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INTRODUCTION |
The most common form of senile dementia, Alzheimer's disease
(AD),1 is characterized by a
progressive loss of neurons from particular regions of the brain, by
the formation of neurofibrillary tangles in neurons, and by numerous
senile plaques in affected brain regions. The major component of the
senile plaques is the
-amyloid protein (A
), a 39-43-amino acid
peptide, derived from a larger transmembrane glycoprotein called
-amyloid precursor protein (1). The A
of the senile plaques
consists of fibrils that are 4-10 nm in diameter and exhibit green
birefringence under polarized illumination when stained with Congo Red.
According to spectroscopic and x-ray diffraction studies, the amyloid
fibril assembly is correlated with the adoption of a
-sheet
structure (2, 3).
According to the "
-amyloid cascade" hypothesis, the excessive
deposition of A
is the key pathogenetic event in AD (4-8) and would
be responsible for neurodegenerative changes of neurons. In the plaques
the central deposit of extracellular amyloid fibrils (the core) is
surrounded by dystrophic neurites (dendrites and axon terminals) and by
activated microglia and reactive astrocytes (9, 10). Furthermore, it
has been demonstrated that A
is neurotoxic in vitro (7,
11, 12) and that this toxicity correlates with
-sheet structure and
fibrillary state (Ref. 13; Ref. 7 and references therein). Data have
been also provided showing that it is the amphiphilic nature of the
peptides rather than their
structure per se that causes toxicity
(14).
Studies of A
cytotoxicity have resulted in some broad concepts of
mechanism that are not mutually exclusive (7, 15). One proposes that
A
can directly injure neurons by specifically or unspecifically
interacting with molecules of cell surface (11). This direct toxicity
could be due to free radicals oligopeptides derived from A
itself
(16) or stimulation of intracellular production of reactive oxygen
intermediates (ROI). Two groups of experimental data demonstrate this
last mechanism. The first is that the interaction in vitro
of A
(1-40) or A
(25-35) with PC12 cells, B12 cells, cultured
rat cortical (14, 17), or hippocampal neurons (18) induces the
stimulation of intracellular production of ROI. It has also been
demonstrated that A
causes the appearance of markers of oxidative
stress in neurons adjacent to senile plaque and in distrophic neurites
contiguous to A
deposits (19). The second group of experimental data
is that antioxidants prevent the cytotoxicity by A
(14, 17, 18, 20,
21). Another proposal is that A
causes little cytotoxicity by itself but enhances the vulnerability of neurons to a variety of common insults, such as excitotoxicity, hypoglycemia, or peroxidative damage
(22, 23). Finally, it has been recently suggested by us and others that
A
can also be indirectly toxic to neurons by activating the
production of toxic and inflammatory mediators such as NO and cytokines
in microglia (24, 25) and in astrocytes (26) and ROI in monocytes and
macrophages (27, 28) and in microglia (29-31). The mechanism by which
A
stimulates the production of ROI is not known. Potential main
sources of ROI are mitochondria, microsomal enzymes, xanthine oxidase,
classical NADPH oxidase of phagocytes, some unidentified NADH-NADPH
oxidases, lipoxygenase, and cycloxygenase (Ref. 32 and references
therein). Following previous work of our laboratory concerning the
indirect mechanism of neurotoxicity mediated by activated microglia
(24, 26, 27) and on the basis of recent data that A
is able to
stimulate the production of O
2 in microglia (29, 31), we have
investigated the involvement of NADPH oxidase in this stimulation (33,
34). This oxidase is an enzymatic system, present in phagocytic and few
other nonphagocytic cell types, which is dormant in resting cell and
becomes active upon contact with appropriate
receptor-dependent or -independent agonists. Microglial
cells belong to the mononuclear phagocytic system and as monocytes and
neutrophils present the classical burst of ROI production when
stimulated by appropriate agonists, such as cytokines and PMA (35). In
this paper we have investigated the effect of A
(25-35), A
(1-39), and A
(1-42) on rat primary cultures of microglia, human
blood monocytes, and human neutrophils. The data presented support the
concept that A
can be indirectly neurotoxic and clarify a further
mechanism, in addition to the production of TNF-
and stimulation of
inducible NO synthase (24-26), by which microglial cells are involved
in the generation of toxic mediators. In fact, the results demonstrate that A
peptides cause the activation of the classical NADPH oxidase, which results in the stimulation of ROI production. This activation of
the oxidase requires that A
peptides are in fibrillary form, is
potentiated by IFN-
and TNF-
, and is inhibited by inhibitors of
tyrosine kinase or phosphatidylinositol 3-kinase and by dibutyryl cyclic AMP.
