There are three subtypes of NOS in the nervous system. The two constitutive forms of NOS-neuronal and endothelial NOS are mainly sources of NO production. The third subtype-iNOS as its name indicates is induced by acute inflammatory stimuli. iNOS is induced mainly by activated astrocytes and microglia cells in various neurodegenerative diseases. Once iNOS is expressed, it produces neurotoxic amounts of NO chronically, without any requirement for further activation (Saha and Pahan 2006). In ALS, increased tissue levels of NO in the lumbar spinal cord of mutant ALS transgenic mice are mainly produced by iNOS located in astrocytes, seeing as nNOS-positive motor neurons are depleted, while iNOS-positive activated glial cells are increased in ALS mice (Lee et al. 2009). Furthermore, pharmacological inhibition of iNOS shows the significant effects of delaying disease-onset and extending survival in ALS mice (Chen et al. 2010). The findings of our study demonstrate that iNOS protein expression is up-regulated in the spinal cords of both G93A and G37R SOD1 mutant transgenic mice compared with non-transgenic mice and wild-type SOD1 transgenic mice. In addition, the level of iNOS increased during the progression of disease, and it reached a peak at the end-stage of ALS. These observations demonstrate that iNOS is involved in the causal mechanisms of motor neuron degeneration in ALS mice. The role of iNOS in the pathogenesis of ALS is less clear (than nNOS), probably because study on its effect is very controversial. For example, iNOS-knockout mice are protected against neuronal injury in several models of neurodegenerative diseases, including Alzheimer's disease (Nathan et al. 2005) and Parkinson's disease (Dehmer et al. 2000). In ALS, the G93A-high-mutant SOD1 mouse, iNOS gene deletion has a significantly prolonged survival (Martin et al. 2007). However, a recent study of three different strains of iNOS-knockout showed no effect of iNOS on the infarct size after transient focal brain ischemia (Pruss et al. 2008). This may suggest that iNOS is more likely to be toxic to chronic neurodegeneration, rather than to acute neuronal injury (Brown 2010). We focused on iNOS because earlier studies on ALS mice (Almer et al. 1999; Sasaki et al. 2001; Martin et al. 2007) and human patients (Phul et al. 2000) have indicated that this isoform of NOS could be vital in ALS, and its role in the neuropathology of ALS has been under appreciated. The importance of iNOS comes from its properties that are different from endothelial NOS and nNOS. Homodimeric iNOS is catalytically active when expressed and it is active for extended periods with a maximum of 10-fold greater than other NOS isoforms, generating a very high output of NO (Lowenstein and Padalko 2004).
We also studied the concentrations of NO in the spinal cord extracts in mutant SOD1 transgenic mice (both G93A and G37R), wild-type SOD1 transgenic mice, and non-transgenic mice. As predicted, compared with the wild-type SOD1 transgenic mice and non-transgenic mice, the mutant SOD1 transgenic mice had a higher net amount of NO production, which was probably because of the increase in inflammation and gliosis with a concomitant up-regulation of iNOS expression. Furthermore, NO concentration was significantly increased in the spinal cord extracts of mutant SOD1 transgenic mice at end-stage of ALS. The up-regulation of NO level during disease progression presumably resulted from activation of astrocytes and microglia, which induced iNOS up-regulation in the spinal cords of mutant SOD1 transgenic mice. The unaltered NO levels in both non-transgenic mice and wild-type SOD1 transgenic mice indicated that the constitutive level of NO was probably because of nNOS residued in motor neurons. Our results imply an interesting connection between robust iNOS expression and excessive NO generation in mutant transgenic mice at end-stage of ALS. Up-regulation of iNOS may account for the large amount of NO generation at disease end-stage.
