Cocaine and human immunodeficiency virus type 1 gp120 mediate neurotoxicity through overlapping signaling pathways

Abstract

Although it has been well documented that drugs of abuse such as cocaine cause enhanced progression of human immunodeficiency virus (HIV)-associated neuropathological disorders, the underlying mechanisms mediating these effects remain poorly understood. The present study demonstrated that exposure of rat primary neurons 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 both caspase-3 activity and expression of the proapoptotic protein Bax. Furthermore, increased neurotoxicity 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. Increased neurotoxicity mediated by cocaine and gp120 was ameliorated by NADPH oxidase inhibitor apocynin, thus underscoring the role of oxidative stress in this cooperation. Signaling pathways including c-jun N-teminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK), and nuclear factor (NF)-kappaB were also identified to be critical in the neurotoxicity induced by cocaine and gp120. These findings thus underscore the role of oxidative stress, mitochondrial and MAPK signal pathways in cocaine and HIV gp120-mediated neurotoxicity.

Figure 1

Effects of cocaine and gp120 on cell viability in rat neurons. (A) Effects 1 μM cocaine in the presence or absence of gp120 for 24 h on the cell survival of rat neurons using the MTT assay. All the data are presented as mean±SD of four individual experiments. *P<.05;** P<.01 versus control group; #P<.05 versus gp120 group; ^P<.05 versus cocaine group. (B) Rat primary neurons treated with cocaine and/or gp120 for 24 h were stained with Hoechst 33342 by fluorescence microscopy. (C) Quantification of Hoechst-positive cells as percentage of control untreated cells. All the data are presented as mean±SD of four individual experiments. *P<.05; **P<.01 versus control group; ##P<0.01 versus gp120 group; ^P<.05 versus cocaine group.

Figure 2

Effects of cocaine and gp120 exposure on caspase-3 activation in rat neurons. (A) Primary neurons treated with cocaine and/or gp120 for 24 h were monitored for caspase-3 activity assay in cell lysates. All data in these figures are presented as mean ± SD of four individual experiments. *P<.05; ***P<.001 versus control group; #P<.05 versus gp120 group; ^P<.05 versus cocaine group. (B) Primary neurons treated with cocaine and/or gp120 for 24 h were monitored for active caspase-3 by immunostaining using anti-cleaved caspase-3 anti-body. Immunocytochemical images showing active caspase-3 staining (left panel) and differential interference contrast (DIC; right panel) were captured using Nikon inverted fluorescence microscope TE02000-E. (C) Quantification of caspase-3-positive cells as percentage of control untreated cells using NIH ImageJ software. All the data are presented as mean ± SD of four individual experiments. *P<.05 versus control group; #P<.05 versus gp120 group; ^P<.05 versus cocaine group.

Figure 3

Cocaine enhanced gp120-mediated induction of Bax expression. Rat primary neurons were treated with gp120 and cocaine for varying times (5 to 30 min), followed by cell lysis and detection of proapoptotic (Bax) and antiapoptotic proteins (Bcl-xL) on a Western blot. Cells cotreated with both the agents demonstrated up-regulation of Bax protein, resulting in increased Bax/Bcl-xL ratio. Densitometry scan of the ratio of band intensities of Bax/Bcl-xL from three independent experiments. *P<.05; **P<.01 versus control group; #P<.05 versus gp120 group; ^P<.05 versus cocaine group.

Figure 4

Effects of cocaine and/or gp120 on intracellular ROS production in rat neurons. (A) Cells treated with cocaine in the absence or presence of gp120 for 3 h were assessed for production of ROS using DCFH-DA assay. Quantification of ROS fluorescence intensity in different treated groups. The data are presented as mean ± SD of four individual experiments. **P<.01; ***P<.001 versus control group; #P<.05; versus cocaine + gp120 group. (B) Inhibition of the NADPH oxidase by apocynin resulted in abrogation of cocaine and gp120 neurotoxicity. The data are presented as mean ± SD of four individual experiments. *P<.05 versus control group; #P<.05 versus cocaine+ gp120 group.

