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, 65, 11-20

GSK3β-activation Is a Point of Convergence for HIV-1 and Opiate-Mediated Interactive Neurotoxicity

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GSK3β-activation Is a Point of Convergence for HIV-1 and Opiate-Mediated Interactive Neurotoxicity

Ruturaj R Masvekar et al. Mol Cell Neurosci.

Abstract

Infection of the CNS with HIV-1 occurs rapidly after primary peripheral infection. HIV-1 can induce a wide range of neurological deficits, collectively known as HIV-1-associated neurocognitive disorders. Our previous work has shown that the selected neurotoxic effects induced by individual viral proteins, Tat and gp120, and by HIV(+) supernatant are enhanced by co-exposure to morphine. This mimics co-morbid neurological effects observed in opiate-abusing HIV(+) patients. Although there is a correlation between opiate drug abuse and progression of HIV-1-associated neurocognitive disorders, the mechanisms underlying interactions between HIV-1 and opiates remain obscure. Previous studies have shown that HIV-1 induces neurotoxic effects through abnormal activation of GSK3β. Interestingly, expression of GSK3β has shown to be elevated in brains of young opiate abusers indicating that GSK3β is also linked to neuropathology seen with opiate-abusing patients. Thus, we hypothesize that GSK3β activation is a point of convergence for HIV- and opiate-mediated interactive neurotoxic effects. Neuronal cultures were treated with supernatant from HIV-1SF162-infected THP-1 cells, in the presence or absence of morphine and GSK3β inhibitors. Our results show that GSK3β inhibitors, including valproate and small molecule inhibitors, significantly reduce HIV-1-mediated neurotoxic outcomes, and also negate interactions with morphine that result in cell death, suggesting that GSK3β-activation is an important point of convergence and a potential therapeutic target for HIV- and opiate-mediated neurocognitive deficits.

Keywords: GSK3β; HIV-1; Morphine; NeuroAIDS; Neurodegeneration; Synaptodendritic injury.

