 |
 Previous Article | Next Article 
The Journal of Neuroscience, January 1, 1999, 19(1):64-71
Mechanisms and Structural Determinants of HIV-1 Coat Protein,
gp41-Induced Neurotoxicity
D. Cory
Adamson1, 2,
Kathy L.
Kopnisky1,
Ted M.
Dawson1, 2, and
Valina L.
Dawson1, 2, 3
Departments of 1 Neurology, 2 Neuroscience,
and 3 Physiology, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21287
 |
ABSTRACT |
Of the individuals with human immunodeficiency virus type 1 (HIV-1)
infection, 20-30% will develop the neurological complication of
HIV-associated dementia (HAD). The mechanisms underlying HAD are
unknown; however, indirect immunologically mediated mechanisms are
theorized to play a role. Recently, the HIV-1 coat protein gp41 has
been implicated as a major mediator of HAD through induction of
neurocytokines and subsequent neuronal cell death. Using primary mixed
cortical cultures from neuronal nitric oxide synthase (NOS) null
(nNOS /) mice and immunological NOS null
(iNOS/) mice, we establish iNOS-derived NO as a
major mediator of gp41 neurotoxicity. Neurotoxicity elicited by gp41 is
markedly attenuated in iNOS/ cultures compared
with wild-type and nNOS/ cultures. The NOS
inhibitor L-nitroarginine methyl ester is neuroprotective in wild-type and nNOS/ cultures, confirming the
role of iNOS-derived NO in gp41 neurotoxicity. Confirming that
iNOS/ cultures lack iNOS, gp41 did not induce
iNOS in iNOS/ cultures, but it markedly induced
iNOS in wild-type and nNOS/ cultures. We
elucidate the region of gp41 that is critical for iNOS induction and
neuronal cell death by monitoring iNOS induction with overlapping
peptides spanning gp41. We show that the N-terminal region of gp41,
which we designate as the neurotoxic domain, induces iNOS protein
activity and iNOS-dependent neurotoxicity at picomolar concentrations
in a manner similar to recombinant gp41 protein. Our experiments
suggest that gp41 is eliciting the induction of iNOS through potential
cell surface receptors or binding sites because the induction of iNOS
is dose dependent and saturable and occurs at physiologically relevant
concentrations. These data confirm that the induction of iNOS by gp41
and the production of NO are primary mediators of neuronal damage and
identify a neurotoxic domain of gp41 that may play an important role in HAD.
Key words:
HIV-1; HIV-associated dementia; neurotoxicity; gp41; immunological nitric oxide synthase; nitric oxide
| |
INTRODUCTION |
The most common cause of
neurological disease in young adults in the United States today is
human immunodeficiency virus type 1 (HIV-1) infection (Janssen et al.,
1992 ). Among HIV-1-infected children and adults, 20-30% will develop
HIV-associated dementia (HAD) during the course of their illness (Navia
et al., 1986; Price et al., 1988). The mechanisms by which HIV-1 causes
HAD are not known. However, indirect mechanisms are most likely to be
involved in the pathogenesis of HAD (Wesselingh et al., 1993). There is
increasing evidence that HIV-1 infection in the brain is associated
with significant neuronal loss as well as loss and complexity of
dendritic arborization, loss of synaptic densities, and vacuolization
of dendritic spines (Everall et al., 1991; Masliah et al., 1992; Wiley
et al., 1992). Although neuronal damage occurs in HAD, it is not
attributable to direct infection by HIV-1. Localization of HIV-1 in the
CNS is almost exclusively in blood-derived macrophages, microglia, and
multinucleated giant cells (Wiley et al., 1986; Rosenblum, 1990;
Watkins et al., 1990). A paradox seems to exist between the small
number of productively HIV-1-infected cells and the resulting HAD and
pathological brain deficits. Recent studies indicate that
HIV-1-infected cells in the CNS are making pro-inflammatory cytokines
that induce a local immune or cytokine reaction, ultimately leading to
neuronal dysfunction and neuronal cell death (Merrill and Chen, 1991).
Colocalization studies show expression of mRNA for pro-inflammatory
cytokines such as TNF- and immunological nitric oxide synthase
(iNOS) and for macrophage inflammatory protein-1 (MIP-1) and
MIP-1 in uninfected cells that are spatially localized near
HIV-1-infected cells (Nuovo and Alfieri, 1996; Seilhean et al.,
1997).
Various viral proteins have been implicated as mediators of
neurodegeneration in HAD (Sabatier et al., 1991; Werner et al., 1991;
Dawson et al., 1993). A particularly attractive candidate protein is
the HIV-1 coat protein gp160. gp160 is cleaved by intracellular proteases into gp120 and gp41 (Willey et al., 1988; Haseltine, 1989; Earl et al., 1991), which remain noncovalently associated. gp120
is soluble and can be shed from infected cells, and it is thought to be
quickly degraded by extracellular proteases. gp41 is an integral
membrane protein that remains inserted in the membrane of infected
cells (Willey et al., 1988, 1996; Bird et al., 1990). Early
studies demonstrated the synergistic activity of gp120 with glutamate
that results in neurotoxicity mediated by stimulation of NMDA receptor
(Brenneman et al., 1988; Lipton et al., 1991) and subsequent activation
of neuronal nitric oxide synthase (nNOS) (Dawson et al., 1993).
Although gp120 may contribute to neurodegeneration in HAD, it is
unlikely to be the sole or primary insult in HAD because a number of
host and viral factors have been shown to modulate or contribute to
HAD. In addition, gp120 has not been detected in the brains of
individuals affected with HIV-1 (Hill et al., 1993; Toggas et al.,
1994; Adamson et al., 1996b). In contrast, gp41 is readily detected in
the brains of HIV-1-infected individuals (Kure et al., 1990a,b; Dickson
et al., 1993), and the levels of gp41 correlate with the severity and
progression of HAD in adults (Adamson et al., 1996b, 1998; Rostasy et
al., 1998) and children (Dickson et al., 1989). Recombinant gp41, in a
manner identical to vaccinia virus-expressed gp41 (Koka et al., 1995a,b), efficiently induces the expression of pro-inflammatory cytokines, including TNF-, interleukin 1 (IL-1),
platelet-activating factor (PAF), and iNOS, in both human and rodent
cultures (Merrill et al., 1992; Koka et al., 1995a,b), as well as IL-10
in human monocytes (Barcova et al., 1998). gp41 is active in the
mid-nanomolar range, consistent with the levels that are readily
detected in HIV-1-infected brain tissue (Glass et al., 1995).
