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The Journal of Neuroscience, May 1, 2002, 22(9):3484-3492
Peroxynitrite Mediates Neurotoxicity of Amyloid
 -Peptide1-42- and Lipopolysaccharide-Activated
Microglia
Zhong
Xie1,
Min
Wei1,
Todd E.
Morgan1,
Paola
Fabrizio1,
Derick
Han2,
Caleb E.
Finch1, *, and
Valter D.
Longo1, *
1 Division of Biogerontology, Andrus Gerontology
Center, and Department of Biological Sciences, and
2 Department of Molecular Pharmacology and Toxicology,
School of Pharmacy, University of Southern California, Los Angeles,
California 90089
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ABSTRACT |
The amyloid  -peptide (A) activates microglia and promotes the
generation of cytokines and oxygen species, including nitric oxide (NO)
and tumor necrosis factor  (TNF-), which can be either neurotoxic
or neuroprotective. We show that neuron death in cocultures of rat
cortical microglia and neurons activated by lipopolysaccharide (LPS) or
A1-42 plus interferon  (IFN) is caused by
short-lived diffusible molecules and follows the generation of
superoxide and/or peroxynitrite as determined by electron
paramagnetic spectroscopy. Neurotoxicity induced by LPS or
A1-42 plus IFN is blocked by inhibitors of NO
synthesis and by the peroxynitrite (ONOO )
decomposition catalysts FeTMPyP
[5,10,15,20-tetrakis(n-methyl-4'-pyridyl)porphinato iron
(III) chloride] and FeTPPS
[5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III)
chloride] but not by the TNF- inhibitor pentoxifylline. The
specificity of FeTMPyP for ONOO was confirmed by
its ability to block the toxicity of a peroxynitrite donor but not of
NO donors or of high levels of superoxide in a yeast mutant lacking
superoxide dismutase 1. These results implicate peroxynitrite as a
mediator of the toxicity of activated microglia, which may play a major
role in A1-42 neurotoxicity and Alzheimer's disease.
Key words:
peroxynitrite; A; LPS; microglia; neurons; superoxide; nitric oxide; TNF-
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INTRODUCTION |
Microglial activation is implicated
in neurodegenerative disorders, including Alzheimer's disease (AD),
multiple sclerosis, Parkinson's disease, and stroke. AD, the most
common form of senile dementia, is accompanied by a progressive loss of
neurons and synapses in brain regions characterized by senile plaques
and neurofibrillary tangles. The major components of senile plaques are
the -amyloid (A) peptides, which in experimental models can
damage neurons directly or indirectly through the activation of
microglia (Yankner et al. 1990 ; Pike et al., 1991; Meda et al., 1995;
Combs et al., 1999; Klein et al., 2001).
Activated microglia are capable of releasing neurotoxic molecules, such
as proinflammatory cytokines and toxic oxygen and nitrogen species
(Colton and Gilbert, 1987; Klegeris and McGeer, 1994). Accumulating
evidence shows that activated microglia can damage or kill neurons
in vitro by generating nitric oxide (NO) (Boje and Arora,
1992; Chao et al., 1992; Goodwin et al., 1995; Meda et al., 1995),
tumor necrosis factor- (TNF-) (Wood, 1995), various toxic oxygen
species (Tanaka et al., 1994), L-cysteine (Yeh et
al., 2000), phenolic amine (Giulian et al., 1995), and tissue
plasminogen activator (Flavin et al., 2000). NO and superoxide react to
form the neurotoxic peroxynitrite (Estevez et al., 1998a,b; Koppal et
al., 1999), which has been implicated in AD, in part because the levels
of nitrotyrosine, a product of the reaction of peroxynitrite with
tyrosine, increase in AD (Smith et al., 1997). However, a role of
peroxynitrite in the toxicity of A-activated microglia has not been demonstrated.
Although NO can be neurotoxic, NO is also an important signaling
molecule that can protect PC12 cells and primary neurons against A
toxicity (Troy et al., 2000; Wirtz-Brugger and Giovanni, 2000).
Furthermore, the protective effect of inhibitors of NO synthase (NOS)
against A toxicity (Ii et al., 1996) may be attributable to the
inhibition of neuronal instead of microglial inducible NOS (iNOS)
(Combs et al., 2001). Therefore, the mechanisms of A and microglial
neurotoxicity remain unclear.
Here we identify the mediator of A and lipopolysaccharide (LPS)
neurotoxicity by measuring the generation of toxic oxygen and nitrogen
species by microglia and by studying the role of inhibitors and
decomposition catalysts of specific molecules released by activated
microglia in preventing neuron death. Neurotoxicity is studied in a
cocultures system in which microglia and neurons can be separated
before cell death analysis.
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MATERIALS AND METHODS |
Reagents. LPS (Escherichia coli strain
O26:B6), superoxide dismutase (SOD), catalase, sodium nitroprusside
(SNP), and fluorescein diacetate are from Sigma (St. Louis, MO).
Recombinant mouse interferon (IFN) is from R & D Systems
(Minneapolis, MN).
