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JOURNAL OF VIROLOGY, Oct. 2000, p. 9717–9726 0022-538X/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 20

Human Immunodeficiency Virus Type 1 Vpr Induces Apoptosis in Human Neuronal Cells CHARVI A. PATEL, MUHAMMAD MUKHTAR,

AND

ROGER J. POMERANTZ*

The Dorrance H. Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received 18 May 2000/Accepted 24 July 2000

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) causes AIDS dementia complex (ADC) in certain infected individuals. Recent studies have suggested that patients with ADC have an increased incidence of neuronal apoptosis leading to neuronal dropout. Of note, a higher level of the HIV-1 accessory protein Vpr has been detected in the cerebrospinal fluid of AIDS patients with neurological disorders. Moreover, extracellular Vpr has been shown to form ion channels, leading to cell death of cultured rat hippocampal neurons. Based on these previous findings, we first investigated the apoptotic effects of the HIV-1 Vpr protein on the human neuronal precursor NT2 cell line at a range of concentrations. These studies demonstrated that apoptosis induced by both Vpr and the envelope glycoprotein, gp120, occurred in a dose-dependent manner compared to protein treatment with HIV-1 integrase, maltose binding protein (MBP), and MBP-Vpr in the undifferentiated NT2 cells. For mature, differentiated neurons, apoptosis was also induced in a dose-dependent manner by both Vpr and gp120 at concentrations ranging from 1 to 100 ng/ml, as demonstrated by both the terminal deoxynucleotidyltransferase (Tdt)-mediated dUTP-biotin nick end labeling and Annexin V assays for apoptotic cell death. In order to clarify the intracellular pathways and molecular mechanisms involved in Vpr- and gp120-induced apoptosis in the NT2 cell line and differentiated mature human neurons, we then examined the cellular lysates for caspase-8 activity in these studies. Vpr and gp120 treatments exhibited a potent increase in activation of caspase-8 in both mature neurons and undifferentiated NT2 cells. This suggests that Vpr may be exerting selective cytotoxicity in a neuronal precursor cell line and in mature human neurons through the activation of caspase-8. These data represent a characterization of Vpr-induced apoptosis in human neuronal cells, and suggest that extracellular Vpr, along with other lentiviral proteins, may increase neuronal apoptosis in the CNS. Also, identification of the intracellular activation of caspase-8 in Vpr-induced apoptosis of human neuronal cells may lead to therapeutic approaches which can be used to combat HIV-1-induced neuronal apoptosis in AIDS patients with ADC. Fas-associated death domains (FADD or MORT), which in turn bind to analogous domains of caspase-8 (also known as FLICE, MACH, and Mch5) through caspase recruitment domains (4, 5, 9). Together Fas, FADD, and caspase-8 form a complex, entitled the death-induced signaling complex, and recently a new component, the FLICE-associated huge protein, has been identified as a necessary component in the activation of caspase-8-mediated apoptosis (18). Upon recruitment by FADD, caspase-8 undergoes proteolytic cleavage into smaller subunits and activates downstream effector caspases, resulting in apoptosis. However, caspase-8 can also be activated through the tumor necrosis factor alpha-related apoptosis-inducing ligand pathway or through the mitochondria following an irreversible commitment to cell death. It has been reported that in HIV-1-infected individuals, the tumor necrosis factor alpha-related apoptosis-inducing ligand but not the Fas ligand mediates apoptosis of activated T cells (21). Apoptosis is also frequently dependent on the p53 tumor suppressor protein (11). Although the underlying mechanisms of HIV-1-induced dysfunctions of the CNS remain enigmatic, the HIV-1 regulatory protein, Vpr, has been implicated in the induction of apoptosis of T-lymphocytes in HIV-1 infections. Studies have reported that Vpr induces apoptosis in human fibroblasts, T-cell lines, and primary peripheral blood lymphocytes following arrest of the G2 cell cycle (44). HIV-1 Vpr was shown recently to cause apoptosis following G2 cell cycle arrest via the activation of caspases and independent of p53 stimulation in T-lymphocytes (43). Even though most of the evidence for the induction of

