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: 10404860
• 负责人: JEAN-CHRISTOPHE ROCHET
• 金额: $ 7.35万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10404860
• 关键词: ATP sensitive potassium channel complex; Address; Affect; Age; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease patient; Antibodies; Autopsy; Binding; Brain; Central Nervous System Diseases; Clustered Regularly Interspaced Short Palindromic Repeats; Cognitive deficits; Confocal Microscopy; Coupled; Cyclic AMP-Dependent Protein Kinases; Data; Defect; Down Syndrome; Down-Regulation; Foundations; Gene Deletion; Glutamates; Goals; Hippocampus (Brain); Human; Immunoblotting; Immunohistochemistry; Impaired cognition; Individual; Knock-out; Knowledge; Lead; Length; Lentivirus; Link; Liquid Chromatography; Mediating; Membrane; Mission; Modeling; Molecular; Monitor; Mus; National Institute of Neurological Disorders and Stroke; Nerve Degeneration; Neurites; Neurocirculatory Asthenia; Neuronal Dysfunction; Neurons; Neurotransmitters; Outcome; Parkinson Disease; Pathology; Phosphoproteins; Phosphorylation; Potassium; Proteins; Public Health; Publishing; Reporter; Research; Role; Sampling; Serine; Signal Transduction; Syndrome; Temporal Lobe; Testing; Therapeutic; Variant; base; cognitive function; design; endosulfine; evidence base; expectation; improved; induced pluripotent stem cell; innovation; insight; multi-electrode arrays; nervous system disorder; neural circuit; neural network; novel therapeutics; programs; screening; sulfonylurea receptor; tandem mass spectrometry; therapeutic candidate; therapeutic target; therapy design; therapy development

Efforts to develop therapies that improve cognitive function in Down syndrome (DS) have so far been unsuccessful largely because there continue to be gaps in knowledge of molecular mechanisms underlying neurocircuitry defects in the brains of DS individuals. A hallmark of DS brains is a decrease in glutamatergic neuronal activity, similar to changes observed in the brains of Alzheimer’s disease (AD) patients. The phosphoprotein endosulfine-alpha (ENSA) could potentially stimulate neuronal activity in DS brains by inhibiting ATP-sensitive potassium channels (KATP) channels through its interaction with the SUR1 regulatory subunit. ENSA phosphorylation at serine 109 (S109) by protein kinase A has been shown to influence the protein’s interactions with various binding partners and is predicted to favor ENSA-SUR1 binding. Understanding the impact of ENSA or pS109-ENSA on neuron function in DS is essential in order to validate ENSA as a therapeutic target in the brains of DS individuals. The long-term goal of this research is to define molecular mechanisms that contribute to neuronal dysfunction and neurodegeneration in CNS disorders such as Parkinson’s disease (PD), AD, and DS, with a view towards developing new therapies. The overall objective in this application is to determine the effects of ENSA expression and S109 phosphorylation on neuronal activity in DS. The rationale for this research is that its successful completion would provide a strong evidence- based foundation to justify screening for agents that favor ENSA-mediated KATP inhibition as new therapeutic candidates for DS. The central hypothesis, formulated on the basis of extensive published data, is that ENSA down-regulation and a decrease in S109 phosphorylation lead to a reduction in neuronal firing in DS brains. This hypothesis will be addressed with the following specific aims: (1) Determine the effect of ENSA knockout on neuron activity in a cellular DS model; and (2) Determine the relative abundance of pS109-ENSA in DS brains. Aim 1 studies will involve comparing neurons derived from human trisomy-21 iPSCs, with or without a CRISPR-mediated ENSA gene deletion, in terms of activity monitored using a multi-electrode array and via confocal microscopy using a fluorescent Ca2+ reporter. A subset of ENSA knockout neurons will be transduced with lentivirus encoding the non-phosphorylatable and phosphomimetic variants S109A and S109E. Aim 2 studies will involve characterizing homogenates prepared from DS and control cortical samples in terms of relative levels of ENSA S109 phosphorylation via quantitative LC-MS/MS, and validating the MS data via immunoblotting and immunohistochemistry using a new anti-pS109-ENSA antibody. This approach is innovative because it is focused on new directions related to the role of ENSA in DS. The research is significant because the new knowledge from this study would set the stage for developing therapeutic strategies to ameliorate cognitive impairment in DS (and potentially AD) by enhancing ENSA expression and/or S109 phosphorylation.

迄今为止 不成功 DS个体的大脑中的神经记录缺陷。 DS大脑的标志是谷氨酸能的降低 神经元活性，类似于阿尔茨海默氏病（AD）患者大脑中观察到的变化。 磷蛋白内硫硫酸盐α（ENSA）可能会通过DS大脑中的神经元活性。 通过与SUR1调节的相互作用，抑制ATP敏感的钾通道（KATP）通道 亚基。蛋白激酶A在丝氨酸109（S109）处的ENSA磷酸化已被证明会影响 蛋白质与各种结合伴侣的相互作用，预计有利于ENSA-SUR1结合。 了解ENSA或PS109-ENSA对DS中神经元功能的影响对于验证至关重要 ENSA是DS个体大脑中的治疗靶标。这项研究的长期目标是定义 在CNS疾病中导致神经元功能障碍和神经退行性的分子机制 作为帕金森氏病（PD），AD和DS，以期开发新疗法。总体目标 在此应用中，是确定ENSA表达和S109磷酸化对神经元的影响 DS的活动。这项研究的理由是，其成功完成将提供有力的证据 - 基于基础，以证明筛查促进ENSA介导的KATP抑制作用的代理的合理基础 DS的候选人。根据广泛发布的数据提出的中心假设是 S109磷酸化的下调和降低会导致DS大脑中神经元发射的降低。 该假设将以以下特定目的解决：（1）确定ENSA敲除的影响 细胞DS模型中的神经元活性； （2）确定ds中ps109-ensa的相对丰度 大脑。 AIM 1研究将涉及将来自人类三体三体21 IPSC的神经元与OR进行比较 没有CRISPR介导的ENSA基因删除，就使用多电极监测的活动而言 阵列和通过共聚焦显微镜使用荧光Ca2+报告基因。 ENSA敲除神经元的子集将 用编码非磷酸化和磷酸化变体S109A和 S109E。 AIM 2研究将涉及表征由DS制备和对照皮质样品制备的匀浆 就通过定量LC-MS/MS的ENSA S109磷酸化的相对水平而言，并验证MS 使用新的抗PS109-ENSA抗体通过免疫印迹和免疫组织化学进行数据。这种方法是 创新性是因为它专注于与ENSA在DS中的作用相关的新方向。研究是 意义重大，因为这项研究的新知识将为发展治疗奠定阶段 通过增强ENSA表达和/或 S109磷酸化。