Bcl-2 Transfection via Herpes Simplex Virus Blocks Apoptosis-Inducing Factor Translocation after Focal Ischemia in the Rat

Abstract

Apoptosis plays a critical role in many neurologic diseases, including stroke. Cytochrome c release and activation of various caspases are known to occur after focal and global ischemia. However, recent reports indicate that caspase-independent pathways may also be involved in ischemic damage. Apoptosis-inducing factor (AIF) is a novel flavoprotein that helps mediate caspase-independent apoptotic cell death. AIF translocates from mitochondria to nuclei where it induces caspase-independent DNA fragmentation. Bcl-2, a mitochondrial membrane protein, protects against apoptotic and necrotic death induced by different insults, including cerebral ischemia. In the present study, Western blots confirmed that AIF was normally confined to mitochondria but translocated to nuclei or cytosol 8, 24, and 48 hours after onset of ischemia. Overall, AIF protein levels also increased after stroke. Confocal microscopy further demonstrated that nuclear AIF translocation occurred in the peri-infarct region but not in the ischemic core where only some cytosolic AIF release was observed. Our data also suggest that AIF translocated into nuclei after cytochrome c was released into the cytosol. Bcl-2 transfection in the peri-infarct region blocked nuclear AIF translocation and improved cortical neuron survival.

Influence of duration of focal cerebral ischemia and neuronal nitric oxide synthase on translocation of apoptosis-inducing factor to the nucleus

“…An increase in AIF in the cytosolic fraction and a decrease in the mitochondrial fraction have been reported by 8 hours of permanent ischemia (Zhao et al, 2004). However, in the present study, AIF was undetectable in the cytosolic fraction and was unchanged in the mitochondrial fraction.…”

“…Progressive increases in nuclear AIF have also been reported over 48 hours of permanent distal MCAO in the rat (Zhao et al, 2004). A recent study in mice reported progressive increases in nuclear AIF over the first 24 hours of permanent MCAO, and the increase observed at 4 hours was attenuated by an antioxidant (Lee et al, 2005).…”

“…After permanent occlusion of the distal middle cerebral artery (MCA) in rats, significant increases in AIF in nuclear subcellular fractions occurred at 48 hours, although localized increases in cortical border regions were evident at 8 hours (Zhao et al, 2004). With 45 minutes of transient MCA occlusion (MCAO) in mice, significant increases in nuclear AIF in cortex occurred by 1 hour (Plesnila et al, 2004) and 2 hours (Culmsee et al, 2005).…”

“…This design permitted evaluation at early reperfusion, at a mid-range of reperfusion, when many neurons show ischemic morphologic changes, and at late reperfusion, when cell death is widespread throughout the maturing infarct. These timepoints spanned the 1-8 hour range for AIF translocation reported by others (Ferrer et al, 2003;Plesnila et al, 2004;Zhao et al, 2004;Culmsee et al, 2005). Two hemispheres were used per lane.…”

Hypothermia blocks β-catenin degradation after focal ischemia in rats

“…The ischemic penumbra was defined as the tissue saved by hypothermia 2 days post-stroke, and the corresponding region from the control ischemic brain was dissected for comparison. Whole cell protein was extracted from the fresh brain tissue, and Western blot was performed as described with modification [14,17,18]. In each lane 20 μg protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4-15% Ready Gel (BIO-RAD Laboratories, Cat# L050505A2) for 1.5 hours.…”