HIV and Cocaine Impact Glial Metabolism: Energy Sensor AMP-activated protein kinase Role in Mitochondrial Biogenesis and Epigenetic Remodeling

Abstract

HIV infection and cocaine use have been identified as risk factors for triggering neuronal dysfunction. In the central nervous system (CNS), energy resource and metabolic function are regulated by astroglia. Glia is the major reservoir of HIV infection and disease progression in CNS. However, the role of cocaine in accelerating HIV associated energy deficit and its impact on neuronal dysfunction has not been elucidated yet. The aim of this study is to elucidate the molecular mechanism of HIV associated neuropathogenesis in cocaine abuse and how it accelerates the energy sensor AMPKs and its subsequent effect on mitochondrial oxidative phosphorylation (OXPHOS), BRSKs, CDC25B/C, MAP/Tau, Wee1 and epigenetics remodeling complex SWI/SNF. Results showed that cocaine exposure during HIV infection significantly increased the level of p24, reactive oxygen species (ROS), ATP-utilization and upregulated energy sensor AMPKs, CDC25B/C, MAP/Tau and Wee1 protein expression. Increased ROS production subsequently inhibits OCR/ECAR ratio and OXPHOS, and eventually upregulate epigenetics remodeling complex SWI/SNF in CHME-5 cells. These results suggest that HIV infection induced energy deficit and metabolic dysfunction is accelerated by cocaine inducing energy sensor AMPKs, mitochondrial biogenesis and chromatin remodeling complex SWI/SNF activation, which may lead to neuroAIDS disease progression.

Figure 1. HIV-1 gp120 protein and cocaine exposure impact ATP utilization, oxygen consumption and extracellular acidification in microglia.

CHME-5 (10,000 cells/ml) were treated with HIV-1 gp120 (0–100 ng), cocaine (0.5 μM) and combination of HIV-1 gp120 with cocaine for 24 h. Controls were maintained by drug free medium. At the end of the incubation period; the OCR and ECAR were measured by Seahorse Bioscience-XF96 extracellular flux analyzer. Data presented is the average of three independent experiments conducted under the same experimental conditions.

Figure 2. Effects of HIV infection and cocaine co-morbidity impact energy sensor AMPK and epigenetic remodeling complex SWI/SNF in microglia.

CHME-5 (50 × 10⁵cells/ml) were infected with the HIV Bal strain TCID₅₀ for 18 hours. After that, treated with cocaine (0.5 μM) at every 72 h and the supernatants were collected at 7^th day and used to estimate p24 antigen by ELISA (A) and infected cells total RNA was extracted and reverse transcribed followed by quantitative real time PCR for LTR (B), AMPK-α (C) and SWI/SNF-1 expression (D). Data are expressed as mean ± SE of TAI values of three independent experiments conducted under the same experimental conditions.

Figure 3. Effects of HIV-1 gp120 protein and cocaine co-morbidity induced oxidative stress altered energy sensor AMPK and epigenetic remodeling complex SWI/SNF in microglia.

CHME-5 (1 × 10⁶ cells/ml) were treated with HIV-1 gp120 (50 ng), cocaine (0.5 μM) and combination of HIV-1 gp120 with cocaine for 24 h. Controls were maintained by drug free medium. At the end of the incubation, ROS production was analyzed by flow cytometry (A), the total RNA was extracted and reverse transcribed followed by quantitative real time PCR for AMPK-α (B), MAP/Tau (C), Wee1 (D) and SWI/SNF-1 (E) and housekeeping β-actin specific primers. Data are expressed as mean ± SE of TAI values of three independent experiments conducted under the same experimental conditions.

Figure 4. HIV-1 gp120 and cocaine impacts AMPK signaling network.

CHME-5 (1 × 10⁶ cells/ml) were treated with HIV-1 gp120 (50 ng), cocaine (0.5 μM) and combination of HIV-1 gp120 with cocaine for 24 h. Controls were maintained by drug free medium. At the end of the incubation, equal amount of protein lysate were resolved by 4–15% SDS-PAGE and protein expression were analyzed by Western blot showing AMPK-α (A), AMPK-β (B), P-ACC (C), CDC25B (D), CDC25C (E), MAP/Tau (F) and WEE1 (G). (H–N) represent % densitometric values of protein levels (% control), respectively. Data are expressed as mean ± SE of three independent experiments conducted under the same experimental conditions.

Figure 5. Effect on BRSKs and oxidative damage on mitochondrial proteins.

CHME-5 (1 × 10⁶cells/ml) were treated with HIV-1 gp120 (50 ng), cocaine (0.5 μM) and combination of HIV-1 gp120 with cocaine for 24 h. Controls were maintained by drug free medium. At the end of the incubation, equal amount of protein lysate were resolved by 4–15% SDS-PAGE and protein expression were analyzed by Western blot showing neuronal cells level in BRSK1 (A), and CHEM-5 cells in BRSK 1 (B), BRSK-2 (C), and mitochondrial proteins (D). (E–G) represent % densitometric values of CV-ATPase, CIII-UQCRC2 and CII-SDHB protein levels (% control). Data are expressed as mean ± SE of three independent experiments conducted under the same experimental conditions.

Figure 6. HIV infection and HIV-1 gp120 co-morbidity with cocaine effect on epigenetics remodeling protein.

CHEM-5 (1 × 10⁶ cells/ml) were infected with the HIV Bal strain TCID₅₀ for 18 hours. After that, treated with cocaine (0.5 μM) every 72 h. In another set of experiment, CHME-5 (1 × 10⁶ cells/ml) were treated with HIV-1 gp120 (50 ng), cocaine (0.5 μM) and combination of HIV-1 gp120 with cocaine for 24 h. At the end of the incubation, equal amount of protein lysate were resolved by 4–15% SDS-PAGE and protein expression were analyzed by Western blot showing HIV infected with cocaine effect on SWI/SNF protein ARID1A (A) and HIV-1 gp120 protein with cocaine (B). (C,D) represent % densitometric values protein levels (% control), respectively. Data are expressed as mean ± SE of three independent experiments conducted under the same experimental conditions.

Figure 7. Schematic pathway of HIV infection and cocaine impact on microglial energy resource and influence neuronal toxicity.

A comprehensive model showing how HIV-gp120 and cocaine effect energy sensor AMPKs signaling mechanism and epigenetic signature lead neurotoxicity. HIV infection and HIV envelop protein (gp120) leads to altered AMP/ATP ratio and AMPKs activations’ which influence mitochondrial biogenesis and subsequent induction of MAP/Tau and CDC25B/C mediated epigenetic remodeling SWI/SNF complex, and that may lead neurotoxicity.