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) 疾病改变疗法的一个主要障碍是 了解 PD 中脑多巴胺能神经元丧失的分子机制 患者。多种证据表明突触前蛋白 α-突触核蛋白 (aSyn) 的聚集 膜表面在帕金森病中神经元细胞死亡中起着核心作用。最近发表的研究结果表明 蛋白质endosulfine-alpha (ENSA)，先前已被证明可以选择性地与脂质结合的aSyn相互作用，干扰 具有膜诱导的 aSyn 聚集和 aSyn 神经毒性，而 ENSA 的模拟物磷酸化 蛋白激酶A对残基S109没有抑制活性。此外，ENSA 被发现表现不佳—— 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是否会下调 促进黑质多巴胺能细胞死亡，或 PD 中 ENSA 是否发生 S109 磷酸化变化 脑。解决这些问题对于为开发疗法奠定坚实的科学前提至关重要 旨在通过调节蛋白质的表达和/或增强 ENSA 神经保护活性 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。该应用程序的总体目标是确定 ENSA 下调和磷酸化对 aSyn 神经毒性的影响。这项研究的理由是 它的成功完成将为证明筛选药物的合理性提供强有力的循证基础 上调 ENSA 表达或抑制 ENSA 磷酸化作为 PD 的新治疗候选者。这 根据大量初步数据制定的中心假设是 ENSA 在其未磷酸化状态下 形式干扰 PD 患者大脑中膜诱导的 aSyn 聚集和 aSyn 神经毒性。 该假设将通过以下具体目标来解决：（1）确定ENSA的影响 降低 aSyn 神经毒性； (2)确定PD中脑中pS109-ENSA的相对丰度。 目标 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shRNA，在多巴胺能细胞活力和神经突长度方面对大鼠ENSA具有特异性。 其他实验将包括检查单侧黑质注射 rAAV 病毒的大鼠 编码 ENSA 靶向 shRNA，作为运动功能障碍和 PD 相关神经病理学的证据。目标2 研究将涉及对从患者和对照中脑样本制备的匀浆进行表征 通过定量 LC/MS/MS 分析 ENSA S109 磷酸化的相对水平，并通过以下方式验证 MS 数据 使用新型抗 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。研究意义重大 因为这项研究的新知识将为制定治疗策略奠定基础 刺激 PD 患者大脑中 ENSA 介导的神经保护活性。