HIV-1 Tat neurotoxicity: A model of acute and chronic exposure, and neuroprotection by gene delivery of antioxidant enzymes☆
Highlights
► HIV-1 gene product Tat elicits reactive oxygen species and cause neuron apoptosis. ► We studied acute exposure by injecting recombinant Tat into the caudate-putamen. ► Ongoing Tat-induced neurotoxicity was studied using a SV40-derived vector, SV(Tat). ► Acute and chronic Tat injection induced lipid peroxidation and neuronal apoptosis. ► Gene delivery of antioxidant enzymes protected from Tat-induced lesions.
Introduction
HIV-1 enters the Central Nervous System (CNS) soon after it enters the body. There, it is largely impervious to highly active anti-retroviral therapeutic drugs (HAART). As survival with chronic HIV-1 infection improves, the number of people harboring the virus in their CNS increases. The prevalence of HIV-associated neurocognitive disorder (HAND) therefore continues to rise, and less fulminant forms of HAND such as minor neurocognitive/motor disorder (MCMD) have become more common than their more fulminant predecessors. HAND remains a significant independent risk factor for AIDS mortality (Major et al., 2000, Mattson et al., 2005, McArthur et al., 2005, Nath and Sacktor, 2006, Ances and Ellis, 2007, Antinori et al., 2007).
The pathogenesis of HAND largely reflects the neurotoxicity of HIV-1 proteins. Neurons themselves are rarely infected by HIV-1, and neuronal damage is felt to be mainly indirect. HIV-1 infects resident microglia, periventricular macrophages and some astrocytes (Ranki et al., 1995), leading to increased production of cytokines and to release of HIV-1 proteins, the most likely neurotoxins, among which are the envelope (Env) proteins and Tat (Kaul et al., 2001, van de Bovenkamp et al., 2002). The HIV-1 trans-acting protein Tat, an essential protein for viral replication, is a key mediator of neurotoxicity. Brain areas that are particularly susceptible to Tat toxicity include the CA3 region and the dentate gyrus of the hippocampus and the striatum. Tat is internalized by neurons primarily through lipoprotein related protein receptor (LRP) and by activation of NMDA receptor (Eugenin et al., 2003). It also interacts with several cell membrane receptors, including integrins, VEGF receptor in endothelial cells and possibly CXCR4 (Ghezzi et al., 2000).
Tat can directly depolarize neuron membranes, independently of Na+ flux (Magnuson et al., 1995) and may potentiate glutamate- and NMDA-triggered calcium fluxes and neurotoxicity (Magnuson et al., 1995). It promotes excitotoxic neuron apoptosis (Bonavia et al., 2001, Haughey et al., 2001) by activating endoplasmic reticulum pathways to release intracellular calcium ([Ca2 +]i) (Norman et al., 2008). Consequent dysregulation of calcium homeostasis (Kruman et al., 1998, Nath et al., 2000, Bonavia et al., 2001) leads to mitochondrial calcium uptake, caspase activation and, finally, neuronal death. Tat also increases levels lipid peroxidation (Haughey et al., 2004) by generating the reactive oxygen species (ROS) superoxide (O2−) and hydrogen peroxide (H2O2). It activates inducible nitric oxide synthase (iNOS) to produce nitric oxide (NO), which binds superoxide anion to form the highly reactive peroxynitrite (ONOO) (Bonfoco et al., 1995). The latter may attack lipids, proteins and DNA, enhancing oxidant-related injury.
Tat neurotoxicity has been reported in cultured cells, but fewer studies have demonstrated its neurotoxic properties in vivo (Jones et al., 1998, Bansal et al., 2000, Askenov et al., 2001, Askenov et al., 2003). Tat-induced protein oxidation is well documented (Askenov et al., 2001, Askenov et al., 2003) but little is known about its effects on lipid peroxidation. We recently demonstrated that Tat activates multiple signaling pathways. In one of these, Tat-induced superoxide is an intermediate, while the other utilizes peroxide as a signal transducer (Agrawal et al., 2007). We show here that directly injecting Tat into the caudate putamen (CP) can induce neuron apoptosis and lipid peroxidation, and that Tat can be transported retrograde from the CP to the Substantia Nigra. We also tested the consequences of protracted exposure to Tat, by expressing Tat over time in CNS cells. Finally, prior gene delivery of the antioxidant enzymes Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) into the CP before injecting Tat protected against Tat-induced injury.
Section snippets
Animals
Female Sprague–Dawley rats (300–350 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 Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) standards. Because estrogens can influence inflammation in the brain and attenuate HIV-1 gp120- and Tat-induced oxidative stress, experiments
Injection of Tat into the CP elicits apoptosis
After injection of 10 ng Tat into the CP, TUNEL assays were performed to detect apoptosis between 1 h and 8 weeks. TUNEL-positive cells were counted in the whole CP, at least in 5 different sections for each animal. There were extremely rare TUNEL-positive cells when the CP was injected with saline or rat IgG (negative controls). Numbers of TUNEL-positive cells peaked 2 days after injection of Tat (Figs. 1A, B). TUNEL staining was localized to the nuclei (Fig. 1C).
Tat-induced apoptotic cells are mainly neurons
Sections from the CP obtained 2
Discussion
Oxidative stress plays a role in the development of HAND, and other neurodegenerative diseases (Beal, 1995, Smith et al., 1995, Cao et al., 1998, Mollace et al., 2001, Turchan et al., 2003). Oxidative stress in HIV-1 dementia has been documented by analyses of brain tissue, including increased levels of lipid peroxidation product (i.e., HNE) and the presence of oxidized proteins. Membrane-associated oxidative stress correlates with HIV-1 dementia pathogenesis and cognitive impairment (Mattson
Conclusion
Injection of Tat and SV(Tat) into the rat CP causes respectively acute and ongoing apoptosis and oxidative stress in neurons. These approaches to Tat administration may therefore represent useful animal models for studying the pathogenesis and treatment of HIV-1 Tat-related damage. Gene delivery of antioxidant enzymes by recombinant SV40-derived vectors protects against Tat-induced oxidative stress and neuronal apoptosis.
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