Published online before print May 10, 2004
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* Laboratoire de Pathologie des Communications entre Cellules Nerveuses et Musculaires, EA 3429, Faculté de Pharmacie, and
Clinique Neurologique 2, Service des maladies du Nerf et du Muscle, Hôpitaux Universitaires, Faculté de Médecine, Université Louis Pasteur de Strasbourg, Cedex, France
1Correspondence: Laboratoire de Pathologie des Communications entre Cellules Nerveuses et Musculaires, EA 3429, Faculté de Pharmacie, Université Louis Pasteur, 74, route du Rhin-BP 24, F-67401 Illkirch, Cedex, France. E-mail: poindron{at}pharma.u-strasbg.fr
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Key Words: amyloid ß-protein senile plaque Alzheimers disease scavenger receptor A phagocytosis endocytosis
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To prevent neurotoxicity of Aß peptides, whichever their conformation and physical state, an organism can clear them using four mechanisms: transport of nfAß and fAß peptides in excess through the blood-brain barrier [5
]; clearance of fAß peptides through receptor-mediated endocytosis [6
]; extracellular catabolism of nfAß and fAß peptides [4
, 7
], especially through the action of neprilysin, a membrane-anchored, neutral endoprotease [8
]; and pinocytosis of extracellular nfAß peptides [9
]. Improper clearance of Aß may play a major role in AD pathogenesis [4
, 10
]. Microglial cells migrate and surround the Aß deposits [11
]. The presence of activated (possibly by Aß) microglial cells within senile plaques suggests that these cells may contribute to AD pathogenesis by removing or producing amyloid fibrils. Microglial cells are cerebral, phagocytic mononuclear cells. They are primarily involved in inflammatory response of the brain. Activated microglial cells, indeed, are able to release cytotoxic agents such as cytokines [12
], proteases [13
], nitric oxide [14
], or reactive oxygen intermediates [15
]. Although the available data suggest that activated microglial cells are mainly harmful in AD, one cannot exclude that they are also involved in the removal and possibly digestion of amyloid fibers. The role of microglial cells in AD pathogenesis would be ambivalent. It has been proved that cultured microglial cells can internalize small aggregates of fAß [6
, 9
, 16
17
18
19
]; injection of fAß aggregates into the rat striatum lead to their phagocytosis by microglial cells [14
]; and microglial cells from brains of patients with AD are stained with anti-Aß peptide antibodies [20
21
22
23
]. Uptake of Aß is mediated by various receptors, such as class A scavenger receptors (SR-A) [17
], Fc receptors [24
], and low-density lipoprotein receptor-related protein (LRP), provided that Aß is complexed to
2-macroglobulin (
2M) [25
], lactoferrin [25
], or apolipoprotein E (APO-E) [26
], which are ligands of LRP.
To study the interactions of Aß peptides with microglial cells in vitro, we have developed an experimental system consisting of heat-killed yeast particles opsonized with Aß 140 or Aß 142 peptides (subsequently, shortly referred to as Aß 140- and Aß 142-opsonized, heat-killed yeast particles, respectively). We have used four different forms of Aß peptides: soluble peptides (aqueous solution); aggregates of fibrillar peptides (heated aqueous solution); deposits of nf peptides (nfAß-opsonized, heat-killed yeast particles); and deposits of f peptides (fAß-opsonized, heat-killed yeast particles). nfAß-opsonized, heat-killed yeast particles mimicked diffuse senile plaques, and fAß-opsonized, heat-killed yeast particles mimicked amyloid core of neuritic senile plaques. We supposed that microglial cells could internalize Aß peptides through receptor-mediated endocytosis or through phagocytosis, depending on the size (aggregates or deposits) of the preparation used. By using competitive ligands, it was possible to explore interactions between fAß aggregates or deposits and several kinds of microglial cell receptors and follow the fate of opsonized, heat-killed yeast particles.
In this work, we present results, which validate the use of Aß-opsonized, heat-killed yeast particles as artificial diffuse or amyloid plaques; indicate that these artificial deposits could be ingested by microglial cells using different receptors; and show that LRP is partly involved in phagocytosis of amyloid deposits.
