Molecular Analysis of Endoplasmic Reticulum Stress Response After Global Forebrain Ischemia/Reperfusion in Rats: Effect of Neuroprotectant Simvastatin

Abstract

Simvastatin is a cholesterol-lowering agent whose functional significance and neuroprotective mechanism in ischemic brain injury is not yet solved. The purpose of this study is to evaluate the effect of simvastatin on ischemic brain injury. We examined the endoplasmic reticulum stress response (UPR/unfolded protein response), by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The results from the group of naïve ischemic rats were compared with results from the group of pre-treated animals with simvastatin. The results of the experiments showed significant increase in all genes at the mRNA level in ischemic phase (about 43% for XBP1, 58% for GRP78, and 39% for ATF6 more than control). The protein level of XBP1 was decreased in pre-treated animals at ischemic phase and first hour of reperfusion (about 15% less), and did not reach control levels. The protein levels of GRP78 were maximal at third hour of reperfusion in statin group with a small decrease at 24 h of reperfusion in both groups. The levels of ATF6 mRNA in statin-treated animals was higher in comparison to non-statin animals at the ischemic phase and the third hour of reperfusion (about 35% higher), which was also translated into the higher protein level. This could indicate that one of the main proteins targeted to enhance neuroprotective effect to ER during the first two hours of reperfusion was ATF6 protein, the levels of which were 60% higher than in non-treated animals. These data suggest that simvastatin, in addition to the proposed neuroprotective effect, exerts a neuroprotective role in the attenuation of ER stress response after acute ischemic/reperfusion insult.

Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans

“…In particular, given that activation of small GTPases such as RAS contributes to about 20% of human cancers, the ability of statins to inhibit GTPases holds the potential for their use as anti-cancer therapeutics (1,32). Another example is the in vivo use of statins to induce the UPR in a rat model where it had neuroprotective effects under ischemic conditions (33). Indeed, induction of the UPR is likely to be protective in several disease contexts, including Parkinson disease (34) and diabetes (35), where misfolded proteins or increased protein production impose a burden on the ER.…”

Role of the ubiquitin–proteasome system in brain ischemia: Friend or foe?

“…These proteins translocate to the nucleus, where they induce the expression of genes coding for molecular chaperones and folding enzymes, including GRP78 itself, HERP (homocysteine-inducible, ER-stress inducible protein), GRP94, calnexin and PDI (protein disulphide isomerase) (Kokame et al, 2000;Kokame et al, 2001). An increase in the expression of the grp94, grp78, herp, calnexin and pdi genes was observed after transient ischemia using different models (Aoki et al, 2001;Paschen et al, 2003;Truettner et al, 2009;Urban et al, 2009), and this effect was correlated with a small upregulation of GRP78 and GRP94 protein levels in the hippocampus of rats subjected to the two-vessel occlusion brain ischemia model (Truettner et al, 2009). A significant upregulation of GRP78 protein levels was also observed in the ischemic territory after transient MCAO in mice (Shibata et al, 2003).…”

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance

“…On the other hand 6-10-minutes long global ischemia and three days of reperfusion caused death of almost all pyramidal neurons in the same hippocampal area (Coimbra and Wieloch, 1994). Urban et al, (2009) showed a significant increase of XBP1 mRNA level in ischemic phase in comparison to control (about 43% more). These findings are similar to those observed by Paschen (2003a), which showed a marked increase in processed XBP1 mRNA levels using semi-quantitative RT-PCR after focal ischemia.…”

“…As shown by previous studies, the changes of the UPR gene expression induced by transient ischemia occur mostly during the first 24 h (Paschen 2003b) or the first few days after the insult (Qi et al, 2004). In line with this, Urban et al (2009) have decided to measure changes in mRNA and protein levels of GRP78, ATF6, and XBP1 after 15 min of global ischemia and 1, 3, and 24 h reperfusion (UPR reaction). In addition, they have focused their attention on the effect of simvastatin pretreatment on the stress reaction of endoplasmic reticulum induced by ischemic/reperfusion insult.…”

“…The changes in mRNA levels were not projected onto protein levels, which, in contrast to control I/R animals, was found to be significantly lower (about 37.4 ± 2.2% in ischemic phase and about 36.3 ± 5.7% lower in first hour of reperfusion). In this paper, Urban et al (2009) were interested in finding whether global ischemia induced by four vessel occlusion followed by reperfusion at different time points would initiate the unfolded protein response of ER in cortical neurons. In addition, they have proved that prophylactic simvastatin therapy affects expression of gene coding for the main proteins involved in UPR.…”