The cells in the human brain that are productively infected with human immunodeficiency virus type 1 (HIV-1) are macrophages and microglia; however, neuropathological abnormalities in the brains of infected patients include neuronal dropout and apoptosis of neurons, which seem to be the most probable cause of central nervous system (CNS) injury in AIDS dementia complex (ADC) (1, 13, 33, 36, 41). Thus, indirect causes for neuronal apoptosis during ADC may include expression of select chemokines and cytokines from HIV-1-infected CNS macrophages and microglia, or apoptosis may be induced by specific viral proteins. Other CNS cell types (e.g., microvascular endothelial cells, neurons, and astrocytes) may also be restrictively infected by HIV-1 in vivo and, therefore, it also remains formally possible but less likely that apoptosis of certain neuronal populations may be a secondary effect of direct HIV-1 infection of these cells (3, 24). Apoptosis has been considered one of the mechanisms for CD4⫹ T-lymphocyte depletion during AIDS progression (31, 34). The Fas/Fas ligand (also known as CD95 and APO-1) apoptotic pathway was recognized as one of the pathways of T-cell death during HIV-1 infection (2, 6, 10, 20). The Fas/Fas ligand pathway activates death domains which are linked to * Corresponding author. Mailing address: The Dorrance H. Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107. Phone: (215) 503-8575. Fax: (215) 923-1956. E-mail: [email protected] .edu. 9717

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apoptosis by HIV-1 in T-lymphocytes implicates the Tat and gp120 proteins, recently it has been suggested that Vpr, when encapsidated within virions, is involved in the direct cell-killing effects of HIV-1. Vpr expressed during a lentiviral infection, in the absence of other viral components, was capable of inducing apoptosis. In the same study, the synthetic caspase inhibitor z-VAD-fmk reduced Vpr-induced apoptosis, indicating that Vpr induced apoptosis through caspase activation (45a). Thus, caspase activation seems to be the most likely mechanism for induction of apoptosis in hematopoietic cells secondary to HIV-1 infection. However, which specific caspase(s) is involved remains to be fully identified. Several functional properties have been associated with HIV-1 Vpr, including its incorporation, at molar quantities, into the virus particle, ability to oligomerize, localization in the nucleus, induction of G2 cell cycle arrest, and positive effects on virion production and replication (14, 15, 17, 19, 28, 46, 48). As noted above, Vpr has also been demonstrated to have deleterious effects on cell cycle kinetics, leading to Vpr-induced apoptosis in T cells (44, 47). Nevertheless, one study, using extracellular HIV-1 Vpr, has demonstrated that Vpr not only is capable of binding to cells extracellularly but also increased cellular permissiveness to HIV-1 replication at concentrations of ⬍100 pg/ml to 100 ng/ml in promonocytic and lymphoid cell lines (26). Of importance, extracellular recombinant Vpr has been shown to cause a large inward current leading to death of cultured rat hippocampal neurons (38). Based on these previous studies, it can be hypothesized that although most of the direct cell-killing effects of HIV-1 have been attributed to the envelope gene product, gp120, and the Tat regulatory protein, Vpr may also be involved in the induction of CNS-based cell death (32, 33, 42, 44, 47). Of note, Levy and colleagues detected relatively high levels of Vpr protein, which may be as high as the nanogram-per-milliliter range, in the cerebrospinal fluid of AIDS patients with neurological disorders (26). Based on these findings, the effects of extracellular Vpr protein at concentrations ranging from 1 to 100 ng/ml on mature human neurons and the undifferentiated NT2 human teratocarcinoma cell line were studied. Apoptotic cell death was measured using both Annexin V and the terminal deoxynucleotidyltransferase (Tdt)-mediated dUTP-biotin nick end labeling (TUNEL) assays, as complementary approaches, to detect early and late apoptotic events, respectively. Caspase-8 activity was then measured as an indication of the intracellular events involved in Vpr-induced apoptosis. NT2 cells are derived from a human teratocarcinoma cell line with a phenotype resembling that of committed CNS neuronal precursor cells. Following treatment with retinoic acid, NT2 cells undergo an irreversible commitment to terminally differentiate into stable postmitotic neurons (39, 40). Neurons generated from differentiating NT2 cells express all ubiquitous neuronal markers, as well as phenotypically elaborating extensive neuritic processes identifiable as axons and dendrites (40). NT2 cells were grown in Dulbecco’s modified Eagle’s medium-high growth containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% glutathione. For the present experiments, NT2 cells were grown on chamber slides (Falcon), and protein treatments were administered on undifferentiated as well as mature, differentiated neuronal cultures, following daily microscopic evaluation for morphological changes occurring in the cells. Mature, differentiated neurons were induced from undifferentiated NT2 cells through an extensive differentiation, replating, and neuronal harvesting protocol. Undifferentiated NT2 cells were treated with 10 ␮M retinoic acid for a period of 5 to 6 weeks and harvested by selective trypsinization.

