Restoring neurofilaments to axons in a mouse model of CMT2E

摘要 这是一项新的R21探索性研究提案，旨在建立临床前治疗药物的概念验证 腓骨肌萎缩症2 E型（CMT 2 E）治疗策略。CMT 2 E由NEFL基因突变引起， 其编码神经丝蛋白L（NEFL）。NEFL是神经丝（NF）的亚基，其 细胞骨架聚合物的作用是扩大轴突口径，从而增加轴突传导速度。一个 CMT 2 E的一个有趣的特征是它可以由无效突变（遗传性rectification）或错义突变引起。 突变（显性遗传）。初步数据表明，隐性遗传和 优势CMT 2 E聚集在外周有髓轴突中缺乏NF，导致轴突减少， 口径和神经传导缺陷。我们的长期目标是开发一种基因治疗策略， 这些缺乏神经纤维的轴突。核心前提是这将拯救轴突口径和神经传导 从而改善疾病。作为第一步，我们建议在NEFL测试这一战略的可行性- /-小鼠，其是由NEFL蛋白完全缺失引起的隐性CMT 2 E的模型。初步 数据表明，NEFL-/-小鼠在4周时表现出外周神经传导的严重损伤， 年龄或更早。这个小鼠模型的逻辑很简单：这些小鼠缺乏NF，我们试图取代它们。到 为了实现这一目标，我们建议在俄亥俄州州立大学的四个PI之间进行多学科合作， 全国儿童医院，在NF细胞生物学、病毒基因 治疗、神经退行性疾病和神经肌肉功能的电生理学评估，包括 在临床前和临床基因治疗研究中取得了成功。在目标1中，我们将检验以下假设： NEFL-/-小鼠神经元中NEFL蛋白的表达将恢复NF至NF缺陷轴突， 增加轴突口径。我们将NEFL通过脑室注射到P0 NEFL-/-小鼠的神经元中 重组腺相关病毒。在目标2中，我们将检验以下假设： NEFL-/-小鼠的轴突将拯救外周神经传导。拟议的研究将解决以下问题 基本问题：（1）我们能否在运动和感觉神经元中有效表达NEFL蛋白，（2） NEFL蛋白组装成NF多聚体（3）是被转运到周围神经轴突的NF多聚体 以及在什么时间过程中，（4）这是否导致部分或完全恢复有髓鞘轴突口径， G-比率，和（5）这是否恢复轴突神经传导？这些目标的成功实现将建立 一个概念验证，即NF可以被递送到NF缺陷的轴突，以拯救轴突形态和生理功能。 功能这将为今后制定一项主导和 隐性CMT 2 E，为患有这种难治性疾病的患者提供了希望。

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.