HIV AND OPIATES-MEDIATED NEUROTOXICITY: GSK3β IS A POTENTIAL THERAPEUTIC TARGET

HIV AND OPIATES-MEDIATED NEUROTOXICITY: GSK3β IS A POTENTIAL THERAPEUTIC TARGET By Ruturaj R. Masvekar A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience at Virginia Commonwealth University. Virginia Commonwealth University, 2014 Director: Pamela E. Knapp, PhD. Professor, Department of Anatomy and Neurobiology. HIV-1 enters the CNS soon after initial systemic infection. HIV-1 can induce a wide range of neurological deficits, collectively known as HIV-1-associated neurocognitive disorders (HAND). Mature neurons are not infected by HIV-1; instead, infected and/or activated glial cells release various viral and cellular factors that induce direct and/or indirect neuronal toxicity, leading to HAND. Injection drug abuse is a significant risk factor for HIV-infection, and opiate drug abusers show increased HIV-neuropathology, even with anti-retroviral treatments. Our previous work has largely modeled HIV-neuropathology using the individual viral proteins Tat or gp120, with murine striatal neurons as targets. To model disease processes more closely, the current study uses supernatant from HIV-1-infected cells. Supernatant from HIV-1SF162 (R5-tropic)-infected differentiated-U937 cells (HIV + sup) was collected and p24 level was measured by ELISA to assess the infection. We assessed HIV + sup effects on neuronal survival and neurite growth/pruning with or without concurrent exposure to morphine, an opiate that preferentially acts through μ-opioid receptors. Effects of HIV + sup ± morphine were assessed on neuronal populations, and also by time-lapse imaging of individual cells. HIV + sup caused dose-dependent toxicity over a range of p24 levels (10-500 pg/ml). Significant interactions occurred with morphine at lower p24 levels (10 and 25 pg/ml). In the presence of glia, selective neurotoxic measures were significantly enhanced and interactions with morphine were also augmented. Importantly, the arrest of neurite growth that occurred with exposure to HIV + sup was reversible unless neurons were continuously exposed to morphine. Thus, while reducing HIV-infection levels may be protective, ongoing exposure to opiates may limit recovery. During early stage of HIV-infection R5-tropic viruses are predominant, but during later stages of disease X4-tropic viruses are more predominant; co-receptor usage switch from CCR5 to CXCR4 is crucial in disease progression to AIDS. Some previous studies have shown that drugs of abuse interact with virus or viral proteins in strain/tropism-dependent manner. Therefore, we also assessed neurotoxic effects and interactions with opiates by supernatant from HIV-1LAI (X4tropic)-infected H9 cells. Neurotoxic effects and the interactions with opiates of HIV-1LAIsupernatant are quantitatively similar to that of HIV-1SF162. Surprisingly, the cytokine/chemokine release profile of HIV-1LAI-infected H9 cells is similar to that of HIV-1SF162-infected U937 cells. Only in the presence of glia, HIV-1LAI virion induced neurotoxic effects, but no interactions with morphine were seen. Also our studies have shown that HIV-1LAI virions are slightly more neurotoxic than HIV-1SF162. Altogether, largely our results suggest that HIV + sup mediated neurotoxicity and the interactions with opiates are majorly attributed to cytotoxic factors released from infected and activated cells instead of viral strain specific factors. Although there is a correlation between opiate drug abuse and progression of HAND, the mechanisms that underlie 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 the brains of young opiate abusers suggesting that GSK3β is also linked to neuropathology seen with opiate abusing patients. Thus, we hypothesized that GSK3β activation is a point of convergence for HIVand opiate-mediated interactive neurotoxic effects. Cultures of striatal neurons were treated with HIV + sup (R5-tropic), in the presence or absence of morphine and GSK3β inhibitors. Our results show that multiple GSK3β inhibitors significantly reduce HIV-1-mediated neurotoxic outcomes, and also negate interactions with morphine that result in cell death. This suggests that GSK3βactivation is an important point of convergence and a potential therapeutic target for HIVand opiate-mediated neurocognitive deficits.