Intracellular Human Immunodeficiency Virus Tat Expression in Astrocytes Promotes Astrocyte Survival but Induces Potent Neurotoxicity at Distant Sites via Axonal Transport*

Ashok ChauhanDagger , Jadwiga TurchanDagger , Chava Pocernich§, Anna Bruce-Keller, Susan Roth||, D. Allan Butterfield§, Eugene O. Major**, and Avindra NathDagger DaggerDagger

From the Dagger  Department of Neurology, Johns Hopkins University, Baltimore, Maryland 21287, the Departments of § Chemistry, Anatomy and  Neurobiology and || Microbiology and Immunology, University of Kentucky, Lexington, Kentucky 40536, and the ** Laboratory of Molecular Medicine and Neuroscience, NINDS, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, September 12, 2002, and in revised form, December 19, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human immunodeficiency virus (HIV)-Tat protein has been implicated in the neuropathogenesis of HIV infection. However, its role in modulating astroglial-neuronal relationships is poorly understood. Astrocyte infection with HIV has been associated with rapid progression of dementia. We thus initially transfected astrocytes with HIV proviral DNA and confirmed Tat production in these cells. Subsequently, using stably Tat-producing asytocyte cell lines, we observed that Tat promoted astrocyte survival by causing a prominent antioxidant effect and resistance to cell injury in these cells. Tat was released extracellularly where it could be taken up by other cells. Tat remained functionally active following uptake and caused long terminal repeat (LTR) transactivation in lymphocytic and astrocytic cell lines. Tat released from astrocytes caused mitochondrial dysfunction, trimming of neurites, and cell death in neurons. Tat neurotoxicity was attenuated by anti-Tat antibodies, kynurenate or heparan sulfate. The neurotoxic effects of Tat were caused at concentrations lower than that needed to cause LTR transactivation. When Tat-expressing cells were injected into the rat dentate gyrus, Tat was taken up by granule cells and transported along neuronal pathways to the CA3 region where it caused glial cell activation and neurotoxicity. The arginine-rich domain of Tat was essential for both the LTR transactivation and the neurotoxic properties of Tat. Thus HIV-Tat is a potent neurotoxin that may act at distant sites while at the same time it assures its production by preventing cell death in astrocytes where it is produced.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The brain is a frequent target in patients with human immunodeficiency virus (HIV)1 infection resulting in a dementing illness termed HIV dementia (HIVD). The pathological features associated with HIVD include microglial cell activation, astrocytosis, decreased synaptic and dendritic density, and selective neuronal loss (1). Neuronal damage occurs through toxic substances released from infected microglia or macrophages and possibly astrocytes (2). The virus predominantly infects microglia and macrophages where it causes a productive infection. Astrocytes are also frequently infected with HIV but produce limited viral replication (2). The relevance of this viral infection remains unknown. Astrocytes maintain a barrier between blood and brain and provide neuronal support functions. Therefore, astrocytes may serve as a reservoir for the virus or induce neuronal damage by loss of neuronal support functions or release of cellular and viral products. Hence, infection of these cells could potentially have long term consequences on cerebral function.

A striking feature is the presence of Tat transcripts in brains of HIV-infected individuals (3, 4). HIV Tat is a non-structural regulatory protein of 15 kDa that transactivates viral and cellular genes. It is produced in the early phase of infection and is actively released from infected lymphoid cells extracellularly (5). Extracellular Tat may be internalized by uninfected cells, or it may interact with the cell membrane initiating a cascade of events (6). Tat protein has both direct and indirect neurotoxic activity mediated by interaction with glutamate receptors, by disruption of cytokine network, and by reducing the neuroprotective effects of astrocytes (7-11). However, neither is the role of intracellularly expressed Tat on astrocyte function known, nor is the relative role of Tat uptake by neurons or its membrane interactions understood.

Tat can produce oxidative stress in microglia cells (12), monocytes (13), and T lymphocytes. In the latter, intracellular expression of Tat leads to down-regulation of mitochondrial superoxide dismutase, thereby causing impaired mitochondrial membrane potential (14, 15). Oxidative damage is difficult to measure directly; however, oxidized endogenous macromolecules such as free protein carbonyl and 4-hydroxynonenal (HNE) can serve as indicators of oxidative damage (16). Lipid peroxidation leads to the formation of HNE, a lipophilic alkenal that forms stable adducts on mitochondrial proteins (17). It has been suggested to be the key mediator of oxidative stress-induced cell death (18). Hence, in this study, we have determined the effect of Tat on mitochondrial function and other indicators of oxidative stress in astrocytes and neurons.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- Tat-86 from HIVHXB-2 was cloned from pGEX-Tat into pcDNA3 vector at BamHI/EcoRI driven by a cytomegalovirus promoter. Further, Tat-86 and a deletion mutant of Tat-86 from which amino acids 48-56 were deleted by PCR (Delta Tat), was cloned in-frame upstream of green fluorescent protein (GFP) gene driven by cytomegalovirus promoter in pEGFP-N1 (Clonetech) (Fig. 1A). The HIV long terminal repeat (LTR)-driven GFP construct was made in pEGFP vector by deleting the cytomegalovirus promoter and inserting the LTR at SalI and SmaI. The PCR-cloned sequences were verified by double strand DNA sequencing. Tetracycline (tet) "on" system was used for generation of inducible constructs. Tat-86 was cloned downstream of a tet responsive element in pTREX vector (Clonetech). Reverse tetracycline transactivator (rtta) was first cloned in pEGFP vector (Clonetech) at BamHI/EcoRI and further subcloned in GFAP promoter-driven vector pGfaLac-1 at BamHI/BglII (Fig. 1B).


