![]() ![]() Indeed, past studies underscore a complex and seemingly disparate relationship between mitochondrial dynamics and mitophagy, i.e., inhibiting fission during ischemia may attenuate cell death 8, 13, 18, 19, or, conversely, may worsen outcomes by compromising the clearance of damaged mitochondria that accumulate during reperfusion. Second, there is no agreement on whether the upregulation of mitophagy observed in the setting of I/R is beneficial or detrimental to neuronal viability 12, 13, 14, 15, 16, 17. First, it is unknown whether fission is necessary for the upregulation of mitophagy and subsequent disposal of damaged mitochondria following I/R injury. However, despite the importance of these insights, two major gaps in knowledge remain. Moreover, these stressors, and the accompanying increase in mitochondrial fission, are also reportedly associated with an upregulation in mitophagy 10, 11. Multiple stressors (including cerebral I/R) have been demonstrated to initiate a transition to a fragmented mitochondrial phenotype, and there is a consensus that stress-induced activation of mitochondrial fission is directly linked to cell death 5, 6, 7, 8, 9. Therefore, it is critical to have stringent quality control mechanisms in place to sequester and dispose of damaged mitochondria. Mitochondrial damage and subsequent dysfunction have been well characterized as precursors to cell death following I/R 1, 2, 3, 4. Mitochondrial quality control (MitoQC), consisting of mitochondrial dynamics and mitophagy, has been a major area of focus in the context of many disease conditions including cerebral ischemia/reperfusion (I/R) injury. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems however, the role of this relationship in the context of I/R injury remains unclear. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Mitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. ![]()
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