Abnormal Mitochondrial Trafficking and Cytoskeletal Organization in a Human Induced Pluripotent Stem Cell and a Mouse Model of Charcot-Marie-Tooth Disease Type 2E (IN7-2.001)

Abstract

Objective: To characterize human neuronal cells derived from induced pluripotent stem cells of a patient with CMT2E and a mouse model of the same mutation. Background Induced pluripotent stem cells (iPS) offer an alternative human model for the study of neurogenetic conditions. Previous studies have demonstrated the feasibility of generating and differentiating iPS cells from patients with neurological diseases into functional neurons and glia. This approach could be particularly useful in the pathophysiological study of distinct forms of inherited neuropathies. Charcot-Marie-Tooth disease type 2E (CMT2E) is caused by heterozygous point mutations in the NEFL gene, which encodes the intermediate filament neurofilament light chain, an essential component of the neuronal cytoskeleton. Design/Methods: We developed iPS cell lines from skin fibroblasts of a patient with a N98S NFL point mutation. Knockin mice carrying the same N98S mutation were also generated. Following a differentiation protocol for generating spinal cord motor neurons, we obtained human CMT2E neuronal cultures consisting of at least 50% ISLET1 immunoreactive cells. These neuronal cultures were characterized by time-lapse microscopy and axonal mitochondrial kinetics was analyzed. Results: Immunostaining for intermediate filaments revealed an increased content of these proteins in the neuronal body of CMT2E spinal cord motor neurons. Immunostaining of both the spinal cord and brain of a N98S mouse shows abundant NFL positive aggregates in the cell bodies and axons of the motor neurons, as well as in the cerebellum. Multiple filamentous spheroid-like structures were also observed in the brain stem of the mice. An increased proportion of paused mitochondria was found in axons from human CMT2E neurons, compared to an age-matched control. Conclusions: This study demonstrates disease relevant phenotypes in human CMT2E motor neurons that were supported by a mouse model of the same mutation, demonstrating the potential of iPS cell technology to model neurogenetic diseases. Supported by: M.S. was funded by a Rare Disease Clinical Research Network Fellowship from the National Institutes of Health. This study was also supported by iPierian Inc. Disclosure: Dr. Saporta has nothing to disclose. Dr. Volfson has received personal compensation for activities with iPierian Inc. Dr. Volfson has received research support from iPierian Inc. Dr. Liem has nothing to disclose. Dr. Shy has nothing to disclose. Dr. Liem has nothing to disclose. Dr. Dimos has received personal compensation for activities with iPierian Inc. as an employee.Dr. Dimos has received research support from iPierian Inc.