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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：
8212196
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 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8212196
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）：转基因表达突变形式的人超氧化物歧化酶I（SOD 1）的小鼠产生家族性肌萎缩侧索硬化症（fALS），其发生运动神经末梢的进行性丧失，这通常先于脊髓中运动神经元细胞体的最终死亡。除了这种对fALS诱导的变性的早期易变性之外，我们发现症状前SOD 1-G93 A小鼠（高拷贝数）的运动终末也比野生型终末更易受短暂缺血/再灌注应激引起的变性的影响。在健康的运动终端，线粒体是必不可少的处理大，刺激诱导的Ca 2+负荷。拟议的研究将测试的假设，不同的fALS小鼠模型的运动终端变得越来越容易受到压力，由于缺乏线粒体Ca 2+处理。待测试的应激包括运动神经的高频刺激、能量应激（缺血/再灌注、氧/葡萄糖剥夺）和过氧化氢。我们假设fALS小鼠终末的线粒体最终失去了充分增加呼吸的能力，以隔离与这些应激相关的Ca 2+过载，导致线粒体去极化，细胞溶质[Ca 2 +]和钙蛋白酶介导的变性的毒性增加。我们将使用具有（G93 A）或缺乏（G85 R）歧化酶活性的SOD 1-fALS模型，并且它们的SOD 1表达水平不同（高和低拷贝数G93 A），以测试这些fALS突变体是否将表现出比表达野生型人SOD 1的小鼠更低的对这些应激的阈值。将使用活终端中荧光指示染料的成像来测定运动终端胞质和线粒体[Ca 2 +]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Ca 2+的过度积累、线粒体渗透性转换孔的开放、钙蛋白酶激活和活性氧的产生。应力诱导的肌肉去神经支配的程度和施加的治疗的任何保护作用将使用运动终末进行测定，其中黄色荧光蛋白被转基因表达，并且肌肉终板用荧光标记的1-银环蛇毒素鉴定。拟议的实验是重要的，因为他们将测试是否有缺陷的线粒体Ca 2+处理是一个主要的上游机制介导的运动终端damage. PROJECT叙述性代理，可以保护运动终端对这种damage.PROJECT叙述性小鼠模型的家族性肌萎缩侧索硬化症（fALS）表现出早期退化的运动神经末梢。拟议的实验将测试这样的假设，即多个fALS小鼠品系的运动终端变得特别容易受到能量应激的影响，并且这种脆弱性涉及线粒体对钙负荷的处理缺陷。我们将测试多种策略来保护运动终端免受这些压力。保留剩余运动神经末梢的治疗应该减缓瘫痪的进展，因此可能成为治疗ALS的重要新组成部分。