Intracellular Human Immunodeficiency Virus Tat
Expression in Astrocytes Promotes Astrocyte Survival but Induces Potent
Neurotoxicity at Distant Sites via Axonal Transport*
Ashok
Chauhan
,
Jadwiga
Turchan
,
Chava
Pocernich§,
Anna
Bruce-Keller¶,
Susan
Roth
,
D. Allan
Butterfield§,
Eugene O.
Major**, and
Avindra
Nath

From the
Department of Neurology, Johns Hopkins
University, Baltimore, Maryland 21287, the Departments of
§ Chemistry, Anatomy and ¶ Neurobiology and
Microbiology and Immunology, University of Kentucky,
Lexington, Kentucky 40536, and the ** Laboratory of Molecular
Medicine and Neuroscience, NINDS, National Institutes of Health,
Bethesda, Maryland 20892
Received for publication, September 12, 2002, and in revised form, December 19, 2002
 |
ABSTRACT |
The human immunodeficiency virus
(HIV)-Tat protein has been implicated in the neuropathogenesis
of HIV infection. However, its role in modulating astroglial-neuronal
relationships is poorly understood. Astrocyte infection with HIV has
been associated with rapid progression of dementia. We thus
initially transfected astrocytes with HIV proviral DNA and
confirmed Tat production in these cells. Subsequently, using stably
Tat-producing asytocyte cell lines, we observed that Tat promoted
astrocyte survival by causing a prominent antioxidant effect and
resistance to cell injury in these cells. Tat was released
extracellularly where it could be taken up by other cells. Tat remained
functionally active following uptake and caused long terminal repeat
(LTR) transactivation in lymphocytic and astrocytic cell lines.
Tat released from astrocytes caused mitochondrial dysfunction, trimming
of neurites, and cell death in neurons. Tat neurotoxicity was
attenuated by anti-Tat antibodies, kynurenate or heparan sulfate. The
neurotoxic effects of Tat were caused at concentrations lower than that
needed to cause LTR transactivation. When Tat-expressing cells were
injected into the rat dentate gyrus, Tat was taken up by granule cells and transported along neuronal pathways to the CA3 region where it
caused glial cell activation and neurotoxicity. The arginine-rich domain of Tat was essential for both the LTR transactivation and the
neurotoxic properties of Tat. Thus HIV-Tat is a potent
neurotoxin that may act at distant sites while at the same time it
assures its production by preventing cell death in astrocytes where it is produced.
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INTRODUCTION |
The brain is a frequent target in patients with human
immunodeficiency virus (HIV)1
infection resulting in a dementing illness termed HIV
dementia (HIVD). The pathological features associated with HIVD include microglial cell activation, astrocytosis, decreased synaptic and dendritic density, and selective neuronal loss (1). Neuronal damage
occurs through toxic substances released from infected microglia or
macrophages and possibly astrocytes (2). The virus predominantly
infects microglia and macrophages where it causes a productive
infection. Astrocytes are also frequently infected with HIV but produce
limited viral replication (2). The relevance of this viral infection
remains unknown. Astrocytes maintain a barrier between blood and brain
and provide neuronal support functions. Therefore, astrocytes may serve
as a reservoir for the virus or induce neuronal damage by loss of
neuronal support functions or release of cellular and viral products.
Hence, infection of these cells could potentially have long term
consequences on cerebral function.
A striking feature is the presence of Tat transcripts in brains of
HIV-infected individuals (3, 4). HIV Tat is a non-structural regulatory
protein of 15 kDa that transactivates viral and cellular genes. It is
produced in the early phase of infection and is actively released from
infected lymphoid cells extracellularly (5). Extracellular Tat may be
internalized by uninfected cells, or it may interact with the cell
membrane initiating a cascade of events (6). Tat protein has both
direct and indirect neurotoxic activity mediated by interaction with
glutamate receptors, by disruption of cytokine network, and by reducing
the neuroprotective effects of astrocytes (7-11). However, neither is
the role of intracellularly expressed Tat on astrocyte function known,
nor is the relative role of Tat uptake by neurons or its membrane interactions understood.
Tat can produce oxidative stress in microglia cells (12), monocytes
(13), and T lymphocytes. In the latter, intracellular expression of Tat
leads to down-regulation of mitochondrial superoxide dismutase, thereby
causing impaired mitochondrial membrane potential (14, 15). Oxidative
damage is difficult to measure directly; however, oxidized endogenous
macromolecules such as free protein carbonyl and 4-hydroxynonenal (HNE)
can serve as indicators of oxidative damage (16). Lipid peroxidation
leads to the formation of HNE, a lipophilic alkenal that forms stable
adducts on mitochondrial proteins (17). It has been suggested to be the
key mediator of oxidative stress-induced cell death (18). Hence, in
this study, we have determined the effect of Tat on mitochondrial
function and other indicators of oxidative stress in astrocytes and neurons.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Tat-86 from HIVHXB-2 was cloned
from pGEX-Tat into pcDNA3 vector at
BamHI/EcoRI driven by a cytomegalovirus
promoter. Further, Tat-86 and a deletion mutant of Tat-86 from which
amino acids 48-56 were deleted by PCR (
Tat), was cloned in-frame
upstream of green fluorescent protein (GFP) gene driven by
cytomegalovirus promoter in pEGFP-N1 (Clonetech) (Fig.
