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INTRODUCTION |
Activated microglia have been observed in patients suffering from
both acute (stroke) and chronic (Alzheimer's disease) neurological disorders (1, 2). Microglia are believed to contribute to the
progression of Alzheimer's disease
(AD)1 because these cells can
release pro-inflammatory substances known to induce neurotoxicity (3).
Reactive oxygen intermediates (ROIs), one of several pro-inflammatory
substances released by microglia (4), are likely to play a very
important role in AD because hallmark modifications of ROI damage such
as lipid peroxidation and nitrotyrosine conjugates are characteristic
of post-mortem AD brains (3). Hence, pro-inflammatory stimuli that
promote microglial ROI production might contribute to the pathogenesis
of AD.
ATP is an important messenger in the brain and can be released from
cells by both lytic and non-lytic mechanisms (5). ATP evokes a variety
of biological responses in microglia (6-9). The effects of ATP are
mediated through interactions with the P2 purinoceptors, broadly
classified into P2Y metabotropic and P2X ionotropic receptors (10). The
P2Y receptors are G protein-coupled and P2X receptors are ligand-gated
ion channels (10). Whereas the P2Y receptors are responsible for
Ca2+ release predominantly from intracellular stores, P2X
receptors are responsible for Ca2+ influx from
extracellular sources.
Microglia possess both P2Y and P2X receptors (11-13). The
P2X7 receptor is highly expressed by cells of the
macrophage lineage, such as dendritic cells, alveolar macrophages, and
microglia. Activation of the P2X7 receptor is unique in
triggering the formation of large nonselective membrane pores,
permeable to molecules up to 900 Da which ultimately results in death
of the cell (9, 14). ATP and ATP analogs have been used to characterize
the role of P2 receptors in microglial activation. Micromolar
concentrations of ATP are required to activate the P2Y receptors,
whereas millimolar (1-5 mM) concentrations of ATP are
required to activate the P2X receptors. The ATP analog BzATP is a
selective agonist at the P2X receptor and does not bind P2Y receptors
(15, 16). Oxidized ATP (oATP) is a specific antagonist of
P2X7 that binds irreversibly to the receptor and prevents
its activation by ATP (17). In this study, these pharmacological tools
were used to determine the purinergic receptors involved in O
production in microglia.
The P2X7 receptor plays a role in the generation of
superoxide in microglia. Our studies elucidate a putative signal
transduction pathway that mediates this response. These studies also
demonstrate that BzATP- and ATP-activated microglia can mediate
neurotoxicity. Finally, a distinct alteration was detected in the
staining pattern for P2X7 receptor in a transgenic mouse
model of AD, suggesting that P2X7 receptor activation could
play a contributing role in AD.
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MATERIALS AND METHODS |
Reagents--
Reagents not specified otherwise were obtained
from Sigma. PD98059, SB203580, LY294002, and DPI were obtained from
Biomol (Plymouth Meeting, PA). p38 MAPK and p42/44 MAPK
phospho-antibody kits were obtained from New England Biolabs (Beverly,
MA). P2X7 and p67phox antibodies were obtained
from Pharmingen. Anti-CD45 was purchased from Serotec (Oxford, UK).
Amplex red kit and Fluo-4 were from Molecular Probes (Eugene, OR).
Hematoxylin kit obtained from Shandon, Inc. (Pittsburgh, PA).
The Tg2576 transgenic mice overexpressing mutant APP
(K670N,M671L) and control mice were purchased from the
Mayo Clinic (Jacksonville, FL).
Isolation of Microglia--
Rat microglia were prepared from
2-day-old Sprague-Dawley rat pups. The rat cortices were separated from
meninges and minced, triturated, and centrifuged (200 × g for 10 min) to remove dead cells. The pellet was
resuspended in media and triturated, and two brains were transferred to
a 175-mm2 flask containing medium and incubated at
37 °C, 95% relative humidity in a 5% CO2 atmosphere.
The medium was changed after 3-4 days and twice a week thereafter.
Microglia were isolated on day 10 by shaking the flasks on an orbital
shaker (VWR Scientific) at 125 rpm for 15 min. The supernatant was
passed through a sterile nylon mesh (20 µM) (VWR
Scientific), and cells were collected by centrifugation (200 × g for 10 min) and used the same day. The purity of the
cultures was 98-100% as determined by immunostaining with ED-40 antibody.
Isolation of Cortical Neurons--
Primary cortical cell
cultures were prepared from embryos of timed pregnant Sprague-Dawley
rats at E14 (18). Briefly, the cortex triturated in DNase/Protease
dissociation buffer was centrifuged and resuspended in PC-1 SF medium
(BioWhittaker). The cells (2 × 105/ml) were plated
onto poly-L-ornithine-coated 24-well plates, and 4 days
later the media were replaced with Neurobasal Medium containing B-27
supplement (Invitrogen), 1% penicillin/streptomycin, and 10 mM L-glutamine. Neuronal cells constituted
90-95% of the total cells and were used on day 10 for experiments.
Isolation of Neutrophils--
Neutrophils were isolated from
peripheral blood of healthy human donors as reported previously
(19).
