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

母公司补助金摘要1-R 03 NS 116433 -01 腓骨肌萎缩症（CMT）包括一组异质性周围神经病变， 超过90个基因的突变。编码Na+，K+-ATP酶（NKA）α1亚基的ATP 1A 1突变， 最近与CMT 2相关，CMT 2是一种以轴突变性为特征的CMT形式。 NKA是一种异源二聚体（αβ）蛋白，可水解ATP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同工酶中的突变诱导几种 神经系统疾病ATP 1A 1广泛表达，与CMT 2相关的突变导致NKA丢失， 功能在与其他疾病相关的ATP 1A 1突变体中也观察到NKA功能丧失，包括 原发性醛固酮增多症和一种伴有癫痫发作和认知延迟的低镁血症。 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 ATP 1A 1突变相关疾病的研究我们实验室的长期目标是了解 NKA的功能机制以及NKA在生理和疾病状态中的作用。的目的 一项建议是开发和评估动物模型，以研究ATP 1A 1相关疾病，重点是CMT 2。 目标1。对杂合子ATP 1A 1基因敲除小鼠进行全面的神经病理学评估， 中心假设，即在CMT 2患者中观察到的功能丧失突变的严重影响，包括高度 变量症状强度和发病年龄，应在ATP 1A 1 +/-小鼠中重现。 目标2.使用CRE-LoxP技术开发新型ATP 1A 1功能缺失突变模型， 假设在成年期缺失一个ATP 1A 1等位基因会加速CMT 2症状的发作， 神经元单倍不足足以诱导CMT 2。通过组织的生成和时间 使用他莫昔芬诱导的CreERT 2系的依赖性条件敲除小鼠，我们将能够确定年龄- 依赖性补偿机制，以及CMT 2诱导的局部或全身单倍不足的必要性。 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CMT 2机制。该项目的结果将为未来测试特定的 NKA连锁的CMT 2的治疗。此外，这里概述的实验可能会提供模型， ATP 1A 1突变相关疾病科学上，我们的研究结果将揭示NKA α1的功能作用， 神经元生理学阐明了它在神经系统中的重要性。这些老鼠模型将被制作出来 通过标准的多机构多边贸易协定向科学界提供。

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.