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This enriched culture was replated (replate 1) into T175 flasks at a cell density of ⬃100 ⫻ 106 cells per flask, and neurons were allowed to attach to the layer of supporting cells for 2 to 3 days. During the replating procedure, cell clumps were dispersed with plugged Pasteur pipettes with a reduced bore size that was achieved by fire polishing the pipette tip. The neurons and stem cells were replated again following mild trypsinization, which selectively harvests the neurons (replate 2) and, depending on the degree of cell recovery, neurons were reseeded into a T25 flask at 10 ⫻ 106 to 13 ⫻ 106 cells per flask. At this stage ⬎10% of the cells had begun to differentiate into postmitotic neurons. To prevent the undifferentiated cells from overgrowing the postmitotic neurons, mitotic inhibitors (10 ␮M 5-fluoro2⬘-deoxyuridine, 10 ␮M uridine, 1 ␮M cytosine ␤-D-arabinofuranoside) were added to the culture medium. After further enrichment by treatment with conditioned medium consisting of growth factors, the cells were replated once more (replate 3) to obtain 90 to 95% pure neuronal cultures (25, 39, 40). NT2 cells differentiate into a committed, irreversible human neuronal phenotype exhibiting a stable, postmitotic neuronal state which possesses the characteristics of mature human neurons, making them an excellent model for primary neurons. Furthermore, previous studies have demonstrated that HIV-1 replicates in differentiated but not in undifferentiated NT2 cells, although our recent studies showed some HIV-1 growth in both differentiated and undifferentiated NT2 cells (25, 32a). Thus, NT2 cells are a unique and potent cellular model system in which to study neuronal dysfunction secondary to HIV-1 infection. Expression and purification of recombinant HIV-1 Vpr from the MBP fusion system. Recombinant Vpr was expressed in the maltose binding protein (MBP) fusion system (Fig. 1A). Briefly, the MBP-Vpr protein was expressed and induced using 1 mM isopropyl-␤-D-thiogalactopyranoside, after which bacterial cells were lysed and protein was extracted. Extracted MBPVpr protein was purified by affinity chromatography using amylose resin (New England Biolabs), and the Vpr portion was cleaved from the fusion protein using factor Xa. The cleavage product was further purified by affinity chromatography using amylose resin and benzamidine agarose column purification to bind MBP and factor Xa, respectively. After the pure protein was obtained, it was further subjected to dialysis (Dialysis Cassettes; Pierce) at 4°C to ensure complete removal of any possible salts and contaminants from the purification process. Purified Vpr was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis, using polyclonal anti-Vpr antibodies raised in rabbits (kindly provided by Nathaniel Landau, Salk Institute). HIV-1 integrase (IN) protein containing the central region, amino acids 58 to 201, was cloned into pMAL-c to produce the MBP-IN fusion product. This central region of the IN protein, consisting of ⬃140 amino acids, includes the highly conserved catalytic domain D,D(35),E (22a). IN protein was expressed and purified identically to Vpr, as described above. Recombinant HIV-1IIIB gp120 was obtained (Intracel Inc., Issaquah, Wash.), and working dilutions were prepared in 1⫻ phosphatebuffered saline prior to treatment of cells. In order to ascertain the purity and size of the recombinant Vpr protein used in these studies, SDS-PAGE and then Western blotting were performed for cleaved products before and after purification, and the products were probed with a Vprspecific antibody (Fig. 1B). Western blot analysis demonstrated MBP-Vpr (⬃58 kDa) and its cleaved partner Vpr (⬃14 kDa), as well as the purity of free Vpr protein probed by the polyclonal anti-Vpr antibody (Fig. 1B).

