ARTICLE |
Correspondence to: Marcel M. Verbeek, University Medical Center Nijmegen, Dept. of Neurology, 319 LKN, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: m.verbeek@ckslkn.azn.nl
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Summary |
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It has been suggested that the successive proteolytic events leading to the production of the amyloid-ß protein from its precursor may take place at different intracellular locations. Using cultured human leptomeningeal smooth muscle cells and brain pericytes, we modulated the intracellular localization of the amyloid-ß precursor protein (APP) to study possible effects on its processing. By using immunofluorescence and immunoelectron microscopy we demonstrated that, under normal conditions, the APP is found in small intracellular vesicles, some of which were characterized as lysosomes. Both the cytokine interferon- and the lysosomotropic drug chloroquine, but not the cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor-
(TNF-
), induced an accumulation of APP in newly formed multivesicular body-like organelles. The secretion of the amyloid-ß precursor protein was slightly reduced by interferon-
or chloroquine. Double-labeling and tracer molecule uptake experiments showed that the multivesicular body-like organelles were part of the endocytic pathway. Our findings suggest that the multivesicular body-like organelles function as an intermediate organelle in the intracellular trafficking of the APP. Accumulation of the APP in this organelle is reflected by its reduced secretion from the cell.
(J Histochem Cytochem 50:681690, 2002)
Key Words: Alzheimer's disease, amyloid-ß precursor protein, endosomes, multivesicular bodies, pericyte, smooth muscle cell, lysosomes
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Introduction |
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BRAINS of patients with dementia of the Alzheimer type (DAT) are characterized by deposits of the amyloid-ß protein (Aß) in senile plaques and in the cerebral microvessels (
The intracellular localization and trafficking of APP may be crucially important for its metabolism. This has been studied mostly in neuronal cells (
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Materials and Methods |
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Cells
Human brain pericytes (HBPs) were isolated and cultured from human autopsy brain tissue from both DAT and control patients as described previously (-smooth muscle cell actin, the high molecular weight melanoma-associated antigen and vascular cell adhesion molecule-1, as described previously for SMCs isolated from umbilical artery (
(IFN-
), interleukin (IL)-1, and IL-6 were purchased from Boehringer Mannheim (Almere; The Netherlands) and chloroquine from Sigma Chemical (St Louis, MO). Tumor necrosis factor-
(TNF-
) was a kind gift of Boehringer Ingelheim (Ingelheim, Germany). Cells were treated with IFN-
at 200 U/ml for the indicated time periods, with chloroquine at 1025 µM overnight or 50100 µM for 14 hr, with IL-1 and IL-6 at 50 U/ml or with TNF-
at 50 ng/ml. Levels of cellular and secreted APP were analyzed by Western blotting as described previously (
Antibodies
The following antibodies were kindly donated to us: monoclonal antibody (MAb) P2-1 (anti-APP, N-terminal; Dr. W.E. Van Nostrand, Stony Brook, NY) (
Immunofluorescence Staining of Cultured Cells
Cultured cells were grown on glass slides that were previously coated with gelatin and treated with glutaraldehyde. At confluence, they were washed once in PBS and then fixed in acetone for 10 min and air-dried. Fixed cells were stained at room temperature (RT) as follows. After incubation with primary antibodies for 1 hr, cells were incubated with fluorescein-isothiocyanate (FITC)-conjugated swine anti-rabbit or sheep anti-mouse antibodies (Cappel; Boxtel, The Netherlands) for 30 min for primary PAbs or MAbs, respectively. Alternatively, biotinylated horse anti-mouse antibodies in combination with Texas Red-conjugated avidin (Vector; Burlingame, CA) were used for staining with primary MAbs. All dilutions were made in PBS supplemented with 0.5% BSA and 0.1% cold water fish skin gelatin (PBS-C) (Aurion; Wageningen, The Netherlands), which also served as a negative control. Each incubation was followed by extensive washing with PBS. For double immunostaining, cells were incubated with primary MAbs and PAbs simultaneously, and secondary antibodies were also incubated simultaneously. Finally, cells were incubated with Texas Red-conjugated avidin as above. Secondary antibodies were tested for lack of crossreactivity and for nonspecific binding. No differences were observed among cells from different donors.
