HIV-1 Tat neurotoxicity: A model of acute and chronic exposure, and neuroprotection by gene delivery of antioxidant enzymes

Neurobiology of Disease 45 (2012) 657–670

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HIV-1 Tat neurotoxicity: A model of acute and chronic exposure, and neuroprotection by gene delivery of antioxidant enzymes☆ Lokesh Agrawal a, Jean-Pierre Louboutin a,⁎, Beverly A.S. Reyes b, Elisabeth J. Van Bockstaele b, David S. Strayer a a b

Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA Department of Neurosurgery, Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA 19107, USA

a r t i c l e

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Article history: Received 29 March 2011 Revised 14 September 2011 Accepted 8 October 2011 Available online 17 October 2011 Keywords: HIV-1 Tat Antioxidant enzymes Gene therapy SOD1 HIV-1 associated neurocognitive disorders

a b s t r a c t HIV-associated neurocognitive disorder (HAND) is an increasingly common, progressive disease characterized by neuronal loss and progressively deteriorating CNS function. HIV-1 gene products, particularly gp120 and Tat elicit reactive oxygen species (ROS) that lead to oxidant injury and cause neuron apoptosis. Understanding of, and developing therapies for, HAND requires accessible models of the disease. We have devised experimental approaches to studying the acute and chronic effects of Tat on the CNS. We studied acute exposure by injecting recombinant Tat protein into the caudate-putamen (CP). Ongoing Tat expression, which more closely mimics HIV-1 infection of the brain, was studied by delivering Tat-expression over time using an SV40-derived gene delivery vector, SV(Tat). Both acute and chronic Tat exposure induced lipid peroxidation and neuronal apoptosis. Finally, prior administration of recombinant SV40 vectors carrying antioxidant enzymes, copper/zinc superoxide dismutase (SOD1) or glutathione peroxidase (GPx1), protected from Tat-induced apoptosis and oxidative injury. Thus, injection of recombinant HIV-1 Tat and the expression vector, SV(Tat), into the rat CP cause respectively acute or ongoing apoptosis and oxidative stress in neurons and may represent useful animal models for studying the pathogenesis and, potentially, treatment of HIV-1 Tat-related damage. © 2011 Elsevier Inc. All rights reserved.

Introduction HIV-1 enters the Central Nervous System (CNS) soon after it enters the body. There, it is largely impervious to highly active antiretroviral therapeutic drugs (HAART). As survival with chronic HIV1 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

☆ This work was supported by NIH grant MH70287. ⁎ Corresponding author at: Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Room 255, Philadelphia, PA 19107, USA. E-mail address: [email protected] (J.-P. Louboutin). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2011.10.005

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 glutamateand 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 ([Ca 2 +]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).

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The latter may attack lipids, proteins and DNA, enhancing oxidantrelated 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, 2003). Tatinduced protein oxidation is well documented (Askenov et al., 2001, 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. Material and methods 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 gp120and Tat-induced oxidative stress, experiments were done in female rats at similar points of their estrous cycle determined by vaginal smears. Animals were preferably injected during the diestrus stage of the estrous cycle. Estrogens are typically low during this stage. In any case, animals were not injected during the estrus stage of the cycle when estrogens levels are elevated. The diet that the animals received was a standard commercial, regular powdered rodent diet without any component that might cause oxidative stress (e.g., such as high fat diet, or high manganese) and was not folate/methyl or iron deficient. Animals had free access to water and diet. Numbers of animals used in experiments are indicated in the “Experimental design” section. Reagents Recombinant Tat was obtained through the NIH AIDS Research & Reference Reagent Program, Division of AIDS, NIAID, NIH, Germantown, MD. Tat was reconstituted in PBS containing 1 mg/ml BSA and 0.1 mM DTT, then aliquoted and stored in dark bottles to avoid oxidation. Endotoxin levels of Tat were also determined using E-Toxate kit (ET0200) (Sigma Chemical Co., St Louis, USA). Tat protein was found to be endotoxin free with concentration below 0.015 Endotoxin units (EU)/ml, which was below the lowest concentration of endotoxin standard. Antibodies Diverse primary antibodies were used: rabbit anti-Iba1 (IgG; 1:100), a marker of quiescent and active microglia (Waco Chemicals, Osaka, Japan), mouse anti-glial fibrillary acidic protein (GFAP) (IgG2b; 1:100) (BD Pharmingen Franklin Lakes, NJ), mouse anti-NeuN (IgG1; 1:100), mouse anti-Tau-1 (IgG2a); 1:100) (Chemicon International, Temecula, CA), Fluorescein IsoThioCyanate (FITC)-conjugated mouse anti-rat CD11b, a marker of quiescent microglial cells (IgG; 1:100; clone OX-42 recognizes the receptor for iC3b component of the complement) (Accurate Chemicals, Westbury, NY), mouse anti-rat