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MATERIALS AND METHODS |
Reagents--
The A
peptides A
(25-35) and A
(1-39)
were synthesized, purified, and characterized by Prof. E. Peggion
(Dipartimento di Chimica Organica, Università di Padova, Italia)
or by Dr. R. Gennaro (Dipartimento di Biochimica, Biofisica e Chimica
delle Macromolecole, Università di Trieste, Italia) as described
previously (24); A
(1-42) and A
(25-35) scrambled, IMLKGNGASIG,
were a gift by Dr. L. Otvos (Wistar Institute, Philadelphia, PA).
Lyophilized peptides were dissolved in sterile PBS or double-distilled
H2O at a concentration of 2-4 mg/ml, aged 5-7 days at
37 °C, aliquoted, and stored at +4 °C. Fibril formation was
monitored by examining the birefringence after staining with Congo Red
and quantified by measuring the fluorescence intensity after staining
with Thioflavine T (36). Fibrillogenesis by A
(25-35) was rapid
(minutes) in either PBS or H2O at room temperature, whereas
that by A
(1-39) and A
(1-42) was induced by aging 5-7 days at
37 °C. When the experimental protocol required A
peptides in
nonfibrillary form, they were dissolved in Me2SO or kept at
4 °C after solution in PBS. In these conditions the fluorescence by
Thioflavine T was negative (see Fig. 2).
Recombinant rat IFN-
was from BIOSOURCE
International and mouse TNF-
was from PeproTech Inc. Wortmannin (WT)
was kindly provided by Dr. M. Thelen (Theodor Rocher Institute, Bern),
and glycated human serum albumin was kindly provided by Dr. P. J. Thornally (Department of Biological and Chemical Sciences, University of Essex, UK). Dihydrorhodamine 123 (DHR) was purchased from Molecular Probes (Eugene, OR). 4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo [3,4-d] pyrimidine (PPI) was from Calbiochem (La Jolla, CA).
Dibutyryl cAMP, homovanillic acid, heparan sulfate (HS), PMA,
formyl-methionyl-leucyl-phenylalanine (fMLP), horseradish
peroxidase (HRP) (type1), catalase (from bovine liver), trypsin,
diphenyleneiodonium chloride (DPI), Thioflavine T, Congo Red, and
cytochalasin B were from Sigma. The markers Griffonia
simplicifolia isolectin B4, anti-galactocerebroside, and anti-glia
fibrillary acidic protein were from Sigma, and mouse anti rat myeloid
cell (CR3) was from Serotec. Penicillin, streptomycin, and RPMI were
from BioWhittaker (Verviers Belgium). Dulbecco's modified Eagle's
medium was from Life Technologies, Inc., and fetal bovine serum was
from Seromed (Biochrom KG, Germany).
Anti-p47phox, anti-p67phox, and anti-p40phox
affinity-purified rabbit policlonal antibodies were a generous gift of
Dr. F. Wientjes (Department of Medicine, University College, London,
UK); anti-gp91phox and anti-p22phox antibodies were
kindly provided by Dr. D. Roos (Central Laboratory of the Netherlands
Red Cross Blood Transfusion Service and Laboratory of Experimental and
Clinical Immunology, University of Amsterdam). Solutions used
throughout our experiments were prepared with endotoxin-free water for
clinical use.
