Aims will be performed at Massachusetts Institute of Technology
in Dr. Troy Littleton’s lab.

## Key facts

- **NIH application ID:** 10462313
- **Project number:** 1F31NS127420-01
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Ellen Guss
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $46,752
- **Award type:** 1
- **Project period:** 2022-04-07 → 2025-09-06

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10462313

## Citation

> US National Institutes of Health, RePORTER application 10462313, Establishing the cohort of early active zone proteins and their role in synaptic strength and maturation at the Drosophila neuromuscular junction. (1F31NS127420-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10462313. Licensed CC0.

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