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Fig. 1.   Tat expression systems. A, Tat constructs. Top panel, wild type Tat with demarcation of nuclear localization signal (NLS); middle panel, Tat-GFP; bottom panel, Tat GFP with deletion of 48-56 amino acids (Delta Tat-GFP). B, tetracycline (Tet)-inducible expression. a, GFAP promoter-driven reverse tet transactivator (rTtA) expression; b, rTtA induces tet responsive element (TRE)-driven Tat expression in the presence of doxycycline.

Cell Culture-- Human fetal brain specimens were obtained from fetuses of 12-14 weeks gestational age. Neuronal cultures were prepared as described previously (19). Briefly, the cells were mechanically dissociated, suspended in Opti-MEM with 5% heat inactivated fetal bovine serum, 0.2% N2 supplement, and 1% antibiotic solution (penicillin G 104 units/ml, streptomycin 10 mg/ml, and amphotericin B 25 µg/ml). The dissociated cells were cultured for at least one month prior to use in experiments. Human fetal astrocytes, rat C6 glioma cells, SVGA (a human astrocytic subclone of the SVG cells) (20), and HeLa cells were maintained in Dulbecco's modified Eagle's medium with 2 mM L-glutamine, 10% fetal bovine serum, and antibiotic solution.

Cell Transfection-- C6 (rat glioma cell line), SVGA (human fetal astrocytes cell line), HeLa, or primary human astrocytes were transfected with endotoxin-free plasmids (Qiagen) using LipofectAMINE Plus (Invitrogen) as per supplier's protocol. Cells were monitored for GFP fluorescence or stained for Tat using a monoclonal antibody (APR 352, National Institute for Biological Standards and Control, Herts, UK). For establishing stably transfected cell lines, 72 h post transfection, the cells were cultured in Dulbecco's modified Eagle's medium containing G418 (Invitrogen) at 1.5 mg/ml. After 1 week, GFP florescent clones were picked under a florescent microscope using trypsin-soaked filter discs and sub-cultured for few weeks in G418 containing selection media. The cells were used within 30 passages and maintained in 500 µg/ml G418. We established SVGA-Tat, doxycycline-inducible SVGA-Tat (SVGA13), SVGA-LTR-CAT, SVGA-LTR-GFP, SVGA-pcDNA (SVGA-neo), C6-Tat, C6-Tat-GFP, C6-Delta Tat-GFP, C6-pcDNA (C6-neo), and HeLa-Tat cell lines for the present study. Tat levels in culture supernatants were determined by a sandwich ELISA, where a Tat monoclonal antibody was coated at the bottom of the plate and Tat polyclonal antisera (raised in rabbit against HIV-1 Tat-72) was used as the secondary antibody (4). The sensitivity of detection was 50 pg/ml. Highly purified recombinant Tat protein produced in our laboratory was used as a standard (6).

RNA Isolation and RT-PCR-- In each case, total RNA was extracted from 3-4 × 106 cells by Trizol reagent (Invitrogen) and treated with RNase-free DNase. 1 µg of RNA was reverse transcribed as described previously (21) into cDNA using random hexamer primers and 200 units of superscript reverse transcriptase (Invitrogen) at 45 °C for 1 h and 70 °C for 10 min in 20 µl total volume. 50 ng of cDNA was used in PCR amplification with 25 pM of each primer, 0.1 mM dNTP mix, 1.25 units platinum Taq polymerase, and 1.2 mM Mg2+. The following primers were used: Tat, Tat-AC1 5'-ATGGAGCCAGTAGATCCTAG-3' and Tat-AC2, 5'-TCATTGCTTTGATAGAGAAACTTG-3'; rTta, rTta1 and 5'-AATCGAAGGTTTAACCCG-3'; rTta2, 5'-TTGATCTTCCAATACGCAACC-3'; iNOS, iNOS1 5'-TGTGCCACCTCCAGTCCAGTGACA-3' and iNOS2 5'-GCTCATCTCCCGTCAGTTGGTAGG-3'; FASL, FASL1 5'-TGGGGATGTTTCAGCTCTTC-3' and FASL2 5'-TCATCATCTTCCCCTCCATC-3'; GAPDH, GAPDH1 5'-ACCACAGTCCATGCCATCAC-3' and GAPDH2 5'-TCCACCACCCTGTTGCTGTA-3'. The amplified products were analyzed on 1% agarose gels with ethidium bromide and visualized by a UV transilluminator.

Immunofluorescence-- Cells grown in 60 mm dishes 24-48 h post transfection were fixed in 4% paraformaldehyde for 15 min. The cells were washed with phosphate-buffered saline (PBS), treated with 0.2% Triton X-100 for 10 min at room temperature, and then blocked with 5% bovine serum albumin in PBS for 1 h. The cells were incubated at 4 °C for 18 h with either of the following antisera diluted in blocking buffer: monoclonal anti-Tat (1:1000), anti-microtubule-associated protein (MAP-2, a neuronal marker, 1:400; Chemicon), and anti-GFAP (astrocyte marker, 1:400; Sigma). The cells were washed 4 times in PBS followed by incubation with anti-mouse antisera labeled with Alexa 488 or 568 (Molecular Probes) for 30 min at room temperature. After 4 washes in PBS, specimens were visualized by fluorescent confocal microscopy.