CD68/ED1 (IgG1; 1:100), a marker of activated microglial cells in a phagocytic state (Serotec, Oxford, UK), sheep anti-Tat (IgG; 1:100) (Santa Cruz, Santa Cruz, CA). Secondary antibodies were used at 1:100 dilution: FITC and Tetramethyl Rhodamine IsoThioCyanate (TRITC)-conjugated goat anti-mouse IgG (γ-chain specific and against whole molecule respectively), TRITC-conjugated goat antirabbit IgG (whole molecule), FITC-conjugated sheep anti-rabbit IgG (whole molecule), FITC-conjugated rabbit anti-goat IgG (whole molecule), Cy3-conjugated rabbit anti-goat IgG (whole molecule) (Sigma, Saint-Louis, MO), FITC and TRITC-conjugated donkey antimouse IgG (whole molecule), FITC and TRITC-conjugated donkey antisheep IgG (whole molecule), Cy3-conjugated donkey anti-rabbit IgG (whole molecule) and anti-goat IgG (whole molecule) (Jackson ImmunoResearch Laboratories, Inc, WestGrove, PA). Vector production The general principles for making recombinant, Tag-deleted, replication-defective SV40 viral vectors have been previously reported (Strayer, 1999; McKee and Strayer, 2002). Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) transgenes were subcloned into pT7[RSVLTR], in which transgene expression is driven by the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Tat expression in SV(Tat) is driven by RSV-LTR. The cloned rSV40 genome was excised from its carrier plasmid, gel-purified and recircularized, then transfected into COS-7 cells. These cells supply large T-antigen (Tag) and SV40 capsid proteins in trans, which are needed to produce recombinant replication-defective SV40 viral vectors (Strayer et al., 1997). Crude virus stocks were prepared as cell lysates, then bandpurified by discontinuous sucrose density gradient ultracentrifugation and titered by quantitative (Q)-PCR (Strayer et al., 2001). SV(human bilirubin-uridine 5′-diphosphate-glucuronosyl-transferase) (BUGT), which was used here as negative control vector, with a non-toxic byproduct, has been reported (Sauter et al., 2000). Experimental design Tat injection It is difficult to know what concentration of Tat in the brain is sufficient to produce pathogenesis (Bansal et al., 2000). The amount of Tat injected was decided after previous experiments showing that 10 ng Tat was the optimal amount of Tat necessary for inducing neuronal apoptosis in vitro (Agrawal et al., 2007). In this study, cells were incubated with 0, 1, 10 and 100 ng/ml of recombinant soluble Tat for 48 h. Apoptotic bodies were analyzed using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL). Neurons were identified by their expression of MAP-2. The intensity and frequency of TUNEL+ cells were significantly higher (P b 0.001) as compared to untreated cultures. Increasing Tat concentration above 10 ng/ml did not increase apoptosis significantly. Thus 10 ng/ml of Tat was used in all subsequent in vitro studies. For in vivo studies, Tat was suspended in saline at a concentration of 10 ng/μl. The volume of solution containing Tat that was injected into the striatum was 1 μl. By comparison, the concentration of Tat injected in the striatum ranged from 1–50 μg/μl (Bansal et al., 2000), to 20 μg/μl (Theodore et al., 2006), and 50 μg/μl (Askenov et al., 2003). In order to study Tat-induced abnormalities, 1 μl saline containing 10 ng Tat was injected stereotaxically into the caudate-putamen (CP) of rats whose brains were harvested at different time points after the injection (1, 2, 6, 24, 48 h and 1, 2, 4 and 8 weeks, with 4 rats at each time point; total: n = 36 rats). Controls (n = 2 for each time point) received saline instead of Tat in the CP (total: n = 18 rats). In order to test the specificity of the effects of Tat, 1 μl saline containing 10 ng rat IgG (Sigma) was injected into the CP as a control unrelated protein (n = 2 for each time point,

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total = 18). Saline and saline containing rat IgG were used as negative controls, as was the contralateral side of the unilaterally injected brains. Injection of SV(Tat) To assess the effects of more protracted exposure to Tat, we used a system in which Tat is expressed over time in CNS cells. Rats were injected intra-CP with SV(Tat), which allows for continued Tat production by transduced cells. The brains from the animals were then studied 1, 2 and 4 weeks after injection of SV(Tat) for Tat expression and apoptosis. SV(Tat) was injected into the CP of Sprague–Dawley rats (n = 5 at each time, total =15). Controls received SV(BUGT) instead of SV(Tat) in the CP (n= 5 at each time point, n =15). Challenge with Tat after administration of SV(GPx1) or/and SV(SOD1 To study possible protection by rSV40-mediated overexpression of SOD1 and/or GPx1 from Tat-related injury, we first injected the CP of rats with SV(SOD1) (n = 5), SV(GPx1) (n = 5) and a mixture 50:50 SV (SOD1)/SV(GPx1) (n = 5). One month later, the CP in which SV (SOD1) or/and SV(GPx1) has been administered was injected with 10 ng Tat. Brains were harvested 2 days after injection of Tat into the CP. They were studied for apoptosis and MDA level at d2 (n = 5 for each vector, total = 15 rat). In all cases, controls received SV (BUGT) in the CP instead of SV(SOD1), SV(GPx1) or SV(SOD1)/SV (GPx1) (n = 5). In vivo injection of Tat and vectors Rats were anesthetized with isofluorane UPS (BaxterHealthcare Corp., Deerfield, IL) (1.0 unit isofluorane/1.5 l O2/min) and placed in a stereotaxic apparatus (Stoelting Corp., Wood Dale, IL) for cranial surgery. Body temperature was maintained at 37 °C by using a feedback-controlled heater (Harvard Apparatus, Boston, MA). Glass micropipettes (1.2 mm outer diameter; World Precisions Instruments, Inc., Sarasota, FL) with tip diameters of 15 μm were backfilled with either 5 μl of SV(BUGT), SV(SOD1), SV(GPx1), or a mixture 50:50 SV (SOD1)/SV(GPx1) viral vector, which contains approximately 10 7 particles. The vector-filled micropipettes were placed in the CP using coordinates obtained from the rat brain atlas of Paxinos and Watson (1986). For injection into the CP, a burr hole was placed +0.48 mm anterior to bregma and −3.0 mm lateral to the sagittal suture. Once centered, the micropipette was placed 6.0 mm ventral from the top of the brain. Same coordinates were used for injecting 10 ng Tat in 1 μl saline, as well as for injecting saline and 1 μl saline containing 10 ng rat IgG. Tat or the vector were given by a Picospritzer II (General Valve Corp., Fairfield, NJ) pulse of compressed N2 duration 10 ms at 20 psi until the fluid was completely ejected from the pipette. Following surgery, animals were housed individually with free access to water and food. Procedure for harvesting the tissue After a variable survival period, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (Abbott Laboratories, North Chicago, IL) at 60 mg/kg and perfused transcardially though the ascending aorta with 10 ml heparinized saline followed by ice cold 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in 0.1 M phosphate buffer (pH 7.4). Immediately following perfusion-fixation, the rat brains were removed, placed in 4% paraformaldehyde for 24 h, then in a 30% sucrose solution for 24 h, then frozen in methyl butane cooled in liquid nitrogen. Samples were cut on a cryostat (10 μm sections).