1A). The HIV long terminal repeat (LTR)-driven GFP construct was made in pEGFP vector by deleting
the cytomegalovirus promoter and inserting the LTR at SalI
and SmaI. The PCR-cloned sequences were verified by double strand DNA sequencing. Tetracycline (tet) "on" system was used for
generation of inducible constructs. Tat-86 was cloned downstream of a
tet responsive element in pTREX vector (Clonetech). Reverse tetracycline transactivator (rtta) was first cloned in pEGFP vector (Clonetech) at BamHI/EcoRI and further subcloned
in GFAP promoter-driven vector pGfaLac-1 at
BamHI/BglII (Fig. 1B).

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Fig. 1.
Tat expression systems. A,
Tat constructs. Top panel, wild type Tat with
demarcation of nuclear localization signal (NLS);
middle panel, Tat-GFP; bottom panel, Tat
GFP with deletion of 48-56 amino acids ( Tat-GFP). B,
tetracycline (Tet)-inducible expression. a, GFAP
promoter-driven reverse tet transactivator (rTtA)
expression; b, rTtA induces tet responsive element
(TRE)-driven Tat expression in the presence of
doxycycline.
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Cell Culture--
Human fetal brain specimens were obtained from
fetuses of 12-14 weeks gestational age. Neuronal cultures were
prepared as described previously (19). Briefly, the cells were
mechanically dissociated, suspended in Opti-MEM with 5% heat
inactivated fetal bovine serum, 0.2% N2 supplement, and 1% antibiotic
solution (penicillin G 104 units/ml, streptomycin 10 mg/ml,
and amphotericin B 25 µg/ml). The dissociated cells were cultured for
at least one month prior to use in experiments. Human fetal astrocytes,
rat C6 glioma cells, SVGA (a human astrocytic subclone of the SVG
cells) (20), and HeLa cells were maintained in Dulbecco's
modified Eagle's medium with 2 mM L-glutamine,
10% fetal bovine serum, and antibiotic solution.
Cell Transfection--
C6 (rat glioma cell line), SVGA (human
fetal astrocytes cell line), HeLa, or primary human astrocytes were
transfected with endotoxin-free plasmids (Qiagen) using LipofectAMINE
Plus (Invitrogen) as per supplier's protocol. Cells were monitored for
GFP fluorescence or stained for Tat using a monoclonal antibody (APR
352, National Institute for Biological Standards and Control, Herts,
UK). For establishing stably transfected cell lines, 72 h
post transfection, the cells were cultured in Dulbecco's modified
Eagle's medium containing G418 (Invitrogen) at 1.5 mg/ml. After
1 week, GFP florescent clones were picked under a florescent microscope
using trypsin-soaked filter discs and sub-cultured for few weeks in
G418 containing selection media. The cells were used within 30 passages
and maintained in 500 µg/ml G418. We established SVGA-Tat,
doxycycline-inducible SVGA-Tat (SVGA13), SVGA-LTR-CAT,
SVGA-LTR-GFP, SVGA-pcDNA (SVGA-neo), C6-Tat, C6-Tat-GFP,
C6-
Tat-GFP, C6-pcDNA (C6-neo), and HeLa-Tat cell lines for the
present study. Tat levels in culture supernatants were determined by a
sandwich ELISA, where a Tat monoclonal antibody was coated at the
bottom of the plate and Tat polyclonal antisera (raised in rabbit
against HIV-1 Tat-72) was used as the secondary antibody (4). The
sensitivity of detection was 50 pg/ml. Highly purified recombinant
Tat protein produced in our laboratory was used as a standard (6).
RNA Isolation and RT-PCR--
In each case, total RNA was
extracted from 3-4 × 106 cells by Trizol reagent
(Invitrogen) and treated with RNase-free DNase. 1 µg of RNA was
reverse transcribed as described previously (21) into cDNA using
random hexamer primers and 200 units of superscript reverse
transcriptase (Invitrogen) at 45 °C for 1 h and 70 °C for 10 min in 20 µl total volume. 50 ng of cDNA was used in PCR amplification with 25 pM of each primer, 0.1 mM
dNTP mix, 1.25 units platinum Taq polymerase, and 1.2 mM Mg2+. The following primers were used: Tat,
Tat-AC1 5'-ATGGAGCCAGTAGATCCTAG-3' and Tat-AC2,
5'-TCATTGCTTTGATAGAGAAACTTG-3'; rTta, rTta1 and
5'-AATCGAAGGTTTAACCCG-3'; rTta2, 5'-TTGATCTTCCAATACGCAACC-3';
iNOS, iNOS1 5'-TGTGCCACCTCCAGTCCAGTGACA-3' and iNOS2
5'-GCTCATCTCCCGTCAGTTGGTAGG-3'; FASL, FASL1 5'-TGGGGATGTTTCAGCTCTTC-3' and FASL2 5'-TCATCATCTTCCCCTCCATC-3'; GAPDH, GAPDH1
5'-ACCACAGTCCATGCCATCAC-3' and GAPDH2
5'-TCCACCACCCTGTTGCTGTA-3'. The amplified products were analyzed
on 1% agarose gels with ethidium bromide and visualized by a UV transilluminator.
Immunofluorescence--
Cells grown in 60 mm dishes 24-48 h
post transfection were fixed in 4% paraformaldehyde for 15 min. The
cells were washed with phosphate-buffered saline (PBS), treated with
0.2% Triton X-100 for 10 min at room temperature, and then blocked
with 5% bovine serum albumin in PBS for 1 h. The cells were
incubated at 4 °C for 18 h with either of the following
antisera diluted in blocking buffer: monoclonal anti-Tat (1:1000),
anti-microtubule-associated protein (MAP-2, a neuronal marker,
1:400; Chemicon), and anti-GFAP (astrocyte marker, 1:400; Sigma). The
cells were washed 4 times in PBS followed by incubation with anti-mouse
antisera labeled with Alexa 488 or 568 (Molecular Probes) for 30 min at
room temperature. After 4 washes in PBS, specimens were visualized by
fluorescent confocal microscopy.