Measurement of Superoxide Production--
Superoxide
(O
) was measured indirectly through the detection of hydrogen
peroxide (H2O2) by the method of Mohanty
et al. (20). O
production was measured in initial
experiments by O
-dependent superoxide
dismutase-sensitive reduction of ferricytochrome c (21).
However, microglia released very little O
, and this procedure
required a large number of cells. In subsequent experiments the more
sensitive method of H2O2 detection using conversion of 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to highly
fluorescent resorufin in the presence of horseradish peroxidase was
followed (20). Briefly, 5 × 105/ml microglia in Hanks'
balanced salt solution (HBSS) were preincubated with the inhibitors for
either 2 (oATP, PPADS) or 1 h (SB203580, PD98059, or LY294002,
Brilliant Blue G) or 30 min (DPI, AEBSF, apocyanin) in a 96-well plate.
The various stimuli were directly added into the plate containing cells
in the presence of 0.2 units/ml horseradish peroxidase, 50 µM Amplex Red, and the change in fluorescence was
measured at 590 nm after excitation at 544 nm every 2.5 min using a
fluorometric plate reader (Fluostar, BMG Labtechnologies, Durham, NC).
The H2O2 released was calculated as picomoles
of H2O2, 1 × 105 cells using a
standard curve generated using known amounts of H2O2.
Intracellular Measurement of Superoxide Production--
The
conversion of nitro blue tetrazolium (NBT) to formazan was used to
detect the intracellular generation of O
(21, 22). Briefly,
NBT at a final concentration of 1 mg/ml was added to wells containing
cells. After treatment, the cells were applied to a glass slide using a
Cytospin III (Shandon Southern, Sewickley, PA) and
counterstained with safranin. The number of purple granules of formazan
was counted microscopically to give a qualitative measure O
generation.
Measurement of Calcium--
Isolated rat microglia were plated
onto 384-well plates (Falcon, part 353961) at ~50% confluence. Cells
were loaded with Fluo-4,AM (5 µM) in HBSS containing 10 mM HEPES (pH 7.4) for 1 h before the experiment at
room temperature and washed with the buffer. ATP and BzATP were used to
stimulate the [Ca2+]i signal. In the experiments
where oATP and PPADS were used, cells were pretreated with the
inhibitors for 2 h at 37 °C and then loaded with Fluo-4,AM. The
fluorescent signal from ~104 cells per well was measured
using a fluorometric plate reader (FLIPR, Molecular Devices). Fluo-4
was excited at 488 nm, and fluorescence was measured at 510 nm in a
time-resolved mode (1-Hz frequency). Relative
f/f0 intensity (in counts/ms) was used as an
indication of [Ca2+]i signal. Data acquisition
and preliminary analysis were done using FLIPR software (Molecular
Devices). All calcium measurements were done at room temperature.
Membrane Translocation of p67phox--
Fractionation
of microglia was performed according to the method of Zhao et
al. (23). Briefly, microglia (14 × 106 cells/ml) in
HBSS were treated with or without 500 µM BzATP for 5-10
min. Cells were centrifuged, and the cell pellet was resuspended in 0.5 ml of relaxation buffer (100 mM KCl, 3 mM NaCl,
3.5 mM MgCl2, 1.25 mM EGTA, 10 mM PIPES (pH 7.3), 500 µM
phenylmethylsulfonyl fluoride, and 1:100 dilution of protease inhibitor
mixture), sonicated (3 × 10 s, 4 °C using a microprobe
sonicator), and centrifuged (500 × g for 10 min) to
remove nuclei and unbroken cells. The post-nuclear lysates were then
ultracentrifuged (100,000 × g for 60 min, 4 °C),
and the resulting supernatant was designated the cytosolic fraction.
The membrane/particulate pellet was resuspended in 200 µl of
relaxation buffer containing 1% Triton X-100. Protein concentration
was estimated using the Bio-Rad DC Protein Assay, and 25 µg of protein (for both the cytosolic and membrane/particulate fraction) was loaded onto a gel.
A spontaneously occurring rat microglial cell line was used for the
above experiment because we were unable to generate sufficient numbers
of primary microglia required for this experiment. The spontaneously
occurring rat microglial cell line was isolated from primary rat
microglia growing in LADMAC-conditioned media (ATCC, Manassas, VA). The
cells were propagated in media (Dulbecco's modified Eagle's medium,
1% penicillin/streptomycin, 10 mM L-glutamine, 0.1 mM nonessential amino acids, 10% fetal bovine serum)
containing 20% LADMAC-conditioned media. The cells were positive for
ED-1, a microglial marker. The microglial cells responded to both LPS and BzATP as demonstrated by the generation of TNF
with LPS and ROIs
with BzATP (data not shown).
