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.

父母赠款1-R03 NS116433-01的摘要 charcot-marie-tooth病（CMT）包括一组异质的周围神经病 通过90多个基因中的突变。 ATP1A1的突变，该突变编码为Na+，K+-ATPase（NKA）α1亚基的突变， 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，可以水解ATP以构建和维持Na+和K+梯度 跨越所有人类细胞的质膜。不同NKA同工酶中的突变会影响几个 神经疾病。 ATP1A1无处不在，与CMT2相关的突变导致NKA的NKA损失 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 原发性高醛烷和通过癫痫发作和认知延迟完成的低磁性血症的一种形式。 但是，缺乏适当的模型系统阻止了对病理生理学的详细了解 这些ATP1A1突变连接疾病。我们实验室的长期目标是了解 NKA功能的机制以及NKA在物理和疾病状态中的作用。这个目的 提案是开发和评估动物模型以研究ATP1A1连接疾病，重点是CMT2。 目标1。杂合子ATP1A1敲除小鼠的全面神经性评估以测试我们 中心假设，即在CMT2患者中看到功能丧失突变的严重影响，包括高度 变量症状强度和发作年龄应在ATP1A1 +/-小鼠中概括。 目标2。使用Cre-loxp技术测试新颖的ATP1A1丧失功能突变模型 在成年期删除一个ATP1A1等位基因的假设加速了CMT2症状和 这种神经元的单倍不足足以诱导CMT2。通过组织和时间的产生 使用他莫昔芬诱导的creert2系的依赖性有条件敲除小鼠，我们将能够确定年龄 依赖的补偿机制，以及CMT2诱导的局部或全身单倍不足的必需机制。 这些研究的成功完成将导致可行的NKA连接病理生理学的小鼠模型 了解CMT2机制。该项目的结果将为未来测试特定的工具提供 NKA连接CMT2的处理。此外，此处概述的实验可能会为 其他ATP1A1突变连接疾病。从科学上讲，我们的结果将发现NKAα1在 神经元的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可供科学界。