Transactivation Assay--
HIV-1 LTR-CAT-based transactivation
assays were performed using either HeLa-LTR-CAT or SVGA-LTR-CAT cells.
A stable SVGA-LTR-CAT cell line was established using the LTR-CAT
plasmid and selection with G418 for 3 weeks. The presence of LTR-CAT
was confirmed following a transient transfection with pcDNA-Tat86.
Alternatively, C6-Tat-GFP, C6-
Tat-GFP, SVGA-Tat, and SVGA13 were
transfected with LTR-CAT plasmid. CAT activity was monitored by
quantitative ELISA. The supernatants from these cells were collected at
24, 48, and 96 h, which was then added to LTR-CAT or LTR-GFP cells
by a complete media exchange. The supernatants were used fresh without
undergoing any freeze thaw cycles. For detecting transcellular Tat
activity, Tat-expressing cells were co-cultured with SVGA-LTR-GFP cells or a lymphocytic cell line D3R5 LTR-GFP (22) in equal ratios for 48-96
h in 1.5% fetal bovine serum containing medium with 50 µM chloroquine (to minimize the degradation of
endocytosed Tat). In select experiments polyclonal Tat antisera at
1:200 final concentration was included for neutralization of Tat
activity and observed by fluorescent microscopy.
mRNA Profiles of Apoptotic and Chemokine-related
Genes--
Total RNA was isolated at different time intervals using
Trizol reagent (Invitrogen) from the Tat- or pcDNA-transfected
primary astrocytes. 6-7 µg RNA was labeled using reverse
transcription step, and labeled cDNA was hybridized at 68 °C for
18 h with superarray nylon membranes for chemokine and apoptotic
gene profile (Super Array). The signals were quantified by a
phospho-imager. The signals were normalized to GAPDH, and ratios of
test and control were expressed as fold increase or decrease.
Protein Carbonyl and 4-Hydroxynonenol Assays--
SVGA-Tat and
SVGA-neo cells were treated with 10 µM 3-nitroproprionic
acid (3NP) for varying amounts of time at 37 °C. Cells were
collected in Locke's buffer and sonicated for 5 s. The level of
protein oxidation was determined by an oxidized protein detection kit
(OxyblotTM, ONCOR) (23). Samples were incubated for 20 min
with 12% SDS and 2,4-dinitrophenylhydrazine (DNPH) in 10%
trifluoroacetic acid, vortexing every 5 min, and then neutralized with
OxyblotTM Neutralization solution. 600 ng of protein was
blotted onto nitrocellulose paper by the slot blotting technique.
Membranes were incubated with blocking buffer (PBS with 3% bovine
serum albumin, 0.01% sodium azide, and 0.2% Tween 20) for 30 min at
room temperature, exposed to rabbit anti-DNPH protein antibody (1:150)
for 90 min, followed by anti-rabbit IgG coupled to alkaline phosphatase
(1:15,000) for 2 h at room temperature. Membranes were washed
after each step in PBS with 0.2% Tween 20 and developed with
SigmaFastTM chromogen. Blots were analyzed by
computer-assisted imaging software, Scion Imaging. Samples for HNE
detection were similarly analyzed by slot blotting technique except
that rabbit-anti-HNE antibody (Calbiochem; 1:4,000) was used as a
primary antibody.
Mitochondrial Trans-membrane Potential (
) Activity in
Neurons--
Mitochondrial function was monitored by a fluorescent dye
JC-1 to measure mitochondrial
as previously described (24). When loaded in the cells, JC-1 produces a green florescence at low
membrane potential and red at high membrane potential. At the end of
the experimental treatment, the cells were incubated with JC-1 (10 µM) at 37 °C for 30 min and then washed in Locke's solution. Fluorescence measurements were made with excitation at 485 nm
and emission at 527 and 590 nm. The levels of fluorescence at both
emission wavelengths were expressed as ratios of fluorescence measurements from which the percentage of the mean ratios compared with
the untreated samples was calculated and analyzed using analysis of variance.
Neurotoxicity Assay--
Stable cell lines expressing Tat as
SVGA-Tat, C6-Tat, C6-Tat-GFP, C6-
Tat-GFP, SVGA13, and control cell
lines SVGA-neo and C6-neo were maintained in Dulbecco's modified
Eagle's medium + 10% fetal bovine serum. These cells were placed in
the upper chambers of transwells and neurons in the bottom of the 6- or
24-well plate in medium containing 0.5-1.0% fetal bovine serum.
Tet-inducible cells (SVGA13) were induced 1 day prior to and up to 6 days after co-culture with 1 mg/ml doxycycline. In select experiments
heparan sulfate (1.3 µM), dextran sulfate (0.5 µM), or kynurenate (10 µM) were added to
the bottom chamber of the transwell. Cell death was monitored by trypan
blue exclusion assay as described previously (19). Five random fields
were photographed and neuronal counts were determined by an
investigator blinded to the experimental design. At least 200 cells were counted in each field. Each experiment was done in
triplicate, and three independent experiments were conducted. The
results are expressed as mean percentage of dead cells.
