HIV-1 Tat Causes Apoptotic Death and Calcium Homeostasis Alterations in Rat Neurons

Biochemical and Biophysical Research Communications 288, 301–308 (2001) doi:10.1006/bbrc.2001.5743, available online at http://www.idealibrary.com on

HIV-1 Tat Causes Apoptotic Death and Calcium Homeostasis Alterations in Rat Neurons Rudy Bonavia,* ,§ Adriana Bajetto,* ,§ Simone Barbero,* ,§ Adriana Albini,† Douglas M. Noonan,‡ and Gennaro Schettini* ,§ ,1 *Pharmacology and Neuroscience, †Molecular Biology Laboratory, and ‡Tumour Progression Section, National Cancer Research Institute, c/o Advanced Biotechnology Center, Genoa, Italy; and §Section of Pharmacology, Department of Oncology, Biology, and Genetics, University of Genova, Genoa, Italy

Received September 18, 2001

We investigated the role of the HIV-1 protein Tat in AIDS-associated dementia, by studying its toxicity on rat cortical and hippocampal neurons in vitro. We evaluated the involvement of astroglial cells and of caspase transduction pathway in determining Tat toxicity. Here we report that synthetic Tat 1– 86 induced apoptotic death on cultured rat neurons in a timedependent manner that was not influenced by glial coculture, and that was abolished by blocking caspase transduction pathway. A microfluorimetric analysis on the Tat excitatory properties on neurons, and its effect on intracellular calcium concentrations, revealed that Tat 1– 86 induced increase in cytoplasmic free calcium concentrations in rat hippocampal and cortical neurons. This effect required extracellular calcium and was differently reduced by voltage dependent calcium channel blockers and both NMDA and non-NMDA glutamate receptors antagonists. Furthermore, we observed that Tat 1– 86-treated neurons showed increased sensitivity to the glutamate excitotoxicity. Thus, the Tat-induced neuronal injury seems to occur through a direct interaction of the protein with neurons, requires activation of caspases, and is likely to derive from Tat 1– 86-induced calcium loads and disruption of glutamatergic transmission. © 2001 Academic Press

Key Words: Tat; AIDS Dementia Complex; caspases; neuronal apoptosis; intracellular calcium; glutamate ionotropic receptors; voltage-gated calcium channels.

HIV-1 infection is often associated with a neurological syndrome known as AIDS Dementia Complex (ADC), characterized by deficits in cognition, motor 1

To whom correspondence should be addressed Prof. Gennaro Schettini, Pharmacology and Neuroscience Unit, National Cancer Research Institute, c/o Advanced Biotechnology Centre, Largo Rosanna Benzi 10, 16132 Genova, Italy. Fax: 0039-10-5737257. E-mail: [email protected].

control and behavior (1). Brains from ADC patients show loss of neurons, astrogliosis, demyelination, formation of multinucleated giant cells (2). The cell types infected by HIV-1 in the CNS are macrophages/ microglia, multinucleated giant cells (3) and astrocytes (4), while there is no evidence for direct infection of neurons. Therefore it has been hypothesized that cellular factors and/or viral-encoded proteins released by infected cells may be responsible for neurotoxicity. Tat is the transactivator of the HIV-1 long terminal repeat transcription, and its activity is necessary for the viral replication (5). Despite the fact that Tat belongs to the leader-less proteins, it can be secreted (6) and taken up (7) by several cell types. These characteristics allow Tat to exert a broad range of both extraand intracellular activities: it has been reported to regulate activation of cellular transcription factors (8, 9), stimulate the secretion of cytokines (10 –12) and chemokines (13, 14), stimulate angiogenesis (15), promote chemotaxis of leukocytes (16, 17), upregulate expression of adhesion molecules in endothelial cells (18), induce proliferation of Kaposi’s sarcoma-derived cells (19). Tat mRNA has been detected in brains of AIDS patients and its presence in the CNS seems to correlate with HIV encephalitis (20). It has been reported that extracellular Tat induces depolarization (21) and intracellular calcium elevation (22–24) in several cell types, including neurons, and it has been suggested that it could be the cause of neuronal death (25–27). In the present study we tried to identify a link between Tat and ADC, and to elucidate the mechanisms by which extracellular Tat could give rise to neuronal death. We found that Tat 1– 86 induces apoptotic death in rat hippocampal and cortical neurons in vitro, and that this toxic action needs the activation of caspases. Moreover, we show that Tat 1– 86 is able to stimulate calcium currents in neurons through both voltage-gated cal-