We recently observed a correlation between gp41 protein levels, iNOS
expression. and severity and rate of progression of HAD (Adamson et
al., 1996b, 1998). We showed that gp41 induces a cascade of events that
is toxic to primary neuronal cultures through NO-dependent mechanisms.
In the present study we examined the detailed mechanisms and structural
determinants regulating NO mediation of gp41 neurotoxicity.
| |
MATERIALS AND METHODS |
Cell cultures. For the murine culture experiments,
primary cortical cultures were prepared from gestational day 16 fetal
wild-type, nNOS/, and
iNOS/ mice in a procedure modified from that
described previously (Dawson et al., 1996). The genetic background of
nNOS/ and iNOS/ mice
originated from crosses between 129/SVev and C57B6 parental strains.
Wild-type cultures were obtained from both 129/SVev and C57B6 mice. We
did not observe any difference in the susceptibility to gp41 toxicity
in either 129/SVev or C57B6 wild-type mice (data not shown). Thus, all
data shown for wild-type mice are a mixture of data from both 129/SVev
and C57B6 mice. Briefly, the cortex was dissected and the cells were
dissociated by trituration in modified Eagle's medium (MEM), 20%
horse serum, 25 mM glucose, and 2 mM
L-glutamine after a 30 min digestion in 0.027%
trypsin/saline solution (Life Technologies, Gaithersburg, MD). The
cells were plated on 15 mm multiwell plates coated with polyornithine.
Cells were maintained in MEM, 10% horse serum, 25 mM
glucose, and 2 mM L-glutamine in an 8%
CO2 humidified 37°C incubator. Cultures were treated on
day 5 with 100 nM recombinant HIV-1 gp41IIIB
(amino acids 1 through 241; Intracel, Cambridge, MA), examined, and
harvested over a 7 d period. Peptides were added in the presence
or absence of L-nitro-arginine (N-Arg),
L-nitroarginine methyl ester (L-NAME), and/or
L-arginine (L-Arg). This model was chosen because it
eliminates the use of mitotic inhibitors that may interfere with the
induction of cytokines and to allow the co-culture of neurons and glia
so that the neurons would not be receiving inappropriate signals from
mature glia that may interfere with the neuronal-glial signaling (Dawson et al., 1993; Samdani et al., 1997). Mixed
neuronal/glial rodent cultures were similarly obtained and prepared
from fetal rats at gestational day 16. After 7 d of maturation,
these cultures were exposed to one of each of the peptide fragments
(100 nM) spanning the entire extracellular domain of HIV-1
gp41MN (National Institute of Allergy and Infectious
Diseases AIDS Research and Reference Reagent Program, Rockville, MD).
On the basis of our immunoblot data, the highly active peptide 4 and
inactive peptide 12 were subsequently used to assess the dose-response
relationship and time course of iNOS induction. The specificity of
peptide 4 was further assessed via the use of a purified peptide of the same forward and backward sequence generated at the Johns Hopkins University Department of Biological Chemistry Peptide Synthesis Laboratory.
Immunoblot analysis. For immunoblot analysis of iNOS,
equivalent amounts of cell lysate prepared from the murine and
rodent culture cells were separated on a 10% SDS-PAGE gel (Bio-Rad,
Hercules, CA) in Tris-glycine buffer under reducing conditions. After
electrophoresis, proteins were electroblotted onto nitrocellulose and
incubated with anti-macNOS antibody (1:500; Transduction Laboratories,
Lexington, KY). Immunoblots were developed by enhanced
chemoluminescence (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
For iNOS protein-positive controls, rodent glial cultures stimulated
with 100 ng/ml lipopolysaccharide for 24 hr produced a robust
signal on immunoblot at 130 kDa.
Neurotoxicity assessment. The cultured murine and rat cells
were exposed to recombinant gp41 and gp41 peptides as described previously (Adamson et al., 1996b). Toxicity was assayed as described previously (Gonzalez-Zulueta et al., 1998). Briefly, total and dead
cells were counted by computer-assisted microscopic examination after
staining of all nuclei with 1 µg/ml Hoescht 33342 and staining of
dead cell nuclei with 7 µM propidium iodide. Glial nuclei
fluoresce at a different intensity than neuronal nuclei and can be
gated out. Percentage of cell death was determined as the ratio of live to dead cells as compared with the percentage of cell death in control
wells to account for cell death attributable to mechanical stimulation
of the cultures. At least two separate experiments using four separate
wells were performed with a minimum of 15,000-20,000 neurons counted
per data point. We consider this cell counting method to be the gold
standard in determining cell death. This method allows assessment of
the majority of the culture well, which eliminates potential observer
bias (Gonzalez-Zulueta et al., 1998).
NOS activity assay. After culture media was removed from the
cells, 100 µl of sample buffer (50 mM Tris-HCl, pH 7.4, 1 mM EGTA, and 1 mM EDTA) was added to each well.
Cells were homogenized in the wells, collected, and centrifuged at
15,000 × g at 4°C. The supernatant was then
incubated with excess [3H]arginine and 1 mM NADPH in the presence or absence of 5 mM
N-Arg and in the presence or absence of 1 µM
CaCl2 for 15 min at room temperature. The reaction was
stopped with 20 mM HEPES, pH 5.5, plus 2 mM
EDTA, and loaded into 5 inch chromatography columns (Evergreen
Scientific, Los Angeles, CA) containing an anion exchange resin.