Immunoblotting--
Immunoblotting was performed as described
previously (18). Briefly 25 µg of protein was fractionated on a 10%
SDS-PAGE gel, transferred to polyvinylidene difluoride membrane, and
blocked in 5% milk/Tris-buffered saline containing 0.1% Tween 20 for
2 h. The membrane were washed and incubated overnight with
antibodies specific for phospho-p42/44 MAPK (Thr-202/Tyr-204),
phospho-p38 MAPK (Thr-180/Tyr-182) diluted 1:1000 in TBST containing
5% bovine serum albumin. Membranes were washed with TBST and incubated
with an horseradish peroxidase-conjugated secondary antibody (1:2000) for 2 h. The membrane was washed extensively, and bands were
detected using LumiGLO. The membranes were stripped using RESTORE
Western blot stripping buffer (Pierce), washed several times, and
blocked for 1 h. Membranes were incubated with antibodies specific
for either unphosphorylated p42/44 MAPK or p38 MAPK diluted 1:1000 in
blocking buffer. The next day membranes were incubated with the
secondary antibody and visualized using LumiGLO.
The P2X7 (1:500), p67phox (1:500), and actin
(1:750) antibodies were used according to the manufacturer's
recommendation. In some experiments a P2X7 control peptide
corresponding to amino acid 576-595 of rat P2X7 (the
immunogen used to generate the antibody) was utilized to determine
specificity of the bands. The P2X7 antibody was
preincubated with the control peptide at a 1:1 dilution (v/v) for
1 h at room temperature prior to the addition to the membrane.
Neurotoxicity Assay--
Primary rat microglia (1 × 105) in Neurobasal Medium containing B-27, 1%
penicillin/streptomycin, and 10 mM L-glutamine
were seeded into a 48-well plate containing 1 × 105
primary cortical neurons. The cells were allowed to settle for 2 h
prior to the addition of stimuli. After a 72-h incubation, the
supernatant was assayed for lactate dehydrogenase (LDH). Microglia and
cortical cells were also independently cultured for 72 h in the
presence of stimuli, and LDH released from microglia alone ± stimuli were subtracted out from the values obtained from the combination of microglia and cortical neurons. The LDH was measured with a commercial kit obtained from Promega (WI). In one experiment a
WST-1 cell survival assay was performed on the cells remaining in the
well with a commercial kit obtained from Roche Diagnostics. WST-1
is a modified
3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium assay. The WST-1 assay enables colorimetric measurement of cell viability based on the cleavage of tetrazolium salts by mitochondrial dehydrogenase in viable cells.
TNF
Enzyme-linked Immunosorbent Assay--
Supernatants were
assayed for TNF
using OPtEIA Rat TNF
kit (Pharmingen).
Nitrite Assay--
Nitrite assay was performed in a 96-well
plate using modified Griess Reagent. In brief, 100 µl of Griess
Reagent was added to 100 µl of supernatant in a 96-well plate.
Samples were read at 540 nm, and values were calculated against a
sodium nitrite standard curve.
Tissue Processing and Immunohistochemistry--
The mice were
sedated, perfused with 4% paraformaldehyde and decapitated. The brains
(2-year Tg2576 mice and aged-matched controls) were removed and fixed
in 4% paraformaldehyde. The brains were dehydrated in graded alcohol
solutions followed by Histoclear and embedded in paraffin. Longitudinal
serial sections were cut at 6-µm thickness.
The sections were deparaffinized in Histoclear, rehydrated through a
series of graded alcohols, washed in deionized water, and incubated in
1:5 methanol/water solution containing 3% H2O2 for 30 min to quench endogenous peroxidase activity. The slides were
rinsed in deionized water for 5 min followed by blocking in 5% normal
goat serum in phosphate-buffered saline containing 0.01% Triton-X-100
for 1 h. Sections were incubated with primary antibody (Pan A-
1:1000 (QCB), 4G8 1:1000 (Signet), P2X7 1:100 (Pharmingen),
CD45 1:200 (Serotec), or GFAP 1:1000 (Chemicon)) in 1% normal goat
serum in phosphate-buffered saline overnight at 4 °C.
Immunohistochemistry was completed with appropriate biotinylated secondary antibody (1:500) in 2% normal goat serum/phosphate-buffered saline followed by avidin-biotin complex and visualized by
diaminobenzidine development (Vector Laboratories). Primary or
secondary antibodies were omitted from some sections to serve as
negative controls. After the enzyme substrate (Vector Laboratories) was
added, the manufacturer's protocol was followed. The slides were
washed in water and counter-stained with Shandon-Lippshaw hematoxylin
stain for 2 min at room temperature. Sections were washed in water for 1 min, incubated for 10 s in 50% ethanol/ H2O + 1%
HCl to remove residual hematoxylin, and then washed in water for 1 min.
The slides were then dehydrated in a series of alcohol washes and sealed with a coverslip using DPX mounting medium.
Immunofluorescence was used to detect dual antigen labeling. Tissue
sections were deparaffinized via a series of xylenes and alcohols.
Sections were blocked in 10% donkey serum for 1 h and then
incubated in primary antibodies using 1% serum in Tris-buffered saline
overnight at 4 °C. Primary antibodies (1:1000) specific for GFAP and
P2X7 were pooled for dual immunofluorescence. Slides were
then incubated in pooled secondary antibodies using donkey anti-mouse
Cy-2 (1:100) and donkey anti-rabbit Cy-3 (1:400) in 2% normal donkey
serum in Tris-buffered saline for 1 h in the dark. Slides were
mounted and coverslip sealed with Vectashield (Vector Laboratories,
Burlingame, CA). Images were captured using a Zeiss Axiovert S100TV
microscope with the Zeiss KS400 imaging system.
