Defining the relative roles of pre- and post-synaptic events in the initiation and progression of sarcopenia

 描述(申请人提供)：布鲁克斯博士和范雷曼博士在氧化应激在石棺减少症中的作用的研究中有很强的合作记录。我们先前的工作表明，缺乏超氧阴离子清除剂CuZnSOD的年轻成年小鼠(Sod1KO小鼠)表现出与老年野生型(WT)小鼠非常相似的增龄性肌肉萎缩/虚弱，包括神经肌肉接头(NMJ)退化、运动神经元收缩、肌肉线粒体活性氧簇(MtRO)生成增加以及钙稳态改变。在Sod1KO小鼠的神经元中特异性地替换CuZnSOD可以逆转肌肉萎缩和虚弱、NMJ中断和肌肉氧化应激，暗示运动神经元缺陷是Sod1KO小鼠骨骼肌减少的起始事件。然而，在小鼠中，神经元特异性SOD1基因敲除(NSod1KO)不会导致腓肠肌萎缩，尽管肌肉特异性SOD1基因敲除小鼠表现出收缩能力的丧失，但它们没有表现出肌肉萎缩。因此，SOD1的缺失和单独在神经元或肌肉中诱导氧化应激并不能复制Sod1KO小鼠的石棺减少表型，这表明石棺减少是需要两种组织的交互作用的结果。这项研究的目标是界定这种相互作用。我们 假设突触前氧化应激和损伤引起NMJ结构和功能的改变，从而触发突触后mtROS生成的增加，钙调节失调，以及肌肉中的氧化应激/损伤，从而进一步扰乱神经元和NMJ的功能，产生肌纤维减少的表型。我们将在三个具体目标中阐述这一假设。首先，我们将检查SOD1缺陷对神经元和肌肉纤维的影响，期望这些双基因敲除小鼠将重现Sod1KO小鼠的表型。我们还将通过在成年期有条件地删除SOD1来确定肌肉或神经元特异性SOD1缺失的影响，以确定其对骨质疏松症的影响，而不考虑可能的发育代偿变化。接下来，使用其他条件基因敲除模型，我们将测试没有突触前氧化应激的肌肉mtROS升高是否足以导致骨骼减少，相反，我们将确定清除Sod1KO中突触后mtROS是否会延缓和/或减少肌肉萎缩和功能衰退，尽管Sod1KO小鼠存在神经元变化。在每个模型中，我们将测量坐骨神经、脊髓和肌肉的氧化损伤和氧化还原状态，NMJ的形态和功能，运动单位属性，以及骨骼肌结构，线粒体功能，钙处理和收缩能力。这些研究将明确表明，神经启动的NMJ破坏是否足以导致肌肉萎缩/虚弱，或者是否需要肌肉氧化应激的额外改变来诱导表型。确定神经元和肌肉在骨质疏松症的发生和发展中的协调作用将为骨质疏松症的发生和传播提供新的见解。

 DESCRIPTION (provided by applicant): Drs. Brooks and Van Remmen have a strong record of collaboration in studies on the role of oxidative stress in sarcopenia. Our previous work demonstrated that young adult mice lacking the superoxide anion scavenger CuZnSOD (Sod1KO mice) exhibit age-related muscle atrophy/weakness that closely mimics the sarcopenia phenotype of old wild type (WT) mice, including degeneration of neuromuscular junctions (NMJ), retraction of motor neurons, elevated generation of muscle mitochondrial reactive oxygen species (mtROS), and altered calcium homeostasis. Replacing CuZnSOD specifically in neurons of Sod1KO mice reverses muscle atrophy and weakness, NMJ disruption and muscle oxidative stress, implicating motor neuron deficits as the initiating event in sarcopenia in Sod1KO mice. However, neuronal specific Sod1 knockout (nSod1KO) in mice does not result in atrophy of the gastrocnemius muscle, and although muscle specific Sod1 knockout mice show a loss in contractile force, they show no muscle atrophy. Thus, deletion of Sod1 and induction of oxidative stress in either neurons or muscle alone does not replicate the sarcopenia phenotype of the Sod1KO mice, suggesting that sarcopenia results from an interactive effect requiring both tissues. The goal of this study is to define this interaction. We hypothesize that pre-synaptic oxidative stress and damage initiates alterations in NMJ structure and function that trigger postsynaptic increases in mtROS generation, calcium dysregulation, and oxidative stress/damage in the muscle that further disrupt neuronal and NMJ function to generate the sarcopenia phenotype. We will address this hypothesis in three Specific Aims. First, we will examine the effect of Sod1 deficiencies in both neurons and muscle fibers with the expectation that these double knockout mice will recapitulate the phenotype of Sod1KO mice. We will also determine the effect of muscle or neuronal specific deficiency of Sod1 using conditional deletion of Sod1 during adulthood to determine the impact on sarcopenia, independent of possible developmental compensatory changes. Next, using additional conditional knockout models, we will test whether elevated muscle mtROS without presynaptic oxidative stress is sufficient to induce sarcopenia, and conversely, we will determine whether scavenging post- synaptic mtROS in Sod1KO will delay and/or reduce muscle atrophy and functional declines, despite neuronal changes present in the Sod1KO mice. In each model, we will measure oxidative damage and redox status in sciatic nerve, spinal cord, and muscle, NMJ morphology and function, motor unit properties, and skeletal muscle structure, mitochondrial function, calcium handling, and contractility. These studies will definitively show whether neuronal initiation of NMJ disruption is sufficient for muscle atrophy/weakness or if additional alterations in muscle oxidative stress are required to induce the phenotype. Identifying coordinated roles of neurons and muscle in the initiation and progression of sarcopenia will provide new insights into the pathways that are involved in the onset and propagation of sarcopenia.