From the Departments of a Neurobiology and Anatomy, b Dental Research, d Neurology (Child Neurology Division), e Pediatrics, c Microbiology and Immunology, i Pharmacology and Physiology, and the g Cancer Center, University of Rochester Medical Center, Rochester, New York, 14642
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ABSTRACT |
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Human immunodeficiency virus type 1 (HIV-1)
infection of the central nervous system may result in neuronal
apoptosis in vulnerable brain regions, including cerebral cortex and
basal ganglia. The mechanisms for neuronal loss are likely to be
multifactorial and indirect, since HIV-1 productively infects
brain-resident macrophages and microglia but does not cause cytolytic
infection of neurons in the central nervous system. HIV-1 infection of
macrophages and microglia leads to production and release of diffusible
factors that result in neuronal cell death, including the HIV-1
regulatory protein Tat. We demonstrate in this report that recombinant
Tat1-86 and Tat peptides containing the basic region
induce neuronal apoptosis in approximately 50% of vulnerable neurons
in both rat and human neuronal cultures, and this apoptotic cell death
is mediated by release of the pro-inflammatory cytokine tumor necrosis
factor , and by activation of glutamate receptors of the
non-N-methyl-D-aspartate subtype. Finally, we
show that Tat-induced apoptosis of human neuronal cell cultures occurs
in the absence of activation of the transcription factor NF
B. These
findings further define cellular pathways activated by Tat, that
dysregulate production of tumor necrosis factor
, and lead to
activation of glutamate receptors and neuronal death during HIV-1
infection of the central nervous system.
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INTRODUCTION |
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It has been proposed that the mechanisms by which human
immunodeficiency virus type 1 (HIV-1)1 induces widespread
neuronal dysfunction and death in the developing and adult central
nervous system may involve activation of glutamate receptor subtypes
(1, 2). However, the sequence of events that lead to glutamate receptor
activation in the setting of HIV-1 infection and low-level chronic
inflammation in the central nervous system are complex and poorly
understood. Many previous studies on the neuropathogenesis of HIV-1
infection have focused on the neurotoxic effects of the HIV-1 envelope
protein, gp120 (3, 4). These studies have revealed that gp120 causes
neuronal toxicity in large part via indirect mechanism(s), possibly
mediated by intermediary glial cells that in turn produce cellular
metabolites that ultimately lead to excitotoxic activation of glutamate
receptors. In contrast, the HIV-1 regulatory protein Tat is soluble,
secreted, and efficiently taken up by many cell types, including
astrocytes and neurons (5, 6). Several studies have implicated both full-length Tat and basic Tat peptides (i.e. Tat amino acid
residues 31 to 61 (7)) as mediators of neuronal death. In
vitro studies have demonstrated that Tat-induced neuronal cell
death occurs via apoptosis (8), and involves activation of a non-NMDA
subtype of glutamate receptors (9). In addition, in vivo
experiments have revealed that Tat-mediated neurotoxicity can be
prevented by pentoxifylline, an agent that blocks the transcription of
the pro-inflammatory cytokine tumor necrosis factor (TNF
)
(10).
Previous studies from this laboratory have shown that TNF-induced
neurotoxicity can be prevented in part by blockade of AMPA receptors
(11), and that application of TNF
to neuronal cultures results in
apoptosis through a NF
B-independent mechanism (12). Hence, we
theorized that Tat might up-regulate synthesis and/or release of TNF
in neural cell cultures, which in turn could lead to activation of AMPA
receptors and neuronal death. In support of this hypothesis, we now
demonstrate that application of Tat to neural cultures induces neuronal
apoptosis that can be blocked in part by antibodies to TNF
, and by a
non-NMDA receptor antagonist, but not a NMDA or metabotropic
glutamate receptor antagonist.
