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

开发帕金森氏病(PD)改变疾病疗法的一个主要障碍是 帕金森病患者中脑多巴胺能神经元丢失的分子机制研究 病人。多条证据表明，突触前蛋白α-突触核蛋白(ASyn)的聚集 膜表面AT在帕金森病神经细胞死亡中起中心作用。最近公布的研究结果表明， 先前被证明选择性地与脂质结合的aSyn相互作用的蛋白质Enosulfinine-Alpha(ENSA)干扰 膜诱导的aSyn聚集和aSyn的神经毒性，而ENSA的模拟物则被磷酸化 At残基S109与蛋白激酶A结合后没有抑制活性。此外，ENSA被发现出现了故障- 在突触核病患者的大脑中进行调节。然而，目前尚不清楚ENSA是否下调了监管 促进黑质多巴胺能细胞死亡，或ENSA是否在帕金森病患者中经历S109磷酸化的变化 大脑。解决这些问题对于为开发治疗方法建立强有力的科学前提至关重要。 旨在通过调节蛋白的表达和/或增强ENSA的神经保护活性 磷酸化。这项研究的长期目标是定义潜在的分子机制 导致帕金森病患者的神经退行性变。此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。这项研究的基本原理是 它的成功完成将提供一个强有力的证据基础，以证明筛选代理人的合理性 上调ENSA表达或抑制ENSA磷酸化是帕金森病新的候选治疗方案。这个 在大量初步数据的基础上形成的中心假设是，ENSA在其非磷酸化过程中 Form干扰膜诱导的帕金森病患者脑内aSyn聚集和aSyn神经毒性。 这一假设将以以下具体目标加以解决：(1)确定ENSA的效果 (2)测定PS109-ENSA在帕金森病中脑中的相对丰度。 目的1研究将涉及与腺病毒共转导的大鼠中脑原代培养物的特征 在多巴胺能细胞活性和突起长度方面编码aSyn和针对大鼠ENSA的shRNA。 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，寻找运动功能障碍和帕金森病相关神经病理的证据。目标2 研究将涉及从患者和对照中脑样本制备的匀浆的特性 通过定量LC/MS/MS分析ENSA S109的相对磷酸化水平，并通过 一种新的抗PS109-ENSA抗体的免疫印迹。这种方法是创新的，因为它专注于 黑质ENSA在神经退行性疾病中作用的新方向。这项研究具有重要意义 因为这项研究的新知识将为制定治疗策略奠定基础 激发ENSA介导的帕金森病患者脑内神经保护活性。

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.