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

与心肌梗死和卒中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)的开放，a 致命的线粒体功能障碍，导致坏死。由此导致的心肌或神经元坏死是 与线粒体钙处理功能障碍和氧化应激有关。虽然PTP开放的概念 PTP已经被研究了几十年，到目前为止，人们还不知道PTP的分子组成 除正向调节剂亲环素D(CypD)外。在生理条件下，PTP可以 通过瞬间开孔来释放累积的有毒线粒体代谢物。在病理上 状态，特别是那些涉及缺氧的状态，钙离子和ROS积累促使PTP打开，导致 线粒体肿胀。因为孔洞的打开扰乱了电子和质子在线粒体上的流动 作为能量生产所必需的细胞膜，PTP活性会导致细胞能量水平的灾难性下降。 使用基于RNA干扰(RNAi)的筛选来识别调节钙离子和ROS诱导的开放的基因 在PTP中，我们确定了痉挛截瘫7(SPG7)作为PTP的一个组成部分的必要和保守的作用 多种细胞类型中依赖CypD的PTP开放。我们最近公布的这一长期寻找的发现 SPG7分子使我们处于一个独特的位置来定义SPG7诱导的坏死启动机制。这 该提案旨在界定SPG7构成PTP组装和在 线粒体水平及其与ROS动态平衡关系的研究 低氧/复氧(H/R)等生理和病理生理条件下PTP的诱导 损坏。由于SPG7对于多种细胞类型的PTP复合体的形成是必不可少的，本提案将利用 体内遗传靶向条件性基因敲除(SPG7cKO)和敲入突变小鼠(SPG7*ID2Ki) 应用CRISPR/Cas9介导的基因打靶研究线粒体钙/ROS依赖的PTP 参与线粒体功能障碍的信号网络。这些型号将使我们能够将我们的 体外H/R导致体内小鼠I/R损伤模型。我们假设SPG7的坏死会减轻。 敲除和敲入功能障碍的PTP点突变SPG7(SPG7*ID2)模型。实现这些目标 我们新开发的小鼠模型将真实地证明SPG7在CypD依赖中的作用 PTP组装和打开。我们建议的研究将表征SPG7在线粒体依赖中的作用 坏死性细胞死亡，为I/R相关疾病的治疗提供新的治疗靶点 损坏。

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.