Flow-through was collected and radioactivity was counted. Reactions
without CaCl2 detected only calcium-independent NOS isoform
(iNOS) activity, whereas reactions with CaCl2 allowed calcium-dependent activity (nNOS and endothelial NOS) to be assessed. Subtraction of the latter activity from the former revealed the true
amount of iNOS, calcium-independent activity. Reactions with N-Arg, a
competitive inhibitor of all NOS isoforms, revealed background radioactivity in the assay.
| |
RESULTS |
Neuronal cultures from iNOS/ animals are
resistant to gp41 neurotoxicity
In primary cerebral cortical cultures from fetal rats, 100 nM gp41 kills 50-60% of neurons after 7 d of
continuous treatment. gp41-induced cell death is markedly reduced by
NOS inhibitors (Adamson et al., 1996b). To evaluate the source of NO
generation causing gp41 neurotoxicity, we examined gp41-mediated cell
death in primary neuronal cultures from wild-type mice versus mice
lacking the gene for nNOS (nNOS/) and mice
lacking the gene for iNOS (iNOS/). We showed
previously that gp41 can induce iNOS in rat neuronal cultures. In
wild-type cortical cultures gp41 begins to induce iNOS at approximately
day 5 of continuous exposure and through day 7 of treatment (Fig.
1A). gp41 has no effect
on astrocyte proliferation in these mixed cultures (data not shown).
Although we did not determine the cell type in which iNOS is expressed, it is likely to be expressed in both astrocytes and microglia. Accompanying the increase in iNOS protein is gp41-mediated
neurotoxicity that is blocked by N-Arg, and its protective effect is
reversed by the NOS substrate L-Arg, as reported previously in rat
cultures (Adamson et al., 1996b). Typically 0-10% of neurons die in
control-treated cultures over the 7 d treatment period (data not
shown). In addition, the absolute number of neurons did not change
significantly in control-treated cultures. However, the non-neuronal
cells approximately double in both gp41 and control cultures
because of the lack of mitotic inhibitors (data not shown). gp41
induces the expression of iNOS in nNOS/ cultures
in a manner that is indistinguishable from that of wild-type animals
(Fig. 1B). Cortical cultures from
nNOS/ mice are not protected against
gp41-mediated neurotoxicity. As in wild-type mice cultures, N-Arg
blocks gp41 neurotoxicity, and this protection is reversed by excess
substrate L-Arg (Fig. 1B). In cultures from
iNOS/ mice, gp41 fails to induce the expression
of iNOS as assessed by Western blot analysis (Fig. 1C). The
50-60% cell death induced by gp41 in wild-type and
nNOS/ cultures is almost completely abolished in
iNOS/ cultures (Fig. 1C). Thus,
iNOS-derived NO is a major mediator and source of gp41-induced
neurotoxicity.
View larger version (31K):
[in this window]
[in a new window]
|
Figure 1.
Neuronal cortical cultures from
iNOS/ mice are resistant to gp41 neurotoxicity.
A, Wild-type mice cultures were exposed to 100 nM recombinant gp41IIIB in the presence or
absence of 500 µM L-NAME, a competitive NOS inhibitor, ± 5 mM L-Arg. Cell death was assayed by microscopic
examination with computer-assisted cell counting after staining of all
nuclei with 1 µg/ml Hoescht 33342 and staining of dead cell nuclei
with 7 µM propidium iodide (Gonzalez-Zulueta et al.,
1998). Percentage of cell death was assessed on days 3, 5, and 7 and
was normalized to percentage of cell death in untreated control
cultures. The presence of iNOS protein in gp41-treated (41) and
untreated control cultures was assessed on the same days via immunoblot
analysis. B, Neuronal NOS/
cultures were similarly treated and assessed for percentage of cell
death and the presence of iNOS protein on days 3, 5, and 7. C, iNOS/ cultures were similarly
treated and assessed for percentage of cell death and the presence of
iNOS protein on days 3, 5, and 7. For all conditions, at least two
separate experiments with four separate wells were performed, with a
minimum of 15,000-20,000 neurons counted per data point. Data are
means ± SEM for n  8 from at least two
experiments. For each set of culture data, significance was determined
by a 3 × 3 ANOVA repeated measures, with differences between
groups ascertained by Fisher's PLSD ANOVA post hoc
test. Wild-type (F = 43.05) and
nNOS/ (F = 12.03) culture
data: *p  0.0001 for gp41 (Day 3)
versus gp41 (Day 5), gp41 (Day 3) versus
gp41 (Day 7);  p 0.0001 for gp41 (Day 5) versus gp41 + L-NAME (Day
5), gp41 (Day 7) versus gp41 + L-NAME
(Day 7);  p 0.0001 for
gp41 + L-NAME (Day 5) versus gp41 + L-NAME + LArg
(Day 5), gp41 + L-NAME (Day 7)
versus gp41 + L-NAME + LArg (Day 7).
iNOS/ culture data: F = 0.34 (p = 0.85).
|
|
Epitope mapping of gp41 induction of iNOS
To identify the structural determinants and critical region of
gp41 that are potentially responsible for the induction of iNOS, we
examined the ability of a number of overlapping peptides spanning the
region of gp41 from the gp120/gp41 junction to the transmembrane domain
of gp41 (Fig. 2A). We
show that a number of peptides are capable of inducing iNOS (Fig.
2B), including peptides 2-4, 6-11, 13, and 15. Peptide 4 is the most potent inducer of iNOS. Peptide 1, which is part
of the fusion domain of gp41, fails to induce iNOS as well as peptides
5, 12, 14, and 16 (Fig. 2B). Because a number of
peptides spanning the entire extracellular domain of gp41 were found to
induce iNOS, we conducted dose-response relationships for all of the
active peptides in an attempt to identify the most active region of
gp41 capable of inducing iNOS (Table 1).
Peptides 2-4 were found to induce iNOS at a gp41 concentration of 1 pM, whereas all other peptides failed to induce iNOS at
this concentration. Based on this set of data we have designated amino acids 530-559, which are contained within peptides 2-4, as the neurotoxic domain of gp41 (Fig. 3).
