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

• 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
• 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

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）：转基因表达产生家族性肌萎缩性侧索硬化症（fALS）的人超氧化物歧化酶I （SOD1）突变形式的小鼠会发生运动神经末梢的进行性丧失，通常在脊髓运动神经元细胞体最终死亡之前。除了对fals诱导的变性的早期易感性外，我们发现症状前SOD1-G93A小鼠（高拷贝数）的运动末端也比野生型末端更容易受到短暂缺血/再灌注应激引起的变性。在健康的运动终末，线粒体对于处理大的、刺激诱导的Ca2+负荷是必不可少的。拟议的研究将验证不同fALS小鼠模型的运动终端由于线粒体Ca2+处理缺陷而变得越来越容易受到压力的假设。需要测试的压力包括运动神经的高频刺激、能量压力（缺血/再灌注、缺氧/葡萄糖剥夺）和过氧化氢。我们假设fALS小鼠末端的线粒体最终失去了增加呼吸的能力，不足以隔离与这些应激相关的Ca2+过载，导致线粒体去极化，细胞质[Ca2+]毒性增加和calpain介导的变性。我们将使用SOD1-fALS模型，具有（G93A）或缺乏（G85R）歧化酶活性，并且SOD1表达水平不同（高拷贝数G93A和低拷贝数G93A），以测试这些fALS突变体是否比表达野生型人类SOD1的小鼠表现出更低的应激阈值。活端荧光指示染料成像将用于检测运动端细胞质和线粒体[Ca2+]和线粒体膜电位的变化。抑制剂将用于阻断假设的损伤途径，包括胞质Ca2+的过度积累、线粒体通透性过渡孔的打开、钙蛋白酶的激活和活性氧的产生。应力诱导的肌肉去神经支配程度和应用处理的任何保护作用将使用运动终端进行检测，其中黄色荧光蛋白转基因表达，肌肉终板用荧光标记的1- bungarotoxin进行鉴定。提出的实验很重要，因为它们将测试线粒体Ca2+处理缺陷是否是介导运动终端损伤的主要上游机制，并将确定可以保护运动终端免受这种损伤的药物。家族性肌萎缩性侧索硬化症（fALS）小鼠模型表现为运动神经末梢的早期变性。拟议的实验将验证一种假设，即多种fALS小鼠的运动终端特别容易受到能量压力的影响，这种脆弱性涉及线粒体处理钙负荷的缺陷。我们将测试多种策略来保护电机端子免受这些应力的影响。保留剩余运动神经末梢的治疗可以减缓瘫痪的进展，因此可能成为治疗ALS的重要新组成部分。