Cells--
Neutrophils and monocytes (95-98% purity) were
isolated from buffy coats of healthy donors as previously reported (37,
38). Primary microglial cell cultures were prepared from 2-day-old postnatal Wistar rat cerebral cortices as described previously (39)
with some modifications. Briefly, the rats were decapitated, and the
brains taken out and put into a Petri dish containing PBS and
antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). The
meninges were next removed, and the cortices were isolated and
mechanically dissociated by chopping and trituration in PBS containing
0.25% trypsin and antibiotics at room temperature. The mixed glial
suspension was collected by filtration through gauze. Trypsin was next
neutralized by adding an equal volume of Dulbecco's modified Eagle's
medium containing 20% fetal bovine serum. The cell suspension was
centrifuged, and the collected cells were seeded into
160-cm2 flasks (Costar), in Dulbecco's modified Eagle's
medium 20% fetal bovine serum. 3 days later the growth medium was
replaced with fresh one. After 6-8 days microglial cells were
separated from the astroglial cells monolayer by shaking the flask for
60 min at 120 rpm on a rotatory shaker. The released microglial cells were then suspended in Hanks' balanced salt solution and used for the
experiments. When the effect of IFN-
or TNF-
was investigated, these cytokines were added to the cell culture 4-5 days before the
detachment of microglial cells by shaking. The purity of microglial cell preparations (>98%) was confirmed by testing their positivity for markers like G. simplicifolia isolectin B4 and mouse
anti-rat myeloid cell (CR3). The anti-galactocerebroside and the
anti-glial fibrillary acidic protein were used to confirm the
concurrent absence of oligodendrocytes and astrocytes, respectively.
Stimulation of Production of ROI--
The stimulation of
production of ROI, usually called respiratory burst, was measured by
adding the agonists (A
peptides, PMA) to stirred cell suspension in
Hanks' balanced salt solution. In the first experiments the production
of ROI was investigated with the procedures normally used in our
laboratory, that is as H2O2-HRP-dependent oxidation of
homovanillic acid or as O
2-dependent SOD-sensitive reduction of cytochrome c (40, 41). However, because the respiratory burst by A
peptides in monocytes and microglia was very low and the above procedures required a large number
of cells and consequently high concentrations of A
peptides, in most
experiments we adopted the more sensitive method of
H2O2 determination using DHR oxidation
(42).
The procedure was substantially that previously reported (42), modified
by the addition of HRP. The reason for this addition was the following.
Henderson and Chappell (42) have demonstrated that in leukocytes
stimulated by PMA, the oxidation of DHR occurred within the cells and
was catalyzed by endogenous peroxidase and H2O2
generated outside the cell by O
2 from NADPH oxidase and crossing the plasma membrane of the cells. By adopting their procedure we have noted that upon stimulation of the cells with PMA the increase
in fluorescence due to oxidation of DHR was very low but was markedly
increased by addition of exogenous HRP. This finding demonstrated that
a correct estimation of the quantity of H2O2
produced by activated NADPH oxidase, which is located on the plasma
membrane, required the trapping of all the peroxide released outside
the cells. On this basis we have modified the procedure of Henderson
and Chappell by adding HRP to cell suspension. HRP does not enter the
cells but rather catalyzes the oxidation of DHR outside the cells by
trapping all H2O2 as soon as it is generated by
spontaneous dismutation of O
2, the first product of NADPH
oxidase. The procedure that we have used was the following. Stirred
cell suspension in Hanks' balanced salt solution was pre-incubated with 200 units/ml of catalase for 2 min at 37 °C to degrade all the
peroxide present in the solutions and cell suspension. After this time
2 mM NaN3, to inhibit the exogenous catalase,
0.5-2.0 µM DHR, and HRP (500 milliunits/ml) were added.
One minute after HRP, the stimulus or the vehicle was added, and the
changes in fluorescence at 534 nm after excitation at 505 nm were
continuously recorded for the time required. Calibration was made by
adding different amounts of exogenous H2O2.
This highly sensitive procedure enabled the measurement of the
respiratory burst using 0.5-3 × 105 cells/ml. In
these conditions the relationship between free radicals production and
number of cells was linear. When the effects of various drugs on the
stimulation of the respiratory burst was studied, preliminary
investigations were performed to calculate the eventual quenching of
rhodamine 123 fluorescence.
