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Previous Article | Next Article 
Journal of Virology, October 2000, p. 9717-9726, Vol. 74, No. 20
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.
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 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 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 µM retinoic acid for a period of 5 to 6 weeks
and harvested by selective trypsinization. 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 µM 5-fluoro-2'-deoxyuridine, 10 µM
uridine, 1 µM cytosine 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-
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Human Immunodeficiency Virus Type 1 Vpr Induces
Apoptosis in Human Neuronal Cells
ABSTRACT
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-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.
-D-thiogalactopyranoside, after which
bacterial cells were lysed and protein was extracted. Extracted MBP-Vpr
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).
View larger version (68K):
[in a new window]
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'-ACGTCTAGAATGGAACAAGCCCCAGAAG-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 recombinant 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).
|
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 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 phase-contrast 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 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 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. MBP-Vpr caused a modest increase in caspase-8 stimulation at 50 ng/ml in undifferentiated NT2 cells.
|
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 half-life 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 concentration 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 gp120-induced apoptosis.
However, it is important to note that the 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.
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ACKNOWLEDGMENTS |
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We are grateful to Joseph Kulkosky for providing the plasmid MBP-IN. 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.
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FOOTNOTES |
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* 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: roger.j.pomerantz{at}mail.tju.edu.
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