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

使用 Cre-LoxP 技术制作和评估 ATP1A1 功能丧失突变的 CMT2 小鼠模型

• 批准号: 10351607
• 负责人: Pablo Artigas
• 金额: $ 6.04万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10351607
• 关键词: ATP1A1 gene; Address; Adult; Age; Age of Onset; Alleles; Animal Model; Axon; Behavioral; Biological Models; Cell membrane; Cells; Characteristics; Charcot-Marie-Tooth Disease; Cognitive; Communities; Complement; Cre-LoxP; Data; Development; Discrimination; Disease; Electrophysiology (science); Evaluation; Exercise; Family; Fiber; Functional disorder; Future; Generations; Genes; Genetic Recombination; Genetic Transcription; Goals; Hereditary Motor and Sensory-Neuropathy Type II; Histologic; Human; Human body; Hyperaldosteronism; Hypomagnesemia; In Vitro; Induced Mutation; Inherited; Isoenzymes; Knock-out; Knockout Mice; Laboratories; Light; Link; Longevity; LoxP-flanked allele; Masks; Messenger RNA; Modeling; Motor; Mus; Mutation; Myocardium; Na(+)-K(+)-Exchanging ATPase; Nervous system structure; Neuraxis; Neurofilament Proteins; Neurologic; Neurons; Neuropathy; Patients; Peripheral; Peripheral Nervous System Diseases; Phenotype; Physiological; Physiology; Primary Hyperaldosteronism; Production; Protein Isoforms; Proteins; Role; Secondary Hypertension; Seizures; Sensory; Severities; Skeletal Muscle; Symptoms; Tamoxifen; Techniques; Technology; Testing; Time; Tissues; age related; axonal degeneration; conditional knockout; experimental study; insight; loss of function; loss of function mutation; mouse model; mutant; nervous system disorder; novel; parent grant; prevent; protein expression; tool

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.

家长补助金摘要 1-R03 NS116433-01 夏科-马里-图思病 (CMT) 包括一组异质性周围神经病，其原因是 超过 90 个基因发生突变。 ATP1A1 突变，编码 Na+,K+-ATP 酶 (NKA) α1 亚基， 最近与 CMT2 相关，CMT2 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，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 同工酶的突变会诱导多种 神经系统疾病。 ATP1A1 普遍表达，与 CMT2 相关的突变导致 NKA 缺失 功能。在与其他疾病相关的 ATP1A1 突变体中也观察到 NKA 功能的丧失，包括 原发性醛固酮增多症和一种低镁血症，伴有癫痫发作和认知迟缓。 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 这些 ATP1A1 突变相关疾病。我们实验室的长期目标是了解 NKA 功能机制以及 NKA 在生理和疾病状态中的作用。此举的目的 该提案的目的是开发和评估动物模型来研究 ATP1A1 相关疾病，重点是 CMT2。 目标 1. 对杂合 ATP1A1 敲除小鼠进行全面的神经病理评估，以测试我们的 中心假设，CMT2 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 ATP1A1+/- 小鼠中重现。 目标 2. 使用 CRE-LoxP 技术开发新型 ATP1A1 功能丧失突变模型进行测试 假设成年期删除一个 ATP1A1 等位基因会加速 CMT2 症状的发作， 神经元单倍体不足足以诱导 CMT2。通过组织和时间的生成 使用他莫昔芬诱导的 CreERT2 系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 CMT2 诱导局部或系统单倍体不足的必要性。 这些研究的成功完成将产生可行的 NKA 相关病理生理学小鼠模型 深入了解 CMT2 机制。该项目的结果将为未来测试特定的 NKA 相关的 CMT2 的治疗。此外，这里概述的实验可能会提供模型 其他 ATP1A1 突变相关疾病。从科学角度来说，我们的结果将揭示 NKA α1 在 神经元生理学阐明了其在神经系统中的重要性。这些鼠标模型将被制作 通过标准的多机构 MTA 向科学界提供。