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FIG. 1. (A) Plasmid map of MBP-Vpr fusion protein and its cleaved counterparts. HIV-1 Vpr (strain 89.6) was cloned into the pMAL-c expression vector (New England Biolabs) using DNA fragments amplified by PCR. Vpr 89.6 was inserted between the XbaI and HindIII sites in the polylinker region of the pMAL-c vector. Both the forward and backward primers were 28-bp oligonucleotides, and their sequences were 5⬘-ACGTCTAGAATGGAACAAGCCCC AGAAG-3⬘ and 5⬘-ATGCCAAGCTTTAGGATCTACTGGCTCC-3⬘, respectively. When the PCR products were obtained, the insert was cloned into the vector, and all recombinant plasmids were verified by restriction enzyme cleavage and DNA sequence analysis. (B) Left panel, SDS-PAGE of purified MBP-Vpr. MBP alone is shown in lane 2, and MBP-Vpr is shown in lane 3. Protein molecular mass standards were run for lanes 1 and 4 (molecular masses are shown to the left). Right panel, Western blot analysis of recombinant HIV-1 Vpr. MBP-Vpr, cleaved by factor Xa, is located in lanes 1 and 2, alongside MBP alone, treated with factor Xa, in lane 3. The molecular mass of MBP-Vpr was 58 kDa, and that of free Vpr was 14 kDa. Purified Vpr was identified in the Western blot analysis using polyclonal anti-Vpr antibodies raised in rabbits.

Extracellular protein application to undifferentiated NT2 cells and mature, differentiated neurons in culture. Treatments for undifferentiated NT2 cells were the following: Vpr, gp120 (HIV-1IIIB strain), MBP, MBP-Vpr, and IN at concentrations of 1, 10, 50, and 100 ng/ml for each protein (for Vpr, 1 ng/ml ⬇ 69 pM and 100 ng/ml ⬇ 6.9 nM concentrations; for gp120, 1 ng/ml ⬇ 8.3 pM and 100 ng/ml ⬇ 0.83 nM concentrations). Cells treated solely with retinoic acid and completely untreated cultures were used as negative controls. On day 4, the slides were assayed for early and late events of apoptosis, using Annexin V and TUNEL assays. For differentiated neurons in culture, Vpr, gp120, and IN proteins were added extracellularly to the media at concentrations of 1, 50, and 100 ng/ml for each protein. The medium was changed every 24 h, with fresh recombinant proteins added at their respective concentrations for 4 days. Apoptosis was analyzed on day 4, as it has been shown previously by Meucci and colleagues that treatment of rat hippocampal neuron cultures with recombi-

nant gp120 led to maximum apoptosis in 4 days (30). Microscopic observations in the present studies also demonstrated increasing apoptosis over the first 4 days of culture based on the morphological changes in the cell cultures (not illustrated). Purified HIV-1IIIB gp120 was used as a positive control, since studies have demonstrated that HIV-1IIIB gp120 causes apoptosis and neurotoxicity in rat hippocampal neurons (16, 30). Since there have been no reports of IN protein resulting in apoptosis, we used purified IN protein as a negative viral protein control, where IN protein was expressed as a fusion product of MBP, using precisely the same system utilized for Vpr protein expression and purification. MBP alone and the MBPVpr fusion proteins were also applied extracellularly, since Vpr was expressed in this protein system. The effects of Vpr on undifferentiated NT2 cells and mature human neurons were compared using both the Annexin V and TUNEL assays to detect early and late apoptotic events, respectively. The principle of the Annexin V assay is based on