NG-Monomethyl-L-arginine
(L-NMMA), 3-morpholinosydnonimine (SIN-1), 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride
(FeTPPS), 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphinato iron (III) chloride (FeTMPyP),
Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP),
and NOC-18 (a nitric oxide donor; also known as DETA-NONOate)
are from Calbiochem (San Diego, CA); thalidomide and pentoxifylline are
from Research Biochemicals (Natick, MA). All reagents were tested to be
not neurotoxic at the concentrations applied in neuron cultures.
A1-42 (US Peptide, Fullerton, CA) was
dissolved in DMSO (Sigma) to obtain a 5 mM stock and kept
at  70°C. Before treating the cultures, a 50 µM
A1-42 solution was prepared in F12K medium as
10× solution and incubated at 37°C for 1 d to obtain aggregated
A. For all treatments with A1-42, 10 ng/ml IFN was also added as a priming factor (Meda et al., 1995; Ii et
al., 1996).
Cell culture. Rat primary glial cells were derived from
cerebral cortices of neonatal (postnatal day 3) Fisher 344 rat
(Giulian and Baker, 1986). Dispersed cells were grown in
DMEM-F12 (Cellgro; Mediatech, Herndon, VA) supplemented with
10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan,
UT), 50 U/ml penicillin (Sigma), and 0.05 mg/ml streptomycin (Sigma),
at 37°C in a humidified 95%-5% (v/v) mixture of air and
CO2. Culture media were renewed twice per week.
After 14-21 d in culture, microglia were detached from monolayer by
gentle shaking and replated into cell culture inserts (Costar,
Cambridge, MA; Corning, Corning, NY) or 96-well (3 × 104 cells per well) cell culture plates
(Falcon; Becton Dickinson, Franklin Lakes, NJ). The microglia
homogeneity achieved by this procedure was >98%, as determined by
immunocytochemistry for microglial marker complement receptor type 3 (CR3) using mouse anti-rat CR3 antibody OX42 (dilution 1:50; Serotec,
Raleigh, NC) (Morgan et al., 1995).
Neuron cultures were derived from fetal (embryonic day 17) Fisher 344 rat cerebral cortices as detailed previously (Banker and Goslin,
1988; Rozovsky et al., 1994) and plated at 5 × 104 viable cells per well in
poly-D-lysine (Sigma) -coated 24-well plates (Costar).
Culture media were renewed after 1 hr and not changed until the time of
experiment at 6-7 d in culture. Microglia were harvested from
mixed-glia cultures, plated in 9 mm cell culture inserts (membrane pore
size 0.4 µm; Costar) at 105 cells per
insert, and placed into the culture wells containing neurons. The
porous membrane allows free diffusion of molecules. The distance
between neuron layer on the culture plate and microglia layer on the
insert membrane is 1 mm, according to the description of the
manufacturer. Treatment started 3-4 hr afterward. Neuron-microglia cocultures were maintained in glial medium as described above.
Neuron viability assay. After treatment, culture inserts
containing microglia were removed, and neurons were stained with 10 µg/ml fluorescein diacetate (FDA) (Sigma) for 10 min. FDA is membrane
permeable and freely enters intact cells, in which it is hydrolyzed by
cytosolic esterase and converted to membrane-impermeable fluorescein
with a green fluorescence, exhibited only by live cells. Because neuron
deaths occur primarily in the region directly underneath the
microglia-containing culture inserts (see Fig. 6D),
for quantification, eight images at the center of each well were taken
with a Nikon (Tokyo, Japan) TE300 fluorescence microscope and analyzed
with the IP Lab imaging software (version 3.54; Scanalytics, Fairfax,
VA). Viable neurons were quantified by the area covered by green
fluorescence after the establishment of a linear relationship between
the numbers of stained cells and the green fluorescent area. The total
area analyzed occupied 30% of the area in which neuron death occurred.
Nitrite measurement. NO production was determined indirectly
through the assay of nitrite
(NO2), a stable
metabolite of NO, based on the Griess reaction (Huygen, 1970;
Green et al., 1982; Ding et al., 1988). Briefly, a 50 µl aliquot of conditioned media was mixed with an equal volume of Griess
reagent [0.1% N-(1-naphthyl)ethylenediamine
dihydrochloride, 1% sufanilamide, and 2.5% phosphoric acid (all from
Sigma)] and incubated for 10 min at 22°C, and the absorbance was
read at 550 nm on a microtiter plate reader (Spectra MAX 250; Molecular
Devices, Sunnyvale, CA). Nitrite concentrations were calculated from a standard curve of NaNO2 (Sigma) ranging from 0 to
100 µM. Background NO2 was subtracted from
the experimental values.
Detection of superoxide-peroxynitrite by electron paramagnetic
resonance. For electron paramagnetic resonance (EPR) measurements, microglia cells (100,000) with or without LPS treatment were incubated in 200 µl of culture medium containing 120 mM
5,5-dimethyl-1-pyrroline-N-oxide (DMPO). After 15 min, media
were removed and analyzed by EPR. EPR spectra were recorded on a Bruker
ECS106 spectrometer with the following settings: receiver gain, 5 × 105; microwave power, 20 mW; microwave
frequency, 9.81 GHz; modulation amplitude, 1 G; time constant, 1.3 sec;
scan time, 87 sec; and scan width, 80 G. The DMPO-OH signal generated
from activated microglia was quantified by comparison with
4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy standard after
double integration of both signals. All scans shown are an accumulation
of seven scans.