Generation of Hippocampal Lysates--
Hippocampi from Tg2576
mice and age-matched controls (19 months, 1 male and 2 female) were
excised and snap-frozen in liquid nitrogen and stored at
80 °C.
Tissues were homogenized for 20 s on ice in TNE buffer (50 mM Tris, 150 mM NaCl) at 20% weight/volume using a Polytron homogenizer. Samples were then diluted 1:1 in TNE
buffer containing 2% SDS, 1% Nonidet P-40, and 1% deoxycholate, and
sonicated (2 × 15 s, 4 °C). Protein concentration was
estimated using the Bio-Rad DC Protein Assay, and 25 µg
of protein from each hippocampus was loaded onto a gel.
Statistics--
Student's t test was performed to
determine group differences (p < 0.05).
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RESULTS |
Generation of Reactive Oxygen Intermediates by
Microglia--
Primary rat microglia stimulated with ATP and BzATP
rapidly generate ROIs, superoxide (O
) in particular, measured
indirectly as hydrogen peroxide (H2O2). Fig. 1 shows time- and dose-response curves of
H2O2 generated by microglia stimulated with ATP
or BzATP. The response to ATP was maximal at 1 mM. The
production of H2O2 continued slowly for at
least 90 min after stimulation. The H2O2
production by microglia stimulated with BzATP was rapid and peaked by
about 30 min. The maximal stimulus was ~250 µM. The
magnitude of the response was higher in cells treated with BzATP
compared with ATP. The total amount of H2O2 produced (picomoles) with stimulation by ATP and BzATP was lower than
that generated by 10 ng/ml phorbol 12-myristate 13-acetate (PMA).
However at early time points (5 min) 250 µM BzATP
generated 9-10-fold more H2O2 than PMA (Fig.
1C). These results demonstrate there is a distinct
difference in both the magnitude and duration of
H2O2 production depending on the stimulus.

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Fig. 1.
Effect of ATP and BzATP on
H2O2 production in microglia. Primary rat
microglia (1 × 105) were plated onto a 96-well plate in
HBSS. The H2O2 production in microglia
stimulated with various concentrations of ATP
(A), various concentrations of BzATP
(B), or various stimuli (C) was
determined every 5 min for 90 min. The data are the means of
quadruplicate samples repeated three times. O production in
the presence and absence of superoxide dismutase (SOD) was
determined in 2 × 105 microglia (D) or 1 × 105 neutrophils (E) stimulated with BzATP
(100-250 µM) or PMA (20 ng/ml) for 30 min. The data are
the mean ± S.D. of triplicate samples repeated at least
twice.
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The conversion of Amplex Red to highly fluorescent resorufin in the
presence of H2O2 is an indirect measure of
O
generation. Hence the production of O
was
confirmed by a more direct but less sensitive method. The inhibition of
reduction of ferricytochrome c by
O
-dependent superoxide dismutase was used to
detect the generation of O
. As shown in Fig. 1D,
microglia treated with BzATP showed a significant increase in
superoxide dismutase-inhibitable reduction of ferricytochrome c compared with untreated microglia. O
production
by neutrophils treated with BzATP was examined as a control. Similar
increases in O
were observed when neutrophils were stimulated
with BzATP suggesting that BzATP activates a similar cascade in
neutrophils (Fig. 1E). To determine whether BzATP generated
any intracellular O
, the reduction of NBT in neutrophils
treated with BzATP was examined. In neutrophils treated with BzATP or
PMA but not control cells, formations of purple granules of formazan
were visible microscopically indicating that NBT was being reduced to
formazan (data not shown). These results confirm that cells stimulated
with BzATP generate O
. Because microglia produce little O
, the conversion of Amplex Red to highly fluorescent
resorufin, a more sensitive but indirect indicator of O
production (20), was used as the choice reagent in the remaining experiments.
Effect of ATP and BzATP on Calcium--
Treatment of microglia
with ATP or BzATP resulted in a very rapid increase in the level of
intracellular calcium (Fig. 2). ATP
stimulated a transient increase of intracellular calcium (Fig. 2A). BzATP caused a sustained increase in the level of
intracellular free calcium ([Ca2+]i) that was
maintained for more than 6 min (Fig. 2B). The concentration
of ATP or BzATP required for maximal [Ca2+]i
change was lower than required for maximal H2O2
production. Maximal [Ca2+]i changes were
stimulated by 30-100 µM of both ATP and BzATP, whereas
maximal ROI production required 1 mM ATP and
250µM BzATP (Figs. 1 and 2). To determine whether ATP and
BzATP were mobilizing intracellular or extracellular sources of
Ca2+ or both, additional experiments were carried out in
Ca2+-free media containing
1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Ca2+ chelator). In the absence of extracellular
Ca2+, the BzATP response was completely blocked indicating
that BzATP was mobilizing only extracellular Ca2+ (Fig.
