Study of the mitochondrial-cellular response to environmental stress by flourescence imaging

 描述(由申请人提供)：一系列环境因素导致的组织损伤被归因于线粒体产生的活性氧物种(ROS)。然而，由于很难直接监测或具体干扰ROS，导致ROS的致病途径在很大程度上仍不清楚。在R21阶段，这项提议将努力开发一种新的、以基因为目标的工具包，以敏感和特定的方式干扰和测量ROS和钙(Ca2+)信号。此外，这个工具箱将允许记录ROS和Ca~(2+)，直到线粒体特定亚室的水平，这可能在ROS失调中做出不同的贡献。R33阶段将使用腺相关病毒和转基因表达将新的工具箱引入小鼠，以便研究各种环境因素对肝脏、心脏和骨骼肌中原位ROS和钙信号的影响。在第一和第二阶段，该项目将研究ROS和钙离子在砷(As)、镉(Cd)和二恶英(Dioxin)引发的应激信号通路中的具体参与。研究人员将专门测试这一新的假设，即由这些因素诱导的环境应激导致线粒体-内质网/肌浆网(ER/SR)功能和结构耦合受损，为包括肝脏、心肌和骨骼肌在内的各种组织的细胞损伤提供一个重要机制。该团队已经开发出操纵线粒体-ER/SR界面的方法，并表明这对局部的钙信号有影响。这些研究将使研究环境应激的线粒体发病机制的方式发生范式转变，并将为环境因素导致组织损伤的机制提供新的线索，从而为创新治疗策略的开发提供独特的机会。

 DESCRIPTION (provided by applicant): A range of environmental agents causes tissue injury that has been attributed to reactive oxygen species (ROS) produced by mitochondria. However, the causative pathways remain largely unknown because it has been difficult to directly monitor or specifically perturb ROS. In the R21 phase, this proposal brings together efforts to develop a new, genetically-targeted toolkit to perturb and measure ROS and calcium (Ca2+) signals in a sensitive and specific manner. Furthermore, this toolkit will allow recording of ROS and Ca2+ down to the level of specific subcompartments of the mitochondria, which likely make differential contributions in ROS dysregulation. The R33 phase will use adeno-associated viruses and transgene expression to bring the novel toolkit into mice to enable study of the effect of various environmental agents on ROS and Ca2+ signals in situ in the liver, heart, and skeletal muscle. Within phases one and two, the project will study the specific involvement of ROS and Ca2+ in the stress pathways triggered by arsenic (As), cadmium (Cd), and dioxin. The investigators will specifically test the novel hypothesis that environmental stress induced by these agents causes impaired mitochondria- endoplasmic/sarcoplasmic reticulum (ER/SR) functional and structural coupling, providing an important mechanism underlying cell injury in various tissues, including the liver, cardiac and skeletal muscle. This team has developed methods to manipulate the mitochondrial-ER/SR interface and has shown that this has consequences for localized Ca2+ signaling. These studies will allow for a paradigm shift in the way mitochondrial pathogenesis of environmental stress is studied, and will shed new light on the mechanisms by which environmental agents can cause tissue damage, leading to unique opportunities for the development of innovative treatment strategies.