Abstract 161: Molecular Mechanisms of Cytochrome c Release following Ischemia-Reperfusion: Role of Drp1 Translocation and Increased Mitochondrial Fission
Global brain ischemia/reperfusion results in death of vulnerable neurons. Although it is well-recognized that mitochondrial release of cytochrome c (cyto c) is a hallmark of neuronal death, the molecular events underlying this ‘spill’ of cyto c into the cytosol are largely unknown. Our aim was to test the hypothesis that cyto c spill following ischemia/reperfusion is associated with: (i) translocation of the mitochondrial fission protein Drp1 from the cytosol to the mitochondria; and (ii) a subsequent increase in mitochondrial fission. To investigate this concept, cultured HT22 neurons underwent no intervention (normoxic controls) or 3 h simulated ischemia (achieved by oxygen-glucose deprivation) and 3–48 h of reoxygenation. Expression of cyto c and Drp1 in the cytosol versus mitochondria was quantified by immunoblotting, and, in parallel experiments, cellular distribution of mitochondria was visualized by confocal imaging of neurons labeled with MitoTracker® and DAPI (staining mitochondria [red] and nuclei [blue], respectively). In control cells, expression of cyto c was confined to the mitochondria (only 3+2% of total cyto c seen in the cytosol), while Drp1 was robustly expressed in the cytosol versus ∼absent in mitochondria. Moreover, mitochondrial were dispersed throughout the neurons (figure: left). As expected, ischemic-reperfused cells displayed progressive spill of cyto c with increasing durations of reoxygenation (23+4% of total cyto c present in cytosol with 48 h reflow). This was accompanied by: (i) progressive translocation of Drp1 (i.e., localization in mitochondria versus negligible expression in the cytosol at 48 h post-reoxygenation); together with (ii) punctuate mitochondrial staining (indicative of mitochondrial fission) confined to the peri-nuclear region (figure: right). Thus, we show novel evidence of an association between release of cyto c from mitochondria, translocation of Drp1 and increased mitochondrial fission.
- © 2010 by American Heart Association, Inc.