Transactivation Assay-- HIV-1 LTR-CAT-based transactivation assays were performed using either HeLa-LTR-CAT or SVGA-LTR-CAT cells. A stable SVGA-LTR-CAT cell line was established using the LTR-CAT plasmid and selection with G418 for 3 weeks. The presence of LTR-CAT was confirmed following a transient transfection with pcDNA-Tat86. Alternatively, C6-Tat-GFP, C6-Delta Tat-GFP, SVGA-Tat, and SVGA13 were transfected with LTR-CAT plasmid. CAT activity was monitored by quantitative ELISA. The supernatants from these cells were collected at 24, 48, and 96 h, which was then added to LTR-CAT or LTR-GFP cells by a complete media exchange. The supernatants were used fresh without undergoing any freeze thaw cycles. For detecting transcellular Tat activity, Tat-expressing cells were co-cultured with SVGA-LTR-GFP cells or a lymphocytic cell line D3R5 LTR-GFP (22) in equal ratios for 48-96 h in 1.5% fetal bovine serum containing medium with 50 µM chloroquine (to minimize the degradation of endocytosed Tat). In select experiments polyclonal Tat antisera at 1:200 final concentration was included for neutralization of Tat activity and observed by fluorescent microscopy.

mRNA Profiles of Apoptotic and Chemokine-related Genes-- Total RNA was isolated at different time intervals using Trizol reagent (Invitrogen) from the Tat- or pcDNA-transfected primary astrocytes. 6-7 µg RNA was labeled using reverse transcription step, and labeled cDNA was hybridized at 68 °C for 18 h with superarray nylon membranes for chemokine and apoptotic gene profile (Super Array). The signals were quantified by a phospho-imager. The signals were normalized to GAPDH, and ratios of test and control were expressed as fold increase or decrease.

Protein Carbonyl and 4-Hydroxynonenol Assays-- SVGA-Tat and SVGA-neo cells were treated with 10 µM 3-nitroproprionic acid (3NP) for varying amounts of time at 37 °C. Cells were collected in Locke's buffer and sonicated for 5 s. The level of protein oxidation was determined by an oxidized protein detection kit (OxyblotTM, ONCOR) (23). Samples were incubated for 20 min with 12% SDS and 2,4-dinitrophenylhydrazine (DNPH) in 10% trifluoroacetic acid, vortexing every 5 min, and then neutralized with OxyblotTM Neutralization solution. 600 ng of protein was blotted onto nitrocellulose paper by the slot blotting technique. Membranes were incubated with blocking buffer (PBS with 3% bovine serum albumin, 0.01% sodium azide, and 0.2% Tween 20) for 30 min at room temperature, exposed to rabbit anti-DNPH protein antibody (1:150) for 90 min, followed by anti-rabbit IgG coupled to alkaline phosphatase (1:15,000) for 2 h at room temperature. Membranes were washed after each step in PBS with 0.2% Tween 20 and developed with SigmaFastTM chromogen. Blots were analyzed by computer-assisted imaging software, Scion Imaging. Samples for HNE detection were similarly analyzed by slot blotting technique except that rabbit-anti-HNE antibody (Calbiochem; 1:4,000) was used as a primary antibody.

Mitochondrial Trans-membrane Potential (delta  psi ) Activity in Neurons-- Mitochondrial function was monitored by a fluorescent dye JC-1 to measure mitochondrial delta  psi  as previously described (24). When loaded in the cells, JC-1 produces a green florescence at low membrane potential and red at high membrane potential. At the end of the experimental treatment, the cells were incubated with JC-1 (10 µM) at 37 °C for 30 min and then washed in Locke's solution. Fluorescence measurements were made with excitation at 485 nm and emission at 527 and 590 nm. The levels of fluorescence at both emission wavelengths were expressed as ratios of fluorescence measurements from which the percentage of the mean ratios compared with the untreated samples was calculated and analyzed using analysis of variance.

Neurotoxicity Assay-- Stable cell lines expressing Tat as SVGA-Tat, C6-Tat, C6-Tat-GFP, C6-Delta Tat-GFP, SVGA13, and control cell lines SVGA-neo and C6-neo were maintained in Dulbecco's modified Eagle's medium + 10% fetal bovine serum. These cells were placed in the upper chambers of transwells and neurons in the bottom of the 6- or 24-well plate in medium containing 0.5-1.0% fetal bovine serum. Tet-inducible cells (SVGA13) were induced 1 day prior to and up to 6 days after co-culture with 1 mg/ml doxycycline. In select experiments heparan sulfate (1.3 µM), dextran sulfate (0.5 µM), or kynurenate (10 µM) were added to the bottom chamber of the transwell. Cell death was monitored by trypan blue exclusion assay as described previously (19). Five random fields were photographed and neuronal counts were determined by an investigator blinded to the experimental design. At least 200 cells were counted in each field. Each experiment was done in triplicate, and three independent experiments were conducted. The results are expressed as mean percentage of dead cells.