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EXPERIMENTAL PROCEDURES |
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Neural Cell Cultures
Primary Human Neuron Cultures-- Human fetal brain tissue between gestational ages of 13 to 15 weeks was obtained, with consent, from women undergoing elective termination of pregnancy, under the guidelines of the National Institutes of Health, the University of Rochester Human Subjects Review Board. Adherent blood vessels and meninges were removed and the brain tissue was prepared as described previously, in medium containing N1 components as well as 5% heat-inactivated fetal bovine serum, PSN antibiotic mixture (penicillin at 50 mg/liter, streptomycin at 50 mg/liter, and neomycin at 100 mg/liter), and amphotericin B (Fungizone; 2.5 mg/liter) (8). Cells were plated at a density of 105/ml on 12-mm diameter glass coverslips precoated with polylysine (70,000-150,000 Mr; Sigma), and placed in 24-well culture dishes. Cells were cultured 14 to 28 days at 37 °C in a humidified atmosphere of 5% CO2, 95% air, and the medium was changed every 3 days. Sample cultures were stained for the neuroendocrine-specific protein, PGP 9.5, neuron-specific enolase, microtubule-associated protein-2, synaptophysin, and glial fibrillary astrocyte protein; under these culture conditions neuronal cultures were >70% homogenous for neurons. The remaining cells were predominantly astrocytes and <5% were microglia-macrophages, as determined by RCA-1 lectin and CD68 staining (13).
SK-N-MC Cell Cultures-- Human neuroblastoma SK-N-MC cells were obtained from the American Tissue Culture Collection (14) and cultured in plastic tissue culture dishes (Corning). Cells were grown in a humidified incubator at 37°C with 5% CO2, fed every 2 days with minimal essential complete medium, and differentiated to a neuronal phenotype, as necessary, by addition of 5 µM retinoic acid (Sigma) for 4-5 days, as described previously (12). Under these conditions the baseline level of apoptosis in neuronal SK-N-MC cells was identical to vehicle-treated cells when measured by TUNEL staining.
Rat Cerebellar Granule Neuronal Cultures-- 7-Day-old Sprague-Dawley rats were sacrificed and cerebellar brain tissue was harvested according to the guidelines established by the Animal Welfare Act (1987) and NIH policies. Briefly, cerebellum was collected and washed in cold phosphate-buffered saline containing trypsin at 0.25 mg/ml and 0.1% DNase (about 10-ml volume per cerebellum), then minced into 2-mm3 pieces, and triturated with a fire polished pipette, followed by incubation for 20 min at 37 °C (15). The tissue was filtered through nylon mesh and the cell suspension was loaded over a two-step Percoll gradient and centrifuged 1 × g for 15 min at 4 °C, to remove glia. The neurons were collected and washed twice in sterile medium without serum, then resuspended in fresh Dulbecco's modified Eagle's medium:F-12 medium with 10% horse serum. Cells were gently triturated and plated at a density of 2 × 105 cells/12-mm glass coverslip pre-coated with poly-L-lysine (70,000-150,000 Mr; Sigma) in 24-well culture dishes or at a density of 3 × 106 in poly-L-lysine coated 100-mm culture dishes. After 1-2 days in culture, 5-fluorodeoxyuridine was added to the cultures at 20 mg/ml and uridine at 50 mg/ml, to eliminate proliferative cells (astrocytes), and the purity of the resulting neuronal population was verified by immunocytochemical staining for neuronal markers. Neurons were cultured up to 7 days at 37 °C in a humidified atmosphere containing 5% CO2; media (serum-free Dulbecco's modified Eagle's medium:F-12) was replaced every 3 days.
RN46A Cells-- Immortalized neural cells from rat dorsal raphe were obtained as a generous gift from the Miami Project (Dr. S. R. Whittemore, Miami, FL) and grown at 33 °C as described previously (16) in central nervous system culture media (17) supplemented with 10% fetal bovine serum and 250 µg/ml G418. In some experiments, cells were differentiated by changing the media to 50% Dulbecco's modified Eagle's medium, 50% Hams F-12 (D/F) containing 1% (w/v) bovine serum albumin and serum-free media components, and shifting the temperature to 39°C, all as described previously (16).