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cium channels and ionotropic glutamate receptors, and to increase neuronal sensitivity to excitotoxic stimuli. MATERIALS AND METHODS Materials. Synthetic Tat peptide corresponding to amino acids 1– 86 was purchased from Tecnogen (Caserta, Italy); it was resuspended in PBS containing 0.1% bovine serum albumine and 0.1 mM DTT to prevent oxidation. Z-VAD-fmk was purchased from Calbiochem, MK801 and CNQX from RBI, ␻-Conotoxin GVIA from Sigma; Nicardipine was provided by Novartis (Milan, Italy). Cell cultures. Primary cultures of rat hippocampal and cortical neurons were obtained from 18- to 19-day old Sprague–Dawley rat embryos according to the method of Goslin and Banker (28) with slight modifications. Briefly, hippocampi or cortices were excised, trypsinized, mechanically dissociated and resuspended in Neurobasal medium supplemented with 2% B27 (Gibco/Life Technologies, Rockville, MA), 0.5 mM glutamine and antibiotics, and seeded in poly-L-lysine (50 ␮M) coated wells. Primary cultures of rat cortical type-1 astrocytes were prepared from 2-day-old Sprague–Dawley pups as previously described (29). Briefly, cortices were dissected out, trypsinized and mechanically dissociated and plated in 75 mm 2 flasks in MEM/F12 (1:1) supplemented with 10% heat-inactivated FBS (Gibco/Life Technologies, Rockville, MA) and antibiotics. Astrocyte-enriched cultures were obtained from mixed glial cultures by the shaking off method and were composed of greater than 98% glial fibrillary acidic protein-positive cells. Determination of cell death. Percentage of apoptotic neurons was evaluated by an immunoenzymatic method for the detection of cytoplasmic histone-associated DNA fragments (Cell Death Detection ELISA Plus kit, Roche, Mannheim, Germany). A morphological discrimination between normal and apoptotic cells was provided by the nuclear staining with Hoechst 33258: cells plated on glass coverslips were fixed in 4% paraformaldehyde in PBS, rinsed, and stained with Hoechst 33258 (1 ␮g/ml) for 30 min at room temperature. Moreover, in glutamate sensitivity experiments, we evaluated mitochondrial function, as an index of cell viability, by performing the MTT test, as described in (30). Calcium measurements. Cells plated on poly-L-lysine-coated 25 mm glass coverslips were loaded with 4 ␮M fura-2 acetoxymethyl ester (Calbiochem) in Locke’s standard buffer (NaCl 154 mM, KCl 5.6 mM, NaHCO 3 3.6 mM, MgCl 2 1.2 mM, glucose 5.6 mM, Hepes 5 mM, CaCl 2 1.5 mM, pH 7.4). After 30 min, cells were washed twice and the coverslip was mounted on a coverslip chamber for fluorescence measurements. Fluorescence emissions at 340 and 380 nm excitation wavelengths were recorded and analyzed by a digital imaging system (QuantiCell, VisiTech, UK). The [Ca 2⫹] i was determined according to the equation of Grynkiewicz (31), where R max and R min are ratios at saturation and zero [Ca 2⫹] i, and were obtained, respectively, by perfusing cells with standard buffer containing 10 mM CaCl 2 and 5 ␮M ionomycin, and subsequently with a Ca 2⫹ free solution containing 20 mM EGTA and 40 mM Tris. All measurements were performed at room temperature. Each cell in the image was independently analyzed for each time point in the capture sequence.