Implantation of Tat-expressing Cells in Rat Brain-- C6Tat-GFP cells were sorted by a flow sorter. 7.5 × 104 C6-Tat-GFP or C6-neo cells were unilaterally injected into the molecular layer of the dentate gyrus of 12-week-old Sprague-Dawley rats using the following stereotactic coordinates: 3.8 mm anterior and 1.5 mm lateral to Bregma, and 3.2 mm dorsal to the dural surface. Animals were monitored for 10 days post-surgery and then euthanized under deep anesthesia by perfusion with normal saline followed by 4% paraformaldehyde. The brains were removed, post-fixed for 16 h in 4% paraformaldehyde, and then cryopreserved in 30% sucrose for 72 h. 30 µm sections were prepared from the hippocampus. Sections at the needle tract and 300 µm posterior to the injection site were processed for Nissl stain (25). Sections at the injection site were also immunostained for Tat using a monoclonal Tat antibody.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intracellular Expression of Tat in Astrocytes-- To determine whether astrocytes can support the expression of Tat protein and to explore the subcellular localization of Tat, we transfected primary human astrocytes or SVGA cells with full-length HIV-1 infectious molecular DNA clone pNL4-3. Tat expression was demonstrated by immunostaining (Fig. 2A, a and b) and was further confirmed by LTR transactivation in pNL4-3-transfected SVGA-LTR-GFP cells (Fig. 2A, d). No immunostaining for Tat or LTR transactivation was noted in pcDNA-transfected cells (Fig. 2A, c and e). Pattern of Tat expression was also monitored in primary human fetal astrocytes transfected with Tat-GFP plasmid (Fig. 2B). The expression of Tat in astrocytes was confirmed by immunostaining for GFAP and Tat-GFP fluorescence (Fig. 2B, b). Tat expression occurred within 24 h of transfection. Tat was expressed in the cytoplasm and nucleus with a strong signal in the nucleolus as revealed by green fluorescence (Fig. 2B, a, d, and e). The nuclei were counter-stained red with propidium iodide (c). Primary astrocytes transfected with Delta Tat-GFP revealed Delta Tat expression diffusely throughout the cell with no demarcation of nuclear boundary or no nucleolar pattern (Fig. 2B, f and g).


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Fig. 2.   A, Tat production by HIV-1 (pNL4-3) proviral DNA transfection in astrocytes. Immunostaining for Tat after 48 h of transfection in primary astrocytes (a), SVGA cells transfected with infectious pNL4-3 DNA (b), and in pcDNA-transfected control SVGA cells (c). LTR-based GFP expression in SVGA cells co-transfected with pNL4-3 and HIV-1 LTR-GFP (d) or pcDNA and HIV LTR-GFP control (e). B, expression of wild type and mutant Tat in astrocytes. Cells were transfected with wild type Tat-GFP or Delta Tat-GFP and observed for GFP fluorescence and immunostaining for GFAP after 24 h of transfection. a, primary astrocytes reveal Tat-GFP expression (green) predominantly in nucleus but also in cytoplasm; b, same cells immunostained for GFAP (red). c, propidium iodide staining for nuclei (red) in SVGA Tat-GFP cells. d, the same cell as in c, showing nucleolar Tat-GFP (green) and same cell with dual red and green filter (e). f, primary astrocytes express Delta Tat-GFP diffusely throughout the cell with no nuclear demarcation (green); g, same cells as in f stained for GFAP.

Stable Transfection of C6 and SVGA Cells with Tat-- C6-Tat-GFP and C6-Delta Tat-GFP clones were established by G418 selection and multiple subcloning by fluorescence-activated cell sorter. The pattern of Tat and Delta Tat expression in these stable cell lines was similar to that of transiently transfected astrocytes with the respective plasmids and did not change with prolonged culture. The expression of Tat in stable cell lines was further confirmed by detection of Tat mRNA by RT-PCR (Fig. 3A) or LTR-CAT transactivation assay following transfection of these cells with the LTR-CAT plasmid (Fig. 3B). Delta Tat-expressing cells showed basal level of transactivation activity (Fig. 3B). Clones of C6-Tat, SVGA-Tat, and SVGA13 cells that showed high CAT activity were used for further studies.


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Fig. 3.   A, Tat mRNA expression in astrocytes. Each cell line was analyzed for Tat mRNA expression by RT-PCR, and PCR products were shown by ethedium bromide-stained agarose gel. Lane 1, C6-Tat; lane 2, SVGA-Tat; lane 3, SVGA-Tat13 in the absence of doxycycline; lane 4, SVGA-Tat (tet) in the presence of doxycycline; lane 5, HeLa Tat; lane 6, HeLa; lane 7, positive control (Tat plasmid); lane 8, molecular weight markers. B, transactivation of HIV-1 LTR-CAT in Tat-expressing astrocytes. Tat-expressing astrocytes (SVGA (S) or C6 cells) were transfected with HIV LTR-CAT plasmid and quantitated for CAT activity by ELISA. Values represent mean optical density readings of two experiments. Bars represent CAT activity in the following cell types: S-Tat, S-Tat13 with doxycycline for 72 h, S-Tat13, S-Neo with Delta , C6-Tat, C6-Tat-GFP, and C6-Delta Tat-GFP (mutant Tat).

