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.
Ischemia-mediated cell death in stroke and heart diseases are devastating clinical conditions for which effective treatments are currently unavailable. We have uncovered new mitochondrial permeability transition pore (PTP) complex components that are promising therapeutic targets for the treatment of ischemic organ injury. The objective of this proposal is to investigate the molecular mechanisms that regulate mitochondrial PTP opening and necrotic cell death. Once the assembly and the function of PTP complex are identified, this information will be valuable for future development of therapeutic strategies for the treatment of cardiac and neurological disorders.