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events in the early stages of apoptosis, wherein the phosphatidyl serine group which is normally located on the cytoplasmic surface of the cell membrane is caused by the enzymes translocase and flipase to “flip” to the exposed outside surface of the cell membrane upon induction of apoptosis (29). This exposed phosphatidyl serine has a high affinity for Annexin V. Annexin V assays were performed as per specifications in the Annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit (Oncogene Research Products). Samples were analyzed immediately by fluorescence microscopy. Annexin V staining was observed using a filter cube, which is a dual-band filter designed for viewing simultaneous fluorochromes. DNA fragmentation occurs at a later stage in apoptosis, and this assay utilizes the generated 3⬘ free hydroxyl groups at the ends of the fragmented DNA, to be labeled with fluorescein dUTP in the presence of Tdt (27). TUNEL assays were performed using the in situ cell death detection kit, TMR red (Boehringer-Mannheim). All studies were performed in duplicate (see Fig. 2 and 3). HIV-1 Vpr induces apoptosis in a dose-dependent manner in undifferentiated NT2 cells. In initial studies, extracellular Vpr and other control proteins were used to study their potential apoptosis-inducing effects on undifferentiated NT2 cells. These experiments demonstrated that addition of extracellular Vpr began to induce apoptosis in undifferentiated NT2 cells at a concentration of 10 ng/ml, as measured by both TUNEL (Fig. 2A) and Annexin V (Fig. 3A) assays. At increasing concentrations of Vpr, 50 and 100 ng/ml, the induction of apoptosis increased in a dose-dependent manner, as observed in both the TUNEL and Annexin V assays (Fig. 2A and 3A). HIV-1 gp120 also induced apoptosis in a dose-dependent manner with concentrations ranging from 1 to 100 ng/ml, in the undifferentiated NT2 cells. The apoptosis induced in the undifferentiated NT2 cells by various concentrations of gp120 was somewhat more potent than Vpr-induced apoptosis at the same concentrations (Fig. 2A and 3A). MBP and IN showed low to negligible induction of apoptosis at the concentrations studied, whereas MBP-Vpr maintained the ability to induce apoptosis modestly in the undifferentiated NT2 cells (Fig. 2A and 3A). It is important to note that in the two complementary assay systems, TUNEL and Annexin V, results with various treatment regimens were well correlated (Fig. 2A and 3A). The semiquantitation of percent apoptosis in undifferentiated NT2 cells was analyzed by counting the mean number of cells staining positive by the TUNEL assay, and these results are depicted in Table 1. The percentage of apoptotic cells was higher for Vpr and gp120 than for other protein treatments. Also, MBP-Vpr showed a slight induction of apoptosis at 50 and 100 ng/ml. As such, recombinant extracellular HIV-1 Vpr can induce programmed cell death (i.e., apoptosis) in undifferentiated NT2 cells. In a recent report, Stewart and colleagues examined the cytostatic and apoptotic effects of lentiviral vector delivery of the HIV-1 Vpr protein in a wide range of nonneuronal target cells (45). They observed that virion-associated Vpr significantly induced apoptosis, and moreover, analysis of their An-

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TABLE 1. Semiquantitation of apoptosis in undifferentiated NT2 cells by TUNEL assaya Protein

Vpr gp120 MBP MBP-Vpr IN

% Apoptosis at protein concn (ng/ml) of: 1

10

50

100

7 8 3 4 3

12 23 4 5 3

26 39 3 14 4

41 56 3 9 4

a The percentage of cells undergoing apoptosis was analyzed by counting the mean number of cells staining positive by the TUNEL assay (in situ cell death detection kit, TMR red; Boehringer-Mannheim), in four fields of view, by epifluorescence microscopy.

nexin V data suggests that virion-containing Vpr revealed a higher percentage of Annexin V-positive cells than mock-infected and other negative controls. Results of the Annexin V experiments in the present study compare well with their data for apoptosis induction, keeping in consideration that analysis of Annexin V and delivery of Vpr were significantly different in the two studies. HIV-1 Vpr induces apoptosis in mature human neurons. To then investigate whether Vpr could induce apoptosis in mature human neurons, fully differentiated NT2 cells were treated with various concentrations of the HIV-1 Vpr protein and selected control proteins. After differentiation, NT2 cells mature into neurons and tend to form aggregates. The neurons grow in aggregates on top of a supporting cell layer which is needed for attachment. Upon differentiation, the neurons spread their dendritic and axonal processes, as observed in phase-contrast photomicrographs in Fig. 2B. Apoptosis in the differentiated neuron cultures was distinctively and selectively observed in the mature neurons compared to the supporting cells. Also, apoptosis was induced in the mature, differentiated NT2 cells in a dose-dependent manner in both Vpr and gp120 treatments (Fig. 2B and 3B). At a protein concentration of 1 ng/ml, only a few labeled nuclei of neurons were detected with both of these proteins, as determined by a comparison of the TUNEL staining and the phasecontrast photomicrographs of the same fields. Vpr at a concentration of 50 ng/ml showed some induction of apoptosis, although it was more apparent in these mature neurons at 100 ng/ml. At 100 ng/ml of Vpr and gp120, the degrees of induction of apoptosis in mature neurons were approximately equivalent. These results were also corroborated using a separate and distinct TUNEL assay system (Oncogene Research Products), and the fragmented DNA labeled at the free hydroxyl ends was shown for all protein treatments at 100 ng/ml using this complementary assay system (Fig. 2B, bottom panel). Little to no apoptotic cell death was noted in cell cultures left untreated or treated with HIV-1 IN. Of note, the aggregating growth pattern of mature neurons, as compared to undifferentiated NT2 cells, makes strict quantitation of numbers of apoptotic cells difficult. Thus, these studies demonstrate that extracellular