Functional Expression of Tat and Cell to Cell Transmission-- To determine whether biologically active Tat can be released from Tat-expressing astrocytes and taken up by non-expressing cells, SVGA-Tat cells were overlaid with SVGA-LTR-GFP cells. GFP expression occurred in 48-96 h of co-culture (Fig. 4A, a). No GFP expression was noted when these cells were co-cultures with SVGA-neo cells (Fig. 4A, b). Co-culture of C6-Tat or SVGA-Tat with D3R5-LTR-GFP cells also revealed GFP fluorescence (Fig. 4A, c), whereas no effect was noted when co-cultured with SVGA-neo or C6-neo cells (Fig. 4A, d). SVGA cells when directly co-transfected with LTR-GFP and Tat plasmids revealed higher levels of LTR-based GFP expression within 24 h of transfection (Fig. 4A, e and f). Together, these observations demonstrate the LTR transactivation in both astrocytic and lymphocytic cells was specific for the Tat-expressing cells. To further confirm that the effect was due to release of Tat from the Tat-expressing cells and uptake by the LTR-containing cells we repeated the above experiments in the presence of Tat antisera. The cell to cell Tat-mediated LTR transactivation activity was partially inhibited by Tat antisera, which is strongly suggestive of release of Tat extracellularly. The inability of the antisera to block the LTR transactivation completely is most likely related to poor access to Tat by the antisera in areas of cell to cell contact. The transactivation signal was specific for Tat because no effect was noted with preimmune rabbit sera or by co-incubation of the LTR-GFP cells with SVGA-neo cells (Fig. 4B). Tat secretion in a 48-h post transfection period from 8 × 105 cells was estimated as 100 ng/ml by ELISA. However, culture supernatants containing Tat from the stable Tat-expressing cells collected at 24, 48, and 96 h post media change (Tat concentration less than 50 pg), did not reveal transactivation of LTR when incubated with the LTR-CAT or LTR-GFP cells, suggesting that small amounts of Tat secreted may not be sufficient for LTR transactivation. This is consistent with previous observations where the minimum Tat concentration required for LTR transactivation and HIV replication was determined to be > 100 ng/ml (26).


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Fig. 4.   Cell to cell migration of functionally active Tat. A, LTR transactivation in astrocytic and lymphocytic cells following co-culture with Tat-expressing cells. C6-Tat or SVGA-Tat cells were co-cultured with D3R5-LTR-GFP or SVGA-LTR-GFP cells. Green fluorescence indicates LTR transactivation. a, SVGA LTR-GFP cells overlaid on a monolayer of SVGA-Tat cells; b, control where SVGA LTR-GFP cells were overlaid on SVGA-neo cells; c, lymphocytic D3R5-LTR-GFP cells were incubated with C6-Tat cells; d, control where D3R5-LTR-GFP cells were incubated with C6-neo cells; e, SVGA-LTR-GFP cells transfected with Tat plasmid; f, SVGA LTR-GFP cells transfected with pcDNA-3 plasmid. B, effect of Tat antisera on cell to cell LTR-GFP activation assay. SVGA cells transfected with 6.0 µg tat or neo plasmid were overlaid with SVGA LTR-GFP cells followed by addition of Tat antisera (aTat) or preimmune rabbit serum (PRS). Florescent cells were counted 24 h later and calculated as a percentage of the mean number of fluorescent cells in the wells without aTat or PRS. aTat partially blocked Tat transactivation activity, whereas PRS did not show blocking potential.

Effect of Intracellularly Expressed Tat on Astrocyte mRNA Profile for Apoptosis and Chemokine Genes-- Because Tat was stably expressed in astrocytes, we determined the effect of Tat expression on apoptotic genes as well as chemokine and cytokine gene expression profiles. Interestingly, primary astrocytes transfected with Tat plasmid for 24-36 showed that tumor necrosis factor-alpha and DAXX mRNAs were elevated 2-4-fold (Table I), whereas others were unchanged. Surprisingly, we did not see any effect on monocyte chemoattractant protein-1 or macrophage inflammatory protein-1alpha , which was previously shown to increase in astrocytes after extracellular treatment with Tat protein (27). Other mRNAs like, Caspase gene family, iNOS, tumor necrosis factor-related apoptosis-inducing ligand, Bcl gene family, and chemokine genes were unaffected. The results were also confirmed by RT-PCR (not shown). Further, tumor necrosis factor-alpha and MCP-1 were undetectable in the 48 h conditioned medium from C6-Tat and SVGA-Tat cells by quantitative ELISA. To rule out the under-expression of Tat, Tat expression was monitored in these cells by RT-PCR and immunostaining. Further cells expressing the Tat-GFP construct were used as a control. Nearly 95% of cells showed green fluorescence.


                              
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Table I
mRNA profile of Tat transfected primary human fetal astrocytes
Caspase gene family, iNOS, TRAIL, Bcl gene family, and Chemokine genes were unaffected.

Protein Carbonyl and HNE Levels in Tat-transfected Cell Lines-- To further explore the possible effects of Tat on vulnerability of astrocytes to oxidative stress, we analyzed protein and lipid peroxidation products after challenging the cells with mitochondrial toxin 3NP. Oxidized endogenous products, protein carbonyl, and HNE were measured as markers of oxidative stress (16). Tat-expressing astrocytic cells showed decreased oxidation products (HNE and protein carbonyl) when compared with the neo-cells (Fig. 5). These effects were specific for astrocytes as HeLa cells expressing Tat did not have a similar protective effect (Fig. 5).