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Immunocytochemistry For immunofluorescence, coronal cryostat sections (10 μm thick) were processed for indirect immunofluorescence. Blocking was performed by incubating 60 min with 10% goat, or 10% donkey, serum in phosphate buffer saline (PBS; pH 7.4). Then, sections were incubated with antibodies diluted according to manufacturer's recommendations: 1 h with primary antibody, then 1 h with secondary antibody diluted 1:100, all at room temperature. Double immunofluorescence was performed as previously described (Rouger et al., 2001). All incubations were followed by extensive washing with PBS. To stain nuclei, we used mounting medium containing 4′,6-diamidino-2phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Specimens were finally examined under a Leica DMRBE microscope (Leica Microsystems, Wetzlar, Germany). Negative controls performed each time immunostaining was done consisted of preincubation with PBS, substitution of non-immune isotype-matched control antibodies for the primary antibody, and/or omission of the primary antibody. Staining of neurons using NeuroTrace NeuroTrace (NT) staining has been used as a neuronal marker in studies focusing on the characterization of neurons (Morinville et al., 2004; Nikonov et al., 2005) and NT staining has been performed as previously reported (Agrawal et al., 2006; Louboutin et al., 2006, 2007a, 2011a). After rehydration in 0.1 M PBS, pH 7.4, sections were treated with PBS plus 0.1% Triton X-100 10 min, washed twice for 5 min in PBS then stained by NT (Molecular Probes, Inc., Eugene, OR) (1:100), for 20 min at room temperature. Sections were washed in PBS plus 0.1% Triton X-100 then ×2 with PBS, then let stand for 2 h at room temperature in PBS before being counterstained with DAPI. Combination NT+ antibody staining was performed using primary and secondary antibodies staining first (see above), followed by staining with the NT fluorescent Nissl stain. All experiments were repeated 3 times and test and control slides were stained the same day. TUNEL assay Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay was performed according to the manufacturer's recommendations (Roche, Indianapolis, IN) and following a previously described protocol (Agrawal et al., 2006). We used two assays: end-labeling of DNA with fluorescein-dUTP and tetramethylrhodaminedUTP (TMR-dUTP). To quantitate the TUNEL assay, TUNEL-positive cells were expressed as a total number per CP measured in at least 5 consecutive sections, using the same computerized system as described in the Morphometry section. The final number was an average of results measured in the different sections. Morphometry Tat-, NeuN-, NT-, CD68/ED1-, GFAP-, CD11b- and Iba-1-positive cells were enumerated manually on the injected and uninjected sides in the whole CP of animals injected with Tat, or saline, in at least 5 consecutive sections using a computerized imaging system (Image-Pro Plus, MediaCybernetics, Bethesda, MD) as previously described (Louboutin et al., 2007a, 2010c). In all cases, the final number was an average of results measured in the different sections. This procedure already described for assessment of numbers of transgene-positive cells in the brain (Mandel et al., 1998; Louboutin et al., 2007a) allows quantitative and relative comparisons among different time points, although it does not reflect the total number of transduced cells in vivo. The results were expressed as percentages of NT-positive cells.

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Tat- and TUNEL-positive cells were enumerated as previously described in SV(Tat) recipients. Neuronal loss was estimated by calculating the ratio of the number of NT-positive cells on the injected side compared with the number of NT-positive cells on the uninjected side. The longitudinal distribution of the transgene away from the injection site was assessed as the distance between the most distant sections anterior and posterior to the injection site demonstrating transgene expression (Louboutin et al., 2007a).

Measurement of malondialdehyde Measurement of malondialdehyde (MDA) was used as an indicator of lipid peroxidation. The MDA assay was performed using lipid peroxidation kit (Oxford Biochemical Research, Oxford, MI). The assay is based on reaction of chromogenic reagent N-methyl-2phenylindole, with MDA and 4-hydroxyalkenals at 45 °C to form a stable chromophore at 586 nm. MDA standards were prepared with target concentration in reaction mixture ranging from 0 to 4 μM, and the assay was performed following manufacturer's instructions and as previously reported (Louboutin et al., 2009a). Cryostat sections (50 μm thick) of the striatum areas injected with Tat or saline were harvested then homogenized in buffer, and lysates were prepared as previously reported (Agrawal et al., 2006; Louboutin et al., 2009a). Briefly, for 200 μL of sample, 650 μL of reagent R1 and 150 μL of 12 N HCL were added and incubated at 45 °C for 60 min. The absorbance was measured at 586 nm, and the MDA levels of unknown samples were deduced from the standard curve. MDA levels of samples from CPs injected with Tat after prior administration of SV(BUGT), SV(SOD1), SV(GPx1) or SV(SOD1) and SV(GPx1) were measured the same way.

Statistical analysis Comparison of medians between 2 groups was achieved by using the Mann–Whitney test (with a two-tail p value). Comparison of medians between more than 2 groups was done by using the Kruskall–Wallis test. The difference between the groups was considered significant when P b 0.05. On graphs, values are represented as means ±s.e.m. For correlations studies, we used Spearman nonparametric test.

Results 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 TUNELpositive 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 days after Tat injection were double-stained for TUNEL and NeuN, a marker of neurons, or Iba-1 or CD11b, both markers of microglial cells. Numerous TUNELpositive cells were seen, most of which were immunopositive for NeuN, identifying them as neurons. Rare TUNEL-positive cells also co-immunostained for Iba-1- or CD11b, both microglial markers (Fig. 2). About 96% of TUNEL-positive cells were NeuN-positive, 4% were microglial cells immunostained for Iba-1, or/and CD11b.