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-mannans (extracted from Saccharomyces cerevisiae), lactoferrin,
2M, and methylamine were purchased from Sigma (LIsle dbeau-Chesnes, France). 125I-laleled Aß 140 and Aß 142 peptides were obtained from Amersham France SA (Les Ulis). Tannic acid was obtained from Merck (Darmstadt, Germany). Receptor-associated protein (RAP) was a kind gift from S. K. Moestrup (Department of Clinical Chemistry, AKH Aarhus University Hospital, Aarhus, Denmark).
Activation of
2M
To expose its receptor recognition site,
2M was incubated in aqueous solution consisting of methylamine (200 mmol/l), sodium phosphate (50 mmol/l), sodium chloride (150 mmol/l), pH 7.4, for 1 h at room temperature. Methylamine was removed by extensive dialysis against a 0.1 mmol/l sodium bicarbonate, 0.5 mmol/l sodium chloride, pH 8.3, aqueous solution. The methylamine-treated
2M will be subsequently referred to as
2M+.
Electron microscopy
Amyloid fibrils were obtained by incubation of Aß in aqueous solutions at 37°C. Formation of fibrils was checked at regular intervals by negative staining of the heated preparations with a 2% uranyl acetate aqueous solution and observation with a Philipps EM 410 electron microscope.
Congo red staining
fAß preparations were mixed with a 6.6% solution of Congo red (C.I 22120, Sigma) in a mixture (v/v) of phosphate-buffered saline (PBS; 60%) and ethanol (40%) in such a way that the final concentration of Congo red was 6 µM and that of Aß, 90 µg/ml, as described [27
]. The mixture is made directly in wells of 96-well plates. The plates were maintained for 15 min at room temperature, and the absorbance of the mixture was recorded at 340 nm, 405 nm, 450 nm, 492 nm, 540 nm, 570 nm, 600 nm, and 620 nm.
Yeast
Heat-killed yeast suspension was prepared from a local strain of S. cerevisiae. Briefly, yeast was grown in Sabourauds broth for 48 h at 28°C under agitation and then autoclaved for 45 min at 120°C in the culture medium. Heat-killed yeast suspension was washed three times in PBS without calcium and magnesium (PBS). The stock suspension contained
4 x 109 particles/ml. It was aliquoted and stored at 4°C until use.
Preparation of Aß-opsonized, heat-killed yeast particles
Heat-killed yeast suspension (200x106 particles) was centrifuged for 6 min at 3200 g. Supernatant was removed, and the pellet of heat-killed yeast particles was mixed with 20 µl fAß (or in some cases, nfAß) peptide aqueous solution. After an overnight incubation at room temperature, the heat-killed yeast suspension was washed three times in PBS and stored in PBS. To study the stability of the Aß peptide binding to the heat-killed yeast particles, aqueous solutions of 125I-labeled nfAß 140 or nfAß 142 peptides were mixed to cold, aqueous solutions of nfAß 140 or nfAß 142 peptides in a molar ratio of 1 to 20,000 at a total concentration of 1 mg/ml and allowed to transform into fAß as described above. Heat-killed yeast particles were then opsonized with these radioactive preparations. Heat-killed, yeast-associated radioactivity was measured at regular intervals by
-counting, after three washes in PBS.
Microglial cells
The microglial cell line used throughout this study was established from a newborn BALB/c mouse cortex freed of meninges, as described for obtaining peritoneal macrophage cell lines [28
]. After cortices were cut in pieces less than 1 mm3, the explants were transferred into 50 ml centrifugation tube, and the volume was adjusted to 30 ml per brain with RPMI-1640 medium (Life Technologies, Cergy-Pontoise, France) containing 10% inactivated fetal calf serum (FCS; Life Technologies), 105 U/l penicillin, 50 mg/ml streptomycin (Laboratoires Diamant, Puteaux, France). The resuspended explants were transferred into 25 cm2 plastic-culture flasks (Poly Labo Paul Block et Cie, Strasbourg, France) and incubated at 37°C under 5% CO2. Cells were regularly subcultured as described [28
]. Briefly, cultured cells were mostly adherent. However, some cells spontaneously passed into the culture medium (
50,000 cells/ml culture medium; viability exceeding 98%, as assessed by trypan blue exclusion test). These cells were identified as microglial cells (Fig. 1
) by their reactivity with the biotin-conjugated Griffonia (Bandeiraea) simplicifolia isolectin B4 (Sigma) [29
]. Cells passed into suspension were allowed to adhere to 14-mm-diameter, sterile, glass coverslips placed in 24-well plates (Nunclon, Poly Labo Paul Block et Cie). Under these conditions, cells adhered very rapidly to the support. The plates were incubated for 24 h at 37°C in an air (95%)CO2 (5%)-humidified incubator. Microglial cell preparations were then treated or not with cytochalasin B (Sigma) before phagocytosis. For cytochalasin treatment, they were washed with PBS and incubated for 20 min in PBS containing the appropriate cytochalasin B concentration.