2C). However, with ATP, the initial transient peak was reduced by about 75%, suggesting that ATP mobilizes both intracellular (via inositol 1,4,5-trisphosphate-induced Ca2+ release) and
extracellular sources of Ca2+ (possibly via capacitative
Ca2+ influx) (24, 25).

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Fig. 2.
Intracellular free Ca2+ changes
in cells treated with ATP and BzATP in the presence and absence of
extracellular Ca2+. Primary rat microglia (1 × 104) were plated onto a 384-well plate and stimulated
10 s after beginning the experiment with various concentrations of
ATP in HBSS (A), various concentrations of BzATP in HBSS
(B), 100 µM ATP or 100 µM BzATP
in HBSS without Ca2+ or Mg2+ containing 0.5 mM
1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (C). The response was measured using a fluorescence
plate reader. The black lines represent responses in the
presence and the gray lines represent responses in the
absence of Ca2+/Mg2+ ions. The data are the
means of quadruplicate samples repeated at least twice.
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Effect of Extracellular Calcium on ROI Production--
Because ATP
appeared to stimulate Ca2+ release from intracellular
stores and Ca2+ influx from extracellular sources, whereas
BzATP appeared to stimulate only Ca2+ influx from
extracellular sources, the effect of removal of extracellular Ca2+ on H2O2 production was
examined. Both ATP- and BzATP-stimulated H2O2
production was blocked to below control levels in the absence of
extracellular Ca2+ (Fig. 3).
These results suggest that despite the differences in Ca2+
mobilization, both ATP and BzATP required only extracellular Ca2+ to generate H2O2.

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Fig. 3.
Effect of EGTA on ATP- and BzATP-induced
H2O2 production. Primary rat microglia (1 × 105) were plated onto a 96-well plate in either HBSS or
HBSS without Ca2+ and Mg2+ containing 500 µM EGTA and stimulated with various concentrations of ATP
or BzATP. The data are the means ± S.E. of two experiments each
done in quadruplicate.
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Receptors Involved in the Generation of ROI--
The ability of
BzATP, an agonist of P2X receptors, to stimulate
H2O2 production and the requirement of
extracellular Ca2+ for this response suggest P2X receptors
mediate the production of H2O2 in microglia. To
determine whether the production of H2O2 was
mediated through the P2X7 receptor, two selective
inhibitors of P2X7, PPADS and oATP, were tested. Both PPADS
and oATP blocked H2O2 production by BzATP
treatment (Fig. 4A) suggesting
that BzATP activates H2O2 production primarily
through the P2X7 receptor. Further support for the role of
P2X7 in H2O2 production was
obtained by treating cells with Brilliant Blue G, a potent and highly
selective inhibitor of P2X7, at nanomolar concentrations
(26). Brilliant Blue G (500 nM) inhibited BzATP (250 µM)-induced H2O2 production by
more than 80% (Fig. 4A).

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Fig. 4.
Effect of P2X selective inhibitor oATP and
PPADS on BzATP-induced H2O2 production and
calcium changes. Primary rat microglia (1 × 105) were
plated and pretreated with oATP or PPADS for 2 h or Brilliant
Blue-G (BB) for 1 h. H2O2
production (A) or Ca2+ changes (B)
were measured in cells treated with BzATP. The data are the means ± S.E. of five (PPADS, oATP) or three (Brilliant Blue-G
(BB)) experiments each done in quadruplicate. The data are
presented as a percent of H2O2 produced by
microglia stimulated with 250 µM BzATP for 30 min. *
indicates p < 0.05 by Student's t
test.
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To determine whether oATP and PPADS affected Ca2+ responses
similarly, Ca2+ changes were measured in cells pretreated
with oATP and PPADS in the presence or absence of BzATP. oATP (100 µM) inhibited BzATP-induced Ca2+ flux to near
control levels (Fig. 4B). Similar results were obtained with
PPADS (Fig. 4B). These results suggest that P2X7
is the primary receptor stimulated by BzATP to generate
H2O2.
Source of ROI--
Several sources can contribute to the
production of ROI. These include the classical NADPH oxidase, the
mitochondrial respiratory chain, and microsomal enzymes.
Pharmacological inhibitors of the NADPH oxidase were used to determine
whether P2X7 receptor activates NADPH oxidase. Three
selective inhibitors with different mechanisms of action,
diphenyleneiodonium chloride (DPI), apocyanin, and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) were used (27, 28).
As shown in Fig. 5A, all three
inhibitors completely inhibited BzATP-induced
H2O2 release from microglia. To confirm the
activation of NADPH oxidase by BzATP in microglia, a functional change
in NADPH oxidase was examined. A critical step in the activation of the
NADPH oxidase is the translocation of p67phox from the
cytosol to the membrane. In BzATP-stimulated microglia, p67phox, which is primarily cytosolic, rapidly translocated
to the particulate/membrane fraction (Fig. 5B). These
results suggest that BzATP stimulates the release of ROI in microglia
via the activation of the NADPH oxidase.

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Fig. 5.
NADPH oxidase is activated by BzATP.