Studies on the Activation State of NADPH Oxidase--
The
activation of NADPH oxidase was investigated by evaluating the
translocation of cytosolic components p47phox, p67phox,
and p40phox from cytosol to the plasma membrane after cell
stimulation as described previously (40). Briefly, neutrophils or
monocytes (5 × 106/ml) were incubated at 37 °C
under stirring with and without the agonists. At the indicated time
samples containing 1.5 × 107 cells were withdrawn and
disrupted by sonication (two 15 s cycles at 200 W at 4 °C). The
postnuclear supernatants were loaded on a discontinuous sucrose
gradient of 1.5 ml of 15% (w/w) sucrose on 1.5 ml of 34% (w/w)
sucrose made up in relaxation buffer. After centrifugation at
150,000 × g (45 min, 4 °C), the light membranes were collected at the 15%/34% sucrose interface, washed with
relaxation buffer, and after centrifugation at 150,000 × g (30 min, 4 °C) resuspended in 150 µl of sample buffer
and boiled for 5 min. Aliquots of samples containing the same amount of
proteins were subjected to SDS/polyacrylamide gel electrophoresis on
12% gel and incubated overnight with 1:500 diluted
anti-p47phox, anti-p67phox, and anti-p40phox
antibodies. All of the subsequent steps for ECL Western blotting detection were performed as described in detail elsewhere (40). In
separate samples under the same conditions of incubation, the stimulation of H2O2 production was also
measured to control the effect of the agonists. In this case, due to
the high number of cells employed the production of
H2O2 was measured as HRP-dependent oxidation of homovanillic acid as described previously (41).
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RESULTS |
A
Peptides Stimulate the Production of ROI--
The results of
Table I and Fig.
1 show that A
(25-35), A
(1-39),
and A
(1-42) were able to stimulate the production of ROI, measured
as H2O2, in rat primary culture of microglial
cells, human blood monocytes, and neutrophils. The data of Fig. 1 show that the stimulation of H2O2 production by A
(25-35), A
(1-39), and A
(1-42) was detectable already at 1 µM and was maximal between 10 and 20 µM.
From the data of Table I and Fig. 1, it can be seen that the response
of human neutrophils was much greater than that of human monocytes and
rat microglia and that the latter cells were the less responsive. In
all cell types the magnitude of the response to 10 µM
A
peptides was smaller than that to 100 ng/ml of PMA, a dose that in
the experimental conditions employed was the maximal one. The
stimulation of H2O2 production was detectable within 1-2 min after the addition of the A
peptides and lasted for
30-60 min (not shown). Cells exposed for 30 min to 10 µM
A
(25-35) scrambled peptide did not present any stimulation of
H2O2 production (Table I).
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Table I
Stimulation of H2O2 production by A peptides
The values are expressed as increases in H2O2
production upon the addition of stimulants. H2O2
production in the absence of stimulants was <0.02 nmol
H2O2/30 min in all cell types. Data are the means ± S.D. of the experiments indicated in parentheses.
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Fig. 1.
Stimulation of H2O2
production by different doses of A peptides in
primary culture of rat microglia, human blood monocytes, and
neutrophils. The results are the means ± S.D. of three
experiments.
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A
Peptides Are Active in Fibrillary State--
It is widely
accepted that A
is active on neurons in fibrillary state (7, 12, 13,
43, 44). We present here two findings that demonstrate that this was
the case also in our experiments. First of all, the preparations of
A
(25-35), A
(1-39), and A
(1-42) that we have used were in
fibrillary state as demonstrated by the birefringence under polarized
illumination when stained with Congo Red and by the fluorescence when
stained with Thioflavine T. A direct demonstration that only A
peptides in fibrillary state was able to stimulate the production of
H2O2 is reported in Fig.
2. It has been shown that Thioflavine T
associates with fibrils of A
peptides giving rise to an enhanced
emission at 482 nm with excitation at 450 nm (36). Fig. 2A
shows that this occurred when Thioflavine T was added with A
(1-39)
dissolved in PBS and aged 5 days at 37 °C. On the contrary, when
Thioflavine T was added with the peptide dissolved in PBS and kept at
4 °C, the fluorescence emission spectrum of Thioflavine T did not
change, indicating that in this preparation A
(1-39) was not in
fibrillary state. When these two preparations were tested for their
agonistic activity in microglia, the results have shown that only the
aged fibrillary A
peptide was able to stimulate the production of H2O2 (Fig. 2B). Similar results were
obtained in one experiment made with A
(1-42). Furthermore, when
A
(25-35), A
(1-39), and A
(1-42) were dissolved in
Me2SO they did not form fibrils and were unable to
stimulate the production of H2O2 in all cell
types. Fig. 2 reports the result obtained with A
(1-39).

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Fig. 2.
Correlation between the fibrillary state of
A (1-39) and the stimulation of
H2O2 production in microglia.
