O-linked β-N-acetylglucosamine modification of proteins is activated in post-ischemic brains of young but not aged mice: Implications for impaired functional recovery from ischemic stress

Abstract

To evaluate the effect of age on the response of brains to an ischemic challenge, we subjected young and aged mice to transient forebrain ischemia, and analyzed the heat shock response and unfolded protein response, ubiquitin conjugation and SUMO conjugation, and O-linked β-N-acetylglucosamine modification of proteins (O-GlcNAcylation). The most prominent age-related difference was an inability of aged mice to activate O-GlcNAcylation. Considering many reports on the protective role of O-GlcNAcylation in various stress conditions including myocardial ischemia, this pathway could be a promising target for therapeutic intervention to improve functional recovery of aged patients following brain ischemia.

Keywords: Aging; O-GlcNAc modification; SUMO conjugation; brain ischemia; ubiquitin conjugation; unfolded protein response.

Figure 1.

Activation of stress response pathways in post-ischemic brains of young and aged mice. Two- and 22-month-old male mice were subjected to 10 minutes forebrain ischemia and 1 hour of reperfusion. Sham-operated mice served as controls. (a and b) Effects of age on ischemia-induced changes in levels of phosphorylated eIF2α, total eIF2α, GRP78, and PDI as measures of UPR induction. Ischemia-induced phosphorylation of eIF2α was significantly more pronounced in cortex of aged than in young mice (a). (c and d) Effects of age on ischemia-induced changes in total ubiquitin conjugation and levels of polyubiquitin with K48 and K63 linkage. In cortex and hippocampus of aged mice the post-ischemic increase in levels of ubiquitin-conjugated proteins was significantly suppressed, particularly polyubiquitin with K63 linkage. (e and f) Effects of age on ischemia-induced activation of SUMO1 and SUMO2/3 conjugation and changes in Ubc9 levels. In aged mice, the post-ischemic activation of SUMO1 and SUMO2/3 conjugation was markedly suppressed, both in the cortex (e) and hippocampus (f). The high-molecular-weight regions marked by brackets were used to quantify levels of conjugates. All individual data were normalized to β-actin. For calculation of fold change, mean values of sham groups were set to 1.0. Data are presented as means ± SD (n = 4 per group). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2.

Activation of O-GlcNAc modification in brains of young and aged mice. Two- and 22-month-old male mice were subjected to 10 minutes forebrain ischemia and 1 hour of reperfusion. Sham-operated mice served as controls. (a) Ischemia-induced changes in O-GlcNAc-modified proteins and levels of OGT and OGA in cortex of young and aged mice. (b) Ischemia-induced changes in O-GlcNAc-modified proteins and levels of OGT and OGA in hippocampus of young and aged mice. The high-molecular-weight regions marked by brackets were used to quantify levels of O-GlcNAc-modified proteins. All individual data were normalized to β-actin. For calculation of fold change, the mean values of sham groups were set to 1.0. Data are presented as means ± SD (n = 4 per group). *p < 0.05; **p < 0.01; ***p < 0.001.