Preparation of HIV-1 Recombinant Tat
Recombinant HIV-1 Tat1-86 was expressed and
purified as a glutathione S-transferase fusion protein as
described previously (8). A thrombin proteolytic digestion procedure
was performed to remove the glutathione S-transferase domain
from the purified glutathione S-transferase-Tat protein, and
Tat preparations were then stored at 70 °C until use. Purified
Tat1-86 was further characterized and quantified by
protein assay (Lowry method), SDS-polyacrylamide gel electrophoresis,
and by immunoblot analysis, using a polyclonal antibody (AIDS Research
and Reference Reagent Program, NIAID, National Institutes of Health),
all as described previously (8). To control for the possible presence
of contaminating bacterial proteins which might elicit neurotoxicity,
an Escherichia coli strain containing a glutathione
S-transferase expression plasmid without the
Tat1-86 sequence was used to prepare control extracts,
which were purified in the same manner as the Tat fusion protein. This
sham extract was used as a vehicle control. For some experiments,
recombinant Tat peptides (Tat46-60: SYGRKKRRQRRRPPQ;
Tat65-80: HQVSLSKQPTSQPRGD) were obtained from Intracel
Corp. (Cambridge, MA). To control for the possibility of
lipopolysaccharide contamination of neurotoxic species of Tat, both
Tat1-86 and Tat46-60 were heated to 60 °C
for 30 min (lipopolysaccharide is heat-stable), then applied to
neuronal cultures for 24 h. TUNEL immunostaining of apoptotic
neurons in Tat-treated cultures was the same as control vehicle-treated
cultures (data not shown).
In Situ Detection of Apoptotic Neurons by TUNEL Stain
Neurons cultured on 12-mm poly-L-lysine-coated coverslips were treated with Tat or other reagents, and apoptotic cells were then stained using an in situ terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end labeling (TUNEL) assay method (Oncor, Gaithersburg, MD), as described (8). Briefly, neurons stained by the TUNEL assay were fixed in 4% paraformaldehyde, rinsed with phosphate-buffered saline, then post-fixed with a 100% ethanol:acetic acid solution (2:1), and rinsed again with phosphate-buffered saline. Neurons were pretreated with 2% H2O2 to quench endogenous peroxidase, prior to the addition of the terminal deoxynucleotidyl transferase. Anti-digoxigenin peroxidase was then added, and catalytically reacted with 0.05% diaminobenzidine in phosphate-buffered saline. TUNEL-stained neurons were then counted from 15 randomly selected fields. Each field of at least 100 neurons was counted for positively stained versus negatively stained cells.
TNF ELISA
Human TNF--
A solid phase sandwich ELISA kit (Biosource
International, Camarillo, CA) was employed to measure human TNF
in
culture media from human fetal neuronal cultures and SK-N-MC cultures.
Media samples were diluted until within the range of the standard
curve.
Murine TNF--
A solid phase sandwich ELISA (Biosource
International, Camarillo, CA) was employed to measure TNF
in rat
cerebellar granule cell cultures.
Transient Transfections and Luciferase Assays
Terminally differentiated SK-N-MC cells (1.5 × 105) were transfected using liposome-mediated gene
transfer. One microgram of NF-B luciferase reporter plasmid
(p(
B)3conaLUC; kind gift of Dr. Alaïn Israel,
Refs. 12 and 19) and 3.5 µl of LipofectAMINETM reagent
(Life Technologies, Inc.) were used for each transfection. At 40 h
after transfection, the recipient cells were either left untreated or
incubated with the recombinant Tat protein (500 nM) or
rh-TNF
(40 ng/µl; Genzyme, Cambridge, MA) for 8 h. Cells were then subjected to extract preparation using a reporter lysis buffer (Luciferase reagent; Promega, Madison, WI) at about 40 µl/105 cells, and luciferase activity was measured with
an LKB Wallace 1250 Luminometer.