TNF- ELISA. Levels of secreted TNF- in culture
supernatants were determined by an ELISA kit following the
instructions of the manufacturer (BioSource, Camarillo, CA).
Yeast sod mutants and growth assay. EG118
(sod1 ) (DBY746 wild type with
sod1:: URA3) yeast were grown in liquid media
in synthetic complete media (SDC) with 2% glucose, supplemented
with amino acids, adenine, and uracil, as well as a fourfold excess of
the supplements tryptophan, leucine, histidine, lysine, and methionine
(Longo et al., 1996). Overnight cultures were grown in selective
media and inoculated with a flask volume/medium volume ratio of 5:1 at
30°C with shaking at 220 rpm. After overnight cultures in SDC medium,
sod1 cells were diluted to an optical density at 600 nm
(OD600) of 0.1 and inoculated in 3 ml of
yeast complete media containing 2% ethanol (YPE) (2% ethanol)
containing MnTMPyP (25 µM) or FeTMPyP (25 µM) with or without paraquat (10 µM). Cell density was determined after 48 hr by OD.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP
nick end labeling staining. DNA cleavage was detected
with the In Situ Cell Death Detection kit as described by
the manufacturer (Roche Molecular Biochemicals, Indianapolis, IN).
Briefly, cells were fixed with paraformaldehyde (4% in PBS, pH7.4) and
permeabilized (0.1% Triton X-100 in 0.1% sodium citrate). After 1 hr
at 37°C incubation in terminal deoxynucleotidyl transferase reaction
mixture, signals were visualized under fluorescence microscope
(excitation/emission wavelengths, 450-500 nm/515-565 nm). Samples
were further blotted with alkaline phosphatase-conjugated
anti-fluorescein antibody. After color reaction, samples were analyzed
under light microscope.
Statistical analysis. Data were analyzed by one-way ANOVA,
followed by post hoc tests of Newman-Keuls multiple
comparison to determine whether there were significant differences
between individual groups. Statistical significance was established
when p < 0.05.
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RESULTS |
LPS-dependent neuronal death follows the generation of
superoxide and/or peroxynitrite and NO-nitrite by microglia
Microglia, the resident macrophages of the CNS, during activation
can produce large quantities of nitric oxide synthesized by iNOS.
Activated microglia also produce abundant superoxide through the
membrane-associated NADPH oxidase. In microglia- neuron cocultures, LPS induced dose-dependent nitrite generation and neuronal
death after a 48 hr treatment with 0.2-100 ng/ml LPS (Fig.
1A). LPS-activated
microglia caused the death of 18 ± 2% of neurons by 24 hr, 50 ± 3% at 36 hr, and 78 ± 3% at 48 hr. Treatment of
neurons with LPS in the absence of microglia does not cause toxicity or
nitrite generation (data not shown). Cell death was observed only in
neurons cultured directly under the filter insert (membrane pore size
0.4 µm; Costar) containing the microglia (1 mm distance), suggesting
that neurotoxicity is mediated by short-lived diffusible molecules (see
Fig. 6D). To determine whether neurotoxicity correlated with NO generation, we first investigated the time course of
nitric oxide and superoxide generation. NO generation was estimated by
measuring the concentration of nitrite, its stable metabolite, released
into the medium (Ding et al., 1988). LPS at 100 ng/ml induced nitrite
generation beginning at 6 hr (reported as micromolar generated per
hour), with a peak at ~14 hr and a gradual decline until 48 hr
(Fig. 1B).
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Figure 1.
LPS-activated microglia kill neurons in a
dose-dependent manner. A, Neuron survival and nitrite
accumulation after a 48 hr treatment of rat microglia-neuron
cocultures with 0.2-100 ng/ml LPS. Viable neurons were detected by
fluorescein diacetate staining. Data represent mean ± SEM from a
representative experiment. The experiment was repeated three times with
similar results. B, Time course of nitrite and
superoxide-peroxynitrite (DMPO-OH) generation determined by Griess
reaction and EPR, respectively. Repeated three times with similar
results. A representative experiment is shown. C,
Detection of superoxide-peroxynitrite generation by microglia cells by
EPR spectra of DMPO-OH. Arrows indicate the quartet
generated by DMPO-OH. Microglia cells (100,000) with or without LPS
treatment (24 hr) were incubated with 120 mM DMPO with or
without 186 U/ml SOD for 15 min and analyzed by EPR. The figures shown
are an accumulation of seven scans.
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The generation of superoxide-peroxynitrite by activated microglia
cells was monitored by EPR with the spin trap DMPO. A DMPO-OH signal (a
quartet signal with signal intensity ratios of 1:2:2:1; aN = aH = 14.9) was observed in the
medium of activated microglia cells but not in control cells (Fig.