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Fig. 5.   Tat production protects against oxidative stress in astrocytes. Protein carbonyl and HNE products were measured in each cell line with or with out treatment with 3NP. Top left, protein carbonyl levels were increased in HeLa cell lines following 3NP treatment. No significant difference was present between the HeLa-neo (HeLa) and HeLa-Tat cells. Top right, 3NP treatment failed to induce any significant change in HNE products in the HeLa cell lines. Bottom left, Tat-expressing SVGA cells showed significantly lower production of protein carbonyl following 3NP treatment. Bottom right, Tat-expressing SVGA cells showed significantly lower production of HNE products following 3NP treatment. Data represents mean ± S.E. from 3 experiments. *, p < 0.05.

Dissipation of Neuronal Mitochondrial delta  psi  by Astrocyte-expressed Tat-- To determine the effect of Tat-expressing cells on neurons, stable Tat-producing or neo-C6 cells were co-cultured with primary neurons using transwells and analyzed at various time points. The mitochondrial delta  psi  of neurons was unaffected within the first 48 h but after 72 h membrane potential decreased significantly in neurons co-cultured with C6-Tat cells as compared with C6-neo cells or C6-Delta Tat cells (Fig. 6). These observations were confirmed by the use of doxycycline-regulated expression of Tat in SVGA cells (Fig. 6). Doxycycline takes ~72 h for induction of Tat. Hence, in keeping with the above observation, we noted a significant decrease in mitochondrial delta  psi  at about 7 days after adding doxycycline to the media. No significant toxicity was noted in similar cultures where doxycycline was not added or when doxycycline was added to the neuronal cultures similarly incubated with SVGA neo cells where the Tat gene was not present (Fig. 6).


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Fig. 6.   Effect of Tat-expressing astrocytes on neurons. Neuronal cultures were exposed to Tat-expressing astrocytes in transwells. Left panel, mitochondrial delta  psi  in neurons after co-culture with C6-Tat cells in transwells. Significant decrease in mitochondrial delta  psi  was noted following incubation with C6-Tat cells. No significant change was noted with C6-neo or C6-dTat cells. 3NP was used as a control. Middle panel, mitochondrial delta  psi  in neurons after co-culture with tet-inducible SVGA Tat cells in transwells. Significant decrease in mitochondrial delta  psi  was observed in neurons following induction of Tat in SVGA13 Tat cells by doxycycline. No significant change in neuronal mitochondrial delta  psi  was noted following incubation with SVGA-neo cells similarly treated with doxycycline or SVGA13 cells without treatment with doxycycline. 3NP was used as positive control. Right panel, effect of kynurenate, dextran sulfate, and heparan sulfate on C6-Tat induced neuronal mitochondrial effects. Kynurenate, dextran sulfate, and heparan sulfate blocked decreases in mitochondrial delta  psi  in neurons induced by C6-Tat cells in transwells. Heparan sulfate did not block staurosporine-induced decrease in neuronal mitochondrial delta  psi . In each figure, data represents mean ± S.E. of 4 experiments. *p < 0.05.

These observations were further confirmed by collecting supernatants from stably Tat-producing cells after 48 h of culture and applying it to primary neurons for different times. The C6-Tat conditioned media decreased the neuronal mitochondrial membrane potential at 6 h post treatment (not shown).

Attenuation of Astrocyte-expressed Tat Effects on Neuronal Mitochondrial delta  psi  by Kynurenate, Heparan Sulfate, or Dextran Sulfate-- Previous studies (6, 19) have shown that Tat-induced neurotoxicity is mediated via excitatory amino acid receptors and Tat binds to heparan sulfate and dextran sulfate. Hence we incubated Tat-expressing astrocytes with neurons in transwells in the presence of each of these compounds. All these compounds attenuated the neurotoxicity measured as change in mitochondrial delta  psi . In control cultures where staurosporine (mitochondrial toxin) was incubated in the presence of heparan sulfate, no protective effect was noted (Fig. 6), thus indicating that extracellular Tat is responsible for the neurotoxicity.

Astrocyte-expressed Tat Induces Neuronal Cell Death-- To determine the effect of Tat-expressing astrocytes on neuronal survival, we exposed neuronal cultures to C6-Tat cells in transwells. Significant neuronal cell death occurred with C6-Tat cells (p < 0.05). However, C6-Delta Tat cells did not produce significant neurotoxicity (Fig. 7), indicating that amino acids 48-56 are essential for neurotoxicity. To confirm the neurotoxic properties of Tat, SVGA-13 cells were cultured in transwells with neurons. Doxycycline-treated SVGA-13 cells induced significant neuronal cell death first noted at 4 days of induction (p < 0.001) with maximal neurotoxicity at 7 days post induction (p < 0.001) compared with controls where doxycycline was omitted or with SVGA-neo cells similarly treated with doxycycline (Fig. 7). Further, neuronal cell death could be neutralized using polyclonal Tat antisera but not preimmune antisera (Fig. 7), indicating that extracellular release of Tat is required for neurotoxicity.


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Fig. 7.   Tat cells induced significant cell death in neurons. Left panel, C6-Tat cells induced significant cell death in neurons (p < 0.05). No significant neuronal cell death was noted with C6-neo or C6-Delta Tat cells. Middle panel, doxycycline-induced Tat-expressing SVGA13 cells caused significant neuronal cell death. No significant cell death was noted with SVGA-neo cells similarly treated with doxycycline or with SVGA13 cells untreated with doxycycline. Right panel, Tat antisera but not preimmune rabbit sera (prs) significantly blocked neurotoxicity induced by C6-Tat cells in transwells.

