Cocaine potentiates astrocyte toxicity mediated by human immunodeficiency virus (HIV-1) protein gp120

Abstract

It is becoming widely accepted that psychoactive drugs, often abused by HIV-I infected individuals, can significantly alter the progression of neuropathological changes observed in HIV-associated neurodegenerative diseases (HAND). The underlying mechanisms mediating these effects however, remain poorly understood. In the current study, we explored whether the psychostimulant drug cocaine could exacerbate toxicity mediated by gp120 in rat primary astrocytes. Exposure to both cocaine and gp120 resulted in increased cell toxicity compared to cells treated with either factor alone. The combinatorial toxicity of cocaine and gp120 was accompanied by an increase in caspase-3 activation. In addition, increased apoptosis of astrocytes in the presence of both the agents was associated with a concomitant increase in the production of intracellular reactive oxygen species and loss of mitochondrial membrane potential. Signaling pathways including c-jun N-teminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK), and nuclear factor (NF-κB) were identified to be major players in cocaine and gp120-mediated apoptosis of astrocytes. Our results demonstrated that cocaine-mediated potentiation of gp120 toxicity involved regulation of oxidative stress, mitochondrial membrane potential and MAPK signaling pathways.

Figure 1. Effects of cocaine and/or gp120 on cell viability in rat primary astrocytes.

(A) Effects of 1, 10, 100 µM cocaine in the presence or absence of gp120 IIIB (200 ng/ml) for 48 h on the survival of rat primary astrocytes using the MTT assay. (B) Effects of gp120 IIIB, heated inactivated gp120 IIIB and gp120 Bal (200 ng/ml) on the survival of rat primary astrocytes using the MTT assay. All the data are presented as mean ± SD of four individual experiments. *p<0.05; **p<0.01; ***p<0.001 versus control group; # p< 0.05 versus cocaine group; ∧ p< 0.05 versus gp120 group.

Figure 2. Effects of cocaine and/or gp120 exposure on caspase-3 activation in rat primary astrocytes.

(A) Rat primary astrocytes treated with cocaine (10 µM) and/or gp120 IIIB (200 ng/ml) for 24 h were monitored for active caspase-3 by immunostaining using anti-cleaved caspase-3 antibody. Scale bars indicate 20 µm. (B) Rat primary astrocytes were treated with cocaine and/or gp120 for 24 h, followed by cell lysis and detection of anti-cleaved caspase-3 protein on a Western blot. (C) Densitometry scans of the ratio of band intensities of cleaved caspase-3/β-actin from three separate experiments. * p<0.05 versus control group; # p< 0.05 versus cocaine group; ∧p< 0.05 versus gp120 group.

Figure 3. Effects of cocaine and/or gp120 on intercellular ROS production in rat astrocytes.

(A) Cells treated with cocaine in the absence or presence of gp120 for 1 h were assessed for production of ROS using DCFH-DA assay. Treatment of astrocytes with cocaine and/or gp120 resulted in increased ROS production. Scale bars indicate 200 µm. (B) Quantification of ROS fluorescence intensity in cells treated with gp120 and/or cocaine using fluorescence plate reader. All the data are presented as mean ± SD of four individual experiments. ** p<0.01; ***p<0.001 versus control group; ## p< 0.01 versus cocaine group; ∧∧ p< 0.01 versus gp120 group.

Figure 4. Effects of cocaine and/or gp120 on mitochondrial membrane potential in rat astrocytes.

(A) Cells treated with cocaine and/or gp120 for 18 h were assayed for mitochondrial membrane potential by staining with JC-1 dye. Treatment with cocaine/gp120 resulted in reduction of the aggregation of JC-1 dye in the mitochondria (red fluorescence) and decreased ratio of the aggregate (red fluorescence) to monomer JC-1 (green fluorescence) in the cells. Scale bars indicate 200 µm. (B) Quantification of Δψm expressed as a ratio of J-aggregate to JC-1 monomer (red∶ green) fluorescence intensity. All the data are presented as mean±SD of three individual experiments. ** p<0.01; ***p<0.001 versus control group; #p< 0.05 versus cocaine group; ∧p< 0.05 versus gp120 group.

Figure 5. Cocaine and gp120 induced phosphorylation of MAPKs.

Western blot analysis of time-dependent activation of ERK, JNK and p38 kinases in astrocytes treated with cocaine and gp120. Representative immunoblots are presented from 4 separate experiments.

Figure 6. Involvement of MAPK pathway in cocaine and/or gp120-induced toxicity in rat primary astrocytes.

(A–C) Western blot analysis of cytosolic lysates from cocaine (10 µM) and/or gp120 IIIB (200 ng/ml) or heat-inactivated gp120 IIIB)-treated cells (15 min) using antibodies specific for the phosphorylated forms of JNK, p38, ERK/MAPK respectively. (D–F) Densitometric analyses of phos-JNK/β-actin, phos-p38/β-actin, phos-ERK/ERK from three separate experiments. Representative immunoblot and the densitometric analysis of phos-JNK/β-actin, phos-p38/β-actin, phos-ERK/ERK from three separate experiments is presented. All the data in these figures are presented as mean±SD of three individual experiments. **p<0.01; ***p<0.001 versus control group; #p< 0.05, ## p<0.01 versus cocaine group; ∧ p<0.05 versus gp120 group. (G–I) Pharmacological inhibition of the MAPK pathway in astrocytes resulted in abrogation of cocaine and gp120-induced toxicity. All the data are presented as mean±SD from three individual experiments. **p<0.01 versus control group. #p<0.05, ##p<0.01 versus cocaine + gp120 group.

Figure 7. NF-κB is involved in cocaine and gp120-induced astrocyte apoptosis.

(A) Rat primary astrocytes grown on coverslips were treated with cocaine (10 µM) and/or gp120 IIIB (200 ng/ml) for 15 min and stained with an anti-NF-κB p65 antibody, followed by treatment with an Alexa Flour 488-conjugated secondary antibody. Slides were mounted in Slow Fade antifade reagent (with DAPI, blue nuclear stain) and images were captured by fluorescence microscope. Scale bars indicate 20 µm. (B) Western Blot analysis of nuclear extracts from cocaine and gp120-treated cells for varying times (5 to 60 min) using an antibody specific for p65 subunit of NF-κB. Representative immunoblot and the densitometric analysis of p NF-κB/histone from three separate experiments is presented. All the data in these figures are presented as mean±SD of three individual experiments. *p<0.05 versus control group. (C) Inhibition of NF-κB using the specific inhibitor TPCK (10 µM) resulted in abrogation of cocaine and gp120 toxicity. **p<0.01 versus control group. # p<0.05 versus cocaine + gp120 group.