Cell Extracts and Immunoblotting
Whole cell extracts were prepared from differentiated SK-N-MC
cells (5 × 105) by in situ lysis using ELB
buffer (50 mM HEPES, pH 7.0, 250 mM NaCl, 0.1%
Nonidet P-40, 5 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride) supplemented with
various phosphatase inhibitors. Cell lysates containing equal amounts
of total protein (15-25 µg) were immediately supplemented with 1%
SDS, heated for 5 min at 100 °C, fractionated by reducing 10%
SDS-polyacrylamide gel electrophoresis, and electrophoretically
transferred to HybondTM ECLTM nitrocellulose
membrane (Amersham Life Sciences, Arlington Heights, IL). Rabbit
polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
raised against peptides corresponding to the carboxyl terminus of
IB
(C-21) and I
B
(C-20), were used to form immunocomplexes on these membranes and were further analyzed by using an enhanced chemiluminescence detection system (Amersham).
Nuclear Extract Preparation and Electrophoresis Mobility Shift Assay (EMSA)
SK-N-MC cells were treated with the indicated species of Tat for varying time periods, followed by collection in phosphate-buffered saline by centrifugation 800 × g for 5 min, and then subjected to nuclear extract preparation as described previously (20). EMSA was performed by incubating the nuclear extracts (15 µg) with 32P-radiolabeled probes at 20-22 °C for 10 min, followed by resolution of DNA-protein complexes on native 4% polyacrylamide gels as described previously (18). The oligonucleotide probe used in this EMSA had the following sequence: CAACGGCAGGGGAATTCCCCTCTCCTT.
Computerized Morphometry and Statistical Analysis
Digitized images of TUNEL-stained neurons in 15 or more microscopic fields were analyzed for numbers of positively stained neuronal nuclei, and for total numbers of neurons, per × 50 field using computerized morphometry (Imaging Research Inc., Ontario, Canada). Data were expressed as % TUNEL-positive neurons, and arithmetic mean values, and standard errors of mean values were calculated; significance was determined by one-way ANOVA.
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RESULTS |
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Tat has previously been shown to induce TNF mRNA and
protein production in macrophages and astrocytes (21), and results from
this laboratory have shown that TNF
is neurotoxic to terminally differentiated (i.e. neuronal) SK-N-MC neuroblastoma cells,
but not to undifferentiated SK-N-MC cells (12). We therefore
investigated whether recombinant Tat1-86 was toxic to
terminally differentiated SK-N-MC cells. Fig.
1A demonstrates that when
Tat1-86 or Tat46-60 peptide containing the
basic domain is applied to undifferentiated SK-N-MC cells with a glial
phenotype (i.e. cells not treated with retinoic acid), no
significant cell death is observed. In contrast, when either
Tat1-86 or Tat46-60 peptide is applied to
differentiated SK-N-MC cells with a neuronal phenotype (i.e.
retinoic acid treated cells), 20-25% of neuronal cells are
TUNEL-stained, indicative of apoptotic death (Fig. 1B). However, Tat65-80 peptide, which lacks the basic,
neurotoxic region, is not neurotoxic to neuronal SK-N-MC cells, in
agreement with previously published studies (7).
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We further investigated the role of TNF in the neuronal cell death
that occurred after exposure to Tat, by co-incubating neuronal SK-N-MC
cells with Tat46-60 peptide, plus a neutralizing monoclonal antibody directed against human TNF
(clone cA2; Ref. 22).
Fig. 2A shows that the
neuronal cell death that occurs after exposure to Tat46-60
peptide can be markedly reduced with cA2. Similar results were obtained
in primary cultures of human fetal neurons when full-length Tat was
incubated with cA2 (Fig. 2B), suggesting that both
Tat1-86 and Tat46-60 peptide induce neuronal
apoptosis in part through synthesis and/or release of TNF
. Because
TNF
may be neuroprotective in some rodent neuronal culture systems
(23), and because Tat has been demonstrated to have anti-apoptotic
effects when applied to serum-deprived rodent PC12 neuronal cells (24),
we tested the effects of Tat1-86 on primary cultures of
rodent cerebellar granule neurons and immortalized rat dorsal raphe
neurons (RN46A). In rodent cerebellar granule neuronal cultures,
application of Tat1-86 resulted in apoptosis in 60-70%
of primary cerebellar granule neurons, and in approximately 15% of
differentiated RN46A neurons (data not shown).
