Cellular Mechanisms of Neuropathology in Parkinson's Disease

描述（由申请人提供）： 含有α-突触核蛋白（ASYN）的路易体的积累和相关的多巴胺（DA）神经元变性是帕金森病（PD）的主要病理标志。自噬-溶酶体途径（ALP）是重要的细胞内降解和再循环途径，并且ALP功能的妥协促进毒性ASYN种类的积累。由于ALP在PD中发生改变，因此鉴定和验证促进ASYN清除的新型ALP靶标是开发新型PD治疗剂的及时方法。ATP 6V 0 C形成V-ATP酶的质子转运通道，V-ATP酶是一种调节溶酶体酸化和ALP底物清除的酶复合物。高浓度的巴弗洛霉素（一种以高亲和力结合ATP 6V 0 C的大环内酯类抗生素）抑制V-ATP酶并产生与其抑制溶酶体功能和ASYN清除相关的神经毒性。然而，巴弗洛霉素的浓度太低而不能抑制V-ATP酶会减弱溶酶体功能障碍导致的细胞死亡和内源性ASYN积累，并抑制体内ASYN过表达导致的DA神经元死亡。虽然巴弗洛霉素具有狭窄的治疗指数，这限制了其在人类PD患者中的长期使用，但我们的数据表明ATP 6V 0 C是促进ASYN清除和细胞存活的新靶标。然而，尚未测试ATP 6V 0 C是否介导巴弗洛霉素的这些保护作用以及ATP 6V 0 C本身的过表达是否可以在不存在巴弗洛霉素的情况下提供神经保护益处。我们假设ATP 6V 0 C介导毒性ASYN物质的巴弗洛霉素依赖性清除，并减弱ASYN相关的神经毒性。在目的1中，我们将在培养的哺乳动物细胞中遗传过表达ATP 6V 0 C，以确定其在调节内源性ASYN、ALP功能和细胞死亡的清除中的相对重要性。我们还将确定小鼠黑质（SN）中人ATP 6V 0 C的过表达是否减弱由野生型人ASYN的过表达引起的ASYN积累和相关的神经变性。在目标2中，我们将在基因上敲低ATP 6V 0 C或在ATP 6V 0 C中引入点突变，从而赋予对巴弗洛霉素介导的V-ATP酶抑制的抗性，以确定ATP 6V 0 C在培养的哺乳动物细胞中调节巴弗洛霉素的这种神经保护功能的需要。我们还将开发具有降低的V-ATP酶抑制活性的巴弗洛霉素类似物，以确定巴弗洛霉素介导的神经保护是否随着V-ATP酶抑制活性的降低而增强。这些研究将验证ATP 6V 0 C作为新一代PD治疗剂的新型ALP依赖性靶标的效用，其促进ASYN清除和DA神经元存活。 公共卫生相关性： 帕金森病（PD）是一种主要的年龄相关性神经退行性疾病，我们的实验室研究了毒性α突触核蛋白物质在PD中积累并导致PD神经病理学和神经退行性变的机制。具体来说，我们的实验室专注于如何调节蛋白质降解的细胞内途径可能会对PD中DA神经元的存活产生负面影响。对于该应用，我们假设蛋白ATP 6V 0 C（溶酶体和其他酸性细胞器的空泡ATP酶（V-ATP酶）复合物的亚基）是PD治疗的有效靶标，并且其过表达或药理学调节可通过其增加毒性α突触核蛋白种类从细胞中清除的能力来促进DA神经元存活。总之，我们的研究将验证ATP 6V 0 C的过表达是否是促进α突触核蛋白清除的有效策略，以及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.