Neurite Trimming in Neurons by Tat-- Neurite fragmentation is a common phenomenon before the neurons collapse in response to toxic substances, and loss of dendrites is a prominent histopathological feature of patients with HIV encephalitis (28). Hence we monitored morphological changes in neurons following culture with Tat-expressing astrocytes. Direct co-culture of C6-Tat GFP cells with primary human fetal neurons for 72 h induced prominent neurite trimming or loss as shown in Fig. 8, following MAP-2 staining (red florescence). Tat production was confirmed by direct visualization of GFP florescence.


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Fig. 8.   Neurite trimming by Tat-producing astrocytes. C6-Tat or C6-neo cells were co-cultured with primary fetal neurons and immunostained for MAP-2. a, neurons co-cultured with C6-Tat cells showing neurite shortening or trimming; b, neurons co-cultured with C6-neo cells showing long and extended neurites.

Hippocampal Neurotoxicity Induced by Tat-producing C6 Cells in Vivo-- To determine whether Tat-producing C6 cells could elicit neurotoxicity in vivo, C6-Tat or -neo cells were unilaterally injected into the rat dorsal hippocampus. Nissl-stained sections from the injection site revealed that C6-Tat or C6-neo cells produced local damage of the dentate granule cells, but only animals with C6-Tat cells had extensive damage in the CA4/3 pyramidal cell layers (Fig. 9a). 300-µm posterior to the injection site, only minimal signs of neuronal injury were observed in animals with C6-neo cells, whereas extensive damage to the dentate gyrus and CA4/3 pyramidal cells was observed in animals with C6-Tat cells (Fig. 9, b and c). Tat immunoreactivity at the injection site was localized to the C6 cells (Fig. 9d), indicating that these cells are able to survive and produce Tat in vivo. Additionally, Tat immunoreactivity was present in the CA4/3 region in cells that morphologically resembled both microglial cells and neurons (Fig. 9d), raising the possibility that constitutively produced Tat from the C6 cells could be secreted and taken up by adjacent cells in the brain.


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Fig. 9.   Effect of Tat-producing astrocytes on hippocampus in vivo. Adult male rats were stereotactically injected with C6-neo cells (left panel) or C6Tat-GFP cells (right panel) into the dorsal hippocampus. At 10 days, sections were processed for Nissl stain and Tat immune reactivity. Nissl-stained images (panels a-c) depict hippocampal neurons at the injection site or 300 µm posterior to the injection site (-300 µm) (magnification 4× or 20×). Arrows indicate damaged, pyknotic CA3 pyramidal neurons observed only in animals injected with C6Tat-GFP cells. Immunoreactivity for Tat protein (Tat IHC, panel d) was seen at the injection site and in the CA3 region. Images are representative of results obtained from 4 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To determine the effect of HIV-1 Tat on astroglia-neuronal relationships, we used constitutively and inducible expressed Tat in astrocytes in contrast to previous studies that used recombinant Tat protein (29). We used two different astrocytic cell lines and two approaches for intracellular Tat expression, i.e. direct expression through cytomegalovirus promoter and tet-inducible expression using a human GFAP promoter. Similar results were obtained with both cell lines and both approaches for Tat expression. Our experimental model took advantage of a unique observation that intracellularly expressed Tat was not toxic to astrocytes. In fact it had increased resistance to oxidizing agents such as 3NP. We also noted overexpression of DAXX mRNA. Activation of physiological levels of DAXX has been associated with induction of apoptosis; however, overexpression has an anti-apoptotic effect (30). This is in keeping with observations that HIV infection of astrocytes is non-cytopathic (31). We were thus able to produce stably Tat-expressing astrocytic cell lines. We expressed Tat in astrocytes because Tat can be detected in HIV-infected astrocytes in vivo (4) and in vitro as shown in this study. Production of Tat was monitored by mRNA analysis, immunostaining, HIV-LTR transactivation assays, and ELISA. Further, Tat released from these cells was biologically active because it transactivated HIV-LTR in lymphocytic and astrocytic cell lines. These observations are consistent with a previous study (10) where Tat was taken up by neurons and was able to transactivate the HIV-LTR. A unique pattern of Tat expression was noted intracellularly in astrocytes. Tat was highly concentrated in the nucleolus, followed by the nucleus and cytoplasm. Deletion of the amino acids 48-56 resulted in diffuse staining throughout the cytoplasm and nucleus, confirming that this region of Tat is required for its nuclear localization (32-34). Previous studies (35-37) have shown that the intact arginine-rich domain of Tat (amino acids 49-57) is required for nuclear or nucleolar expression or localization. Despite the diffusion of Delta Tat into the nucleus, it failed to transactivate HIV-LTR. In astrocytic cells, LTR transactivation occurs by two mechanisms; interaction of Tat with transactivation response element RNA of LTR or via interaction of Tat with NFkappa B-binding sites in the LTR (38). The arginine-rich domain is thus essential for both modes of LTR transactivation and as discussed below also essential for Tat-induced neurotoxicity. Hence this region may be an important target for development of therapeutic approaches. The significance of Tat localization to the nucleolus remains undetermined. Interestingly, we were unable to detect an effect of intracellularly expressed Tat on iNOS, tumor necrosis factor-µ, MCP-1, cytokines, and chemokines production in astrocytes using super array, which was further confirmed by RT-PCR and ELISA for tumor necrosis factor-µ and MCP-1 (data not shown). Recently, it was shown that transiently transfected Tat plasmids induced iNOS and produced NO in an astrocytoma cell line U373MG (39). However, our gene array and RT-PCR data on primary astrocytes as well as the astrocyte cell line SVGA do not support these observations. Similarly, a macrophage cell line stably expressing Tat inhibited induction of iNOS by gamma interferon (40). In previous studies (41), others and we have shown that extracellular Tat has profound effects on cytokine and chemokine production by astrocytes. Thus intra- and extracellular Tat may have opposing effects.

