Ca2+ mishandling and ischemia-vulnerability in fALS model motor terminals

fALS 模型运动终端中的 Ca2 处理不当和缺血脆弱性

基本信息

批准号：
8029525
负责人：
GAVRIEL DAVID
金额：
$ 26.24万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-02-01 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8029525
关键词：
Age Animal Model Animals Antioxidants Biological Assay Blood Bungarotoxins Calcium Calpain Cell membrane Cells Cessation of life Collaborations Contralateral Cyclosporine Cytosol Data Denervation Development Dextrans Disease Progression Dyes EUK-134 Equipment Exhibits Familial Amyotrophic Lateral Sclerosis Frequencies Functional Imaging Functional disorder Generations Glucose Head Hindlimb Human Hydrogen Peroxide Image In Vitro Ischemia Knowledge Label Laboratories Life Limb structure Link Measures Mediating Membrane Membrane Potentials Mitochondria Modeling Monitor Motor Motor Neurons Mus Muscle Muscle Fibers Muscle denervation procedure Mutate Mutation Natural regeneration Nerve Neurons Oligomycins Onset of illness Oregon Oxygen Paralysed Peptides Physiological Preparation Presynaptic Terminals Principal Investigator Production Property Proteins Publications Reactive Oxygen Species Reperfusion Injury Reperfusion Therapy Research Research Personnel Respiration Rhodamine 123 Rotenone Route Solutions Spinal Cord Stress Superoxide Dismutase Symptoms Techniques Testing Tetrodotoxin Transgenic Mice axonal sprouting calpain inhibitor catalase deprivation dextran extensor digitorum in vivo inhibitor/antagonist innovation mitochondrial membrane mitochondrial permeability transition pore mouse model mutant neuronal cell body operation programs protective effect reinnervation research study response tetramethylrhodamine methyl ester therapy design uptake voltage

项目摘要

DESCRIPTION (provided by applicant): Mice that transgenically express mutated forms of human superoxide dismutase I (SOD1) that produce familial amyotrophic lateral sclerosis (fALS) develop progressive loss of motor nerve terminals that often precedes the eventual death of motor neuron cell bodies in the spinal cord. In addition to this early vulnerabiltiy to fALS-induced degeneration, we found that motor terminals of pre- symptomatic SOD1-G93A mice (high-copy number) are also more susceptible than wild-type terminals to degeneration caused by brief ischemia/reperfusion stress. In healthy motor terminals, mitochondria are essential for handling large, stimulation-induced Ca2+ loads. The proposed studies will test the hypothesis that motor terminals of different fALS mouse models become increasingly vulnerable to stresses due to deficient mitochondrial Ca2+ handling. Stresses to be tested include high frequency stimulation of motor nerves, energy stresses (ischemia/reperfusion, oxygen/glucose deprivation) and hydrogen peroxide. We hypothesize that mitochondria of fALS mouse terminals eventually lose their capacity to increase respiration sufficiently to sequester the Ca2+ overload associated with these stresses, resulting in mitochondrial depolarization, toxic increases in cytosolic [Ca2+] and calpain-mediated degeneration. We will use SOD1-fALS models that possess (G93A) or lack (G85R) dismutase activity, and that differ in their level of SOD1 expression level (high and low copy number G93A), to test if these fALS mutants will exhibit a lower threshold to these stresses than mice expressing wild-type human SOD1. Imaging of fluorescent indicator dyes in living terminals will be used to assay changes in motor terminal cytosolic and mitochondrial [Ca2+] and mitochondrial membrane potential. Inhibitors will be used to block hypothesized routes of damage, including excessive accumulation of cytosolic Ca2+, opening of the mitochondrial permeability transition pore, calpain activation and generation of reactive oxygen species. The extent of stress-induced muscle denervation and any protective effect of applied treatments will be assayed using motor terminals in which yellow fluorescent protein is transgenically expressed and muscle endplates are identified with fluorescently-labelled 1- bungarotoxin. The proposed experiments are important because they will test whether defective mitochondrial Ca2+ handling is a major upstream mechanism mediating motor terminal damage, and will identify agents that can protect motor terminals against this damage.PROJECT NARRATIVE Mouse models of familial amyotrophic lateral sclerosis (fALS) exhibit early degeneration of motor nerve terminals. The proposed experiments will test the hypothesis that motor terminals in multiple strains of fALS mice become especially vulnerable to energy stresses, and that this vulnerability involves defective mitochondrial handling of calcium loads. We will test multiple strategies for protecting motor terminals against these stresses. Treatments to preserve remaining motor nerve terminals should slow the progression of paralysis, and thus may become an important new component of therapies for treating ALS.

描述（由申请人提供）：跨传感器表达突变形式的人类超氧化物歧化酶I（SOD1）的小鼠产生家族性肌萎缩性侧面硬化症（FALS）会发展出运动神经终末的逐渐丧失，这通常是脊髓中运动神经元细胞最终死亡的运动神经终末。除了对伪造引起的变性的早期脆弱性外，我们还发现，症状性SOD1-G93A小鼠的运动末端（高拷贝数）也比野生型末端更容易受到野生型末端的影响，从而引起了由简短的缺血/再灌注胁迫引起的变性。在健康的运动端子中，线粒体对于处理大型刺激诱导的Ca2+载荷至关重要。提出的研究将检验以下假设：由于线粒体CA2+处理不足，不同fal的小鼠模型的运动末端变得越来越容易受到应力的影响。要测试的应力包括高频刺激运动神经，能量应力（缺血/再灌注，氧/葡萄糖剥夺）和过氧化氢。我们假设线粒体最终失去了足够增加呼吸的能力，以隔离与这些应力相关的Ca2+过载，从而导致线粒体去极化，毒性增加了胞质[Ca2+]和Calpain介导的毒性。我们将使用具有（G93A）或缺乏（G85R）歧化酶活性的SOD1-FALS模型，并且它们的SOD1表达水平（高和低拷贝数G93A）的水平有所不同，以测试这些fals突变体是否表现出比表达野生型人类SOD1的小鼠的阈值。活末端中荧光指示剂染料的成像将用于测定运动末端胞质和线粒体[Ca2+]和线粒体膜电位的变化。抑制剂将用于阻止假设的损伤途径，包括胞质CA2+的过度积累，线粒体渗透性过渡孔的打开，加仑蛋白酶激活和反应性氧物种的产生。应使用型黄色荧光蛋白转基因表达黄色荧光蛋白，并用荧光标记的1-bungarotoxin鉴定出黄色荧光蛋白的运动末端测定应力诱导的肌肉神经支配的程度和应用处理的任何保护作用。提出的实验很重要，因为它们将测试有缺陷的线粒体Ca2+处理是否是介导运动末端损伤的主要上游机制，并将确定可以保护运动末端的药物。提出的实验将检验以下假设：多种菌株小鼠中的运动终端变得特别容易受到能量应力的影响，并且该脆弱性涉及钙负载的线粒体处理缺陷。我们将测试多种保护运动终端免受这些应力的策略。保留剩余运动神经末端的治疗方法应减缓瘫痪的进展，因此可能成为治疗ALS治疗的重要组成部分。