View larger version (50K):
[in this window]
[in a new window]
|
Figure 2.
Epitope mapping of gp41 induction of iNOS reveals
a number of regions in the extracellular domain of gp41 capable of
inducing iNOS. A, A number of overlapping peptides
(1-16) spanning the region of gp41 from the gp120/gp41 junction
(slash seen to the left of peptide 1) to
the transmembrane domain (TMD) of gp41 were obtained.
B, Rodent mixed cortical cultures were exposed to 100 nM each peptide, harvested on day 7, and assessed for iNOS
protein via immunoblot analysis. Positive controls (PC)
were obtained from rodent glial cultures stimulated with 100 ng/ml
lipopolysaccharide.
|
|
View larger version (20K):
[in this window]
[in a new window]
|
Figure 3.
Epitope mapping of gp41 induction of iNOS reveals
a neurotoxic domain. Based on the dose-response relationships of
induction of iNOS protein, we have designated amino acids 530-559,
which are contained within peptides 2 (amino acids 534-524), 3 (amino
acids 543-533), and 4 (amino acids 559-539), as the neurotoxic domain
( ). This domain lies adjacent to the putative fusion peptide domain
of gp41.
|
|
Characterization of the neurotoxic domain of gp41
Because peptide 4 was found to be the most potent inducer of iNOS
within the neurotoxic domain of gp41, we elected to further study
peptide 4-mediated induction of iNOS and its relationship to
gp41-mediated neurotoxicity. We first examined dose-response relationships of peptide 4 induction of iNOS by Western blot analysis and NOS catalytic activity as assessed by
[3H]arginine to
[3H]citrulline conversion (Fig.
4). Peptide 4 maximally induces iNOS at
100 nM, as assessed by Western blot analysis, which
parallels the induction of calcium-independent NOS catalytic activity.
Peptide 4 fails to influence calcium-dependent NOS catalytic activity (data not shown). To control for possible nonspecific effects caused by
possible contaminants in the peptide synthesis, we also examined in
more detail peptide 12, an inactive peptide identified in our initial
screen that was synthesized and purified in a manner identical to
peptide 4. Peptide 12 fails to induce iNOS at a concentration ranging
from 100 pM to 100 nM as assessed by both
Western blot analysis for iNOS and calcium-independent NOS catalytic
activity (Fig. 4B). Peptide 12 also fails to
influence calcium-dependent NOS catalytic activity (data not shown). We
next evaluated the time course of induction of iNOS by peptide 4 (Fig.
4C). Peptide 4 begins to induce iNOS at day 3, with maximal
effects occurring at day 7 as assessed by Western blot analysis. There
is a slight delay in the induction of NOS catalytic activity because
NOS catalytic activity is not detected until day 5 of peptide 4 treatment. NOS catalytic activity is blocked by the NOS inhibitor
L-NAME, and this inhibition of NOS catalytic activity is reversed by
excess substrate L-Arg. The inactive peptide 12 fails to induce iNOS as
assessed by Western blot analysis and NOS catalytic activity at all
days examined (Fig. 4D). The dose-response and time
course relationships of iNOS induction by peptide 4 closely parallel those as described previously for full-length recombinant gp41 (Adamson
et al., 1996b). To further assess the specificity of the induction of
iNOS by peptide 4, and to control for possible nonspecific effects such
as contaminants in the synthesis and purification of the peptides, and
to control for amino acid composition and charge, we obtained peptide 4 from another peptide synthesis facility and also synthesized it in the
reverse direction. Peptide 4 from the alternative source potently
induces iNOS in a manner identical to its counterpart, and the reverse
peptide fails to induce iNOS (Fig. 5).
Thus, the induction of iNOS by peptide 4 does not seem to be
attributable to nonspecific effects.
View larger version (41K):
[in this window]
[in a new window]
|
Figure 4.
Active gp41 peptide 4 induces iNOS protein and
activity. A, Rodent mixed cortical cultures were treated
with 0.1, 1, 10, and 100 nM peptide 4 or peptide 12 (B) and assessed for NOS activity and protein on
day 7 of exposure. C, Similar cultures were treated with
100 nM peptide 4 or peptide 12 (D)
and assessed for NOS activity and protein each day for a 7 d
period. Untreated control cultures (C) were also
examined. Catalytic activity (cpm) assessments are
means ± SEM for n 3 experiments. For each
set of data, significance was determined by a one-way ANOVA with
differences between groups ascertained by Fisher's PLSD ANOVA
post hoc test. A, F = 9.00, *p 0.02. B,
F = 0.18, p = 0.95. C, F = 3.96, *p 0.02. D, F = 1.92, p = 0.13.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Figure 5.
Alternatively synthesized gp41 peptide 4 induces
iNOS protein. Rodent mixed cortical cultures were treated with 100 nM peptide 4 (F, forward sequence) from a
different peptide synthesis facility or a reverse peptide 4 (B, backward sequence), harvested on day 7 of exposure
and assessed for iNOS protein via immunoblot analysis. Untreated
control cultures (C) were also assessed. Positive
controls (PC) were obtained from rodent glial cultures
stimulated with 100 ng/ml lipopolysaccharide.
|
|
NOS inhibitors block neuronal cell death elicited by a gp41
neurotoxic domain peptide
To establish the physiological relevance of the induction of iNOS
by peptide 4 in neuronal cultures, we examined the ability of peptide 4 to cause neuronal killing in primary neuronal cultures. Peptide 4 begins to kill neurons at day 5 of treatment, which is consistent with
the induction of NOS catalytic activity at this same time point (Fig.
6). Peptide 4-mediated neurotoxicity occurs through the full course of treatment, with maximal effects observed on day 7. Neurotoxicity elicited by peptide 4 is blocked by
L-NAME, and the protective effect of L-NAME is reversed by L-Arg (Fig.
6A). The inactive peptide 12 fails to elicit cell death at day 1 through day 7 (Fig. 6B).
View larger version (29K):
[in this window]
[in a new window]
|
Figure 6.
