HIV-1 gp120 neurotoxicity proximally and at a distance from the point of exposure: Protection by rSV40 delivery of antioxidant enzymes

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Abstract

Toxicity of HIV-1 envelope glycoprotein (gp120) for substantia nigra (SN) neurons may contribute to the Parkinsonian manifestations often seen in HIV-1-associated dementia (HAD). We studied the neurotoxicity of gp120 for dopaminergic neurons and potential neuroprotection by antioxidant gene delivery. Rats were injected stereotaxically into their caudate-putamen (CP); CP and (substantia nigra) SN neuron loss was quantified. The area of neuron loss extended several millimeters from the injection site, approximately 35% of the CP area. SN neurons, outside of this area of direct neurotoxicity, were also severely affected. Dopaminergic SN neurons (expressing tyrosine hydroxylase, TH, in the SN and dopamine transporter, DAT, in the CP) were mostly affected: intra-CP gp120 caused approximately 50% DAT+ SN neuron loss. Prior intra-CP gene delivery of Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) protected SN neurons from intra-CP gp120. Thus, SN dopaminergic neurons are highly sensitive to HIV-1 gp120-induced neurotoxicity, and antioxidant gene delivery, even at a distance, is protective.

Introduction

Human Immunodeficiency Virus (HIV)-associated Dementia (HAD), the most severe form of HIV-related CNS dysfunction, and the most common cause of dementia in adults under 40 was estimated to affect as many as 30% of patients with advanced Acquired Immune Deficiency Syndrome (AIDS) (Mattson et al., 2005, Major et al., 2000). It has become less common since Highly Active AntiRetroviral Therapy (HAART) was introduced (McArthur et al., 1993). This reduction probably reflects better ongoing entry of HIV-infected cells from the blood into the central nervous system (CNS), since antiretroviral drugs penetrate the CNS poorly. However, as survival improves, the prevalence of HIV encephalopathy (HIVE) continues to rise, and a less fulminant form of HIV-related neurological dysfunction, minor neurocognitive/motor disorder (MCMD), is more commonly seen and remains a significant independent risk factor for AIDS mortality (Ellis et al., 1997, Mattson et al., 2005, McArthur et al., 2005). The brain may also be an important reservoir for the virus, and neurodegeneration and inflammation may progress despite the use of HAART (McArthur et al., 2003, Nath and Sacktor, 2006, Ances and Ellis, 2007). Before the introduction of HAART, most patients with HIVE showed subcortical dementia, with predominant basal ganglia involvement, manifesting as psychomotor slowing, Parkinsonism, behavioral abnormalities and cognitive difficulties (Koutsilieri et al., 2002). Several studies stressed the role of basal ganglia in HIV encephalopathy and suggested that dopaminergic neurotransmission could be impaired in brains of HIV-1 patients, reflecting decreased dopamine (Sardar et al., 1993), and neuronal degeneration in the Substantia Nigra (SN) in brains of AIDS patients (Itoh et al., 2000). Similar findings have been reported in Simian Immunodeficiency Virus (SIV)-infected monkeys (Scheller et al., 2005). HIV-positive patients may show an extreme sensitivity to dopamine receptor antagonists and can develop side effects to antipsychotic agents (Koutsilieri et al., 2002). Neurons themselves are rarely infected by HIV-1, and neuronal damage is felt to be mainly indirect (Kaul et al., 2001). HIV-1 infects periventricular macrophages, resident microglia, and some astrocytes (Ranki et al., 1995), leading to increased production of cytokines, such as TNF-alpha, IL-1beta and IL-6, and chemokines such as MCP-1 (van de Bovenkamp et al., 2002). Macrophages and microglial cells also release HIV-1 proteins, several of which are neurotoxins: envelope (Env) proteins gp41 and gp120, and the nonstructural proteins Tat, Nef, Vpr and Rev. Gp120-induced apoptosis has been demonstrated in studies in cortical cell cultures, in rat hippocampal slices and by intracerebral injections in vivo (Meucci et al., 1998). We recently reported that exposure to HIV-1 Env gp120 by injection in vivo, induced neuronal apoptosis in the caudate putamen (CP). Gp120-induced apoptosis was prevented by prior local recombinant Simian Virus 40 (rSV40)-based delivery of anti-oxidant enzymes Cu/Zn superoxide dismutase (SOD1) and glutathione peroxidase (GPx1). These vectors were respectively named SV(SOD1) and SV(GPx1) (Agrawal et al., 2006, Louboutin et al., 2007a). However, tissue loss has also been observed in the brain after direct injection of gp120 (Bansal et al., 2000, Nosheny et al., 2004). Here we document gp120-mediated tissue and neuron loss in the CP as a function of time after injection of gp120 and for different doses of the neurotoxin. We examined the consequences of gp120-induced injury on the dopaminergic pathway in the striatum, dopamine transporter (DAT)-immunoreactivity in the CP, and tyrosine hydroxylase (TH)-immunoreactivity in the SN. Finally, we tested the extent to which prior rSV40 delivery of anti-oxidant enzymes SOD1 and GPx1 to the CP could protect striatal and nigral neurons from gp120-induced injury.

Section snippets

Animals

Female Sprague–Dawley rats (200–250 g) were purchased from Charles River Laboratories (Wilmington, MA). Protocols for injecting and euthanizing animals were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee (IACUC), and are consistent with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) standards. Because estrogens can regulate microglial activation in some conditions, experiments were done in female rats at similar

Direct injection of gp120 into the CP induces tissue loss

Following the administration of different doses of gp120 in 1 μl saline into the CP, a partly hemorrhagic lesion was already present 6 h after injection. At later time points, striatal tissue loss occurred with gp120 doses of 250 and 500 ng gp120, and by 14 days after injection an empty cavity was seen in the CP. Injection of 100 ng gp120 caused a much smaller lesion. No significant tissue loss was observed when saline was injected instead of gp120 (Fig. 1A). By 2 days, gp120 caused an area of

Discussion

Basal ganglia are often affected in patients with HIV-1 infection, leading to some of the manifestations of HIV-1-related neurological disorders. A reduction of dopamine and homovanillic acid, its major metabolite, has been shown in the caudate nucleus of patients with AIDS, consistent with a loss of nigrostriatal dopaminergic neurons (Sardar et al., 1996). The highest levels of HIV RNA in AIDS patients were found in the basal ganglia (Wiley et al., 1998). Abnormal extrapyramidal symptomatology

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