Immunoelectron Microscopy (IEM) of Cultured Cells
Cells were cultured on Transwells (Costar; Cambridge, MA) and washed twice with serum-free medium before fixation. For morphological examination, cells were fixed with 2% glutaraldehyde in PBS for 60 min, washed with PBS, and treated with OsO4 as indicated below. For IEM, cells were fixed in periodatelysineparaformaldehyde (PLP) for 15 min, washed three times with PBS, and briefly dipped in 10% methanol in PBS. After a wash in PBS, cells were subsequently incubated with P2-1 diluted in PBS-C for 24 hr at RT, washed with PBS, and incubated with ultrasmall (1-nm) gold-labeled goat anti-mouse F(ab')2 IgG(H+L) antibodies (Aurion) diluted in PBS-C overnight at 4C and again washed in PBS. Subsequently, cells were treated with 0.5% glutaraldehyde in PBS, washed in PBS, incubated with 0.5% OsO4 in PBS, washed again in PBS, and thoroughly washed with distilled water. Silver enhancement was performed according to
Endocytosis of Marker Molecules
HBPs or HLSMCs cultured on glass slides were incubated with FITC-labeled dextran (10 mg/ml; Molecular Probes, Leiden, The Netherlands) for 1 hr at 4C before incubation at 37C for 3 hr. Cells were washed with PBS, fixed with 2% paraformaldehyde (10 min) and, for P2-1 staining, treated with acetone for 5 min. Cell preparations were stained with P2-1 or lamp-1 antibodies in combination with biotinylated secondary antibodies and Texas Red-conjugated avidin as described above. Alternatively, cells grown on Transwells were allowed to ingest 6-nm gold particles coupled to BSA (Aurion) for similar time periods. Afterwards, cells were washed with PBS, fixed with 2% glutaraldehyde (1 hr), treated with 0.5% OsO4 (30 min), dehydrated in alcohol, and embedded in Epon 812. Ultrathin sections (70 nm) were stained with uranyl acetate (20 min) and lead nitrate (1 min) and were examined in a JEOL electron microscope at 60 kV.
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Results |
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Intracellular Localization of APP
Cultured cells were immunocytochemically stained with the anti-APP MAb P2-1. This epitope was sensitive to paraformaldehyde and glutaraldehyde fixation but after paraformaldehyde fixation the antigen could be retrieved by brief methanol (10%) or acetone treatment. In resting HBPs and HLSMCs, APP was predominantly observed in small organelles (Fig 1A and Fig 1C) with variable but generally faint reactivity of the ER and Golgi compartment. In HMEC-1 the perinuclear staining for APP of these cells was suggestive of a localization in the ER and Golgi compartment (Fig 1E). In addition, few intracellular organelles were stained. Staining for APP with MAb F12 was similar to P2-1 staining (not shown).
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Intracellular Localization of APP is Altered by IFN- and Chloroquine
After 1 day of exposure of the cells to the cytokine IFN- (200 U/ml, 3 days) large APP-positive intracellular organelles were observed in addition to the staining described above (Fig 1B, Fig 1D, and Fig 1F). They became more abundant after 3 days and increased in number even more after prolonged exposure. The number of these organelles was dependent on the dose of IFN-
(25400 U/ml). Incubation with 200 U/ml IFN-
resulted in a consistently robust response of the cells. In cells treated for 3 days with IFN-
and subsequently incubated in medium without the cytokine, the large APP-positive organelles gradually disappeared. In contrast, incubation for 172 hr with the cytokine IL-1, IL-6, or TNF-
, did not affect the intracellular distribution of APP in either cell type (not shown). Furthermore, there was no difference in the response by cells derived from different donors.
Treatment of HBPs and HLSMCs with chloroquine resulted in an increase in the number and size of intracellular organelles stained for APP (see below). Similar treatment of HMEC-1 also resulted in the appearance of large APP-positive organelles, but fewer than in the other cell types.