1C). The coincubation with exogenous SOD resulted in a
complete loss of the DMPO-OH signal in LPS-treated microglia cells
(Fig. 1C), indicating the EPR signal was caused by
superoxide, peroxynitrite, or both but not by hydroxyl radical (although hydroxyl radical may be generated from peroxynitrite). Similar to NO, the superoxide and/or peroxynitrite generated by microglia cells peaked at 12 hr (Fig.
1B,C).
It is difficult to distinguish between the EPR signal of
superoxide and that of peroxynitrite because both species generate the
DMPO-OH either through DMPO-OOH decomposition (superoxide) or by the
peroxynitrite-dependent generation of hydroxyl radical, which
subsequently reacts with DMPO (Augusto et al., 1994). Furthermore, both
superoxide- and peroxynitrite-dependent quartet EPR signal can be
prevented by SOD. We incubated LPS-activated microglia in the presence
of both DMPO and the hydroxyl radical scavenger DMSO (200 mM). Whereas the quartet signal caused by superoxide would
not be affected by DMSO, peroxynitrite is expected to generate a
quartet, as well as a sextet, signal in the presence of hydroxyl radical scavengers. However, the weakness of the signal did not allow
us to establish whether DMSO resulted in a sextet signal. This may be
caused by the peroxynitrite-dependent decomposition of DMPO-OH (Augusto
et al., 1994). Although this method cannot distinguish between
superoxide and peroxynitrite, the near diffusion-limited reaction rate
between nitric oxide and superoxide and the high concentrations of
these two molecules in the medium would unavoidably result in the
generation of peroxynitrite.
Nitric oxide is required for the neurotoxicity of
activated microglia
To test whether NO or TNF- mediate neuron-killing by activated
microglia, the NO synthase inhibitor L-NMMA and the two
inhibitors of TNF- production pentoxifylline and thalidomide were
added at the same time with LPS. A 48 hr treatment of LPS killed 78 ± 3% of neurons (Fig.
2A).
L-NMMA completely blocked LPS-induced NO
synthesis and neuron death (viable neurons, 92 ± 7% of control) (Fig.
2).
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Figure 2.
The neurotoxicity of LPS-activated
microglia is nitric oxide dependent. Neuron survival
(A), nitrite accumulation
(B), and TNF- secretion
(C) in microglia-neuron cocultures treated with
100 ng/ml LPS with or without 1 mM NOS inhibitor
L-NMMA for 48 hr. Nitrite, a metabolite of nitric oxide,
and TNF- are quantified from the same cultures used to measure
neuron survival. Mean ± SEM from five independent experiments.
*p < 0.05 compared with LPS alone.
CTL, Control.
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Pentoxifylline is a nonselective phosphodiesterase inhibitor that
blocks the release of TNF- from microglia (Chao et al., 1992).
Pentoxifylline did not affect nitrite accumulation and provided only
minor protection against activated microglia (Fig. 3). In contrast, thalidomide, another
TNF- inhibitor that also inhibits NO production, completely blocked
neuron death (Fig. 3). These results suggest that inhibition of NO
synthesis is sufficient to completely block the neurotoxicity of
activated microglia.
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Figure 3.
TNF- released from activated microglia has a
minor neurotoxic effect. Neuron survival (A),
nitrite accumulation (B), and TNF- secretion
(C) in microglia-neuron cocultures treated with
100 ng/ml LPS for 48 hr with or without the two TNF- inhibitors
pentoxifylline (PEN; 500 µM) and
thalidomide (THA; 200 µM) and 100 ng/ml
LPS. Pentoxifylline inhibits TNF- secretion but has little effect on
nitrite accumulation and neuron killing. Thalidomide inhibits both
TNF- secretion and NO production. Mean ± SEM from five to
eight independent experiments *p < 0.05 compared
with LPS alone. CTL, Control.
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Peroxynitrite mediates the neurotoxicity of microglia activated
by LPS
Nitric oxide reacts with superoxide
(O2·) at a near
diffusion-limited rate with a constant of 6.7 × 109 M/sec, producing
the highly reactive and toxic peroxynitrite (ONOO) (Ischiropoulos et al., 1992).
Furthermore, unlike superoxide, ONOO is
membrane permeable (Marla et al., 1997) and is able to reach and damage
intracellular neuronal targets (Estevez et al., 1998b). Because
activated microglia generate both NO and superoxide, peroxynitrite is
hypothesized to be a major mediator in microglia-induced neuronal injury (Van Dyke, 1997; Combs et al., 2001).
To test the role of superoxide and peroxynitrite in the toxicity of
activated microglia, we treated neurons-microglia cocultures with the
membrane-permeable iron porphyrin peroxynitrite decomposition catalysts
FeTMPyP and FeTPPS or the superoxide dismutase mimetic MnTMPyP (Misko
et al., 1998). FeTMPyP and FeTPPS at 2 µM blocked LPS-induced microglial neurotoxicity without decreasing nitrite production (Fig.
4A,B).
The protective action of FeTMPyP is dose-dependent with an optimal
concentration of 2 µM (Fig.
