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

描述(由申请人提供)：Jean Martin Charcot，Pierre Marie和Howard Henry Tooth在1886年描述了Charcot-Marie-Tooth(CMT)的特征。今天，CMT是最常见的周围神经系统遗传性疾病，影响着大约15万美国人。现在认识到CMT的四种主要形式(称为CMT 1-4)。形式之间的主要区别是连接的基因和受影响的原始细胞类型(雪旺细胞与神经)。Charcot-Marie-Tooth Type 2E(CMT2E)是CMT2的亚型。CMT2E是一种常染色体显性遗传病，影响周围神经轴突。最近的一系列报道将神经细丝LIGH(NF-L)的16个突变与CMT2E联系起来。具有CMT2E连锁突变的核因子-L在细胞培养中表达，扰乱神经丝的组织和运输。此外，突变型核因子-L在原代神经元培养中以显性方式发挥作用。有趣的是，通过靶向缺失小鼠的核因子-L，完全丧失所有轴突神经丝，并不会导致明显的病理。因此，目前对核因子-L连锁CMT2E的发病机制(S)知之甚少。我们的目的是建立两种CMT2E的动物模型，以便分析核因子-L连锁的CMT的发病机制。我们将通过培育两个独立的基因敲入小鼠品系来实现这一点。一个品系的小鼠将表达的核因子-L与8个突变为精氨酸，另一个品系将表达的核因子-L与谷氨酸突变为赖氨酸。我们将在细胞和机体水平上分析这些小鼠的病理变化。在细胞水平上，我们将寻找轴突中神经丝聚集和组织的变化，放射状轴突生长的变化和髓鞘形成的变化。我们还将监测老鼠是否出现类似CMT的症状。具体地说，我们将分析肌肉消耗、步态、热感觉和神经传导速度。产生两个基因靶向的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位于蛋白质的不同功能区域，这些突变是否通过不同的机制导致疾病？不同的核因子-L突变是否会导致不同的疾病发病或严重程度？此外，我们将独立分析运动神经和感觉神经轴突的细胞表型，以确定这两种神经元类型是否同样容易受到突变的核因子-L表达的影响。时间进程的使用将使我们能够识别与表达突变的核因子-L相关的早期变化。建立和分析CMT2E的动物模型是确定新的治疗干预部位和策略的第一个关键步骤。公共卫生相关性：夏科-玛丽牙(Charcot-Marie-Tooth，CMT)是最常见的周围神经系统遗传性疾病，影响大约150,000美国人，CMT2的严重病例在婴儿中表现为呼吸功能障碍，在成年人中表现为喉咙无力、声音嘶哑和呼吸困难。细胞骨架蛋白神经丝光蛋白(NF-L)的突变与CMT2E有关。我们有兴趣建立CMT2E的动物模型，以确定核因子-L突变是如何导致疾病发展的，并为治疗干预寻找新的部位和策略。

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.