Basal activation of astrocytic Nrf2 in neuronal culture media: Challenges and implications for neuron-astrocyte modelling.

The transcription factor Nrf2 is a stress-responsive master regulator of antioxidant, detoxification and proteostasis genes. In astrocytes, Nrf2-dependent gene expression drives cell-autonomous cytoprotection and also non-cell-autonomous protection of nearby neurons, and can ameliorate pathology in several acute and chronic neurological disorders associated with oxidative stress. However, the value of astrocytic Nrf2 as a therapeutic target depends in part on whether Nrf2 activation by disease-associated oxidative stress occludes the effect of any Nrf2-activating drug. Nrf2 activation classically involves the inhibition of interactions between Nrf2's Neh2 domain and Keap1, which directs Nrf2 degradation. Keap1 inhibition is mediated by the modification of cysteine residues on Keap1, and can be triggered by electrophilic small molecules such as tBHQ. Here we show that astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling. Keap1 deficiency elevates basal Nrf2 target gene expression in astrocytes and occludes the effects of tBHQ, oxidative stress still induced strong Nrf2-dependent gene expression in Keap1-deficient astrocytes. Moreover, while tBHQ prevented protein degradation mediated via Nrf2's Neh2 domain, oxidative stress did not, consistent with a Keap1-independent mechanism. Moreover the effects of oxidative stress and tBHQ on Nrf2 target gene expression are additive, not occlusive. Mechanistically, oxidative stress enhances the transactivation potential of Nrf2's Neh5 domain in a manner dependent on its Cys-191 residue. Thus, astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling, meaning that further Nrf2 activation by Keap1-inhibiting drugs may be a viable therapeutic strategy.

Astrocytes may modulate the survival of motor neurons in amyotrophic lateral sclerosis (ALS). We have previously shown that fibroblast growth factor-1 (FGF-1) activates astrocytes to increase secretion of nerve growth factor (NGF). NGF in turn induces apoptosis in co-cultured motor neurons expressing the p75 neurotrophin receptor (p75NTR) by a mechanism involving nitric oxide (NO) and peroxynitrite formation. We show here that FGF-1 increased the expression of inducible nitric oxide synthase and NO production in astrocytes, making adjacent motor neurons vulnerable to NGF-induced apoptosis. Spinal cord astrocytes isolated from transgenic SOD1G93A rats displayed increased NO production and spontaneously induced apoptosis of co-cultured motor neurons. FGF-1 also activates the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in astrocytes. Because Nrf2 increases glutathione (GSH) biosynthesis, we investigated the role of GSH production by astrocytes on p75NTR-dependent motor neuron apoptosis. The combined treatment of astrocytes with FGF-1 and t-butylhydroquinone (tBHQ) increased GSH production and secretion, preventing motor neuron apoptosis. Moreover, Nrf2 activation in SOD1G93A astrocytes abolished their apoptotic activity. The protection exerted by increased Nrf2 activity was overcome by adding the NO donor DETA-NONOate to the co-cultures or by inhibiting GSH synthesis and release from astrocytes. These results suggest that activation of Nrf2 in astrocytes can reduce NO-dependent toxicity to motor neurons by increasing GSH biosynthesis.

Activation of the transcription factor Nrf2 in astrocytes coordinates the upregulation of antioxidant defenses and confers protection to neighboring neurons. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. Non-neuronal cells, including astrocytes, shape motor neuron survival in ALS and are a potential target to prevent motor neuron degeneration. The protective effect of Nrf2 activation in astrocytes has never been examined in a chronic model of neurodegeneration. We generated transgenic mice over-expressing Nrf2 selectively in astrocytes using the glial fibrillary acidic protein (GFAP) promoter. The toxicity of astrocytes expressing ALS-linked mutant hSOD1 to cocultured motor neurons was reversed by Nrf2 over-expression. Motor neuron protection depended on increased glutathione secretion from astrocytes. This protective effect was also observed by crossing the GFAP-Nrf2 mice with two ALS-mouse models. Over-expression of Nrf2 in astrocytes significantly delayed onset and extended survival. These findings demonstrate that Nrf2 activation in astrocytes is a viable therapeutic target to prevent chronic neurodegeneration.

