Cytoplasmic,mitochondrial or ER stress response after transient cerebral ischemia

Background

Cellular proteins in non-native states, i. e., newly synthesized, misfolded, denatured, or damaged, expose their sticky hydrophobic surfaces, and are highly prone to toxic aggregation. There are several cellular defense systems to cope toxic aggregation of non-native proteins in normal cells. Molecular chaperones can shield hydrophobic surfaces, thereby blocking their toxic aggregation. Irreparably damaged proteins must be quickly eliminated from cells, mostly by the ubiquitin-proteasomal system. Brain ischemia disables ATP-dependent molecular chaperone-assisted folding and ubiquitin-proteasome-mediated degradation, resulting in accumulation of non-native proteins during the postischemic phase. Non-native proteins activate cell defense systems, i.e., cellular stress responses, to induce expression of molecular chaperones, folding enzymes and components of the ubiquitin-proteasomal system during the postischemic phase. There are different signal pathways regulating expression of molecular chaperones residing in the cytoplasm, ER or mitochondria, respectively. This study investigated induction of major chaperones of different cellular compartments in order to figure out in which cellular compartments non-native proteins may be overproduced after brain ischemia.

Methods

Fifteen min of transient cerebral ischemia followed by 30 min, 4, 24 h of reperfusion in rats was utilized to investigate induction of major cytoplasmic, ER-luminal, and mitochondrial chaperones, respectively, by in situ hybridization, confocal microscopy and western blot analysis.

Results

Major ER luminal chaperones Herp, GRP78, GRP94, calnexin and PDI, major mitochondrial chaperone HSP60, and major cytoplasmic chaperone HSP70 were studied. The most dramatically induced chaperone was HSP70 in the cytoplasm. Mitochondrial chaperone HSP60 was relatively less induced, whereas the ER-luminal stress genes Herp, GRP78 and GRP94 were least induced after ischemia. In addition, induction of cytoplasmic HSP70 was started as early as 30 min of reperfusion, peaked at 4 h and declined thereafter in ischemic surviving neurons, but induction was continuously increased in ischemic vulnerable neurons until the onset of delayed neuronal death after ischemia. Induction of mitochondrial and ER-luminal chaperones was started late, at 4 h of reperfusion, and was seen only in ischemic vulnerable neurons after ischemia. Western blot analysis and confocal microscopy further confirmed that different signaling pathways regulate induction of chaperones in different neuronal compartments after ischemia.

Discussion

Induction of molecular chaperones is a common endogenous cellular defense response to stress conditions that lead to cellular overproduction of non-native proteins. Overproduced non-native proteins in the cytoplasm activate cytoplasmic heat-shock transcription factors, thus promoting expression of cytoplasmic stress genes such as HSP70. Whereas overproduced non-native proteins in the ER lumen trigger the ER-stress response via activation of ER membrane-associated transcription factors ATF-6 and Ire-1, thus inducing expression of ER stress genes Herp, GRP78, GRP94, calnexin and PDI. Therefore, activation of either cytoplasmic stress gene or the ER stress gene signaling pathway reflects in which cellular compartments non-native proteins are overproduced. This study suggests that non-native proteins are predominantly overproduced in the cytoplasm, and to a much lesser degree, in the mitochondria and the ER lumen after ischemia. These results support the notion that cytoplasmic non-native proteins may play an important role in delayed neuronal death after brain ischemia.