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

• 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
• 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

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.

与心肌梗塞和中风有关的主要器官损伤之一是缺血/再灌注损伤。 I/R损伤从应力引起的线粒体通透性过渡孔（PTP）的开口表现出来 线粒体故障的致命形式导致坏死。由此产生的心肌或神经元坏死是 与线粒体Ca2+处理和氧化应激的功能障碍有关。虽然PTP开放的概念 已经检查了几十年，迄今为止，PTP的分子成分尚不清楚 除了阳性调节剂环蛋白D（CYPD）。在生理条件下，PTP可能 通过瞬态孔口开放的功能，释放累积的有毒线粒体代谢产物。在病理中 状态，特别是涉及缺氧的州，Ca2+和ROS积累，促使PTP开放，导致 线粒体肿胀。因为孔的打开会破坏电子和质子在线粒体上的流动 能源生产所需的膜，PTP活性导致细胞能级的灾难性下降。 使用基于RNA的干扰（RNAi）屏幕来识别调节Ca2+和ROS诱导开口的基因 在PTP中，我们确定了痉挛性截瘫7（SPG7）的必要和保守作用 CYPD依赖性PTP在多种细胞类型中开放。我们最近出版的有关这个长期追求的发现 分子SPG7使我们处于定义SPG7诱导的坏死起始机制的独特位置。这 提案旨在描述SPG7构成PTP组装和开放的机制 线粒体水平并表征线粒体Ca2+与ROS稳态与与ROS的关系 PTP在生理和病理生理条件下诱导，例如低氧/二氧化（H/R） 损害。由于SPG7对于多种单元类型的PTP复合物是必不可少的，因此该建议将使用 体内遗传靶向有条件敲除（SPG7CKO）和敲除突变小鼠（SPG7*ID2 KI） 使用CRISPR/CAS9介导的基因靶向线粒体Ca2+/ros依赖性PTP的研究 涉及线粒体功能障碍的信号网络。这些模型将使我们能够翻译我们 体外H/R导致I/R损伤的体内鼠模型。我们假设坏死将在SPG7中减弱 敲除和敲除功能失调的PTP点突变体SPG7（SPG7*ID2）模型。实现这些目标 借助我们新开发的鼠标模型将实际证明SPG7在CYPD依赖性中的作用 PTP组件和开放。我们提出的研究将表征SPG7在线粒体依赖性的作用 坏死细胞死亡，并为治疗与I/R相关的疾病提供新的治疗靶标 损害。