Glutathione in mitochondrial dysfunction and disease progression in ALS-models

描述(申请人提供)：ALS，俗称Lou Gehrig病，是最常见的成人运动神经元病。这种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ALS病例是遗传性的(家族性)，但大多数病例没有确定的遗传成分(零星)，暴露于尚未确定的环境毒物可能是这些病例的原因。在家族性病例中，大约20%是由铜锌超氧化物歧化酶(SOD1)的显性遗传突变引起的。过度表达人类突变型SOD1的啮齿动物通常会发展成类似ALS的表型。已经提出了几种假说，包括SOD1异常催化引起的氧化应激和线粒体功能障碍，以解释突变的SOD1的毒性作用。为了探索抗氧化防御在肌萎缩侧索硬化症中的作用，使用谷氨酸-半胱氨酸连接酶修饰亚单位(GCLM-/-)的基因敲除小鼠，与野生型小鼠相比，总谷胱甘肽(GSH)减少了70%-80%。尽管GCLM-/-小鼠可以生育和存活，但与GCLM(+/+)/hSOD1G93A小鼠相比，GCLM(-/-)/hSOD1G93A小鼠的寿命缩短了50%-60%。寿命的缩短似乎与线粒体病理的加重有关，在过度表达保持超氧化物歧化酶活性的突变形式的hSOD1的转基因小鼠中常见。有趣的是，当GCLM-/-动物与另一种ALS动物模型(hSOD1H46R/H48Q)交配时，GCLM(-/-)/hSOD1H46R/H48Q小鼠的发病或存活没有任何影响。此外，在这些动物中很少或根本没有观察到线粒体病理。在上述背景下，该建议的具体目的是：1：评估GSH水平降低对hSOD1G93A、hSOD1H46R/H48Q和hSOD1WT鼠疾病发生和发展的影响；2：确定hSOD1诱导的线粒体功能障碍在表达ALS连锁突变SOD1的星形胶质细胞对共培养运动神经元毒性中的作用；以及3.评估线粒体过氧化氢解毒增加在hSOD1G93A和hSOD1H46R/H48Q小鼠疾病发生和发展中的作用。该提案旨在确定谷胱甘肽含量降低对ALS线粒体病理和疾病进展的影响。由于特定的修饰物，如GSH缺乏，可能只影响某些SOD1突变体，这些实验的结果将有助于了解不同SOD1突变体致病的分子途径的潜在差异。由于周围的神经胶质细胞在疾病进展中也起着关键作用，所获得的结果还应该确定GSH缺乏和hSOD1毒性的细胞类型特异性影响，这将对理解ALS的发病机制至关重要。最后，从两种不同的提高线粒体过氧化解毒能力的策略获得的结果将有助于描述线粒体靶向抗氧化剂作为ALS可行的临床治疗方法的价值。 公共卫生相关性：肌萎缩侧索硬化症(ALS)或Lou Gehrig病的特征是控制肌肉运动的神经细胞的特定死亡，导致瘫痪和最终死亡。线粒体是细胞的动力源，它的故障可能是观察到的神经变性的原因。这项提案将调查谷胱甘肽作为线粒体功能的主要保护因素的作用，谷胱甘肽是细胞中发现的一种天然抗氧化剂。其中一人还将研究恢复正常线粒体功能的可能方法，作为ALS的治疗方法。

DESCRIPTION (provided by applicant): 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, knockout mice were employed using 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 overexpresses a mutant hSOD1 with undetectable dismutase activity (hSOD1H46R/H48Q), no effect was observed in the 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: To evaluate the effect of reduced GSH levels on the onset and progression of the disease in hSOD1G93A, hSOD1H46R/H48Q and hSOD1WTmice; 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; and 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. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease is characterized by the specific death of the nerve cells that control muscle movement, leading to paralysis and eventual death. Malfunction of the mitochondria, the powerhouse of the cell, may be responsible for the observed neurodegeneration. This proposal will investigate the role of glutathione, a natural antioxidant found in the cells, as a major protective factor for mitochondrial function. One will also investigate possible ways to restore normal mitochondrial function as a therapy for ALS.