Implantation of Tat-expressing Cells in Rat Brain--
C6Tat-GFP
cells were sorted by a flow sorter. 7.5 × 104
C6-Tat-GFP or C6-neo cells were unilaterally injected into the
molecular layer of the dentate gyrus of 12-week-old Sprague-Dawley rats using the following stereotactic coordinates: 3.8 mm anterior and 1.5 mm lateral to Bregma, and 3.2 mm dorsal to the dural surface. Animals
were monitored for 10 days post-surgery and then euthanized under deep
anesthesia by perfusion with normal saline followed by 4%
paraformaldehyde. The brains were removed, post-fixed for 16 h in
4% paraformaldehyde, and then cryopreserved in 30% sucrose for
72 h. 30 µm sections were prepared from the hippocampus.
Sections at the needle tract and 300 µm posterior to the injection
site were processed for Nissl stain (25). Sections at the injection site were also immunostained for Tat using a monoclonal Tat antibody.
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RESULTS |
Intracellular Expression of Tat in Astrocytes--
To determine
whether astrocytes can support the expression of Tat protein and to
explore the subcellular localization of Tat, we transfected primary
human astrocytes or SVGA cells with full-length HIV-1 infectious
molecular DNA clone pNL4-3. Tat expression was demonstrated by
immunostaining (Fig. 2A,
a and b) and was further confirmed by LTR
transactivation in pNL4-3-transfected SVGA-LTR-GFP cells (Fig.
2A, d). No immunostaining for Tat or LTR
transactivation was noted in pcDNA-transfected cells (Fig.
2A, c and e). Pattern of Tat
expression was also monitored in primary human fetal astrocytes transfected with Tat-GFP plasmid (Fig. 2B). The expression
of Tat in astrocytes was confirmed by immunostaining for GFAP and Tat-GFP fluorescence (Fig. 2B, b). Tat expression
occurred within 24 h of transfection. Tat was expressed in the
cytoplasm and nucleus with a strong signal in the nucleolus as revealed
by green fluorescence (Fig. 2B, a, d,
and e). The nuclei were counter-stained red with propidium
iodide (c). Primary astrocytes transfected with
Tat-GFP revealed
Tat expression diffusely throughout the cell with no demarcation of nuclear boundary or no nucleolar pattern (Fig. 2B, f and g).

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Fig. 2.
A, Tat production by HIV-1 (pNL4-3)
proviral DNA transfection in astrocytes. Immunostaining for Tat after
48 h of transfection in primary astrocytes (a), SVGA
cells transfected with infectious pNL4-3 DNA (b), and in
pcDNA-transfected control SVGA cells (c). LTR-based GFP
expression in SVGA cells co-transfected with pNL4-3 and HIV-1 LTR-GFP
(d) or pcDNA and HIV LTR-GFP control (e).
B, expression of wild type and mutant Tat in astrocytes.
Cells were transfected with wild type Tat-GFP or Tat-GFP and
observed for GFP fluorescence and immunostaining for GFAP after 24 h of transfection. a, primary astrocytes reveal Tat-GFP
expression (green) predominantly in nucleus but also in
cytoplasm; b, same cells immunostained for GFAP
(red). c, propidium iodide staining for nuclei
(red) in SVGA Tat-GFP cells. d, the same cell as
in c, showing nucleolar Tat-GFP (green) and same
cell with dual red and green filter (e).
f, primary astrocytes express Tat-GFP diffusely
throughout the cell with no nuclear demarcation
(green); g, same cells as in f
stained for GFAP.
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Stable Transfection of C6 and SVGA Cells with
Tat--
C6-Tat-GFP and C6-
Tat-GFP clones were established
by G418 selection and multiple subcloning by fluorescence-activated
cell sorter. The pattern of Tat and
Tat expression in these stable cell lines was similar to that of transiently transfected astrocytes with the respective plasmids and did not change with prolonged culture.
The expression of Tat in stable cell lines was further confirmed by
detection of Tat mRNA by RT-PCR (Fig.
3A) or LTR-CAT transactivation
assay following transfection of these cells with the LTR-CAT plasmid
(Fig. 3B).
Tat-expressing cells showed basal level of
transactivation activity (Fig. 3B). Clones of C6-Tat, SVGA-Tat, and SVGA13 cells that showed high CAT activity were used for
further studies.

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Fig. 3.
A, Tat mRNA expression in
astrocytes. Each cell line was analyzed for Tat mRNA expression by
RT-PCR, and PCR products were shown by ethedium bromide-stained agarose
gel. Lane 1, C6-Tat; lane 2, SVGA-Tat; lane
3, SVGA-Tat13 in the absence of doxycycline; lane 4,
SVGA-Tat (tet) in the presence of doxycycline; lane 5, HeLa
Tat; lane 6, HeLa; lane 7, positive control
(Tat plasmid); lane 8, molecular weight markers.
B, transactivation of HIV-1 LTR-CAT in Tat-expressing
astrocytes. Tat-expressing astrocytes (SVGA (S) or C6 cells)
were transfected with HIV LTR-CAT plasmid and quantitated for CAT
activity by ELISA. Values represent mean optical density readings of
two experiments. Bars represent CAT activity in the
following cell types: S-Tat, S-Tat13 with doxycycline for
72 h, S-Tat13, S-Neo with , C6-Tat, C6-Tat-GFP, and
C6- Tat-GFP (mutant Tat).
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Functional Expression of Tat and Cell to Cell Transmission--
To
determine whether biologically active Tat can be released from
Tat-expressing astrocytes and taken up by non-expressing cells,
SVGA-Tat cells were overlaid with SVGA-LTR-GFP cells. GFP expression
occurred in 48-96 h of co-culture (Fig.
