Elsevier

Brain Research

Volume 1306, 8 January 2010, Pages 116-130
Brain Research

Research Report
Dopaminergic neurotoxicity of HIV-1 gp120: Reactive oxygen species as signaling intermediates

https://doi.org/10.1016/j.brainres.2009.09.113Get rights and content

Abstract

We examined the role of reactive oxygen species (ROS) in loss of dopaminergic neurons (DNs) from the substantia nigra (SN) in neuroAIDS. The frequency of Parkinson-like symptomatology, and DN loss, in neuroAIDS is often attributed to nonspecific DN fragility to oxidative stress. Cultured DN are more sensitive to ROS than non-dopaminergic neurons (RN): DN underwent apoptosis at far lower H2O2 concentrations than RN. Gene delivery of glutathione peroxidase (GPx1), which detoxifies H2O2, largely protected both neuron types. HIV-1 envelope, gp120, which elicits oxidative stress in neurons, caused apoptosis more readily in DN than in RN. However, unlike apoptosis caused by H2O2, gp120-induced DN apoptosis was specific: DNs were specifically more sensitive than RN to receptor-mediated [Ca2+]i fluxes triggered by gp120. Gp120-induced Ca2+ signaling in both neuron types was inhibited by GPx1 or Cu/Zn superoxide dismutase (SOD1), implicating superoxide and peroxide in ligand (gp120)-induced signaling upstream of Ca2+ release from intracellular stores. In vivo, rats given 10 ng of gp120 stereotaxically showed rapid DN loss within the SN, while loss of RN in the SN and caudate–putamen (CP) was slower and required ≥ 100 ng of gp120. Furthermore, gp120 injected into the CP was transported axonally retrograde to the SN, causing delayed DN loss there. This, too, was prevented by SOD1 or GPx1. DNs are therefore specifically hypersensitive to gp120-induced apoptosis, signaling for which involves ROS intermediates. These findings may help explain why DN loss and Parkinson's-like dysfunction predominate in neuroAIDS and may apply to other neurodegenerative diseases involving the SN.

Introduction

Parkinson's disease (PD) is characterized by motor, cognitive, and psychiatric disturbances. These include apathy, slow movement and thought, impaired gait, rigidity and others, and largely reflect the consequences of loss of dopaminergic neurons (DNs) from the substantia nigra (SN). The causes of most cases of PD are unknown, but progressive loss of DNs often involves continuing oxidant damage to DN proteins, accumulation of these abnormally modified proteins and consequent disruption of neuronal function (Bernheimer et al., 1973, Hornykiewicz, 2001, Zhang and Kaufman, 2006, Gorman, 2008, Levy et al., 2009).

Ongoing oxidative neuronal injury is also characteristic of symptomatic HIV-1 infection in the CNS, i.e., neuroAIDS (Steiner et al., 2006). HIV-1 enters the CNS shortly after entering the body and replicates in macrophages and microglia there (Gartner, 2000, Rausch et al., 1999). Although highly active antiretroviral therapy (HAART) generally does not penetrate the CNS well and may not greatly impair replication of HIV-1 within the CNS (McArthur et al., 1999), HAART exposure may decrease CNS virus load (Sinclair et al., 2008). The consequences of CNS HIV-1 infection reflect in a major way the neurotoxicity of two HIV-1 protein products, envelope gp120 (Env) and Tat (Kruman et al., 1998, Shi et al., 1998, Corasaniti et al., 1998a, Rappaport et al., 1999, Nath et al., 2000a, Corasantini et al., 2001). Although frank HIV-1-associated dementia is now rare in the US, minor cognitive and motor dysfunction (MCMD) is common. As life expectancies improve for HIV-1-positive patients whose peripheral HIV-1 loads are controlled with HAART, prevalence of MCMD is rising (Sacktor et al., 2002, McArthur et al., 2005).

