Glutathione in mitochondrial dysfunction and disease progression in ALS-models

项目摘要 ALS，俗称卢伽雷氏病，是最常见的成人运动神经元疾病。的 疾病的主要标志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ALS病例是遗传的（家族性的），但大多数病例没有遗传性。 成分确定（零星）和暴露于尚未确定的环境毒物可能负责 对于这些案件。在家族性病例中，大约20%是由显性遗传突变引起的， Cu，Zn超氧化物歧化酶（SOD 1）。过表达人类突变体SOD 1的啮齿动物通常会产生一种 ALS样表型。几种假说，包括SOD 1异常催化诱导的氧化应激， 线粒体功能障碍已被提出来解释突变SOD 1的毒性作用。为了探索 在ALS抗氧化防御中，我们使用谷氨酸-半胱氨酸连接酶修饰亚基敲除小鼠， GCLM-/-），当与野生型同窝仔相比时，其具有70-80%的总谷胱甘肽（GSH）减少。 尽管GCLM-/-小鼠是可生育的且存活的，但是GCLM（-/-）/hSOD 1G 93 A小鼠的寿命减少了50-60%。 与GCLM（+/+）/hSOD 1G 93 A小鼠相比，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线粒体病理学恶化， hSOD 1保留超氧化物歧化酶活性。有趣的是，当GCLM-/-动物与 过表达具有不可检测的歧化酶活性的突变hSOD 1的不同ALS动物模型 在GCLM（-/-）/hSOD 1H 46 R/H48 Q小鼠的发病或存活中未观察到影响。此外，本发明还提供了一种方法， 在这些动物中观察到很少或没有线粒体病理。在上述背景下，具体 该提案的目标是：1-具体目标1。评估GSH水平降低对发病的影响， hSOD 1G 93 A、hSOD 1H 46 R/H48 Q和hSOD 1 WT小鼠中疾病的进展。2-具体目标2。以确定 hSOD 1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 SOD 1对共培养的运动神经元的影响。3-目标3.为了评估线粒体过氧化物增加的影响， 在hSOD 1G 93 A和hSOD 1H 46 R/H48 Q小鼠中的疾病的发作和进展中的解毒。该提案 旨在确定GSH含量降低对线粒体病理学和疾病进展的影响 在ALS中。因为一个特定的修饰物，如GSH缺乏，可能只影响某些SOD 1突变体， 这些实验将有助于了解分子途径中的电势差， 不同的SOD 1突变体产生疾病。由于周围的神经胶质细胞在疾病中也起着关键作用 进展，所获得的结果还应确定GSH缺乏和hSOD 1的细胞类型特异性效应 这对理解ALS发病机制至关重要。最后，从两个方面得到的结果 旨在提高线粒体过氧化物解毒能力的不同策略将有助于描述 作为ALS可行的临床治疗的神经靶向抗氧化剂的价值。

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.