Nitric oxide mediates cellular signaling pathways that regulate broad aspects of physiological processes. NO has been implicated in neurotransmission, synaptic plasticity, and neuromodulation in CNS (Garthwaite and Boulton 1995). Excessive generation of NO and its derivatives has also been implicated in the pathogenesis of neurodegenerative disorders (Calabrese et al. 2000). For example, high levels of NO induce neuronal death by causing inhibition of mitochondrial cytochrome oxidase in neurons (Brown and Cooper 1994). The inhibition of neuronal respiration leads to depolarization and glutamate release, followed by excitotoxicity via the NMDA receptor (Jekabsone et al. 2007; Golde et al. 2002). Normally, NO mediates the physiological and pathophysiological effects via stimulation of guanylate cyclase to form cyclic guanosine – 3′,5′-monophosphate (cGMP) or through S-nitrosylation of regulatory protein thiol groups (Isaacs et al. 2006). S-nitrosylation involves the covalent addition of a NO group to a critical cysteine thiol/sulfhydryl to form an S-nitrosothiol derivative. This S-nitrosylated modification can influence the function of a broad spectrum of proteins as well as the protein–protein interaction (Hara et al. 2005). Our studies have found that PDI is S-nitrosylated in spinal cord tissues of end-stage mutant SOD1 transgenic mice (both G93A and G37R). However, the S-nitrosylated PDI was virtually undetectable in our pre-symptomatic stage mice. In addition, in non-transgenic mice and wild-type SOD1 transgenic mice, there was no S-nitrosylation of PDI. This finding suggests that S-nitrosylation of PDI probably inactivates the normal properties of PDI and it may contribute to the pathogenesis of ALS. Consistent with this result, we also found that PDI expression levels at the end-stage of disease were significantly up-regulated in spinal cords of both G93A and G37R SOD1 mutant transgenic mice as compared with the non-transgenic and wild-type transgenic mice. Similarly, a previous study confirmed significant up-regulation of PDI expression at disease end-stage in both G93A mutant mice and rats as compared with that of the non-transgenic controls (Atkin et al. 2006). However, there are some varieties of PDI expression at different disease stages in mutant transgenic rats relative to the corresponding non-transgenic rat. Ferri and colleagues found that PDI expression was up-regulated at the pre-symptomatic stage (8 weeks), but at 16 weeks and end stage, PDI levels were not significantly higher in the spinal cords of G93A mutant transgenic rats as compared with non-transgenic rats (Ahtoniemi et al. 2008). Another study showed that PDI levels in CSF of G93A mutant transgenic rats were most prominently elevated at the disease onset stage when compared with non-transgenic rats (Atkin et al. 2008). All these studies set the age-matched non-transgenic mice or rats as control. To examine the change of PDI expression during disease progression, absolute PDI levels were quantified as a ratio relative to the corresponding β-actin anounts at each stage. We found that the PDI level increased gradually with the progression of disease and peaked at disease end-stage in both G93A and G37R mutant transgenic mice. Even though the up-regulation of PDI represents an adaptive response to provide potential neuroprotection (Conn et al. 2004; Hetz et al. 2005; Ko et al. 2002; Tanaka et al. 2000), NO-mediated S-nitrosylation of PDI probably affects its normal function and promotes aggregate formation. The formation of SOD1 aggregates in ALS was probably through destabilizing mutant SOD1 with reactive reduced cysteine residues (Ahtoniemi et al. 2008). Cysteine residues are crucial for SOD1 stability, and a non-physiological intermolecular disulfide bond between cysteine 6 and 111 in mutant SOD1 was found to be associated with mutant SOD1 aggregates' formation (Niwa et al. 2007a). PDI may get involved in rearranging the cysteine residues of SOD1, and S-nitrosylation of PDI toward the end-stage of ALS may facilitate the formation of non-physiological disulfide bonds and promote SOD1 aggregates formation. This could be a possible explanation to the finding that cysteine-reduced SOD1 levels increased with disease progression and reach the peak at end-stage in G93A mutant transgenic rats (Ahtoniemi et al. 2008). Furthermore, an association of PDI and mutant SOD1 aggregates was identified, seeing as high levels of PDI that recruit to abnormal inclusions were observed in both G93A transgenic mice and ALS patients (Atkin et al. 2006, 2008). In addition, PDI was found to be co-localized with SOD1 in neuronal cytoplasmic inclusions (Honjo et al. 2011). Given the chaperone activity of PDI, it is possible that PDI may interact with insoluble mutant SOD1 to form abnormal inclusions; this may partially explain the findings of other studies that PDI levels were not most prominently up-regulated at the disease end-stage in mutant transgenic rat when compared with non-transgenic mice (Atkin et al. 2008; Ahtoniemi et al. 2008). A greater portion of PDI may be associated with mutant SOD1 in abnormal inclusion at end-stage of ALS, and then it cannot be detected in the soluble fraction of tissue lysates.
