Essential Role for SPG7 in Mitochondrial Permeability Transition Pore Assembly and Function

SPG7 在线粒体渗透性转变孔组装和功能中的重要作用

基本信息

批准号：
10241316
负责人：
MADESH MUNISWAMY
金额：
$ 30.72万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10241316
关键词：
Ablation Accidents Affect Area Attenuated Blood Vessels Body Regions Brain CRISPR/Cas technology Cardiac Myocytes Cell Death Cell Proliferation Cell physiology Cessation of life Cicatrix Clinical Complex Cyclophilin A Cysteine Dependence Disease Drops Electrons Event Fibroblasts Functional disorder Future Gene Targeting Genes Genetic Goals Heart Heart Diseases Homeostasis Hypoxia ID2 gene In Vitro Injury Ischemia Isomerase Knock-in Knock-out Link Maintenance Mediating Membrane Membrane Potentials Methodology Mitochondria Mitochondrial Swelling Modeling Molecular Mutant Strains Mice Myocardial Myocardial Infarction Necrosis Neurons Outcome Outer Mitochondrial Membrane Oxidation-Reduction Oxidative Stress Oxygen Pathogenicity Pathologic Pattern Permeability Physiological Positioning Attribute Process Production Proteins Protons Publishing RNA Interference RNA interference screen Reperfusion Injury Reperfusion Therapy Role Signal Transduction Spastic Paraplegia Specificity Stimulus Stress Stroke Tissues Translating Variant base cell regeneration cell type conditional knockout conditional mutant cyclophilin D defined contribution effective therapy gain of function in vivo mitochondrial dysfunction mitochondrial membrane mitochondrial permeability transition pore mouse model mutant nervous system disorder new therapeutic target organ injury prevent protein expression regenerative solute therapeutic development therapeutic target treatment strategy voltage

项目摘要

One of the major organ injuries associated with myocardial infarction and stroke is ischemia/reperfusion injury. I/R injury manifests from the stress-induced opening of the mitochondrial permeability transition pore (PTP), a lethal form of mitochondrial malfunction leading to necrosis. The resultant myocardial or neuronal necrosis is linked to dysfunction in mitochondrial Ca2+ handling and oxidative stress. Although the concept of PTP opening has been examined for several decades, the molecular components of the PTP have been unknown until now with the exception of a positive regulator cyclophilin D (CypD). Under physiological conditions, the PTP may function through transient pore opening to release accumulated toxic mitochondrial metabolites. In pathological states, particularly those involving hypoxia, Ca2+ and ROS accumulate prompting the PTP to open, resulting in mitochondrial swelling. Because pore opening disrupts the flow of electrons and protons across the mitochondrial membranes necessary for energy production, PTP activity results in a catastrophic drop in cellular energy levels. Using a RNA interference (RNAi)-based screen to identify genes that modulate Ca2+ and ROS-induced opening of the PTP, we identified a necessary and conserved role for spastic paraplegia 7 (SPG7) as a component of CypD-dependent PTP opening in multiple cell types. Our recently published discovery of this long-sought molecule, SPG7, places us in a unique position to define SPG7-induced necrotic initiation mechanisms. This proposal aims to delineate the mechanisms by which SPG7 constitutes PTP assembly and opening at the mitochondrial level and characterize the relationship between mitochondrial Ca2+ and ROS homeostasis with PTP induction under physiological and pathophysiological conditions such as hypoxia/reoxygenation (H/R) damage. Since SPG7 is essential for the PTP complex formation in multiple cell types, this proposal will utilize in vivo genetically targeted conditional knockout (SPG7cKO), and knock-in mutant mice (SPG7*ID2 KI) using CRISPR/Cas9 mediated gene targeting for the study of mitochondrial Ca2+/ROS-dependent PTP signaling networks involved in mitochondrial dysfunction. These models will allow us to translate our in vitro H/R results to an in vivo murine model of I/R injury. We hypothesize that necrosis will be attenuated in SPG7 knockout and knock-in dysfunctional PTP point mutant SPG7 (SPG7*ID2) models. Accomplishment of these goals with our newly developed mouse models will authentically demonstrate the role of SPG7 in CypD-dependent PTP assembly and opening. Our proposed studies will characterize the role of SPG7 in mitochondria-dependent necrotic cell death and provide new therapeutic targets for the treatment of conditions associated with I/R damage.

与心肌梗塞和中风相关的主要器官损伤之一是缺血/再灌注损伤。 I/R 损伤表现为应激诱导的线粒体通透性转换孔 (PTP) 打开，这是一种致命形式的线粒体功能障碍导致坏死。由此产生的心肌或神经元坏死是与线粒体 Ca2+ 处理和氧化应激功能障碍有关。虽然PTP开放的概念经过数十年的研究，PTP 的分子成分至今仍未知正调节亲环蛋白 D (CypD) 除外。在生理条件下，PTP可能通过短暂的孔开放来释放积累的有毒线粒体代谢物。病理状态下状态，特别是那些涉及缺氧、Ca2+和ROS积累的状态，促使PTP打开，导致线粒体肿胀。因为孔的开放破坏了电子和质子穿过线粒体的流动细胞膜是产生能量所必需的，PTP 活性会导致细胞能量水平灾难性下降。使用基于 RNA 干扰 (RNAi) 的筛选来识别调节 Ca2+ 和 ROS 诱导的开放的基因在 PTP 的基础上，我们确定了痉挛性截瘫 7 (SPG7) 的一个必要且保守的作用，作为 CypD 依赖性 PTP 在多种细胞类型中开放。我们最近发表了这个长期寻找的发现 SPG7 分子使我们处于一个独特的位置来定义 SPG7 诱导的坏死起始机制。这该提案旨在描述 SPG7 构成 PTP 大会和在线粒体水平并表征线粒体 Ca2+ 和 ROS 稳态之间的关系生理和病理生理条件下的 PTP 诱导，例如缺氧/复氧 (H/R) 损害。由于 SPG7 对于多种细胞类型中 PTP 复合物的形成至关重要，因此该提案将利用体内基因靶向条件敲除 (SPG7cKO) 和敲入突变小鼠 (SPG7*ID2 KI) 使用 CRISPR/Cas9 介导的基因靶向研究线粒体 Ca2+/ROS 依赖性 PTP 信号网络参与线粒体功能障碍。这些模型将使我们能够将我们的体内 I/R 损伤小鼠模型的体外 H/R 结果。我们假设 SPG7 中的坏死会减弱敲除和敲入功能失调的 PTP 点突变 SPG7 (SPG7*ID2) 模型。这些目标的实现我们新开发的小鼠模型将真实地证明 SPG7 在 CypD 依赖性中的作用 PTP 组装和打开。我们提出的研究将描述 SPG7 在线粒体依赖性中的作用坏死细胞死亡并为 I/R 相关疾病的治疗提供新的治疗靶点损害。