Tat is internalized in neurons To determine if Tat is internalized in vivo, sections from the CP obtained 24 h and 48 h after injection were double stained for Tat and either NT, a marker of neurons, CD11b, a marker of quiescent microglial cells, or GFAP, a marker of astrocytes. In these rats, Tat was detected along the needle tract but also at some distance of it (Fig. 3A). The majority (97%) of Tat-positive cells were also NTpositive. A small fraction (b3%) of CD11b-positive cells, mostly localized along the needle track, were also Tat-immunoreactive. Comparable results were seen at 24 and 48 h for neurons and CD11b-positive cells. No GFAP-positive cells were Tat-positive at 24 h but rare astrocytes were Tat-immunopositive at 48 h (Fig. 3B). In saline- or rat IgG-treated animals, there was no evidence of Tat-immunoreactivity (not shown). Axonal transport of Tat to the Substantia Nigra To determine if Tat, once internalized, is transported retrograde to cell bodies, Tat-immunoreactivity was examined in neurons of the Substantia Nigra (SN) at different times after its injection. Dopaminergic neurons and fibers of the SN were visualized by immunoreactivity for tyrosine hydroxylase (TH, the rate limiting enzyme for dopamine synthesis). By 2 days post-injection, a few Tat-positive cells were detected in the nigrostriatal bundle and in the SN pars compacta but more such Tat-containing cells were detected in these areas by day 7, and remained Tat-immunopositive up to 2 weeks post-intra-CP injection (Figs. 4A, B). Tat-IR cells in the SN pars compacta were immunopositive for TH (Fig. 4B′). Tat was not detected in the SN of the uninjected control side, or after injection of saline or rat IgG in the CP. HIV-1 Tat elicits increases in different populations of microglial cells as well as in astrocytes We first used immunocytochemistry to characterize different populations of microglial cells to assess if Tat induced an increase of microglial cells. Immunostaining for Iba-1, a marker of quiescent and activated microglial cells, and for CD68/ED1, a marker of activated microglial cells (mainly in the phagocytic state), showed the numbers of Iba-1- and CD68/ED1-positive cells had increased 7 and 14 days after injection of Tat into the CP. No increase in the number of microglial cells was seen in the contralateral uninjected side or after injection of saline into the CP (P b 0.01: Tat vs. saline, both for Iba-1 and CD68/ED1, at d7 and d14). We then determined whether tissue damage and microglial cell proliferation induced by Tat led to proliferation of astrocytes. We observed an increase in the number of GFAP-positive cells in the CPs injected with Tat. There were more astrocytes 14 days after injection of Tat into the CP than at 7 days (P b 0.05). Astrocytes were significantly less numerous in the contralateral CP or after injection of saline into the CP (P b 0.01: Tat vs. saline, at d7 and d14) (Fig. 5). Injection of Tat induces oxidative damage Because oxidative injury is seen in the brains of patients with HAND, we tested for oxidative damage occurring after Tat injection. Lipid peroxidation was measured by calorimetric MDA assay. Injection of Tat elicited more MDA than did the control saline at the different times (P b 0.001) (Fig. 6). Transgene expression of Tat by intra-CP injection of SV(Tat) is related to apoptosis HIV-1 infection of the brain is a long-term disease that is characterized by production of HIV-1 proteins for extended periods of

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Fig. 1. Intra-CP injection of Tat induces apoptosis. To detect apoptosis, TUNEL assays [tetramethylrhodamine-dUTP (TMR-dUTP)] were performed from 1 h to 8 weeks after injection of 10 ng Tat into the CP. Numerous TUNEL-positive cells were seen after injection of Tat into the CP, particularly 2 days after administration. Extremely rare TUNEL-positive cells were detected when the CP was injected with rat IgG or saline (negative controls). The site of injection is indicated by *. Note that no TUNEL-positive cells were seen on the uninjected side or when sections were incubated with label solution only without the enzyme (terminal deoxynucleotidyl transferase). B. TUNEL-positive cells were counted in the whole CP, at least in 5 different sections for each animal. The number of TUNEL-positive cells peaked 2 days after Tat injection. C. TUNEL (fluorescein-dUTP) staining was localized to the nuclei visualized by 4′,6-diamidino-2-phenylindole (DAPI). Bar: A, B: 40 μm.

time. We have previously reported the use of an SV40-derived expression vector, SV(gp120), to provide ongoing expression of HIV-1 envelope glycoprotein as a model to study more chronic CNS consequences of continuing production of neurotoxic HIV-1 gene products. Since Tat is also neurotoxic, we tested whether a similar system could be used to study the effects of Tat production in the CNS for a protracted period of time. Therefore, we injected SV(Tat), from which long-term HIV-1 Tat expression is expected, into the CNS. Expression of Tat protein, which is produced as a result of

transduction, was demonstrated by immunocytochemistry at different time points after injection of SV(Tat). One month after SV (Tat)injection, Tat-positive cells were still present in the CP. Tat was localized mainly within neurons, immunopositive for Tau-1 (Fig. 7A). No Tat was detected in the contralateral uninjected side (not shown) or after injection of SV(BUGT), a control vector, into the CP. Apoptotic cells were detected at different times by TUNEL assay in SV(Tat) recipients. Rare TUNEL-positive cells were seen after

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Fig. 2. The majority of apoptotic cells following intra-CP injection of Tat are neurons. Cryostat sections from the CP obtained 2 days after injection of 10 ng Tat were double stained for TUNEL [fluorescein-dUTP, upper two rows or tetramethylrhodamine-dUTP (TMR-dUTP), lower row] and NeuN, a marker of neurons, Iba-1 or CD11b, both markers of microglial cells. Secondary antibodies were TRITC-conjugated (upper two rows) or FITC-conjugated (lower row). The majority of TUNEL-positive cells were also immunoreactive for NeuN identifying them as neurons. By contrast, occasional TUNEL-positive cells were also Iba-1- or CD11b-positive, and so were consistent with microglia. Bars: upper row: 100 μm, inset: 30 μm, lower two rows: 30 μm.

injection of SV(BUGT) (P b 0.001). One month after SV(Tat) injection, TUNEL-positive cells were mainly Tat-expressing cells. Not all Tatpositive cells were apoptotic 4 weeks after intra-CP injection of SV (Tat) (Fig. 7B). Four weeks after injection, some TUNEL-staining colocalized with NT, some other staining did not. CD11b-positive cells were rarely apoptotic (Fig. 7C). No TUNEL-positive astrocytes were seen (not shown). No apoptotic cells were detected on the contralateral uninjected side (not shown). The expression of Tat protein was sustained after injection of SV (Tat) (Fig. 8A). However, there was a decrease in the number of Tat-positive cells with time, probably because of the neuronal death following Tat expression. The longitudinal extent of transgene expression in the CP [estimated as previously reported (Louboutin et al., 2007a)] was 3.7 mm. We also quantitated the number of apoptotic cells at different times following SV(Tat) injection (Fig. 8B). At all time points, the number of apoptotic cells in SV(Tat) recipients was significantly higher than in SV(BUGT) injected rats (P b 0.001). The neuronal loss, expressed as a percentage of NT-positive cells compared with the contralateral side, was between 26.7 and 34.3% after injection of SV(Tat) in the CP and was significantly higher than after injection of SV(BUGT) in the same area (P b 0.001) (Fig. 8C). Lipid peroxidation, measured by calorimetric MDA assay, showed that injection of SV(Tat) induced significantly more MDA than was seen in the recipients of saline control (saline: 0.4 μM vs. SV(Tat): 1.4 μM; P b 0.01). Thus, the injection of SV(Tat) induces substantial expression of Tat in neurons, lipid peroxidation, and neuron death.