A, Thioflavine T fluorescence emission spectra: Thioflavine
T (10 µM) in 50 mM potassium phosphate
buffer, pH 6.0, in the absence of peptide (line 1) and in
the presence of 2.5 µM peptide dissolved in
Me2SO (line 2) or dissolved in PBS and kept at
4 °C (line 3) or aged 5 days at 37 °C (line
4). Excitation was 450 nm. B, stimulation of
H2O2 production by 10 µM A
peptide dissolved in Me2SO (bar 2) or in PBS and
kept at 4 °C (bar 3) or aged 5 days at 37 °C
(bar 4) in microglia. Bar C, unstimulated cells.
Results are from one representative of three experiments.
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The second finding was the effect of HS. On the basis of the previous
demonstrations that sulfate or sulfonate compounds bound to the
-pleated sheet conformation of A
fibrils and inhibited the
neurotoxicity of A
peptides (13, 43-45), we have investigated the
effect of these compounds on the ability of A
(25-35), A
(1-39), and A
(1-42) to stimulate the production of
H2O2. The results reported in Fig.
3 show that HS inhibited the stimulation of H2O2 production by A
peptides in
microglia, monocytes, and neutrophils. An unspecific inhibitory effect
of HS can be excluded by the finding that HS did not modify the
response to PMA (Fig. 3).

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Fig. 3.
Effect of HS (final concentration, 2 µg/ml) on the stimulation of
H2O2 production by 10 µM A peptides
and by 2 ng/ml PMA. HS dissolved in double-distilled
H2O was added to the solution of A peptides. The mixture
was kept at room temperature for 5-10 min and then added to the cell
suspension. Results are shown as the means ± S.D. from four
experiments. When PMA was the stimulant, the cells were preincubated
with HS for 5 min, and the results are the means of two
experiments.
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Sources of ROI--
Many sources could be responsible for the
increase in the production of ROI by exposure to A
peptides. The
most common cellular sources of ROI include mitochondrial respiratory
chain, microsomal enzymes, xanthine oxidase, arachidonic acid cascade,
the classical NADPH oxidase of phagocytes, and some unidentified
NADH-NADPH-dependent enzymes (32). Three findings show that
the exposure of the cells to A
peptides induced the stimulation of
ROI production by activating the classical NADPH oxidase. The first was
the direct demonstration of the activated state of the oxidase in cells
treated with A
peptides. This oxidase is composed of several
proteins, and when the cells are in resting state the oxidase is
inactive, and some components are located on the plasma membrane (the
two subunits gp91phox and p22phox of the
flavocytochrome b558), whereas others are in the
cytosol (the p47phox, p67phox, p40phox, and
Rac1/Rac2). The activation of the NADPH oxidase involves multiple
signal transduction pathways, all resulting in the translocation of the
cytosolic components on the plasma membrane where, in association with
the subunits of cytochrome b558, they allow the
oxidation of cytosolic NADPH and the univalent reduction of oxygen with formation of O
2 to take place (34). To identify the NADPH
oxidase as a source of ROI, we investigated whether the stimulation of H2O2 production induced by A
peptides was
associated with the translocation of its cytosolic components on the
plasma membrane. For this purpose we fractionated neutrophils and
monocytes in resting state and after stimulation with A
peptides or
other stimulants and immunoblotted the light membrane fraction with antibodies raised against the cytosolic components p47phox,
p67phox, and p40phox of NADPH oxidase. The results
reported in Fig. 4 show that although the
cytosolic components are practically absent or present in very low
amounts in the plasma membrane of unstimulated neutrophils and
monocytes, they were translocated on the plasma membrane upon stimulation with A
peptides, as occurred after the treatment of the
cells with fMLP alone, fMLP plus cytochalasin B, or PMA. The
stimulation of the production of H2O2 in the
same experiment by various agonists is also reported in the legend of
Fig. 4. It can be seen that despite the facts that the ECL system for detection of proteins is semiquantitative and a very precise
correlation between translocation and activation of NADPH oxidase
cannot be made, the entity of the translocations paralleled those of
the stimulation of H2O2 production. The
translocation of NADPH oxidase components was not feasible on rat
microglial cells because antibodies against the components of the NADPH
oxidase are species-specific and antibodies against components of rat
oxidase are not available.

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Fig. 4.
Translocation on the plasma membrane of
cytosolic components of NADPH oxidase. A, neutrophils:
unstimulated (lane 1), stimulated for 5 min with 10 µM A (25-35) (lane 2), 100 nM
fMLP (lane 3), 100 nM fMLP plus 5 µg/ml
cytochalasin B (lane 4), or 2 ng/ml PMA (lane 5).