A, primary rat microglia (1 × 105) were plated
and pretreated with AEBSF, apocyanin, or DPI for 30 min, and
H2O2 production was measured in cells
stimulated with 250 µM BzATP. The data are the means ± S.D. of quadruplicate samples repeated three times. * indicates
p < 0.05 by Student's t test.
B, microglia were left untreated or activated with 500 µM BzATP for 5-10 min. The cells were then collected and
fractionated as described under "Materials and Methods."
p67phox was detected using a polyclonal antibody.
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Signal Transduction Cascade Involved in the Generation of
H2O2--
The production of
O
/H2O2 is stimulated through several
different signal transduction pathways. Two kinases implicated in the
activation of the NADPH oxidase are p42/44 ERK and p38 MAPK (29-31).
Both p42/44 ERK and p38 MAPK were rapidly activated in cells stimulated
with 250 µM BzATP (Fig. 6).
However, pretreatment with SB203580 (a selective p38 MAPK inhibitor)
but not PD98059 (a selective p42/44 MAPK inhibitor) attenuated
H2O2 production in cells stimulated with BzATP
(Fig. 6). Nonetheless, PD98059 did attenuate BzATP-induced TNF
release suggesting that p42/44 MAPK was involved in cytokine signaling but not in the generation of H2O2 (data not
shown). LY294002, a selective phosphatidylinositol 3-kinase inhibitor
(PI3-K) also significantly inhibited H2O2
stimulated by BzATP. LY294002 at 50 and 10 µM inhibited
H2O2 production by 74.7 ± 2 (n = 3) and 47 ± 5% (n = 3),
respectively. The inhibitors used were not toxic to the cells at the
concentrations used. These results suggest that whereas both p38 MAPK
and p42/44 ERK are rapidly activated in microglia stimulated with
BzATP, only the inhibition of p38 MAPK attenuates
H2O2 production. Furthermore, PI3-K may play a more important role in the release of H2O2 than
p38 MAPK because the PI3-K inhibitor blocks
H2O2 release to a greater extent than the p38
MAPK inhibitor.

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Fig. 6.
The signal transduction cascade activated by
BzATP in the generation of H2O2.
A, primary rat microglia (1 × 105) were plated
and pretreated with SB203580, PD98059, or LY294002 for 1 h, and
then H2O2 production was measured in cells
stimulated with 250 µM BzATP. The data represent the
means ± S.E. of three experiments each done in quadruplicate. The
data are presented as a percent of H2O2
produced by microglia stimulated with 250 µM BzATP for 30 min. * indicates p < 0.05 by Student's t
test. Lysates of cells stimulated with 250 µM BzATP or
100 ng/ml of LPS for various periods were probed with antibodies
against phosphorylated p38 or unphosphorylated p38 MAPK antibodies
(B) against phosphorylated p42/44 MAPK or unphosphorylated
p42/44 MAPK (C).
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Microglia Stimulated with ATP or BzATP Are Neurotoxic--
To
determine whether activation of microglia with ATP and BzATP is
neurotoxic, a co-culture system using highly purified primary rat
cortical neurons and primary rat microglia was employed. Stimulation of
microglia with LPS resulted in massive production of TNF
and nitric
oxide but no H2O2 release (Figs. 1 and
7, B and C). LPS did not stimulate LDH release from microglia/cortical neurons co-cultures (Fig. 7A). Conversely, ATP or BzATP stimulated
very little TNF
and nitric oxide production but induced
neurotoxicity at 72 h (Fig. 7). There was no significant
neurotoxicity up to 48 h post-stimulation with either ATP or
BzATP. The amount of neurotoxicity was much greater with BzATP compared
with ATP, which is consistent with the observation that BzATP is a more
potent stimulus compared with ATP in the generation of
H2O2. The values shown in Fig. 7A
represent LDH released from both microglia and cortical cells minus LDH
released from microglia alone. A WST-1 cell survival assay was used to
confirm that the LDH release is a measure of neuronal toxicity. Neither
ATP nor BzATP had a significant neurotoxic effect on cortical neurons
(Fig. 7A). Results comparing LPS-treated co-cultures to
ATP/BzATP-treated co-cultures show that factors other than TNF
or
nitric oxide are contributing to toxicity in our system.

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Fig. 7.
Toxicity of microglia stimulated with ATP or
BzATP toward primary rat cortical neurons. A, primary
rat microglia were co-cultured with primary rat cortical neurons
without any stimuli or in the presence of 100 ng/ml LPS, 1 mM ATP, or 250 µM BzATP. The viability of
cortical neurons after 72 h of incubation was assessed by LDH
release into the supernatant. The data represent the mean ± S.E.
of three separate experiments each done in triplicate expressed as a
percentage of LDH released into the supernatant compared with control
(LDH released from the co-culture of microglia and cortical neurons in
the absence of any stimuli). * indicates p < 0.05 by
Student's t test. B and C, microglia
were treated with LPS (100 ng/ml), BzATP (500 µM), and
ATP (3 mM) for various times. Media were assayed for TNF
release by enzyme-linked immunosorbent assay (B) and nitrite
release by modified Greiss reagent (C). The data represents
the mean ± S.D. of triplicate samples repeated at least three
times.