4C,D). Because the superoxide anion is
required to form peroxynitrite, its decrease should also attenuate
neuron death. We tested the protective effect of the manganese
porphyrin SOD mimetic MnTMPyP (membrane-permeable) in the coculture
treated with LPS. MnTMPyP attenuated LPS-induced microglial
neurotoxicity without compromising nitrite production (Fig.
4A,B). In contrast, treatment with
SOD plus catalase, or each scavenger alone (data not shown), did not
protect against microglial neurotoxicity (Fig.
4A,B), suggesting that
peroxynitrite may be generated inside microglia, where superoxide
cannot be reached by the nonmembrane-permeable SOD. Peroxynitrite may
be also formed extracellularly as a result of the inability of
SOD to efficiently compete with NO for superoxide because of the very fast rate of reaction between NO and superoxide. The inability of
catalase to block neuronal death suggests that hydrogen peroxide is not
a major mediator of microglial neurotoxicity. Only the neurons cultured
directly under the insert containing the microglia died (data not
shown) (see Fig. 6D). This is consistent with
peroxynitrite toxicity, because its half-life of only 1.9 sec at pH 7.4 would prevent high levels of ONOO to
reach distant neuronal targets.
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Figure 4.
Peroxynitrite mediates the neurotoxicity
of LPS-activated microglia. Neuron survival (A)
and nitrite accumulation (B) after a 48 hr
treatment of microglia-neuron cocultures with 100 ng/ml LPS with or
without the membrane-permeable peroxynitrite decomposition catalysts
FeTMPyP (2 µM) or FeTPPS (10 µM), the SOD mimetic MnTMPyP (5 µM), or
50 U/ml SOD plus 100 U/ml catalase (SOD/CAT).
Values in A and B show mean ± SEM of five independent experiments. Dose-dependent effect of the
peroxynitrite decomposition catalyst FeTMPyP on LPS-induced
microglial neurotoxicity (C) and nitrite
accumulation (D) in microglia-neuron
cocultures. Effect of the NO donors SNP and NOC-18 on neuronal survival
(E) and nitrite generation
(F) in the presence or absence of the
peroxynitrite decomposition catalysts FeTMPyP or FeTPPS. Cocultures are
treated with LPS (100 ng/ml), the nitric oxide donor SNP (300 µM), or 100 µM NOC-18 plus FeTMPyP (2 µM) or FeTPPS (10 µM) for 48 hr. Mean ± SEM from five independent experiments. *p < 0.05 compared with LPS. CTL, Control.  p < 0.01 compared with LPS plus FeTMPyP or LPS plus FePPS.
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To test whether peroxynitrite is formed in neurons or microglia from
the reaction of exogenous nitric oxide with neuronal superoxide, we
treated cells with the NO donor SNP (300 µM) at a
concentration that generates nitrite-NO at levels similar to those
generated by LPS-activated microglia (Fig. 4F). At
300 µM, SNP killed <50% of the neurons,
significantly below the 85% cell death caused by LPS-induced
activation of microglia (Fig. 4E) (p < 0.05). These results suggest that the
superoxide and peroxynitrite that mediates the neurotoxicity of
microglia activated by LPS are generated by the microglia. However, we
have not ruled out that a portion of the superoxide that reacts to form
peroxynitrite may be generated within the neurons (Longo et al.,
2000).
The inability of FeTMPyP to block NO-induced neuron death (Fig.
4E) also confirms that this decomposition catalyst is
specific for peroxynitrite. To test further the specificity of the iron porphyrins for peroxynitrite, we treated neurons with the NO donor NOC-18, which releases NO· during decomposition (Maragos et al., 1991). NOC-18 at 100 µM resulted in the
generation of high levels of nitrite and caused the death of 99% of
the neurons (Fig. 4E,F). FeTPPS, which completely blocked the toxicity of peroxynitrite generated by activated microglia (Fig. 4A), did not
block the toxic effect of NOC-18.
FeTMPyP is an efficient decomposition catalyst of peroxynitrite but
not superoxide
To validate further the specificity and effectiveness of the
peroxynitrite decomposition catalyst FeTMPyP, we tested it in neuron
cultures treated with SIN-1, an exogenous NO and superoxide donor that,
consequently, generates peroxynitrite under physiological conditions
(Hogg et al., 1992). FeTMPyP effectively protected neurons against
SIN-1-induced peroxynitrite toxicity (Fig.
5A).
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Figure 5.
FeTMPyP and MnTMPyP are efficient and specific
inhibitors of peroxynitrite and superoxide, respectively. Effect of
FeTMPyP on the neurotoxicity of the NO-O2
donor SIN-1. Primary cortical neurons are treated with 50 µM SIN-1 or 50 µM SIN-1 plus 2 µM FeTMPyP for 48 hr. A, Neuron survival;
B, nitrite accumulation. Data represent mean ± SEM from three independent experiments. C,
Effect of FeTMPyP or MnTMPyP on the growth defects (ethanol) of
Saccharomyces cerevisiae cells lacking cytosolic
superoxide dismutase (sod1). S.
cerevisiae cells are inoculated in SDC medium and diluted in
YPE (ethanol) medium (3 ml) containing MnTMPyP (25 µM) or
FeTMPyP (25 µM) with or without the intracellular
superoxide generator paraquat (10 µM). Cell density is
measured after 48 hr. Average ± SEM of two independent
experiments with duplicate samples. *p < 0.05 compared with control plus paraquat; **p < 0.05 compared with control. CTL, Control.