4A, a). No GFP
expression was noted when these cells were co-cultures with SVGA-neo
cells (Fig. 4A, b). Co-culture of C6-Tat or
SVGA-Tat with D3R5-LTR-GFP cells also revealed GFP fluorescence (Fig.
4A, c), whereas no effect was noted when
co-cultured with SVGA-neo or C6-neo cells (Fig. 4A,
d). SVGA cells when directly co-transfected with LTR-GFP and
Tat plasmids revealed higher levels of LTR-based GFP expression within
24 h of transfection (Fig. 4A, e and
f). Together, these observations demonstrate the LTR
transactivation in both astrocytic and lymphocytic cells was specific
for the Tat-expressing cells. To further confirm that the effect was
due to release of Tat from the Tat-expressing cells and uptake by the
LTR-containing cells we repeated the above experiments in the presence
of Tat antisera. The cell to cell Tat-mediated LTR transactivation
activity was partially inhibited by Tat antisera, which is strongly
suggestive of release of Tat extracellularly. The inability of the
antisera to block the LTR transactivation completely is most likely
related to poor access to Tat by the antisera in areas of cell to cell
contact. The transactivation signal was specific for Tat because no
effect was noted with preimmune rabbit sera or by co-incubation of the
LTR-GFP cells with SVGA-neo cells (Fig. 4B). Tat secretion
in a 48-h post transfection period from 8 × 105 cells
was estimated as 100 ng/ml by ELISA. However, culture supernatants containing Tat from the stable Tat-expressing cells collected at 24, 48, and 96 h post media change (Tat concentration less than 50 pg), did not reveal transactivation of LTR when incubated with the
LTR-CAT or LTR-GFP cells, suggesting that small amounts of Tat secreted
may not be sufficient for LTR transactivation. This is consistent with
previous observations where the minimum Tat concentration required for
LTR transactivation and HIV replication was determined to be > 100 ng/ml (26).

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Fig. 4.
Cell to cell migration of functionally active
Tat. A, LTR transactivation in astrocytic and
lymphocytic cells following co-culture with Tat-expressing cells.
C6-Tat or SVGA-Tat cells were co-cultured with D3R5-LTR-GFP or
SVGA-LTR-GFP cells. Green fluorescence indicates LTR transactivation.
a, SVGA LTR-GFP cells overlaid on a monolayer of SVGA-Tat
cells; b, control where SVGA LTR-GFP cells were overlaid on
SVGA-neo cells; c, lymphocytic D3R5-LTR-GFP cells were
incubated with C6-Tat cells; d, control where
D3R5-LTR-GFP cells were incubated with C6-neo cells; e,
SVGA-LTR-GFP cells transfected with Tat plasmid; f, SVGA
LTR-GFP cells transfected with pcDNA-3 plasmid. B,
effect of Tat antisera on cell to cell LTR-GFP activation assay. SVGA
cells transfected with 6.0 µg tat or neo plasmid were
overlaid with SVGA LTR-GFP cells followed by addition of Tat antisera
(aTat) or preimmune rabbit serum (PRS).
Florescent cells were counted 24 h later and calculated as a
percentage of the mean number of fluorescent cells in the wells without
aTat or PRS. aTat partially blocked Tat transactivation activity,
whereas PRS did not show blocking potential.
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Effect of Intracellularly Expressed Tat on Astrocyte mRNA
Profile for Apoptosis and Chemokine Genes--
Because Tat was stably
expressed in astrocytes, we determined the effect of Tat expression on
apoptotic genes as well as chemokine and cytokine gene expression
profiles. Interestingly, primary astrocytes transfected with Tat
plasmid for 24-36 showed that tumor
necrosis factor-
and DAXX mRNAs were elevated 2-4-fold (Table I), whereas others were unchanged. Surprisingly, we did not see any effect on monocyte chemoattractant protein-1 or
macrophage inflammatory protein-1
, which was previously shown to
increase in astrocytes after extracellular treatment with Tat protein
(27). Other mRNAs like, Caspase gene family, iNOS, tumor necrosis
factor-related apoptosis-inducing ligand, Bcl gene family, and
chemokine genes were unaffected. The results were also confirmed by
RT-PCR (not shown). Further, tumor necrosis factor-
and MCP-1 were
undetectable in the 48 h conditioned medium from C6-Tat and
SVGA-Tat cells by quantitative ELISA. To rule out the under-expression
of Tat, Tat expression was monitored in these cells by RT-PCR and
immunostaining. Further cells expressing the Tat-GFP construct were
used as a control. Nearly 95% of cells showed green fluorescence.
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Table I
mRNA profile of Tat transfected primary human fetal astrocytes
Caspase gene family, iNOS, TRAIL, Bcl gene family, and Chemokine genes
were unaffected.
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Protein Carbonyl and HNE Levels in Tat-transfected Cell
Lines--
To further explore the possible effects of Tat on
vulnerability of astrocytes to oxidative stress, we analyzed protein
and lipid peroxidation products after challenging the cells with
mitochondrial toxin 3NP. Oxidized endogenous products, protein
carbonyl, and HNE were measured as markers of oxidative stress
(16). Tat-expressing astrocytic cells showed decreased oxidation
products (HNE and protein carbonyl) when compared with the neo-cells
(Fig. 5). These effects were specific for
astrocytes as HeLa cells expressing Tat did not have a similar
protective effect (Fig. 5).

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Fig. 5.
Tat production protects against oxidative
stress in astrocytes. Protein carbonyl and HNE products were
measured in each cell line with or with out treatment with 3NP.
