Glutaredoxin, a Critical Regulator of Parkinson Disease Pathogenesis

描述（由申请人提供）：我们研究的总体目标是识别和验证治疗帕金森病（PD）的新靶点。PD的特征在于多巴胺能神经元的损失，多巴胺能神经元特别容易受到氧化应激的影响。谷氧还蛋白（Glutaredoxin，Grx）是一种抗氧化防御酶，通过催化半胱氨酸残基上的蛋白-谷胱甘肽混合二硫化物（蛋白-SSG）的可逆形成来对抗氧化应激并维持巯基稳态。这种氧化修饰与许多参与细胞存活的蛋白质的功能改变有关，包括先前已被鉴定为家族性PD的蛋白质。利用C.作为模式生物，我们已经发现Grx 1的线虫同源物（哺乳动物中Grx的主要同种型）的缺陷导致由突变体LRRK 2（G2019 S或R1441 C）、γ-突触核蛋白或酪氨酸羟化酶的过表达引起的多巴胺能变性的加剧。这些发现表明，Grx 1缺陷可以在动物模型中易患PD相关表型，表明Grx 1缺陷有助于PD发病机制。支持这些遗传模型的相关性，我们已经获得了初步证据，Grx 1含量在PD患者死后的大脑中减少。因此，我们建议表征和验证Grx 1在哺乳动物PD模型中的神经保护作用，并研究与家族性PD有关的关键蛋白的谷胱甘肽化状态的变化是否也对疾病发病机制有重要贡献。为了研究Grx 1缺陷是否会导致哺乳动物多巴胺能变性，我们在目标1中提出了一种新的小鼠模型，该模型是通过将现有的转基因人LRRK 2突变动物与Grx 1敲除动物杂交而产生的。Grx 1基因敲除的转基因LRRK 2突变小鼠代表了一种氧化应激升高和巯基稳态特异性缺陷的遗传工程哺乳动物模型。我们将确定这种新的小鼠模型是否表现出PD特征性多巴胺能神经元的丧失。这项研究的结果将验证Grx 1是否作为PD发病机制的关键调节因子。在目标2中，我们将鉴定与PD发病机制有关的蛋白质的氧化半胱氨酸修饰和功能变化，包括<$-突触核蛋白、parkin、UCH-L1、DJ-1、PINK 1和LRRK 2。半胱氨酸修饰对PD样表型的贡献将通过使用哺乳动物细胞和C. elegans模型的PD。家族性PD中神经变性易感性和蛋白质氧化修饰之间的对应关系将为了解散发性PD的致病机制提供重要的进展。

DESCRIPTION (provided by applicant): The overall objective of our research is to identify and validate novel targets for the treatment of Parkinson's disease (PD). PD is characterized by loss of dopaminergic neurons, which are especially vulnerable to oxidative stress. Glutaredoxin (Grx) is an antioxidant defense enzyme that counteracts oxidative stress and maintains thiol homeostasis by catalyzing the reversible formation of protein-glutathione mixed disulfides on cysteine residues (protein-SSG). This oxidative modification is implicated in change of function for many proteins involved in cell survival, including proteins that have been previously identifie with familial forms of PD. Using C. elegans as a model organism, we have found that deficiency in the nematode homolog of Grx1, the major isoform of Grx in mammals, leads to exacerbation of dopaminergic degeneration elicited by overexpression of mutant LRRK2 (G2019S or R1441C), ¿-synuclein, or tyrosine hydroxylase. These findings indicate that Grx1 deficiency can predispose to PD-relevant phenotype in an animal model, suggesting that Grx1 deficiency contributes to PD pathogenesis. Supporting the relevance of these genetic models, we have obtained preliminary evidence that Grx1 content is decreased in postmortem brains of PD patients. Accordingly, we propose to characterize and validate the neuroprotective role of Grx1 in mammalian models of PD, and to investigate whether changes in glutathionylation status of key proteins implicated in familial PD also contribute importantly to the disease pathogenesis. To examine if Grx1 deficiency confers vulnerability to dopaminergic degeneration in mammals, we propose in Aim 1 to characterize a novel mouse model generated from crossing existing transgenic human LRRK2 mutant animals with Grx1-knockout animals. The transgenic LRRK2 mutant mice with Grx1-knockout represent a genetically engineered mammalian animal model of elevated oxidative stress and specific deficiency in thiol homeostasis. We will determine whether this novel mouse model manifests loss of dopaminergic neurons characteristic of PD. The outcome of this study would validate if Grx1 serves as a critical regulator of PD pathogenesis. In Aim 2, we will identify oxidative cysteine modifications and changes in function of the proteins implicated in PD pathogenesis including ¿-synuclein, parkin, UCH-L1, DJ-1, PINK1, and LRRK2. The contribution of cysteine modifications to PD-like phenotype will be examined by the use of non-oxidizable mutant forms of the proteins in mammalian cell and C. elegans models of PD. Correspondence between susceptibility to neurodegeneration and oxidative modification of proteins implicated in familial PD would provide an important advance in understanding pathogenic mechanisms underlying sporadic PD.