# New Mouse Models to Investigate Neurological Defects Caused by Exocyst Mutations

> **NIH NIH R21** · VANDERBILT UNIVERSITY · 2020 · $459,872

## Abstract

Project Summary
The development of functional neurons requires extensive polarized membrane traffic, to drive axonal and
dendritic tree extension and organize synaptic connections. An essential factor in the delivery of proteins to the
plasma membrane is the exocyst, which is a complex of 8 subunits that functions to tether vesicles to the
membrane prior to their fusion. Exocyst accumulates in the growth cone during neurite outgrowth of
hippocampal neurons, and in puncta along the axon prior to synaptogenesis. Disruption of an exocyst gene in
Drosophila blocks neurite outgrowth, although neurotransmitter release persists. Functional exocyst is required
for viability in all organisms tested. Remarkably, however, human patients have recently been found with
mutations in an exocyst subunit (EXOC2) that cause severe neurological defects including micro- or
macrocephaly and mental retardation. Nothing is known about structural deficits in the mutant exocyst. Indeed,
the structure of the mammalian exocyst complex remains unknown, and the dynamics of
assembly/disassembly and vesicle tethering are still not fully understood. In this project we will develop
powerful tools for structural and live cell analysis of the exocyst in primary neurons, and to investigate
molecular mechanisms underlying the neurological defects caused by exocyst subunit mutation.
First, we will create new knock-in mouse lines that express fluorophore-tagged exocyst subunits, using
CRISPR strategy to generate GFP-Exo70 and Halo-Sec8. (We have shown that gene-edited murine cell lines
expressing tagged endogenous exocyst subunits are viable and the exocyst is functional). These mice will
enable for the first time the analysis of exocyst protein dynamics in primary neurons. Embryonic cortical
neurons and brain slices will be used to investigate exocyst expression, localization and dynamics during
neurite extension and synaptogenesis.
Second, we will edit the human EXOC2 mutation into the mouse genome. We will use homozygotes, if viable,
to investigate the molecular defect that causes the neurological phenotype in patients. If the mutant is late
embryonic lethal, we will isolate primary neurons from embryos and use brain slices to image exocyst
localization and measure vesicle docking and fusion efficiencies. If early embryonic lethal we will create a
floxed mutant strain, to enable conditional expression of the mutation in the developing brain, using a nestin
Cre. Finally, the new mouse strains described above will provide sufficient purified mammalian exocyst for
cryo-EM and other structural studies. Exocyst complexes will be purified from sfGFP-exocyst brain tissue using
GFP nanobody beads. Mass spectrometry will determine the purity of the complex and identify associated
accessory proteins under different purification conditions. Biochemical studies will investigate the stoichiometry
and stability of the WT and mutant complex; and cryo-EM imaging will test the feasibility of derivin...

## Key facts

- **NIH application ID:** 9998205
- **Project number:** 1R21NS117160-01
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** IAN G MACARA
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $459,872
- **Award type:** 1
- **Project period:** 2020-04-01 → 2022-09-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9998205

## Citation

> US National Institutes of Health, RePORTER application 9998205, New Mouse Models to Investigate Neurological Defects Caused by Exocyst Mutations (1R21NS117160-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9998205. Licensed CC0.

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