Neuroprotective effects of the drug GVT (monosodium luminol) are mediated by the stabilization of Nrf2 in astrocytes

Abstract

Oxidative stress is implicated in various kinds of neurological disorders, including human immunodeficiency virus (HIV) associated dementia (HAD). Our laboratory has been studying the murine retrovirus ts1, a pathogenic mutant of the Moloney murine leukemia virus (MoMuLV), as a model for HAD. Like HIV in humans, ts1 induces oxidative stress and progressive neurodegeneration in mice. We have shown previously that an antioxidant and anti-inflammatory drug GVT or MSL (monosodium luminol) suppresses ts1-induced oxidative stress, attenuates the development of spongiform encephalopathy, and delays hind limb paralysis in infected mice. It is known that upregulation of the nuclear transcription factor NF-E2-related factor 2 (Nrf2) is involved in upregulating cellular antioxidant defenses. Since Nrf2 is associated with elevation of antioxidant defenses in general, and since GVT suppresses ts1-induced neurodegeneration, our aim in this study was to determine whether GVT neuroprotection is linked to Nrf2 upregulation in the brain. We report here that GVT upregulates the levels of Nrf2, both in primary astrocyte cultures and in brainstem of ts1-infected mice. Significant upregulation of Nrf2 expression by GVT occurs in both the cytosolic and nuclear fractions of cultured astrocytes and brainstem cells. Notably, although GVT treatment increases Nrf2 protein levels in cultured astrocytes and brainstem tissues, Nrf2 mRNA levels are not altered. This suggests that the neuroprotective effects of GVT may be mediated by the stabilization of the Nrf2 protein, allowing continuous upregulation of Nrf2 levels in the astrocytes.

Fig.1. Upregulation of Nrf2 by GVT and tBHQ in C1 astrocytes

C1 astrocytes were treated with various concentrations of GVT (0.2, 5 and 20 μg/ml) for 16h after which the levels of Nrf2 protein were analyzed. The western blots show that GVT treatment resulted in a upregulation and accumulation of Nrf2. Treatment with tertiary-butyl hydroquinone (tBHQ), a known positive regulator of Nrf2 (10 and 20 μg/ml), also resulted in upregulation of Nrf2 (A). Quantification of the band intensities of Nrf2 normalized against actin (B). Data in (B) were obtained from three independent experiments and analysis of variance was done by employing Tukey test. *p<0.05 vs. control was considered statistically significant. Error bars represent mean ± SEM.

Fig.2. GVT induces Nrf2 upregulation in primary astrocyte cultures

Primary cultures of astrocytes derived from mouse pups were exposed to GVT for 2-16 h and then immunoblotted to compare their Nrf2 content. GVT treatment resulted in increased expression of Nrf2 as early as 2h and this increase in Nrf2 levels was maintained up to 16h of post treatment (A). Nrf2 band was quantified and normalized against actin (B). Data were obtained from three independent experiments and analysis of variance was done by employing Tukey test. *p<0.05 vs. control was considered statistically significant. Error bars represent mean ± SEM.

Fig.3. GVT increases Nrf2 levels in the cytosolic and nuclear fractions of cultured astrocytes

Primary astrocytes were treated with GVT for 0, 2 and 4 h. Following treatment, the cells were harvested and lysates of cytosolic and nuclear fractions were isolated. Immunoblots run against anti-Nrf2 showed a significant upregulation in the Nrf2 protein levels both in cytosol (A) as well as in nucleus (B). Quantification of the band intensities of Nrf2 normalized against actin and lamin in cytosol (lower panel of A) and nuclear fractions (lower panel of B) Data were obtained from three independent experiments and analysis of variance was done by employing Tukey test. *p<0.05 vs. control was considered statistically significant. Error bars represent mean ± SEM.

Fig.4

Primary cultured astrocytes were treated with GVT for 2, 4 and 8h, after which time their mRNA was isolated and the levels of Nrf2 mRNA quantitated by RT-PCR. There was no significant alteration in average ct values, indicative of levels of Nrf2-specific mRNAs, after GVT treatment. Three independent experiments and analysis of variance was done by employing Tukey test. *p<0.05 vs. control was considered statistically significant. Error bars represent mean ± SEM.

Fig.5. GVT treatment stabilizes existing Nrf2 protein, but does not increase de novo production of Nrf2

Primary cultured astrocytes were either left untreated or pretreated with GVT, followed by CHX (10μg/ml). In cells treated with CHX alone, loss of Nrf2 protein, presumably by proteasomal degradation in the absence of new protein synthesis, occurred within 2h (A). However, in cells pretreated with GVT, Nrf2 was levels remained elevated (B). The relative intensities of the Nrf2 protein bands were compared using densitometry followed by normalization of the band values (C). Data from (C) were obtained from three independent experiments (n=3). *p<0.05 vs. control was considered statistically significant. Error bars represent mean ± SEM.

Fig.6

Primary cultured astrocytes were pretreated with GVT followed by treatment with a proteasomal inhibitor MG-132. Treatment with GVT alone increased the Nrf2 protein to certain extent. However the MG132 treated cells expressed increased levels of Nrf2 protein (A). Quantification of the band intensities of Nrf2 normalized against actin (B). Data were obtained from three independent experiments and analysis of variance was done by employing Tukey test. *p<0.05 vs. control was considered statistically significant. Error bars represent mean ± SEM.

Fig.7

In lysates of brain tissue from ts1-infected untreated and ts1-infected GVT-treated mice, Western blots show a slight decrease in levels of cytoplasmic Nrf2 protein, compared to brainstem lysates from uninfected control mice. While increased levels of Nrf2 protein were present in the nuclear fractions isolated from the GVT-treated uninfected, ts 1-infected, and ts 1-infected-GVT treated mice, nuclear localization of Nrf2 was most evident in the ts1-infected, GVT-treated brainstem lysates. For these tissues, the ratio of Nrf2 in nuclear/Nrf2 in cytosol is increased by 2 fold in ts1-infected GVT-treated brainstems, relative to levels for control uninfected mouse tissues.

Fig.8. Increased levels of Nrf2 in the brainstem of ts1-infected vs. GVT-treated-ts1-infected mice

The photomicrographs show Nrf2 immunoreactivity (brown) in the cytoplasm and nucleus of neurons and glial cells in sectioned brainstem tissues from control (A), ts1-infected untreated (B) and GVT-treated-ts1-infected mice (C). Note the intense cytoplasmic and nuclear Nrf2 immunoreactivity in the neurons (blue arrows) and glial cells (green arrows) in the ts1-infected and GVT-treated-ts1-infected mice brainstems, compared with the same cells in sectioned uninfected mouse brainstem tissue. The black arrowhead is pointing at a blood vessel.