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Up-regulation of P2X7 Receptor in a Transgenic Mouse
Model of Alzheimer's Disease--
By having demonstrated that ATP-
and BzATP-stimulated microglia release H2O2 and
kill cortical neurons in vitro, we examined the brains of a
transgenic mouse model of AD (Tg2576 carrying a
APP(K670N,M671L) double mutation) (32) to determine whether
the mechanism is important in vivo. In 24-month-old
transgenic mouse brains but not in aged-matched control brains, plaques
were evident in the hippocampus and surrounding outer cortical region
when stained with two different antibodies for A
(Fig.
8, A, B, and E;
data not shown). P2X7 immunostaining gave a ring-like
pattern around plaques only in transgenic mice suggesting that cells
staining for P2X7 were surrounding the plaques (Fig. 8,
C, D, and F). This staining was not evident in
the absence of the primary antibody indicating that the staining was
not due to nonspecific binding of the secondary antibody to the
plaques. There was some basal staining with the P2X7
antibody in both control and transgenic animals suggesting low levels
of P2X7 are expressed in the brain.

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Fig. 8.
P2X7 expression and
localization in APP(K670N,M671L) transgenic and age-matched
control mice. Two-year-old Tg2576 (B and
D-F) and age-matched control mice (A and
C) were stained for -amyloid with a pan-A antibody
(A, B, and E) and P2X7
with a polyclonal anti-P2X7 antibody (C,
D, and F). Magnification bar = 300 µm for A-D and 50 µm for E-F.
G, hippocampal lysates from three 19-month-old Tg2576 mice
or age-matched controls were probed with an antibody against
P2X7. The specificity of the P2X7 band was
confirmed by the use of a blocking peptide as described under
"Materials and Methods." The blot was then stripped and reprobed
with an antibody against actin.
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To determine whether the increased staining of P2X7 in the
transgenic mice was due to increased expression of P2X7
receptor, lysates from the hippocampi (region with higher concentration of plaques) of three 19-month Tg2576 mice and age-matched controls were separated by SDS-PAGE, electrophoretically transferred, and probed
with a polyclonal antibody specific for P2X7 (Fig.
8G). The signal for the 55-kDa isoform was higher in the
transgenic mice compared with age-matched controls. Preincubation of
the P2X7 antibody with the P2X7 peptide (amino
acid 576-595) resulted in the disappearance of the P2X7
band and had no effect on the actin band, demonstrating the specificity
of the antibody (Fig. 8G).
To determine the identity of cells expressing P2X7 around
the plaques, serial sections were stained with markers selective for
either pan-A
(Fig. 9A),
P2X7 (Fig. 9, B and E), microglia (CD45 (Fig. 9C)), astrocytes (GFAP (Fig. 9F)), or
P2X7 and GFAP (Fig. 9D). Microglial and
astrocytic staining were seen surrounding plaques in the transgenic
animals (Fig. 9, C and F, respectively). It could
not be determined if P2X7 immunoreactivity was restricted specifically to either microglia or astrocytes; however, very little
co-localization was detected by dual immunofluorescence with anti-GFAP
and anti-P2X7 (assessed by the number of dual labeled yellow cells (Fig. 9D)). These results show that
P2X7 is up-regulated in Tg2576 mice around amyloid plaques
and is regionally localized with activated microglia and
astrocytes.

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Fig. 9.
P2X7 immunoreactivity is associated with
GFAP- and CD45-positive cells around plaques in
APP(K670N,M671L) transgenic mice. Two-year-old Tg2576
mice (A-F) were stained with pan-A antibody
(A), P2X7 (B and E), CD45
(C), GFAP (F), or GFAP + P2X7 (D).
Magnification bar = 50 µm. Photomicrographs
A-C were obtained using immunohistochemistry using DAB
substrate; D-F utilizing dual
immunofluorescence (Cy-2-GFAP (green)/Cy-3-P2X7
(red)). Dual labeled cells are represented as
yellow.
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DISCUSSION |
The P2X7 receptor has been implicated in the
activation of transcription factors, apoptosis, and in the release of
pro-inflammatory substances like TNF
and interleukin-1
in
microglia (8, 9, 14, 33). In this report we demonstrate that
P2X7 is the primary receptor involved in
H2O2 production in primary rat microglia stimulated with ATP or BzATP. The P2X-selective agonist BzATP was a
more potent stimulus than ATP, a P2Y/P2X agonist. Functionally, the
activation of microglia with ATP or BzATP induced cell death in primary
cortical neurons. In vivo there was a striking association of P2X7 receptor-positive cells around plaques in a
transgenic mouse model of Alzheimer's disease.
No detailed study has profiled the expression of P2Y and P2X receptors
in microglia, but microglial expression of both P2X and P2Y is
supported by electrophysiological studies (13, 34). Our results
demonstrating Ca2+ changes induced by ATP or BzATP also
support the existence of functional P2X and P2Y receptors on primary
rat microglia.
Several lines of evidence point to P2X receptors and P2X7,
in particular, as the primary receptor involved in the generation of
H2O2 in ATP- or BzATP-stimulated microglia.