Figure 5

Effects of cocaine and/or gp120 on mitochondria membrane potential in rat neurons. (A) Cells treated with cocaine and/or gp120 for 18 h were assayed for mitochondrial membrane potential by staining with JC-1 dye. Cotreatment of cells with cocaine and/or gp120 exposure 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. (B) Quantification of Δ ψm expressed as a ratio of J-aggregate to JC-1 monomer (red:green) fluorescence intensity using fluorescence plate reader. *P<.05; **P<.01 versus control group; ##P<0.01 versus gp120 group; ^^P<0.01 versus cocaine group.

Figure 6

The JNK/MAPK pathway mediated cocaine- and/or gp120-induced neurotoxicity in rat primary neurons. (A) Western blot analysis of cytosolic lysates from cocaine and gp120–treated cells for varying times (5 to 30 min) using antibodies specific for the phosphorylated forms of JNK. An antibody against total JNK was used to reprobe the blots for normalization. Densitometry scan of the ratio of band intensities of pJNK/JNK from three different experiments. *P<.05 versus control group. (B) Inhibition of the JNK pathway by SP600125 resulted in abrogation of cocaine and gp120 neurotoxicity. Values are mean ± SD from three independent experiments. *P<.05 versus control group; #P<.05 versus cocaine + gp120 group.

Figure 7

The p38/MAPK pathway mediated cocaine- and/or gp120–mediated neurotoxicity. (A) Western blot analysis of cytosolic lysates from cocaine and gp120-treated cells for varying times (5 to 30 min) using antibodies specific for the phosphorylated forms of p38. An antibody against total p38 was used to reprobe the blots for normalization. Densitometric scan of the ratio of band intensities of p38/p38 from three different experiments. *P<.05; **P<.01 versus control group. (B) Inhibition of the pp38/MAPK pathway by SB203580 resulted in abrogation of cocaine and gp120 neurotoxicity. Values are mean ± SD from three independent experiments. *P<.05 versus control group; #P<.05 versus cocaine + gp120 group.

Figure 8

The ERK/MAPK pathway mediated cocaine- and/or gp120-induced neurotoxicity. (A) Western Blot analysis of cytosolic lysates from cocaine and gp120–treated cells for varying times (5 to 30 min) using antibodies specific for the phosphorylated forms of ERK. An antibody against total ERK was used to reprobe the blots for normalization. Densitometry scan of the ratio of band intensities of pERK/ERK from three independent experiments. *P<.05; **P<.01; ***P<.001 versus control group. (B) Inhibition of the ERK pathway by U0126 resulted in abrogation of cocaine and gp120 neurotoxicity. Values are mean ± SD from three independent experiments. *P<.05 versus control group; #P<.05 versus cocaine + gp120 group.

Figure 9

NF-κB plays a role in the cocaine and gp120–induced neurotoxicity. (A) Western Blot analysis of nuclear extracts from cocaine and gp120–treated cells for varying times (5 to 60 min) using an antibody specific for the p65 subunit of NF-κB. (B) Rat primary neurons grown on coverslips were treated with cocaine and gp120 for 30 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 confocal microscopy (magnification × 250). After 30 min, nuclear translocation of NF-κB was clearly evident in the treated cells, as show by the green (NF-κB) and blue (DAPI) stains overlapping. (C) Inhibition of the NF-κB using the specific inhibitor TPCK resulted in abrogation of cocaine and gp120 neurotoxicity. Values are mean ± SD from three independent experiments. *P<.05 versus control group; ##P<.01 versus cocaine + gp120 group.

Figure 10

Schematic illustration of signaling pathways involved in the neurotoxicity mediated by cocaine and gp120 in rat primary neurons. Treatment of primary neurons with cocaine and gp120 results in increased intracellular ROS production, mitochondrial membrane potential loss, increased proapoptotic protein (Bax) expression, and increased caspase-3 activation. Other signaling pathways activated by both gp120 and cocaine involve the JNK, p38, and ERK/MAPK pathways converging in the activation of NF-κB and ultimately culminating into neuronal death.