Changes in ubiquitin and ubiquitin-protein conjugates in the CA1 neurons after transient sublethal ischemia

Abstract

ABSTRACTlibiquitin is involved in the degradation of denatured proteins in the recovery process after various stresses. To clarify the different responses of the ubiquitin system in the hippocampal neurons after ischemia, we chose 7.5 min of sublethal forebrain ischemia in the rat. After 7.5 min of ischemia, ubiquitin-like immunoreactivity (UIR) in most of the hippocampal pyramidal cells, except for the interneurons, diminished after 3 h of reperfusion, but enhanced UIR and subsequent recovery of UIR were observed in the different hippocampal regions after 24 h of reperfusion. The most prolonged recovery of UIR in the hippocampal cells was observed in the CAl neurons after 72 h of reperfusion. Immunoblot analysis of the proteins extracted from CAI region showed that high-mol-wt ubiquitin conjugates (HMWUC) above 40 kDa increased, whereas free ubiquitin and ubiquitinated histone 2A decreased slightly after 4 h and 24 h of reperfusion. At 72 h of reperfusion, HMWUC decreased to the original level and free ubiquitin slightly increased beyond the control level. These results suggested that (1) diminished UIR does not always mean depletion of entire ubiquitin-protein conjugates; (2) even after sublethal ischemia, damaged proteins in the CAI neurons may increase, and it may take a long time for elimination of these proteins.

Ubiquitin‐protein conjugates in different structures of the central nervous system of the rat

“…The major anatomical difference between both structures is the abundance of the different type of cells present in the CNS, neuronal bodies predominating in the cerebral cortex, while glial cells, axons, and myelin predominate in the brain stem. These results are in agreement with those of Hayashi et al (1991), who have recently described that after sublethal ischemia, the Ub responses are present in the CA1 and CA2 hippocampal neurons compared to the other regions of the brain. To definitely prove that the formation of UPC in the CNS is mainly present, under normal circumstances, in structures containing neuronal cell bodies, we investigated the formation of UPC in the optic nerve from adult rats.…”

Transient Ischemia Depletes Free Ubiquitin in the Gerbil Hippocampal CA1 Neurons

“…The other involves a multi-ubiquitin chain, which comprises a Lys-48-linked ubiquitin-ubiquitin conjugate and functions as a strong degradation signal.1' 2 Multi-ubiquitin chain-conjugated proteins are efficiently degraded by the 26S proteasome.34 Because DF2 and MAB1510 can recognize both mono-ubiquitinated and multi-ubiquitinated chain-conjugated proteins (unpublished observations), the present immunocytochemical results suggest that multi-ubiquitination is not significantly affected in the CA1, whereas mono-ubiquitination may be greatly affected after 48 hours of reperfusion, as shown with 5-25 immunostaining ( Figure 4). A slight decrease in the level of ubiquitinated histone H2A after ischemia was already reported by Hayashi et al 26 Overall, transient ischemia depletes free ubiquitin but not conjugated ubiquitin in the post-ischemic CAl. In other words, the response of CAl to ischemia is characterized by a profound reduction in the free ubiquitin level and an unaffected level of conjugated ubiquitin.…”

Characterization of the Ubiquitin-Modified Proteome Regulated by Transient Forebrain Ischemia

“…3 Aggregates of ubiquitylated proteins start to appear after ischemia during early reperfusion when cells are still morphologically intact. 5 Several groups have reported postischemic accumulation of ubiquitin-conjugated proteins in Triton X-100-insoluble aggregates, 2,3,[5][6][7][8][9][10] but the significance of this process for the fate and functions of postischemic neurons has not yet been uncovered. No attempts have been made to perform proteomics analysis to characterize the ubiquitin-modified proteome in postischemic Triton X-100-insoluble aggregates.…”