HIV proteins (gp120 and Tat) and methamphetamine in oxidative stress-induced damage in the brain: potential role of the thiol antioxidant N-acetylcysteine amide

Abstract

An increased risk of HIV-1 associated dementia (HAD) has been observed in patients abusing methamphetamine (METH). Since both HIV viral proteins (gp120, Tat) and METH induce oxidative stress, drug abusing patients are at a greater risk of oxidative stress-induced damage. The objective of this study was to determine if N-acetylcysteine amide (NACA) protects the blood brain barrier (BBB) from oxidative stress-induced damage in animals exposed to gp120, Tat and METH. To study this, CD-1 mice pre-treated with NACA/saline, received injections of gp120, Tat, gp120+Tat or saline for 5days, followed by three injections of METH/saline on the fifth day, and sacrificed 24h after the final injection. Various oxidative stress parameters were measured, and animals treated with gp120+Tat+Meth were found to be the most challenged group, as indicated by their GSH and MDA levels. Treatment with NACA significantly rescued the animals from oxidative stress. Further, NACA-treated animals had significantly higher expression of TJ proteins and BBB permeability as compared to the group treated with gp120+Tat+METH alone, indicating that NACA can protect the BBB from oxidative stress-induced damage in gp120, Tat and METH exposed animals, and thus could be a viable therapeutic option for patients with HAD.

Fig 1

Schematic representation of the study protocol. Male CD1 mice were injected with gp120 and Tat for 5 consecutive days. Animals in the methamphetamine treated group were injected with 3 doses, at 2 h intervals on the fifth day. All of the animals were pretreated with either NACA or saline, 30 min before exposure to HIV viral proteins and methamphetamine, as mentioned in the Materials and Methods Section. The mice were sacrificed by urethane injection 24 h after the last methamphetamine injection.

Fig 2

Glutathione levels in the brain. The GSH levels in the brain of CD1 mice exposed to (A) HIV viral proteins (gp120 and Tat), methamphetamine and the antioxidant NACA for 5 days as described in the Materials and Methods section. (B) Comparison of the GSH levels in the brain of animals treated with only NACA and heat inactivated gp120 and Tat.* Values significantly different from the control. # Values significantly different from the gp120+Tat+METH treated group.

Fig 3

Glutathione peroxidase (GPx) levels in the brain. GPx levels in the brain of mice treated with HIV viral proteins (gp120 and Tat), methamphetamine and the antioxidant NACA for 5 days as described in the Materials and the Methods section. * Values significantly different from the control. # Values significantly different from the gp120+Tat+METH treated group.

Fig 4

Lipid peroxidation in the brain. MDA levels in the brain of mice treated with HIV viral proteins (gp120 and Tat), methamphetamine and the antioxidant NACA for 5 days as described in the Materials and the Methods section. * Values significantly different from the control. # Values significantly different from the gp120+Tat+METH treated group.

Fig 5

Protein carbonyl levels in the brain. Protein carbonyl was measured as an indicator of protein oxidation in the brain of mice treated with HIV viral proteins (gp120 and Tat), methamphetamine and the antioxidant NACA for 5 days as described in the Materials and the Methods section. * Values significantly different from the control. # Values significantly different from the gp120+Tat+METH treated group. ## Values significantly different from the gp120+Tat treated group.

Fig 6

Effect of HIV viral proteins and methamphetamine on tight junction proteins. Shown are the representative western blots of ZO1 (A), ZO2 (B), Occludin (C) and Claudin5 (D) proteins in total brain homogenates of CD1 mice treated with HIV viral proteins (gp120 and Tat), Methamphetamine and the thiol antioxidant NACA. The graphs represent relative densitometric analysis of treated animals over the controls. The results are representative of three individual experiments. * Values significantly different from the control. ! Values significantly different from the gp120+Tat+METH treated group.

Fig 6

Effect of HIV viral proteins and methamphetamine on tight junction proteins. Shown are the representative western blots of ZO1 (A), ZO2 (B), Occludin (C) and Claudin5 (D) proteins in total brain homogenates of CD1 mice treated with HIV viral proteins (gp120 and Tat), Methamphetamine and the thiol antioxidant NACA. The graphs represent relative densitometric analysis of treated animals over the controls. The results are representative of three individual experiments. * Values significantly different from the control. ! Values significantly different from the gp120+Tat+METH treated group.

Fig 7

Effect of HIV viral proteins and methamphetamine on blood brain barrier permeability. CD1 mice pretreated with NACA or saline, were injected with HIV viral proteins (gp120 and Tat) and methamphetamine for 5 days. BBB permeability was evaluated by administration of the sodium fluorescein (Na-F) tracer (i.p), as described in the Materials and Methods section. After perfusion, the level of Na-F in the brain tissue was measured. The results are expressed as ng of Na-F/ gm of brain tissue. * Values significantly different from the control and NACA-alone group. # Values significantly different from the gp120+Tat+METH+NACA treated group.

Fig 8

Immunoprecipitation assay to detect the oxidative modification of the tight junction protein Occludin. Total brain homogenates were immunoprecipitated with Occludin antibody, separated by SDS-PAGE and immunoblotted with anti rabbit HNE and 3NT antibodies as mentioned in the Materials and Methods section. IgG was used as a negative control, and Occludin served as the positive control. Data shown are representative of three independent experiments.

Fig 9

Immunoprecipitation assay to detect the oxidative modification of the tight junction protein Claudin 5. Total brain homogenates were immunoprecipitated with Claudin 5 antibody, separated by SDS-PAGE and immunoblotted with anti rabbit HNE and 3NT antibodies as mentioned in the Materials and Methods section. IgG was used as a negative control, and Claudin 5 served as the positive control. Data shown are representative of three independent experiments.

Fig 10

Schematic representation of the mechanism of HIV viral proteins and methamphetamine-induced oxidative damage to the blood brain barrier, and the protective role of the thiol antioxidant N-acetylcysteineamide (NACA). HIV viral protein gp120 and Tat, along with methamphetamine, synergistically increases oxidative stress induced damage by lowering the level of antioxidant enzymes GSH and GPx and increasing oxidative modification of proteins and lipids in the brain. This leads to decrease in the expression of tight junction proteins in the blood brain barrier (BBB), as a result of which the BBB permeability increases. Further, oxidative modification of the tight junction proteins also aids in increase in permeability of the BBB, leading to increased passage of toxins and leukocytes in to the brain leading to severe dementia in HIV patients abusing methamphetamine. However, pretreatment with the novel antioxidant, NACA partially restores the oxidative balance in the brain and maintains the BBB permeability, thus protecting the brain from toxins and inflammation.