RESULTS

with synthetic Tat 1– 86 100 nM. At 1, 3, and 5 posttreatment days, we quantified the rate of Tat 1– 86-induced neuronal death by an ELISA test, that specifically detects cells dead by apoptosis, whose results are summarized in Fig. 1a. While at day 1 we did not observe significant toxicity, a slight induction of apoptosis was detectable 3 days after Tat 1– 86 stimulation: at this time we measured an increment of 35 ⫾ 7.2% in the number of apoptotic cells (P ⬍ 0.01). At day 5 the Tat 1– 86induced apoptosis rose up to 74 ⫾ 16% (P ⬍ 0.005) (Fig. 1A). To determine a possible role carried by glial cells in Tat neurotoxicity, we performed parallel toxicity experiments in the presence of an astrocyte feeder-layer added at the beginning of the treatments. In these conditions, we could detect a significant induction of apoptosis after a 3-day treatment with Tat 1– 86 (30 ⫾ 1.7%, P ⬍ 0.01), that substantially increased at day 5 (75 ⫾ 11%, P ⬍ 0.005) (Fig. 1A). In agreement with these results, the Hoechst staining (Fig. 1B) revealed that a prolonged exposure of primary hippocampal cultures to Tat 1– 86 produces an increase in the number of cells with condensed and fragmented nuclei (arrows), a cytological hallmark of apoptotic death, although condensed nuclei are occasionally present in control cultures too. The morphological analysis by Hoechst staining did not reveal in treated cultures the presence of an amount of necrotic cells significantly different from controls. Similar results were found by performing the same treatments on cultures of rat cortical neurons (data not shown). Tat 1– 86 Toxicity Is Inhibited by Z-VAD-fmk To understand the intracellular mechanisms that could be responsible for the transduction of Tatinduced neurotoxic stimulus, we investigated the involvement of caspases, one of the most common pathways involved in the transduction of apoptotic stimuli in neurons. We analyzed the Tat 1– 86-induced toxicity in the presence of a pharmacological blocker of this enzymatic cascade, Z-VAD-fmk, a broad range inhibitor of caspase activity. Hippocampal neurons were incubated for 30 min with the inhibitor or vehicle and then treated with Tat 1– 86 100 nM. After 3 days, Tat-treated cultures showed a 30 ⫾ 1.7% increase in apoptotic cells vs control, while no significant increase in apoptosis was seen in neurons pre-treated with Z-VAD-fmk (81.9 ⫾ 0.8%) (Fig. 1C).

Tat 1– 86 Induces Apoptosis in Cultured Hippocampal Neurons Independently of Glial Coculture

Tat 1– 86 Stimulates Ca 2⫹ Entry in Neurons through Voltage-Gated Calcium Channels and Glutamate Ionotropic Receptors

To evaluate Tat-induced neuronal damage, we treated primary rat hippocampal neurons at 7– 8 DIV

Since most processes that lead to neuronal death imply massive increases of cytoplasmic free calcium

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FIG. 1. (A) Time-course analysis of Tat 1– 86-induced apoptosis in primary rat hippocampal cultures. 7– 8 DIV neurons cultured in Neurobasal Medium supplemented with B-27 were treated with synthetic Tat 1– 86 100 nM or vehicle, both in the presence and the absence of glial feeder-layer. At day 1, 3, and 5 the percentage of apoptotic cells was evaluated by ELISA (*P ⬍ 0.01, and **P ⬍ 0.005 vs control). (B) Neurons treated as above stained with Hoechst 33258; white arrows indicate apoptotic neurons with condensed and fragmented chromatine (scale bars: 20 ␮m). (C) Inhibition of Tat 1– 86 neurotoxicity by blockade of caspase activity. Hippocampal neurons were pretreated with 0.2% dimethylsulfoxide or Z-VAD-fmk (100 ␮M) for 30 minutes, and then exposed to Tat 1– 86 100 nM. ELISA test was performed 3 days after the treatment (*P ⬍ 0.005 vs Tat 1– 86 alone).

