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

点击翻译按钮获取中文摘要

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.