NOS inhibitors block neuronal cell death elicited
by a gp41 neurotoxic domain peptide. A, Rodent mixed
cortical cultures were treated with 100 nM peptide 4 in the
presence or absence of 500 µM L-NAME, a competitive NOS
inhibitor, ± 5 mM L-Arg. Cell death was assayed by
microscopic examination with computer-assisted cell counting after
staining of all nuclei with 1 µg/ml Hoescht 33342 and staining of
dead cell nuclei with 7 µM propidium iodide
(Gonzalez-Zulueta et al., 1998). Percentage of cell death was assessed
on each day for a 7 d period and normalized to untreated control
cultures. B, Similar cultures were treated with 100 nM peptide 12 and similarly assayed for cell death over a
7 d period. For all conditions, at least three separate
experiments with four separate wells were performed, with a minimum of
15,000-20,000 neurons counted per data point. Data are means ± SEM for n 3 experiments. For each set of data,
significance was determined by ANOVA repeated measures, with
differences between groups ascertained by Fisher's PLSD ANOVA
post hoc test. A, F = 3.63, *p 0.01 for Peptide 4 (day
1) versus Peptide 4 (days 5, 6, 7); p 0.01 for Peptide 4 (day
5) versus Peptide 4 + L-NAME (day 5),
Peptide 4 (day 6) versus Peptide 4 + L-NAME (day
6), Peptide 4 (day 7)
versus Peptide 4 + L-NAME (day 7);
p 0.01 for Peptide 4 + L-NAME (day
5) versus Peptide 4 + L-NAME + L-Arg (day
5), Peptide 4 + L-NAME (day 6)
versus Peptide 4 + L-NAME + L-Arg (day 6),
Peptide 4 + L-NAME (day 7) versus Peptide 4 + L-NAME + L-Arg (day 7).
|
|
| |
DISCUSSION |
Our study using primary neuronal cultures from
iNOS/ and nNOS/ mice
clarifies considerably the role of NO in gp41-mediated neurotoxicity of
cortical neurons. Previous studies have implicated NO as a mediator of
gp41 neurotoxicity based on the neuroprotective properties of NOS
inhibitors (Adamson et al., 1996b). iNOS was believed to be the source
of NO because gp41 potently induces the expression of iNOS (Adamson et
al., 1996b). Despite these data indicating NO and iNOS in gp41
neurotoxicity, questions could be raised that these studies are based
on the use of drugs that may elicit nonspecific effects and that the
induction of iNOS is an epiphenomenon that is unrelated to the
toxicity of gp41. The use of mice lacking the gene for nNOS and
iNOS overcomes many of these problems. The pronounced attenuation of
gp41 neurotoxicity in iNOS/ cortical cultures
and the preservation of gp41 neurotoxicity in
nNOS/ cortical cultures establishes a major role
for iNOS and NO in gp41 neurotoxicity. Our epitope mapping of gp41
induction suggests that there may be a putative neurotoxic domain of
gp41 that leads to the induction of iNOS. Through dose-response
relationships we were able to identify a region of gp41 that is
extremely potent in inducing iNOS in rodent cultures. This region of
gp41 corresponds to amino acids 530-559.
We cannot exclude the possibility that the protection against gp41 in
iNOS/ cortical cultures is caused by
compensatory processes attributable to the gene-targeting strategy.
However, we believe that the absence of gp41 toxicity truly reflects
the absence of iNOS and NO formation and is not caused by a general
lack of response to neurotoxic agents because the
iNOS/ cortical cultures are susceptible to
glutamate-mediated excitotoxicity (our unpublished
observations). In addition, our results do not necessarily exclude the
role of other host factors, particularly cytokines such as TNF-,
although TNF- is likely to act upstream of iNOS in the death cascade.
Recently the crystal structure of gp41 has been determined (Chan et
al., 1997). gp41, a trimer of molecules, has an -helical coiled coil
structure with an extracellular amino terminus tip. The C-terminal
-helix packs antiparallel against the outside of the coiled coil
placing the amino and C termini near each other. Based on our epitope
mapping, the neurotoxic domain of gp41 is located near the amino
terminal fusion peptide at a site where the gp41 would be in close
contact with the cell surface. Our current understanding of HIV-1 entry
into target cells is that at least two cell surface molecules are
necessary (Zhang et al., 1996). HIV-1 viral strains use the CD4
receptor as the primary virus receptor through high-affinity
interactions with the gp120 viral envelope protein. However, CD4 alone
is not sufficient for viral entry. At least one additional surface
protein, a co-receptor, is required (Alkhatib et al., 1996; Deng et
al., 1996; Dragic et al., 1996). Numerous co-receptors of the chemokine
receptor family have been identified, and these various receptors
determine cell type-specific tropisms for HIV-1 infection. A model of
HIV-1 interactions with CD4 and chemokine co-receptors is depicted in Figure 7. The close proximity of the
putative neurotoxic domain of gp41 to the fusion peptide places it in
an ideal position for cell/cell interaction. Our experiments suggest
that gp41 is eliciting the induction of iNOS through potential cell
surface receptors or binding sites because the induction of iNOS by
gp41 and its neurotoxic domain peptides is dose dependent and saturable
and occurs at physiologically relevant concentrations. Consistent with
the notion that this putative neurotoxic domain is important for gp41
interactions with cell surface receptors is the observation that a
peptide from this region is able to completely block HIV-1-mediated fusion (Kliger et al., 1997).
View larger version (29K):
[in this window]
[in a new window]
|
Figure 7.
