Mitochondrial dynamics in acute lung injury

描述(申请人提供)：意义。急性肺损伤(ALI)是导致死亡率和致残率的主要原因。我们的目标是确定可能导致治愈的机制。我们将专注于肺泡线粒体机制，这是一个知之甚少的机制。在ALI中，线粒体功能障碍可能是肺泡功能障碍的基础，从而导致肺损伤。这种功能障碍可能与线粒体钙缓冲的丧失有关，在缓冲中，钙通过线粒体钙单一转运体(MCU)从胞浆扩散到线粒体基质，阻止胞浆钙(细胞钙)的促炎增加，从而保护细胞免受损伤。更好地了解这些机制可能会导致MCU恢复线粒体钙缓冲，作为治疗内毒素-ALI的方法。求婚。我们提出了线粒体钙决定表面活性物质分泌的新假设，因为线粒体钙摄取增加了ATP和H_2O_2的产生。在内毒素-ALI中，持续的细胞钙升高导致线粒体钙缓冲失效，导致钙依赖的磷酸酶钙调神经磷酸酶激活，进而使线粒体分裂蛋白Drp1去磷酸化并激活。由此产生的线粒体分裂导致MCU的废除。钙调神经磷酸酶也促进肌动蛋白解聚。这些影响共同导致肺泡功能障碍，这反映在表面活性物质分泌的丧失上。明确的目标。其具体目的是确定MCU在肺泡表面活性物质分泌中的作用(目标1)，并确定MCU的缺失在多大程度上加剧了内毒素诱导的ALI(目标2)的肺损伤。我们将确定MCU作为一种特定的钙通道的作用，以及它在表面活性物质分泌中的作用。我们将评估在内毒素-ALI中线粒体钙缓冲丧失的分子机制，我们将考虑以骨髓间充质基质细胞(BMSC)为基础的治疗方法，旨在通过线粒体受损的肺泡恢复钙缓冲作为肺修复的策略。接近。我们将通过对分离的小鼠肺进行活的共聚焦和双光子显微镜以及对分离的肺泡2型(AT2)细胞的研究来实现这些目标。我们的检测将包括：(1)完整肺泡和分离的AT2细胞中的细胞钙和丝裂原钙；(2)单细胞成像通过测定钙、三磷酸腺苷、过氧化氢和F-肌动蛋白栅栏调节表面活性物质的分泌；(3)MCU和钙通过肺和AT2细胞在内毒素ALI中的缓冲；(4)MCU突变体在BMSCs中的表达，以验证MCU恢复可恢复线粒体钙缓冲的假说；(5)MCU通过线粒体转移过度表达，作为一种保护内毒素-ALI的手段。初步数据。我们发现：(1)MCU通过激活DRp1下调MCU，从而抑制线粒体的钙缓冲；(3)当突变的MCU在内毒素ALI中的表达恶化了小鼠在内毒素ALI中的存活率时，填充长度的MCU的表达增加了小鼠的存活率。这些和其他有关我们测量策略的初步数据支持该项目的可行性。冲击力。该项目将首次系统地了解MCU作为肺泡功能决定因素的作用。失去MCU在多大程度上加剧了ALI，以及MCU恢复在多大程度上促进了肺修复，这将是第一次了解。将对ALI的发病机制有全新的认识。

DESCRIPTION (provided by applicant): Significance. Acute lung injury (ALI) is major cause of mortality and morbidity. Our goal is to define mechanisms that might lead to a cure. We will focus on alveolar mitochondrial mechanisms, which are poorly understood. In ALI, mitochondrial dysfunction might underlie alveolar dysfunction, leading to lung injury. The dysfunction might follow loss of mitochondrial Ca2+ buffering in which Ca2+ diffuses from cytosol to mitochondrial matrix across the mitochondrial Ca2+ uniporter (MCU), preventing proinflammatory increase of the cytosolic Ca2+ (cytCa2+), hence protecting against injury. Better understanding of these mechanisms might lead to restoration of mitochondrial Ca2+ buffering by MCU as a therapy for LPS-ALI. Proposal. We propose the novel hypothesis that mitochondrial Ca2+ (mitCa2+) determine surfactant secretion, because the mitochondrial Ca2+ uptake increases ATP and H2O2 production. In LPS-ALI, sustained cytCa2+ increase causes failure of mitochondrial Ca2+ buffering leading to the activation of the Ca2+-dependent phosphatase, calcineurin, which in turn, dephosphorylates and activates the mitochondrial fission protein, DRP1. The resulting mitochondrial fission leads to abrogation of MCU. Calcineurin also promotes actin depolymerization. Together these effects induce alveolar dysfunction as reflected in loss of surfactant secretion. Specific Aims. The specific aims are to determine the role of the MCU in alveolar surfactant secretion (Aim 1) and to determine the extent to which loss of MCU exacerbates lung injury in LPS-induced ALI (Aim 2). We will establish the role of the MCU as a specific Ca2+ channel, as well as its role in surfactant secretion. We will evaluate molecular mechanisms underlying the loss of mitochondrial Ca2+ buffering in LPS-ALI and we will consider bone-marrow-derived mesenchymal stromal cell (BMSC)-based therapeutic approaches aimed at reinstating Ca2+ buffering by mitochondria damaged alveoli as a strategy for lung repair. Approach. We will achieve these aims by live confocal and two-photon microscopy of isolated perfused mouse lungs and by studies in isolated alveolar type 2 (AT2) cells. Our determinations will include: (1) cytCa2+ and mitCa2+ in intact alveoli and in isolated AT2 cells; (2) regulation of surfactant secretion by single cell imaging through determinations of Ca2+, ATP, H2O2 and the F-actin fence; (3) MCU and Ca2+ buffering in endotoxin ALI through lung and AT2 cell expressions of specific mutants and siRNA; (4) Expression of MCU mutants in BMSCs to test the hypothesis that MCU restitution re-instates mitochondrial Ca2+ buffering; (5) MCU overexpression by mitochondrial transfer as a means to protect against LPS-ALI. Preliminary data. We show (1) MCU regulates cytCa2+ and mitCa2+ and surfactant secretion, (2) endotoxin ALI blocks mitochondrial Ca2+ buffering by downregulating MCU through activation of DRP1, and that (3) while alveolar transfection of a mutant MCU worsens mouse survival in endotoxin ALI, expression of a fill length MCU increases mouse survival. These and other preliminary data on our measurement strategies support feasibility of the project. Impact. This project will provide the first systematic understanding of the role of the MCU as a determinant of alveolar function. The extent to which loss of the MCU exacerbates ALI, and the extent to which MCU reinstatement promotes lung repair will be understood for the first time. Outstandingly new understanding of the pathogenesis of ALI will be achieved.