Regulator of calcineurin 1 (Rcan1) has a protective role in brain ischemia/reperfusion injury

Abstract

Background: An increase in intracellular calcium concentration [Ca2+]i is one of the first events to take place after brain ischemia. A key [Ca2+]i-regulated signaling molecule is the phosphatase calcineurin (CN), which plays important roles in the modulation of inflammatory cascades. Here, we have analyzed the role of endogenous regulator of CN 1 (Rcan1) in response to experimental ischemic stroke induced by middle cerebral artery occlusion.

Methods: Animals were subjected to focal cerebral ischemia with reperfusion. To assess the role of Rcan1 after stroke, we measured infarct volume after 48 h of reperfusion in Rcan1 knockout (KO) and wild-type (WT) mice. In vitro studies were performed in astrocyte-enriched cortical primary cultures subjected to 3% oxygen (hypoxia) and glucose deprivation (HGD). Adenoviral vectors were used to analyze the effect of overexpression of Rcan1-4 protein. Protein expression was examined by immunohistochemistry and immunoblotting and expression of mRNA by quantitative real-time Reverse-Transcription Polymerase Chain Reaction (real time qRT-PCR).

Results: Brain ischemia/reperfusion (I/R) injury in vivo increased mRNA and protein expression of the calcium-inducible Rcan1 isoform (Rcan1-4). I/R-inducible expression of Rcan1 protein occurred mainly in astroglial cells, and in an in vitro model of ischemia, HGD treatment of primary murine astrocyte cultures induced Rcan1-4 mRNA and protein expression. Exogenous Rcan1-4 overexpression inhibited production of the inflammatory marker cyclo-oxygenase 2. Mice lacking Rcan1 had higher expression of inflammation associated genes, resulting in larger infarct volumes.

Conclusions: Our results support a protective role for Rcan1 during the inflammatory response to stroke, and underline the importance of the glial compartment in the inflammatory reaction that takes place after ischemia. Improved understanding of non-neuronal mechanisms in ischemic injury promises novel approaches to the treatment of acute ischemic stroke.

Figure 1

Regulator of calcineurin (Rcan)1-4 protein and mRNA expression increase at sites of transient focal brain ischemia, paralleling increases in inflammatory cytokine mRNA levels. (A), panel (i) Representative immunoblot showing expression of endogenous Rcan1-1 and Rcan1-4 proteins in infarcted cortical tissue (I) and the corresponding region of the contralateral hemisphere (C) of rats subjected to middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion for the indicated times. Sh = sham operated animals. A total of 100 μg protein was analyzed per lane. Panel (ii), band density quantification from four independent experiments as in (A), normalized to α-tubulin. (B) Histograms showing relative quantification of mRNA expression by TaqMan real time qRT-PCR. Rat mRNAs for Rcan1-4 (i), TNF α (ii), IL-6 (iii) and Cox-2 (iv) were amplified from total RNA from infarcted and corresponding contralateral hemispheres of rats subjected to MCAO (1 h) followed by reperfusion (I/R) as indicated. Transcript amounts are normalized to 18S rRNA, and are expressed relative to contralateral samples from sham-operated animals after reperfusion for 5 h (Sh). Real time qPCR reactionwas conducted in triplicate for each condition, and data are the means ± SD of four experiments. **P < 0.01, *P < 0.05 (analysis of variance (ANOVA)) versus contralateral Sh samples.

Figure 2

Regulator of calcineurin 1 (Rcan1) expression colocalizes with glial fibrillary acidic protein (GFAP)-positive cells during transient focal brain ischemia. Coronal brain sections of animals subjected to 1 h middle cerebral artery occlusion (MCAO) followed by 24 h blood reperfusion. (A) Nissl staining revealing a hypochromatic area of ischemic tissue in the infarcted (I) neocortex. (B) Immunohistochemistry showing expression of GFAP protein (red) in the contralateral hemisphere (a) and the infarcted region (b). Nuclei are revealed by staining adjacent sections with TO-PRO 3 (blue; c and d). (C) Immunohistochemistry showing the GFAP-positive area around the infarcted brain region. The boxed region is magnified in the lower panels: (a) GFAP immunostaining (red); (b) TO-PRO 3 nuclear staining (blue); (c) Rcan1 immunostaining (green); (d) overlay of (a-c). Yellow indicates GFAP-Rcan1 colocalization.

Figure 3

Hypoxia (3%) and glucose deprivation induces regulator of calcineurin (Rcan)1-4 protein and mRNA expression in primary cortical murine astrocytes. (A, B) Immunoblots showing endogenous Rcan1-4, nuclear factor of activated T cells (NFAT)c1 and NFATc3 protein expression, with α-tubulin and PSF (PTB-associated splicing factor) expression detected as loading controls. (A) Rat primary cortical astrocytes were non-stimulated (ns), treated with phorbol ester (phorbol 12-myristate 13-acetate) plus A23187 calcium ionophore (PIo) (4 h) as a positive control, or subjected to combined hypoxia (3% O2) and glucose deprivation (HGD) for 1 to 6 h. (B) Mouse primary astrocyte cultures were non-stimulated (ns) or subjected to HGD for 30 minutes to 4 h. (C) Rcan 1-4 mRNA was quantified by TaqMan real time qRT-PCR on total RNA from primary cortical mouse astrocytes stimulated as in (B). Transcript amounts are normalized to 18S rRNA and TATA-binding protein (TBP) endogenous controls, and are expressed relative to the level in non-stimulated control cells (ns). Data are the means ± SD of triplicate real time qRT-PCR determinations for each condition; n = 4. **P < 0.01 (analysis of variance (ANOVA)) versus ns.