Schematic diagram of the relationship of HIV-1
coat proteins to extracellular receptors. The close proximity of the
putative neurotoxic domain of gp41 to the fusion peptide places it in
an ideal position for cell/cell interaction.
|
|
Recent studies have reported that the C-terminal fusion domain of gp41
can create pores and thus elicit nonspecific toxicity. Our observations
that the fusion peptide region of gp41 is inactive in inducing iNOS
excludes the pore-forming region of gp41 as mediating the induction of
iNOS and subsequent cell death. The induction of iNOS by recombinant
gp41 as well as neurotoxic domain peptides has been described in human
mixed glial cultures with a response identical to that observed in our
rodent cultures (Koka et al., 1995a,b). Furthermore, gp41 can induce
iNOS- and NO-dependent toxicity in human neural cell aggregate
cultures that develops over 7 d and peaks at day 14 of exposure
(L. Pulliam, personal communication). These findings suggest
that the mechanism by which gp41 stimulates iNOS and induces cell death
is similar in rodent and human tissue and that production of NO from
iNOS is toxic to human cells.
It is unlikely that glycosylation is required for biological activity
of gp41 because the four or five potential glycosylation sites on gp41
are located more than 50 amino acids away from the region we found to
be critical in eliciting NO-dependent neurotoxicity. Consistent with
this notion is the observation that HIV-1-mediated fusion does not
require glycosylation of gp41 (Perrin et al., 1998) and the previous
observations that recombinant gp41 behaves in a manner identical to
full-length glycosylated gp41 in inducing pro-inflammatory cytokines
and cell death (Koka et al., 1995a,b).
Recently, we observed a correlation between gp41 levels and severity
and rate of progression of HAD (Adamson et al., 1996b, 1998). Our
observations that gp41 can induce a cascade of events that is toxic to
neurons suggests that gp41 may play an important role in the
pathogenesis of HAD. Consistent with this notion is the observation
that the localization of neuroinflammatory cytokines is in close
proximity to gp41-expressing cells (Nuovo and Alfieri, 1996). In
addition, markers of apoptosis in postmortem studies of HAD and simian
immunodeficiency virus (SIV)-infected monkeys is in close proximity to
gp41 expression (Adamson et al., 1996a). The correlation of gp41 levels
and severity of HAD has been observed in HIV-1-infected children
(Dickson et al., 1989) and adults (Rostasy et al., 1998). In
SIV-infected macaques, neurological disease also correlates with gp41
expression (Zink et al., 1997).
The ability of NOS inhibitors and the importance of iNOS in gp41
neurotoxicity and the correlation of gp41 levels with severity and rate
of progression of HAD suggest that inhibitors of iNOS might have
therapeutic potential in the treatment of HIV-1-associated neurological
illness. The identification of a neurotoxic domain of gp41 may direct
future studies toward identification of cell surface receptors or
binding sites and identification of potential therapeutic agents that
may prevent this interaction and offer therapeutic benefits.
| |
FOOTNOTES |
Received July 22, 1998; revised Oct. 13, 1998; accepted Oct. 19, 1998.
This work was supported by United Public Health Service Grants T32 NS
07392 (to D.C.A. and K.L.K.) and NS 26643 (to V.L.D.). T.M.D. is an
Established Investigator of the American Heart Association and is
supported by the Paul Beeson Faculty Scholar Award in Aging Research.
We thank Dr. Allen Mandir for his suggestions regarding our statistical
analyses, and Brian Hoffman for his technical help with generating our
mice cultures.
Under an agreement between the Johns Hopkins University and Guilford
Pharmaceuticals, T.M.D. and V.L.D. are entitled to a share of sales
royalty received by the University from Guilford. T.M.D. and the
University also own Guilford stock, and the University stock is subject
to certain restrictions under University policy. The terms of this
arrangement are being managed by the University in accordance with its
conflict of interest policies.
Correspondence should be addressed to Dr. Valina L. Dawson, Department
of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe
Street, Carnegie 2-214, Baltimore, MD 21287.
| |
REFERENCES |
-
Adamson DC,
Dawson TM,
Zink MC,
Clements JE,
Dawson VL
(1996a)
Neurovirulent simian immunodeficiency virus infection induces neuronal, endothelial and glial apoptosis.
Mol Med
2:417-428[Web of Science][Medline].
-
Adamson DC,
Wildemann B,
Sasaki M,
Glass JD,
McArthur JC,
Christov VI,
Dawson TM,
Dawson VL
(1996b)
Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 coat protein, gp41.
Science
274:1917-1921[Abstract/Free Full Text].
-
Adamson DC, McArthur JC, Dawson TM, Dawson VL (1999) Course
of HIV-associated dementia: correlations with gp41, iNOS and
macrophage/microglial activation. Mol Med, in press.
-
Alkhatib G,
Combadiere C,
Broder CC,
Feng Y,
Kennedy PE,
Murphy PM,
Berger EA
(1996)
CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1.
Science
272:1955-1958[Abstract].
-
Barcova M,
Kacani L,
Speth C,
Dierich MP
(1998)
gp41 envelope protein of human immunodeficiency virus induces interleukin (IL)-10 in monocytes, but not in B, T, or NK cells, leading to reduced IL-2 and interferon-gamma production.
J Infect Dis
177:905-913[Web of Science][Medline].
-
Bird C,
Burke J,
Gleeson PA,
McCluskey J
(1990)
Expression of human immunodeficiency virus 1 (HIV-1) envelope gene products transcribed from a heterologous promoter. Kinetics of HIV-1 envelope processing in transfected cells.
J Biol Chem
265:19151-19157[Abstract/Free Full Text].
-
Brenneman DE,
Westbrook GL,
Fitzgerald SP,
Ennist DL,
Elkins KL,
Ruff MR,
Pert CB
(1988)
Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide.
Nature
335:639-642[Medline].
-
Chan DC,
Fass D,
Berger JM,
Kim PS
(1997)
Core structure of gp41 from the HIV envelope glycoprotein.
Cell
89:263-273[Web of Science][Medline].
-
Dawson VL,
Dawson TM,
Uhl GR,
Snyder SH
(1993)
Human immunodeficiency virus type 1 coat protein neurotoxicity mediated by nitric oxide in primary cortical cultures.
Proc Natl Acad Sci USA
90:3256-3259[Abstract/Free Full Text].
-
Dawson VL,
Kizushi VM,
Huang PL,
Snyder SH,
Dawson TM
(1996)
Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide synthase-deficient mice.
