Testing the mechanisms by which NMJ disruption contributes to sarcopenia

描述（由申请人提供）： 肌肉减少症（肌肉质量和功能的丧失）普遍影响老年人，并对老年退伍军人的生活质量、独立生活、残疾和医疗费用产生巨大影响。氧化应激已被认为在许多与年龄相关的退行性疾病（包括肌肉减少症）的潜在机制中发挥作用。在我的实验室以前的研究中，我们已经表明，缺乏抗氧化酶CuZnSOD的小鼠（Sod 1-/-小鼠）具有高水平的氧化应激，并表现出肌肉质量和功能的损失，类似于在老年野生型小鼠中观察到的退行性变化。因此，Sod 1-/-小鼠是一个强大的模型，以获得独特的洞察肌肉减少症的机制。衰老和Sod 1-/-小鼠中最引人注目的表型之一是神经肌肉接头（NMJ）的破坏和断裂。NMJ是运动神经元和骨骼肌之间相互作用的部位，对肌肉活力和性能至关重要。目前，运动神经元和肌肉在NMJ恶化和年龄相关性肌肉萎缩中的相对贡献知之甚少。这些知识对于我们设计有效干预措施以延迟或预防肌肉减少症的能力至关重要。因此，为了解决这个重要问题，我们生成了一个条件性Sod 1-/-小鼠模型，其中我们可以分别删除肌肉和神经中的Sod 1，以评估这两种组织对NMJ恶化和肌肉萎缩的相对贡献。我们证明，Sod 1的删除仅限于骨骼肌组织不会导致肌肉萎缩或NMJ的改变，这表明肌肉萎缩是由运动神经元的变化引起的。在目前的研究中，我们将测试的假设，在神经肌肉接头的改变，通过启动骨骼肌下游退行性过程在肌肉减少症中发挥关键作用。为此，我们将通过突触前和突触后改变来负性和正性调节NMJ，并确定对肌肉中导致萎缩和肌无力的下游通路的影响。首先，我们将确定是否增加突触前氧化应激产生的神经元特异性缺失的Sod 1在小鼠（nSod 1-/-小鼠）导致NMJ变性和肌肉萎缩途径的启动。我们将测量年龄匹配的野生型、nSod 1-/-小鼠和Sod 1-/-小鼠以及老年野生型小鼠的NMJ形态、组成和功能以及乙酰胆碱受体（AchR）片段。此外，我们将采取无偏见的方法在基因表达的变化，以响应NMJ中断使用微阵列和更有偏见的方法来测量肌肉退行性通路的变化，表明在我们以前的研究中改变神经支配的损失在Sod 1-/-小鼠（钙蛋白酶蛋白酶和蛋白酶体活性，线粒体功能和ROS的产生和肌肉蛋白的氧化修饰）。相反，我们将确定是否通过在Sod 1-/-小鼠（nTgSod 1-/-小鼠）中的人Sod 1转基因的神经元特异性表达来逆转突触前氧化应激，从而挽救NMJ变性并防止基因表达变化和肌肉萎缩途径的启动。这些研究将使我们能够确定突触前变化对NMJ和肌肉萎缩通路的影响。最后，我们将测量由聚集蛋白缺失引起的突触后NMJ中断的影响。因为聚集蛋白对于NMJ聚集和稳定性是必不可少的，聚集蛋白的损失导致突触后NMJ破坏和肌肉萎缩。虽然目标1和2中使用的模型直接靶向突触前神经元的作用，但该模型允许我们靶向突触后侧的NMJ，以确定在NMJ破坏的突触后模型中基因表达变化和肌肉退行性途径是否相同或不同。这些研究将共同定义NMJ在肌肉萎缩中的作用，并指出受NMJ破坏影响的共同途径，这可能是干预的重要目标。

DESCRIPTION (provided by applicant): Sarcopenia (loss of muscle mass and function) universally affects the elderly and has a tremendous impact on quality of life, independent living, disability and healthcare costs in aging veterans. Oxidative stress has been implicated to play a role in the underlying mechanisms of a number of age-related degenerative diseases including sarcopenia. In previous studies from my laboratory, we have shown that mice lacking the antioxidant enzyme CuZnSOD (Sod1-/- mice) have high levels of oxidative stress and exhibit loss of muscle mass and function similar to the degenerative changes seen in old wild type mice. Thus, the Sod1-/- mice are a powerful model to gain unique insight into the mechanisms underlying sarcopenia. One of the most striking phenotypes in aging and in the Sod1-/- mice is the disruption and fragmentation of neuromuscular junctions (NMJs). The NMJ is the site of interaction between the motor neurons and skeletal muscle and is critical for muscle viability and performance. At present, the relative contributions of the motor neuron and muscle in deterioration of the NMJ and age-related muscle atrophy are poorly understood. This knowledge is essential to our ability to design effective interventions to delay or prevent sarcopenia. Therefore, to address this important question, we generated a conditional Sod1-/- mouse model in which we can delete Sod1 in muscle and nerve respectively to assess the relative contributions of the two tissues to NMJ deterioration and muscle atrophy. We demonstrated that deletion of Sod1 restricted to skeletal muscle tissue does not result in muscle atrophy or alterations in the NMJ, suggesting that muscle atrophy is initiated by changes in the motor neuron. In the current study, we will test the hypothesis that alterations in the neuromuscular junction play a critical role in sarcopenia through the initiation of downstream degenerative processes in skeletal muscle. To do this, we will negatively and positively modulate the NMJ through presynaptic and postsynaptic alterations and determine the effect on downstream pathways in muscle that contribute to atrophy and muscle weakness. First, we will determine if increased presynaptic oxidative stress generated by neuron specific deletion of Sod1 in mice (nSod1-/- mice) leads to NMJ degeneration and initiation of muscle atrophy pathways. We will measure NMJ morphology, composition and function and acetylcholine receptor (AchR) fragmentation in age-matched wild type, nSod1-/- mice and Sod1-/- mice and in old wild type mice. In addition, we will take an unbiased approach at changes in gene expression in response to NMJ disruption using microarrays and a more biased approach to measure changes in muscle degenerative pathways indicated to be altered in response to loss of innervation in our previous studies in Sod1-/- mice (calpain protease and proteasome activities, mitochondrial function and ROS generation and oxidative modification of muscle proteins). Conversely, we will determine if reversal of presynaptic oxidative stress through neuron specific expression of a human Sod1 transgene in Sod1-/- mice (nTgSod1-/- mice) rescues NMJ degeneration and prevents initiation of gene expression changes and muscle atrophy pathways. Together these studies will allow us to determine the effects of presynaptic changes on the NMJ and muscle atrophy pathways. Finally, we will measure the effect of postsynaptic NMJ disruption initiated by agrin deletion. Because agrin is essential for NMJ clustering and stability, loss of agrin leads to postsynaptic NMJ disruption and muscle atrophy. While the models used in Aims 1 and 2 target the role of the presynaptic neuron directly, this model allows us to target the NMJ at the postsynaptic side to determine if the gene expression changes and muscle degenerative pathways are the same or different in this postsynaptic model of NMJ disruption. Together these studies will define the role of the NMJ in muscle atrophy and point to common pathways affected by NMJ disruption that might be important targets for interventions.