Mitochondrial complex III-derived ROS in astrocytic signaling and Alzheimer's disease-related pathogenesis

项目总结/摘要 线粒体活性氧（ROS）与多种衰老的发病机制密切相关- 相关的神经系统疾病，包括阿尔茨海默病（AD）和额颞叶痴呆。 线粒体在氧化代谢过程中产生ROS，并且线粒体ROS的产生增加是由于 与AD的各种过程有因果关系，包括衰老、淀粉样前体蛋白/淀粉样β（APP/A β） 病理学、tau蛋白病和神经炎症。最近的研究表明，ROS产生的不同 线粒体位点在细胞信号传导和疾病中具有不同的作用。然而，以前的抑制工具 线粒体活性氧没有位点选择性，破坏呼吸，或抑制活性氧释放后，而不是 阻碍生产。因此，线粒体活性氧在AD发病机制中的作用需要调查。 线粒体复合物III具有大的ROS产生能力，并向胞质溶胶产生ROS， 使其平衡以调节细胞内信号传导和疾病机制。为了研究复合物III的作用- 衍生的ROS，我们的实验室已经确定并表征了抑制复合物III ROS产生的小分子 （S3QEL，"后遗症"），但不阻断其他线粒体位点的ROS产生或影响其他线粒体 流程.在我们使用S3QEL的初步研究中，我们发现AD相关的神经免疫因子 增强星形胶质细胞复合物III ROS和复合物III ROS促进JAK-STAT3信号传导和基因 与疾病有关的表达变化。在星形胶质细胞-神经元培养物中，S3QEL可防止神经元功能障碍 与tau蛋白病有关，但不影响分离培养的神经元。此外，S3QELs降低了神经免疫功能， 和表达突变型人tau的小鼠中的神经胶质改变。这些数据暗示了星形胶质细胞中复合物III ROS的表达。 信号传导和AD相关级联。我建议测试我的中心假设，即星形胶质细胞复合物III ROS是 通过特定疾病相关刺激增加并调节星形胶质细胞功能和与痴呆症相关的致病性 通过氧化不同的半胱氨酸靶点，包括那些与STAT3相关的过程。在目标1中，我将使用 靶向原代小鼠特定亚细胞区室的遗传编码比率H2O2传感器 和人iPSC衍生的星形胶质细胞来定义星形胶质细胞复合物III的确切模式和上游触发物 罗斯我还将使用遗传学和药理学工具来确定复合物III ROS在星形胶质细胞中的作用。 信号传导、基因表达和星形胶质细胞-神经元相互作用。在目标2中，我将使用创新的氧化还原蛋白质组学 广泛描述复合物III ROS介导的半胱氨酸氧化的方法， 基因操作以评估特定半胱氨酸位点的氧化如何改变星形胶质细胞信号传导， 与痴呆症有关的病理级联反应这项研究可能会阐明新的氧化机制 调节神经胶质细胞信号传导和疾病级联反应，并可能导致靶向和有效的 老年痴呆症的治疗方法

PROJECT SUMMARY/ABSTRACT Mitochondrial reactive oxygen species (ROS) are strongly implicated in the pathogenesis of diverse aging- associated neurological disorders, including Alzheimer's disease (AD) and frontotemporal dementia. Mitochondria produce ROS during oxidative metabolism and increased production of mitochondrial ROS are causally linked to various processes in AD, including aging, amyloid precursor protein/amyloid-β (APP/Aβ) pathology, tauopathy, and neuroinflammation. Recent work suggests that ROS produced by different mitochondrial sites have distinct roles in cell signaling and disease. However, previous tools to suppress mitochondrial ROS were not site-selective, disrupted respiration, or inhibited ROS only after release rather than blocking production. Thus, the roles of mitochondrial ROS in AD pathogenesis require investigation. Mitochondrial complex III has a large capacity for ROS production and generates ROS toward the cytosol, poising it to regulate intracellular signaling and disease mechanisms. To investigate the effects of complex III- derived ROS, our lab has identified and characterized small molecules that suppress complex III ROS production (S3QELs, “sequels”), but do not block ROS production by other mitochondrial sites or affect other mitochondrial processes. In our preliminary studies using S3QELs, we found that AD-associated neuroimmune factors enhance astrocytic complex III ROS and that complex III ROS promote JAK-STAT3 signaling and gene expression changes linked to disease. In astrocytic-neuronal cultures, S3QELs prevented neuronal dysfunction linked to tauopathy, but did not affect neurons cultured in isolation. In addition, S3QELs reduced neuroimmune and glial alterations in mice expressing mutant human tau. These data implicate complex III ROS in astrocytic signaling and AD-related cascades. I propose to test my central hypotheses that astrocytic complex III ROS are increased by specific disease-related stimuli and modulate astrocytic functions and dementia-linked pathogenic processes through oxidation of distinct cysteine targets, including those related to STAT3. In Aim 1, I will use a genetically-encoded ratiometric H2O2 sensor targeted to specific subcellular compartments in primary mouse and human iPSC-derived astrocytes to define the exact patterns and upstream triggers of astrocytic complex III ROS. I will also use genetic and pharmacological tools to determine the roles of complex III ROS in astrocytic signaling, gene expression, and astrocytic-neuronal interactions. In Aim 2, I will use innovative redox proteomics methods to broadly profile complex III ROS-mediated cysteine oxidation and use targeted and cell-specific genetic manipulations to assess how oxidation of specific cysteine sites alters astrocytic signaling and pathological cascades linked to dementia. The proposed study is likely to elucidate novel oxidative mechanisms that regulate glial signaling and disease cascades and could lead to the development of targeted and effective therapies for aging-related dementias.