FIG. 2. (A) HIV-1 Vpr- and gp120-induced apoptosis in undifferentiated NT2 cells, as measured by the TUNEL assay. Protein treatments were at concentrations of 1, 10, 50, and 100 ng/ml on the undifferentiated NT2 cell line (left sides of panels, fluorescent staining with the TUNEL assay; right side, phase-contrast photomicrograph of the same slide section). The microscope used was an Olympus System microscope, model BX60, with fluorescence attachment BX-FLA. TUNEL assays were performed using the in situ cell death detection kit, TMR red (Boehringer-Mannheim). Magnification, ⫻13.6. (B) HIV-1 Vpr- and gp20-induced apoptosis in mature neurons, as measured by TUNEL assay. Protein treatments were administered at concentrations of 1, 50, and 100 ng/ml on the mature, differentiated neurons. Left sides of panels show fluorescent staining with the TUNEL assay in situ cell death detection kit, TMR red (Boehringer-Mannheim); right sides show a phase-contrast photomicrograph of the same slide section. The lowest panels (line of four photomicrographs) show confirmatory data using a different TUNEL assay kit (Oncogene Research Products) for all protein treatments given at 100 ng/ml. Magnification, ⫻13.6.

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FIG. 3. (A) Apoptosis in undifferentiated NT2 cells, as determined by an Annexin V assay. Protein treatments were administered at concentrations of 1, 10, 50, and 100 ng/ml on the undifferentiated NT2 cell line. Left sides of panels show fluorescent staining with the Annexin V assay (Oncogene Research Products); right sides show a phase-contrast photomicrograph of the same slide section. The microscope used was an Olympus System microscope, model BX60, with fluorescence attachment BX-FLA. Magnification, ⫻7.5. (B) Apoptosis in mature human neurons, as determined by an Annexin V assay. Protein treatments were administered at concentrations of 1, 50, and 100 ng/ml on the mature, differentiated neurons. Left sides of panels show fluorescent staining with the Annexin V assay (Oncogene Research Products); right sides show a phase-contrast photomicrograph of the same slide section. Magnification, ⫻15.

HIV-1 Vpr can specifically induce apoptosis in mature human neurons. Caspase-8 is the intracellular protease involved in HIV-1 Vpr- and gp120-induced apoptosis. To begin to determine the molecular mechanisms involved with Vpr-induced apoptosis in human neural cells, caspase-8 activation was studied in NT2 cells. Caspase-8 activation results from the induction of apoptosis through death effector domains, and due to this activation two active subunits, p18 and p10, are released. The cells that undergo apoptosis activate caspase-8, which exists as an intracellular cysteine protease in the proenzyme form and cleaves a caspase-specific colorimetric substrate, resulting in

the release of chromophore p-NA, which is quantitated spectrophotometrically at a wavelength of 405 nm (8). HIV-1 Vpr exhibited a dose-dependent increase in caspase-8 activation in the undifferentiated NT2 cells (Fig. 4A). gp120 was also observed to display a dose-related increase of caspase-8 in treatments utilizing 10 to 100 ng/ml of recombinant protein. The caspase-8 increase was higher at a concentration of 1 ng/ml than at 10 ng/ml for gp120 treatments (Fig. 4A). Of note, the stimulation of caspase-8 by Vpr and gp120 was higher than that for MBP-, MBP-Vpr-, and IN-treated cells and untreated controls among the undifferentiated NT2 cells. MBP-, MBP-Vpr-, and IN-treated cells had only slightly

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FIG. 3—Continued.