In all cell types, APP could not be demonstrated at the surface of either untreated or IFN-- or chloroquine-treated cells by immunofluorescence. This observation was confirmed by the failure to stain unfixed suspended cells with P2-1 and FITC-conjugated secondary antibodies, and subsequent analysis by flow cytometry, a procedure that we used previously with success to analyze expression of various membrane molecules by HBPs and endothelial cells (
APP Immunoreactivity in Multivesicular Body-like Organelles after Exposure to IFN- or Chloroquine as Demonstrated by IEM
In resting HBPs and HLSMCs, small organelles (diameter 250300 nm) were stained for APP (Fig 2A). In HMEC-1, the ER/Golgi-like staining could not be observed. In IFN--treated cells (200 U/ml, 3 days), especially in HBPs and HLSMCs and to a minor extent in HMEC-1, large organelles (diameter 5001000 nm) were immunogold-labeled with P2-1 (Fig 2B and Fig 2C). In glutaraldehyde-fixed cells, with better preservation of the cell morphology than with PLP-fixed cells, these remarkable large organelles showed many vesicular inclusions, resembling a multivesicular body (MVB)-like appearance (Fig 2E). The immunogold labeling representing APP was not concentrated on the organelle's outer border but was randomly spread all over its interior. By using these ultrathin (70-nm) sections we observed MVB-like organelles in approximately 1020% of the cells. The number of MVB-like organelles observed in a section ranged from one to three per cell. However, both the frequency of occurrence and the number of MVB-like organelles per cell will be higher because only a very small portion of the cell is viewed with this technique. By using immunofluorescence, we observed MVB-like organelles in virtually all cells exposed to IFN-
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Treatment of the cells with chloroquine caused dilatation of the ER, an increased incidence of coated and non-coated vesicles, and formation of irregularly shaped intracellular organelles, probably swollen lysosomes, in HBPs and HLSMCs (Fig 2F), but not in HMEC-1. Large intracellular MVB-like organelles were found in chloroquine-treated cells as well and were immunostained for APP (Fig 2D), especially prominent in HBPs and HLSMCs. In all cell types, the cell's outer membrane appeared to be well-preserved, but membrane expression of APP was absent. Both coated pits and coated vesicles could be identified in all cell types, but these were not immunostained for APP (Fig 2A). The number of coated pits and coated vesicles in IFN--treated cells was markedly increased compared to untreated cells, but all these coated structures remained unstained for APP (Fig 2B).
Characterization of the APP-immunoreactive Intracellular Organelles
Both anti-lamp-1 and anti-lamp-2 antibodies stained small intracellular organelles, putatively lysosomes, in all cell types. Anti-Rab4 staining resembled that of ER and Golgi staining. Anti-Rab5A predominantly stained the cell membrane and few intracellular organelles. All untreated cells failed to express MHC class II molecules. Double immunostaining of untreated HBPs and HLSMCs revealed that lamp-1- or lamp-2-positive lysosomes were more numerous than P2-1-positive organelles. Most of the P2-1-positive organelles were negative for lamp-1 (Fig 3A), but sometimes P2-1 staining overlapped with lamp-1 (or lamp-2) staining. Organelles stained with P2-1 remained negative for Rab4 and Rab5A (not shown).
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Treatment with IFN- or chloroquine resulted in an increase in the number of lamp-1- or lamp-2-positive organelles or a swelling of lamp-1-positive lysosomes, respectively. However, double-labeling experiments, confirmed by EM analysis showed that the large APP-positive MVB-like organelles that were observed after IFN-
or chloroquine treatment of HBPs, HLSMCs, or HMEC-1 remained unstained for lamp-1 or lamp-2 (Fig 3B and Fig 3C).
Exposure to IFN- induced MHC class II expression in a subpopulation of each of the three cell types. Apart from staining of the cell surface, as confirmed by flow cytometric analysis, MHC class II-positive intracellular organelles were observed that were also stained for lamp-1 (Fig 3D). P2-1 staining only partially overlapped with intracellular MHC class II staining in IFN-
-treated HBP and HLSMC cultures, similar to that observed for P2-1/lamp-1 double staining. The APP-positive MVB-like organelles in IFN-
-treated cells remained unstained for MHC class II (not shown) and Rab5A (Fig 3E). Rab4 staining of HBPs or HLSMCs was unaffected by IFN-
treatment and did not co-localize with P2-1 staining (not shown).
The number of intracellular organelles stained for Rab5A was strongly increased after chloroquine treatment. Again, however, the APP-positive MVB-like organelles remained unstained for Rab5A (not shown). In chloroquine-treated cells, P2-1 and lamp-1 staining overlapped more frequently than in untreated cells, suggesting a lysosomal localization of APP. Similar treatment of HMEC-1 revealed that P2-1 staining did not overlap with lamp-1 staining (not shown).