In this study, we investigated whether nuclear factor erythroid 2-related factor 2 (Nrf2) activation in astrocytes contributes to the neuroprotection induced by a single hyperbaric oxygen preconditioning (HBO-PC) against spinal cord ischemia/reperfusion (SCIR) injury. In vivo: At 24 h after a single HBO-PC at 2.5 atmospheres absolute for 90 min, the male ICR mice underwent SCIR injury by aortic cross-clamping surgery and observed for 48 h. HBO-PC significantly improved hindlimb motor function, reduced secondary spinal cord edema, ameliorated the reactivity of spinal motor-evoked potentials, and slowed down the process of apoptosis to exert neuroprotective effects against SCIR injury. At 12 h or 24 h after HBO-PC without aortic cross-clamping surgery, Western blot, enzyme-linked immunosorbent assay, realtime-polymerase chain reaction and double-immunofluorescence staining were used to detect the Nrf2 activity of spinal cord tissue, such as mRNA level, protein content, DNA binding activity, and the expression of downstream gene, such as glutamate-cysteine ligase, γ-glutamyltransferase, multidrug resistance protein 1, which are key proteins for intracellular glutathione synthesis and transit. The Nrf2 activity and downstream genes expression were all enhanced in normal spinal cord with HBO-PC. Glutathione content of spinal cord tissue with HBO-PC significantly increased at all time points after SCIR injury. Moreover, Nrf2 overexpression mainly occurs in astrocytes. In vitro: At 24 h after HBO-PC, the primary spinal astrocyte-neuron co-cultures from ICR mouse pups were subjected to oxygen-glucose deprivation (OGD) for 90 min to simulate the ischemia-reperfusion injury. HBO-PC significantly increased the survival rate of neurons and the glutathione content in culture medium, which was mainly released from asctrocytes. Moreover, the Nrf2 activity and downstream genes expression induced by HBO-PC were mainly enhanced in astrocytes, but not in neurons. In conclusion, our findings demonstrated that spinal cord ischemic tolerance induced by HBO-PC may be mainly related to Nrf2 activation in astrocytes.

Inhibition of cytochrome P450 2E1 and activation of transcription factor Nrf2 are renoprotective in myoglobinuric acute kidney injury

Activation of Nrf2 signaling as a common treatment of neurodegenerative diseases.

Objectives: Nuclear factor erythroid 2-related factor (Nrf2) in astrocyte plays important roles in brain homeostasis. Fermented papaya preparation (FPP) has anti-oxidative, anti-inflammatory, immunoregulatory properties. The present study investigated the effects of FPP on activation of Nrf2 and release of Nrf2-regulated neuroprotective antioxidants and detoxifying molecules.Methods: Primary cultured astrocytes from rat embryos were treated with FPP for 6 or 24 hours. The expression levels of nuclear Nrf2 and cytoplasmic Nrf2-regulated molecules were determined by western blot analysis and immunohistochemistry. Glutathione levels were measured in cells and medium. Dopaminergic neurons were exposed 6-hydroxydopamine (6-OHDA) with/without pre-treatment with FPP astrocytes. Mice were treated orally with FPP for 2 weeks.Results: FPP increased nuclear translocation of Nrf2 in striatal astrocytes, induced up-regulation of NAD(P)H quinine oxidoreductase-1, glutathione-S transferase and hemeoxygenase-1, and increased glutathione level and the percentage of metallothionein-expressing astrocytes. Moreover, FPP suppressed 6-OHDA-induced dopaminergic neuronal loss in not only neuron-astrocyte mixed culture, but also neuron-rich cultures pre-treated with glial conditioned medium. Two-week oral treatment of mice with FPP resulted in Nrf2 activation and increase in glutathione level in striatum.Discussion: The results indicated that FPP enhances the anti-oxidative capacity through activation of Nrf2 in astrocytes, suggesting it may provide neuroprotection in oxidative stress-related neurodegenerative diseases.

