# Dissecting the assembly of vertebrate neurotransmitter release sites

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2020 · $598,877

## Abstract

Project Summary
Neurotransmitter release critically depends on the precise assembly of the secretory machine. Within a
presynaptic nerve terminal, synaptic vesicles exclusively fuse at the active zone, a protein scaffold that forms
release sites opposed to postsynaptic receptors. This scaffold consists of RIM, ELKS, Liprin-α and other active
zone specific proteins. It also contains many proteins that are important for secretion and synaptic structure,
but that are not restricted to the active zone. In the past two decades, research from many laboratories has
started to provide deep insight into the functions of individual proteins at the active zone. However, much less
is known about the assembly mechanisms of this key protein scaffold. This is particularly true for vertebrate
synapses, perhaps because no genetic mutation to date has strongly disrupted the active zone scaffold.
We here overcome this limitation by generating conditional knockout mice to simultaneously delete RIM and
ELKS in hippocampal neurons. This mutation leads to massive disruption of the presynaptic active zone
scaffold with loss of most of its vital components and of vesicle docking. Based on extensive preliminary data,
we hypothesize that RIM and ELKS are redundantly required for release site assembly and function, and that
these scaffolding proteins are recruited to the active zone by Liprin-α. We designed three specific aims to
address our overarching hypothesis. In the first aim, we rigorously test synaptic structure and synaptic vesicle
docking in these active zone disrupted neurons. We propose rescue experiments with individual proteins or
protein domains to evaluate the molecular hierarchy of the recruitment of active zone proteins and to dissect
mechanisms for vesicle docking. In aim 2, we use the mutants with disrupted active zones to test two models
that are prominent in the field: we will determine whether the active zone and docking are required for synaptic
vesicle release and we will test whether the active zone targets release to the membrane domain opposed to
postsynaptic receptors. Our preliminary data reveal that fusion competent vesicles persist upon disruption of
the active zone and loss of vesicle docking, which is surprising given the dogma that fusion competent vesicles
are docked. In aim 3, we address molecular mechanisms of active zone assembly upstream of RIM and ELKS.
The most parsimonious interpretation of the literature and our preliminary data is that Liprin-α recruits RIM and
ELKS for active zone scaffolding. We systematically test this hypothesis in newly generated Liprin-α knockout
mice. This is the first study that addresses vertebrate Liprin-α function using a rigorous genetic approach.
This grant application will generate new knowledge on the mechanisms of vertebrate active zone assembly
and function. Human genetic studies have identified mutations in many active zone proteins, including in RIM
and in ELKS, which contribute to neurological...

## Key facts

- **NIH application ID:** 9839675
- **Project number:** 5R01MH113349-04
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Pascal Simon Kaeser
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $598,877
- **Award type:** 5
- **Project period:** 2017-03-13 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9839675

## Citation

> US National Institutes of Health, RePORTER application 9839675, Dissecting the assembly of vertebrate neurotransmitter release sites (5R01MH113349-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9839675. Licensed CC0.

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