Novel mechanism of neuroprotection against neurotoxins

描述（由申请人提供）： α-突触核蛋白（1-syn）的积累是一组神经退行性疾病（统称为“突触核蛋白病”）的共同病理特征，其中最常见的是帕金森病（PD）。路易体（LB）是PD的组织病理学特征，它们由蛋白质聚集体组成，其中1-syn是最主要的成分。α-syn基因扩增是家族性PD的一小部分的原因。α-syn过表达在多种动物模型中导致神经变性。然而，在大多数散发性PD中，怀疑α-syn积累是蛋白质降解不足的结果。具体到PD发病机制，已发现1-甲基4-苯基1，2，3，6-四氢吡啶（MPTP）在人类中引起帕金森综合征，并且此后广泛用作动物模型中多巴胺能（DA）神经元变性的模型。MPTP抑制线粒体复合物I活性并诱导a-syn积累。α-syn敲除小鼠对MPTP对DA神经元的毒性具有抗性。α-syn的转基因过表达加剧MPTP毒性。在年龄依赖性和/或神经毒素加重的PD中如何调节a-syn水平和毒性是一个关键问题。高容量蛋白质降解途径包括将蛋白质转运至溶酶体的大自噬和分子伴侣介导的自噬。组织蛋白酶D（CD）是完成自噬所需的溶酶体天冬氨酸蛋白酶（21）。我们已经表明，CD敲除导致小鼠脑中广泛的1-syn积累，表明CD在介导α-syn代谢中的作用。在体外，我们已经表明CD减少α-syn聚集并保护免受1-syn介导的毒性。为了检验CD对a-syn积累和毒性具有神经保护作用的假设，并且为了定义CD对a-syn积累和毒性的神经保护作用的机制，我们将进行具有以下目的的实验：检验CD单倍不足导致体内MPTP毒性敏感性增加的假设。2.测试将AAV-CD立体定位注射至SN在体内减弱MPTP毒性的假设。这些研究的完成将确定CD的完全和部分损失在体内神经毒素诱导的DA神经元死亡和α-syn代谢中的作用，以及CD的功能获得在体内神经保护中的作用。未来的机制研究将确定CD的上调或下调如何影响a-syn代谢、聚集和毒性，以及a-syn毒性是否受线粒体功能障碍调节。这些研究将有助于确立CD作为PD治疗的潜在靶点。 公共卫生相关性： 包括帕金森病在内的神经退行性疾病影响着美国400多万人，其中包括4万多名退伍军人。我们将研究帕金森病模型中溶酶体酶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基于溶酶体的治疗帕金森病。

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.