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组织损伤，这归因于线粒体产生的活性氧（ROS）。然而，致病途径仍然在很大程度上是未知的，因为它一直难以直接监测或专门干扰活性氧。在R21阶段，该提案汇集了开发新的遗传靶向工具包的努力，以灵敏和特异的方式干扰和测量ROS和钙（Ca 2+）信号。此外，该工具包将允许记录ROS和Ca 2+下降到线粒体的特定亚区室的水平，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工具包带入小鼠体内，以研究各种环境因子对肝脏，心脏和骨骼肌中ROS和Ca 2+信号的原位影响。在第一和第二阶段，该项目将研究ROS和Ca 2+在砷（As），镉（Cd）和二恶英引发的应激途径中的具体参与。 研究人员将专门测试新的假设，即这些药物诱导的环境应激导致线粒体-内质网/肌浆网（ER/SR）功能和结构偶联受损，提供了各种组织（包括肝脏，心脏和骨骼肌）细胞损伤的重要机制。该团队已经开发出了操纵细胞-ER/SR界面的方法，并表明这对局部Ca 2+信号传导有影响。这些研究将使环境应激的线粒体发病机制的研究方式发生范式转变，并将揭示环境因子可能导致组织损伤的机制，从而为创新治疗策略的发展提供独特的机会。

 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.