increased levels of caspase-8 compared to untreated controls, which was a negligible increase compared to the increase in caspase-8 activation from Vpr and gp120 treatments. MBPVpr caused a modest increase in caspase-8 stimulation at 50 ng/ml in undifferentiated NT2 cells. Mature neurons demonstrated a dose-dependent increase in caspase-8 activation for Vpr and for gp120, except for Vprtreated cells at 100 ng/ml. The lack of caspase-8 stimulation for Vpr at the highest dose in differentiated, mature NT2 cells may be a result of high levels of cell death, already present at the time of the analyses. The caspase-8 levels of the negative controls (i.e., HIV-1 IN) were maintained at an unstimulated baseline level in the differentiated NT2 cells (Fig. 4B). The caspase-8 stimulatory activity of Vpr protein treatments was as high as 64 and 56% of that with gp120 at 100 ng/ml in the undifferentiated and differentiated cells, respectively. gp120 at 100 ng/ml had the highest caspase increase measured in both undifferentiated and differentiated neurons. Doxorubicin-treated Jurkat cells were used as a positive control, and they exhibited 17- and 9.4-fold increases of caspase-8 activity in the undifferentiated and differentiated NT2 cell studies, respectively, compared to negative control values. These studies demonstrated that analogous to that by gp120, apoptosis of human neural cells induced by HIV-1 Vpr occurs via the caspase activation pathway. In previous studies, Piller and colleagues used extracellular Vpr at concentrations of 0.6 nM, which was ⬵7 ng/ml, and observed apoptosis in cultured rat hippocampal neurons (38). Their results are comparable to the apoptotic effects observed in the present studies at similar concentrations of extracellular Vpr, 10 ng/ml in human NT2 cells. In addition to neuronal

cells, in human bone marrow cultures, Kulkosky and colleagues from our laboratories reported on the deleterious effects of extracellular HIV-1 Vpr at a concentration of 250 ng/ml, at which it induced macrophage activation and erythrophagocytosis to a marked degree (23). Paradoxically, in a study by Fukumori and coworkers, it was reported that Vpr displayed strong antiapoptotic activity in the human epidermoid carcinoma cell line HEp-2 (12). It is likely that in our studies, extracellular recombinant Vpr, in solution, exists in a properly folded structural form and therefore possesses the ability to activate certain specific receptors, ultimately causing apoptosis through a direct or an indirect mechanism(s). HIV-1 Vpr may cause direct neurotoxicity leading to cell death, as has been proposed, via ion channel formation or indirectly through the release of toxic moieties from the microglia and macrophages of the brain (26, 38). It seems more likely, though, that Vpr’s effects on neurons may have a direct mechanism, since it was neurotoxic to both isolated mature, neuronal cultures and undifferentiated cultures of NT2 cells in the present studies, and no other CNS-based cells were present in these culture systems. Furthermore, Zhao and colleagues have proposed that the Vpr protein possesses the ability to oligomerize (48). Thus, it is possible that the oligomeric structure of Vpr may have the ability to act as a ligand for inducing apoptosis through receptor interactions. Kewalramani and coworkers have demonstrated that the halflife of recombinant HIV-1 Vpr expressed in 293 T cells was 20 h (22). In our studies, fresh medium with recombinant Vpr protein was applied every 24 h in the NT2 cell cultures. Thus, at no time during the course of 4 days did the average concen-

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FIG. 4. (A) Caspase-8 activity in undifferentiated NT2 cells. Treatments with 1, 10, 50, or 100 ng/ml of each respective protein (x axis) and percent caspase-8 activity for each treatment relative to caspase-8 activity in gp120 (100 ng/ml)-treated cell cultures, which were considered as 100% (y axis), are indicated. Jurkat T cells treated with 0.2-␮g/ml doxorubicin for 48 h (positive control) exhibited a 17-fold increase in caspase-8 activity over that of control cultures. (B) Caspase-8 activity in mature, differentiated human neurons. Treatment with 1, 50, or 100 ng/ml of respective protein (x axis) and percent caspase-8 activity for each treatment relative to caspase-8 activity in gp120 (100 ng/ml)-treated cell cultures, which were considered as 100% (y axis), are shown. Jurkat T cells treated with 0.2-␮g/ml doxorubicin for 48 h exhibited a 9.4-fold increase in caspase-8 activity over that of control cultures (positive control).