Overexpression of antioxidant enzymes protects against Tat-induced lesions To determine whether rSV40-delivered antioxidant enzymes Cu/Zn SOD1 and GPx1 strongly could protect against Tat-induced CNS injury, we administered SV(SOD1), SV(GPx1), and a mixture SV(SOD1)/SV (GPx1) to the CP 1 month before we injected Tat. We then assayed for apoptotic cells after Tat inoculation. Prior administration of SV (SOD1) and SV(GPx1) protected the brains from the damage elicited by Tat. That is, there were fewer Tat-induced apoptotic cells in recipients of SV(SOD1) or SV(GPx1). However, the greatest protection from Tat-related neuronal apoptosis was seen when both vectors were administered into the CP together (Fig. 9A). Prior SV40 delivery of these antioxidant enzymes also reduced Tat-induced lipid peroxidation (Fig. 9B). In this case, the reduction in lipid peroxidation was seen in all antioxidant recipient groups, with animals receiving SV(GPx1), whether alone or together with SV(SOD1), being the best protocole. 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

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Fig. 3. Tat is internalized in neurons. Sections from the CP obtained after injection of 10 ng Tat into the CP were double stained for Tat and either NT, a marker of neurons, CD11b, a marker of quiescent microglial cells, or GFAP, a marker of astrocytes. Secondary antibody against sheep Tat antibody was TRITC-conjugated, secondary antibody against rat CD11b was FITC-conjugated, and secondary antibody against mouse GFAP antibody was FITC-conjugated. The NeuroTrace (NT) green fluorescent stain used here exhibits bright, green fluorescence that is visible with filters appropriate for fluorescein. There was no evidence of Tat-immunoreactivity (IR) in saline- or rat IgG-treated animals (not shown). In Tatinjected rats, Tat-IR was detected along the needle tract but also at some distance of it (A). Images 1 and 2 correspond to areas close to the lateral ventricle and the corpus callosum respectively. The majority (97%) of Tat-positive cells were NT-positive, while a small fraction (3%) of CD11b-positive cells were also Tat-immunoreactive (B). Unlike neurons, these cells were localized along the needle track only. No GFAP-positive cells were Tat-positive at 24 h but rare astrocytes were Tat-immunopositive at 48 h. Bars: A: 30 μm; B: 1st row: 30 μm, 2nd row: 15 μm, 3rd row: 20 μm, and 4th row: 15 μm.

cognitive impairment (Mattson et al., 2005). In the case of HIV-1 infection, Tat and gp120 can elicit such oxidative stress (Mattson et al., 2005; Agrawal et al., 2007), as has been shown with gp120induced lipid peroxidation and production of hydroxynonenal (HNE) (Louboutin et al., 2009a). HNE-positive neurons have been demonstrated in the brains of patients with HIV-1 encephalitis (Turchan et al., 2003; Haughey et al., 2004; Cutler et al., 2004). Such oxidative stress can induce apoptosis in cultured neurons (Kruman et al., 1997). It can also damage neurons and cause cognitive

dysfunction in vivo (Bruce-Keller et al., 1998). Tat and gp120 induce ceramide production in cultured neurons by triggering sphingomyelinase activity via a mechanism that involves induction of oxidative stress by CXCR4 activation (Mattson et al., 2005). It is still unclear whether oxidative stress is the primary initiating event associated with neurodegeneration. However, a growing body of evidence implicates it as being involved in at least the propagation of cellular injury that leads to neuron death (Andersen, 2004). Earlier reports support the hypothesis that oxidative modifications of

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Fig. 4. Tat is retrograde transported to the Substantia Nigra. Tat-immunoreactivity was examined in the Substantia Nigra after administration of 10 ng Tat into the CP. Dopaminergic neurons and fibers of the SN were visualized by tyrosine hydroxylase (TH) immunoreactivity. Secondary antibodies against sheep Tat and mouse TH antibodies were respectively FITC- and TRITC-conjugated. Several Tat-IR cells were observed in the nigrostriatal bundle and in the SN pars compacta by day 7 and up to 2 weeks post-intra-CP injection (A, B). Tat-IR cells in the SN pars compacta were immunopositive for TH (B′). Tat-IR was not seen in the SN in the uninjected control side, one day after injection of Tat in the CP, or after injection of rat IgG or saline in the CP. Bars: A: 30 μm, inset: 15 μm; B: 3 upper rows: 30 μm, inset: 15 μm; lower row: 8 μm; B′: 8 μm.

macromolecular cell components (lipids, proteins and nucleic acids) may be an early step in the mechanism of Tat neurotoxicity (Askenov et al., 2001, 2003). Tat-mediated neurotoxicity may be associated with increased oxidative modifications of proteins (Askenov et al., 2001). For example, increased protein carbonyl formation, a well-known marker of protein oxidative damage, occurred early after Tat injection and coincided with the earliest changes in the amount of degenerating striatal neurons (Askenov et al., 2003). When the number of degenerating neurons reaches its peak 1 day after Tat administration, protein oxidation in striatal extracts decreased back to control levels, probably because oxidized proteins are prone to proteolytic

degradation (Askenov et al., 2003). There was a later increase in protein carbonyl levels 7 days after Tat injection, possibly caused by Tat-induced compromise astrocytic functions (Askenov et al., 2003). However, astrocytosis and associated changes in protein oxidation were not sufficient to cause an additional neuronal cell death (Askenov et al., 2003). Tat increase in levels of protein oxidation may result from Tat-mediated increase of the production of prooxidants, as Tat can trigger the production of inflammatory products, which, in turn, may cause an excess of ROS (Bruce-Keller et al., 2001; Askenov et al., 2003). Tat can also mediate neurotoxicity through lipid peroxidation. For proper functioning of the brain, the ROS has to be counter-balanced

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Fig. 5. Microglial and astroglial proliferation elicited by Tat injection into the CP. Brain sections were immunostained for Iba-1, CD68/ED1 and GFAP, 7 (A) and 14 (B) days after intra-CP injection of 10 ng Tat, or control saline. Injection of saline did not elicit a microglial or astroglial response. The graphs show an increase in the numbers of Iba-1- and CD68/ED1-positive cells as well as GFAP-positive cells.