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Figure 1. Identification of microglial cells by staining with biotin-conjugated G. simplicifolia isolectin B4. (a) Aspect of microglial cells after incubation with biotin-conjugated lectin and revelation with streptavidin-conjugated peroxidase. (b) Control cells: aspect of a cell not treated with lectin before adjunction of streptavidin-conjugated peroxidase. Note the stellate outlines of these cells.
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Phagocytosis
In a previous study, we had observed that heat-killed yeast particles were ingested by murine macrophages in culture through a phagocytic pathway involving mannose and ß-glucan receptors and called lectinophagocytosis [30
]. To investigate the specific phagocytosis of Aß-opsonized, heat-killed yeast particles by microglial cells, it was therefore necessary to block the normal phagocytic pathway of heat-killed yeast particles by ligands of mannose and ß-glucan receptors, namely soluble
-mannans and laminarin, respectively.
Just before addition of Aß-opsonized, heat-killed yeast suspension, the cells were washed once with PBS and treated with 450 µl PBS containing laminarin (0.4 mg/ml),
-mannans (0.4 mg/ml), and the competitor ligand to be studied at the required concentration. Ten minutes later, 30 µl Aß-opsonized, heat-killed yeast (
50 particles per cell) was added to each well. The plates were incubated for 45 min at 37°C in an air (95%)CO2 (5%)-humidified incubator. The cells were then washed three times with PBS. To discriminate adherent and ingested Aß-opsonized, heat-killed yeast particles, the preparations were stained as described [31
]. Briefly, the cells were treated for 1 min with a 1% tannic acid solution in PBS, washed with PBS, stained with May-Grünwald Giemsa solutions, and then mounted, cell-side down, on glass slides. Adherent, noningested Aß-opsonized, heat-killed yeast appeared as violet particles and ingested Aß-opsonized, heat-killed yeast, as pink particles (Fig. 2
). At least 100 microglial cells were observed per coverslip at a magnification of 1,000. Data were expressed in terms of mean number of Aß-opsonized, heat-killed yeast particles ingested (MNI) or adhering (MNA) per cell, that is, the total number of ingested or adhering particles counted divided by the total number of cells observed.
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Figure 2. Discrimation between adhering (dark) and ingested (clear), heat-killed yeast particles by the Giaimis method [31
].
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-mannan and laminarin. After a 45-min incubation, the cells were washed with PBS and mechanically lysed. Cell-associated, heat-killed yeast particles were then collected and washed three times with PBS. Yeast-associated radioactivity was measured by
-counting.
Internalization of free fAß
To verify that free fAß were readily ingested, microglial cells were incubated for 55 min with radioactive fAß. Unbound amyloid fibrils were collected by washing three times the preparations, cell-surface-bound fibrils were removed by treating the cells for 10 min with a solution of 0.2 mol/l acetic acid containing 0.5 M NaCl, and internalized fibrils were collected by dissolving the cells in boiling 1 mol/l NaOH for 1 h. Then, internalized, cell-surface-bound and unbound Aß were determined by
-counting.
Opsonization of Aß-opsonized, heat-killed yeast particles by free fAß during competitive experiments
To verify that Aß-opsonized, heat-killed yeast particles do not adsorb the free fAß used as competitor in some experiments, fAß-opsonized, heat-killed yeast particles (5x106 particles) were incubated for 45 min with radioactive amyloid fibril (total concentration of fAß=106 mol/l). Particles were washed three times with PBS to remove fibrils. Supernatant radioactivity and yeast-associated radioactivity were evaluated by
-counting.
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15 µm [14
].
