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Open Access Research

SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI)

Xueping Chen12, Teng Guan1, Chen Li1, Huifang Shang2, Liying Cui3, Xin-Min Li4 and Jiming Kong1*

Author Affiliations

1 Department of Human Anatomy and Cell Science, University of Manitoba, 745 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0J9, Canada

2 Department of Neurology, West China Hospital, Sichuan University, Chengdu, 610041, China

3 Department of Neurology, Beijing Union Hospital, Beijing, China

4 Department of Psychiatry, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

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Journal of Neuroinflammation 2012, 9:237  doi:10.1186/1742-2094-9-237

Published: 12 October 2012

Abstract

Background

Ubiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration.

Methods

To study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI) and copper-zinc superoxide dismutase (SOD1) were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining.

Results

Our data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the formation of ubiquitinated-protein aggregates in cultured astrocytes following oxygen glucose deprivation and reperfusion was also suppressed by 1400W. Interestingly, these aggregates were colocalized with SOD1, which was found to co-immunoprecipitate with PDI.

Conclusions

NO-mediated S-nitrosylation of PDI may be involved in the formation of the SOD1-linked ubiquitinated-protein aggregates in cerebral ischemia/reperfusion injury.

Keywords:
Nitrosative stress; PDI; S-nitrosylation; SOD1; Ubiquitinated-protein aggregates