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确定和验证治疗帕金森病（PD）的新靶点。帕金森病的特点是多巴胺能神经元的丧失，这些神经元特别容易受到氧化应激的影响。Glutaredoxin （Grx）是一种抗氧化防御酶，通过催化半胱氨酸残基（protein-SSG）上蛋白-谷胱甘肽混合二硫化物的可逆形成来抵消氧化应激并维持硫醇稳态。这种氧化修饰与许多与细胞存活有关的蛋白质的功能改变有关，包括先前已确定与家族性帕金森病有关的蛋白质。我们将秀丽隐杆线虫作为模式生物，发现Grx1（哺乳动物中Grx的主要亚型）的线虫同源物缺乏，会导致突变体LRRK2 （G2019S或R1441C）、¿-synuclein或酪氨酸羟化酶的过度表达引发的多巴胺能变性加剧。这些发现表明，在动物模型中，Grx1缺乏可诱发PD相关表型，表明Grx1缺乏有助于PD的发病。为了支持这些遗传模型的相关性，我们已经获得了PD患者死后大脑中Grx1含量降低的初步证据。因此，我们拟对Grx1在PD哺乳动物模型中的神经保护作用进行表征和验证，并探讨家族性PD相关关键蛋白谷胱甘肽化状态的改变是否也在疾病发病机制中起重要作用。为了研究Grx1缺乏是否会导致哺乳动物对多巴胺能变性的易感性，我们在Aim 1中提出了一种新的小鼠模型，该模型是由现有的转基因人类LRRK2突变动物与Grx1敲除动物杂交产生的。grx1基因敲除的转基因LRRK2突变小鼠代表了氧化应激升高和硫醇稳态特异性缺乏的基因工程哺乳动物模型。我们将确定这种新型小鼠模型是否表现出PD特征的多巴胺能神经元的丧失。本研究的结果将验证Grx1是否在PD发病机制中起关键调节作用。在Aim 2中，我们将鉴定与PD发病机制相关的蛋白质的氧化半胱氨酸修饰和功能变化，包括¿-synuclein， parkin, UCH-L1, DJ-1， PINK1和LRRK2。半胱氨酸修饰对PD样表型的贡献将通过在哺乳动物细胞和秀丽隐杆线虫PD模型中使用非氧化突变形式的蛋白质来检查。神经变性易感性与家族性帕金森病相关蛋白的氧化修饰之间的对应关系将为理解散发性帕金森病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.