The pathogenesis of neuronal injury leading to symptomatic neuroAIDS is complex and involves neuronal loss (Navia and Rostasy, 2005, Everall et al., 2005, Langford et al., 2003, Gonzalez-Scarano and Martin-Garcia, 2005, Kaul et al., 2005, Rumbaugh and Nath, 2006, Lawrence and Major, 2002) and dysfunction due to excitotoxic activation of synaptic glutamate receptors, proinflammatory cytokines, proapoptotic signaling elicited by gp120 and Tat in neurons and other factors (Kruman et al., 1998, Shi et al., 1998, Corasaniti et al., 1998b, Rappaport et al., 1999, Nath et al., 2000a, Corasantini et al., 2001, Watanabe et al., 2003, Betz et al., 1998). Several of these mechanisms involve generation of reactive oxygen (ROS) and nitrogen (RNS), causing oxidative damage to many cellular macromolecules, especially lipids and proteins (Turchan et al., 2003, Bandaru et al., 2007, Mattson et al., 2005, Steiner et al., 2006). Gene delivery of Cu/Zn superoxide dismutase (SOD1) and/or glutathione peroxidase (GPx1) to neurons using recombinant SV40-derived vectors (rSV40s) can protect neurons from the toxicity of gp120 and Tat in vitro and in vivo (Agrawal et al., 2006, Agrawal et al., 2007, Louboutin et al., 2007a, Louboutin et al., 2007b).

Interestingly, the symptomatology and pathology of neuroAIDS both reflect disproportionate damage to the dopaminergic system of the SN. Thus, the bradykinesia, bradyphrenia, abnormalities of gait, rigidity, and other characteristics of Parkinson's disease also characterize neuroAIDS (Nath et al., 2000b, Berger and Arendt, 2000, Koutsilieri et al., 2002a, Koutsilieri et al., 2002b, Chang et al., 2008, Khanlou et al., 2009). Disproportionate DN loss is common in neuroAIDS, as are decreased dopamine (DA) and DA metabolites (Reyes et al., 1991, Silvers et al., 2006). Tyrosine hydroxylase (TH), which is critical for DA synthesis, and DA transporter (DAT), which is important for DA reuptake into DN, are often decreased also (Itoh et al., 2000, Wang et al., 2004, Silvers et al., 2007).

Compared to non-dopaminergic neurons (RNs), DNs are reported to be nonspecifically more sensitive to oxidative damage (Fahn and Cohen, 1992, Jenner, 2003), particularly via damage to their mitochondria (Ben-Shachar et al., 1995, Gluck and Zeevalk, 2004). However, the basis for the predilection for DN injury and loss in neuroAIDS has been unclear. We report here that DNs are nonspecifically more susceptible than non-dopaminergic neurons to ROS-induced apoptosis but that, as well, gp120 toxicity for DN is largely specific for gp120, reflects gp120-initiated proapoptotic signaling, localizes mainly to the cytosol, and occurs in DN far more readily than in other neuron types.

ROS are involved in HIV-1 gp120-induced neurotoxicity as signaling intermediates activated by gp120–DN interaction. They may also be direct effectors of oxidant damage. Gp120 is toxic to DN at levels that are innocuous to RN. In addition, it is specifically transported from other areas of the brain to the SN, where it causes selective loss of DN. Antioxidant gene delivery blocks gp120-induced proapoptotic signaling in vitro and protects DN viability in vitro and in vivo.

Section snippets

Cell lines and chemicals

COS-7 cell line was obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum (Hyclone, Logan, UT, USA), 2 mm l-glutamine and containing 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 1.0 mm sodium pyruvate, penicillin (200 U/ml), and streptomycin (100 g/ml). H2O2 was purchased from Sigma Chemical Co. (St. Louis, MO). Growth factors used as media supplements, epidermal growth factor (EGF), fibroblast growth

Cultured DNs express tyrosine hydroxylase as a marker for DNs and are more sensitive than RNs to oxidative stress

Fully differentiated, DNs were prepared as described in Methods, from 12 to 16-week-old fetal brain, then compared to RNs for expression of the DN marker tyrosine hydroxylase (TH) by immunohistology (Fig. 1a) and Western blotting (Fig. 1b). DNs were positive for TH by both assays, compared to control cultures. On Western analysis, TH was visualized as a prominent band around 65 kDa. These cells were also positive for protein markers such as nestin, NCAM (neuronal cell adhesion molecule), and

Discussion

Gp120 binds neuron cell membrane coreceptors (CCR3, CCR5, CXCR4) and elicits apoptosis (Garden et al., 2004) via G protein-coupled pathways (Kruman, 1998; Kaul and Lipton, 1999). Soluble gp120 also increases glial cell release of arachidonate, which impairs astrocyte and neuron reuptake of glutamate (Lipton et al., 1993) leading to prolonged activation of N-methyl-d-aspartate (NMDA) receptor with consequent disruption of cellular Ca2+ homeostasis (Kaul and Lipton, 2006). A second kind of

Acknowledgments

This work was supported by NIH grant MH70287. We are indebted to the Human Fetal Tissue Bank at Albert Einstein College of Medicine for the primary tissue from which neuron cultures were prepared.

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