B, monocytes: unstimulated (lane 6) or stimulated
for 20 min with 10 µM A (25-35) (lane 7),
10 µM A (1-42) (lane 8), or 2 ng/ml PMA
(lane 9). The cells were fractionated, and the plasma
membrane fractions were processed for the detection of translocated
cytosolic components as described under "Materials and Methods."
Results shown are from one representative of three experiments. In this
experiment the stimulated production of H2O2 in
neutrophils (nmol/5 min/5 × 106 cells) was: plus A
(25-35), 15.4; plus fMLP, 2.6; plus fMLP + cytochalasin B, 13.0; plus
PMA, 12.8. In monocytes the stimulated production of
H2O2 (nmol/20 min/5 × 106
cells) was: plus A (25-35), 9.3; plus A (1-42), 8.9; plus PMA,
15.1.
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The second demonstration of the involvement of the NADPH oxidase was
obtained by using neutrophils and monocytes of the CGD patients. CGD is
an inherited condition wherein the absence of cytochrome b
(X-linked form) or of p47phox or p67phox (autosomal
forms) results in an inactive NADPH oxidase. Thus the leukocytes of CGD
patients are unable to produce ROI in response to all the agonists of
this enzyme. The disease is characterized clinically by recurrent and
sometime life-threatening bacterial and fungal infections due to the
inability of leukocytes to produce ROI and efficiently kill
microorganisms. We analyzed the effect of A
on leukocytes from two
CGD patients (G. N. and Z. N.) affected by X-linked form
lacking cytochrome b558 and one patient (A. M.) affected by an autosomal form lacking p47phox. As shown in
Fig. 5, neutrophils and monocytes of all
the CGD patients did not respond with an increase in
H2O2 production to the exposure to A
peptides or PMA.

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Fig. 5.
The lack of stimulation of
H2O2 production by 10 µM A (25-35),
10 µM A
(1-39), or 100 ng/ml PMA in neutrophils and monocytes of three
CGD patients.
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The last result concerning the involvement of the NADPH oxidase was the
finding reported in Fig. 7 that 10 µM DPI markedly suppressed the stimulation of H2O2 production
by A
peptides in all cell types. This result is not an absolute
demonstration of the involvement of the NADPH oxidase because DPI is
also an inhibitor of other flavoprotein dehydrogenases (46), but it may
be a support to the demonstration that NADPH oxidase is the real source
of H2O2 in cells stimulated with A
peptides.
Mechanism of Activation of NADPH Oxidase by A
Peptides--
It
is known that the activation of NADPH oxidase can be the result of many
signal transduction pathways involving receptor-dependent and -independent agonists, different forms of G proteins, second messengers, protein kinase activation, and phospholipid turnover (33,
47). To understand the mechanisms of NADPH oxidase activation by A
peptides, we investigated the effects of PP1, a Src family-selective tyrosine kinase inhibitor (48), and of WT, an inhibitor of
phosphatidylinositol 3-kinase (49). The results reported in Figs.
6 and 7
show that the stimulation of H2O2 production by
A
(25-35), A
(1-39), and A
(1-42) was markedly sensitive to
micromolar concentrations of PP1 and to nanomolar concentrations of WT.
The results reported in Fig. 7 show also that the stimulation of NADPH
oxidase by A
(25-35), A
(1-39), and A
(1-42) was inhibited
by 1 mM dibutyryl cyclic AMP, indicating that protein
kinase A could play a negative role in the transduction pathway of
NADPH oxidase activation by A
peptides. All the inhibitors used were
not toxic for the cells as shown by the lack of effect on the
stimulation of H2O2 production by PMA (data not
shown).

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Fig. 6.
Effects of different doses of PP1 and WT on
the stimulation of H2O2 production by 5 µM A (1-42) in
microglia. Cells were preincubated with vehicle
(Me2SO, 1 µl/ml), PP1, or WT for 5 min before
stimulation. 100% value is 0.16 nmol
H2O2/3 × 105 cells/30 min.
Results are the means of two experiments.
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Fig. 7.
Effect of 5 µM PP1, 20 nM WT, 1 mM dibutyryl cAMP, and 10 µM DPI on the stimulation of
H2O2 production by 10 µM A (25-35),
A (1-39), or A
(1-42), in microglia, monocytes, and neutrophils. The cells
were incubated with all the compounds 5 min prior to stimulation.