by an antioxidant defense system. The tripeptide glutathione (γ-Lglutamyl-L-cysteinylglycine, GSH) is the key low molecular thiol antioxidant involved in the defense of brain cells against oxidative stress. A decrease in GSH levels has been connected to physiological processes such as aging and neurological disorders like Alzheimer's disease, epilepsy, and Parkinson's disease (Banerjee et al., 2011). Although GSH is the primary molecule involved in detoxification of ROS in the body, antioxidant enzymes like GPx1, are also known to play a role in this process (Steiner et al., 2006). During detoxification of peroxides, the enzyme GPx1 converts GSH to GSSG (glutathione disulphide). Membrane lipids undergo oxidation, producing cytotoxic lipid peroxidation products like MDA and 4-hydroxynonenal (4-HNE). Serum levels of GSH and GPx1 are decreased in HIV-1 patients while MDA levels are increased (Steiner et al., 2006). There are few reports concerning MDA levels in the brain after Tat injection, and there are no data concerning late time points. Repeated intravenous injection of 50 ng Tat during 5 days decreases brain levels of GSH and GPx1 and increases levels of MDA (Banerjee et al., 2011). Pretreatment of the animals with thiol antioxidant N-acetylcysteine amide (NACA) increased the GSH levels significantly, indicating that the antioxidant NACA was able to partially abrogate oxidative stress induced damage in these animals. A significant decrease in the activity of GPx1 was observed in animals treated with Tat, as compared to the controls, indicating that the overwhelming oxidative stress induced by these toxins deplete the antioxidant enzyme in the brain. Animals pretreated with NACA had GPx-1 levels similar to that of the control.

Fig. 6. Intra-CP injection of Tat induces oxidative damage. Lipid peroxidation was measured by calorimetric MDA assay at different time points after injection of 10 ng Tat into the CP. Injection of Tat elicited statistically more MDA than did the control saline at the different times (P b 0.001 Tat vs. saline for the different times examined).

In the present study, we observed elevated MDA levels 4 weeks after Tat administration. The sustained MDA levels might be due to the long-term neuroinflammation, because it is known that increased production of inflammatory products induced by Tat may cause an excess formation of ROS (Askenov et al., 2003). However, persisting elevated MDA levels at 4 weeks were not associated with continuing apoptosis. These data resemble to the ones observed when an increase in protein carbonyl levels seen 7 days after Tat injection was not accompanied by neuron death (Askenov et al., 2003). The present study did not identify the reason(s) why the number of apoptotic neurons does not increase with time, despite the inflammatory response and astrocytosis on one hand, and the persisting lipid peroxidation on the other hand; the origin of the lack of neuron death despite persisting oxidative stress (either lipid peroxidation or protein oxidative damage) is not clear. Oxidative stress is intimately linked with an integrated series of cellular phenomena, which all seem to contribute to neuronal death. Interaction between these various components is not necessarily a cascade but might be a cycle of events, of which oxidative stress is a major component (Andersen, 2004). Consequently, if one of the events, besides oxidative stress, is missing, neuronal death might be limited or not occur. Inhibition of oxidative stress therapeutically, as we did it in the present study, might act to ‘break the cycle’ of cell death. Our results might also suggest that a direct interaction of Tat with neurons is necessary for inducing early neuron death. However, it is difficult to directly answer the question whether Tat directly induces oxidative stress in neurons or promotes it through activation of proinflammatory responses. It remains plausible that direct interactions of Tat with neurons play the role of a triggering mechanism in the process of the development of oxidative stress and neurodegeneration (Askenov et al., 2003). Numerous neurotoxic effects of Tat injection can be transient and can be observed only at early time points after its administration. It has been reported that Tat decreases levels of GSH available to relieve oxidant stress (Banerjee et al., 2011). The presence of Tat in the striatum is short-lived after its injection. It is thus possible that after degradation of Tat, the levels of GSH are restored to normal levels. GSH could then protect neurons against ROS directly and indirectly, and could bind to lipid peroxidation products such as HNE, thereby providing neuroprotection (Steiner et al., 2006). Tat can also induce a lipid imbalance in neurons, resulting in an overproduction of sphingomyelin and ceramide, followed by increased levels of HNE (Steiner et al., 2006). Once Tat is degraded, the levels of ceramide and sphingomyelin return to control levels, limiting cellular dysfunction and death due to lipid imbalance. Tat can also trigger the expression of

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Fig. 7. Transgene expression of Tat by intra-CP injection of SV(Tat) induces apoptosis. A. Expression of Tat protein, which is produced as a result of transduction, was demonstrated by immunocytochemistry 4 weeks after injection of SV(Tat). Secondary antibodies against sheep Tat and mouse TAU-1 antibodies were respectively TRITC- and FITC-conjugated. Tat was localized mainly within neurons, immunopositive for Tau-1. No Tat was detected after injection of SV(BUGT), a control vector, into the CP, or in the contralateral uninjected side (not shown). B. Apoptotic cells were detected by TUNEL assay (fluorescein-dUTP) 4 weeks after injection of SV(Tat) into the CP. Secondary antibody against sheep Tat antibody was TRITC-conjugated. Nuclei were stained by DAPI. TUNEL-positive cells were mainly Tat-expressing cells. Not all Tat-positive cells were apoptotic 4 weeks after intra-CP injection of SV(Tat). Rare TUNEL-positive cells were seen after injection of SV(BUGT) (P b 0.001). C. TUNEL assay [tetramethylrhodamine-dUTP (TMR-dUTP)] was performed four weeks after injection of SV(Tat) into the CP. The NeuroTrace (NT) green fluorescent stain used here exhibits bright, green fluorescence that is visible with filters appropriate for fluorescein. Secondary antibody anti-rat CD11b was FITC-conjugated. Some TUNEL-positive colocalized with NT, whereas some others did not. CD11b-positive cells were rarely apoptotic. Bars: A: 45 μm, inset: 20 μm; B: 70 μm, inset: 30 μm; C: upper row: 60 μm; lower row: 30 μm.

iNOS, leading to the overproduction of NO, which can react with superoxide anion to form peroxynitrite, a neurotoxic compound. NO can increase glutamate release from astrocytes, enhancing NMDA excitotoxicity (Steiner et al., 2006). If NO production can be increased shortly after Tat treatment, NO levels would decrease once Tat is degraded. Thus, if persisting increased MDA levels are observed at 4 weeks (possibly linked to continued neuroinflammation), they might not be enough, by themselves, to induce neuronal death, because: 1) there is no direct interaction of Tat with neurons and no Tat-mediated dysregulation of calcium homeostasis 4 weeks after the injection; 2) some protective mechanisms (i.e., GSH) are probably restored at that

time; and 3) neurotoxic compounds like NO and peroxynitrite are probably not present at that time. As HIV-1 infection of the brain lasts the lifetime of affected individuals, and as eradication of CNS HIV-1 is currently not possible, control of the damage caused by the virus may represent a useful approach to treatment. This could entail limiting oxidative stressrelated neurotoxicity. Gene transfer of antioxidant enzymes has been studied in numerous models of neurological disorders by using diverse viral vectors (Watanabe et al., 2003; Hoehn et al., 2003; Ridet et al., 2006). We previously demonstrated that SV40-derived vectors deliver long-term transgene expression to brain neurons and microglia, when