concentration ([Ca 2⫹] i), we studied the effects of Tat on intracellular calcium levels in cultured hippocampal and cortical neurons. The traces extrapolated from mi-

crofluorimetric measurements show that the exposure of neurons to Tat 1– 86 100nM brings to an immediate elevation of [Ca 2⫹] i when the experiment is carried out

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FIG. 2. Calcium currents in neurons exposed to Tat 1– 86 100nM. (A–D) Representative traces showing the cytoplasmic free-calcium concentration in hippocampal (A, C) and cortical (B, D) neurons stained with fura-2, obtained by microfluorimetric analysis, in buffer containing 1.5 mM Ca 2⫹ (A, B), or in Ca 2⫹-free buffer with 50 ␮M EGTA (C, D). (E) Mean values of cytoplasmic calcium concentration recordings at baseline and at plateau in the presence or absence of extracellular calcium in hippocampal neurons. Values are means of 7 different experiments and are expressed as percentage of basal concentration.

in the presence of extracellular calcium (1.5 mM) (Figs. 2A and 2B), while, in calcium-free conditions, there is no significant response (Figs. 2C and 2D). In the typical time-course curve in calcium-containing buffer (Figs. 2A and 2B) the [Ca 2⫹] i rises gradually starting from the point of stimulation until it reaches a sustained plateau. In these conditions we evaluated the average increase in [Ca 2⫹] i at the steady-state plateau to be 143 ⫾ 24% of the baseline level, while in the experiments with calcium-free buffer the induced [Ca 2⫹] i registered after the addition of Tat 1– 86 was 5 ⫾ 15% of basal (Fig. 2E).

To understand which membrane channels could mediate the Tat-induced calcium elevation in neurons, we set up a series of experiments in which we alternately blocked calcium-permeable channels highly represented in neurons: L- and N-type voltage sensitive calcium channels with nicardipine and ␻-conotoxin GVIA (␻-CTX) respectively, AMPA/kainate glutamate receptors with CNQX and NMDA glutamate receptors with MK801. The specific pharmacological inhibitor was either added after the Tat 1– 86 stimulation (Fig. 3A) or 10 minutes before the experiment (Fig. 3B): in the former case we obtained a partial recovery of the cal-

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or 50 ␮M for 30 min in Locke’s buffer without Mg 2⫹. After additional 24 h in fresh medium the cell survival was measured by MTT test. The results obtained revealed that Tat 1– 86-treated cultures exhibit increased sensitivity to glutamate toxicity in a dose-dependent manner. In agreement with the apoptosis analysis (Fig. 1a), neurons treated with Tat 1– 86 alone (25–100 nM, 24 h) did not show significant decrease in the survival in comparison with untreated cells. However, the treatment with Tat 1– 86 50 or 100 nM (24 h) significantly potentiated the glutamate 25 and 50 ␮M toxicity (from 91.3 ⫾ 4.6% (control) to 68.9 ⫾ 0.54% (Tat 1– 86 50 nM) and to 67.5 ⫾ 0.68% (Tat 1– 86 100 nM), and from 80.8 ⫾ 0.54% to 54.9 ⫾ 2.6% and 49.2 ⫾ 3.3%, respectively). DISCUSSION

FIG. 3. Inhibition of Tat 1– 86-induced increases of cytoplasmic free-calcium levels in hippocampal neurons by nicardipine (1 ␮M), ␻-conotoxin GVIA (1 ␮M), CNQX (10 ␮M), MK801 (10 ␮M). (A) The indicated inhibitor was added following Tat 1– 86 stimulation in calcium-containing buffer (data are expressed as % of [Ca 2⫹] i at plateau). (B) The ability of the same drugs to prevent the elevation of [Ca 2⫹] i when added 10 min before the stimulation with Tat 1– 86 (values expressed as % of [Ca 2⫹] i at plateau without inhibitors).

