HIV-1 Tat and methamphetamine co-induced oxidative cellular injury is mitigated by N-acetylcysteine amide (NACA) through rectifying mTOR signaling

Highlights

• Meth and Tat synergistically increased cell death in dose- and time-dependent manners.

• Meth and Tat-induced autophagy and apoptosis in vitro and in vivo.

• NACA confers protection against Tat- and/or Meth-induced and/or autophagy through mTOR signaling in vitro and in vivo.

Abstract

Methamphetamine (Meth) is an addictive psychostimulant whose abuse is intimately linked to increased risks for HIV-1 infection. Converging lines of evidence indicate that Meth also aggravates the symptoms of HIV-associated neurocognitive disorders (HAND), though the underlying mechanisms remain poorly understood. By using the lipophilic antioxidant N-acetylcysteine amide (NACA) as an interventional agent, we examined the roles of oxidative stress in autophagy and apoptosis induced by HIV-Tat (the transactivator of transcription), Meth or their combined treatment in human SH-SY5Y neuroblastoma cells and in the rat striatum. Oxidative stress was monitored in terms of the production of intracellular reactive oxygen species (ROS) and antioxidant reserves including glutathione peroxidase (GPx) and Cu,Zn-superoxide dismutase (SOD). NACA significantly reduced the level of ROS and restored GPx and SOD to levels comparable to that of normal control, implying a cytoprotective effect of NACA against oxidative stress elicited by Tat- and/or Meth. Protein expression of mammalian target of rapamycin (mTOR) was measured in SH-SY5Y cells and in the rat striatum to further explore the underlying mechanism of NACA protect against oxidative stress. The results support a beneficial effect of NACA in vivo and in vitro through rectification of the mTOR signaling pathway. Collectively, our study shows that NACA protects against Meth and/or Tat-induced cellular injury in vitro and in the rat striatum in vivo by attenuating oxidative stress, apoptosis and autophagy, at least in part, via modulation of mTOR signaling.

Introduction

Methamphetamine (Meth) is a widely abused psychostimulant with high addictive potential. Higher prevalence of Meth abuse (ranging from 40 to 60%) was consistently found in human immunodeficiency virus (HIV)-positive patients (Rajasingham et al., 2012; Shoptaw et al., 2003). Multiple lines of evidence from neuroimaging, neurochemical and neuropsychological studies suggest that Meth aggravates the symptoms of HIV infection in the brain (Clemens et al., 2007; Hoefer et al., 2015; Jernigan et al., 2005; Soontornniyomkij et al., 2016). Notably, HIV-positive Meth users are more likely to have cognitive deficits and profound abnormalities in white matter integrity when compared with cohorts of HIV-negative Meth users or HIV infection without Meth use (Chang et al., 2005; Rippeth et al., 2004; Soontornniyomkij et al., 2016). Several neurotransmitter systems including dopaminergic, serotonergic and GABAergic systems are disrupted by Meth use in conjunction with HIV infection. In particular, Tat and Meth synergize to cause enhanced loss of dopaminergic neurons in the striatum (Maragos et al., 2002; Theodore et al., 2006). In our previous in vitro study, we observed that HIV Tat and Meth synergistically induced autophagy and apoptosis in dopaminergic neurons (Qi et al., 2011). Other viral components such as the HIV-1 envelope protein gp120 were also recently shown to alter autophagic signaling in the presence of Meth (Sanchez et al., 2015), suggesting multiple host-virus interactions converge on this self-degradative process. Autophagy is a vital homeostatic mechanism for maintaining cellular health in neurons. Dysregulation of autophagy is implicated in the etiology and progression of many neurodegenerative disorders (Ghavami et al., 2014). Thus, in the present study, the synergic effects of Tat and Meth on autophagy and/or apoptosis were investigated in vivo in the rat striatum and in vitro by using SH-SY5Y human neuroblastoma cells, which are known to display the morphological, biochemical, and electrophysiological properties resembling that of dopaminergic neurons. Specific inhibitors and activators of autophagy were employed to interrogate potential mechanisms underlying the synergic effects of Tat and Meth on autophagy in SH-SY5Y cells.

HIV-1 transactivator of transcription (Tat) is essential to HIV-1 viral replication. It is neurotoxic in its own right and has been speculated to exert such effects in an oxidative stress-dependent manner. For instance, HIV-1 Tat was found to trigger mitochondrial membrane depolarization (Lecoeur et al., 2012; Regulier et al., 2004), nitrosative stress accompanied by elevated inflammation in HIV-1 transgenic rats (Cho et al., 2017), and accumulation of cellular reactive oxygen species (ROS) in hippocampal neurons (Aksenov et al., 2006). The pathophysiology of HAND associated with Meth use and HIV-1 infection has been described in terms of oxidative stress, cytokine production and neurotoxicity elicited by the condition (Flora et al., 2003; Nath and Geiger, 1998; Silverstein et al., 2011). Like the case of HIV-1 Tat-induced cytotoxicity, the mechanisms underlying Meth neurotoxicity are complex and may involve redox dyshomeostasis (Cubells et al., 1994; Imam et al., 2001), glial cell activation (Pu et al., 1994; Stadlin et al., 1998) and cytokine mobilization (Asanuma and Cadet, 1998; Ladenheim et al., 2000). However, relatively little is known about the molecular mechanisms responsible for Meth’s ability to augment neuronal injury in HIV-1 infection. Therefore, we ventured to examine the link between oxidative stress and autophagy in SH-SY5Y cells under exposure to Tat and/or Meth in this study.

N-acetylcysteine amide (NACA), a novel thiol antioxidant, displays a strong capacity for ameliorating oxidative stress, dampening excessive immuno-inflammatory responses and preventing subsequent programmed cell death in neurons (Banerjee et al., 2009; Paul et al., 2015; Schimel et al., 2011). Given that NACA has a superior BBB (blood brain barrier) permeability (Zhang et al., 2009), reproducing its therapeutic effects on Tat and/or Meth-induced autophagy in vivo in animal models is practically more conceivable. In addition, it was recently reported that Meth-induced autophagy in endothelial cells was triggered by inactivation of the mammalian target of rapamycin (mTOR) pathway, which is a canonical negative regulator of autophagy (Ma et al., 2014). Inactivation of the mTOR pathway triggered by the production of oxidative stress mediators, in particular ROS, results in white matter dysfunction in schizophrenia patients, whose symptoms are also characterized by neurochemical abnormalities (Maas et al., 2017). A priori, we predicted that NACA would attenuate HIV-1 protein Tat and Meth-induced cellular injury resulting from excessive autophagy in vivo and in vitro, at least in part, through restoring of normal mTOR signaling.