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Despite differentiation with retinoic acid, SK-N-MC cultures still
contain some cells with glial phenotypes, as do primary human fetal
cortical neuronal cultures. Additionally, primary human fetal cortical
neuronal cultures contain <5% macrophages (13). Potentially, this
population of glial cells and/or macrophages could serve as a source of
TNF, particularly since Chen et al. (21) have
demonstrated that Tat1-72 markedly elevated production of
TNF
by highly purified cultures of macrophages and astrocytes. We
therefore set out to examine whether Tat could induce TNF
production
by macrophage and glial cells in a variety of primary neuronal culture
systems. Using an ELISA detection method to measure soluble TNF
in
culture media, we determined that basal levels of TNF
in a primary
human fetal cortical neuronal culture 24 h after the application
of vehicle control were below detectable levels of the assay, while
treatment with 500 nM Tat1-86 elevated TNF
levels to 176.6 ± 2.3 pg/ml (Fig.
3). In contrast to the full-length
protein, the shorter Tat peptide (Tat46-60) containing the
basic region elicited a lesser response (TNF
levels to 32.5 ± 0.3 pg/ml), while the non-neurotoxic Tat65-80 had no
effect on TNF
production 24 h after addition to neuronal cultures (data not shown). As expected, co-incubation of human neuronal
cultures with Tat1-86 or Tat46-60 plus the TNF
neutralizing monoclonal antibody cA2 completely reversed this
effect (Fig. 3). Additional experiments were performed to test whether
Tat1-86 could elevate TNF
levels in the media of
postnatal rodent cerebellar granule neuronal cultures. Here Tat1-86 elevated TNF
only 2-fold, and cA2 was not used to reverse this effect because it does not bind or neutralize rodent
TNF
(Ref. 22, data not shown).
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We have previously shown that TNF induces neuronal apoptosis through
a mechanism that involves oxidative stress, and is blocked by
overexpression of either bcl-2 or the caspase inhibitor, crmA, but is
independent of NF
B activation (12). NF
B, present in the cytoplasm
of most cell types, is normally bound to a member of a family of
inhibitor proteins known as I
B. The best characterized member of
this family I
B-
, binds specifically to the
P65/P50 heterodimer of NF
B in the cytoplasm.
When cells are exposed to inducers of NF
B, such as TNF
, the
mitogen-activated protein kinase-NIK signaling pathway (25, 26) is
activated, which results in stimulation of the multiprotein I
B
kinase complex (27-31), followed by serine phosphorylation
(Ser32 and Ser36) of I
B-
and subsequent
degradation of this protein by the 26 S proteasome complex (32, 33).
NF
B is thus released to translocate to the nucleus and activate
transcription of target genes. Because Tat1-86 and
Tat46-60 peptide induce neuronal apoptosis in part through
production of TNF
and activate NF
B in other cell types (21), we
wondered whether NF
B activation might play a significant role in
mediating Tat-induced neuronal death.
To investigate whether Tat1-86 could transduce a signal
for activation of NFB in neuronal SK-N-MC cells, studies were performed to test the effect of Tat1-86 on the fate of
I
B. In untreated SK-N-MC cells, a single 37-kDa form of I
B-
was detected with an I
B-
-specific antiserum (Fig.
4A). Incubation of the cells
with Tat1-86 (500 nM, at the LD50
for neuronal apoptosis; see Ref. 8) for 2-6 h did not induce any
detectable change in steady state levels of I
B-
(Fig.