Arsenic trioxide (As2O3) is a potent drug for the treatment of acute promyelocytic leukemia (APL) and has achieved remarkable remissions in patients. Unfortunately, clinical reports have shown that the treatment is associated with cardiotoxicity. Many efforts have been made to mitigate drug-mediated cardiac damage using naturally occurring antioxidant compounds possessing free radical scavenging activity. The present investigation aims to explore protective role of L-ascorbic acid (L-AA) and α-tocopherol (α-TOC) from As2O3-induced oxidative stress in H9c2 cardiomyocytes through the evaluation of Nrf2 (nuclear factor erythroid 2-related factor 2) and Bcl-2 (B-cell lymphoma 2) transcription factors. The in vitro study was conducted using H9c2 cardiomyocytes. The evaluation of total antioxidant capacity, mitochondrial membrane potential, cellular calcium concentration and reactive oxygen species generation was performed. Oxidative stress (Nrf2) and anti-apoptotic (Bcl2) signaling indicators were measured by reverse transcriptase polymerase chain reaction. A depletion of the total antioxidant capacity and mitochondrial transmembrane potential were observed in As2O3-treated cardiomyocytes. In addition, the cellular calcium concentration and ROS generation were found to be increased on treatment with As2O3 with the alterations in the activity of transcription factors, Nrf2 and Bcl2. Co-treatment of antioxidant vitamins with As2O3 resulted in a significant reversal of oxidative stress and alteration on the antioxidant defense through the activation of Nrf2 and Bcl2. L-AA and α-TOC alleviates As2O3-induced oxidative stress in cardiac cells by activating Nrf2 and Bcl2 transcription factors that results in increased cell survival and prevents apoptosis.

Pristane-induced lupus is associated with nonresolving inflammation and deficiency of proresolving macrophages. Proresolving nonclassic macrophages (NCMs) are less responsive to type I interferon (IFN) than classic macrophages (CMs; which are proinflammatory), reflecting their relative expression levels of the type I IFN receptor (IFNAR). This study was undertaken to investigate the regulation of IFNAR expression in macrophages. We carried out gene expression profiling of purified CMs and NCMs from mice treated with pristane (which develop lupus) or mineral oil (non-lupus controls). Macrophage differentiation and IFNAR expression were examined in mice treated with NF-E2-related factor 2 (Nrf2) activators and inhibitors and in Nrf2-deficient mice. Nrf2 activity was also assessed in blood cells from patients with systemic lupus erythematosus (SLE). Significant differences were determined by Student's t-test. RNA sequencing revealed increased expression of genes regulated by the transcription factor Nrf2 in NCMs from mineral oil-treated versus pristane-treated mice and in NCMs versus CMs. The Nrf2 activator CDDO-imidazole (CDDO-Im) decreased CMs (P < 0.0001) and promoted the development of proresolving NCMs (P = 0.06), whereas the Nrf2 inhibitor brusatol increased CMs (P < 0.05) and decreased NCMs (P < 0.001). CDDO-Im decreased Ifnar1 (P < 0.001) and IFN-stimulated gene (ISG) expression in macrophages and alleviated oxidative stress (P < 0.05), whereas brusatol had the opposite effect (P < 0.01). Moreover, Ifnar1 and ISG expression levels were higher in Nrf2-knockout mice than controls (P < 0.05). As seen in mice with lupus, SLE patients showed evidence of low Nrf2 activity. Our findings indicate that Nrf2 activation favors the resolution of chronic inflammation in lupus. Since autoantibody production and lupus nephritis depend on IFNAR signaling, the ability of Nrf2 activators to repolarize macrophages and reduce the INF signature suggests that these agents may warrant consideration for treating lupus.

Acute expression of the transcription factor Nrf2 after treatment with quinolinic acid is not induced by oxidative stress in the rat striatum

