ER mitochondrial signaling and alcoholic tissue injury

描述(申请人提供)：这项建议汇集了酒精研究和完整组织中细胞和亚细胞结构的实时动态荧光成像、高分辨率电子断层扫描、复杂的分子生物学和计算生物学的领先专家组成的独特多学科团队，以解决一个基本问题，即解除对细胞器之间局部相互作用的管制如何导致全球细胞功能障碍、组织损伤和疾病。本研究探讨线粒体-内质网(ER)/肌质网(SR)界面的调节及其与局部钙信号、活性氧(ROS)形成和内质网应激的关系。需要检验的中心假设是，长期酒精暴露会导致ER/SR-线粒体结构和功能偶联受损，从而增加肝脏和骨骼肌细胞损伤的易感性。我们的团队已经开发出操纵ER/SR-线粒体界面的方法，并表明这对局部的钙信号有影响。我们的第一个目标是进一步开发这些新的实验工具，以便能够a)操纵和测量ER/SR-线粒体界面，b)监测线粒体受控定位对局部[Ca~(2+)]信号和局部ROS形成的影响，以及c)在单细胞水平实时评估ER/SR应激反应。这将有助于我们分析ER/SR应激与局部钙和ROS信号变化的关系。然后，我们将检验长期酒精暴露对ER/SR-线粒体相互作用的影响会导致ER/SR-线粒体结构、钙信号的损害，并诱导肝脏和骨骼肌中ER/SR-线粒体的应激反应。最后，我们将在体内确定内质网线粒体形态和功能的改变是否与酒精诱导的代谢功能障碍和细胞损伤有关。通过这些方法，我们可以回答关于ER-线粒体网络的亚细胞组织及其被乙醇破坏的基本问题。这些研究将为酒精滥用导致组织损伤的机制提供新的线索，并将为开发创新的治疗策略提供独特的机会。 公共卫生相关性：慢性酒精中毒与内质网应激和线粒体功能障碍以及多个组织中内质网和线粒体形态的变化有关。ER/SR-线粒体连接对ER/SR和线粒体的功能和结构都很重要，被认为是酒精对肝脏和骨骼肌损伤作用的靶点和重要的介导物。利用酒精研究和完整组织中细胞和亚细胞结构的实时动态荧光成像、高分辨率电子断层扫描、复杂的分子生物学和计算生物学方面的专业知识的多学科方法将有助于解决一个根本问题，即解除对细胞器之间局部相互作用的管制如何导致全球细胞功能障碍、组织损伤和疾病。

DESCRIPTION (provided by applicant): This proposal brings together a unique multidisciplinary team of leading experts in alcohol studies and real time dynamic fluorescence imaging of cells and subcellular structures in intact tissues, high resolution electron tomography, sophisticated molecular biology and computational biology to address a fundamental question of how a deregulation of local interactions between organelles can result in global cell dysfunction, tissue damage and disease. The study addresses the regulation of the mitochondria-endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) interface and its relationship to localized Ca2+ signaling, formation of reactive oxygen species (ROS) and ER stress. The central hypothesis to be tested is that chronic alcohol exposure causes impaired ER/SR-mitochondrial structural and functional coupling and thereby increases the susceptibility to cell injury in liver and skeletal muscle. Our group has developed methods to manipulate the ER/SR-mitochondrial interface and has shown that this has consequences for localized Ca2+ signaling. Our first aim is to further develop these new experimental tools to be able a) to manipulate and measure the ER/SR-mitochondrial interface, b) to monitor the impact of controlled mitochondrial localization on local [Ca2+] signals and local ROS formation and c) to assess ER/SR stress responses in real time at the single cell level. This will help us analyze the relationship between ER/SR stress and changes in localized Ca2+ and ROS signaling. We will then test the concept that the effect of chronic alcohol exposure on ER/SR-mitochondrial interactions leads to impairments in ER/SR-mitochondrial structure, calcium signaling and induces ER/SR- mitochondrial stress responses in the liver and skeletal muscle. Finally, we will determine whether the alterations in ER-mitochondrial morphology and function contribute to alcohol-induced metabolic dysfunction and cell injury in vivo. With these approaches, we can answer fundamental questions about the subcellular organization of the ER-mitochondrial network and its disruption by ethanol. These studies will shed new light on the mechanisms by which alcohol abuse can cause tissue damage and will provide unique opportunities for the development of innovative treatment strategies. PUBLIC HEALTH RELEVANCE: Chronic alcoholism is associated with ER stress and mitochondrial dysfunction and changes in ER and mitochondrial morphology in multiple tissues. The ER/SR-mitochondrial junctions are important for the function and structure of both ER/SR and mitochondria and are envisioned as a target and an important mediator of the alcohol's effect on liver and skeletal muscle injury. A multidisciplinary approach utilizing expertise in alcohol studies and real time dynamic fluorescence imaging of cells and subcellular structures in intact tissues, high resolution electron tomography, sophisticated molecular biology and computational biology will help to address a fundamental question of how a deregulation of local interactions between organelles can result in global cell dysfunction, tissue damage and disease.