cium load with all the compounds except for ␻-CTX, with the greatest effect provided by nicardipine (11 ⫾ 6.5% of Tat 1– 86-induced [Ca 2⫹] i rise at plateau); in the latter case all tested drugs were able to reduce the height of the calcium peak: again, the smallest inhibition of the Tat 1– 86 effect was obtained with ␻-CTX (48.0 ⫾ 12.0% of control) and a complete inhibition of the [Ca 2⫹] i elevation with nicardipine (⫺2.0 ⫾ 7.0%). Tat 1– 86 Increases Neuronal Sensitivity to Excitotoxic Stimuli Tat ability to activate calcium fluxes through glutamate receptors prompted us to study whether this action could lead neurons to an increased vulnerability to excitotoxic stimuli. For this purpose, we studied the survival to glutamate in primary rat cortical neurons previously exposed to different concentrations of Tat 1– 86 (Fig. 4). The cells were treated with Tat 1– 86 25, 50 or 100 nM for 24 h, then pulsed with glutamate 25

HIV-1 Tat plays a key role in AIDS pathogenesis, not only because of its transactivation function, but also by its ability to impair host immune defenses and to facilitate several AIDS-associated disorders such as Kaposi’s sarcoma. In fact, despite its small dimensions, Tat has multiple functional domains, and it has been demonstrated to interact with cell receptors such as integrin receptors through its RGD sequence (32), the Flk-1/kinase insert domain receptor (Flk-1/KDR) through its basic domain (33), and chemokine receptors through its cysteine-rich domain (24). The role of Tat in AIDS dementia has been discussed in the last few years: Tat can easily enter the CNS by secretion from infected leukocytes, and several studies in vitro and in vivo have indicated Tat as a potential neurotoxin. Our experimental data demonstrate that extracellular Tat 1– 86 can exert a toxic action on primary rat neuronal cultures: we found that the Tat 1– 86-induced neuronal death occurs with apoptotic features and correlates with the time of treatment, being strongly enhanced after prolonged exposures (up to 5 days). Since many authors have supposed that AIDS dementia represents the result of the activation of nonneural cells within the CNS (34), and some studies in vitro indicated that another HIV-1 protein, the capside glycoprotein gp120, induces neuronal death through a mechanism that requires (or is enhanced by) the presence of glial cells (30, 35), we analyzed the role of astroglial cells as modulators of Tat toxicity. Parallel treatments, with or without astrocyteenriched (more than 98%) coculture, indicated that the glial presence neither enhances nor prevents the Tat 1– 86 toxic activity on neurons in both short and long term experiments. Other authors demonstrated that Tat is able to modulate cytokine expression and secretion (12, 14) and stimulate calcium elevations in astrocytes (22), but, in our experimental model, the modulation of glial cell

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FIG. 4. Dose-dependent increase in glutamate sensitivity in cortical neurons exposed to Tat 1– 86. Cells were treated 24 h with Tat 1– 86 25, 50, or 100 nM, and then 30 min with glutamate 25 or 50 ␮M. The graph represents cell survival evaluated by MTT test 24 h after glutamate pulse (% of neurons not treated with glutamate) (*P ⬍ 0.01, and **P ⬍ 0.001 vs corresponding control values).

activity by Tat does not seem to be directly correlated with neuronal mortality, although astrocytes might play a relevant role in the onset of AIDS dementia in vivo, in particular with regard to monocyte/macrophages recruitment and activation. This finding suggests that the toxicity exerted by Tat occurs through a mechanism that is, at least in part, different from that of gp120, that was proposed to indirectly kill neurons by inducing the release of neurotoxins from non-neural cells as macrophages/ microglia, monocytes, and astrocytes (34). The effect of extracellular Tat on the activation of caspases has been reported in both neural and lymphoid cells (25, 36): in these works is shown the activation, respectively, of caspase-3 and caspase-8. We found that a broad range inhibitor of caspase activity, Z-VAD-fmk, is sufficient to completely prevent Tat 1– 86 toxicity. The Tat ability to increase cytoplasmic calcium concentration has been described in several cell types including monocytes (24), microglia (23), astrocytes (22) and neurons (21, 22, 25). In most cases, the calcium elevation is attributed to release from IP3-regulated stores or to delayed entry from extracellular medium (10, 22, 25). However, our data show that the calcium increase appear immediately after the pulse, last over the longest exposure times (up to 6 minutes), and are completely abolished when the extracellular calcium is removed. These findings are in agreement with Cheng et al. (21) who showed that Tat 1– 86 triggers a rapid, non-desensitizing membrane depolarization in neurons, dependent on the presence of extracellular calcium. Thus, our results indicate that Tat 1– 86 is able to