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Fig. 8. Time course of Tat expression and neuron apoptosis after SV(Tat) injection. A. Graph showing that SV(Tat) induces a sustained Tat expression in the CP. B. Graph showing the number of apoptotic neurons after injection of SV(Tat) into the CP. C. Neuronal loss was expressed as a percentage of NT-positive cells compared with the contralateral side. It was significantly higher than after injection of SV(BUGT) in the same area (P b 0.001).

administered by several different routes. Intracerebral injection of SV (SOD1) or SV(GPx1) carrying the antioxidant enzymes, Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) respectively, into the rat caudate putamen (CP), significantly protects neurons from apoptosis caused by subsequent inoculation of recombinant HIV-1 envelope glycoprotein, gp120 at the same location (Agrawal et al., 2006; Louboutin et al., 2007b, 2009b, 2010a, 2010b, 2010d). Vector

Fig. 9. Overexpression of antioxidant enzymes protects against Tat-induced lesions. A. SV(SOD1), SV(GPx1), and a mixture SV(SOD1)/SV(GPx1) were administered into the CP 1 month before 10 ng Tat injection into the same structure. Then, we assayed for apoptotic cells. Fewer apoptotic cells were seen when SV(SOD1) and SV(GPx1) were administered 1 month before Tat inoculation into the CP. However, the best results were obtained when both vectors were injected together into the CP. Prior administration of SV(SOD1) and SV(GPx1) protected the brains from apoptosis elicited by Tat. B. Lipid peroxidation was measured by calorimetric MDA assay. Tat-induced lipid peroxidation was reduced by prior SV40 delivery of these antioxidant enzymes. *: P b 0.05, **: P b 0.01.

administration into the lateral ventricle (LV) or cisterna magna, particularly if preceded by intraperitoneal mannitol, protects from intra-CP gp120-induced neurotoxicity comparably to intra-CP vector administration (Louboutin et al., 2007b, 2011b). The safety of SV(SOD1) and SV(GPx1) delivered intra-CP has been demonstrated in rats and in Rhesus macaques monkeys, and resulting transgene expression is very durable (Louboutin et al., 2011c). rSV40s were employed in the current study because they transduce a wide range of cell types from humans and other mammals and deliver genes to cells in Go efficiently, including neurons, to achieve long-term transgene expression in vitro and in vivo (Strayer, 1999). These vectors transduce >95% of cultured human NT2-derived neurons, primary human neurons (Cordelier et al., 2003a,b) and microglia (Cordelier and Strayer, 2006) without detectable toxicity. Tat activates multiple signaling pathways, in one of which superoxide acts as an intermediate, while the other utilizes peroxide (Agrawal et al., 2007). Tat elicits lipid peroxidation (induced by generation of ROS), one of the elements leading to cell death. SV(SOD1) and SV(GPx1) not only increase levels of antioxidant enzymes and decrease lipid peroxidation but also have an effect on Tat-induced cytosol calcium fluxes (Agrawal et al., 2007). Neuroprotection from apoptosis caused by Tat requires detoxification of both O2− and H2O2via SOD1 and GPx1. Combining SV(SOD1) and SV(GPx1) provides such protection. Neuroinflammation has been reported after Tat injection. The increase in macrophage/microglial activation might reflect a response to Tat-induced neuronal cell death and as the dead neurons are degraded, the number of infiltrating cells returns to the control level (Askenov et al., 2003). Microglia can be activated by Tat to produce IL-6, TNF-α and IL-1, as well as a number of chemokines: MCP-1, IP-10, MIP-1α, MIP-1β, and RANTES. Tat-induced expression of these chemokines is mediated by activation of different pathways: p38 MAPK, PI3K, and ERK1/2 MAPK. After they transmigrate into the CNS, HIV-infected monocytes release toxic and chemotactic molecules, such as Tat and chemokines that might trigger migration of uninfected microglia to HIV infected areas through autocrine signaling. Following transmigration of HIVinfected monocytes, activation and migration of microglia occurs, then the enhancement of inflammation leads to neurodegeneration (King et al., 2006). Astrogliosis represents a generic response of astrocytes to all types of injuries of the CNS. Activation of astrocytes has been shown in Tat-injected brains (Jones et al., 1998; Bansal et al., 2000; Askenov et al., 2003). Astrogliosis becomes evident several days only after a single Tat injection and follows accumulation of microglia (Askenov et al., 2003). These results are consistent with the idea that cytokines and chemokines serve as a stimulus for induction of astrogliosis. In vitro studies indicate that astrocytes are responsive to a variety of growth factors, chemokines and cytokines. They can be activated

