Background
Although definitive mechanisms underlying ischemic tolerance remain incompletely understood, activation of endogenous cellular defense machinery has been proposed to contribute to the acquisition of ischemic tolerance. Molecular chaperones and the ubiquitin-proteasomal system defend virtually all cell types from diverse pathological conditions by preventing proteotoxicity from newly synthesized polypeptides and denatured proteins, and by eliminating irreparably damaged proteins. Despite the fact that neuronal ATP production is gradually recovered during reperfusion, inhibition of the overall rate of protein translation continues. Irreversible inhibition of protein biosynthesis after ischemia is a most accurate indicator for delayed neuronal death, and is most probably caused by translational complex aggregation-mediated destruction of molecular chaperone-mediated translational folding and folding-coupled degradation. Despite such close ties between preconditioning and translational complex aggregation, it has not been studied whether ischemic preconditioning is able to prevent translational complex aggregation, thereby protecting neurons from ischemia.
Methods
Ischemic preconditioning was introduced by 3 min of sublethal ischemia followed by 48 h of recovery. Brains with either ischemic preconditioning or sham-surgery were subjected to a second 7 min of ischemia followed by 30 min, 4, 24, 48 and 72 h of reperfusion. Translational complex aggregation and neuronal death were studied by electron and confocal microscopy, as well as by biochemical analyses.
Results
The EPTA-ribosome-selective EM staining in brain sections clearly show that ribosomes are clumped into large aggregates only in the cytoplasm of neurons destined to undergo delayed neuronal death after brain ischemia. The translational complex components consisting of ribosomal small subunit protein S6, large subunit protein L28, co-translational chaperones HSC70 and HSP40, and co-translational ubiquitin ligase CHIP, are all highly deposited into a detergent/salt-insoluble protein aggregate-containing fraction in vulnerable neurons after ischemia. Immunofluorescence of ubi-proteins becomes highly aggregated only in neurons destined to undergo delayed neuronal death after ischemia. Ischemic preconditioning abolishes ribosomal aggregation after lethal ischemia under EM and ubi-protein aggregation under confocal microscopy, and reverses deposition of translational complex components into the detergent/salt-insoluble protein aggregate-containing fraction in vulnerable neurons after ischemia.
Discussion
Translational complex aggregation in ischemic vulnerable neurons may account for irreversible inhibition of protein biosynthesis which is a most accurate indicator for delayed neuronal death after ischemia. Ischemic preconditioning activates endogenous molecular chaperones and the ubiquitin-proteasomal defense systems to counter ischemia-mediated disabilities of molecular chaperone-mediated translational folding and folding-coupled degradation, thereby preventing translational complex aggregation after ischemia.
Footnotes
Acknowledgements
Supported by: NS040407