Cellular Mechanisms of Neuropathology in Parkinson's Disease

描述(由申请人提供)： 含α-突触核蛋白(ASYN)的路易体积聚和伴随的多巴胺(DA)神经元变性是帕金森病(PD)的主要病理特征。自噬-溶酶体途径(ALP)是一条重要的细胞内降解和循环途径，ALP功能的折衷促进了有毒ASYN物种的积累。随着碱性磷酸酶在帕金森病中的改变，识别和验证促进ASYN清除的新的碱性磷酸酶靶点是开发新的帕金森病治疗药物的及时途径。ATP6V0C形成V-ATPase的质子转运通道，V-ATPase是一种调节溶酶体酸化和ALP底物清除的酶复合体。巴菲霉素是一种大环内酯类抗生素，能与ATP6V0C高亲和力结合，高浓度的巴菲霉素可抑制V-ATPase，并产生与其抑制溶酶体功能和ASYN清除有关的神经毒性。然而，巴非霉素浓度过低，不能抑制V-ATPase，则可减轻溶酶体功能障碍引起的细胞死亡和内源性ASYN积聚，并抑制体内ASYN过度表达导致的DA神经元死亡。虽然巴非霉素的治疗指数很窄，限制了它在人类PD患者中的长期使用，但我们的数据表明，ATP6V0C是促进ASYN清除和细胞存活的新靶点。然而，ATP6V0C是否介导了巴非霉素的这些保护作用，以及ATP6V0C的过度表达本身是否可以在没有巴菲霉素的情况下提供神经保护作用，还没有得到测试。我们假设ATP6V0C介导了巴非霉素依赖的有毒ASYN物种的清除，并减轻了ASYN相关的神经毒性。在目标1中，我们将在培养的哺乳动物细胞中过表达ATP6V0C，以确定其在调节内源性ASYN清除、ALP功能和细胞死亡中的相对重要性。我们还将确定在小鼠黑质(SN)中过表达人ATP6V0C是否可以减少由野生型人ASYN过度表达引起的ASYN积累和相关的神经退行性变。在目标2中，我们将从基因上敲除ATP6V0C，或在ATP6V0C中引入点突变，使其对巴菲霉素介导的V-ATPase抑制产生抵抗，以确定ATP6V0C在调节巴菲霉素对培养的哺乳动物细胞的这种神经保护功能中的需求。我们还将开发V-ATPase抑制活性降低的巴非霉素类似物，以确定是否随着V-ATPase抑制活性的降低，巴非霉素介导的神经保护得到加强。这些研究将验证ATP6V0C作为一种新的ALP依赖靶点的实用性，用于下一代促进ASYN清除和DA神经元存活的PD治疗。 公共卫生相关性： 帕金森病(PD)是一种主要的与年龄相关的神经退行性疾病，我们的实验室研究了有毒的α-突触核蛋白在帕金森病中蓄积并导致帕金森病神经病理和神经退行性变的机制。具体地说，我们的实验室专注于调节蛋白质降解的细胞内途径如何对帕金森病患者的DA神经元存活产生负面影响。对于这一应用，我们假设蛋白质ATP6V0C是溶酶体和其他酸性细胞器的空泡ATPase(V-ATPase)复合体的一个亚单位，是PD治疗的有效靶点，其过度表达或药物调节可能通过增加有毒α突触核蛋白从细胞中的清除来促进DA神经元的存活。总之，我们的研究将验证ATP6V0C的过度表达是否是促进α突触核蛋白清除的有效策略，以及对该靶点的药理学调节是否也提供类似的神经保护。

DESCRIPTION (provided by applicant): The accumulation of alpha-synuclein (ASYN)-containing Lewy bodies and associated degeneration of dopamine (DA) neurons are major pathological hallmarks of Parkinson's disease (PD). The autophagy- lysosome pathway (ALP) is an important intracellular degradation and recycling pathway and a compromise in ALP function promotes the accumulation of toxic ASYN species. As the ALP is altered in PD, identification and validation of novel ALP targets which promote ASYN clearance is a timely approach in the development of novel PD therapeutics. ATP6V0C forms the proton translocating channel of V-ATPase, an enzyme complex that regulates lysosomal acidification and clearance of ALP substrates. High concentrations of bafilomycin, a macrolide antibiotic that binds ATP6V0C with high affinity, inhibits V-ATPase and produces neurotoxicity related to its inhibition of lysosome function and ASYN clearance. However, concentrations of bafilomycin too low to inhibit V-ATPase attenuate cell death and endogenous ASYN accumulation resulting from lysosome dysfunction, and inhibit DA neuron death resulting from ASYN over-expression in vivo. While bafilomycin has a narrow therapeutic index which limits its long-term use in human PD patients, our data point to ATP6V0C as a novel target for promoting ASYN clearance and cell survival. However, whether ATP6V0C mediates these protective effects of bafilomycin and if over-expression of ATP6V0C itself can provide neuroprotective benefit in the absence of bafilomycin has not been tested. We hypothesize that ATP6V0C mediates the bafilomycin- dependent clearance of toxic ASYN species and attenuates ASYN-associated neurotoxicity. In Aim 1, we will genetically over-express ATP6V0C in cultured mammalian cells to determine its relative importance in regulating the clearance of endogenous ASYN, ALP function and cell death. We will also determine if the over-expression of human ATP6V0C in mouse substantia nigra (SN) attenuates ASYN accumulation and associated neurodegeneration resulting from over-expression of wild-type human ASYN. In Aim 2, we will genetically knockdown ATP6V0C or introduce point mutations in ATP6V0C that confer resistance to bafilomycin-mediated inhibition of V-ATPase, to determine the requirement of ATP6V0C in regulating such neuroprotective functions of bafilomycin in cultured mammalian cells. We will also develop bafilomycin analogs with reduced V-ATPase inhibitory activity to determine if bafilomycin mediated neuroprotection is enhanced with a reduction in V-ATPase inhibitory activity. These studies will validate the utility of ATP6V0C as a novel ALP-dependent target for the future generation of PD therapeutics which promotes ASYN clearance and DA neuron survival. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a leading age-related neurodegenerative disease and our laboratory studies the mechanisms by which toxic alpha synuclein species accumulate in PD and contribute to PD neuropathology and neurodegeneration. Specifically, our laboratory focuses on how intracellular pathways that regulate protein degradation may negatively influence DA neuron survival in PD. For this application we hypothesize that the protein ATP6V0C, a subunit of the vacuolar ATPase (V-ATPase) complex of lysosomes and other acidic organelles, is a valid target for PD therapeutics and that its over-expression or pharmacological regulation may promote DA neuron survival through its ability to increase the clearance of toxic alpha synuclein species from cells. In summary, our research will validate whether over-expression of ATP6V0C is an effective strategy for promoting alpha synuclein clearance, and if pharmacological regulation of this target also affords similar neuroprotection.