Protein disulfide isomerase is a ubiquitous, highly conserved redox enzyme from the thioredoxin superfamily, and it is mainly located in the ER (Noiva 1999). During protein folding in the ER, PDI facilitates proper protein folding and helps to maintain the structural stability of the mature protein (Lyles and Gilbert 1991). As a consequence, PDI is considered a molecular chaperone capable of stabilizing the correct folding of substrate proteins. It also facilitates ER-associated degradation of misfolded proteins (Lee et al. 2010). Through interacting with the ER transmembrane protein Derlin-1, PDI is involved in retro-translocation of misfolded cholera toxin from the ER to the cytoplasm. (Moore et al. 2010). As we know, aberrant protein folding and further protein aggregates are associated with various neurodegenerative diseases, including ALS. The accumulation of misfolded protein in the ER results in ER stress that triggers the protective unfolded proteins response (UPR). The UPR entails the induction of chaperone molecules, the degradation of misfolded proteins, and inhibition of protein translation (Zhang and Kaufman 2006). Prolonged ER stress can nonetheless lead to activation of apoptosis (Xu et al. 2004). Studies involving pancreatic β cells, macrophages (Gotoh and Mori 2006), and cerebellar granule cells (He et al. 2004) have demonstrated that NO can induce ER stress. However, the molecular basis remains unclear. Furthermore, although the involvement of NO in neurodegeneration has been widely accepted, the chemical relationship between nitrosative stress and formation of 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 excitotoxic activation of nNOS leads to excessive NO generation, which causes S-nitrosylation of the active-site thiols of PDI, inhibiting its isomerase and chaperone activities (Uehara et al. 2006). In this regard, NO blocks the protein's protective effect via S-nitrosylation of PDI, which leads to accumulation of misfolded and polyubiquitinated proteins, resulting in prolonged UPR activation, and thus persistent ER stress, which induces apoptosis. Our study showed that, when SH-SY5Y cells were expressing mutant SOD1G93A, iNOS expression was significantly increased in compared with cells expressing SOD1wt. This finding was relatively inconsistent with the previous study that had showed expressing SOD1wt or mutant SOD1G93A in NSC-34 cells did not alter iNOS expression, which remained at a constitutive level. This variation may be because of the controlled SOD1wt or mutant SOD1G93A expression in NSC-34 cells. In that study, the cell clones expressing low and similar amounts of various SOD1 proteins upon induction. The induced human SOD1 expression was lower than murine endogenous SOD1 expression; this low expression of human expression may not be extensive enough to trigger the possible correlation between mutant SOD1 expression and iNOS expression. Furthermore, the increased iNOS level in mitochondria has been observed in G93A mutant transgenic mice and the deletion of iNOS gene significantly extended the survival of mice. However, the activity of iNOS is more crucial to its biological role in ALS pathogenesis. The effect of mutant SOD1 in alteration of iNOS activity was further investigated. We found that cells expressing mutant SOD1G93A had much higher NO generation, and this increased NO generation could be reversed by the use of NNA (non-selective inhibitor of NOS) (data not shown). Consequently, ALS-linked SOD1 is highly associated with up-regulated iNOS expression and increased iNOS-derived NO generation. However, another enzyme responsible for NO production, nNOS, was found to be down-regulated in SH-SY5Y cells with mutant SOD1G93A expression in a study (Aquilano et al. 2003). The down-regulation of nNOS-derived NO production was also evidenced. It has been suggested that NO released from nNOS activity keeps iNOS inhibited under normal conditions. However, under