Critical Roles of Glutaredoxin in Brain Cells-Implications for Parkinson's Disease.

SIGNIFICANCE: Glutaredoxin (Grx)1, an evolutionarily conserved and ubiquitous enzyme, regulates redox signal transduction and protein redox homeostasis by catalyzing reversible S-glutathionylation. Grx1 plays different roles in different cell types. In Parkinson's disease (PD), Grx1 regulates apoptosis signaling in dopaminergic neurons, so that loss of Grx1 leads to increased cell death; in microglial cells, Grx1 regulates proinflammatory signaling, so that upregulation of Grx1 promotes cytokine production. Here we examine the regulatory roles of Grx1 in PD with a view toward therapeutic innovation. Recent Advances: In postmortem midbrain PD samples, Grx1 was decreased relative to controls, specifically within dopaminergic neurons. In Caenorhabditis elegans models of PD, loss of the Grx1 homologue led to exacerbation of the neurodegenerative phenotype. This effect was partially relieved by overexpression of neuroprotective DJ-1, consistent with regulation of DJ-1 content by Grx1. Increased GLRX copy number in PD patients was associated with earlier PD onset; and Grx1 levels correlated with levels of proinflammatory tumor necrosis factor-α in mouse and human brain samples. In vitro studies showed Grx1 to be upregulated on proinflammatory activation of microglia. Direct overexpression of Grx1 increased microglial activation; silencing Grx1 diminished activation. Grx1 upregulation in microglia corresponded to increased neuronal cell death in coculture. Overall, these studies identify competing roles of Grx1 in PD etiology. CRITICAL ISSUES: The dilemma regarding Grx1 as a PD therapeutic target is whether to stimulate its upregulation for neuroprotection or inhibit its proinflammatory activity. FUTURE DIRECTIONS: Further investigation is needed to understand the preponderant role of Grx1 regarding dopaminergic neuronal survival.

Novel chloroacetamido compound CWR-J02 is an anti-inflammatory glutaredoxin-1 inhibitor.

Glutaredoxin (Grx1) is a ubiquitously expressed thiol-disulfide oxidoreductase that specifically catalyzes reduction of S-glutathionylated substrates. Grx1 is known to be a key regulator of pro-inflammatory signaling, and Grx1 silencing inhibits inflammation in inflammatory disease models. Therefore, we anticipate that inhibition of Grx1 could be an anti-inflammatory therapeutic strategy. We used a rapid screening approach to test 504 novel electrophilic compounds for inhibition of Grx1, which has a highly reactive active-site cysteine residue (pKa 3.5). From this chemical library a chloroacetamido compound, CWR-J02, was identified as a potential lead compound to be characterized. CWR-J02 inhibited isolated Grx1 with an IC50 value of 32 µM in the presence of 1 mM glutathione. Mass spectrometric analysis documented preferential adduction of CWR-J02 to the active site Cys-22 of Grx1, and molecular dynamics simulation identified a potential non-covalent binding site. Treatment of the BV2 microglial cell line with CWR-J02 led to inhibition of intracellular Grx1 activity with an IC50 value (37 µM). CWR-J02 treatment decreased lipopolysaccharide-induced inflammatory gene transcription in the microglial cells in a parallel concentration-dependent manner, documenting the anti-inflammatory potential of CWR-J02. Exploiting the alkyne moiety of CWR-J02, we used click chemistry to link biotin azide to CWR-J02-adducted proteins, isolating them with streptavidin beads. Tandem mass spectrometric analysis identified many CWR-J02-reactive proteins, including Grx1 and several mediators of inflammatory activation. Taken together, these data identify CWR-J02 as an intracellularly effective Grx1 inhibitor that may elicit its anti-inflammatory action in a synergistic manner by also disabling other pro-inflammatory mediators. The CWR-J02 molecule provides a starting point for developing more selective Grx1 inhibitors and anti-inflammatory agents for therapeutic development.

Upregulation of Glutaredoxin-1 Activates Microglia and Promotes Neurodegeneration: Implications for Parkinson's Disease.

AIMS: Neuroinflammation and redox dysfunction are recognized factors in Parkinson's disease (PD) pathogenesis, and diabetes is implicated as a potentially predisposing condition. Remarkably, upregulation of glutaredoxin-1 (Grx1) is implicated in regulation of inflammatory responses in various disease contexts, including diabetes. In this study, we investigated the potential impact of Grx1 upregulation in the central nervous system on dopaminergic (DA) viability. RESULTS: Increased GLRX copy number in PD patients was associated with earlier PD onset, and Grx1 levels correlated with levels of proinflammatory tumor necrosis factor-alpha (TNF-α) in mouse and human brain samples, prompting mechanistic in vitro studies. Grx1 content/activity in microglia was upregulated by lipopolysaccharide (LPS), or TNF-α, treatment. Adenoviral overexpression of Grx1, matching the extent of induction by LPS, increased microglial activation; Grx1 silencing diminished activation. Selective inhibitors/probes of nuclear factor κB (NF-κB) activation revealed glrx1 induction to be mediated by the Nurr1/NF-κB axis. Upregulation of Grx1 in microglia corresponded to increased death of neuronal cells in coculture. With a mouse diabetes model of diet-induced insulin resistance, we found upregulation of Grx1 in brain was associated with DA loss (decreased tyrosine hydroxylase [TH]; diminished TH-positive striatal axonal terminals); these effects were not seen with Grx1-knockout mice. INNOVATION: Our results indicate that Grx1 upregulation promotes neuroinflammation and consequent neuronal cell death in vitro, and synergizes with proinflammatory insults to promote DA loss in vivo. Our findings also suggest a genetic link between elevated Grx1 and PD development. CONCLUSION: In vitro and in vivo data suggest Grx1 upregulation promotes neurotoxic neuroinflammation, potentially contributing to PD. Antioxid. Redox Signal. 25, 967-982.

Sulfhydryl-mediated redox signaling in inflammation: role in neurodegenerative diseases.

Posttranslational modifications of cysteine sulfhydryl (-SH) moieties, e.g., S-nitrosylation, S-glutathionylation, or S-sulfuration, play an important role in cellular response to oxidative stress. Reversible cysteine modifications alter protein function and can play a critical role in redox signal transduction. Perturbation of sulfhydryl homeostasis is a hallmark of many diseases, including neurodegenerative disorders. Besides direct oxidative stress within the neurons, inflammation of the central nervous system as well as the periphery is implicated also in the development and progression of neurodegeneration. Therefore, perturbation of redox regulation of key inflammatory mediators is an important component of neurodegenerative diseases. Many proteins involved in inflammation have been shown to undergo S-nitrosylation (-SNO) and/or S-glutathionylation (-SSG) with functional consequences. The mechanistic and functional relationships between these two modifications have yet to be thoroughly investigated. While protein-SNO intermediates in some cases may signal independently of protein-SSG intermediates, the relatively unstable nature of protein-SNO derivatives in the presence of GSH suggests that protein-SNO formation in many cases may serve as a precursor for protein-SSG modifications. In this review, we describe the cysteine modifications of specific inflammation-mediating proteins and their relationship to inflammatory responses such as cytokine and chemokine production. In particular, we consider evidence for sequential protein-SNO â†’ protein-SSG modifications of these proteins. We conclude that cysteine modifications of critical regulatory proteins are likely to play a central role in the onset and progression of neuroinflammatory diseases and thus should be studied thoroughly in this context.