Stimulation of H2O2 production by P2X-selective
agonist BzATP (35, 36) provides the first line of evidence for the
involvement of P2X receptors. P2X receptors mobilize only extracellular
Ca2+, consequently the experiments that demonstrate BzATP
mobilizes only extracellular Ca2+ provide further evidence
for the involvement of this receptor. The inability of BzATP to
stimulate H2O2 production in the absence of
extracellular Ca2+ provides a direct link between
Ca2+ mobilization and H2O2
production. The inhibition of H2O2 production and Ca2+ influx by P2X-selective antagonist PPADS,
P2X7-selective antagonist oATP (17), and
P2X7-selective inhibitor Brilliant Blue-G in BzATP-stimulated cells provide additional lines of evidence for the
involvement of P2X7 receptors. The contribution of other
purinergic receptors cannot be excluded, and can be evaluated only by
measuring H2O2 production in microglia lacking
P2X7 receptors.
The levels of ATP required to stimulate the P2X7 receptor
in vitro suggests that the low concentrations of ATP found
in the extracellular milieu of the brain would not be sufficient for microglia to induce neurotoxicity. However, recent reports (6) suggest
that low concentrations of ATP can act as a chemoattractant for
microglia directing them to a region of injury. Furthermore, ATP
released from activated astrocytes has been shown recently (37) to
activate microglia. Finally, a recent report (38) has documented the
significance, the source, and needed high concentrations of ATP to
activate P2X7 receptors in the brain, as well as the role
of ATP in neurodegeneration.
Endogenous production of O
is a biochemical process that
requires tight regulation. NADPH oxidase, a known generator of
O
, is regulated by an intricate signal transduction cascade
within cell types such as neutrophils (39). Dysregulation of the
oxidase leads to the damage of surrounding tissue and results in
inflammatory conditions. Both p42/44 ERK and p38 MAPK play an important
role in the regulation of various proteins of NADPH oxidase complex in
neutrophils (29-31). However, little is known about the regulation of
the NADPH oxidase in microglia. Although both p42/44 ERK and p38 MAPK
were activated very rapidly in microglia stimulated with BzATP, only
inhibition of p38 MAPK attenuated the release of
H2O2. This suggests that there are subtle
differences in the regulation of the NADPH oxidase complex in
neutrophils compared with microglia. Other kinases like PI3-K promote
NADPH oxidase activity through the PX domain of p47phox and
p40phox (40). Inhibition of H2O2
levels by the PI3-K inhibitor, LY294002, suggests that PI3-K plays a
similar yet more important role in the assembly of the oxidase complex
in microglia compared with p38 MAPK.
Microglia activated by certain stimuli can induce cell death in
cortical neurons. Although the neurotoxicity is attributed to various
individual secretory products like TNF
, nitric oxide, and ROIs,
toxicity is likely due to a combination of factors (41-43). The
inability of LPS-activated microglia to induce cell death in cortical
neurons despite large increases in TNF
and inducible nitric-oxide
synthase shows that these products do not solely induce cell death in
our microglia/cortical neuron co-culture system. Published reports
support this observation. Klegeris et al. (44, 45)
demonstrated that LPS-stimulated microglia are not neurotoxic but in
combination with IFN
induces neurotoxicity. IFN
can prime
microglia to generate O
in the absence and presence of TNF
suggesting that TNF
could be indirectly inducing cell death by
generating O
(46, 47). Moreover, both IFN
/LPS or
IFN
/TNF
can up-regulate P2X7 expression in THP-1
cells and monocytes (48, 49). Given that ROIs can directly induce
neurotoxicity, it will be important to determine the exact mechanism by
which ATP and BzATP are inducing cortical cell death. The link between
LPS, IFN
, and P2X7 expression has yet to be examined in
detail in microglia.
Currently there are no reports on the expression profile of
P2X7 in Alzheimer's disease. Ours is the first paper
demonstrating a remarkable difference in the staining pattern for
P2X7 in brain slices of a transgenic (Tg2576) mice model.
This intense staining for P2X7 around plaques can be the
result of up-regulation of the P2X7 receptor and/or
aggregation of glia around plaques. The fact that P2X7
message and receptor is up-regulated in monocytes treated with
LPS/IFN
or LPS/TNF
(48, 50) raises the possibility that
P2X7 indeed can be up-regulated in the mouse model of AD. The increased P2X7 immunoreactivity in immunoblots
comparing Tg2576 hippocampi lysates to age-matched control hippocampi
lysates and the presence of P2X7-immunopositive cells
around plaques supports this theory. However, the identity of the
P2X7 immunopositive cells around plaques is still not
clear, even though activated microglia and astrocytes are found in the
same vicinity. Although a correlation has been established between
increased P2X7 immunoreactivity and amyloid plaques, the
question of cause and effect is beyond the scope of this paper. Because
P2X7 knock out mice have been made, it will be interesting
to see if microglia from these mice generate
H2O2 in response to BzATP or if a
P2X7 knock out mouse crossed with a Tg2576 mouse would have
any alteration in plaque deposition. It is possible that receptor
antagonists of P2X7 could have therapeutic utility in
treatment of AD by regulating pathologically activated microglia.