Novel Mouse Models for the Peripheral Neuropathy Disorder ACCPN

描述（由申请人提供）：K-Cl协同转运蛋白3（KCC 3）突变导致最严重的早发性周围神经病变之一，称为ACCPN或“与周围神经病变相关的胼胝体增生”。我们通过同源重组在小鼠胚胎干细胞中破坏了KCC 3基因，小鼠外周神经表型与ACCPN患者中观察到的相似。由于在周围神经中研究K-Cl共转运功能的困难，我们建议通过建立额外的小鼠模型来进一步研究KCC 3在ACCPN中的作用。首先，由于除了一个人突变之外的所有人突变都导致具有较短羧基末端的截短的KCC 3蛋白，因此我们建议创建具有单个错义氨基酸取代的敲入小鼠，并评估是否缺乏协同转运蛋白功能或缺乏蛋白质-蛋白质相互作用导致了该病症。第二，由于协同转运蛋白在感觉神经元和雪旺细胞中都有表达，我们建议用协同转运蛋白的组织特异性缺失来创建小鼠。为了显著减少组装构建体所需的时间并提高获得这些新小鼠模型的效率，我们设计了一种通用的模块化靶向构建体。由于我们已经鉴定了使协同转运蛋白无功能而不影响其膜表达的单个残基取代，我们将利用编码该残基的外显子作为我们的靶向区域来构建三个小鼠系。以这种方式，两种构建体将含有相同的重组臂，并允许共同的ES细胞筛选策略来鉴定重组事件。将通过自发活动和外周神经病理学分析小鼠表型。这两种新的小鼠模型将使我们能够进一步了解神经病变的发育与退行性本质，以及这种疾病的分子基础和细胞类型起源。本提案的目标是建立三种新型KCC 3小鼠模型，这将促进我们对与人类KCC 3基因破坏相关的周围神经病变的理解。

DESCRIPTION (provided by applicant): Mutations in K-Cl cotransporter 3 (KCC3) are responsible for one of the most severe, early onset peripheral neuropathy disorders called ACCPN or "Agenesis of Corpus Callosum associated with Peripheral Neuropathy". We have disrupted the KCC3 gene by homologous recombination in mouse embryonic stem cells and the mouse peripheral nerve phenotype is similar to that observed in ACCPN patients. Due to the difficulty in studying K-Cl cotransport function in peripheral nerves, we propose to further examine the role of KCC3 in ACCPN by creating additional mouse models. First, as all but one human mutation results in truncated KCC3 proteins with shorter carboxyl- termini, we propose to create a knock-in mouse with a single missense amino acid substitution and assess whether absence of cotransporter function or absence of protein-protein interaction is causing the disorder. Second, as the cotransporter is expressed in both sensory neurons and Schwann cells, we propose to create mice with tissue-specific deletions of the cotransporter. To significantly reduce the time necessary to assemble the constructs and increase efficiency in obtaining these new mouse models, we have designed a common modular targeting construct. As we have identified a single residue substitution that renders the cotransporter non-functional without affecting its membrane expression, we will utilize the exon encoding this residue as our targeted region to construct the three mouse lines. In this way, both constructs will contain the same arms of recombination and allow for a common ES cell screening strategy to identify recombination events. Mouse phenotype will be analyzed through locomotor activity and peripheral nerve pathology. These two new mouse models will allow us to further understand the developmental versus degenerative nature of the neuropathy, as well as the molecular basis and cell-type origin of the disorder. PUBLIC HEALTH RELEVANCE The goal of this proposal is the creation of three novel KCC3 mouse models that will advance our understanding of the peripheral neuropathy disorder associated with disruption of the KCC3 gene in humans.