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 CMT2 phenotype, and validate our platform as a suitable tool for use in the preclinical assessment of new drugs designed to ameliorate peripheral neuropathic conditions.
This proposal centers on the use of novel stem cell-based bioengineering strategies to study the underlying etiology of a common debilitating peripheral neuropathy (axonopathic Charcot-Marie-Tooth disease; CMT). We will evaluate differences in the functional phenotypes and synapse forming abilities of motor neurons derived from multiple CMT patient stem cell lines. Evaluation of common phenotypic distinctions, as well as mutation specific effects, will help stratify disease subtypes, improve our understanding of the critical pathway defects leading to CMT, and identify suitable targets for future therapeutic strategies.
