Novel Mouse Models for the Peripheral Neuropathy Disorder ACCPN

描述(申请人提供)：K-Cl协同转运蛋白3(KCC3)的突变是最严重、最早发病的周围神经病之一，称为ACCPN或“与周围神经病相关的胼胝体发育不全”。我们已经在小鼠胚胎干细胞中通过同源重组破坏了KCC3基因，小鼠周围神经的表型与在ACCPN患者中观察到的相似。由于研究外周神经K-Cl共转运功能的难度较大，我们建议通过建立更多的小鼠模型来进一步研究KCC3在ACCPN中的作用。首先，由于除了一个人类突变外，所有的突变都会导致KCC3蛋白的羧基末端缩短，我们建议创建一个带有单一错义氨基酸替换的敲入小鼠，并评估是否缺乏协转运体功能或蛋白质-蛋白质相互作用的缺失导致了这种疾病。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，我们建议建立具有组织特异性共转运蛋白缺失的小鼠。为了显著减少组装结构所需的时间并提高获得这些新的小鼠模型的效率，我们设计了一种通用的模块化靶向结构。由于我们已经确定了一个单一的残基替代，使共转运蛋白不起作用，而不影响其膜表达，因此我们将利用编码该残基的外显子作为我们的靶区来构建三个小鼠品系。通过这种方式，这两个结构将包含相同的重组臂，并允许共同的ES细胞筛选策略来识别重组事件。将通过运动活动和周围神经病理来分析小鼠的表型。这两个新的小鼠模型将使我们进一步了解神经病变的发育和退行性本质，以及该疾病的分子基础和细胞类型起源。公共卫生相关性这项建议的目标是创建三个新的KCC3小鼠模型，这将促进我们对与人类KCC3基因中断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.