Brain ischemia/reperfusion injury encompasses all cell types in the central nervous system, including neurons and astrocytes. Astrocytes are believed to play a fundamental role in the pathogenesis of neuronal death. The failure of astrocytes in supporting the essential needs of neurons constitutes a great threat for neuronal survival. The multifaceted and complex role of astrocytes in response to injury includes the enhancement of neuronal survival or regeneration and contributes to further injury [29, 30]. Glial cells, including astrocytes, generate excessive amounts of NO as a result of the activation of iNOS, and NO can induce neuronal apoptosis in ischemia/reperfusion injury. However, the cellular and molecular mechanisms of neuronal death induced by excessive NO have not yet been clearly defined. Brain hypoxic or ischemic injury is associated with an obvious inflammatory reaction that results in the expression and release of several cytokines . These important mediators activate the expression of iNOS in different cell types, including glial cells in the central nervous system [32–34]. Interleukin-1β and tumor necrosis factor are both significantly increased within a few hours of ischemia. Interleukin-1β and tumor necrosis factor trigger transcriptional activation of the iNOS gene and then up-regulate iNOS expression [35, 36]. Oxidative stress induced by ischemia might itself trigger the induction of iNOS. Moreover, the iNOS promoter contains a hypoxia response element, since a specific pathway, the hypoxia-inducible factor-1α pathway, can be activated at the onset of ischemia . Consequently, the generation of NO persists. It is believed that NO produced by de novo expression of iNOS contributes to brain damage caused by hypoxic ischemia . In the present study, we examined whether iNOS expression was enhanced in response to OGD/reperfusion in astrocytes. Consistently with previous research, OGD/reperfusion markedly elevated iNOS protein levels in cultured astrocytes. Our study gives the first demonstration that PDI is S-nitrosylated in cultured astrocytes following ischemia/reperfusion injury, and that this is highly associated with extensive generation of NO, which is induced by up-regulated iNOS expression. This finding suggests that S-nitrosylation of PDI probably inactivates the normal properties of PDI, and that it may contribute to the pathogenesis of ischemia/reperfusion injury.
Protein disulfide isomerase is a ubiquitous, highly conserved redox enzyme from the thioredoxin superfamily, located mainly in the ER . During protein folding in the ER, PDI facilitates proper protein folding and helps to maintain the structural stability of the mature protein . As a consequence, PDI is considered to be a molecular chaperone capable of stabilizing the correct folding of substrate proteins. It also facilitates the ER-associated degradation of misfolded proteins . Protein disulfide isomerase is involved in the retro-translocation of misfolded cholera toxin from the ER to the cytoplasm by interacting with the ER transmembrane protein Derlin-1 . In this study, we found that PDI expression was up-regulated in astrocytes following OGD/reperfusion. This result was consistent with previous studies that have demonstrated the up-regulation of PDI in astrocytes in response to hypoxia or transient forebrain ischemia in astrocytes . A study of ischemic cardiomyopathy indicates that PDI is up-regulated in the viable peri-infarct myocardial region after infarction. This up-regulation of PDI led to a significant decrease in the rate of cardiomyocyte apoptosis . All of this evidence put together indicates that the up-regulation of PDI in ischemia/reperfusion injury represents an adaptive response that promotes correct protein folding and offers potential protection to cells. However, detrimental generation of NO derived from iNOS induces S-nitrosylation of PDI; this posttranslational modification of PDI may attenuate its protective effects in ischemia/reperfusion injury.
As we know, ischemia-reperfusion causes accumulation of high-molecular weight ubiquitinated proteins following forebrain ischemia . These ubiquitinated-protein aggregates are visualized in cultured astrocytes following glucose deprivation/recovery . They are clustered with co-translational chaperones, protein folding enzymes , subcellular structures , proteasomes , and stress granules . These changes may contribute to ischemic dysfunction of astrocytes and lead to neuronal damage. The accumulation of misfolded protein in the ER results in ER stress that triggers the protective unfolded protein response. The unfolded protein response entails the induction of chaperone molecules, the degradation of misfolded proteins, and the inhibition of protein translation . Nonetheless, prolonged ER stress can still lead to activation of apoptosis . Studies on pancreatic β cells, macrophages , and cerebellar granule cells  have demonstrated that NO can also induce ER stress. However, the molecular basis of this remains unknown. Furthermore, although the involvement of NO in the pathology of brain ischemia/reperfusion injury has been widely accepted, the chemical relationship between nitrosative stress and formation of ubiquitinated-protein aggregates has remained obscure. Our findings indicate that S-nitrosylation of PDI may hold some of the answers to these questions. Studies have shown that in Parkinson’s disease, excitotoxic activation of nNOS leads to excessive NO generation, which causes S-nitrosylation of the active-site thiols of PDI and inhibits its corresponding isomerase and chaperone activities . In this way, NO blocks the protein’s protective effects via S-nitrosylation of PDI. S-nitrosylation of PDI leads to the accumulation of misfolded and polyubiquitinated proteins, and results in prolonged unfolded protein response activation. NO-mediated S-nitrosylation of PDI, therefore, participates in persistent ER stress and the induction of apoptosis .
