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

到目前为止，致力于开发改善唐氏综合征(DS)认知功能的治疗方法 失败的主要原因是在分子机制方面仍然存在空白 DS患者大脑中的神经回路缺陷。DS脑的一个特征是谷氨酸能减少 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。这个 磷酸蛋白-硫代巴比妥酸(ENSA)可能通过以下途径刺激DS大鼠脑内神经元的活动 通过与调节因子SUR1的相互作用抑制ATP敏感钾通道 亚单位。蛋白激酶A在丝氨酸109(S109)处的Ensa磷酸化已被证明影响 蛋白质与各种结合伙伴的相互作用，并被预测有利于ENSA-SUR1结合。 为了验证，了解ENSA或PS109-ENSA对DS神经元功能的影响是至关重要的 Ensa作为DS患者脑内的治疗靶点。这项研究的长期目标是确定 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 如帕金森氏病(PD)、AD和DS，以期开发新的治疗方法。总体目标 在这方面的应用是确定ENSA表达和S109磷酸化对神经元的影响 DS中的活动。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- 为筛选支持ENSA介导的KATP抑制作为新的治疗药物提供依据 DS的候选人。根据大量公布的数据提出的中心假设是，ENSA 下调和S109磷酸化的减少导致DS脑中神经元放电的减少。 这一假设将以以下具体目标来解决：(1)确定ENSA基因敲除的效果 关于细胞DS模型中神经元活性的研究；以及(2)确定DS中PS109-ENSA的相对丰度 大脑。目的1研究将比较来自人类21三体ipscs的神经元与或 没有CRISPR介导的ENSA基因缺失，就使用多电极监测的活性而言 阵列和使用荧光钙离子报告程序的共聚焦显微镜。ENSA基因敲除神经元的子集将 被编码非磷酸化和拟磷化变异体S109A和 S109E。目的2研究将包括从DS和对照皮质样品制备的匀浆的特征 通过LC-MS/MS定量测定ENSA S109的相对磷酸化水平，并验证MS 数据通过免疫印迹和免疫组织化学使用新的抗PS109-ENSA抗体。这种方法是 创新是因为它专注于与Ensa在DS中的作用相关的新方向。这项研究是 意义重大，因为这项研究的新知识将为开发治疗药物奠定基础 通过增强ENSA表达和/或改善DS(和潜在的AD)患者认知功能障碍的策略 S109的磷酸化。

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.