4A; compare the intensity of the I
B-
band to that of
the upper, nonspecific band, which provides a control for total protein
content in these extracts). I
B-
levels remained unaltered even
when de novo synthesis of I
B-
was inhibited through
the addition of a protein synthesis inhibitor, cycloheximide. In order
to confirm that neuronal SK-N-MC cells are responsive to other
activators of NF
B, we incubated the cells with human recombinant
TNF
(40 ng/ml) for various time periods. As expected, this led to
the rapid degradation of I
B-
, which was prevented by
preincubation of the cells for 15 min with 50 µM of
proteasome inhibitor MG132 (Fig. 4A). Parallel studies showed that TNF
potently activated NF
B driven luciferase activity in transiently transfected neuronal SK-N-MC cells (12). These results
suggest that the mitogen-activated protein kinase-NIK-I
B kinase
signal transduction pathway is fully functional in neuronally differentiated SK-N-MC cells. Additional experiments were performed to
confirm that Tat1-86 did not activate NF
B in neuronally differentiated SK-N-MC cells. First, immunoblot experiments were conducted using an I
B-
-specific antiserum; no change in I
B-
levels was detected in Tat1-86-treated cells (data not
shown). Second, EMSA studies were performed. Constitutive nuclear
expression of NF
B was detected in unstimulated, undifferentiated
SK-N-MC cells (data not shown). NF
B levels remained unaltered after
exposure of cells to retinoic acid, and were similarly unchanged upon
exposure to Tat1-86 (Fig. 4B). Interestingly,
Tat1-86 treatment in the presence of an anti-TNF
neutralizing antibody (cA2) had no influence on activation of NF
B,
indicating that the low levels of TNF
generated due to
Tat1-86 treatment of neuronally differentiated SK-N-MC
cells are not sufficient to trigger the activation of NF
B.
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These biochemical analyses of NFB activation were also verified by
direct functional analysis of the transactivation ability of NF
B,
with a
B-driven luciferase reporter (p(
B)3conaLUC, Refs. 12 and 19). Briefly, this reporter plasmid was introduced into
differentiated SK-N-MC cells by transient transfection and cells were
then treated with neurotoxic Tat1-86,
Tat46-60 peptide, or non-neurotoxic Tat65-80
peptide, respectively. As shown in Fig. 4C, significant
luciferase activity was detected in untreated differentiated SK-N-MC
cells indicating high constitutive NF
B activity. However, no change
in NF
B-dependent transcription was observed after
treatment with Tat46-60, Tat65-80, or
Tat1-86. Taken together the data in Fig. 4 strongly
suggest that the activation of NF
B does not occur in neuronal
SK-N-MC cells during Tat-induced apoptosis.
Using primary human fetal cortical neuronal cultures, we have
previously demonstrated that TNF activates non-NMDA (i.e.
AMPA) receptors, resulting in neuronal death (11). We therefore
performed analogous experiments, using Tat1-86. Neurons
were treated with full-length Tat in the presence of a non-NMDA
receptor antagonist, to establish whether Tat-mediated neurotoxicity
resulted from non-NMDA receptor activation (as would be predicted for a
TNF
mediated effect). In agreement with our previous results (11), co-incubation of Tat1-86 with the AMPA receptor
antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione resulted in an
approximately 50% reduction in neuronal apoptosis, using primary
cultures of either human fetal cortical neurons (Fig.
5A) or rat cerebellar granule
neurons (Fig. 5B). In both culture systems, the NMDA
receptor channel antagonist dizocilpine (MK-801) was ineffective in
ameliorating Tat1-86-mediated neurotoxicity (Fig. 5,
A and B). Additionally, the metabotropic
glutamate receptor antagonist 2-amino-3-phosphonopropionic acid (AP-3)
was ineffective in antagonizing Tat1-86-mediated neurotoxicity in rodent cerebellar granule neuronal cultures (Fig. 5B).
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DISCUSSION |
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HIV-1 Tat has been shown to cause apoptosis of human peripheral
blood mononuclear cells, and a T cell line (34-37), in addition to
neuronal cells. In earlier work (8), we had speculated that Tat-induced
neuronal apoptosis was due in part to TNF-mediated signaling and
glutamate receptor activation. In the present report, we have
experimentally examined this hypothesis, and we have shown that intact
HIV-1 Tat, or a basic Tat peptide, is able to induce neuronal apoptosis
through a mechanism that involves extracellular release of TNF
, and
activation of neuronal non-NMDA receptors. As previously noted,
Tat-induced neurotoxicity is confined to vulnerable neurons, and
astroglia are spared (8).
