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

描述(申请人提供)：转基因表达突变形式的人类超氧化物歧化酶I(SOD1)导致家族性肌萎缩侧索硬化症(FALS)的小鼠发展为进行性运动神经末梢丧失，通常在脊髓运动神经元细胞体最终死亡之前。除了这种早期易受FALS诱导的变性外，我们还发现有症状前的SOD1-G93A小鼠的运动终末(高拷贝数)也比野生型终末更容易受到短暂的缺血/再灌流应激引起的变性。在健康的运动终末，线粒体是处理大的、刺激诱导的钙负荷所必需的。拟议的研究将检验这一假设，即不同的FALS小鼠模型的运动终末由于线粒体钙处理能力不足而变得越来越容易受到压力的影响。测试的应激包括对运动神经的高频刺激、能量应激(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，足以隔离与这些应激相关的钙超载，导致线粒体去极化，胞浆[Ca~(2+)]毒性增加，并由Calain介导的变性。我们将使用具有(G93A)或缺乏(G85R)歧化酶活性且SOD1表达水平不同(高和低拷贝数G93A)的SOD1-FALS模型，以测试这些FALS突变体是否会比表达野生型人SOD1的小鼠表现出更低的应激阈值。荧光指示剂染料在活体终端的成像将用于分析运动终末、胞浆和线粒体[Ca~(2+)]和线粒体膜电位的变化。抑制剂将被用来阻断假想的损伤途径，包括细胞内钙离子过度积累，线粒体通透性转换孔开放，钙蛋白激活和活性氧物种的产生。应激诱导的肌肉失神经的程度和所用治疗的任何保护效果将使用运动神经末梢进行分析，在运动神经末梢中转基因表达黄色荧光蛋白，并用荧光标记的1-银环蛇毒素识别肌肉终板。拟议中的实验很重要，因为他们将测试线粒体钙处理缺陷是否是介导运动终末损害的主要上游机制，并将确定可以保护运动终末免受这种损害的药物。PROJECT家族性肌萎缩侧索硬化症(FALS)的叙事性小鼠模型显示运动神经末梢早期退化。拟议的实验将检验这样一种假设，即多个品系的FALS小鼠的运动终末在能量压力下变得特别脆弱，并且这种脆弱性与线粒体对钙负载的处理有缺陷有关。我们将测试多种保护电机端子免受这些应力影响的策略。保留残留运动神经末梢的治疗应该会减缓瘫痪的进展，因此可能成为治疗ALS的一个重要的新组成部分。

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.