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by Tat and this activation includes increases cytokine expression and release. Cytokine expression in astrocytes and microglia may be differentially regulated by Tat. Tat-activated astrocytes produce IL-6 (Nath et al., 1999), TNF-α (Chen et al., 1997), and MCP-1 (Conant et al., 1998). MCP-1 is important for the recruitment of monocytes to sites of inflammation and in the CNS, MCP-1 induces monocytes to cross the blood–brain barrier and migrate to the source of secretion. The monocytes recruited into the CNS may produce neurotoxins and mediators (cytokines and chemokines) causing CNS damage (King et al., 2006). Upregulation of MCP-1 by Tat in astrocytes is mediated by several transcription factors (i.e., AP-1, NFκB, Smad3) (King et al., 2006). One of the primary functions of the astrocytes is the regulation of extracellular levels of glutamate, the major excitatory neurotransmitter in the CNS, and Tat may induce glutamate dysregulation through different mechanisms. Release of TNF-α by monocytes impairs glutamate metabolism by astrocytes, leading to extracellular glutamate accumulation and neuronal toxicity (King et al., 2006; Steiner et al., 2006). Extracellular glutamate accumulation was demonstrated after Tat treatment in mixed cultures of human neurons and astrocytes (King et al., 2006). Astrocytes transfected by a viral vector to express Tat exhibited 75% reduction in the glutamate uptake (Zhou et al., 2004). Astrocytes might contribute to neuron cell death by other mechanisms. For example, astrocytes treated with Tat express iNOS through activation of NFκB (Liu et al., 2002). Tat is known to trigger an increased production of inflammatory products, which in turn, may cause an excess formation of ROS (Bruce-Keller et al., 2001; Nath et al., 1999). Tat may induce superoxide and nitrite release in a microglial cell line (Bruce-Keller et al., 2001). An exposure of macrophages and astrocytes to Tat for few minutes in vitro is sufficient for sustained release of cytokines for several hours (Nath et al., 1999). Thus, Tat might promote oxidative stress and its consequences (i.e., neuron death) through activation of proinflammatory responses. Tat interacts with many different receptors on various cell types, including a VEGF receptor (flk1) in endothelial cells, integrins and possibly CXCR4. In neurons, Tat internalization is primarily through the lipoprotein related protein receptor (LRP) expressed on the cell surface. In the brain, LRP is expressed by neurons and activated astrocytes (Rebeck et al., 1993; King et al., 2006). We found that neurons were immunopositive for Tat, but no astrocytes were Tatpositive one day post-injection and rare astrocytes were immunopositive at d2. Astrogliosis becomes evident several days only after Tat injection and follows accumulation of microglia (Askenov et al., 2003). It is thus possible that LRP is not strongly expressed one day after Tat injection because astrocytes are not activated at this time. Consequently, accumulation of Tat in astrocytes might be weak one day after Tat injection. We show here that Tat is transported retrograde from the CP to the Substantia Nigra. Retrograde transport of gp120 from the striatum to the Substantia Nigra with consequent apoptosis of dopaminergic neurons has been documented (Bachis et al., 2006; Louboutin et al., 2009b). When cells stably producing Tat were injected in the rat dentate gyrus, Tat was taken up by granule cells and transported along neuronal pathways to the CA3 region (Chauhan et al., 2003). However, our report of Tat being transported retrograde from the CP to the Substantia Nigra is unique, to the best of our knowledge. Basal ganglia are often affected in patients with HIV-1 infection, leading to some of the manifestations of HIV-1-related neurological disorders. Reductions in dopamine and homovanillic acid, its major metabolite, have been shown in the caudate nucleus of patients with AIDS, consistent with a loss of nigrostriatal dopaminergic neurons (Sardar et al., 1996). Abnormal extrapyramidal symptomatology was exacerbated when patients with AIDS and symptoms of psychosis received dopamine-blocking drugs (Ramachandran et al., 1997; Mirsattari et al., 1998; Koutsilieri et al., 2002).

There are no perfect models for HAND. Several animal systems have been used to study the pathogenesis of HIV-1-induced neurological disease. Many of them are based on other lentiviruses (i.e., simian immunodeficiency virus infection of macaques, feline immunodeficiency virus infection of cats, Visna–Maedi virus infection in sheep) (Hurtrel et al., 1992; Thormar, 2005: Lackner and Veazey, 2007). However, only small percentages of animals develop neurological manifestations in these models and the costs for using these species may be high. Transgenic expression of gp120 in mice has been studied (Toggas et al., 1994), but the gp120 in that model is mainly expressed in astrocytes, whereas in humans HIV-1 chiefly infects microglial cells. Other models based on introduction of HIVinfected macrophages into the brains of SCID mice have been proposed, but they suffer from the fact of human macrophages delivered into a murine brain (Avgeropoulos et al., 1998). We (Agrawal et al., 2006; Louboutin et al., 2007b) and others (Bansal et al., 2000; Nosheny et al., 2004) have used model systems in which recombinant gp120 or, as here, Tat, proteins are directly injected into the striatum. The neurotoxicity of such recombinant proteins is highly reproducible and can be used as an interesting tool for testing novel therapeutic interventions. Administration of recombinant proteins is useful in understanding the effects of HIV-1 gene products, and so their individual contribution to the pathogenesis of HAND. However, HIV-1 infection of the brain is a chronic process, and its study would benefit from a model system allowing longer term exposure to HIV-1 gene product. Despite evidence that Tat has been detected in the striatum of patients with HIV encephalitis (Wiley et al., 1996; Hudson et al., 2000), it is difficult to know the exact levels of Tat generated. Although mRNA for Tat was detected by RT-PCR in brain extracts from half (Hudson et al., 2000) or more (Wiley et al., 1996) of patients with HIV encephalitis, protein levels could not be measured by ELISA (Hudson et al., 2000). There are important differences between the situation in the striatum of patients with AIDS and our model where Tat is directly injected into the CP. In this model, Tat is localized initially in extracellular space following intra-CP injection, then it is internalized in neurons and in some microglial cells, while the sites of production are focal in patients with HAND (i.e., infected macrophages and microglial cells). Direct injection of Tat results in acute injury, while production of Tat is more protracted in the brain of patients with HAND. The lesions of HAND reflect chronic injury caused by ongoing production of Tat, as well as other substances, by HIV-1-infected cells. This is in part the reason why we developed experimental models of chronic HIV-1 neurotoxicity based on recombinant SV40 (rSV40) vector-modified expression of gp120 (Louboutin et al., 2009a) or Tat, as described here, in the brain. Some models of ongoing exposure to Tat have been developed. For example, GFAP-driven, doxycycline-inducible Tat transgenic mice have been useful for mechanistic studies of Tat contribution to HAND. However, the reported data concerning neuronal TUNEL positivity are controversial (Kim et al., 2003; Bruce-Keller et al., 2008). We report here an experimental model of chronic HIV-1 Tatinduced neurotoxicity based on recombinant SV40 vector-mediated expression of Tat in the brain. Injection of SV(Tat) into the CP caused expression of Tat in the same structure associated with continuing neuronal apoptosis for at least 4 weeks. We previously reported a rat model of HIV-1 encephalopathy using Env glycoprotein gp120 expression delivered by SV40 vectors in which intra-CP inoculation of SV(gp120) causes ongoing oxidative stress and apoptosis in neurons (Louboutin et al., 2009a). These animal models may be useful for studying the pathogenesis and treatment of HIV-1 related brain damage. Conclusion Injection of Tat and SV(Tat) into the rat CP causes respectively acute and ongoing apoptosis and oxidative stress in neurons. These

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