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Figure 3. Appearance of fibrils formed upon heating of soluble Aß peptides for 5 days at 37°C. (ac) Unheated solutions. (eg) Heated solutions. (a, e) Aß 2535. (b, f) Aß 140. (c, g) Aß 142. (d, h) Heated solutions of Aß 1525 or Aß 3525, respectively. Note that fibrils (diameter: 0.010.02 µm) formed only with Aß 2535, Aß 140, or Aß 142 peptides. (i) Absorption spectra of Congo red solution ( ) or Congo red in the presence of fAß (Ab) 140 () or fAß (Ab) 142 ( ). Note the characteristic, spectral shift of Congo red, which proves the amyloid structure of the heated fibrillar Aß peptides.
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fAß peptides bind to heat-killed yeast particles
To determine whether Aß peptides were able to bind to heat-killed yeast particles, mixtures of cold and radio-iodinated nfAß or fAß aqueous solutions were added to heat-killed yeast particles initially washed with PBS-. The mixtures were kept at room temperature overnight. The suspensions were then centrifuged at 3,200 g for 6 min, and the pellets were washed three times in PBS to eliminate unbound Aß. Radioactivity of resulting suspensions was assayed on an aliquot, and the results were referred to as zero-time radioactivity. The same operations were done on the remaining suspension every day after zero time. Figure. 4a
and 4b
, shows that fAß 140, fAß 142, and nfAß 142 could bind to heat-killed yeast particles, whereas nfAß 140 could not (zero time). However, binding of nfAß 142 is rather low. After 3 days,
50% of fAß 140 and fAß 142 remained bound. Therefore, it was possible to use fAß 140- and fAß 142-opsonized, heat-killed yeast particles as artificial amyloid plaques and nfAß 142-opsonized, heat-killed yeast particles as artificial diffuse plaques.
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Figure 4. Heat-killed yeast particles can be opsonized by Aß peptides. (a) A mixture of 125I-labeled nfAß (nfAb) 140 and cold nfAß 140 was allowed to transform into amyloid fibrils () or not (fAb; ). Heat-killed yeast particles were then treated by these radioactive preparations. Yeast-associated radioactivity was measured at regular intervals by -counting after three washes in PBS. (b) Same experiment using 125I-labeled nfAß 142 and cold nfAß 142.
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-mannans not only almost completely inhibited lectin-like, receptor-mediated phagocytosis of heat-killed yeast particles but ingestion of Aß 3525- or Aß 1525-treated, heat-killed yeast particles, whether these peptides were heated or not before the treatment. From these results, we concluded that when heat-killed yeast particles were opsonized with fAß 140 or fAß 142, they can be ingested by a process involving specific receptors.
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Figure 5. Ingestion of Aß-opsonized, heat-killed yeast particles or heat-killed yeast particles treated with different Aß-related peptides (Ab). After a 24-h culture on glass coverslips, microglial cells were pretreated with inhibitors of a lectin-like, receptor-mediated ingestion pathway and were then incubated for 45 min with heat-killed yeast particles treated with different Aß-related peptides preincubated (solid bars) or not (open bars) at 37°C. Ingested particles were counted on 100 cells randomly selected, and three wells were observed for each condition. Data are expressed as the MNI ± SEM. ***, P < 0.001; **, P < 0.01.
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-mannan and laminarin. To ensure that Aß-opsonized, heat-killed yeast particles were ingested in their opsonized state, experiments of phagocytosis were performed with radioactive, fAß-opsonized, heat-killed yeast particles in the presence of
-mannan and laminarin, and radioactivity was measured. As shown in Figure 6
, a significant amount of cell-associated radioactivity could be detected, indicating that Aß-opsonized, heat-killed yeast particles were adhering or ingested by microglial cells.
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Figure 6. After a 24-h culture on glass coverslips, microglial cells were pretreated for 10 min with inhibitors of lectinophagocytosis and were then incubated for 45 min with [125I]fAß-opsonized (Ab), heat-killed yeast particles. Microglial cell-associated particles were collected and washed, and radioactivity of suspension was measured by -counting. Error bars represent SEM. **, P < 0.01; *, P < 0.05.
