Mitochondrial calcification in juvenile dermatomyositis

项目摘要 线粒体是参与代谢、炎症和细胞死亡的细胞内细胞器。线粒体 在调节细胞内钙离子水平、清除胞浆钙离子和无机钙离子方面也起重要作用 磷酸盐，将其以晶体形式（羟基磷灰石）捕获在线粒体内，以拯救细胞， 胞浆Ca~（2+）超载。在成人的病例报告中观察到线粒体内钙化， 皮肌炎（DM）以及皮肤钙质沉着症（一种使人衰弱的表现）的实验模型 青少年DM（JDM）然而，线粒体内的潜在机制和后果， 尚未对钙化进行研究。虽然线粒体主要存在于细胞内， 在ROS介导的损伤后，参与炎症的诱导。本申请的前提是， JDM患者存在线粒体钙化，促进线粒体挤出，钙结晶积聚 （钙质沉着症）和炎症。为了研究这个假设，我们有两个具体的目的。第一个目标看起来 在体外研究线粒体钙化和挤压的机制。我们假设 线粒体ROS的产生将支持线粒体钙化和挤压，有助于 炎症和钙质沉着。在实验中，原代人骨骼肌细胞将在钙 在存在应激剂（包括缺氧、TLR激动剂或JDM血清）和线粒体 使用流式细胞术、qPCR、免疫荧光显微术和代谢组学评估功能。质量 基于光谱的磷酸化蛋白质组学将用于确定哪些途径参与线粒体 钙化包括线粒体ROS在内的潜在靶点将通过使用小分子来阻断，例如 mitoTEMPO。第二个目标是研究挤出的线粒体被清除的机制。我们 假设钙化的线粒体被吞噬，但不降解，留在胞质中， 区室触发TLR9和炎性体活化。简而言之，线粒体将与 原代人单核细胞和中性粒细胞，并评估摄取、细胞内定位、信号传导 途径（磷酸蛋白质组学）和细胞因子诱导。参与诱导线粒体- 介导的炎症，例如DNA传感器TLR9和cGAS，以及炎性小体激活，将被 使用化学抑制剂进行靶向。来自JDM儿童和健康个体的细胞支持 将在体外比较线粒体的清除。最后，将分析JDM免疫细胞中是否存在 使用流式细胞术和共聚焦显微镜在体内观察含羟基磷灰石的线粒体。我们认为 我们的建议具有高度的创新性和重要性，因为：i）我们将确定所涉及的基本机制， 线粒体钙化; ii）我们将首次探索挤压的线粒体是否以及如何有助于 炎症和钙质沉着。这些发现也可能适用于其他条件，其中 钙化过程突出，包括动脉粥样硬化。

Project Summary Mitochondria are intracellular organelles involved in metabolism, inflammation and cell death. Mitochondria also play an important role in regulating intracellular calcium levels, scavenging cytosolic calcium and inorganic phosphate, trapping it in its crystalline form (hydroxyapatite) within the mitochondria to rescue the cell from cytosolic Ca2+ overload. Intramitochondrial calcification has been observed in case reports of adult dermatomyositis (DM) as well as in experimental models of cutaneous calcinosis, a debilitating manifestation of juvenile DM (JDM). However, the underlying mechanisms and consequences of intramitochondrial calcification have not been investigated. Though mainly found intracellularly, mitochondria can be extruded upon ROS-mediated damage, partaking in induction of inflammation. The premise of this application is that JDM patients have mitochondrial calcification promoting mitochondrial extrusion, calcium crystal accumulation (calcinosis), and inflammation. To investigate this hypothesis we have two specific aims. The first aim will look into mechanisms contributing to mitochondrial calcification and extrusion in vitro. We hypothesize that mitochondrial ROS generation will support mitochondrial calcification and extrusion, contributing to inflammation and calcinosis. Experimentally, primary human skeletal muscle cells will be incubated in calcium rich medium in presence of stress agents, including hypoxia, TLR agonists, or JDM sera, and mitochondrial function assessed using flow cytometry, qPCR, immunofluorescence microscopy and metabolomics. Mass spectrometry-based phosphoproteomics will be used to establish which pathways are involved in mitochondrial calcification. Potential targets, including mitochondrial ROS, will be blocked by using small molecules, e.g. mitoTEMPO. The second aim will investigate mechanisms by which extruded mitochondria are cleared. We hypothesize that calcified mitochondria are phagocytosed, but not degraded, remaining in the cytosolic compartment triggering TLR9 and inflammasome activation. In brief, mitochondria will be incubated with primary human monocytes and neutrophils and assessed for uptake, intracellular localization, signaling pathways (phosphoproteomics) and cytokine induction. Key pathways involved in induction of mitochondrial- mediated inflammation, e.g. DNA sensors TLR9 and cGAS, as well as inflammasome activation, will be targeted using chemical inhibitors. The capacity of cells from JDM children and healthy individuals to support clearance of mitochondria will be compared in vitro. Finally, JDM immune cells will be analyzed for presence of hydroxyapatite-containing mitochondria in vivo using flow cytometry and confocal microscopy. We believe that our proposal is highly innovative and significant as i) we will define underlying mechanisms involved in mitochondrial calcification; and ii) we will for the first time explore if, and how, extruded mitochondria contribute to inflammation and calcinosis in JDM. These findings may also be applicable to other conditions in which the calcification process is prominent, including atherosclerosis.