Morphine Enhances HIV-1SF162-Mediated Neuron Death and Delays Recovery of Injured Neurites

Abstract

HIV-1 enters the CNS soon after initial systemic infection; within the CNS parenchyma infected and/or activated perivascular macrophages, microglia and astrocytes release viral and cellular toxins that drive secondary toxicity in neurons and other cell types. 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-infected differentiated-U937 cells (HIV+ sup) was collected and p24 level was measured by ELISA to assess the infection. Injection drug abuse is a significant risk factor for HIV-infection, and opiate drug abusers show increased HIV-neuropathology, even with anti-retroviral treatments. We therefore 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), and GSK3β was implicated as a point of convergence. In the presence of glia, selective neurotoxic measures were significantly enhanced and interactions with morphine were also augmented, perhaps related to a decreased level of BDNF. 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. Opiate interactions observed in this HIV-infective environment were similar, though not entirely concordant, with Tat/gp120 interactions reported previously, suggesting unique interactions with virions or other viral or cellular proteins released by infected and/or activated cells.

GSK3β-activation is a point of convergence for HIV-1 and opiate-mediated interactive neurotoxicity

“…We also tested effects of HIV + sup and morphine on cortical and hippocampal neurons. Neurons were cultured as previously described (Masvekar et al, 2014; Podhaizer et al, 2012; Zou et al, 2011a; Zou et al, 2011b). In brief, striata or cortices from E15–E16 or hippocampi from P0–P1 ICR (CD-1) mice (Charles River Laboratories International, Inc., Wilmington, MA) were dissected, minced and incubated with trypsin (2.5 mg/ml; Sigma-Aldrich, St. Louis, MO) and deoxyribonuclease (DNase; 0.015 mg/ml; Sigma-Aldrich) in neurobasal medium (Gibco, Grand Island, NY) for 30 min at 37°C.…”

“…Our previous studies showed that supernatants from HIV-infected cells induce neurotoxic effects in a concentration-dependent manner over a range of p24 levels (10–500 pg/ml), but the interactions with morphine were significant only at lower HIV-exposure levels (p24 = 10 and 25 pg/ml) (Masvekar et al, 2014). Thus, current studies used an HIV + sup exposure level of p24 = 25 pg/ml.…”

“…Thus, current studies used an HIV + sup exposure level of p24 = 25 pg/ml. HIV + sup and Control sup were diluted similarly and added to neuronal cultures in the presence or absence of morphine (morphine sulfate; Sigma-Aldrich), at a dose that maximally stimulates neuronal and glial μ-opioid receptors (MORs) in vitro (500 nM) (Bruce-Keller et al, 2008; El-Hage et al, 2005; Gurwell et al, 2001; Ikeda et al, 2010; Masvekar et al, 2014; Miyatake et al, 2009; Podhaizer et al, 2012; Zou et al, 2011b). To determine the role of GSK3β in HIV- and opiate-mediated neurotoxicity, HIV + sup ± morphine treatments were conducted in the presence of a standard GSK3β inhibitor, valproate (valproic acid, VPA; 1 mM; Sigma-Aldrich), or in the presence of two very selective, small molecule inhibitors, SB415286 (10 μM; Sigma-Aldrich; a competitive inhibitor of the ATP binding site on GSK3β and GSK3α with an IC 50 value of 78 nM (Coghlan et al, 2000)) or GSK3β inhibitor XXVI (XXVI; 1 μM; EMD Millipore, Billerica, MA; a cell-permeable, pyrazolone GSK3β inhibitor with an IC 50 value of 34 nM).…”

“…In digital images, neurons were assessed for viability at 6 h intervals using rigorous morphological criteria confirming cell death, including cytoplasmic swelling, nuclear destruction, cell body fragmentation, excessive neurite degeneration, and loss of phase brightness (Masvekar et al, 2014; Podhaizer et al, 2012; Singh et al, 2004; Zou et al, 2011a; Zou et al, 2011b). Findings were reported as the average percentage of neuronal survival, with respect to pre-treatment neuron count ± the standard error of the mean (SEM).…”

“…Since opiates by themselves promote outcomes involved in CNS dysfunction, such as blood-brain barrier breakdown, immune and glial cell activation, and neuronal damage (Bell et al, 2006; Hauser et al, 2005; Hu et al, 2002; Sheng et al, 1997), it can be predicted that opiate drug abuse might exacerbate HIV-1 pathogenesis in the CNS. Our previous work has shown that certain neurotoxic effects induced by the individual HIV-1 proteins, trans-activator of transcription (Tat) and glycoprotein 120 (gp120) (Fitting et al, 2010a; Gurwell et al, 2001; Podhaizer et al, 2012; Suzuki et al, 2011; Zou et al, 2011b), and by HIV + supernatant (HIV + sup ) (Masvekar et al, 2014), are enhanced by co-exposure to morphine, the major metabolite of heroin in the CNS (Sawynok, 1986). This mimics co-morbid neurological effects observed in opiate-abusing HIV + patients (Anthony et al, 2008; Bell et al, 2002; Byrd et al, 2011; Meijerink et al, 2014; Robinson-Papp et al, 2012; Smith et al, 2014).…”