The pro-apoptotic effect of a basic Tat peptide on SK-N-MC cells
differentiated to a neuronal phenotype (Fig. 1, A and
B) was strongly reminiscent of the effect of TNF on these
same cells (12). This led us to consider the possibility that
Tat-mediated neurotoxicity may be due, at least in part, to TNF
. We
were able to confirm this using a neutralizing monoclonal antibody
(cA2) directed against TNF
, both in neuronal SK-N-MC cell cultures and in primary human fetal neuronal cultures (Fig. 2, A and
B). This finding is consistent with results from earlier
experiments, in which the in vivo neurotoxicity of basic Tat
peptides in murine brain could be partially blocked by
co-administration of a specific inhibitor of TNF
(10).
Chen et al. (21) have recently demonstrated that
Tat1-72 up-regulates production of TNF at the
transcriptional and translational level in human macrophages and fetal
astrocytes. In their study, using highly purified cultures of
macrophages, 100 nM Tat1-72 increased
production of TNF
protein to 15 ng/ml 4 h after exposure, with
levels of TNF
remaining at 10 ng/ml 24 h after exposure. In
contrast, using highly purified cultures of fetal astrocytes, 1 µM Tat1-72 increased production of TNF
to
1 ng/ml transiently 1 h after exposure, with a return to
near-baseline levels by 4 h. We performed a TNF
ELISA 24 h after application of full-length Tat (Tat1-86) and have detected TNF
levels of approximately 0.18 ng/ml. We have previously shown that TNF
levels in this range are neurotoxic to human fetal cortical neurons (11). Furthermore, if one considers the fact that
macrophages and microglia comprise
5% of primary human cortical neuronal cultures grown in defined (N1) medium (13), the present levels
of TNF
(0.18 ng/ml) would be consistent with the production of
approximately 3.5 ng/ml TNF
by a pure population of human macrophages; this would be quite consistent with the results of Chen
and co-workers (21). The findings that Tat46-60 produces 20% of TNF
levels elicited by Tat1-86 in these
neuronal culture systems, yet Tat46-60 is able to induce
neuronal apoptosis with equal efficacy as Tat1-86, and is
blocked to the same degree by cA2 (Fig. 1B), remain to be
reconciled. Possible explanations include different rates of cellular
internalization and different time courses of TNF
production for
Tat46-60 compared with Tat1-86. Additionally,
basal values of murine TNF
in rodent cerebellar granule neuron
cultures were approximately 70-80 pg/ml, and doubled to approximately
140-160 pg/ml with Tat-induced stimulation 24 h later (data not
shown). This may reflect the lower concentration of macrophages and
microglia in rodent cerebellar granule neuron
cultures2 or species-specific
differences in Tat-induced production of murine TNF
.
Chen et al. (21) have also demonstrated that
Tat1-72-mediated production of TNF by monocytoid cells
and astroglia occurs via activation of nuclear factor
B (NF
B). In
contrast, we demonstrate here that Tat1-86 and
Tat46-60-induced neuronal apoptosis is independent of
NF
B activation (Fig. 4, A and B). If
Tat-mediated neurotoxicity is due in part to production of TNF
, with
subsequent pro-apoptotic signaling, this is in good agreement with our
previously published studies demonstrating that TNF
-mediated
neuronal apoptosis is also independent of NF
B activation (12).
Further support for this notion has been provided by the work of Hsu
and co-workers (38), who have shown that TNF
-mediated induction of
apoptosis and activation of NF
B occur via distinct signal
tranduction pathways. Alternatively, the constitutive level of NF
B
activation may be high enough in basal (i.e. control) neuronal SK-N-MC cells to mediate Tat-induced neurotoxicity (Fig. 4).
However, the very low level of apoptosis in untreated neuronal SK-N-MC
cultures mitigates against the idea of a threshold elevation for
NF
B. Furthermore, in the SK-N-MC model system, it appears that
Tat-induced production of TNF
is also independent of NF
B activation; it is unknown whether this is also true for primary human
and rodent neuronal cultures. One possibility may be that Tat-induced
production of TNF
in neuronal SK-N-MC cells occurs via a
post-transcriptional mechanism which is known to occur in lipopolysaccharide-stimulated mononuclear cells (39-41).