Figures

Figure 1
Figure 1. HIV+sup ± morphine-mediated GSK3β activation
At 4 h after treatments, cells were lysed and protein levels were detected by immunoblotting. Findings were reported as average normalized protein levels (% control) ± SEM. Significance was analyzed by one-way ANOVA and Duncan’s post hoc test; n = 3 separate experiments. (A) Immunoblotting for phospho-GSK3β-Ser9 (p-GSKβ-S9) and total GSK3β (t-GSK3β); levels of p-GSKβ-S9 were normalized with t-GSK3β (p-GSKβ-S9/t-GSK3β). HIV+sup significantly reduced p-GSK3β-S9 (*p < 0.05 vs. Control), without any significant morphine interaction. VPA significantly abrogated HIV+sup ± morphine-mediated effects (#p < 0.05). (B) Immunoblotting for β-catenin and GAPDH; levels of β-catenin were normalized to GAPDH (β-catenin/GAPDH). HIV+sup significantly decreased β-catenin (*p < 0.05 vs. Control) without any morphine interaction. VPA restored β-catenin levels to control values (#p < 0.05), although in the case of neurons treated with HIV+sup alone the effect was less significant (HIV + VPA was not different from either HIV or control). Control = controlsup; HIV = HIV+sup; Mor = morphine; VPA = valproate.
Figure 2
Figure 2. HIV+sup- and morphine-mediated interactive effects on GSK3β-activation
At 12, 24, and 72 h after treatments, cells were lysed and immunoblotted for p-GSKβ-S9 and t-GSK3β. Findings were reported as average levels of p-GSKβ-S9 normalized with t-GSK3β (p-GSKβ-S9/t-GSK3β) as a percentage of control values ± SEM. Significance was analyzed by one-way ANOVA and Duncan’s post hoc test; n = 4 separate experiments. At all time points, HIV+sup significantly reduced p-GSK3β-S9 (*p < 0.05 vs. Control). At 24 and 72 h, morphine alone also induced significant loss of p-GSK3β-S9, but a significant interaction with HIV+sup was seen only at 24 h ($p < 0.05). Control = controlsup; HIV = HIV+sup; Mor = morphine.
Figure 3
Figure 3. Role of GSK3β in HIV+sup ± morphine-mediated cell death
(A) Cells were repeatedly imaged for 72 h after treatments. Digital images show the same cells/fields at 0, 24 and 72 h (white arrowheads indicate dead cells that were alive in previous image). (B) Cells were assessed for viability at 6 h intervals in digital images. Findings were reported as the average percentage of neuronal survival as a proportion of pre-treatment neuron count ± SEM. Significance was analyzed by repeated measures ANOVA and Duncan’s post hoc test; n = 3 separate experiments (at least 150 neurons per treatment group). HIV+sup significantly reduced neuronal survival (*p < 0.05 vs. Control), which was significantly augmented by morphine co-treatment ($p < 0.05). HIV+sup ± morphine-mediated effects were partially, but significantly, reversed by VPA (#p < 0.05). VPA also effectively negated HIV+sup-morphine interactions. (C) At 72 h after treatments, cells were fixed, permeabilized, and labeled for TUNEL and Hoechst 33342. The rate of neuronal apoptosis was reported as the average percentage of TUNEL(+) cells ± SEM. Significance was analyzed by one-way ANOVA and Duncan’s post hoc test; n = 3 separate experiments. HIV+sup significantly increased the percentage of TUNEL(+) cells (*p < 0.05 vs. Control), and morphine augmented the effect ($p < 0.05). HIV+sup ± morphine-mediated effects were partially, but significantly, reversed by VPA co-treatment (#p < 0.05), and VPA negated interactions between HIV+sup and morphine. Control = controlsup; HIV = HIV+sup; Mor = morphine; VPA = valproate.
Figure 4
Figure 4. Role of GSK3β in HIV+sup ± morphine-mediated changes in neuritic arborization, MAP-2 and PSD-95
(A) At 72 h after treatments, cells were fixed, permeabilized, and labeled for MAP-2 (green), TUNEL (red) and Hoechst 33342 (blue) (B) Neurite arborization was measured by Sholl analysis in digital images of TUNEL(−) neurons. The findings were reported as average Sholl score ± SEM. HIV+sup significantly reduced the Sholl score (*p < 0.05 vs. Control), without any significant morphine interaction. HIV+sup ± morphine-mediated effects were partially, but significantly, reversed by VPA co-treatment (#p < 0.05). (C) At 72 h after treatments, cells were lysed and immunoblotted for MAP-2 and GAPDH. Findings were reported as average normalized MAP-2 (% control) ± SEM. HIV+sup, with or without morphine, significantly reduced MAP-2 (*p < 0.05 vs. Control). VPA significantly inhibited HIV+sup ± morphine-mediated effects (#p < 0.05). (D) Cell lysates were immunoblotted for PSD-95 and GAPDH. Findings were reported as average normalized PSD-95 (% control) ± SEM. HIV+sup induced a significant loss of PSD-95 (*p < 0.05 vs. Control), which was augmented by morphine co-treatment ($p < 0.05). HIV+sup ± morphine-mediated effects were partially, but significantly, reversed by VPA co-treatment (#p < 0.05). Even in the presence of VPA, morphine significantly augmented the effects of HIV+sup ($p < 0.05). In all studies, significance was analyzed by one-way ANOVA and Duncan’s post hoc test, from n = 3 separate experiments. Control = controlsup; HIV = HIV+sup; Mor = morphine; VPA = valproate.
Figure 5
Figure 5. Role of GSK3β in HIV+sup ± morphine-mediated changes in MAP-2 and PSD-95 in cortical and hippocampal neurons
At 72 h after treatments, cells were lysed and immunoblotted for MAP-2, PSD-95 and GAPDH. Findings were reported as average normalized protein levels (% control) ± SEM. Significance was analyzed by one-way ANOVA and Duncan’s post hoc test; n = 3 separate experiments. (A) Cortical Neurons. MAP-2: HIV+sup ± morphine induced significant loss of MAP-2 (*p < 0.05 vs. Control), VPA significantly inhibited HIV+sup-mediated effects (#p < 0.05). PSD-95: HIV+sup, with or without morphine, significantly reduced PSD-95 (*p < 0.05 vs. Control). HIV+sup-mediated loss of PSD-95 was partially, but significantly, reversed by VPA co-treatment (#p < 0.05). (B) Hippocampal Neurons. MAP-2: HIV+sup induced a significant loss of MAP-2 (*p < 0.05 vs. Control), which was augmented by morphine co-treatment ($p < 0.05). HIV+sup ± morphine-mediated loss of MAP-2 was significantly reversed by VPA co-treatment (#p < 0.05). PSD-95: HIV+sup induced a significant loss of PSD-95 (*p < 0.05 vs. Control), which was augmented by morphine co-treatment ($p < 0.05). HIV+sup ± morphine-mediated effects were partially, but significantly, reversed by VPA co-treatment (#p < 0.05). Control = controlsup; HIV = HIV+sup; Mor = morphine; VPA = valproate.
Figure 6
Figure 6. Effects of small molecule GSK3β-inhibitors
(A) Cell viability was assessed by time-lapse imaging analysis. Neurons exposed to ‘Control + SB’, ‘Control + XXVI’, ‘Mor’, ‘Mor + SB’ and ‘Mor + XXVI’ treatments had survival equivalent to ‘Control’, but are not shown here in order to highlight other groups. HIV+sup significantly reduced neuronal survival (*p < 0.05 vs. Control), with a significant morphine interaction ($p < 0.05). The effects of HIV+sup were partially, but significantly, reversed by both small molecule inhibitors (#p < 0.05 vs. respective HIV or HIV + Mor). Both SB415286 and XXVI also effectively negated interactions between HIV+sup and morphine. (B) Cell death was examined using TUNEL-staining. HIV+sup significantly increased the percentage of TUNEL(+) cells (*p < 0.05 vs. Control), with a significant morphine interaction ($p < 0.05). Both small molecule inhibitors partially reversed HIV+sup ± morphine-mediated effects (#p < 0.05) and also eradicated the HIV+sup-morphine interaction. (C) Neuritic arborization was evaluated using MAP-2 immunostaining and Sholl analysis. HIV+sup significantly reduced the Sholl score (*p < 0.05 vs. Control), without a morphine interaction. All losses in arborization were partially, but significantly, reversed by both small molecule inhibitors (#p < 0.05), except that XXVI completely reversed the effect of HIV+sup. Control = controlsup; HIV = HIV+sup; Mor = morphine; SB = SB415286; XXVI = GSK3β inhibitor XXVI.

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