tration of Vpr drop below an approximation of one intracellular half-life. Although studies in the past have demonstrated that Vpr induced apoptosis in hematopoietic and fibroblastic cells, the mechanism(s) by which it acted has so far remained somewhat elusive. Previous studies have shown that intracellular proteases play a critical role in apoptosis. Shostak and colleagues investigated the roles of p53 and caspases in the induction of

cell cycle arrest and apoptosis by HIV-1 Vpr in T-lymphocytes, and their report indicated that Vpr-induced apoptosis is caused via the activation of caspases (43). In the present studies this phenomenon was investigated, and caspase-8 was identified as the enzyme involved in the mediation of Vpr-induced apoptosis in human neurons. Furthermore, our results demonstrate that caspase-8 also seems to play a major role in gp120induced apoptosis. However, it is important to note that the

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activation of caspase-8 may lead to further activation of other caspases, which results in an irreversible commitment to apoptosis. Thus, it is not surprising that Zheng and coworkers have implicated caspase-3 in apoptosis induced by HIV-1 virions (50). It has been previously demonstrated that the envelope protein of HIV-1, gp120, induces apoptosis in neurons. A key chemokine coreceptor for HIV-1, CXCR4, has been implicated in HIV-1-induced neuronal apoptosis and more specifically in gp120-induced neuronal apoptosis (16, 49). In the study by Hesselgesser and colleagues (16), neuronal apoptosis was shown to be induced by HIV-1IIIB gp120 and was mediated through the chemokine receptor CXCR4. Their studies were performed with the same mature NT2 cell line as was used in the present study. Additionally, undifferentiated NT2 cultures were also utilized for the present report. The present data further corroborate the studies of Hesselgesser and coworkers, since these authors observed a dose-dependent induction of apoptosis in the mature NT2 cells when they combined gp120 with the cognant chemokine SDF-1␣ (16). Based upon the preferential use of the CXCR4 receptor in mediating gp120-induced apoptosis, it can be suggested that gp120-induced apoptosis in the present studies may be due to the notable expression of CXCR4 receptors on the NT2-derived neurons (16). Nonetheless, chronic administration of HIV-1 gp120 in BALB/c mice demonstrated that the adult rodent brain was unaffected by exposure to gp120 alone and that this glycoprotein may not be solely responsible for the severity of disease in the CNS of HIV-1-infected individuals (37). Thus, our results indicate that Vpr, in combination with gp120, may prove to be detrimental to the CNS of AIDS patients. In summary, HIV-1 Vpr can potently induce programmed cell death in undifferentiated NT2 neural cells and mature human neurons. We have also identified caspase-8 as an intracellular messenger involved in both Vpr- and gp120-induced apoptosis. These data clarify the concentrations required for induction of apoptosis by the lentiviral proteins Vpr and gp120. Further studies should be directed towards exposure of neuronal cells to combinations of Vpr and gp120. Also, the potential effects of virion-encapsidated Vpr and intracellular expression of Vpr in both uninfected and HIV-1-infected neuronal cells will require future analysis. Finally, the demonstration of the involvement of caspase-8 in gp120- and Vpr-induced apoptosis may lead to novel therapeutic approaches that could be rationally targeted to combat HIV-1-induced neuronal apoptosis in AIDS patients with ADC. We are grateful to Joseph Kulkosky for providing the plasmid MBPIN. We thank Aaron Geist for assistance with protein purification and express gratitude to Hui Zhang, Dennis Kolson, Satoshi Kubota, Mohamad Bouhamdan, Farida Shaheen, and Eleni Anni for helpful discussions. We thank Nathaniel Landau for the gift of the anti-Vpr antibodies. We also thank Rita M. Victor and Brenda O. Gordon for excellent assistance in the preparation of the manuscript. Research was supported in part by USPHS grants MH58526 and NS27405 and NIH NRSA Training Grant T32-A107532. REFERENCES 1. Adie-Biassette, H., Y. Levy, M. Colombel, F. Poron, S. Natcher, C. Keohane, and F. Gray. 1995. Neuronal apoptosis in HIV infection in adults. Neuropathol. Appl. Neurobiol. 21:218–227. 2. Alderson, M. R., T. W. Tough, T. Davis-Smith, S. Braddy, K. A. Schooley, R. G. Goodwin, C. A. Smith, F. Ramsdell, and D. H. Lynch. 1995. Fas ligand mediates activation-induced cell death in human T lymphocytes. J. Exp. Med. 181:71–77. 3. Bagasra, O., E. Lavi, K. Khalili, J. P. Pestaner, R. Tawadros, and R. J. Pomerantz. 1996. Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: identification by the combination of in situ polymer-

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