Co-localization of APP with Markers of Endocytosis in MVB-like Organelles
HBPs and HLSMCs were allowed to ingest FITC-labeled dextran to determine whether the APP-positive MVB-like organelles are part of the endocytic pathway. Intracellular fluorescent label could be detected only after incubation at 37C for at least 1.5 hr. Staining of cells for APP after incubation with FITC-labeled dextran revealed that many APP-positive organelles, including the APP-positive MVB-like organelles observed in IFN-- and chloroquine-treated cells, contained the FITC label (Fig 4A and Fig 4B). Some APP-positive intracellular organelles did not contain the FITC label. By using EM analysis of cells that were allowed to ingest 6-nm gold particles coupled to BSA, it could be verified that the MVB-like organelles observed in IFN-
-treated cells always contained 6-nm gold particles (Fig 4C and Fig 4D). Furthermore, organelles containing the FITC label but lacking APP reactivity could be observed (long arrows in Fig 4B). These organelles are probably represented by the gold-containing organelles in Fig 4E and Fig 4F. Occasionally, the fluorescent marker and the 6-nm gold particles were found in lamp-1-positive lysosomes (not shown).
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IFN- and Chloroquine Enhance the Levels of Full-length APP
Western blotting analysis of cell lysates with P2-1 showed that all cell types expressed full-length APP as a set of three proteins with a relative molecular weight of 105125 kD. Cellular APP expression in HBPs and HLSMCs was elevated approximately twofold after exposure to IFN-
(Fig 5A). Identical results were obtained with the anti-C-terminal antibodies R57 and F12 (not shown). Chloroquine treatment (1025 µM, overnight) resulted in a two- to fourfold increase in the APP content of HBP and HLSMC cultures. Secreted APP (APPs) appeared as two bands with relative molecular weights of approximately 115 kD in supernatants of the cell cultures. Both IFN-
and chloroquine treatment slightly, but reproducibly, reduced the secretion of APP (Fig 5B). No effect on either APP production or secretion was observed after treatment with IL-1, IL-6, or TNF-
.
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Discussion |
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The predominant subcellular localization of APP in cultured HBPs and HLSMCs was in small intracellular vesicles, some of which were characterized as lamp-1-positive lysosomes. In HMEC, APP was observed in ER/Golgi. After treatment with either IFN- or chloroquine, a unique and remarkable redistribution of intracellular APP was observed. APP accumulated in newly formed multivesicular body (MVB)-like organelles. The shift of APP expression towards these unique organelles was not a nonspecific re-allocation of membrane proteins after IFN-
treatment because induction of MHC class II expression in the three cell types by IFN-
resulted in intracellular accumulation of MHC class II molecules in lysosomes but not in the MVB-like organelles. Unlike "true" MVBs described elsewhere (
or chloroquine has not been previously reported.
We were not able to demonstrate expression of APP on the cell surface membrane of our cells. It seems unlikely that this was a technical problem because the same techniques have previously been used successfully to identify several cell-surface markers of HBPs and other cell types (
In comparison with several other studies in which various cell types were studied, differences in the intracellular localization of APP are noted. First, APP staining in the ER/Golgi compartment has been observed in a number of cell lines, including endothelial cells which we could confirm ( and chloroquine treatment, they were consistently APP-negative. This indicates that re-internalization of APP from the cell surface to the interior is either of minor importance in HBPs and HLSMCs or occurs very rapidly (
In a few other reports, MVBs have been observed in the intracellular routing of APP. Neurons are the only cell type in which APP-containing MVBs were observed so far. Neuronal MVBs are the major intermediates in the transport of endocytosed molecules ( or chloroquine treatment (
or chloroquine treatment on Aß secretion by HBP and HLSMC cultures (unpublished observations). More detailed studies will be needed to reveal the exact role of the MVB-like organelles in APP processing and Aß production.
In conclusion, we have characterized the intracellular localization of APP in cultured human cerebrovascular cells. After stimulation of these cells with either chloroquine or IFN-, but not with other cytokines, we identified the MVB-like organelle as an organelle that may play a role in the trafficking of APP. An intracellular redistribution of APP towards these MVB-like organelles was accompanied by a reduced secretion of APP from the cells, suggesting that modulation of the intracellular localization of APP may, to some extent, be accompanied by alterations in its metabolic pathways.
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Acknowledgments |
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Supported by grants from the Netherlands Organization for Scientific Research (NWO, no. 970-10-010) and from the Internationale Stichting Alzheimer Onderzoek (ISAO).
We thank R. Pieters for technical assistance, Drs R. Koopmans, J.H.M. CoxClaessens, and G. Woestenburg (Psychogeriatric Centers "Joachim en Anna" and "Margriet," Nijmegen, The Netherlands) for their cooperation in autopsy procedures, and Drs W.E. Van Nostrand, W. Tax, J.E. Gardella, J. Buxbaum, H. Ploegh, D.L. Miller, and S. Carlsson for their generous gifts of antibodies.
Received for publication July 12, 2001; accepted November 15, 2001.
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