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Figure 7. Effect of cytochalasin B on ingestion of fAß-opsonized, heat-killed yeast particles by microglial cells. After a 24-h culture on glass coverslips, microglial cells were pretreated for 30 min with variable concentrations of cytochalasin B, then treated with lectinophagocytosis inhibitors for 10 min, and incubated for 45 min with fAß 140-opsonized, heat-killed yeast particles (a) or fAß 142-opsonized, heat-killed yeast particles (b). Ingested and adhering particles were counted on 100 cells randomly selected, and three wells were observed for each condition. Data are expressed as the MNI and the MNA ± SEM. Note that cytochalasin B diminished the number of ingested particles but did not modify the number of adhering particles. ***, P < 0.001; *, P < 0.05. Statistics were performed for each value versus untreated cells.
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Figure 8. Free fAß and nFAß peptides do not apparently inhibit ingestion of fAß-opsonized, heat-killed yeast particles by microglial cells. After a 24-h culture on glass coverslips, microglial cells were pretreated for 10 min with inhibitors of lectinophagocytosis and fAß peptide () or nf Aß peptide ( ), then incubated for 45 min with fAß 140 (Ab 140)-opsonized, heat-killed yeast particles (a) or fAß 142 (Ab 142)-opsonized, heat-killed yeast particles (b). Competitors used are Aß 140 (a) and Aß 142 (b). Ingested particles were counted on 100 cells randomly selected, and three wells were observed for each condition. Untreated cells serve as a control and represent 100% of phagocytosis. Error bars represent SEM. Statistics were performed for each value versus untreated cells, and no significant difference was found. (En: E minus n)
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Figure 9. Ingestion of free amyloid fibrils by microglial cells. After a 24-h culture on glass coverslips, microglial cells were incubated for 55 min with radioactive fAß 140 (a) or fAß 142 (b). Ingested ( ), cell-surface bound ( ), or extracellular ( ) fAß was collected and measured by -counting. Error bars represent SEM.
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Figure 10. Adsorption of free fAß on fAß-opsonized, heat-killed yeast particles during competition experiment. fAß-opsonized, heat-killed yeast particles were incubated for 45 min with free, radioactive fibrils of Aß. Then, fAß-opsonized, heat-killed yeast particles and radiolabeled fAß were separated by centrifugation, and radioactivity of the pellet of fAß-opsonized, heat-killed yeast particles (open bars) or supernatant (solid bars) was measured by -counting. The experiment was done with fAß 140-opsonized, heat-killed yeast particles and fAß 140 (Ab 140) or fAß 142-opsonized, heat-killed yeast particles and fAß 142 (Ab 142). Note that fAß-opsonized, heat-killed yeast particles do not absorb free fAß. Error bars represent SEM. ***, P < 0.001.
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-mannans. Figure 11
shows that none of these SR-A ligands was able to inhibit ingestion of fAß 140- or fAß 142-opsonized, heat-killed yeast particles.
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Figure 11. Ligands of SR-A do not inhibit phagocytosis of fAß-opsonized, heat-killed yeast particles by microglial cells. After a 24-h culture on glass coverslips, microglial cells were pretreated for 10 min with lectinophagocytosis inhibitors and fucoidin (a) or dextran sulfate (b) and were then incubated for 45 min with fAß 140 (Ab 140)-opsonized, heat-killed yeast particles () or fAß 142 (Ab 142)-opsonized, heat-killed yeast particles ( ). Ingested particles were counted on 100 cells randomly selected, and three wells were observed for each condition. Untreated wells serve as a control and represent 100% of phagocytosis. Error bars represent SEM. Statistics were performed for each value versus untreated cells, and no significant difference was found.
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-mannans, and ligands known to recognize LRP, namely lactoferrin,
2M+, or the 39-kD antagonist RAP, were added to the cultures before addition of fAß 140- or fAß 142-opsonized, heat-killed yeast particles. Figure 12a
shows that in the presence of lactoferrin, ingestion of fAß 140-opsonized, heat-killed yeast particles was not impaired, whichever the concentration of the competitor ligand used, whereas that of fAß 142-opsonized was inhibited to a degree depending on that concentration. Similar observations (Fig. 12b) were made when
2M+ was used. At a concentration of 300 nmol/l, ingestion of fAß 142-opsonized, heat-killed yeast particles was inhibited by
50%, whereas that of fAß 140-opsonized, heat-killed yeast particles remained unaffected. In addition, RAP (at a concentration of 100 nmol/l) also partly inhibited (20%) phagocytosis of fAß 142-opsonized, heat-killed yeast particles but did not reduce that of fAß 140-opsonized, heat-killed yeast particles (Fig. 12c)
.