Top left, protein carbonyl levels were increased in
HeLa cell lines following 3NP treatment. No significant difference was
present between the HeLa-neo (HeLa) and HeLa-Tat cells.
Top right, 3NP treatment failed to induce any significant
change in HNE products in the HeLa cell lines. Bottom left,
Tat-expressing SVGA cells showed significantly lower production of
protein carbonyl following 3NP treatment. Bottom
right, Tat-expressing SVGA cells showed significantly lower
production of HNE products following 3NP treatment. Data represents
mean ± S.E. from 3 experiments. *, p < 0.05.
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Dissipation of Neuronal Mitochondrial
by
Astrocyte-expressed Tat--
To determine the effect of Tat-expressing
cells on neurons, stable Tat-producing or neo-C6 cells were co-cultured
with primary neurons using transwells and analyzed at various time
points. The mitochondrial
of neurons was unaffected within the
first 48 h but after 72 h membrane potential decreased
significantly in neurons co-cultured with C6-Tat cells as compared with
C6-neo cells or C6-
Tat cells (Fig. 6).
These observations were confirmed by the use of doxycycline-regulated
expression of Tat in SVGA cells (Fig. 6). Doxycycline takes
~72 h for induction of Tat. Hence, in keeping with the above
observation, we noted a significant decrease in mitochondrial
at about 7 days after adding doxycycline to the media. No significant
toxicity was noted in similar cultures where doxycycline was not added
or when doxycycline was added to the neuronal cultures similarly
incubated with SVGA neo cells where the Tat gene was not present (Fig.
6).

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Fig. 6.
Effect of Tat-expressing astrocytes on
neurons. Neuronal cultures were exposed to Tat-expressing
astrocytes in transwells. Left panel, mitochondrial in neurons after co-culture with C6-Tat cells in transwells.
Significant decrease in mitochondrial was noted following
incubation with C6-Tat cells. No significant change was noted with
C6-neo or C6-dTat cells. 3NP was used as a control.
Middle panel, mitochondrial in neurons after
co-culture with tet-inducible SVGA Tat cells in transwells. Significant
decrease in mitochondrial was observed in neurons following
induction of Tat in SVGA13 Tat cells by doxycycline. No significant
change in neuronal mitochondrial was noted following
incubation with SVGA-neo cells similarly treated with doxycycline or
SVGA13 cells without treatment with doxycycline. 3NP was used as
positive control. Right panel, effect of kynurenate, dextran
sulfate, and heparan sulfate on C6-Tat induced neuronal mitochondrial
effects. Kynurenate, dextran sulfate, and heparan sulfate blocked
decreases in mitochondrial in neurons induced by C6-Tat cells
in transwells. Heparan sulfate did not block staurosporine-induced
decrease in neuronal mitochondrial . In each figure, data
represents mean ± S.E. of 4 experiments. *p < 0.05.
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These observations were further confirmed by collecting supernatants
from stably Tat-producing cells after 48 h of culture and applying
it to primary neurons for different times. The C6-Tat conditioned media
decreased the neuronal mitochondrial membrane potential at 6 h
post treatment (not shown).
Attenuation of Astrocyte-expressed Tat Effects on Neuronal
Mitochondrial
by Kynurenate, Heparan Sulfate, or Dextran
Sulfate--
Previous studies (6, 19) have shown that Tat-induced
neurotoxicity is mediated via excitatory amino acid receptors and Tat
binds to heparan sulfate and dextran sulfate. Hence we incubated Tat-expressing astrocytes with neurons in transwells in the presence of
each of these compounds. All these compounds attenuated the neurotoxicity measured as change in mitochondrial
. In control cultures where staurosporine (mitochondrial toxin) was incubated in the
presence of heparan sulfate, no protective effect was noted (Fig.
6), thus indicating that extracellular Tat is responsible for
the neurotoxicity.
Astrocyte-expressed Tat Induces Neuronal Cell Death--
To
determine the effect of Tat-expressing astrocytes on neuronal survival,
we exposed neuronal cultures to C6-Tat cells in transwells. Significant
neuronal cell death occurred with C6-Tat cells (p < 0.05). However, C6-
Tat cells did not
produce significant neurotoxicity (Fig. 7), indicating that
amino acids 48-56 are essential for neurotoxicity. To confirm the
neurotoxic properties of Tat, SVGA-13 cells were cultured in transwells
with neurons. Doxycycline-treated SVGA-13 cells induced significant
neuronal cell death first noted at 4 days of induction
(p < 0.001) with maximal neurotoxicity at 7 days post
induction (p < 0.001) compared with controls where
doxycycline was omitted or with SVGA-neo cells similarly treated with
doxycycline (Fig. 7). Further, neuronal cell death could be
neutralized using polyclonal Tat antisera but not preimmune antisera
(Fig. 7), indicating that extracellular release of Tat is required for
neurotoxicity.

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Fig. 7.
Tat cells induced significant cell death in
neurons. Left panel, C6-Tat cells induced significant
cell death in neurons (p < 0.05). No significant
neuronal cell death was noted with C6-neo or C6- Tat cells.
Middle panel, doxycycline-induced Tat-expressing SVGA13
cells caused significant neuronal cell death. No significant cell death
was noted with SVGA-neo cells similarly treated with doxycycline or
with SVGA13 cells untreated with doxycycline. Right panel,
Tat antisera but not preimmune rabbit sera (prs)
significantly blocked neurotoxicity induced by C6-Tat cells in
transwells.
|
|
Neurite Trimming in Neurons by Tat--
Neurite fragmentation is a
common phenomenon before the neurons collapse in response to toxic
substances, and loss of dendrites is a prominent histopathological
feature of patients with HIV encephalitis (28). Hence we monitored
morphological changes in neurons following culture with
Tat-expressing astrocytes. Direct co-culture of C6-Tat GFP cells with primary human fetal neurons for
72 h induced prominent neurite trimming or loss as shown in Fig.