Figure 4

Cyclosporin A (CsA) pretreatment and regulator of calcineurin (Rcan)1-4 overexpression cause the appearance of slower migrating nuclear factor of activated T cells (NFAT)c3 forms on SDS-PAGE. (A, B) Total cell lysates were separated on 6% SDS-PAGE gels and immunoblotted with anti-NFATc3 antibody. Ponceau stainings of membranes are presented as loading controls. (A) Mouse primary astrocyte cultures were either left without pretreatment or were pretreated with CsA for 1 h as indicated. Cells then were left non-stimulated (ns) or were subjected to hypoxia and glucose deprivation (HGD) for 30 minutes to 4 h. (B) Three independent mouse astrocyte cultures (A-C) were infected with adenovirus bicistronically encoding green fluorescent protein (GFP) plus Rcan1-4 (Ad Rcan1-4) or encoding GFP alone (Ad GFP). Samples were run in parallel on 10% gels to analyze Rcan1-4 expression and to monitor GFP expression as a control of infection. Immunoblots show the effect of Ad Rcan1-4 infection on Rcan1-4 protein expression and NFATc3 electrophoretic mobility.

Figure 5

Regulator of calcineurin (Rcan)1-4 protein downregulates expression of the proinflammatory gene Cox-2 in mouse primary astrocytes. (A) Astrocytes were infected with adenovirus bicistronically encoding green fluorescent protein (GFP) plus Rcan1-4 (Ad Rcan1-4) or encoding GFP alone (Ad GFP). Immunoblots show the effect of Ad Rcan1-4 infection on Rcan1-4 protein expression and nuclear factor of activated T cells (NFAT)c3 electrophoretic mobility. α-Tubulin and PSF (PTB-associated splicing factor) were used as endogenous loading controls and GFP as a marker of equal infection. (B) Rcan1-4 overexpression inhibits astrocyte cyclo-oxygenase 2 (Cox-2) induction. At 48 h post infection, cells were quiesced, and then stimulated with phorbol ester (phorbol 12-myristate 13-acetate) plus A23187 calcium ionophore (PIo) as indicated. Blots show expression of Cox-2 and Rcan1-4 protein. Identical concentrations of adenoviral particles were used for each infection in all experiments. (C) Densitometry analysis of PIo-Cox2 induction. Data are the means ± SD of densitometry value of n = 4 experiments. The figure of 100% is the Cox2 expression achieved by PIo stimulation for 6 h in Ad GFP cells. PIo-induced Cox2 expression in Ad Rcan1-4 cells is 72 ± 11%. All values are corrected relative to the loading control α-tubulin.

Figure 6

Expression of ischemia/reperfusion-inducible inflammatory markers and infarct volume are increased in regulator of calcineurin (Rcan)1 knockout mice after transient focal cerebral ischemia in mice. (A) TNFα, IL-6 and Cox-2 mRNAs were amplified by TaqMan real time qRT-PCR from total RNA obtained from infarcted (I) and corresponding contralateral hemispheres (C) of wild-type (WT) (black columns) and Rcan1 knockout (KO) (gray columns) mice subjected to 90 minutes middle cerebral artery occlusion (MCAO) followed by 5 h reperfusion. Transcript amounts are normalized to TATA-binding protein (TBP) as an endogenous control, and are expressed relative to the level in contralateral samples from sham-operated animals after reperfusion for 5 h (sham). Real time qPCR was conducted in triplicate for each condition, and data are the means ± SD of four experiments. **P < 0.01, *P < 0.05 (ANOVA) versus contralateral sham samples. (B) Calcineurin (CN) enzyme activity against phosphopeptide RII measured in brain cortices from Rcan1 WT and Rcan1 KO mice. ns = non-significant (Student's t test). (C) Mice were subjected to 90 minutes MCAO followed by 48 h blood reperfusion, and infarct volumes were estimated by Cavalieri's principle from Nissl-stained serial coronal sections. (i) Representative stacks of six Nissl-stained sections, revealing a larger hypochromatic area of ischemic tissue in the infarcted neocortex of Rcan1 KO animals. (ii) Quantification of cerebral infarct volume in WT and Rcan1 KO mice. The percentage of tissue volume infarcted in the right hemisphere = (1 - (R_N/L) ×100), where RN is spared tissue in right (infarcted) hemispheres and L is the left (contralateral) hemisphere; cerebral tissue volume is expressed in mm³. Data are means ± SEM. n = 7 per genotype; *P < 0.05.