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Frontiers in Biology

Front. Biol.    2015, Vol. 10 Issue (5) : 427-438     DOI: 10.1007/s11515-015-1373-z
RESEARCH ARTICLE |
Targeting ERK1/2-calpain 1-NF-κB signal transduction in secondary tissue damage and astrogliosis after spinal cord injury
Xin Xin Yu,Vimala Bondada,Colin Rogers,Carolyn A. Meyer,Chen Guang Yu()
Spinal Cord and Brain Injury Research Center, Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, KY 40536-0509, USA
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Abstract

Neuronal damage, glial inflammation, and astrogliosis/astroglial scar formation are major secondary injury mechanisms that are significant contributors to functional deficits after spinal cord injury (SCI). The objectives of the study were to evaluate the distinct roles of ERK2 vs. ERK1/2 and ERK1/2-calpain 1−NF-κB signal transduction in the tissue damage and astrogliosis/astroglial scar formation following SCI in rats. RNAi approaches, pharmacological intervention (U0126), Western blot analysis, immunofluorescence analysis, and histological assessment were used to target ERK1/2-calpain 1-NF-κB signal transduction pathway for neuroprotection. Histological staining analysis demonstrated that selectively reducing pERK2 using ERK2 siRNA, but not inhibition of pERK1/2 with U0126, significantly reduced lesion volume and improved total tissue sparing, white matter sparing, and gray matter sparing in spinal cord two weeks after contusive SCI. An ERK1/2-calpain 1-NF-κB signal transduction pathway was involved in the astroglial scar formation after SCI. Blockade of ERK1/2 by U0126 decreased calpain 1 expression 4 h following SCI. Selective calpain 1 reduction by lentiviral shRNA attenuated astroglial NF-κB activity and astroglial scar formation after SCI in rats. Taken together, these results demonstrate the involvement of individual ERK2 and calpain 1 signaling pathways in tissue damage and astrogliosis/astroglial scar formation in animal models of SCI. Therefore, targeting individual ERK and its downstream signal transduction of calpain 1-NF-κB may provide greater potential as novel therapeutics for minimizing tissue damage and astroglial scar formation following SCI.

