Abstract
Background: Global brain ischemia/reperfusion induces neuronal damage in vulnerable brain regions. A key event leading to neuronal death is mitochondrial cytochrome c (cyt c) release which we have demonstrated is accompanied by release of mitochondrial Opa1, a key mitochondrial fusion protein. We propose that cyt c release is dependent on disruption of Opa1, thus inadvertently promoting fission. Methods: Mitochondrial dynamics in vitro were analyzed with real-time imaging of neurons transfected with mito-localized GFP and MR-DEVD to detect caspase activation. Ischemia (OGD) was achieved with a media-gas exchange system and confocal microscopy. Mitochondrial dynamics in vivo was assessed with (i) 3D-electron microscopy to quantify mitochondrial volume, shape, and length and (ii) immunofluorescence for ATP synthase and Opa1. Results: Real-time imaging of cells during OGD (A, n=3/group) demonstrated that mitochondria transition from a tubular morphology to a highly fragmented morphology during ischemia. Tubular morphology was rapidly regained in the initial stages of reoxygenation, suggesting fusion remains intact following ischemia. However, mitochondria progressively undergo fragmentation culminating in cell death. Next, we utilized a rat model of cerebral ischemia to evaluate mitochondrial dynamics in vivo. Immunofluorescence (B, n=5/group) and 3D TEM (C, n=3/group) of control brains both exhibit mitochondria with long tubular morphology and Opa1 fluorescence in CA1 neurons was consistent with mitochondrial localization. In contrast, ischemia followed by 6 or 24 hrs of reperfusion resulted in extensive mitochondrial fragmentation (i.e. reduced mitochondrial length<0.05) and loss of Opa1/ATP synthase co-localization (Pearson’s coefficient = 0.63), consistent with Opa1 release and mitochondrial fragmentation. Conclusion: These data provide novel evidence that Opa1 may play a role in cell death during brain reperfusion.
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