An Animal Model of LRSAM1 Peripheral Neuropathy

描述（由申请人提供）：本提案的目的是验证小鼠Lrsam 1基因突变作为人类遗传性周围神经病变模型，并确定LRSAM 1蛋白发挥功能的细胞途径。遗传性感觉和运动神经病（HSMNs或Charcot-Marie-Tooth病，CMT）是一类临床和遗传多样性的疾病，导致周围神经系统功能障碍和轴突变性。越来越多的轴突神经病（2型CMT）的潜在基因正在被鉴定，但这些突变导致外周轴突变性的疾病机制尚不清楚。最近，在加拿大和荷兰家系中分别发现了隐性和显性遗传性周围轴突神经病的LRSAM 1突变。LRSAM 1是一种E3泛素连接酶，具有一种已知底物TSG 101，TSG 101是ESCRT 1复合物的组分，对于晚期内体到溶酶体囊泡分选和运输等功能很重要。其他几种Charcot-Marie-Tooth相关蛋白如SIMPLE、RAB 7和FIG 4也参与内体/溶酶体运输。没有哺乳动物实验模型LRSAM 1相关的神经病变存在，LRSAM 1是否选择性地与TSG 101和功能的内体/溶酶体分选和运输的外周神经元仍然未知。为了解决这些问题，我们提出了两个目标。在目标1中，我们将在小鼠中使用基因陷阱等位基因来产生Lrsam 1突变，并验证这些小鼠作为人类周围神经病变模型。我们目前有来自两个独立插入突变的小鼠。这些插入确实导致Lrsam 1表达的丧失，初步研究表明它们在杂合和纯合小鼠中引起周围神经病变表型，与人类疾病一致。我们将继续对这些小鼠进行分子、电生理、组织学和行为学表征，以确定其作为疾病模型的有效性。在目标2中， 我们将检查LRSAM 1蛋白的亚细胞定位，以确定它是否与内体/溶酶体运输途径的其他组分相关，以及它是否与该途径中的其他CMT相关蛋白共定位。此外，我们将检查Lrsam 1在小鼠中的表达模式，以确定是否限制表达可能占外周神经元特异性的表型。在完成这些目标后，我们将有一个新的人类轴突CMT小鼠模型，将可用于临床前和机制研究。此外，我们将更好地了解LRSAM 1功能的细胞途径，这将为未来的机制研究提供信息，并可能说明LRSAM 1和其他CMT相关蛋白的共享细胞途径。 公共卫生相关性：该提案旨在验证LRSAM 1突变引起的遗传性周围神经病变的动物模型。LRSAM 1突变已在加拿大和荷兰遗传性轴突神经病家系中鉴定;然而，疾病机制以及潜在的治疗策略仍不清楚。使用小鼠资源，我们已经产生了缺乏Lrsam 1表达的小鼠品系。看来这些小鼠具有与患者相当的神经病变，因此我们将建立这些小鼠作为未来临床前和机制研究的模型。

DESCRIPTION (provided by applicant): The goal of this proposal is to validate mutations in the mouse Lrsam1 gene as a model of human inherited peripheral neuropathy, and to determine the cellular pathway in which LRSAM1 protein functions. Heritable sensory and motor neuropathies (HSMNs or Charcot-Marie-Tooth disease, CMT) are a clinically and genetically diverse class of diseases that cause dysfunction and axonal degeneration in the peripheral nervous system. An increasing number of genes underlying axonal neuropathies (type 2 CMTs) are being identified, but the disease mechanisms by which these mutations cause peripheral axon degeneration are not clear. Recently, mutations in LRSAM1 have been identified in both Canadian and Dutch pedigrees presenting with recessive and dominant inherited peripheral axonal neuropathies respectively. LRSAM1 is an E3 ubiquitin ligase with one known substrate, TSG101, which is a component of the ESCRT1 complex and important for late endosome to lysosome vesicular sorting and trafficking, among other functions. Several other Charcot-Marie-Tooth-associated proteins such as SIMPLE, RAB7, and FIG4 are also implicated in endosome/lysosome trafficking. No mammalian experimental model for LRSAM1-associated neuropathy exists, and whether LRSAM1 selectively associates with TSG101 and functions in endosome/lysosome sorting and trafficking in peripheral neurons remain unknown. To address these issues, we propose two aims. In Aim 1, we will use gene trap alleles in mice to produce Lrsam1 mutations and validate these mice as a model of human peripheral neuropathy. We presently have mice derived from two independent insertional mutations. These insertions do result in a loss of Lrsam1 expression and preliminary studies indicate that they cause a peripheral neuropathy phenotype in both heterozygous and homozygous mice, consistent with the human disease. We will continue the molecular, electrophysiological, histological, and behavioral characterization of these mice to establish their validity as a disease model. In Aim 2, we will examine the subcellular localization of LRSAM1 protein to determine if it is associated with other components of the endosome/lysosome trafficking pathway and if it colocalizes with other CMT-associated proteins in this pathway. In addition, we will examine the expression pattern of Lrsam1 in mice to determine if restricted expression may account for the peripheral neuron-specificity of the phenotype. Upon completion of these aims, we will have a new mouse model of human axonal CMT that will be available for preclinical and mechanistic studies. In addition, we will have a better understanding of the cellular pathway in which LRSAM1 functions that will inform future mechanistic studies and possibly illustrate a shared cellular pathway for LRSAM1 and other CMT-associated proteins. PUBLIC HEALTH RELEVANCE: This proposal seeks to validate an animal model of inherited peripheral neuropathy caused by mutations in LRSAM1. Mutations in LRSAM1 have been identified in both Canadian and Dutch pedigrees with inherited axonal neuropathy; however, the disease mechanism, and therefore potential therapeutic strategies, remains unclear. Using mouse resources, we have generated strains of mice lacking Lrsam1 expression. It appears that these mice have a neuropathy comparable to patients, and we will therefore establish these mice as a model for future preclinical and mechanistic studies.