Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.

Focal brain ischemia leads to a slow type of neuronal death in the penumbra that starts several hours after ischemia and continues to mature for days. During this maturation period, blood flow, cellular ATP and ionic homeostasis are gradually recovered in the penumbral region. In striking contrast, protein synthesis is irreversibly inhibited. This study used a rat focal brain ischemia model to investigate whether or not irreversible translational inhibition is due to abnormal aggregation of translational complex components, i.e. the ribosomes and their associated nascent polypeptides, protein synthesis initiation factors and co-translational chaperones. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons, but clumped into large abnormal aggregates in penumbral neurons subjected to 2 h of focal ischemia followed by 4 h of reperfusion. The abnormal ribosomal protein aggregation lasted until the onset of delayed neuronal death at 24-48 h of reperfusion after ischemia. Biochemical study further suggested that translational complex components, including small ribosomal subunit protein 6 (S6), large subunit protein 28 (L28), eukaryotic initiation factors 2a, 4E and 3g, and co-translational chaperone heat-shock cognate protein 70 (HSC70) and co-chaperone Hdj1, were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational complex components were also highly ubiquitinated. This study clearly demonstrates that focal ischemia leads to irreversible aggregation of protein synthesis machinery that contributes to neuronal death after focal brain ischemia. Keywords: brain ischemia, chaperone, heat-shock cognate protein 70, protein aggregation, protein synthesis, ribosomal proteins, stress granule, ubiquitin. Although substantial progress has been made, cellular and molecular mechanisms underlying focal ischemic neuronal damage are still incompletely understood. Focal brain ischemia leads to acute cell death in the core area and a slow type of neuronal death in the penumbra that can begin hours after ischemia and continue to mature for days (Siesjo et al. 1995;Lee et al. 2002;Wang et al. 2004). During this maturation period, protein synthesis is persistently inhibited in these penumbral neurons destined to die (Mies et al. 1991;Hossmann 1993;Kokubo et al. 2003). Activation of PKRlike ER eIF-2a kinase (PERK) may be responsible, at least in part, for the increase in eukaryotic initiation factor 2a (eIF2a) phosphorylation and for the transient suppression of protein synthesis early in reperfusion after transient brain ischemia. However, the nature of the transient change in phosphorylation of PERK and eIF2a may suggest that eIF2a phosphorylation cannot account for irreversible inhibition of protein synthesis after brain ischemia (Hu and Wieloch 1993;Burda et al. 1994;Althausen et al. 2001;DeGracia 2004;Owen et al. 2005).Cellular proteins in non-native states, i.e. those newly synthesized, misfo...

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