8, following MAP-2 staining (red florescence). Tat production was confirmed by direct visualization of GFP florescence.

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Fig. 8.
Neurite trimming by Tat-producing
astrocytes. C6-Tat or C6-neo cells were co-cultured with primary
fetal neurons and immunostained for MAP-2. a, neurons
co-cultured with C6-Tat cells showing neurite shortening or trimming;
b, neurons co-cultured with C6-neo cells showing long and
extended neurites.
|
|
Hippocampal Neurotoxicity Induced by Tat-producing C6 Cells in
Vivo--
To determine whether Tat-producing C6 cells could elicit
neurotoxicity in vivo, C6-Tat or -neo cells were
unilaterally injected into the rat dorsal hippocampus. Nissl-stained
sections from the injection site revealed that C6-Tat or C6-neo cells
produced local damage of the dentate granule cells, but only animals
with C6-Tat cells had extensive damage in the CA4/3 pyramidal cell
layers (Fig. 9a). 300-µm
posterior to the injection site, only minimal signs of neuronal injury
were observed in animals with C6-neo cells, whereas extensive damage to
the dentate gyrus and CA4/3 pyramidal cells was observed in animals
with C6-Tat cells (Fig. 9, b and c). Tat
immunoreactivity at the injection site was localized to the C6 cells
(Fig. 9d), indicating that these cells are able to survive
and produce Tat in vivo. Additionally, Tat immunoreactivity was present in the CA4/3 region in cells that morphologically resembled
both microglial cells and neurons (Fig. 9d), raising the
possibility that constitutively produced Tat from the C6 cells could be
secreted and taken up by adjacent cells in the brain.

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Fig. 9.
Effect of Tat-producing astrocytes on
hippocampus in vivo. Adult male rats were
stereotactically injected with C6-neo cells (left panel) or
C6Tat-GFP cells (right panel) into the dorsal hippocampus.
At 10 days, sections were processed for Nissl stain and Tat immune
reactivity. Nissl-stained images (panels a-c)
depict hippocampal neurons at the injection site or 300 µm posterior
to the injection site ( 300 µm) (magnification 4× or 20×).
Arrows indicate damaged, pyknotic CA3 pyramidal neurons
observed only in animals injected with C6Tat-GFP cells.
Immunoreactivity for Tat protein (Tat IHC, panel
d) was seen at the injection site and in the CA3 region. Images
are representative of results obtained from 4 separate
experiments.
|
|
 |
DISCUSSION |
To determine the effect of HIV-1 Tat on astroglia-neuronal
relationships, we used constitutively and inducible expressed Tat in
astrocytes in contrast to previous studies that used recombinant Tat
protein (29). We used two different astrocytic cell lines and two
approaches for intracellular Tat expression, i.e. direct expression through cytomegalovirus promoter and tet-inducible expression using a human GFAP promoter. Similar results were obtained with both cell lines and both approaches for Tat expression. Our experimental model took advantage of a unique observation that intracellularly expressed Tat was not toxic to astrocytes. In fact it
had increased resistance to oxidizing agents such as 3NP. We also noted
overexpression of DAXX mRNA. Activation of physiological levels of
DAXX has been associated with induction of apoptosis; however,
overexpression has an anti-apoptotic effect (30). This is in keeping
with observations that HIV infection of astrocytes is non-cytopathic
(31). We were thus able to produce stably Tat-expressing astrocytic
cell lines. We expressed Tat in astrocytes because Tat can be detected
in HIV-infected astrocytes in vivo (4) and in
vitro as shown in this study. Production of Tat was monitored by
mRNA analysis, immunostaining, HIV-LTR transactivation assays, and
ELISA. Further, Tat released from these cells was biologically active
because it transactivated HIV-LTR in lymphocytic and astrocytic cell
lines. These observations are consistent with a previous study (10)
where Tat was taken up by neurons and was able to transactivate the
HIV-LTR. A unique pattern of Tat expression was noted intracellularly
in astrocytes. Tat was highly concentrated in the nucleolus, followed
by the nucleus and cytoplasm. Deletion of the amino acids 48-56
resulted in diffuse staining throughout the cytoplasm and nucleus,
confirming that this region of Tat is required for its nuclear
localization (32-34). Previous studies (35-37) have shown that the
intact arginine-rich domain of Tat (amino acids 49-57) is required for
nuclear or nucleolar expression or localization. Despite the diffusion
of
Tat into the nucleus, it failed to transactivate HIV-LTR. In
astrocytic cells, LTR transactivation occurs by two mechanisms;
interaction of Tat with transactivation response element RNA of
LTR or via interaction of Tat with NF
B-binding sites in the LTR
(38). The arginine-rich domain is thus essential for both modes of LTR transactivation and as discussed below also essential for Tat-induced neurotoxicity. Hence this region may be an important target for development of therapeutic approaches. The significance of Tat localization to the nucleolus remains undetermined. Interestingly, we
were unable to detect an effect of intracellularly expressed Tat on
iNOS, tumor necrosis factor-µ, MCP-1, cytokines, and chemokines production in astrocytes using super array, which was further confirmed
by RT-PCR and ELISA for tumor necrosis factor-µ and MCP-1 (data not
shown). Recently, it was shown that transiently transfected Tat
plasmids induced iNOS and produced NO in an astrocytoma cell line
U373MG (39). However, our gene array and RT-PCR data on primary
astrocytes as well as the astrocyte cell line SVGA do not support these
observations. Similarly, a macrophage cell line stably expressing Tat
inhibited induction of iNOS by gamma interferon (40). In previous
studies (41), others and we have shown that extracellular Tat has
profound effects on cytokine and chemokine production by astrocytes.