J Neurosci
16:2479-2487[Abstract/Free Full Text].
-
Deng H,
Liu R,
Ellmeier W,
Choe S,
Unutmaz D,
Burkhart M,
Di Marzio P,
Marmon S,
Sutton RE,
Hill CM,
Davis CB,
Peiper SC,
Schall TJ,
Littman DR,
Landau NR
(1996)
Identification of a major co-receptor for primary isolates of HIV-1.
Nature
381:661-666[Medline].
-
Dickson DW,
Belman AL,
Park YD,
Wiley C,
Horoupian DS,
Llena J,
Kure K,
Lyman WD,
Morecki R,
Mitsudo S
(1989)
Central nervous system pathology in pediatric AIDS: an autopsy study.
APMIS [Suppl]
8:40-57[Medline].
-
Dickson DW,
Lee SC,
Mattiace LA,
Yen SC,
Brosnan C
(1993)
Microglia and cytokines in neurologic disease, with special reference to AIDS and Alzheimer's disease.
Glia
7:75-83[Web of Science][Medline].
-
Dragic T,
Litwin V,
Allaway GP,
Martin SR,
Huang Y,
Nagashima KA,
Cayanan C,
Maddon PJ,
Koup RA,
Moore JP,
Paxton WA
(1996)
HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.
Nature
381:667-673[Medline].
-
Earl PL,
Moss B,
Doms RW
(1991)
Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein.
J Virol
65:2047-2055[Abstract/Free Full Text].
-
Everall IP,
Luthert PJ,
Lantos PL
(1991)
Neuronal loss in the frontal cortex in HIV infection.
Lancet
337:1119-1121[Web of Science][Medline].
-
Glass JD,
Fedor H,
Wesselingh SL,
McArthur JC
(1995)
Immunocytochemical quantitation of human immunodeficiency virus in the brain: correlations with dementia.
Ann Neurol
38:755-762[Web of Science][Medline].
-
Gonzalez-Zulueta M,
Ensz LM,
Mukhina G,
Lebovitz RM,
Zwacka RM,
Engelhardt JF,
Oberley LW,
Dawson VL,
Dawson TM
(1998)
Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity.
J Neurosci
18:2040-2055[Abstract/Free Full Text].
-
Haseltine WA
(1989)
Development of antiviral drugs for the treatment of AIDS: strategies and prospects.
J Acquir Immune Defic Syndr Hum Retrovirol
2:311-334.
-
Hill JM,
Mervis RF,
Avidor R,
Moody TW,
Brenneman DE
(1993)
HIV envelope protein-induced neuronal damage and retardation of behavioral development in rat neonates.
Brain Res
603:222-233[Web of Science][Medline].
-
Janssen RS,
Nwanyanwu OC,
Selik RM,
Stehr-Green JK
(1992)
Epidemiology of human immunodeficiency virus versus encephalopathy in the United States.
Neurology
42:1472-1476[Abstract/Free Full Text].
-
Kliger Y,
Aharoni A,
Rapaport D,
Jones P,
Blumenthal R,
Shai Y
(1997)
Fusion peptides derived from the HIV type 1 glycoprotein 41 associate within phospholipid membranes and inhibit cell-cell fusion. Structure-function study.
J Biol Chem
272:13496-13505[Abstract/Free Full Text].
-
Koka P,
He K,
Camerini D,
Tran T,
Yashar SS,
Merrill JE
(1995a)
The mapping of HIV-1 gp160 epitopes required for interleukin-1 and tumor necrosis factor alpha production in glial cells.
J Neuroimmunol
57:179-191[Web of Science][Medline].
-
Koka P,
He K,
Zack JA,
Kitchen S,
Peacock W,
Fried I,
Tran T,
Yashar SS,
Merrill JE
(1995b)
Human immunodeficiency virus type 1 envelope proteins induce interleukin 1, tumor necrosis factor alpha, and nitric oxide in glial cultures derived from fetal, neonatal, and adult human brain.
J Exp Med
182:941-951[Abstract/Free Full Text].
-
Kure K,
Lyman WD,
Weidenheim KM,
Dickson DW
(1990a)
Cellular localization of an HIV-1 antigen in subactute AIDS encephalitis using an improved double-labelling immunohistochemistry method.
Am J Pathol
136:1085-1092[Abstract].
-
Kure K,
Weidenheim KM,
Lyman WD,
Dickson DW
(1990b)
Morphology and distribution of HIV-1 gp41-positive microglia in subacute AIDS encephalitis. Pattern of involvement resembling a multisystem degeneration.
Acta Neuropathol
80:393-400[Medline].
-
Lipton SA,
Sucher NJ,
Kaiser PK,
Dreyer EB
(1991)
Synergistic effects of HIV coat protein and NMDA receptor-mediated neurotoxicity.
Neuron
7:111-118[Web of Science][Medline].
-
Masliah E,
Ge N,
Morey M,
DeTeresa R,
Terry RD,
Wiley CA
(1992)
Cortical dendritic pathology in human immunodeficiency virus encephalitis.
Lab Invest
66:285-291[Web of Science][Medline].
-
Merrill JE,
Chen ISY
(1991)
HIV-1, macrophages, glial cells, and cytokines in AIDS nervous system disease.
FASEB J
5:2391-2396[Abstract].
-
Merrill JE,
Koyanagi Y,
Zack J,
Thomas L,
Martin F,
Chen IS
(1992)
Induction of interleukin-1 and tumor necrosis factor alpha in brain cultures by human immunodeficiency virus type 1.
J Virol
66:2217-2225[Abstract/Free Full Text].
-
Navia BA,
Jordan BD,
Price RW
(1986)
The AIDS dementia complex: 1. Clinical features.
Ann Neurol
19:517-524[Web of Science][Medline].
-
Nuovo GJ,
Alfieri ML
(1996)
AIDS dementia is associated with massive, activated HIV-1 infection and concomitant expression of several cytokines.
Mol Med
2:358-366[Web of Science][Medline].
