Ca Signaling in Progression of Amyotrophic Lateral Sclerosis in Skeletal Muscle

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种进行性神经肌肉疾病，涉及运动神经元退化和骨骼肌萎缩。超氧化物歧化酶(SOD1)基因的突变与大多数遗传性ALS病例有关，导致线粒体代谢功能受损。目前，对于肌萎缩侧索硬化症进展过程中线粒体功能障碍和钙信号改变如何导致肌肉退行性变缺乏了解。这项由研究者发起的新研究项目将检验一种假说，即在ALS的发展过程中，神经肌肉接头(NMJ)的动作电位活动将导致线粒体局部钙超载，这种超载将进一步破坏线粒体-肌浆网(SR)偶联，导致钙和活性氧(ROS)信号的改变。这种变化反过来将增加局部钙，加强局部缺失，从而构成ALS肌肉病理生理中的一个主要事件。我们研究的中心主题是全面了解导致肌萎缩侧索硬化症肌肉钙稳态能力受损的线粒体-SR偶联进行性下降的细胞机制。我们预计，从我们的研究中获得的信息将适用于其他神经肌肉疾病，如衰老、糖尿病、帕金森病和其他，在这些疾病中，钙信号和细胞内氧化应激与肌肉收缩功能障碍和运动神经元通讯改变有关。在目标1中，我们建议量化线粒体在肌浆网钙信号的局部控制中的作用，并利用这种量化来评估线粒体-SR偶联缺陷如何促进ALS肌肉萎缩的进展。通过对肌萎缩侧索硬化症进展不同阶段骨骼肌中钙信号和线粒体功能变化的研究，我们的研究应该提供一个机制，了解线粒体如何改变肌浆网钙信号的局部控制，这一过程在肌萎缩侧索硬化症肌肉中是如何改变的，以及轴突退出在破坏肌浆网-线粒体偶联导致肌萎缩侧索硬化症肌肉功能进行性下降方面有什么影响。在目标2中，我们建议使用局部线粒体超氧化物(O2-)闪光信号作为ALS肌肉线粒体代谢功能的指标，并研究失神经对ALS肌肉萎缩过程中线粒体-SR控制细胞内钙释放和局部O2-水平相互作用的影响。这些研究将揭示钙和O2-产生的局部偶联在肌肉生理学中的作用，并建立有助于肌萎缩侧索硬化症NMJ重塑和肌肉病理进展的细胞机制(S)。 公共卫生相关性：肌萎缩侧索硬化症(ALS)，通常被称为“卢格里克病”，是一种进行性神经肌肉疾病，涉及大脑和脊髓中神经细胞的丧失，以及骨骼肌萎缩。人们普遍认为，神经退行性变会先于肌肉萎缩。我们问肌肉是否也参与了疾病的发生和发展。肌肉细胞使用钙(Ca)作为信使来控制其收缩活动，而细胞内钙的过度升高会导致细胞死亡。我们在ALS转基因小鼠模型的肌细胞中发现了不受控制的高活性钙信号。两个主要的细胞器参与了细胞内钙水平的严密控制，肌浆网(SR)-肌肉细胞中存储钙的位置，以及线粒体-也可以产生有害的氧化应激的能量屋。在这个项目中，利用最先进的分子工具和转基因小鼠模型，我们将致力于全面了解导致ALS肌肉中钙信号受损的线粒体-SR偶联进行性下降的细胞机制。预计我们的结果将加速开发新的方法来扩展ALS患者的肌肉功能。我们还相信，从我们的研究中获得的信息将普遍应用于其他神经肌肉疾病，如衰老、糖尿病、帕金森氏病等，这些疾病中钙信号的改变和细胞内氧化应激与收缩功能和神经元通讯的功能障碍有关。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder involving degeneration of motor neurons and atrophy of skeletal muscle. Mutations in the superoxide dismutase (SOD1) gene are linked to most cases of inherited ALS, resulting in impaired metabolic function of the mitochondria. Currently, there is a lack of knowledge on how dysfunctional mitochondria and altered Ca signaling contribute to muscle degeneration during ALS progression. This new investigator initiated research project will test the hypothesis that "during the development of ALS, action potential activity at the neuromuscular junction (NMJ) will cause local Ca overload in mitochondria compromised by the presence of mutant SOD1. This overload will further disrupt mitochondria-sarcoplasmic reticulum (SR) coupling and lead to altered Ca and reactive oxygen species (ROS) signaling. The alteration in turn will increase local Ca, reinforcing the local deficit, thus constituting a primary event in the pathophysiology of ALS muscle". The central theme of our studies is to develop a comprehensive understanding of the cellular mechanism that contributes to the progressive decline in mitochondria-SR coupling leading to the compromised Ca homeostatic capacity of ALS muscle. We anticipate that information obtained from our research will apply to other neuromuscular diseases, such as aging, diabetes, Parkinson's disease and others, where altered Ca signaling and intracellular oxidative stress are linked to dysfunctional muscle contractility and altered motor neuron communication. In Aim 1, we propose to quantify the mitochondrial contribution to local control of SR Ca signaling, and use this quantification to evaluate how defective mitochondria-SR coupling contributes to progression of muscle wasting in ALS. By characterizing the changes in Ca signaling and mitochondrial function in skeletal muscle at different stages of ALS progression, our studies should provide a mechanistic understanding of how local control of SR Ca signaling is altered by mitochondria, how this process is altered in ALS muscle and what is the influence of axonal withdrawal in disrupting SR-mitochondria coupling leading to the progressive decline of muscle function in ALS. In Aim 2, we propose to use localized mitochondrial superoxide (O2-) FLASH signal as an index for mitochondrial metabolic function in ALS muscle, and to examine the effect of denervation on the interplay between mitochondria-SR in controlling intracellular Ca release and local O2- levels during the progression of muscle atrophy in ALS. These studies will reveal the role of localized coupling between Ca and O2- production in muscle physiology, and establish the cellular mechanism(s) that contribute to NMJ remodeling and the progressive development of muscle pathology in ALS. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS), often referred to as "Lou Gehrig's Disease," is a progressive neuromuscular disorder that involves loss of nerve cells in the brain and the spinal cord, and atrophy of skeletal muscle. It is generally believed nerve degeneration precedes and then causes the muscle atrophy. We ask if muscle is also involved in the disease onset and progression. Muscle cells use Calcium (Ca) as a messenger to control their contraction activity, while excessive elevation of Ca inside the cell will cause cell death. We have discovered uncontrolled hyperactive Ca signals in muscle cells of ALS transgenic mouse model. Two major organelles are involved for the tight control of the intracellular Ca level, the sarcoplasmic reticulum (SR)-the site of Ca storage in muscle cells, and mitochondria-"the power house" that can also produce detrimental oxidative stress. In this project, using state of the art molecular tools and transgenic mouse models we will aim at a comprehensive understanding of the cellular mechanism that contributes to the progressive decline in mitochondria-SR coupling that leads to the compromised Ca signaling in ALS muscle. It is anticipated that our results will accelerate development of new means to extend muscle function of patients suffering from ALS. We also believe that information obtained from our research will be of general application to other neuromuscular diseases, such as aging, diabetes, Parkinson's disease, etc, where altered Ca signaling and intracellular oxidative stress are linked to dysfunctional properties of contractile function and neuron communication.