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Figure 12. Differential effects of ligands of LRP on phagocytosis of fAß-opsonized, heat-killed yeast particles by microglial cells. After a 24-h culture on glass coverslips, microglial cells were pretreated for 10 min with lectinophagocytosis inhibitors and lactoferrin (a), 2M+ (b), or RAP (c) and were then incubated for 45 min with fAß 140 (Ab 140)-opsonized, heat-killed yeast particles () or fAß 142 (Ab 142)-opsonized, heat-killed yeast particles ( ). Ingested particles were counted on 100 cells randomly selected, and three wells were observed for each experiment. Untreated wells serve as a control and represent 100% of phagocytosis. Error bars represent SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05. Statistics were performed for each value versus untreated cells.
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2M+ effectively competes with fAß 142-opsonized, heat-killed yeast particles for LRP from BV-2, as elimination of LRP ligands before introduction of fAß 142-opsonized, heat-killed yeast particles does not modify the amount of particles ingested by BV-2.
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Figure 13. Ingestion of fAß 142-opsonized, heat-killed yeast particles by BV-2 microglial cell line. After a 24-h culture on glass coverslips, BV-2 were pretreated with inhibitors of lectin-like, receptor-mediated ingestion pathway and ligands of LRP, were then washed three times with PBS+ (open bars) to remove unbound inhibitors, and were incubated for 45 min at 37°C with fAß 142-opsonized, heat-killed yeast particles and lectinophagocytosis inhibitors. Controls were done as follow: BV-2 were pretreated with -mannans, laminarin, and LRP ligand and were then incubated for 45 min with fAß 142-opsonized, heat-killed yeast particles (solid bars). Error bars represent SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
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fAß-opsonized, heat-killed yeast particles could be structured as amyloid deposits. These deposits consist of thin Aß fibers, perpendicularly bound to long fibers of heparan sulfate proteoglycan, the external part of which is enriched in electronegative, superficial charges of heparan sulfate [34 ]. The negative charges probably facilitate binding of Aß fibers. Here, we show that fAß 140 and fAß 142 peptides were able to bind to heat-killed yeast particles deprived in calcium and magnesium by washing with PBS.
The conformational and physical states of Aß peptides are very likely the main features that will determine their fate (pathway of internalization and intracellular processing). We show here that aggregates of Aß peptides do not use the same internalization pathway as deposits to enter microglial cells: Indeed, neither free fAß 140 nor fAß 142 peptides could compete for microglial Aß peptide receptors with fAß 140- or fAß 142-opsonized, heat-killed yeast particles. Therefore, it is probable that the receptors involved in internalization of aggregates (fAß) or deposits (fAß-opsonized, heat-killed yeast particles) of fAß peptides are not identical; fAß 140 and fAß 142 deposits are phagocytosed by microglial cells through different receptors. The molecular mechanisms of receptor-mediated endocytosis and phagocytosis are quite different and depend on the size of elements to be internalized [35 ]. The mode of ingestion probably will determine the intracellular fate of Aß peptides, as these peptides can be directed to different populations of endosomes, depending on their mode of internalization [36 ]. That Aß peptides use the receptor-mediated endocytic pathway is proved by numerous studies. In vitro, Aß peptides can be cleared from culture medium by several cell types, such as smooth muscle cells [37 ] or neuroblastoma cells [38 ], which are unable to phagocytose particulate substances. Monocytes [39 ] and microglial cells [9 , 16 , 40 ] were also proved to clear Aß peptides from culture medium. However, in these studies, the size of aggregates was not always mentioned, so that it is difficult to draw definite conclusions on the mechanism of ingestion. Whatever the way of entry, it seems, however, that Aß peptides can persist several days in cells. Ingested fAß peptides remained 20 days in phagosomes [41 ] and 12 days in endosomes [9 ].