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To determine whether FeTMPyP may be protecting neurons by also
scavenging superoxide, we studied its effect on yeast mutants lacking
cytosolic SOD (sod1) (Gralla and Valentine, 1991). This is a valuable system to test the specificity of these agents (Chiang et
al., 2000) because the primary cause of the defects of yeast sod1 mutants is superoxide toxicity. In contrast,
molecules such as paraquat and menadione generate other toxic oxygen
species in addition to superoxide. FeTMPyP did not reverse the growth defects of sod1 mutants in either the absence or presence
of paraquat (Fig. 5C). In contrast, the superoxide scavenger
MnTMPyP blocked superoxide toxicity and reversed the growth
defects of sod1 mutants whether or not paraquat was
present. These results confirm that FeTMPyP is acting as a specific
decomposition catalyst of peroxynitrite, whereas MnTMPyP functions as a
permeable superoxide dismutase.
Peroxynitrite mediates the neurotoxicity of A in
neurons-microglia cocultures
To test whether peroxynitrite also mediates the toxicity of A,
implicated in AD pathology, we tested the role of agents that decompose
or scavenge specific nitrogen and oxygen species in protecting neurons
against fibrillar A1-42. A and IFN act
synergistically in stimulating the production of nitric oxide from
microglia and in causing microglial neurotoxicity (Meda et al., 1995).
Treatment of microglia-neuron cocultures with 5 µM A1-42 plus 10 ng/ml IFN for 48 hr caused
the death of >80% of neurons. Neuronal death required the generation
of nitric oxide by microglia (Fig.
6A,B),
as shown by Ii et al. (1996). Treatment of neurons with 5 µM A1-42 or IFN
alone did not cause neuronal death in the absence of microglia (data
not shown). Cell death was confined to the region directly underneath the inserts containing microglia, in an area slightly larger (15%) than that of the insert, as shown in Figure 6D. The
spatial proximity of dead neurons to activated microglia (1 mm) is
consistent with the role of a short-lived molecule, peroxynitrite, in
the mediation of A1-42 neurotoxicity. The
peroxynitrite decomposition catalyst FeTMPyP blocked A-induced
microglial neurotoxicity without affecting nitrite production (Fig.
7). The SOD mimetic MnTMPyP had a similar
but attenuated effect (Fig. 7). These results suggest that
peroxynitrite is a major mediator of the toxicity of microglia activated by A.
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Figure 6.
The neurotoxicity of
A1-42-activated microglia is nitric oxide dependent.
Effect of a 48 hr treatment of microglia-neuron cocultures with 5 µM A1-42 plus 10 ng/ml interferon on
neuron survival (A) and nitrite accumulation
(B) with or without 1 mM NOS
inhibitor L-NMMA. Mean ± SEM from four independent
experiments. *p < 0.05 compared with A.
Fluorescein diacetate staining of neurons untreated
(C), treated with 5 µM
A1-42 plus 10 ng/ml interferon (D), or treated with 5 µM
A1-42 plus 10 ng/ml interferon plus 1 mM NOS inhibitor L-NMMA
(E). Note the dark region in the
top portion of D, indicating dead cells
primarily in the region directly underneath the microglia-containing
culture inserts (see Materials and Methods), in contrast to the
homogenous staining of untreated neurons shown in C.
Dashed arcs delineate the projection of the
microglia-containing inserts. Scale bar, 200 µm. CTL,
Control.
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Figure 7.
Peroxynitrite mediates the neurotoxicity of
A1-42-activated microglia. Neuron survival
(A) and nitrite accumulation
(B) after a 48 hr treatment of microglia-neuron
cocultures with 5 µM A1-42 plus 10 ng/ml
interferon , with or without the peroxynitrite decomposition
catalyst FeTMPyP (2 µM) or the SOD mimetic MnTMPyP (5 µM). Mean ± SEM from four independent experiments.
*p < 0.05 compared with A;
p < 0.05 compared with A plus FeTMPyP.
CTL, Control.
|
|
A1-42 causes peroxynitrite-dependent DNA
fragmentation preceding neuron death
Whereas high concentrations of peroxynitrite induce necrotic cell
death in neurons, low levels of peroxynitrite can induce apoptosis
(Bonfoco et al., 1995; Estevez et al., 1998b). DNA fragmentation was
detected in vivo by the terminal deoxynucleotidyl
transferase-mediated biotinylated UTP nick end labeling (TUNEL). DNA
strand breaks were observed at 24 hr in A-treated neurons in the
area below the microglial inserts (Fig.
8A,B),
when <30% of the cells are dead (Fig. 8C). As shown in
Figure 8B, the majority of the neurons treated with
A 1-42 were TUNEL labeled at 24 hr,
suggesting that peroxynitrite induces DNA damage. Although TUNEL assay
can label both apoptotic and necrotic neurons (Adamec et al., 2001), most of the TUNEL-labeled cells at 24 hr were not necrotic, as determined by the fluorescein diacetate staining (Fig. 8C).
