Glutathione in mitochondrial dysfunction and disease progression in ALS-models

项目摘要 ALS，俗称Lou Gehrig病，是成人最常见的运动神经元病。这个 疾病的主要特征是运动通路中神经元的选择性功能障碍和死亡。 虽然大约10%的肌萎缩侧索硬化症病例是遗传的(家族性)，但大多数病例没有遗传 已确定的成分(零星)和暴露在尚未确定的环境毒物中可能是原因 在这些案件中。在家族性病例中，大约20%是由主要的遗传突变引起的 铜锌超氧化物歧化酶(SOD1)。过度表达人类突变SOD1的啮齿动物通常会患上 ALS样表型。几个假说，包括SOD1异常催化引起的氧化应激，以及 线粒体功能障碍被认为可以解释突变型SOD1的毒性作用。探索这一角色 在肌萎缩侧索硬化症的抗氧化防御中，我们使用了谷氨酸-半胱氨酸连接酶修饰亚单位的基因敲除小鼠 (GCLM-/-)，与野生型相比，它们的总谷胱甘肽(GSH)减少了70%-80%。 尽管GCLM-/-小鼠可以生育和存活，但GCLM(-/-)/hSOD1G93A小鼠的寿命缩短了50%-60% 与GCLM(+/+)/hSOD1G93A小鼠比较。寿命的缩短似乎与 在过度表达突变形式的转基因小鼠中常见的加重线粒体病理 保持超氧化物歧化酶活性的hSOD1。有趣的是，当GCLM-/-动物与一个 不同ALS动物模型过表达未检测到歧化酶活性的突变型hSOD1 (hSOD1H46R/H48Q)，对GCLM(-/-)/hSOD1H46R/H48Q小鼠的发病和存活无影响。此外, 在这些动物中很少或根本没有观察到线粒体病理。在上述情况下，具体的 该提案的目的是：1-特定的目标1.评估降低GSH水平对发病和 疾病在hSOD1G93A、hSOD1H46R/H48Q和hSOD1WT3小鼠中的进展。2.具体目标2.确定 HSOD1诱导的线粒体功能障碍在表达ALS连锁突变体的星形胶质细胞毒性中的作用 SOD1s向共培养运动神经元方向发展。3.目的3.评估线粒体过氧化物量增加的效果 解毒在hSOD1G93A和hSOD1H46R/H48Q小鼠疾病发生和发展中的作用。该提案是 旨在确定谷胱甘肽含量降低在线粒体病理和疾病进展中的作用 在肌萎缩侧索硬化症。因为特定的修饰物，如谷胱甘肽缺乏，可能只影响某些SOD1突变体，所以结果是 这些实验将有助于理解分子通路中的潜在差异 不同的SOD1突变体会产生疾病。因为周围的神经胶质细胞在疾病中也起着关键作用 进展，所获得的结果也应该确定GSH缺乏和hSOD1的细胞类型特异性影响 毒性将是理解ALS发病机制的关键。最后，对两种方法得到的结果进行了比较 旨在提高线粒体过氧化解毒能力的不同策略将有助于描绘 线粒体靶向抗氧化剂作为肌萎缩侧索硬化症可行临床治疗的价值。

Project Summary ALS, commonly known as Lou Gehrig's disease, is the most common adult motor neuron disease. The disease's primary hallmark is the selective dysfunction and death of the neurons in the motor pathways. Although approximately 10% of ALS cases are inherited (familial), the majority of cases have no genetic component identified (sporadic) and exposure to yet unidentified environmental toxicants might be responsible for these cases. Among the familial cases, approximately 20% are caused by dominantly inherited mutations in the Cu, Zn superoxide dismutase (SOD1). Rodents overexpressing human mutant SOD1 generally develop an ALS-like phenotype. Several hypotheses, including oxidative stress induced by SOD1 aberrant catalysis, and mitochondrial dysfunction have been proposed to explain the toxic effect of mutant SOD1. To explore the role of antioxidant defenses in ALS we used knockout mice for the glutamate-cysteine ligase modifier subunit (GCLM-/-), which have a 70-80% reduction of total glutathione (GSH) when compared to wild-type littermates. Although GCLM-/- mice are fertile and viable, the life span of GCLM(-/-)/hSOD1G93A mice decreased in 50-60% when compared to the GCLM(+/+)/hSOD1G93A mice. The decrease in life span seems to be associated with aggravated mitochondrial pathology commonly observed in transgenic mice overexpressing mutated forms of hSOD1 that retain superoxide dismutase activity. Interestingly, when the GCLM-/- animals were mated with a different ALS animal model which overexpress a mutant hSOD1 with undetectable dismutase activity (hSOD1H46R/H48Q), no effect was observed in onset or survival of GCLM(-/-)/hSOD1H46R/H48Q mice. In addition, little or no mitochondrial pathology was observed in these animals. On the aforementioned context, the specific aims of the proposal are: 1-Specific Aim 1. To evaluate the effect of reduced GSH levels on the onset and progression of the disease in hSOD1G93A, hSOD1H46R/H48Q and hSOD1WTmice. 2-Specific Aim 2. To determine the role of hSOD1-induced mitochondrial dysfunction in the toxicity of astrocytes expressing ALS-linked mutant SOD1s toward co-cultured motor neurons. 3-Aim 3. To evaluate the effect of increased mitochondrial peroxide detoxification in the onset and progression of disease in hSOD1G93A and hSOD1H46R/H48Q mice. The proposal is designed to determine the effect of reduced GSH content in mitochondrial pathology and disease progression in ALS. Because a specific modifier, such as GSH deficiency, may affect only certain SOD1 mutants, the result of these experiments will contribute to understand the potential difference in the molecular pathways by which different SOD1 mutants produce disease. Since surrounding glial cells also play a key role in the disease progression, the results obtained should also identify cell-type specific effects of GSH deficiency and hSOD1 toxicity that will be critical to the understanding of ALS pathogenesis. Finally, the results obtained from two different strategies designed to increase mitochondrial peroxide detoxification capacity will help delineate the value of mitochondrial-targeted antioxidants as a viable clinical therapy for ALS.