Our results show that nf and fAß 142- and fAß 140-opsonized, heat-killed yeast particles could be internalized by microglial cells through a phagocytic mechanism (effect of cytochalasin B) involving receptors differing from those used by nonopsonized, heat-killed yeast particles (that is, mannose and ß-glucan receptors). We attempted, therefore, to identify the receptors involved in ingestion of fAß-opsonized, heat-killed yeast particles. To date, indeed, the receptors possibly involved in elimination of fibrillar, potentially neurotoxic, Aß peptides are not known. SR-A have been suspected to play a role in Aß peptide clearance: In vitro, indeed, SR-A of microglial cells have been shown to bind aggregated Aß 142 peptides [17 ]. However, other studies did not support this hypothesis. It has been observed, for instance, that fAß peptides did not bind to SR-A of human peripheral blood monocytes in vitro [42 ]. It has also been found that deletion of SR-A in a murine model of AD reduced neither the amount of amyloid deposit nor neurodegeneration [43 ]. A number of other receptors are suspected to play a role in Aß clearance, such as formyl peptide receptor (FPR) or receptor for advanced glycation end-products (RAGE). The role of these receptors is rather indirect, as binding of Aß to FPR or RAGE induces a release of chemokines [44 , 45 ] that recruit phagocytic microglia and improve the uptake of Aß [46 , 47 ]. We observe here that fAß 140- and fAß 142-opsonized, heat-killed yeast particles were not recognized by these receptors, as they did not compete for them with fucoidin or dextran sulfate. It is possible (although unlikely) that recognition of aggregated Aß 140 or Aß 142 peptides observed in vitro resulted from the poor specificity of SR-A, which recognize a broad range of ligands, provided that they are denaturated. SR-A indeed are multiligand receptors that bind various proteins and lipoproteins modified by glycation, acetylation, or oxidation [32 ].
Recent work has shown that LRP is involved in the clearance of soluble Aß 140 and/or Aß 142 peptides, provided that they (it) are (is) complexed to one of their (its) ligands, namely APO-E [26
], lactoferrin, or
2M [25
]. LRP is a multifunctional cell-surface receptor expressed by microglial cells [48
]. It could play a role in AD pathogenesis by participating in uptake and degradation of ß-amyloid precursor protein [49
] and modulating the severity and age of onset of AD in relation to the polymorphism of exons 3 and 6 [50
, 51
]. Moreover, two of its ligands, APO-E [52
] and
2M [53
, 54
], have been identified as genetic risk factors for late-onset AD; both ligands binds Aß in vitro and mediate its internalization, and several ligands of LRP can be detected in senile plaques, using immunostaining, especially APO-E,
2M, lactoferrin, lipoprotein lipase, tissue, and urokinase-like plasminogen activators and plasminogen activator inhibitor-1 [55
, 56
]. Finally, it has been shown that a low expression of LRP in brain is associated with AD [57
]. In these studies, it is postulated that nfAß and fAß are internalized through receptor-mediated endocytosis or possibly fluid-phase endocytosis. It has also been shown that decreased levels of LRP in brain correlated with increased formation of senile plaques, suggesting that LRP modulate Aß peptide deposition and is involved in its clearance [57
]. However, it has never been suggested that LRP was implicated in the direct clearance of fAß 140 or 142 amyloid deposits. fAß 142-opsonized, heat-killed yeast particles could partly compete with lactoferrin,
2M+, or RAP for LRP. This indicated that LRP probably can directly bind amyloid deposits of fAß 142 peptide, provided it is correctly presented. However, competitor ligands never succeeded in totally inhibiting ingestion of fAß 142-opsonized, heat-killed yeast particles. There should therefore exist another receptor responsible for their residual ingestion. By contrast fAß 140-opsonized, heat-killed yeast particles did not compete with lactoferrin,
2M+, or RAP. It is therefore very probable that fAß 140 cannot recognize LRP. Whether the receptors involved in this phenomenon are the same as those responsible for the residual ingestion of fAß 142-opsonized, heat-killed yeast particles in the presence of lactoferrin,
2M+, or RAP remains to be determined. It is the first observation showing that fibrillar Aß 140 and Aß 142 deposits can be processed by microglial cells through different phagocytic pathways. These results confirm the role of LRP in the Aß metabolism.
Received December 8, 2003; revised March 30, 2004; accepted March 31, 2004.
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