Identification of Compounds to treat Charcot-Marie-Tooth type 2E neuropathy

描述（由申请人提供）：本申请旨在鉴定治疗2E型沙科-玛丽- tooth （CMT2E）的化合物。CMT是最常见的遗传性神经系统疾病，据报道全世界每2500人中就有1人患病。它存在于所有种族和民族群体中。CMT进展缓慢，CMT患者伴有控制足/腿和手/臂感觉信息的周围神经退化。神经退行性变导致随后四肢肌肉的退行性变。CMT的症状包括足下垂、步进步态、高足弓、足骨异常和基本的手功能问题，有时还会出现呼吸困难。CMT通常不会危及生命，但会导致严重的残疾。虽然CMT中突变的基因也在中枢神经系统中表达，但这种疾病几乎不会影响大脑功能。CMT根据神经传导速度分为CMT1型和CMT2型两大类，CMT1型神经传导速度降低，CMT2型神经传导速度相对正常。一般来说，CMT1是由髓鞘形成重要基因突变引起的脱髓鞘神经病变，而CMT2是轴突性的。神经中间丝基因NEFL的突变已被证明会导致CMT2的一种亚型，称为CMT2E。NEFL编码神经丝光（NFL）蛋白，我们已经证明该蛋白是神经元中间丝组装的必要成分。该基因突变约占所有CMT病例的2%。神经元中间丝在神经元中形成中间丝网络，是轴突中主要的细胞骨架结构。在NEFL突变患者以及其他神经退行性疾病（如肌萎缩性侧索硬化症）患者中，可以看到神经元细胞体和轴突中的神经丝状聚集体。我们已经证明突变的NFL蛋白在转染的神经元和非神经元细胞中形成聚集体。在所有表达致病性cmt相关NFL突变蛋白的转染细胞中都发现了错误组装的神经丝聚集体，但在表达几种非致病性多态性变体的细胞中却没有发现。我们假设神经丝错误组装的抑制剂将导致CMT的治疗。因此，本提案的目标是鉴定抑制错误组装的小分子，并在CMT2E小鼠模型中测试它们的作用。我们将把注意力集中在两个最早描述的突变NFL蛋白上，P8R NFL和Q333P NFL，我们已经对它们进行了最详细的表征。使用高通量视觉筛选方法，我们将识别抑制神经丝错误组装的小分子。对于项目的第二部分和相关部分，我们将生成Nefl基因突变的CMT2E敲入小鼠模型。这些小鼠将被表征表型，然后我们将测试在视觉屏幕中识别的化合物，以改善这些小鼠模型中周围神经病变的能力。拟议的研究将确定先导化合物，用于开发治疗人类CMT患者的药物，以及潜在的其他神经退行性疾病，它还将生成人类遗传性神经病变的小鼠模型，这将对该领域的许多其他研究人员有价值。

DESCRIPTION (provided by applicant): This application is designed to identify compounds to treat Charcot-Marie-Tooth type 2E (CMT2E). CMT is the most commonly inherited neurological disorder with a reported prevalence of 1 in 2,500 people worldwide. It is found in all races and ethnic groups. CMT is slowly progressive and CMT patients suffer from degeneration of the peripheral nerves that control sensory information of the foot/leg and hand/arm. The nerve degeneration causes the subsequent degeneration of the muscles in the extremities. Among the symptoms of CMT are foot-drop, steppage gait, high arches, foot bone abnormalities, and basic problems with hand function, as well as sometimes breathing difficulties. CMT is not usually life threatening, but can cause severe disabilities. Although the genes mutated in CMT are also expressed in the central nervous system, the disorder almost never affects brain function. CMT is divided in two major types, CMT1 and CMT2, based on nerve conduction velocities, which are reduced in CMT1 and relatively normal in CMT2. In general, CMT1 is a demyelinating neuropathy caused by mutations in genes important in myelin formation, whereas CMT2 is axonal. Mutations in the neuronal intermediate filament gene, NEFL have been shown to cause a subtype of CMT2, called CMT2E. NEFL encodes the neurofilament light (NFL) protein that we have shown to be a necessary component for the assembly of neuronal intermediate filaments. Mutations in this gene are responsible for approximately 2% of all CMT cases. Neuronal intermediate filaments form the intermediate filament network in neurons and are the predominant cytoskeletal structure in the axon. Neurofilamentous aggregates both in the neuronal cell bodies and axons are seen in patients with mutations in NEFL, as well as in other neurodegenerative diseases, such as amyotrophic lateral sclerosis. We have shown that the mutant NFL proteins form aggregates in transfected neuronal and non-neuronal cells. Misassembled neurofilament aggregates are found in all transfected cells expressing the pathogenic CMT-associated NFL mutant proteins, but not in cells expressing several polymorphic variants that are non-pathogenic. We hypothesize that inhibitors of neurofilament misassembly will lead to therapies for CMT. Therefore, the goals of this proposal are to identify small molecules that inhibit misassembly and to test their effects in a mouse model of CMT2E. We will focus our attention on two of the first described mutant NFL proteins, P8R NFL and Q333P NFL that we have characterized in the most detail. Using high-throughput visual screening methodology, we will identify small molecules that inhibit neurofilament misassembly. For the second and related part of the project, we will generate knock-in mouse models of CMT2E with mutations in the Nefl gene. These mice will be characterized phenotypically and we will then test compounds identified in the visual screens for their ability to ameliorate peripheral neuropathy in these mouse models. The proposed research will identify lead compounds for the development of drugs to treat human subjects with CMT, as well as potentially other neurodegenerative diseases and it will also generate mouse models of a human hereditary neuropathy that will be of value to many other investigators in the field. PUBLIC HEALTH RELEVANCE: CMT is the most commonly inherited form of peripheral neuropathy. The goal of this project is to use chemical screening to identify novel drugs to treat one type of CMT and test them in mouse models of the disease.