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

Miller, Olga Gorelenkova; Behring, Jessica B.; Siedlak, Sandra L.; Jiang, Sirui; Matsui, Reiko; Bachschmid, Markus; Zhu, Xiongwei; Mieyal, John J.

doi:10.1089/ars.2015.6598

Cited by 36 publications

(38 citation statements)

References 55 publications

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“…It was diluted in media to a final concentration of 100 ng/mL, which has previously been shown to stimulate the BV-2 cell line [30]. A 10-μg/mL stock solution of TNFα (Sigma-Aldrich) was prepared in water and stored at –20°C; for treatment, a final concentration of 10 ng/mL was obtained, which has been used on the BV-2 cell line [31]. IL-13 (R&D Biosystems, Burlington, ON, Canada) was dissolved in water to 10 or 50 μg/mL and stored at –20°C, and then diluted to 10 or 50 ng/mL, respectively, for treatment in media.…”

Section: Methodsmentioning

confidence: 99%

Palmitate Induces an Anti-Inflammatory Response in Immortalized Microglial BV-2 and IMG Cell Lines that Decreases TNFα Levels in mHypoE-46 Hypothalamic Neurons in Co-Culture

et al. 2018

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Background and Objectives: Elevated levels of saturated fatty acids (SFA) induce a state of neuroinflammation in the hypothalamus. It has been suggested that microglia sense palmitate, a prevalent circulating SFA, and act as mediators of this inflammatory process by communicating with neurons, particularly those involved in appetite regulation. In this study, we examined the inflammatory response to palmitate in immortalized microglial cell lines, BV-2 and IMG, and the subsequent effects on inflammatory gene expression in a model of NPY/AgRP neurons, mHypoE-46. Methods: The BV-2 cells were treated with 50 µM palmitate for 4 and 24 h, and the transcriptional regulation of markers for inflammation and cellular stress was assessed using an RT² Profiler PCR Array. Select genes were verified with qRT-PCR. The BV-2 and IMG cells were then co-cultured using 1.0-µm cell culture inserts with an immortalized hypothalamic cell line, mHypoE-46, to investigate potential intercellular communication between microglia and neurons. Results: We found that palmitate increased the mRNA levels of specific inflammatory genes, and a general anti-inflammatory profile was revealed in the microglia cells. The mRNA changes in TNFα at 4 and 24 h in BV-2 cells were abrogated with the toll-like receptor 4 (TLR4) inhibitor, TAK-242, indicating the involvement of TLR4. Co-culture of mHypoE-46 neurons with microglia pre-treated with palmitate resulted in repression of TNFα expression in the hypothalamic neurons. As palmitate significantly increased IL-13 expression in microglia, the effect of this cytokine was tested in mHypoE-46 neurons. The addition of IL-13 to neuronal cultures normalized the palmitate-mediated increase in IL-6 and AgRP expression, suggesting that microglia may protect surrounding neurons, at least in part, through the release of IL-13. Conclusions: These results suggest a potential anti-inflammatory role of microglia towards the palmitate-induced neuroinflammation, and potentially energy homeostasis, in hypothalamic neurons.

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Section: Methodsmentioning

confidence: 99%

Palmitate Induces an Anti-Inflammatory Response in Immortalized Microglial BV-2 and IMG Cell Lines that Decreases TNFα Levels in mHypoE-46 Hypothalamic Neurons in Co-Culture

et al. 2018

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“…In addition, microvascular endothelial cells and microphages from GRX-1-deficient mice showed enhanced LPS-stimulated glutathionylation of eNOS and, in a model of necrotizing enterocolitis (NEC), GRX-1-deficient mice were more susceptible to NEC development through the activation of TLR4 than the wild type [40]. In contrast, adenoviral overexpression of GRX-1 increased microglia activation and subsequently led to death of neuron cells [41]. GRX-1-deficient mice did not display increased vulnerability to acute injury of heart and lungs in ischemia/reperfusion and hyperoxia mouse model, whereas mouse embryonic fibroblasts from GRX-1-deficient mice were more tolerant to apoptosis induced by TNF-α plus actinomycin D. These findings suggest that variations in GRX-1 function may exist depending on cell types and animal models of disease [42].…”

Section: Discussionmentioning

confidence: 99%

PEP-1-GRX-1 Modulates Matrix Metalloproteinase-13 and Nitric Oxide Expression of Human Articular Chondrocytes