We present several lines of evidence to conclusively show that extracellularly released Tat is neurotoxic. Supernatants from the Tat-expressing cell lines produced neurotoxicity as manifested by destablization of mitochondria, neurite trimming, and neuronal cell death. The neurotoxicity could be blocked by kynurenate, heparan sulfate, dextran sulfate, and antisera to Tat, clearly demonstrating that the neurotoxicity is mediated directly via Tat. Kynurenate blocks the effect of Tat on glutaminergic receptors on neurons (19), whereas heparan sulfate and dextran sulfate bind directly to Tat (5, 44). This confirms previous studies showing that recombinant Tat protein was toxic to neurons (19). However, the concentration of Tat necessary to cause the neurotoxicity was much lower in the current study, suggesting that Tat produced under physiological conditions by mammalian cells is a potent neurotoxin. Another study (24) showed that antisera to Tat could block neurotoxicity produced by supernatants from HIV-infected macrophages, confirming the relevance of these observations.

We noted cell to cell movement of Tat among astrocytes and from astrocytes to lymphocytes using LTR-GFP reporter expression indicator cells that were exposed to Tat-producing astrocytes. This function was also dependent upon amino acids 48-56 because no effect was seen with Delta Tat. Interestingly, this required co-culture of Tat-expressing cells with the indicator cell lines, whereas culture supernatants from the stably Tat-expressing cells failed to induce LTR transactivation. This was most likely due to lower concentrations of Tat in the supernatants. However, Tat released from the same cell lines was able to cause neurotoxicity, suggesting that the concentrations of Tat required for causing neurotoxicity are lower than that needed for transcellular transactivation of LTR. This may in part be explained by the fact that Tat-induced neurotoxicity requires interaction of Tat with the neuronal cell membrane (42), whereas Tat-induced transactivation of LTR requires Tat uptake by the cell (43). Another unique observation in vivo was that Tat released from the astrocyte cell lines was taken up by granule cells in the dentate gyrus and transported anterogradely via mossy fibers to the CA4/3 region where it caused glial cell activation and neurotoxicity. Thus Tat can cause pathological changes at sites distant to the site of production.

Our observations suggest the following sequence of events. During HIV replication in astrocytes, Tat is produced as an early product. It makes the astrocytes resistant to oxidative stress, thus making it an ideal reservoir for long-term survival of the viral genome. Tat localizes to the nucleolus, but small amounts are released extracellularly. Tat may be taken up by resident cells in the brain or monocytic cells trafficking through the brain. If these cells are latently infected with the virus, it will cause transactivation of the viral genome and viral replication. If taken up be neurons it may be transported along neuronal pathways and cause neurotoxicity and glial cell activation at distant sites. Small amounts of Tat are sufficient to cause excitotoxic neuronal damage that includes mitochondrial dysfunction, neurite trimming, and neuronal cell death in select populations. Because mitochondria are concentrated in neurites, the mitochondrial stress may be directly responsible for neurite trimming. Thus, HIV-infected astrocytes likely play a critical role in the neuropathogenesis of HIV infection. The Tat protein, and in particular the arginine-rich region of Tat, deserves close attention as a therapeutic target for HIV infection.

    ACKNOWLEDGEMENTS

We thank Dr. G. Smith, for pGfalac1, Dr. M. Giacca for pGEX Tat, Dr. D. Dorosky for D3R5 cells, Dr. W. Atwood for SVGA cells, and Dr. C. Pardo for confocal microscopy. We thank the National Institutes of Health AIDS Research and Reference Reagent Program for plasmids pNL4-3 and LTR-CAT and HeLa LTR-CAT cells. We thank National Institute for Biological Standards and Control, Herts, UK, for monoclonal antibody to Tat. We thank C. Anderson, R. Reid, and P. Ray for technical assistance.

    FOOTNOTES

* This work was supported by grants from NINDS, National Institutes of Health, National Institute on Drug Abuse, National Institute on Aging, and National Center for Research Resources.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Dept. of Neurology, Johns Hopkins University, Pathology 509, 600 N. Wolfe St., Baltimore, MD 21287. Tel.: 443-287-4657; Fax: 410-614-1008; E-mail: anath1@jhmi.edu.

Published, JBC Papers in Press, January 24, 2003, DOI 10.1074/jbc.M209381200

    ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; LTR, long terminal repeat; HNE, 4-hydroxynonenal; GFP, green fluorescent protein; tet, tetracycline; rtta, reverse tetracycline transactivator; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; 3NP, 3-nitroproprionic acid; GFAP, glial fibrillary acid protein; CAT, chloramphenicol acetyl transferase; iNOS, induced nitric oxide synthetase; MAP-2, microtubule associated protein-2.

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