Thus intra- and extracellular Tat may have opposing effects.
We present several lines of evidence to conclusively show that
extracellularly released Tat is neurotoxic. Supernatants from the
Tat-expressing cell lines produced neurotoxicity as manifested by
destablization of mitochondria, neurite trimming, and neuronal cell
death. The neurotoxicity could be blocked by kynurenate, heparan
sulfate, dextran sulfate, and antisera to Tat, clearly demonstrating
that the neurotoxicity is mediated directly via Tat. Kynurenate blocks
the effect of Tat on glutaminergic receptors on neurons (19), whereas
heparan sulfate and dextran sulfate bind directly to Tat (5, 44). This
confirms previous studies showing that recombinant Tat protein
was toxic to neurons (19). However, the concentration of Tat necessary
to cause the neurotoxicity was much lower in the current study,
suggesting that Tat produced under physiological conditions by
mammalian cells is a potent neurotoxin. Another study (24) showed that
antisera to Tat could block neurotoxicity produced by supernatants from
HIV-infected macrophages, confirming the relevance of these observations.
We noted cell to cell movement of Tat among astrocytes and from
astrocytes to lymphocytes using LTR-GFP reporter expression indicator
cells that were exposed to Tat-producing astrocytes. This function was
also dependent upon amino acids 48-56 because no effect was seen with
Tat. Interestingly, this required co-culture of Tat-expressing cells
with the indicator cell lines, whereas culture supernatants from the
stably Tat-expressing cells failed to induce LTR transactivation. This
was most likely due to lower concentrations of Tat in the supernatants.
However, Tat released from the same cell lines was able to cause
neurotoxicity, suggesting that the concentrations of Tat required for
causing neurotoxicity are lower than that needed for transcellular
transactivation of LTR. This may in part be explained by the fact that
Tat-induced neurotoxicity requires interaction of Tat with the neuronal
cell membrane (42), whereas Tat-induced transactivation of LTR requires Tat uptake by the cell (43). Another unique observation in
vivo was that Tat released from the astrocyte cell lines was taken up by granule cells in the dentate gyrus and transported anterogradely via mossy fibers to the CA4/3 region where it caused glial cell activation and neurotoxicity. Thus Tat can cause pathological changes
at sites distant to the site of production.
Our observations suggest the following sequence of events. During HIV
replication in astrocytes, Tat is produced as an early product. It
makes the astrocytes resistant to oxidative stress, thus making it an
ideal reservoir for long-term survival of the viral genome. Tat
localizes to the nucleolus, but small amounts are released
extracellularly. Tat may be taken up by resident cells in the brain or
monocytic cells trafficking through the brain. If these cells are
latently infected with the virus, it will cause transactivation of the
viral genome and viral replication. If taken up be neurons it may be
transported along neuronal pathways and cause neurotoxicity and glial
cell activation at distant sites. Small amounts of Tat are sufficient
to cause excitotoxic neuronal damage that includes mitochondrial
dysfunction, neurite trimming, and neuronal cell death in select
populations. Because mitochondria are concentrated in neurites, the
mitochondrial stress may be directly responsible for neurite trimming.
Thus, HIV-infected astrocytes likely play a critical role in the
neuropathogenesis of HIV infection. The Tat protein, and in particular
the arginine-rich region of Tat, deserves close attention as a
therapeutic target for HIV infection.
 |
ACKNOWLEDGEMENTS |
We thank Dr. G. Smith, for pGfalac1, Dr. M. Giacca for pGEX Tat, Dr. D. Dorosky for D3R5 cells, Dr. W. Atwood for
SVGA cells, and Dr. C. Pardo for confocal microscopy. We thank the
National Institutes of Health AIDS Research and Reference Reagent
Program for plasmids pNL4-3 and LTR-CAT and HeLa LTR-CAT cells. We
thank National Institute for Biological Standards and Control, Herts, UK, for monoclonal antibody to Tat. We thank C. Anderson, R. Reid, and P. Ray for technical assistance.
 |
FOOTNOTES |
*
This work was supported by grants from NINDS, National
Institutes of Health, National Institute on Drug Abuse, National
Institute on Aging, and National Center for Research Resources.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Neurology,
Johns Hopkins University, Pathology 509, 600 N. Wolfe St., Baltimore,
MD 21287. Tel.: 443-287-4657; Fax: 410-614-1008; E-mail: anath1@jhmi.edu.
Published, JBC Papers in Press, January 24, 2003, DOI 10.1074/jbc.M209381200
 |
ABBREVIATIONS |
The abbreviations used are:
HIV, human
immunodeficiency virus;
LTR, long terminal repeat;
HNE, 4-hydroxynonenal;
GFP, green fluorescent protein;
tet, tetracycline;
rtta, reverse tetracycline transactivator;
ELISA, enzyme-linked
immunosorbent assay;
RT, reverse transcription;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PBS, phosphate-buffered
saline;
3NP, 3-nitroproprionic acid;
GFAP, glial fibrillary acid
protein;
CAT, chloramphenicol acetyl transferase;
iNOS, induced nitric
oxide synthetase;
MAP-2, microtubule associated protein-2.
 |
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