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.

到目前为止，开发改善唐氏综合征（DS）认知功能的疗法的努力已经取得了进展。 这在很大程度上是不成功的，因为在了解潜在的分子机制方面仍然存在差距。 DS患者大脑中的神经回路缺陷。DS大脑的一个标志是多巴胺能神经元减少， 神经元活动，类似于阿尔茨海默病（AD）患者大脑中观察到的变化。的 磷蛋白内亚硫蛋白-α（ENSA）可能通过以下方式刺激DS脑中的神经元活动： 通过与Sur 1调节因子的相互作用抑制ATP敏感性钾通道（KATP）通道 亚单位已显示通过蛋白激酶A在丝氨酸109（S109）处的ENSA磷酸化影响蛋白激酶A的活性。 蛋白质与各种结合伴侣的相互作用，并预测有利于ENSA-SUR 1结合。 了解ENSA或pS109-ENSA对DS中神经元功能的影响对于验证 ENSA作为DS个体大脑中的治疗靶点。这项研究的长期目标是确定 导致CNS疾病中神经元功能障碍和神经变性的分子机制， 帕金森氏病（PD）、AD和DS，以期开发新的治疗方法。总体目标 本申请的目的是确定ENSA表达和S109磷酸化对神经元细胞凋亡的影响。 活动在DS。这项研究的理由是，它的成功完成将提供一个强有力的证据- 为筛选有利于ENSA介导的KATP抑制的药物作为新的治疗药物提供依据 候选人DS在广泛发表的数据基础上提出的中心假设是，ENSA 下调和S109磷酸化的减少导致DS脑中神经元放电的减少。 该假说将通过以下具体目标来解决：（1）确定ENSA敲除的效果 （2）测定DS中pS109-ENSA的相对丰度 大脑目的1研究将涉及比较来源于人21三体iPSC的神经元， 没有CRISPR介导的ENSA基因缺失，就使用多电极监测的活性而言， 阵列，并通过共聚焦显微镜使用荧光Ca 2+报告。ENSA敲除神经元的子集将 用编码不可磷酸化的和拟磷酸化的变体S109 A和 S109 E。目的2研究将涉及表征由DS和对照皮质样品制备的匀浆 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获得数据。这种方法 创新，因为它专注于与ENSA在DS中的作用相关的新方向。这项研究是 重要的是，这项研究的新知识将为开发治疗性药物奠定基础。 通过增强ENSA的表达和/或增强AD的认知功能来改善DS（以及潜在的AD）的认知功能障碍的策略 S109磷酸化。