Results are shown as the means ± S.D. from eight experiments with
A (25-35) and four with A (1-39) and A (1-42). With DPI the
results are the means of two experiments. 100% values (expressed as
nmol H2O2/30 min) in cells stimulated with A
(25-35), A (1-39), and A (1-42), respectively, are: 0.32 ± 0.06, 0.24 ± 0.07, and 0.21 ± 0.04 in 3 × 105 microglia; 0.70 ± 0.11, 0.60 ± 0.09, and
0.69 ± 0.13 in 3 × 105 monocytes; and 1.48 ± 0.38, 1.02 ± 0.22, and 0.98 ± 0.14 in 5 × 104 neutrophils.
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Potentiation of the NADPH Oxidase Response to A
Peptides by
IFN-
or TNF-
--
Previous works in our (50) and other (51, 52)
laboratories have shown that the stimulus-induced activation of the
NADPH oxidase of macrophages can be potentiated by treatment with
cytokines, mostly IFN-
or TNF-
, and that this potentiation is due
to the increased expression of oxidase components (50-54). It has been also shown that the maturation of monocytes to macrophages resulted in
a progressive loss of the capability to produce ROI (51, 53) due to
decreased expression of NADPH oxidase components (50, 51, 54). The
expression of these components and the stimulus-induced activation of
the oxidase were restored by treatment with IFN-
(50, 54).
Microglial cells are resident macrophages derived from blood monocytes,
and their low responsiveness to A
peptides compared with that of
monocytes probably reflects the down-regulation of NADPH oxidase
components during the maturation. We investigated here whether or not
IFN-
or TNF-
were able to potentiate the stimulation of
H2O2 production by A
peptides in microglia.
For this purpose we grew the primary culture of rat microglial cells
for 4-5 days in presence of 50 units/ml IFN-
or 50 units/ml
TNF-
, and then we compared the stimulation of H2O2 production by A
peptides of these cells
with that of cells grown in the absence of cytokines. The results
reported in Table II show that microglial
cells treated with either IFN-
or TNF-
presented a marked
potentiation of the production of H2O2 when exposed to A
peptides.
 |
DISCUSSION |
According to the most accepted theory, the excessive formation of
A
from
-amyloid precursor protein and its deposition in the brain
is the key event in the progressive neuronal damage in AD (4, 5, 8).
The studies performed on the neurotoxic effect of A
peptides (7,
11-26) have resulted in the concept that many mechanisms, direct or
indirect, are responsible for neurotoxicity. In this paper we confirm
the existence of indirect mechanisms involving the stimulation by A
peptides of microglia, a cell type of the mononuclear phagocyte system,
to produce inflammatory and cytotoxic mediators (24-28, 31, 55, 56).
The results presented here indicate that the activation of classical
O
2 forming NADPH oxidase is involved in the stimulation of the
production of ROI in microglia by A
peptides. Two experimental
results supported this indication. One was the finding that the
stimulation of H2O2 production was associated
with the translocation of cytosolic factors p47phox,
p67phox, and p40phox to the plasma membrane, a process
that corresponds to the activation of the oxidase (34, 40). The second
was the finding that neutrophils and monocytes of patients affected by
CGD, a genetic condition lacking components of the NADPH oxidase, did
not respond to the exposure to A
peptides with the stimulation of
H2O2 production. The possibility that
mechanisms other than NADPH oxidase are responsible for the stimulation
of ROI production by A
peptides cannot be ruled out. However, the
finding that leukocytes of CGD patients are unresponsive to A
suggests that the involvement of other mechanisms is unlikely. These
data and those previously obtained in our (24, 26) and other (25)
laboratories support the hypothesis that A
peptides activate two
indirect mechanisms of oxidative damage involving microglia. One would
be the induction of the NO synthase and production of NO, the other is
the stimulation of O
2 and other ROI generation by activated
NADPH oxidase.
