Charcot-Marie-Tooth Disease Type 2E: Mechanism and therapy

Ohio State University, Columbus OH

Investigators

Abstract

ABSTRACT Neurofilaments (NFs) are abundant cytoskeletal polymers in neurons that function as space-filling structures to expand axonal caliber, which is a critical determinant of axonal conduction velocity. NFs assemble in the neuronal soma and are transported into axons, where they accumulate excessively in many neurodegenerative diseases, leading to axonal swelling and enlargement. In addition, mutations in one of the NF subunit proteins, NFL, cause the hereditary sensory and motor neuropathy known as Charcot-Marie-Tooth disease type 2E (CMT2E). Most CMT2E mutations are dominantly inherited missense mutations. Here we propose three aims to elucidate the disease mechanism of CMT2E and develop a pre-clinical therapeutic strategy. Aim 1 is to establish the effect of CMT2E mutations on NFs. Overexpression of CMT2E mutant NFL in cultured cells has been reported to disrupt NF assembly. However, observations on human nerve biopsies and an NFLN98S/+ knock- in mouse model of CMT2E have revealed accumulations of NF polymers, which suggests an impairment of NF transport. We hypothesize that when expressed at physiologically relevant levels, CMT2E mutant NFL assembles into NFs that have altered transport kinetics. We will test this using biochemical and imaging approaches in neurons differentiated from patient-derived and gene-edited human iPSCs. Aim 2 is to elucidate the mechanism of NF accumulation in CMT2E disease. Ultrastructural studies on the NFLN98S/+ knock-in mouse model of CMT2E have revealed massive accumulations of NF polymers in the cell bodies and proximal regions of myelinated axons of motor and sensory neurons. Distally, these axons lack NFs entirely and have a reduced caliber and impaired nerve conduction. We hypothesize that the proximal accumulations represent NF “logjams” which arise due to an impairment of NF transport, preventing NF movement into distal axons. We will test this hypothesis by performing volumetric reconstruction of axons in NFLN98S/+ mice using immunofluorescence array tomography, and by live imaging of NF transport during the formation of NF accumulations in iPSC-derived neurons from CMT2E patients. Aim 3 is to establish a therapeutic strategy for CMT2E in NFLN98S/+ mice. Preliminary data point to a dominant negative mechanism of action. Thus, we hypothesize that the disease can be rescued by a knockdown-and-replace gene therapy strategy using recombinant adeno-associated viral vectors. We will compare the efficacy of this approach to simply overexpressing wild type NFL. An advantage of this approach is that it will be applicable to all forms of CMT2E, independent of the specific mutation, thus broadening the therapeutic applicability. Our goal is to restore normal NFs to neurons, permitting NF delivery to distal axons during postnatal development. The proposed research leverages more than three decades of experience studying NF assembly and transport in the PI’s lab as well as the complementary expertise of co- investigators and collaborators at UCSF and Ohio State University, who have had proven success with iPSCs, gene editing, RNAi, viral vectors, electrophysiological assessments of neuromuscular function, and the translation of viral gene therapy from mice to patients.