Translational complex aggregation after transient cerebral ischemia

Background

Transient cerebral ischemia followed by reperfusion causes delayed neuronal death selectively in ischemic vulnerable neurons after 48 h of reperfusion. Despite the fact that neuronal ATP production is gradually recovered in neurons destined to undergo delayed neuronal death after ischemia, inhibition of the overall rate of protein translation continues. Irreversible inhibition of protein biosynthesis is a most accurate indicator for delayed neuronal death after transient cerebral ischemia, but the underlying mechanism remains unknown. A brief eIF-2alpha phosphorylation is responsible, at least in part, for the transient depression of translation in ischemia-surviving neurons, but cannot account for irreversible translational inhibition in ischemia-vulnerable neurons. This study suggests that translational complex aggregation destroys protein synthesis machinery and contributes to delayed neuronal death after ischemia.

Methods

This study utilized a 15 or 20 min of transient cerebral ischemia followed by 30 min, 4 and 24 h of reperfusion in rats to investigate whether irreversible translational inhibition is due to aggregation of translational complex, i. e., the ribosomes and their associated newly synthesized polypeptides, protein biosynthesis initiation factors, translation-coupled folding-chaperones and degradation enzymes after ischemia. Translational complex aggregation and delayed neuronal death were studied by electron and confocal microscopy, as well as by biochemical analyses.

Results

Both conventional osmium-uranyl-lead and the EPTA-ribosome-selective EM staining methods 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, translational initiation factor eIF-3ç, and co-translational ubiquitin ligase CHIP, are all deposited into a detergent/salt-insoluble protein aggregate-containing fraction in vulnerable neurons after ischemia. Sedimentation analysis further confirms that these translational complex components are aggregated into higher densities of protein aggregate fractions in a sucrose density gradient. Immuno-fluorescence of ubi-proteins, CHIP, S6, L28, and HSC70 under confocal microscopy gradually disappears only in neurons destined to undergo delayed neuronal death after ischemia. These results clearly demonstrate that translational complex is clumped into large abnormal aggregates, resulting in destruction of protein synthesis machinery after brain ischemia.

Discussion

Translational complex aggregation in ischemic vulnerable neurons may represent a new mechanism underlying permanent inhibition of protein synthesis and delayed neuronal death after transient ischemia. An ischemia-induced cascade of energy failure and changes in intracellular homeostasis cumulatively disable ATP-dependent protein quality control machinery for co-translational folding and folding-coupled degradation after brain ischemia. As a result, nascent polypeptides on ribosomes are unable to fold. Consequently, unprocessed nascent polypeptides, together with their associated chaperones, initiation factors, degradation enzymes and ribosomes, are irreversibly aggregated. Translational complex aggregation-induced damage accumulates over time, and when it reaches a critical degree, may eventually lead to delayed cell death after ischemia.