Novel mechanism of neuroprotection against neurotoxins

描述(由申请人提供)： α-突触核蛋白(1-syn)积聚是一组神经退行性疾病的共同病理特征，统称为突触核病，其中最常见的是帕金森病(PD)。路易小体(L B)是帕金森病的组织病理学特征，由以1-syn为主要成分的蛋白质集合体组成。家族性帕金森病的一小部分与A-syn基因扩增有关。在多种动物模型中，A-SYN过度表达导致神经退行性变。然而，在大多数散发性帕金森病中，a-syn的积聚被怀疑是蛋白质降解不足的结果。针对帕金森病的发病机制，1-甲基-4-苯基-1，2，3，6-四氢吡啶(MPTP)被发现可引起人类帕金森病，并被广泛用作动物模型中多巴胺(DA)能神经元变性的模型。MPTP抑制线粒体复合体I活性，诱导α-syn积聚。A-SYN基因敲除小鼠对MPTP对DA神经元的毒性具有抵抗力。转基因过表达a-syn加剧了MPTP的毒性。如何在年龄依赖性和/或神经毒素加重的帕金森病中调节a-syn水平和毒性是一个关键问题。高容量的蛋白质降解途径包括巨型自噬和伴侣介导的自噬，这些自噬将蛋白质运输到溶酶体。组织蛋白酶D(CD)是完成自噬所需的一种溶酶体天冬氨酸蛋白酶(21)。我们已经证明，Cd基因敲除导致小鼠大脑中广泛的1-syn积累，表明Cd在介导a-syn代谢中起作用。体外实验表明，Cd可减少α-syn的聚集，并对1-syn介导的毒性具有保护作用。为了验证Cd对α-syn蓄积和毒性具有神经保护作用的假说，并明确Cd对a-syn蓄积和毒性的神经保护机制，我们将进行以下实验：1.在体内验证Cd单倍体缺乏导致对MPTP毒性敏感性增加的假说。2.在体内验证立体定向注射AAV-CD可减轻MPTP毒性的假说。这些研究的完成将确定Cd的完全和部分丧失在体内神经毒素诱导的DA神经元死亡和a-syn代谢中的作用，以及Cd在体内的神经保护作用中的作用。未来的机制研究将确定CD的上调或下调如何影响a-syn的代谢、聚集和毒性，以及a-syn的毒性是否受线粒体功能障碍的调节。这些研究将有助于建立CD作为帕金森病治疗的潜在靶点。 公共卫生相关性： 包括帕金森氏症在内的神经退行性疾病在美国影响着400多万人，其中包括超过4万名退伍军人。我们将研究溶酶体酶在帕金森氏病模型中的部分功能丧失和功能获得的影响。这项研究将有助于建立以溶酶体为基础的帕金森病治疗方法。

DESCRIPTION (provided by applicant): Accumulation of alpha-synuclein (1-syn) is a shared pathological feature among a group of neurodegenerative diseases collectively referred to as 'synucleinopathies', the most common of which is Parkinson's disease (PD). Lewy bodies (LB) are the histopathological feature of PD and they consist of proteinaceous aggregates with 1-syn as the most prominent component. a-syn gene amplification is responsible for a small subset of familial PD. a-syn overexperssion led to neurodegeneration in a variety of animal models. However, in most sporadic PD, a-syn accumulation is suspected to be a result from insufficient protein degradation. Specific to PD pathogenesis, 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) has been found to cause Parkinsonism in humans and thereafter widely used as a model for dopaminergic (DA) neuron degeneration in animal models. MPTP inhibits mitochondrial complex I activities and induces a-syn accumulation. a-syn knockout mice are resistant to MPTP toxicity to DA neurons. Transgenic overexpression of a-syn exacerbates MPTP toxicity. How a-syn levels and toxicity are regulated in age-dependent and/or neurotoxin exacerbated PD is a critical issue. High capacity protein degradation pathways include macroautophagy and chaperone-mediated autophagy that transport proteins to the lysosomes. Cathepsin D (CD) is a lysosomal aspartic protease required for completion of autophagy(21). We have shown that CD knockout led to extensive 1-syn accumulation in mouse brains, indicating a role for CD in mediating a-syn metabolism. In vitro we have shown that CD reduces a-syn aggregation and protects against 1-syn-mediated toxicity. To test the hypothesis that CD is neuroprotective against a-syn accumulation and toxicity and to define the mechanism of CD neuroprotection against a-syn accumulation and toxicity, we will perform experiments with the following aims: 1. Test the hypothesis that CD haploinsufficiency leads to an increase of sensitivity to MPTP toxicity in vivo. 2. Test the hypothesis that stereotaxic injection of AAV-CD to the SN attenuates MPTP toxicity in vivo. Completion of these studies will determine the effect of complete and partial loss-of-CD in neurotoxin-induced DA neuron death and a-syn metabolism in vivo, and the effect of gain-of-function of CD in neuroprotection in vivo. Future mechanistic studies will determine how up- or down-regulation of CD affects a-syn metabolism, aggregation and toxicity and whether a-syn toxicity is modulated by mitochondrial dysfunction. These studies will help establish CD as a potential target in PD therapy. PUBLIC HEALTH RELEVANCE: Neurodegenerative diseases, including Parkinson's disease, affect more than 4 million people in the US, including more than 40,000 VA veterans. We will study the effect of partial loss-of-function and gain-of- function of a lysosomal enzyme in Parkinson's disease models. This study will help establish lysosomal based therapy for Parkinson's disease.