elicit calcium currents from the extracellular environment, while there is no evidence of calcium release from intracellular stores. A more specific analysis on the identity of the calcium channels involved in these currents revealed a complex scenario. We were able to identify, at least, four components: L- and N-type voltage-gated calcium channels, ionotropic glutamate receptor of both NMDA and non-NMDA type. This wide-range activation seems likely to be the result of a cascade phenomenon: the observation that every inhibitor was able to prevent more than 50% of the peak probably indicates a positive feed-back between voltage-gated channels and glutamate receptors. In effect, the only tested drug that completely prevented the calcium flux was nicardipine, suggesting that the activation of L-type calcium channels might be a primary event, necessary for the beginning of calcium load, and that the activation of glutamate receptors might likely be the effect of the release of glutamate as a consequence of the initial depolarization. This effect on glutamatergic transmission seems to be a crucial event in Tat-caused neuron damage, since glutamate antagonists were found to protect neurons from Tat toxicity (14, 37, 38). A direct interaction of Tat with L-type calcium channels, although with inhibitory effect on the activation of these channels, has already been described in NK cells by Zocchi et al. (39). An important finding to understand the mechanisms that cause disruption of neuron viability is that Tat 1– 86 markedly enhances the neuronal sensitivity to excitotoxic stimuli in a dose-dependent manner. This effect can be demonstrated very soon during the treatment (as soon as 24 h after the stimulation), and already

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occurs when the apoptosis induced by the viral protein is not detectable or is only slightly higher than controls. In this regard, our results share important similarities with the study conducted by Wang et al. (40) who showed that Tat was able to induce neuronal damage in rat hippocampi in vivo only when coinjected with NMDA. An explanation for these phenomena may be provided by Haughey et al. (22) who hypothesized that a Tat-mediated activation of protein kinase C might be responsible for the phosphorylation, with consequent sensitization, of glutamate receptors. Moreover, a Tat-induced increased sensitivity to apoptotic stimuli has been reported by Bartz et al. (36), who described the up-regulation of caspase-8 synthesis in Tat-expressing cells, while Westendorp et al. demonstrated that Tat is able to weaken cellular defenses by down-regulating the expression of Mn-dependent superoxide dismutase, and thus altering the cellular redox state toward pro-oxidative conditions (9). It is important to remark that the presence of Tat in the brain, by raising the threshold of sensitivity to glutamate-induced toxicity, could make a physiological event such as the synaptic release of excitatory amino acids harmful for neurons. Moreover, gp120, according to Vesce et al. (41), is able not only to inhibit astrocyte glutamate reuptake, but also to stimulate its release from the same cells, thus suggesting that the copresence of the two proteins in the CNS, and their interactions with different cell types could lead to an enhanced damage. These observations underlie how much complex could be the interferences of HIV-1 and its proteins with the neuronal homeostasis, and how many factors could contribute to the determination of the cell fate. Comprehensively, our data demonstrate that HIV-1 Tat 1– 86 cause neuronal death in primary rat neuron cultures; the Tat 1– 86 toxicity is likely attributable to a direct interaction of the viral protein with neural membrane that can upset cellular electrophysiological equilibrium and glutamatergic transmission, and, thus, elicit prolonged increases in cytoplasmic calcium concentrations leading to neuronal death.

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ACKNOWLEDGMENTS

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This work was supported by ISS AIDS Grants 30B.74 and 30C.68. Simone Barbero was supported by a fellowship from FIRC.

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