pathophysiological conditions, down-regulation of nNOS is a necessary condition to promote the iNOS expression and the release of large amounts of NO (Qu et al. 2001). For example, in a rat model of inflammatory bowel disease, nNOS down-regulation could induce iNOS over-expression (Porras et al. 2006). Our results could be in agreement with the hypothesis that down-regulation of nNOS is associated with up-regulation of iNOS in SH-SY5Y cells expressing mutant SOD1G93A. However, the nNOS expression and the mechanism for the association between iNOS and nNOS will certainly deserve further investigation. We introduced the physiological NO donor, SNOC to provoke S-nitrosylation of PDI. The dose-dependent manner of SNOC-induced S-nitrosylation of PDI confirmed that PDI was S-nitrosylated by NO-related species. The role of PDI in protecting against mutant protein aggregation in ALS is supported by various studies. For example, when mutant SOD1 expressing NSC-34 cells were treated with bactracin, an inhibitor of PDI, the formation of SOD1 inclusions was increased (Atkin et al. 2006). Another example was siRNA-mediated knockdown of PDI resulted in increased formation of mutant SOD1 inclusion in neuroblastoma cells (Walker et al. 2010). However, the PDI up-regulation in ALS was not enough to protect against mutant SOD1 aggregate formation, as S-nitrosylation of PDI may affect its enzymatic activity and promote the aggregates formation. We found that NO-mediated S-nitrosylation of PDI was probably involved in the formation of mutant SOD1 aggregates, seeing as NO donor SNOC-induced S-nitrosylation of PDI in a dose-dependent manner. Furthermore, the inclusions in mutant SOD1G93A expressing cells were enhanced by SNOC. Our studies also showed that, with the use of NOS inhibitor NNA to suppress S-nitrosylation of PDI, the SOD1 inclusions were inhibited, and the level of insoluble mutant SOD1 in the pellet fraction was decreased as well. This finding indicates that in the cell model of ALS, manipulating the NO levels by using a NO donor or NOS inhibitor, affects the formation of SNO-PDI. S-nitrosylation of PDI may highly correlate with mutant SOD1 aggregates formation. As S-nitrosylation of PDI could inhibit its chaperone activity in rearrangement of protein folding, allow the misfolded proteins to accumulate, and finally contribute to neuronal cell death. In addition, the subcellular redistribution of PDI has recently been implicated in the pathogenesis of ALS. Studies have demonstrated that the reticulon family of proteins can modulate PDI distribution. They found that reticulon over-expression causes a change of PDI localization, from a normal ER distribution to a less homogenous punctuate pattern (Yang et al. 2009). In ALS mice, knocking down the expression of the reticulon-4A, B proteins accelerate disease processes, possibly resulting from the prevention of reticulon-mediated PDI redistribution (Yang et al. 2009). Furthermore, co-localized inclusions of PDI with mutant SOD1, and TAR DNA-binding protein 43 kDa (TDP-43) have been found in ALS patients (Honjo et al. 2011). Under cellular stress, PDI may leave the ER and then accumulate with SOD1 or TDP-43 in the cytosol. PDI also accumulates in the swollen neuritis, the disturbance of axon transport was probably because of the loss of PDI function (Honjo et al. 2011). Another ALS-related protein, fused in sarcoma (FUS), is found to be associated with PDI. Mutant FUS inclusions in human ALS lumbar spinal cords are co-localized with PDI. As FUS contains cysteine residues, similar to mutant SOD1 and mutant TDP-43, it may physically interact with PDI, and the chaperone function of PDI may have a protective role in refolding misfolded FUS protein. Overall, these findings indicate that post-translational modifications and subcellular redistribution of PDI are involved in regulation of PDI's function in ALS, with potential implications for disease pathogenesis.