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的概念验证 Charcot-Marie-Tooth病2E(CMT2E)的策略。CMT2E是由NEFL基因突变引起的， 该基因编码神经丝蛋白L(Nefl)。NEFL是神经细丝的一个亚单位，它是 细胞骨架聚合物，其功能是扩大轴突口径，从而提高轴突传导速度。一个 CMT2E的有趣特征是它可以由零突变(隐性遗传)或错义引起 突变(以遗传为主)。初步数据表明，隐性和隐性遗传的细胞学机制 优势CMT2E汇聚在周围有髓轴突中缺少神经纤维上，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来恢复 这些神经营养因子缺乏的轴突。中心前提是，这将挽救轴突口径和神经传导。 从而改善这种疾病。作为第一步，我们建议在NEFL中测试这一策略的可行性- /-小鼠，这是一种完全缺乏NEFL蛋白导致的隐性CMT2E模型。初步 数据显示，NEFL-/-小鼠在4周后表现出严重的周围神经传导障碍。 年龄或更早。这个小鼠模型的逻辑很简单：这些小鼠缺乏网络功能，我们试图取代它们。至 为了实现这一目标，我们建议俄亥俄州立大学的四名PI和 全国儿童医院在非典型肺炎、病毒基因的细胞生物学方面具有深厚和互补的专业知识 神经肌肉功能的治疗、神经退行性疾病和电生理评估，包括 在临床前和临床基因治疗研究中证明成功。在目标1中，我们将检验假设 NEFL蛋白在NEFL-/-小鼠神经元中的表达将使神经纤维恢复到神经纤维缺陷的轴突，从而 增加轴突口径。我们将通过脑室注射将NEFL传递给P0 NEFL-/-小鼠的神经元 一种重组腺相关病毒。在目标2中，我们将测试以下假设：将NFS恢复为 NEFL-/-小鼠的轴突将挽救周围神经传导。拟议的研究将解决以下问题 基本问题：(1)我们能否在运动神经元和感觉神经元高效表达NEFL蛋白，(2) NEFL蛋白组装成核因子聚合物(3)是运输到周围神经轴突的核因子聚合物 在什么时间过程中，(4)这会导致部分或完全恢复有髓轴突口径和 G-比率，以及(5)这能恢复轴突神经传导吗？这些目标的成功实现将确立 将神经营养因子传递给神经营养因子缺乏的轴突以挽救轴突形态和生理功能的概念验证 功能。这将为未来制定一项总体战略奠定基础， 隐性CMT2E，为这一顽症患者带来了希望。

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.