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

基本信息

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
项目类别：
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&apos s Disease Alzheimer&apos s disease brain Alzheimer&apos 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

项目摘要

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，人们一直在努力开发改善唐氏综合症（DS）认知功能的疗法很大程度上不成功，因为对潜在分子机制的了解仍然存在差距 DS 个体大脑中的神经回路缺陷是 DS 大脑的一个标志，即谷氨酸能减少。神经元活动，类似于在阿尔茨海默病（AD）患者大脑中观察到的变化。磷蛋白硫辛-α (ENSA) 可能通过以下方式刺激 DS 大脑中的神经元活动通过与 SUR1 调节相互作用抑制 ATP 敏感钾通道 (KATP) 通道 ENSA 丝氨酸 109 (S109) 被蛋白激酶 A 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 神经元功能的影响对于验证至关重要 ENSA 作为 DS 个体大脑的治疗靶点这项研究的长期目标是定义。导致中枢神经系统疾病中神经元功能障碍和神经变性的分子机制总体目标是开发新疗法。本申请的目的是确定 ENSA 表达和 S109 磷酸化对神经元的影响 DS 中的活动的理由是它的成功完成将提供强有力的证据- 以此为基础证明筛选有利于 ENSA 介导的 KATP 抑制作为新疗法的药物的合理性 DS 的候选者是根据大量已发表的数据制定的中心假设是 ENSA。 S109 磷酸化的下调和减少会导致 DS 大脑中神经元放电的减少。该假设将通过以下具体目标来解决：（1）确定ENSA敲除的效果 (2) 确定 DS 中 pS109-ENSA 的相对丰度目标 1 研究将涉及将来自人类 21 三体 iPSC 的神经元与或就使用多电极监测的活性而言，没有 CRISPR 介导的 ENSA 基因删除阵列并通过使用荧光 Ca2+ 报告基因的共聚焦显微镜观察 ENSA 敲除神经元的子集。用编码不可磷酸化和拟磷酸化变体S109A的慢病毒转导，并且 S109E。目标 2 研究将涉及对 DS 和对照皮质样本制备的匀浆进行表征。通过定量 LC-MS/MS 确定 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。创新性在于它专注于与 ENSA 在 DS 中的作用相关的新方向。意义重大，因为这项研究的新知识将为开发治疗方法奠定基础通过增强 ENSA 表达和/或改善 DS（以及潜在的 AD）认知障碍的策略 S109磷酸化。