Restoring neurofilaments to axons in a mouse model of CMT2E

点击翻译按钮获取中文摘要

ABSTRACT This is a new R21 exploratory research proposal to establish proof-of-concept of a pre-clinical therapeutic strategy for Charcot-Marie-Tooth disease type 2E (CMT2E). CMT2E is caused by mutations in the NEFL gene, which codes for neurofilament protein L (NEFL). NEFL is a subunit of neurofilaments (NFs), which are cytoskeletal polymers that function to expand axon caliber and thereby increase axonal conduction velocity. An interesting feature of CMT2E is that it can be caused by null mutations (inherited recessively) or missense mutations (inherited dominantly). Preliminary data indicate that the cellular mechanisms of recessive and dominant CMT2E converge on an absence of NFs in peripheral myelinated axons, resulting in reduced axon caliber and nerve conduction defects. Our long-term goal is to develop a gene therapy strategy for restoring NFs to these NF-deficient axons. The central premise is that this will rescue axon caliber and nerve conduction and thereby ameliorate the disease. As a first step, we propose to test the feasibility of this strategy in the NEFL- /- mouse, which is a model of recessive CMT2E caused by a complete absence of the NEFL protein. Preliminary data demonstrate that NEFL-/- mice exhibit profound impairment of peripheral nerve conduction by 4 weeks of age or earlier. The logic in this mouse model is simple: these mice lack NFs and we seek to replace them. To achieve this goal, we propose a multi-disciplinary collaboration between four PIs at Ohio State University and Nationwide Children’s Hospital with deep and complementary expertise on the cell biology of NFs, viral gene therapy, neurodegenerative disease and electrophysiological assessments of neuromuscular function, including proven success in preclinical and clinical gene therapy studies. In Aim 1, we will test the hypothesis that expression of NEFL protein in neurons of NEFL-/- mice will restore NFs to the NF-deficient axons and thus increase axonal caliber. We will deliver NEFL to neurons of P0 NEFL-/- mice by intra-cerebroventricular injection of a recombinant adeno-associated virus. In Aim 2 we will test the hypothesis that the restoration of NFs to axons of NEFL-/- mice will rescue peripheral nerve conduction. The proposed research will address the following fundamental questions: (1) can we express NEFL protein efficiently in motor and sensory neurons, (2) does the NEFL protein assemble into NF polymers (3) are the NF polymers transported into the axons of peripheral nerves and over what time course, (4) does this result in partial or complete restoration of myelinated axon caliber and G-ratio, and (5) does this restore axonal nerve conduction? Successful completion of these aims will establish a proof-of-concept that NFs can be delivered to NF-deficient axons to rescue axon morphology and physiological function. This will lay the groundwork for future development of a general strategy both for dominant and recessive CMT2E, offering hope to patients with this intractable disease.