Mechanisms of age-dependent nigral neuron loss in PINK1 knockout rats

描述（由申请人提供）：帕金森病（PD）是老年人神经变性、残疾和过早死亡的常见原因。黑质多巴胺能神经元的缺失是PD的主要神经病理学标志。目前还没有治疗方法证明可以减缓PD中进行性黑质细胞丢失，这会导致临床症状的严重程度增加。最近人类基因突变如Parkin、DJ-1和PINK 1与复发性遗传形式的PD之间的联系为发现致病机制以及基于与人类帕金森症生理学相关的机制在黑质细胞丧失的动物模型中开发和测试神经保护疗法提供了新的机会。我们已经使用基因敲除（KO）小鼠追求这一策略十多年了。由于未知的原因，Parkin KO、DJ-1 KO和PINK 1 KO小鼠不会重现在这些基因中携带功能丧失突变的人类中发生的黑质细胞损失。我们和其他人在KO小鼠、果蝇和培养细胞中发现了线粒体功能障碍（呼吸链缺陷），这表明这是这些基因中功能丧失突变导致黑质细胞丧失的机制中的常见且可能是早期事件。然而，黑质细胞损失的缺乏排除了使用黑质细胞损失作为结果测量来直接测试Parkin KO、DJ-1 KO或PINK 1 KO小鼠中的这种和其他潜在的关键致病机制。最近开发的PINK 1 KO大鼠显示年龄依赖性黑质细胞损失从6个月大开始，到7个月大达到50%。PINK 1 KO大鼠中的年龄依赖性黑质神经元损失使得首次有可能在再现PD的这种中枢神经病理学特征的哺乳动物脑中直接测试主要候选致病机制，例如线粒体损伤和减少的帕金森介导的线粒体自噬。我们建议使用PINK 1 KO大鼠来实现以下相互关联的特定目标，这些目标是因为它们对治疗开发的重要性而被选择的：1）鉴定PINK 1缺陷导致大鼠黑质细胞损失的机制，2）表征Parkin作为PINK 1抑制剂的特性，3）确定PINK 1抑制剂的作用机制。 PINK 1 KO大鼠中的年龄依赖性黑质细胞损失和3）刺激线粒体呼吸链作为PD的潜在疗法。我们希望通过测试PINK 1缺陷减少帕金森介导的受损线粒体降解的主要假设和通过饮食摄入亚甲蓝的呼吸链刺激在PINK 1 KO大鼠中具有神经保护作用的第二个主要假设，对PINK 1 KO大鼠进行系统表征，作为治疗开发的适当测试床。我们以前已经表明，亚甲蓝防止鱼藤酮大鼠PD模型中的神经变性。使用这种新的黑质细胞丢失大鼠模型是创新的，该提案将通过将重点转移到PD脑组织中存在的疾病机制来显著影响对PD的理解，这些疾病机制因其强大的治疗潜力而被选择。从我们的研究中获得的知识将广泛适用于与线粒体功能障碍和神经变性相关的疾病。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a frequent cause of neurodegeneration, disability and premature mortality in older adults. Loss of dopaminergic neurons in the substantia nigra is the primary neuropathological hallmark of PD. There are currently no treatments proven to slow down the progressive nigral cell loss in PD, which causes increasing severity of the clinical symptoms. The recent linking of human mutations in genes such as Parkin, DJ-1, and PINK1 to recessively inherited forms of PD provides new opportunities to discover pathogenic mechanisms and to develop and test neuroprotective therapies in animal models with nigral cell loss based on mechanisms physiologically relevant to human parkinsonism. We have pursued this strategy for over a decade using knockout (KO) mice. For unknown reasons, Parkin KO, DJ-1 KO and PINK1 KO mice do not reproduce the nigral cell loss that occurs in humans bearing loss-of-function mutations in these genes. We and others identified mitochondrial dysfunction (respiratory chain defects) in KO mice, Drosophila and cultured cells, suggesting that this is a common and perhaps early event in the mechanism by which loss-of-function mutations in these genes cause nigral cell loss. However, the lack of nigral cell loss precludes using nigral cell loss as an outcome measure to directly test this and other potential key pathogenic mechanisms in Parkin KO, DJ-1 KO or PINK1 KO mice. Recently developed PINK1 KO rats show age-dependent nigral cell loss beginning at age 6 months and reaching 50% by age 7 months. The age-dependent nigral neuron loss in PINK1 KO rats makes it possible for the first time to test the leading candidate pathogenic mechanisms such as mitochondrial impairment and diminished Parkin-mediated mitochondrial autophagy directly in a mammalian brain that reproduces this central neuropathological feature of PD. We propose to use PINK1 KO rats to achieve the following interrelated specific aims which have been chosen because of their significance for therapeutic development: 1) To identify mechanisms by which PINK1 deficiency leads to nigral cell loss in the rat, 2) To characterize Parkin as an inhibitor of age-dependent nigral cell loss in PINK1 KO rats and 3) To stimulate the mitochondrial respiratory chain as a potential therapy for PD. We expect to contribute a systematic characterization of PINK1 KO rats as an apt test bed for therapeutic development by testing the principal hypothesis that PINK1 deficiency diminishes Parkin-mediated degradation of impaired mitochondria and the second main hypothesis that respiratory chain stimulation via dietary intake of methylene blue is neuroprotective in PINK1 KO rats. We have previously shown that methylene blue prevents neurodegeneration in the rotenone rat PD model. The use of this novel rat model of nigral cell loss is innovative and the proposal will significantly impact the understanding of PD by shifting emphasis to disease mechanisms present in PD brain tissue selected for their strong therapeutic potential. The knowledge gained from our study will be broadly applicable to diseases associated with mitochondrial dysfunction and neurodegeneration.