Mechanisms of disease pathogenesis in neurofilament linked Charcot-Marie-Tooth di

描述（由申请人提供）：Jean Martin Charcot、Pierre玛丽和霍华德亨利Tooth于1886年对Charcot-玛丽-Tooth（CMT）进行了表征。今天，CMT是周围神经系统最常见的遗传性疾病，影响约15万美国人。CMT的四种主要形式现在被认可（称为CMT 1-4）。不同形式之间的主要区别是相关基因和受影响的主要细胞类型（雪旺细胞与神经）。Charcot-Marie-Tooth 2 E型（CMT 2 E）是CMT 2的一个亚型。CMT 2 E是一种影响周围神经轴突的常染色体显性遗传疾病。最近的一系列报道将神经丝轻（NF-L）中的16个突变与CMT 2 E联系起来。在细胞培养物中表达的具有CMT 2 E连锁突变的NF-L破坏神经丝组织和运输。此外，突变型NF-L在原代神经元培养物中以显性方式起作用。有趣的是，通过小鼠中NF-L的靶向缺失，所有轴突神经丝的完全丧失不会导致明显的病理学。因此，关于NF-L连接的CMT 2 E的发病机制所涉及的机制知之甚少。我们的目的是建立两种CMT 2 E的动物模型，以便我们可以分析NF-L连锁的CMT中的疾病发病机制。我们将通过开发两个独立的基因敲入小鼠系来实现这一目标。一个品系的小鼠将表达脯氨酸8突变为精氨酸的NF-L，另一品系的小鼠将表达谷氨酸397突变为赖氨酸的NF-L。我们将分析这些小鼠在细胞和生物体水平上的病理变化。在细胞水平上，我们将寻找神经丝积累和轴突组织的改变，径向轴突生长的改变和髓鞘形成的改变。我们还将监测小鼠是否出现CMT样症状。具体来说，我们将分析肌肉萎缩，步态，热感知和神经传导速度。产生两种基因靶向小鼠将解决与治疗开发相关的重要问题。例如，由于我们提出的突变是在蛋白质的不同功能域中，这些突变是否通过不同的机制导致疾病？不同的NF-L突变是否会导致不同的疾病发作或严重程度？此外，我们将独立分析运动和感觉轴突的细胞表型，以确定这两种神经元类型是否同样容易受到突变型NF-L表达的影响。使用时程将允许我们鉴定与表达突变NF-L相关的早期变化。生成和分析CMT 2 E动物模型是确定治疗干预的新位点和策略的第一个关键步骤。公共卫生相关性：腓骨肌萎缩症（CMT）是最常见的外周神经系统遗传性疾病，影响约150，000名美国人，严重的CMT 2病例在婴儿中表现为呼吸功能障碍，在成人中表现为喉无力、声音嘶哑和呼吸困难。细胞骨架蛋白神经丝轻（NF-L）的突变与CMT 2 E有关。我们有兴趣开发CMT 2 E的动物模型，以确定NF-L突变如何导致疾病的发展，并确定新的治疗干预位点和策略。

DESCRIPTION (provided by applicant): Jean Martin Charcot, Pierre Marie and Howard Henry Tooth characterized Charcot-Marie-Tooth (CMT) in 1886. Today, CMT is the most common inherited disease of the peripheral nervous system, affecting approximately 150,000 Americans. Four major forms of CMT are now recognized (referred to as CMT 1-4). The major differences between forms are the linked gene and the primary cell type affected (Schwann cells versus nerves). Charcot-Marie-Tooth type 2E (CMT2E) is a sub-type of CMT2. CMT2E is an autosomal dominant disorder that affects peripheral nerve axons. A series of recent reports linked 16 mutations in neurofilament light (NF-L) to CMT2E. NF-L, with CMT2E linked mutations, expressed in cell culture disrupts neurofilament organization and transport. Additionally, mutant NF-L functions in a dominant manner in primary neuronal cultures. Interestingly, complete loss of all axonal neurofilaments, through targeted deletion of NF-L in mouse, does not result in overt pathology. Therefore, little is known about the mechanism(s) involved in the pathogenesis of NF-L linked CMT2E. Our objective is to develop two animal models of CMT2E so that we can analyze disease pathogenesis in NF-L linked CMT. We will achieve this by developing two independent lines of gene knock-in mice. One line of mice will express NF-L with proline 8 mutated to arginine, and the other will express NF-L with glutamate 397 mutated to lysine. We will analyze these mice for pathological changes at both the cellular and organism level. At the cellular level, we will look for alterations in neurofilament accumulation and organization in axons, alterations in radial axonal growth and alterations in myelination. We will, also, monitor the mice for the appearance of CMT-like symptoms. Specifically, we will analyze muscle wasting, gait, thermal perception and nerve conduction velocities. Generating two lines of gene-targeted mice will address important questions relevant to therapy development. For example, as our proposed mutations are in distinct functional domains of the protein, do these mutations result in disease through different mechanisms? Do different NF-L mutations result in variable onset or severity of disease? Additionally, we will analyze cellular phenotypes independently in motor and sensory axons to determine if both neuronal types are equally vulnerable to expression of mutant NF-L. The use of a time course will allow us to identify early changes associated with expressing mutant NF-L. Generating and analyzing animal models of CMT2E is the first key step in identifying novel sites and strategies for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Charcot-Marie-Tooth (CMT) is the most common inherited disease of the peripheral nervous system affecting approximately 150,000 Americans with severe cases of CMT2 presenting with respiratory dysfunction in infants and with laryngeal weakness, hoarseness and respiratory difficulties in adults. Mutations in a cytoskeletal protein, neurofilament light (NF-L), have been linked to CMT2E. We are interested in developing animal models of CMT2E to determine how mutations in NF-L lead to the development of disease and to identify novel sites and strategies for therapeutic intervention.