Keywords calpain 1      ERK1/2      RNAi      neurodegeneration      astrogliosis      spinal cord injury     
Corresponding Authors: Chen Guang Yu   
Just Accepted Date: 16 September 2015   Online First Date: 13 October 2015    Issue Date: 30 October 2015
 Cite this article:   
Colin Rogers,Carolyn A. Meyer,Chen Guang Yu, et al. Targeting ERK1/2-calpain 1-NF-κB signal transduction in secondary tissue damage and astrogliosis after spinal cord injury[J]. Front. Biol., 2015, 10(5): 427-438.
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http://journal.hep.com.cn/fib/EN/10.1007/s11515-015-1373-z
http://journal.hep.com.cn/fib/EN/Y2015/V10/I5/427
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Colin Rogers
Carolyn A. Meyer
Chen Guang Yu
Xin Xin Yu
Vimala Bondada
Fig.1  Effect of U0126 on ERK1/2 phosphorylation in rat spinal cord after contusive SCI. U0126 ip injection (10 mg/kg, gray bar) or iv injection (2 mg/kg, empty bar) 90 min prior to SCI resulted in inhibition of ERK1/2 phosphorylation in the spinal cord 4 h post-injury using Western blotting with antibodies specific for phospho-ERK1/2 (top panel) or total ERK1/2 (bottom panel). Data are presented as the mean±S.E.M. and analyzed with repeated measures ANOVA followed by Bonferroni post-hoc analysis. Differences were considered statistically significant at p<0.05. *p<0.05, compared to vehicle-treated animals, n = 4/group.
Fig.2  Effect of U0126 on calpain 1 expression in spinal cord after contusive SCI in rats. U0126 ip injection (10 mg/kg, gray bar) or iv injection (2 mg/kg, empty bar) 90 min prior to SCI resulted in reduction in calpain 1 expression in the spinal cord 4 h post-injury using Western blot blotting with antibody specific for calpain 1. Data are presented as the mean±S.E.M. and analyzed with repeated measures ANOVA followed by Bonferroni post-hoc analysis. Differences were considered statistically significant at p<0.05. *p<0.05, compared to vehicle-treated animals, n = 4/group.
Fig.3  Effects of U0126 on tissue sparing 1 week following moderate contusive SCI in rats. U0126 IV administration, but not IP administration, starting at 90 min pre-injury, then once daily for 7 days post-SCI (180 kdyn) resulted in a modest non-significant increase in total tissue sparing and white matter sparing 1 week post-injury compared to vehicle-treated animals. Data are presented as the mean±S.E.M. and analyzed with repeated measures ANOVA followed by Bonferroni post-hoc analysis. Differences were considered statistically significant at p<0.05, n = 4/group.
Fig.4  Effect of ERK2 siRNA on expression of ERK2 and pERK1/2 in the spinal cord 2 days after contusive SCI (A, B, C &D). Postinjury ERK2 siRNA intrathecal treatment knocked down the levels of ERK2 protein (A top panel and B) and both ERK2 and ERK1 phosphorylation (A bottom pane, C & D) 2 days following contusive SCI using Western blotting with antibody specific for ERK2 or pERK1/2. These ERK2 siRNAs do not alter pERK1 levels using Western blot with antibody against pERK1 (A bottom panel and D). Data were presented as mean±S.E.M. and analyzed by t-test, *p<0.05, n = 4/group. ERK2 siRNA was mixed with jetPEI and administered by intrathecal infusion 30 min postinjury via minipump for 2 days (1.0 µg/day) after contusive spinal cord injury (IH impactor, 180 kdyn setting) in female Long Evans rats.
Fig.5  Effects ofERK2 siRNA intrathecal treatment on tissue damage 2 weeks after contusive SCI. Histological assessment showed that postinjury administration of ERK2 siRNA/jetPEI via minipump (1.0 µg/d for 7 days) resulted in a significant increase in total tissue sparing (A), tissue sparing at 1 to 4 mm rostral and 1 to 2 mm caudal to the injury epicenter (D), total white matter sparing (B), and white matter sparing at 1 to 4 mm rostral and 1 mm caudal to the injury epicenter (E) following contusion injury to the spinal cord, compared with control siRNA treated group. (F): Photomicrographs of representative transverse spinal cord section 2 weeks following severe contusion SCI at the epicenter and in 1-mm increments rostral and caudal to the lesion epicenter. The sections were stained with eriochrone cyanine for myelin. Scale bar: 500 µm. Contusive SCI was produced using the Infinite Horizons impactor, 180 kdyn setting, at T10. No significant differences in parameters were found between treatment and controls. Data are presented as mean±S.E.M. and analyzed by t-test (A, B, and C) or repeated measures ANOVA & Bonferroni post-hoc test (D & E). *p<0.05, n = 7/group.
Fig.6  Effects of lentiviral-calpain 1 shRNA on astroglial NF-κB activation and astrogliosis/astroglial scar formation 6 weeks after contusion SCI in rats. The spinal tissues at 2 mm rostral and caudal to the lesion epicenter were collected from sham (a), injured spinal cord-treated with LV-calpain 1 shRNA (c) or injured spinal cord-treated with LV-control shRNA (b). The cross-sections were immunofluorescence-stained with antibody against pNF-κBp65 (red) or GFAP (green). The yellow color visualized in the merged images represented colocalization of two proteins. Blue, Hoechst 33342 nuclear stain. Scale bar: 50 µm. Quantification of fluorescence signals for immunofluorescence staining of GFAP (marker for astroglial scar formation) and for triple immunofluorescence staining of nuclear pNFκBp65 plus GFAP in the spinal cord sections was performed. In contrast to the sham-operated group (a), increased expression of GFAP (astrogliosis/astroglial scar formation, A) and astroglial nuclear activity of pNF-κBp65 (B) after SCI with pretreatment of LV-control shRNA were evident by their upregulated nuclear pNF-kBp65 and/or GFAP labeled immunoreactivity at 6 weeks post-injury (b). LV-calpain 1 shRNA intraspinal pretreatment (c, gray bar in A and B) significantly decreased astrocyte proliferation (A) and nuclear pNF-κBp65 activity in astrocytes (B) 6 weeks postinjury, compared to LV-control shRNA-pretreated group (b, dark bar in A and B). SCI condition is described in Material and Method Section. Four designated areas (I: dorsal central area, II: dorsal horn area, III: ventral horn area, and IV: lateral area) in each section were imaged and the total fluorescent signal (histogram) from all four areas were calculated. Data are presented as mean±S.E.M. and analyzed with repeated measures ANOVA followed by Bonferroni post-hoc analysis, #p<0.05, ##p<0.01, compared to SCI+ LV control shRNA and **p<0.01, compared to sham, n = 6/group. Differences were considered statistically significant at p<0.05.
Fig.7  Photomicrographs for representatives of astroglial scar formation/astrogliosis in transverse rat spinal cord sections 42 days following contusive SCI. The cross-sections were immunofluorescence-stained with antibody against GFAP. Blue, Hoechst 33342 nuclear stain. Scale bar: 50 µm. SCI condition is described in Material and Method Section. Four designated areas (I: dorsal central area, II: dorsal horn area, III: ventral horn area, and IV: lateral area) in each section were imaged and the total fluorescent signal (histogram) from all four areas were calculated.
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