These results are consistent with the demonstrated role of
peroxynitrite in inducing macromolecular damage and apoptosis in motor
neurons (Estevez et al., 1998b).
View larger version (68K):
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Figure 8.
A1-42-activated microglia induce
TUNEL staining preceding loss of viability. TUNEL in neurons removed
from microglia-neuron cocultures treated with 5 µM
A1-42 plus 10 ng/ml interferon for 24 hr. Whereas
neurons exposed to inactive microglia are scarcely stained
(A; white arrowheads), neurons exposed to
A1-42-activated microglia (B) are
predominantly positively stained (black arrowheads).
Scale bar, 20 µm. C, Percentage of TUNEL-negative
neurons and surviving neurons after 24 hr treatment of
microglia-neuron cocultures with 5 µM
A1-42 plus 10 ng/ml interferon .
|
|
| |
DISCUSSION |
Cell death in cocultures of cortical microglia and neurons treated
with LPS or A1-42 was preceded by the
generation of superoxide and/or peroxynitrite and nitrite and prevented
by inhibitors of NO synthesis or decomposition catalysts of
peroxynitrite. Together, these results suggest that the short-lived
peroxynitrite molecule generated by activated microglia is the major
mediator of neurotoxicity during treatment of microglia and neurons
with A1-42 or LPS.
AD brains show widespread oxidative damage (Mattson, 1997; Smith et
al., 2000). Both hydrogen peroxide and superoxide have been implicated
in the direct toxicity of A to neurons (Behl et al., 1994; Behl,
1997; Keller et al., 1998; Longo et al., 2000), whereas nitric oxide
has been implicated in the neurotoxicity of activated microglia (Boje
and Arora, 1992; Chao et al., 1992; Goodwin et al., 1995; Meda et al.,
1995). A also stimulates superoxide production in microglia
(Klegeris and McGeer, 1997; McDonald et al., 1997; Colton et al.,
2000), possibly by activating a NADPH oxidase (Bianca et al., 1999).
Thus, the generation of peroxynitrite by microglia activated by A
demonstrated in our study is consistent with previous studies showing
that microglia can generate both nitric oxide and superoxide.
Furthermore, nitrotyrosines, the product of the reaction of
peroxynitrite with tyrosine residues, and NADPH oxidase activity are
elevated in AD brains (Hensley et al., 1998; Shimohama et al., 2000).
Together with ours, these results suggest that peroxynitrite may play a
major role in A toxicity and Alzheimer's disease.
The role of peroxynitrite as the major mediator of the toxicity of
microglia activated by either A or LPS is supported by several
results: (1) in microglia exposed to LPS, the generation of NO and
superoxide-peroxynitrite reaches its peak after 10-20 hr and
continues for over 50 hr; (2) the neurotoxicity of A is confined to
the area directly under microglia; (3) the NO synthase inhibitor NMMA
blocks toxicity as reported previously (Ii et al., 1996); (4) FeTMPyP
and FeTPPS decompose peroxynitrite, but not NO or superoxide, and
block the toxicity of microglia activated by LPS or
A1-42; and (5) the superoxide mimetic
MnTMPyP, which decreases peroxynitrite generation by
scavenging superoxide, partially blocks the toxicity of activated
microglia. However, we cannot rule out that the neurotoxicity is
mediated by a presently unknown short-lived molecule other than
peroxynitrite, which requires NO and superoxide to be formed and which
is neutralized by peroxynitrite-specific decomposition catalysts.
The high second-order rate constant for the reaction between
peroxynitrite and FeTMPyP (5 × 107
M/sec) enables this efficient decomposition catalyst to
protect mammalian cells against high doses of peroxynitrite (Salvemini et al., 1998). The effectiveness of FeTMPyP in blocking peroxynitrite toxicity was confirmed by its protection of neurons against SIN-1, which generates peroxynitrite. Yeast lacking the cytosolic SOD1 show
severe growth defects in the nonfermentable carbon source ethanol. This
is a particularly appropriate system to test the specificity of the
peroxynitrite and superoxide decomposition catalysts and scavengers
because high cytosolic levels of superoxide are the primary cause of
the growth defects. The possibility that FeTMPyP protects neurons by
scavenging superoxide was ruled out by showing that FeTMPyP did not
reverse the defects of yeast SOD mutants (Fig. 5). In contrast, the SOD
mimetic MnTMPyP was effective in reversing superoxide toxicity in yeast.
The lack of a protective effect of SOD against the neurotoxicity of
activated microglia suggest that peroxynitrite is generated within
microglia and therefore out of the reach of the extracellular SOD
protein. However, in view of the effect of SOD in completely abolishing
the EPR DMPO-OH signal caused by superoxide and/or peroxynitrite
released by activated microglia (Fig. 1C), there are
several possible scenarios. (1) The DMPO-OH adduct is only generated by
superoxide through the decomposition of DMPO-OOH, but most
ONOO is generated within the microglia.