Previous studies of Tat-induced neurotoxicity in human fetal neuronal cultures have demonstrated that blockade of non-NMDA receptors results in complete amelioration of this neurotoxicity in a relatively small percentage (~10%) of the total number of neurons (9). However, these studies differ from the present work in at least two important methodological details. In particular, Magnuson and colleagues (9) quantitated cell death by trypan blue exclusion at 3 h after application of Tat and glutamate receptor antagonists. In the present study, TUNEL staining was used to examine apoptosis, at a 24-h time point. The trypan blue exclusion method is not a specific indicator of apoptosis, and would be expected to provide an indication of the number of neurons undergoing necrosis; apoptotic cells would still be expected to exclude this vital dye, particularly at an early time point in the programmed cell death pathway (42). Consistent with this, unpublished studies3 from our laboratory demonstrate that application of basic Tat peptides results in neuronal apoptosis, not necrosis, using the TUNEL-trypan blue double-stain method of Perry et al. (43).
The extent of Tat-induced neuronal apoptosis, and the efficacy with
which the AMPA/kainate receptor antagonist
6-cyano-7-nitroquinoxaline-2,3-dione inhibited this effect (Fig. 5), is
similar to results from our published studies, in which we examined the
effect of exogenous TNF on primary human neurons, in the presence
and absence of 6-cyano-7-nitroquinoxaline-2,3-dione (11). One
interpretation of these findings is that, in approximately half the
neurons that die following exposure to Tat, production of soluble
TNF
is followed by activation of AMPA and/or kainate receptors, and
subsequently by apoptotic death of vulnerable neurons. This occurs in
the absence of NF
B activation. This pro-apoptotic pathway is
presumably distinct from the very rapid Tat-mediated depolarization of
rat hippocampal CA1 neurons, that occurs over a period of seconds, and
which can be blocked by non-NMDA receptor antagonists (9).
Furthermore, an additional pro-apoptotic pathway that is independent of
AMPA receptor activation, but is responsive to neurotoxic species of
Tat and TNF, exists in neuronal SK-N-MC cells, which lack functional
AMPA receptors.4
The results reported here may offer an explanation for the observed
50% reduction in expression of the GluR-A flop subtype of AMPA
receptors in brains of individuals with HIV-1 infection, at both the
mRNA and protein level (44). Thus, Tat-mediated release of TNF
may induce apoptosis in vulnerable neurons expressing AMPA receptors
within the central nervous system of persons with AIDS. Future studies
will be necessary to characterize further the molecular mechanisms of
Tat-mediated neurotoxicity, to better understand how Tat or TNF
can
activate non-NMDA receptors, and to examine whether Tat exerts its
effects in whole or in part through the induction of oxidative stress
(45).
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ACKNOWLEDGEMENTS |
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We thank Dr. S. R. Whittemore and the
Miami Project for the generous gift of RN46A cells. We thank Dr.
Alaïn Israel for the kind gift of the luciferase reporter
plasmid. We thank Dr. David Shealey and Centocor, Inc. for the generous
gift of the cA2 monoclonal antibody to TNF.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants F32-NS10307 (to D. R. N.) and PO1 MH57556 (to S. D., L. G. E., and H. A. G.).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.
j To whom correspondence should be addressed. Tel.: 716-275-4784; Fax: 716-275-3683; E-mail: hgelbard{at}mail.neurology.rochester.edu.
1
The abbreviations used are: HIV-1, human
imunodeficiency virus type 1; NMDA, N-methyl-D-aspartate;
TNF, tumor necrosis factor
; AMPA,
(±)-
-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid; ELISA,
enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift
assay.
2 D. R. New, unpublished data.
3 D. R. New, S. B. Majjirwar, L. G. Epstein, S. Dewhurst, and H. A. Gelbard, unpublished data.
4 D. R. New and H. A. Gelbard, unpublished data.
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REFERENCES |
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