Mitochondrial dynamics in acute lung injury

描述（由申请人提供）：意义。急性肺损伤（acute lung injury，ALI）是导致死亡和致残的主要原因。我们的目标是确定可能导致治愈的机制。我们将集中在肺泡线粒体机制，这是知之甚少。在ALI中，线粒体功能障碍可能是肺泡功能障碍的基础，导致肺损伤。这种功能障碍可能是由于线粒体Ca 2+缓冲功能丧失，其中Ca 2+通过线粒体Ca 2+单向转运体（MCU）从细胞质扩散到线粒体基质，阻止细胞质Ca 2+（cytCa 2+）的促炎性增加，从而保护免受损伤。更好地理解这些机制可能会导致MCU作为治疗LPS-ALI的线粒体Ca 2+缓冲恢复。提议我们提出了新的假设，线粒体Ca 2+（mitCa 2+）决定表面活性剂的分泌，因为线粒体Ca 2+的摄取增加ATP和H2 O2的生产。在LPS-ALI中，持续的cytCa 2+增加导致线粒体Ca 2+缓冲失败，导致Ca 2+依赖性磷酸酶钙调磷酸酶的激活，钙调磷酸酶反过来使线粒体裂变蛋白DRP 1去磷酸化并激活。由此产生的线粒体分裂导致MCU的废除。钙调神经磷酸酶还促进肌动蛋白解聚。这些作用共同诱导肺泡功能障碍，如表面活性剂分泌的损失所反映的。具体目标。具体目的是确定MCU在肺泡表面活性物质分泌中的作用（目的1），并确定MCU的丧失在LPS诱导的ALI中加重肺损伤的程度（目的2）。我们将建立MCU作为特定Ca 2+通道的作用，以及其在表面活性剂分泌中的作用。我们将评估LPS-ALI中线粒体Ca 2+缓冲丧失的分子机制，并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Ca 2+缓冲，作为肺修复的策略。Approach.我们将通过离体灌注小鼠肺的活体共聚焦和双光子显微镜以及离体肺泡2型（AT 2）细胞的研究来实现这些目标。我们的决定将包括：（1）完整肺泡和分离的AT 2细胞中的cytCa ~（2+）和mitCa ~（2+）;（2）通过测定Ca ~（2+）、ATP、H_2 O_2和F-actin fence通过单细胞成像调节表面活性物质的分泌;（3）通过肺和AT 2细胞表达特异性突变体和siRNA来调节内毒素ALI中的MCU和Ca ~（2+）缓冲;（4）MCU突变体在BMSC中的表达以测试MCU恢复恢复线粒体Ca 2+缓冲的假设;（5）通过线粒体转移的MCU过表达作为保护免受LPS-ALI的手段。初步数据。我们发现（1）MCU调节细胞内Ca 2+和mitCa 2+以及表面活性剂的分泌，（2）内毒素ALI通过激活DRP 1下调MCU阻断线粒体Ca 2+缓冲，以及（3）虽然突变MCU的肺泡转染降低了内毒素ALI中小鼠的存活率，但填充长度MCU的表达增加了小鼠的存活率。这些以及其他关于我们测量策略的初步数据支持项目的可行性。冲击这个项目将提供第一个系统的了解的作用，微控制器作为一个决定因素的肺泡功能。首次了解MCU丢失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。对ALI的发病机制将有全新的认识。

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.