Abstract P25: Mitochondrial Opa1 Dissociation Mediates Cristae Remodeling and Cytochrome C Release Following Global Brain Ischemia
Background: Global brain ischemia due to cardiac arrest followed by resuscitation results in delayed death of neurons in susceptible regions of the brain, the most vulnerable being the CA1 hippocampus. A key event leading to neuronal death is release of cytochrome c (cyt c) from intermembranous space of the mitochondria into the cytosol. Current data suggests that cyt c is released by permeablization of the outer mitochondrial membrane. However:
cyt c is predominately localized in the cristae folds within the intermembranous space and;
the cristae junctions are gated by oligomers of Opa1, which may prevent cyt c from crossing the outer membrane.
Hypothesis: We propose that full cyt c release requires disruption of Opa1 oligomers and alteration of cristae junctions. Specifically, we hypothesize that reperfusion results in Opa1 dissociation resulting in altered cristae morphology and release of Opa1 and cyt c into the cytosol of CA1 neurons.
Methods: We tested these hypotheses in a rat 2-vessel occlusion-hypotension model of cerebral ischemia. Opa1 and cyt c release were assayed by both Western blotting and immunofluorescence. Opa1 oligomer dissociation was interrogated with Blue Native-PAGE, and cristae morphology and volume were assayed by TEM tomography.
Results: Sham-operated rats had negligible immunoreactivity of Opa1 and cyt c in cytosolic fractions and punctate Opa1 and cyt c staining in CA1 neurons, consistent with mitochondrial localization. In contrast, ischemia followed by 4, 6, or 24 hrs of reperfusion resulted in:
increases of cytosolic Opa1 (~5 fold at 6 hr) and cyt c (~3 fold at 6 hr) and diffuse immunofluorescence, consistent with Opa1 and cyt c release; and
loss of Opa1 oligomers in mitochondrial fractions (p<0.05 vs Shams).
Moreover, Opa1 release and oligomer dissociation correlated with alterations in mitochondrial cristae morphology and a reduction of total cristae volume. These molecular alterations were limited to vulnerable CA1 neurons; no changes were seen regions of the hippocampus resistant to ischemic damage (Dentate Gyrus).
Conclusion: These data provide novel evidence that Opa1 oligomer dissociation may alter mitochondrial cristae morphology, allowing cyt c and Opa1 release during brain reperfusion.