Hwang¹,

Park²,

Kim³

et al. 2016

Cell Physiol Biochem

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Background: The protein transduction domain (PTD) enables therapeutic proteins to directly penetrate the membranes of cells and tissues, and has been increasingly utilized. Glutaredoxin-1 (GRX-1) is an endogenous antioxidant enzyme involved in the cellular redox homeostasis system. In this study, we investigated whether PEP-1-GRX-1, a fusion protein of GRX-1 and PEP-1 peptide, a PTD, could suppress catabolic responses in primary human articular chondrocytes and a mouse carrageenan-induced paw edema model. Methods: Human articular chondrocytes were isolated enzymatically from articular cartilage and cultured in a monolayer. The transduction efficiency of PEP-1-GRX-1 into articular chondrocytes was measured by western blot and immunohistochemistry. The effects of PEP-1-GRX-1 on matrix metalloproteinases (MMPs) and catabolic factor expression in interleukin (IL)-1β- and lipopolysaccharide (LPS)-treated chondrocytes were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and western blot. The effect of PEP-1-GRX1 on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) signaling pathway were also analyzed by western blot. Finally, the inhibitory effect of PEP-1-GRX-1 on MMP-13 production was measured in vivo in a mouse carrageenan-induced paw edema model. Results: PEP-1-GRX-1 significantly penetrated into human chondrocytes and mouse cartilage, whereas GRX-1 did not. PEP-1-GRX-1 significantly suppressed MMP-13 expression and nitric oxide (NO) production in LPS-stimulated chondrocytes, and NO production in IL-1β-stimulated chondrocytes, compared with GRX-1. In addition, PEP-1-GRX-1 decreased IL-1β- and LPS-induced activation of MAPK and NF-κB. In the mouse model of carrageenan-induced paw edema, PEP-1-GRX-1 significantly suppressed carrageenan-induced MMP-13 production as well as paw edema. Conclusion: These results demonstrate that PEP-1-GRX-1 can be transduced efficiently in vitro and in vivo into human chondrocytes and mouse cartilage tissue and downregulate catabolic responses in chondrocytes by inhibiting the MAPK and NF-κB pathway. PEP-1-GRX-1 thus has the potential to reduce catabolic responses in chondrocytes and cartilage.

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“…Grx1 has been found to promote transcription of pro-inflammatory genes via deglutathionylation of members of the pro-inflammatory NFκB transcription pathway (reviewed in [ 1 , 2 ]). Grx1 has been implicated as a positive regulator of inflammation in numerous contexts, such as diabetic retinopathy, cigarette smoke-induced inflammation and allergic airway response, and microglial activation [ 4 – 6 ]. Adenoviral overexpression of Grx1 alone; i.e., in the absence of pro-inflammatory stimuli, has been shown to increase release of pro-inflammatory markers from model retinal glial cells, epithelial cells, and microglial cells [ 4 – 6 ].…”

Section: Introductionmentioning

confidence: 99%

“…Grx1 has been implicated as a positive regulator of inflammation in numerous contexts, such as diabetic retinopathy, cigarette smoke-induced inflammation and allergic airway response, and microglial activation [ 4 – 6 ]. Adenoviral overexpression of Grx1 alone; i.e., in the absence of pro-inflammatory stimuli, has been shown to increase release of pro-inflammatory markers from model retinal glial cells, epithelial cells, and microglial cells [ 4 – 6 ]. Moreover, Grx1 is upregulated by various inflammatory stimuli in peripheral immune and epithelial cells [ 7 – 9 ], and in microglia [ 6 ], thereby potentially creating a feed-forward loop of inflammatory propagation.…”

Section: Introductionmentioning

confidence: 99%

“…Adenoviral overexpression of Grx1 alone; i.e., in the absence of pro-inflammatory stimuli, has been shown to increase release of pro-inflammatory markers from model retinal glial cells, epithelial cells, and microglial cells [ 4 – 6 ]. Moreover, Grx1 is upregulated by various inflammatory stimuli in peripheral immune and epithelial cells [ 7 – 9 ], and in microglia [ 6 ], thereby potentially creating a feed-forward loop of inflammatory propagation. Grx1 silencing inhibits pro-inflammatory cytokine release in both cell culture and animal models of inflammatory disease [ 4 , 5 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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

Abstract: In vitro and in vivo data suggest Grx1 upregulation promotes neurotoxic neuroinflammation, potentially contributing to PD. Antioxid. Redox Signal. 25, 967-982.

Cited by 36 publications

References 55 publications

Palmitate Induces an Anti-Inflammatory Response in Immortalized Microglial BV-2 and IMG Cell Lines that Decreases TNFα Levels in mHypoE-46 Hypothalamic Neurons in Co-Culture

Palmitate Induces an Anti-Inflammatory Response in Immortalized Microglial BV-2 and IMG Cell Lines that Decreases TNFα Levels in mHypoE-46 Hypothalamic Neurons in Co-Culture

PEP-1-GRX-1 Modulates Matrix Metalloproteinase-13 and Nitric Oxide Expression of Human Articular Chondrocytes

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

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