The oxidative-stress-induced impairment of autophagy plays a critical role in the pathogenesis of Parkinson’s disease (PD). In this study, we investigated whether the alteration of Nrf2 in astrocytes protected against 6-OHDA (6-hydroxydopamine)- and rotenone-induced PD-like phenotypes, using 6-OHDA-induced rat PD and rotenone-induced Drosophila PD models. In the PD rat model, we found that Nrf2 expression was significantly higher in astrocytes than in neurons. CDDO-Me (CDDO methyl ester, an Nrf2 inducer) administration attenuated PD-like neurodegeneration mainly through Nrf2 activation in astrocytes by activating the antioxidant signaling pathway and enhancing autophagy in the substantia nigra and striatum. In the PD Drosophila model, the overexpression of Nrf2 in glial cells displayed more protective effects than such overexpression in neurons. Increased Nrf2 expression in glial cells significantly reduced oxidative stress and enhanced autophagy in the brain tissue. The administration of the Nrf2 inhibitor ML385 reduced the neuroprotective effect of Nrf2 through the inhibition of the antioxidant signaling pathway and autophagy pathway. The autophagy inhibitor 3-MA partially reduced the neuroprotective effect of Nrf2 through the inhibition of the autophagy pathway, but not the antioxidant signaling pathway. Moreover, Nrf2 knockdown caused neurodegeneration in flies. Treatment with CDDO-Me attenuated the Nrf2-knockdown-induced degeneration in the flies through the activation of the antioxidant signaling pathway and increased autophagy. An autophagy inducer, rapamycin, partially rescued the neurodegeneration in Nrf2-knockdown Drosophila by enhancing autophagy. Our results indicate that the activation of the Nrf2-linked signaling pathways in glial cells plays an important neuroprotective role in PD models. Our findings not only provide a novel insight into the mechanisms of Nrf2–antioxidant–autophagy signaling, but also provide potential targets for PD interventions.

Genetic Alteration of Keap1 Confers Constitutive Nrf2 Activation and Resistance to Chemotherapy in Gallbladder Cancer

Nrf2 promotes neuronal cell differentiation

The expression of phase-II detoxification and antioxidant enzymes is governed by a cis-acting regulatory element named the antioxidant response element (ARE). ARE-containing genes are regulated by the nuclear factor erythroid-2-related factor 2 (Nrf2), a member of the Cap'n'Collar basic-leucine-zipper family of transcription factors. ARE-regulated genes are preferentially activated in astrocytes, which consequently have more efficient detoxification and antioxidant defences than neurons. Astrocytes closely interact with neurons to provide structural, metabolic and trophic support, as well as actively participating in the modulation of neuronal excitability and neurotransmission. Therefore, functional alterations in astrocytes can shape the interaction with surrounding cells, such as neurons and microglia. Activation of Nrf2 in astrocytes protects neurons from a wide array of insults in different in vitro and in vivo paradigms, confirming the role of astrocytes in determining the vulnerability of neurons to noxious stimuli. Here, we review the current data supporting Nrf2 activation in astrocytes as a viable therapeutic approach, not only in acute neuronal damage, but also in chronic neurodegeneration related to oxidative stress.

Inorganic arsenic is a well-documented human carcinogen that targets the skin. The induction of oxidative stress, as shown with arsenic, may have a bearing on the carcinogenic mechanism of this metalloid. The transcription factor Nrf2 is a key player in the regulation of genes encoding for many antioxidative response enzymes. Thus, the effect of inorganic arsenic (as sodium arsenite) on Nrf2 expression and localization was studied in HaCaT cells, an immortalized human keratinocyte cell line. We found, for the first time, that arsenic enhanced cellular expression of Nrf2 at the transcriptional and protein levels and activated expression of Nrf2-related genes in these cells. In addition, arsenic exposure caused nuclear accumulation of Nrf2 in association with downstream activation of Nrf2-mediated oxidative response genes. Arsenic simultaneously increased the expression of Keap1, a regulator of Nrf2 activity. The coordinated induction of Keap1 expression and nuclear Nrf2 accumulation induced by arsenic suggests that Keap1 is important to arsenic-induced Nrf2 activation. Furthermore, when cells were pretreated with scavengers of hydrogen peroxide (H 2O 2) such as catalase–polyethylene glycol (PEG-CAT) or Tiron, arsenic-induced nuclear Nrf2 accumulation was suppressed, whereas CuDIPSH, a cell-permeable superoxide dismutase (SOD) mimic compound that produces H 2O 2 from superoxide (·O 2 −), enhanced Nrf2 nuclear accumulation. These results indicate that H 2O 2, rather than ·O 2 −, is the mediator of nuclear Nrf2 accumulation. Additional study showed that arsenic causes increased cellular H 2O 2 production and that H 2O 2 itself has the ability to increase Nrf2 expression at both the transcription and protein levels in HaCaT cells. Taken together, these data clearly show that arsenic increases Nrf2 expression and activity at multiple levels and that H 2O 2 is one of the mediators of this process.