Production and evaluation CMT2 mouse models of ATP1A1 loss-of-function-mutation using Cre-LoxP technology

家长津贴摘要1-R03 NS116433-01 Charcot-Marie-Tooth病(CMT)由一组不同类型的周围神经病变组成 通过90多个基因的突变。编码Na+，K+-ATPaseα1亚基的ATP1A1突变 最近被认为与CMT2有关，这是一种以轴突变性为特征的CMT形式。 Nka是一种异二聚体(αβ)蛋白，它能水解三磷酸腺苷以建立和维持Na+和K+梯度 穿过所有人类细胞的质膜。不同NKA同工酶的突变诱导了几个 神经系统疾病。ATP1A1普遍表达，与CMT2连锁的突变导致NKA丢失 功能。在与其他疾病相关的ATP1A1突变中也观察到NKA功能的丧失，包括 原发性醛固酮增多症和一种形式的低镁血症，伴有癫痫发作和认知障碍。 然而，缺乏适当的模型系统阻碍了对病理生理学的详细了解。 在这些ATP1A1突变相关疾病中。我们实验室的长期目标是了解 NKA的作用机制及其在生理和疾病状态中的作用。这样做的目的是 建议开发和评估动物模型来研究ATP1A1相关疾病，重点是CMT2。 目的1.对ATP1A1杂合基因敲除小鼠进行全面的神经病理学评估，以测试OUR 中心假设，在CMT2患者中看到的功能丧失突变的严重影响，包括高度 ATP1A1+/-小鼠的症状强度和发病年龄等变量应加以概括。 目的2.利用Cre-loxP技术建立新的ATP1A1功能缺失突变模型 假设一个ATP1A1等位基因在成年后缺失会加速CMT2症状的出现，并 神经元单倍体功能不全足以诱发CMT2。通过组织的产生-和时间- 依赖条件基因敲除小鼠使用他莫昔芬诱导的CreERT2系，我们将能够确定年龄- 依赖的代偿机制，以及局部或全身单倍体不足对CMT2诱导的必要性。 这些研究的成功完成将导致NKA相关病理生理学的可行小鼠模型 深入了解CMT2机制。该项目的结果将为未来的特定测试提供工具 NKA连锁CMT2的治疗。此外，这里概述的实验可能会为 其他与ATP1A1突变相关的疾病。科学地说，我们的结果将揭示NKAα1在 神经生理学阐明了它在两个神经系统中的重要性。这些老鼠模型将被制作成 通过标准的多机构多边技术协定提供给科学界。

SUMMARY of parent grant 1-R03 NS116433-01 Charcot-Marie-Tooth disease (CMT) comprises a heterogeneous group of peripheral neuropathies caused by mutations in over 90 genes. Mutation of ATP1A1, which encodes for the Na+,K+-ATPase (NKA) α1 subunit, has been recently associated with CMT2, a CMT form characterized by axonal degeneration. NKA is a heterodimeric (αβ) protein that hydrolyzes ATP to build and maintain the Na+ and K+ gradients across the plasma membrane of all human cells. Mutation in the different NKA isozymes induces several neurological diseases. ATP1A1 is ubiquitously expressed and the mutations linked to CMT2 cause NKA loss of function. Loss of NKA function is also observed in ATP1A1 mutants associated with other diseases, including primary hyperaldosteronism and a form of hypomagnesemia accompanied by seizures and cognitive delay. However, the lack of appropriate model systems has prevented a detailed understanding of the pathophysiology of these ATP1A1 mutation-linked disorders. The long-term goal of our laboratory is to understand the mechanisms of NKA function and the roles of NKA in physiological and disease states. The objective of this proposal is to develop and evaluate animal models to study ATP1A1-linked disease, with an emphasis on CMT2. Aim 1. Comprehensive neuropathic evaluation of heterozygous ATP1A1 knockout mice to test our central hypothesis, that the severe effect of loss-of-function mutations seen in CMT2 patients, including the highly variables symptom intensity and age of onset, should be recapitulated in ATP1A1+/- mice. Aim 2. Develop novel ATP1A1 loss-of-function-mutation models using CRE-LoxP technology to test the hypotheses that deletion of one ATP1A1 allele in adulthood accelerates the onset of CMT2 symptoms and that neuronal haploinsufficiency is sufficient to induce CMT2. Through the generation of tissue- and time- dependent conditional knockout mice using tamoxifen-inducible CreERT2 lines, we will be able to determine age- dependent compensatory mechanisms, and necessity of local or systemic haploinsufficiency for CMT2 induction. Successful completion of these studies will lead to viable mouse models of NKA-linked pathophysiology to gain insight into CMT2 mechanisms. The results from this project will provide a tool for future testing of specific treatments for NKA-linked CMT2. Additionally, the experiments outlined here are likely to provide models for the other ATP1A1 mutation-linked diseases. Scientifically, our results will uncover the functional roles of NKA α1 in neuron physiology elucidating its importance in both the nervous system. These mouse models will be made available to the scientific community through standard multi-institutional MTAs.