# Tissue Engineered Human Neuromuscular Junctions for Modeling Axonal Neuropathy

> **NIH NIH R01** · UNIVERSITY OF WASHINGTON · 2020 · $154,533

## Abstract

PROJECT SUMMARY
Charcot-Marie-Tooth disease type 2 (CMT2) is a severely debilitating axonopathic peripheral neuropathy,
characterized by neuromuscular junction (NMJ) breakdown, axonal transport defects, and neuronal structure
malformations. Although animal models for this condition are available, the wide array of gene mutations known
to cause a CMT2 phenotype (>30 identified to date) makes the study of common pathway deficits problematic.
This in turn makes identification of suitable therapeutic targets, capable of treating a wide range of patients,
extremely difficult. The successful generation of human induced pluripotent stem cell (hiPSC)-derived motor
neurons from patients with CMT2 makes the establishment of patient-specific humanized assays for studying
disease etiology a tangible goal. However, the ability to effectively model CMT2 in vitro using such cells has yet
to be achieved, due to the complexity associated with generating robust and functionally mature NMJs in culture.
We posit that a culture platform integrating correct tissue-level structural organization, physiologically relevant
cell densities, and correct electromechanical conditioning stimuli will promote the development of human
myotube-motor neuron co-cultures capable of supporting mature synapse formation. Using our well-established
nanopatterned cell sheet manipulation techniques, we will generate scaffold-free 3D tissue structures using
hiPSC-derived motor neurons and primary human myoblasts with highly ordered tissue structures. These
constructs will be assessed for their ability to promote NMJ formation, and electromechanical conditioning will
then be investigated as a means to drive synapse development. This system will be developed in conjunction
with motor neurons derived from four CMT2 patients. Co-culture constructs incorporating these cells will be
investigated for their capacity to accurately model the disease’s pathophysiology in vitro, and to highlight
phenotypic similarities across different mutant lines. Given the prominent role of mitochondria in NMJ
development, and the observed breakdown in axonal transport in multiple CMT2-related mutations, we
hypothesize that reduced mitochondrial density in presynaptic terminals leads to malformations in NMJ
development and ultimately breakdown of the synapse. We will use our CMT2 hiPSC-motor neurons to evaluate
axon transport deficits and structural malformations in these cells and correlate these findings with quantified
changes in NMJ development within our bioengineered co-culture platform. Finally, we will investigate whether
improvement in axon transport properties in multiple CMT2 neuron lines (via stabilization of axonal development
with histone deacetylase 6 inhibitors) results in significant improvements in NMJ development and stability in
human cells. Consistency of results across different patient mutations will highlight axonal transport deficits as a
major causal factor in the development of the human CM...

## Key facts

- **NIH application ID:** 9817690
- **Project number:** 5R01NS094388-04
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Deok-Ho Kim
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $154,533
- **Award type:** 5
- **Project period:** 2016-11-15 → 2019-11-16

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9817690

## Citation

> US National Institutes of Health, RePORTER application 9817690, Tissue Engineered Human Neuromuscular Junctions for Modeling Axonal Neuropathy (5R01NS094388-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9817690. Licensed CC0.

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