-
Perrin C,
Fenouillet E,
Jones IM
(1998)
Role of gp41 glycosylation sites in the biological activity of human immunodeficiency virus type 1 envelope glycoprotein.
Virology
242:338-345[Web of Science][Medline].
-
Price RW,
Brew B,
Sidtis J,
Rosenblum M,
Scheck AC,
Cleary P
(1988)
The brain in AIDS: central nervous system HIV-1 infection and AIDS dementia complex.
Science
239:586-592[Abstract/Free Full Text].
-
Rosenblum M
(1990)
Infection of the central nervous system by the human immunodeficiency virus type 1.
Pathobiol Annu
25:117-169.
-
Rostasy K,
Monti L,
Kneissl M,
Bell J,
Hedreen J,
Navia B
(1998)
Proinflammatory mediators and HIV infection in the basal ganglia: their pathogenetic relationship to the AIDS dementia complex.
Neuroscience of HIV Infection
1:30.
-
Sabatier J,
Vives E,
Mabrouk K,
Benjouad A,
Rochat H,
Duval A,
Hue B,
Bahraoui E
(1991)
Evidence for neurotoxic activity of tat from human immunodeficiency virus type 1.
J Virol
65:961-967[Abstract/Free Full Text].
-
Samdani AF,
Newcamp C,
Resink A,
Facchinetti F,
Hoffman BE,
Dawson VL,
Dawson TM
(1997)
Differential susceptibility to neurotoxicity mediated by neurotrophins and neuronal nitric oxide synthase.
J Neurosci
17:4633-4641[Abstract/Free Full Text].
-
Seilhean D,
Kobayashi K,
He Y,
Uchihara T,
Rosenblum O,
Katlama C,
Bricaire F,
Duyckaerts C,
Hauw JJ
(1997)
Tumor necrosis factor-alpha, microglia and astrocytes in AIDS dementia complex.
Acta Neuropathol (Berl)
93:508-517[Medline].
-
Toggas SM,
Masliah E,
Rockenstein EM,
Rall GF,
Abraham CR,
Mucke L
(1994)
Central nervous system damage produced by expression of the HIV-1 coat protein gp120 in transgenic mice.
Nature
367:188-193[Medline].
-
Watkins BA,
Dorn HH,
Kelly WB,
Armstrong RC,
Potts BJ,
Michaels F,
Kufta CV,
Dubois-Dalcq M
(1990)
Specific tropism of HIV-1 for microglial cells in primary human brain cultures.
Science
249:549-553[Abstract/Free Full Text].
-
Werner T,
Ferroni S,
Saermark T,
Brack-Werner R,
Banati RB,
Mager R,
Steinaa L,
Kreutzberg GW,
Erfle V
(1991)
HIV-1 nef protein exhibits structural and functional similarity to scorpion peptides interacting with K+ channels.
AIDS
5:1301-1308[Web of Science][Medline].
-
Wesselingh S,
Power C,
Glass J,
Tyor WR,
McArthur JC,
Farber JM,
Griffin JW,
Griffin DE
(1993)
Intracerebral cytokine mRNA expression in AIDS dementia.
Ann Neurol
33:576-582[Web of Science][Medline].
-
Wiley CA,
Schrier RD,
Nelson JA,
Lampert PW,
Oldstone MB
(1986)
Cellular localization of human immunodeficiency virus infection within the brains of acquired immune deficiency syndrome patients.
Proc Natl Acad Sci USA
83:7089-7093[Abstract/Free Full Text].
-
Wiley CA,
Masliah E,
Morey M,
Lemere C,
DeTeresa R,
Grafe M,
Hansen L,
Terry R
(1991)
Neocortical damage during HIV infection.
Ann Neurol
29:651-657[Web of Science][Medline].
-
Willey RL,
Bonifacino JS,
Potts BJ,
Martin MA,
Klausner RD
(1988)
Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160.
Proc Natl Acad Sci USA
85:9580-9584[Abstract/Free Full Text].
-
Willey RL,
Shibata R,
Freed EO,
Cho MW,
Martin MA
(1996)
Differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary T-lymphocyte and macrophage cultures.
J Virol
70:6431-6436[Abstract].
-
Zhang L,
Huang Y,
He T,
Cao Y,
Ho DD
(1996)
HIV-1 subtype and second-receptor use.
Nature
383:768[Medline].
-
Zink MC,
Amedee AM,
Mankowski JL,
Craig L,
Didier P,
Carter DL,
Munoz A,
Murphey-Cobb M,
Clements JE
(1997)
Pathogenesis of SIV encephalitis: selection and replication of neurovirulent SIV.
Am J Pathol
151:793-803[Abstract].
Copyright © 1999 Society for Neuroscience 0270-6474/99/19164-08$05.00/0
This article has been cited by other articles:
|
|
|
|
 
H. Girouard, G. Wang, E. F. Gallo, J. Anrather, P. Zhou, V. M. Pickel, and C. Iadecola
NMDA Receptor Activation Increases Free Radical Production through Nitric Oxide and NOX2
J. Neurosci.,
February 25, 2009;
29(8):
2545 - 2552.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Li, T. M. Malpica-Llanos, R. Gundry, R. J. Cotter, N. Sacktor, J. McArthur, and A. Nath
Nitrosative stress with HIV dementia causes decreased L-prostaglandin D synthase activity
Neurology,
May 6, 2008;
70(19_Part_2):
1753 - 1762.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. P. Heyes, R. J. Ellis, L. Ryan, M. E. Childers, I. Grant, T. Wolfson, S. Archibald, and T. L. Jernigan
Elevated cerebrospinal fluid quinolinic acid levels are associated with region-specific cerebral volume loss in HIV infection
Brain,
May 1, 2001;
124(5):
1033 - 1042.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Sarter and M. Podell
Preclinical psychopharmacology of AIDS-associated dementia: lessons to be learned from the cognitive psychopharmacology of other dementias
J Psychopharmacol,
May 1, 2000;
14(3):
197 - 204.
[Abstract]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
