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

与心肌梗死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）开放， 导致坏死的线粒体功能障碍的致命形式。所导致的心肌或神经元坏死是 与线粒体Ca 2+处理和氧化应激功能障碍有关。虽然PTP开放的概念 虽然已经研究了几十年，但PTP的分子组成至今仍不清楚 除了正调节剂亲环素D（CypD）。在生理条件下，PTP可以 通过瞬时孔开放释放累积的毒性线粒体代谢物发挥作用。病理 状态，特别是那些涉及缺氧，Ca 2+和ROS积累，促使PTP开放，导致 线粒体肿胀因为毛孔的打开会扰乱电子和质子在线粒体中的流动 由于细胞膜是能量产生所必需的，PTP活性导致细胞能量水平的灾难性下降。 使用基于RNA干扰（RNAi）的筛选来鉴定调节Ca 2+和ROS诱导的开放的基因 在PTP中，我们确定了痉挛性截瘫7（SPG 7）作为PTP的一个组成部分的必要和保守的作用。 CypD依赖性PTP在多种细胞类型中开放。我们最近发表的发现， 分子SPG 7将我们置于一个独特的位置来定义SPG 7诱导的坏死起始机制。这 该提案旨在描述SPG 7构成PTP大会和开幕式的机制， 线粒体水平和线粒体Ca 2+和ROS稳态之间的关系， 在生理和病理生理条件如缺氧/复氧（H/R）下诱导PTP 损害由于SPG 7对于多种细胞类型中PTP复合物的形成是必需的，因此该提议将利用 体内遗传靶向条件性敲除（SPG 7 cKO）和敲入突变小鼠（SPG 7 * ID 2 KI） 使用CRISPR/Cas9介导的基因靶向研究线粒体Ca 2 +/ROS依赖性PTP 参与线粒体功能障碍的信号网络。这些模型将使我们能够将我们的 体外H/R结果与I/R损伤的体内鼠模型一致。我们假设SPG 7中坏死会减弱， 敲除和敲入功能失调的PTP点突变体SPG 7（SPG 7 * ID 2）模型。实现这些目标 我们新开发的小鼠模型将真实地证明SPG 7在CypD依赖性 PTP组装和打开。我们提出的研究将描述SPG 7在依赖于多巴胺的神经细胞中的作用。 坏死性细胞死亡，并为治疗与I/R相关的病症提供新的治疗靶点 损害

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.