In agreement with the data concerning the direct neurotoxicity (7, 12,
13, 43, 44), we have shown here that also for the stimulation of
H2O2 production A
peptides were active in
aggregated fibrillary form. The type of interaction of A
peptides with the cell surface is controversial. Data have been provided that
scavenger receptors (29, 57-60), advanced glycation end product
receptors (19), tachykinin receptors (11), and serpin-enzyme complex
receptors (61) would be involved. In our experiments, the activation of
NADPH oxidase due to the interaction of A
peptides with scavenger
receptors is possible in microglia because in these cells these
receptors are well expressed (29, 57-60). On the contrary the
involvement of scavenger receptors in neutrophils or monocytes should
be excluded because these receptors are absent in neutrophils and
absent or minimally expressed in monocytes (59, 60). The participation
of advanced glycation end product receptors in the activation of
microglia by A
peptides has been denied (31), and our results that
glycated human serum albumin was unable to stimulate the NADPH oxidase
in microglia, monocytes, and neutrophils agree with this (Table I). The
involvement of tachykinin receptors and serpin-enzyme complex receptors
in the stimulation of NADPH oxidase remains to be investigated. Data have been also provided showing that A
peptides interact with membranes to form cation-selective channels (62) or disrupt the
integrity of plasma membrane by virtue of their lypophilic nature
(63-65). Further studies are needed to understand whether the effect
of A
peptides on NADPH oxidase may be attributable to their
detergent properties (63) or to a receptor-mediated interaction.
It is known that various reactions participate in the signal
transduction for NADPH oxidase activation. Some of these,
i.e. the phosphorylation of cytosolic components, are
constantly activated regardless of the agonist used, whereas others,
i.e. the stimulation of phospholipase C, D, and A2, protein
kinase C, mitogen-activated protein kinase, and trimeric G proteins,
are differently recruited depending on the agonist used (33, 34, 47).
The inhibition by PP1, WT, and dibutyryl cAMP supports the concept that
the activation of NADPH oxidase by A
peptides involves the
participation of Src family of tyrosine kinases (31) and of
phosphatidylinositol 3-kinase and is counteracted by the activation of
protein kinase A. Further investigations are needed to clarify the role
of these enzymes.
The activation of NADPH oxidase occurred with A
peptides alone and
did not require previous priming as, on the contrary, occurred for the
induction of NO synthase that required a priming by IFN-
(24-26).
Previous findings in our (50) and other (51-54) laboratories have
shown that in monocytes and macrophages IFN-
or TNF-
increased
the expression of components of NADPH oxidase and potentiated the
production of ROI by various agonists. We have demonstrated here that
microglial cells treated for some days with IFN-
or TNF-
presented a response to A
peptides much greater than that of control
cells (Table II). Microglial cells are resident macrophages, and their
low response to A
peptides compared with that of monocytes reflects
a down-regulation of the components of the oxidase during the
maturation from monocytes to macrophages (50, 51, 54). A
peptides
alone are able to stimulate the production of TNF-
in microglia
(24), so that one may speculate that this process might, by priming
microglial cells with autocrine or paracrine mechanisms, potentiate the
production of ROI in response to A
peptides in AD.
Finally we discuss briefly the finding (Fig. 3) that the binding of
heparan sulfate to A
fibrils inhibited the interaction with the cell
surface and the stimulation of H2O2 production. This result agrees with those showing that sulfate or sulfonate compounds inhibited the direct toxicity of A
peptides on neurons in vitro (13, 43-45). Because in AD heparan sulfate is
associated with A
fibrils in the neurite plaques (66, 67), one
wonders if in vivo the fibrils covered by heparan sulfate
are impeded in their interaction with the cell surface of neurons and
microglial cells and consequently inhibited in their ability to express
their direct neurotoxicity (43-45) and stimulate microglia to produce toxic compounds. If this were the case the amount of heparan sulfate in
the plaques and its interaction with A
fibrils would play a
regulatory role on the biological activity of A
fibrils.
In conclusion, the results presented in this paper agree with the view
(56) that the pathogenesis of Alzheimer's disease includes also a
series of molecular events characteristic of an inflammatory reaction.
These events would be triggered by the interaction of
-amyloid
fibrils with microglial cells resulting in the activation of NF-
B
(68), production of cytokines such as TNF-
(24), interleukin-8 (27),
MCP-1 (55), and interleukin-1 (69, 70), production of ROI (31), and
induction of NO synthase with generation of NO (24). It is widely
accepted that the control of the production of these inflammatory
molecules could be one of the therapeutic strategies of Alzheimer's
disease. On this view, the finding that the O
2 forming NADPH
oxidase is involved adds new tools to this therapeutic design.