Novel Mouse Models for the Peripheral Neuropathy Disorder ACCPN

描述（由申请人提供）：K-Cl共转运蛋白3 （KCC3）突变是最严重的早期周围神经病变之一，称为ACCPN或“胼胝体发育与周围神经病变相关”。我们在小鼠胚胎干细胞中通过同源重组破坏了KCC3基因，小鼠周围神经表型与ACCPN患者相似。由于很难研究K-Cl在周围神经中的共转运功能，我们建议通过建立额外的小鼠模型来进一步研究KCC3在ACCPN中的作用。首先，由于除了一种人类突变外，所有突变都导致KCC3蛋白的羧基末端较短，因此我们建议创建一个具有单个错义氨基酸替换的敲入小鼠，并评估是否缺乏共转运蛋白功能或缺乏蛋白质-蛋白质相互作用导致了这种疾病。其次，由于共转运蛋白在感觉神经元和雪旺细胞中都有表达，我们建议创建具有组织特异性缺失的共转运蛋白的小鼠。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，我们设计了一个通用的模块化靶向构建体。由于我们已经确定了一个单一的残基替代，使共转运蛋白在不影响其膜表达的情况下失去功能，我们将利用编码该残基的外显子作为我们的目标区域来构建三个小鼠系。通过这种方式，两种结构将包含相同的重组臂，并允许共同的胚胎干细胞筛选策略来识别重组事件。小鼠表型将通过运动活动和周围神经病理分析。这两种新的小鼠模型将使我们进一步了解神经病变的发育与退行性，以及这种疾病的分子基础和细胞类型起源。本提案的目标是创建三种新的KCC3小鼠模型，这将促进我们对人类与KCC3基因破坏相关的周围神经病变疾病的理解。

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.