Role of endosulfine-alpha expression and phosphorylation in Parkinson's Disease

内硫氨酸-α 表达和磷酸化在帕金森病中的作用

• 批准号: 10058021
• 负责人: JEAN-CHRISTOPHE ROCHET
• 金额: $ 14.65万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058021
• 关键词: Address; Adenoviruses; Age; Antibodies; Attenuated; Binding; Binding Proteins; Brain; Brain Diseases; Cell Culture Techniques; Cell Death; Cell Survival; Coculture Techniques; Corpus striatum structure; Coupled; Cyclic AMP-Dependent Protein Kinases; Data; Dependovirus; Development; Disease; Dopaminergic Cell; Down-Regulation; Equilibrium; Foundations; Genetic; Goals; Human; Immunoblotting; Knowledge; Lead; Length; Lipid Binding; Lipids; Liquid Chromatography; Mediating; Membrane; Midbrain structure; Mission; Molecular; National Institute of Neurological Disorders and Stroke; Nerve Degeneration; Neurites; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathogenesis; Patients; Pharmaceutical Preparations; Phosphorylation; Play; Proteins; Public Health; Publishing; Rattus; Recombinant adeno-associated virus (rAAV); Research; Role; Sampling; Substantia nigra structure; Surface; Synaptic Vesicles; Testing; Therapeutic; Variant; Vesicle; Virus; alpha synuclein; conformer; design; dopaminergic neuron; endosulfine; evidence base; expectation; experimental study; in vivo; innovation; insight; knock-down; liquid chromatography mass spectrometry; motor deficit; motor disorder; motor symptom; mutant; nervous system disorder; neuron loss; neuropathology; neurotoxic; neurotoxicity; novel therapeutic intervention; novel therapeutics; pars compacta; presynaptic; programs; protein expression; screening; self assembly; small hairpin RNA; synucleinopathy; tandem mass spectrometry; therapeutic candidate; therapy development

A major hurdle to developing disease-altering therapies for Parkinson’s disease (PD) has been a gap in knowledge of the molecular mechanisms underlying the loss of dopaminergic neurons in the midbrains of PD patients. Multiple lines of evidence suggest that aggregation of the presynaptic protein alpha-synuclein (aSyn) at membrane surfaces plays a central role in neuronal cell death in PD. Recent published findings indicate that the protein endosulfine-alpha (ENSA), previously shown to interact selectively with lipid-bound aSyn, interferes with membrane-induced aSyn aggregation and aSyn neurotoxicity, whereas a mimic of ENSA phosphorylated at residue S109 by protein kinase A has no inhibitory activity. Moreover, ENSA was found to be down- regulated in the brains of synucleinopathy patients. However, it is unknown whether ENSA down-regulation promotes nigral dopaminergic cell death, or whether ENSA undergoes changes in S109 phosphorylation in PD brain. Resolving these issues is essential to establish a strong scientific premise for developing therapies aimed at enhancing ENSA neuroprotective activity by modulating the protein’s expression and/or phosphorylation. The long-term goal of this research is to define the underlying molecular mechanisms that contribute to neurodegeneration in PD patients. The overall objective in this application is to determine the effects of ENSA down-regulation and phosphorylation on aSyn neurotoxicity. The rationale for this research is that its successful completion would provide a strong evidence-based foundation to justify screening for agents that up-regulate ENSA expression or inhibit ENSA phosphorylation as new therapeutic candidates for PD. The central hypothesis, formulated on the basis of extensive preliminary data, is that ENSA in its unphosphorylated form interferes with membrane-induced aSyn aggregation and aSyn neurotoxicity in the brains of PD patients. This hypothesis will be addressed with the following specific aims: (1) Determine the effect of ENSA knockdown on aSyn neurotoxicity; and (2) Determine the relative abundance of pS109-ENSA in PD midbrain. Aim 1 studies will involve characterizing rat primary midbrain cultures co-transduced with adenoviruses encoding aSyn and an shRNA specific for rat ENSA in terms of dopaminergic cell viability and neurite lengths. Additional experiments will involve examining rats injected unilaterally in the substantia nigra with rAAV virus encoding an ENSA-targeting shRNA for evidence of motor dysfunction and PD-related neuropathology. Aim 2 studies will involve characterizing homogenates prepared from patient and control midbrain samples in terms of relative levels of ENSA S109 phosphorylation via quantitative LC/MS/MS, and validating the MS data via immunoblotting using a new anti-pS109-ENSA antibody. This approach is innovative because it is focused on new directions related to the role of nigral ENSA in neurodegenerative disease. The research is significant because the new knowledge from this study would set the stage for developing therapeutic strategies to stimulate ENSA-mediated neuroprotective activity in the brains of PD patients.

开发帕金森病（PD）的疾病改变疗法的主要障碍是 了解PD中脑多巴胺能神经元丢失的分子机制 患者多种证据表明突触前蛋白α-突触核蛋白（aSyn）的聚集 在PD中，在膜表面的神经元细胞死亡中起核心作用。最近发表的研究结果表明， 先前显示与脂质结合的α-Syn选择性相互作用的蛋白质内亚硫蛋白-α（ENSA）干扰 具有膜诱导的aSyn聚集和aSyn神经毒性，而模拟ENSA磷酸化 蛋白激酶A在S109残基上的突变没有抑制活性。此外，ENSA被发现下降- 在突触核蛋白病患者的大脑中受到调节。然而，目前尚不清楚ENSA下调是否 促进黑质多巴胺能细胞死亡，或者ENSA是否在PD中经历S109磷酸化的变化， 个脑袋解决这些问题对于为开发治疗建立强有力的科学前提至关重要 旨在通过调节蛋白质的表达来增强ENSA神经保护活性，和/或 磷酸化这项研究的长期目标是确定潜在的分子机制， 导致PD患者的神经退行性变。本申请的总体目标是确定 ENSA下调和磷酸化对aSyn神经毒性的影响。这项研究的基本原理是 它的成功完成将提供一个强有力的循证基础，证明筛选代理人是合理的， 其上调ENSA表达或抑制ENSA磷酸化作为PD的新的治疗候选物。的 根据大量的初步数据，中心假设是ENSA在其未磷酸化的 形式干扰PD患者大脑中膜诱导的aSyn聚集和aSyn神经毒性。 本研究的目的是：（1）确定ENSA的效果 对aSyn神经毒性的敲低;和（2）测定PD中脑中pS109-ENSA的相对丰度。 目的1研究将涉及表征与腺病毒共转导的大鼠原代中脑培养物 编码aSyn和在多巴胺能细胞活力和神经突长度方面对大鼠ENSA特异性的shRNA。 另外的实验将涉及检查在黑质单侧注射rAAV病毒的大鼠 编码ENSA靶向shRNA，用于运动功能障碍和PD相关神经病理学的证据。目的2 研究将涉及表征从患者和对照中脑样品制备的匀浆， 通过定量LC/MS/MS测定ENSA S109磷酸化的相对水平，并通过 使用新的抗pS109-ENSA抗体进行免疫印迹。这种方法是创新的，因为它专注于 与黑质ENSA在神经退行性疾病中的作用相关的新方向。这项研究具有重要意义 因为这项研究的新知识将为开发治疗策略奠定基础， 刺激PD患者脑中ENSA介导的神经保护活性。