In this case, the extracellular SOD would prevent the
superoxide-dependent generation of DMPO-OH but would not prevent the
generation of the neurotoxic peroxynitrite within the microglia. (2)
The DMPO-OH adduct is generated by both superoxide and peroxynitrite,
but the contribution of DMPO-OH by peroxynitrite is below the
detectable level. Furthermore, peroxynitrite can both generate and
decompose DMPO-OH and, in the absence of superoxide-generated DMPO-OH,
may abolish the quartet signal. (3) The DMPO-OH adduct is only
generated by peroxynitrite formed from the reaction of superoxide and
NO outside microglia. In this scenario, SOD may prevent the generation
of the levels of peroxynitrite required to generate a stable DMPO-OH
signal but may be unable to prevent the generation of levels of
peroxynitrite sufficient to kill neurons after a 48 hr exposure. (4)
FeTMPyP and FeTPPS may protect neurons by decomposing peroxynitrite in
the proximity of the neuronal targets in which the concentration of the
decomposition catalyst is high but that of the short-lived
peroxynitrite is much lower than inside microglia. Thus, additional
studies are necessary to demonstrate whether peroxynitrite is generated
inside microglia, extracellularly, or both.
Although NO has been implicated in A1-42
toxicity, its role is controversial because the NO donor
S-nitroso-N-acetyl-penicillamine (SNAP)
was reported by others to be neuroprotective against A toxicity
(Troy et al., 2000; Wirtz-Brugger and Giovanni, 2000). These
differences may be explained by the presence in these studies of A
and of an NO donor, in the absence of microglia. Whereas microglial
activation results in peroxynitrite generation, the concentration of
superoxide in neurons exposed to A and an NO donor in the absence of
activated microglia may be too low to generate toxic levels of
peroxynitrite. The subtoxic levels (100 µM) of the NO
donor SNAP used in these studies may actually protect the cells against
the superoxide generated in neurons treated with A in the absence of
microglia (Keller et al., 1998; Longo et al., 2000). In fact, NO
activates guanylate cyclase and increases the generation of cGMP, which
protects against cell death (Kim et al., 1999; Wirtz-Brugger and
Giovanni, 2000). NO has also been shown to protect mammalian cells
independently of cGMP by inhibiting complex IV activity (Beltran et
al., 2000) or by inhibiting other respiratory enzymes (Paxinou et al.,
2001). Thus, low levels of NO may play a protective role in the absence
of superoxide.
TNF-, a cytokine released by activated microglia, can also be both
neurotoxic (Chao et al., 1993; Wood, 1995) and neuroprotective (Barger
et al., 1995). Our studies suggest that TNF- does not play a major
role in the toxicity of activated microglia because the inhibition of
TNF- secretion by pentoxifylline did not prevent LPS neurotoxicity.
Peroxynitrite is a short-lived molecule with a half-life of <2 sec,
whereas TNF- is a relatively long-lived protein. Therefore, the
pattern of cell death in neurons confined to an area within ~1 mm of
the microglia (Fig. 6D) is not consistent with a
major role for TNF- in acute neurotoxicity. However, TNF- may be
more toxic at higher concentrations or during longer or chronic
treatments. In fact, TNF- from monocytes and microglia stimulated
with 60 µM A1-40 or
A25-35 causes neuronal apoptosis (Combs et
al., 2001). Whereas in our coculture system the neurons are coincubated
with activated microglia during the treatment with LPS or 5 µM A, Combs et al. (2001) have removed the
media from cultures of activated microglia and transferred it to
neuronal cultures. This method would result in the decomposition of
most of the short-lived peroxynitrite. Thus, the low dose of A1-42 (5 µM) used in
our experiments induces both peroxynitrite and TNF- secretion, but
neurotoxicity is mediated by peroxynitrite. However, higher doses of
A or longer treatments may induce the expression of neurotoxic
levels of TNF-. This may explain the role of both NO and TNF- in
the neurotoxicity of microglia treated with
A1-42 or A 25-35 in
which neurons and microglia were cocultured and treated with a higher
concentration of A1-42 (12 µM) for a longer time (72 hr) than in our
experiments (Meda et al., 1995).
In summary, our results with cell-culture models suggest that
peroxynitrite is the major mediator of the neurotoxicity of microglia
activated by LPS or A and indicate that it may also play an
important role in the toxicity of the chronically activated microglia
in AD brains. These findings support the development of drugs that
protect neurons against the toxicity of specific molecules, such as
peroxynitrite, without interfering with the normal functions of glial
cells and neurons.
| |
FOOTNOTES |
Received Dec. 20, 2001; revised Jan. 28, 2002; accepted Feb. 12, 2002.
*
C.E.F. and V.D.L. contributed equally to this work.
This work was supported by National Institutes of Health Grants AG13499
(to C.E.F.) and AG01028 (to V.D.L.) and the John Douglas French
Alzheimer's Foundation (to V.D.L.). We thank John Martin and George
Fenimore for their generous donations.
Correspondence should be addressed to V. D. Longo, Division of
Biogerontology, Andrus Gerontology Center, University of Southern California, 3715 McClintock Avenue, Los Angeles, CA 90089-0191. E-mail:
vlongo{at}usc.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