We further demonstrated that NO-mediated S-nitrosylation of PDI may take part in the formation of ubiquitinated-protein aggregates in cultured astrocytes following OGD/reperfusion, since the aggregate’s formation was blocked by the iNOS inhibitor 1400W, which could efficiently inhibit the S-nitrosylation of PDI. When cultured astrocytes were subjected to OGD/reperfusion, the cells formed smear detergent/salt-insoluble ubiquitinated-protein aggregates. Furthermore, diffuse free ubiquitin staining changed into punctuated staining within perinuclear regions. This conjugated ubiquitin with reduced cytosolic and nuclear free ubiquitin distribution was considered to be an ubiquitinated-protein aggregate. The formation of these aggregates correlated well with the level of S-nitrosylation of PDI. With the use of 1400W to inhibit the activity of iNOS, the generation of NO was consequently decreased, which subsequently led to down-regulation of SNO-PDI levels. With the inhibition of S-nitrosylation of PDI, the formation of ubiquitinated-protein aggregates was decreased, since the detergent/salt-insoluble smear of ubiquitin in the pellet fraction was significantly reduced through the use of 1400W. This finding clearly demonstrates that blocking NO generation reduces the accumulation of ubiquitinated-protein aggregates. This blockade effect of 1400W may, result from reducing NO-mediated S-nitrosylation of PDI.
Free radicals contribute to neuronal death following hypoxic/ischemic brain injury. Not surprisingly, several studies have demonstrated that antioxidant treatment improves neuroprotection and recovery after brain injury [53–55]. SOD1 is an enzyme that detoxifies free radicals under normal physiologic conditions. SOD1 converts the superoxide anion into hydrogen peroxide, which is subsequently detoxified to water by glutathione peroxidase or catalase . Reperfusion following cerebral ischemia leads to an overproduction of free radicals and the consumption of endogenous antioxidants. Neurons are particularly vulnerable to free radical damage, partly because of their relatively low levels of endogenous antioxidants. Studies have shown that non-neuronal cells may participate in free radical scavenging during ischemia/reperfusion . One facet of reactive astrocytes in brain ischemia/reperfusion injury is the chronic secretion of antioxidants for neuronal protection and survival. SOD1 is one of the beneficial antioxidants produced by astrocytes. Prior studies using transgenic animal models have clearly established a beneficial role of SOD1 in adult ischemia/reperfusion injury . Rodents overexpressing SOD1 have a much better outcome following head injury . In our study, the expression of SOD1 was up-regulated in cultured astrocytes following OGD/reperfusion. The increased expression of SOD1 may represent a protective response to ischemic stress that enhances the antioxidant ability. However, studies have shown that SOD1 overexpression offers no protection under OGD conditions in a hippocampal culture model of excitotoxic injury . Our results regarding the S-nitrosylation of PDI in cultured astrocytes following OGD/reperfusion provides an explanation to this finding. First, SOD1 was shown to be one of the PDI molecular targets  in ischemic cardiomyopathy. Second, a physical interaction between SOD1 and PDI has been indicated in cultured cells in familial amyotrophic lateral sclerosis . Protein disulfide isomerase binds to both wild-type and mutant SOD1, and colocalizes with intracellular aggregates of mutant SOD1. Inhibition of the activity of PDI with the use of bacitracin increases aggregate production . In patients with amyotrophic lateral sclerosis, PDI was found to be colocalized with SOD1 in neuronal cytoplasmic inclusions . In this study, PDI and SOD1 were found to bind to one another in astrocyte cultures. Although PDI was up-regulated after OGD/reperfusion treatment, the increased total PDI did not bind more SOD1. Instead, less PDI-SOD1 binding was detected after OGD/reperfusion treatment in immunoprecipitation. It is possible that, despite the induction of PDI after ischemia/reperfusion injury, the SNO-PDI could not bind to SOD1 as efficiently as a normal PDI. In addition, SOD1 was ubiquitinated to form aggregates, and the insoluble SOD1 aggregates could not be detected in the soluble cell lysates used in the experiment. Some proportion of PDI was associated with SOD1 aggregates in the insoluble fraction of the cell lysates. We may suppose that PDI normally binds to SOD1 to form a disulfide-linked dimer. However, if PDI were S-nitrosylated, it could not bind to SOD1 as efficiently; and the disulfide-reduced SOD1 would more easily form aggregates. The diffuse distribution of SOD1 within the cytosol and nucleus under normal conditions changed into punctuated perinuclear and nuclear distribution following treatment with OGD 8 h/reperfusion 16 h. This result suggests abnormal folding of SOD1 in the cytoplasm had occurred. The ubiquitin-proteasome system (UPS) is the major intracellular proteolytic mechanism that controls the degradation of misfolded or abnormal proteins . The colocalization of SOD1 and ubiquitin indicates that the misfolded SOD1 is ubiquitinated for further degradation.