Accumulation of the Amyloid-β Precursor Protein in Multivesicular Body-like Organelles

Abstract

It has been suggested that the successive proteolytic events leading to the production of the amyloid-β 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-β 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-γ and the lysosomotropic drug chloroquine, but not the cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor-α (TNF-α), induced an accumulation of APP in newly formed multivesicular body-like organelles. The secretion of the amyloid-β precursor protein was slightly reduced by interferon-γ 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.

Brains of patients with dementia of the Alzheimer type (DAT) are characterized by deposits of the amyloid-β protein (Aβ) in senile plaques and in the cerebral microvessels (Glenner and Wong 1984). This protein of 39–42 amino acids is proteolytically cleaved from the membrane-spanning amyloid-β precursor protein (APP) (Kang et al. 1987). Normal secretory processing of the full-length 751- and 770-amino-acid isoforms of APP results in the production of soluble APP (APP_s), identical to protease nexin-2 (Van Nostrand et al. 1989). Because the cleavage site is located within the Aβ sequence, this process precludes Aβ generation. However, Aβ may be produced via an intracellular proteolytic degradation pathway (Weidemann et al. 1989; Haass et al. 1991; Shoji et al. 1992). It has been demonstrated in a variety of cells that mature full-length APP inserted in the cell surface membrane functions as a precursor molecule for Aβ production after re-internalization from the cell surface into, e.g., endosomes and lysosomes (Haass et al. 1992,1993; Koo and Squazzo 1994; Koo et al. 1996; Yamazaki et al. 1996). Alternatively, Aβ may accumulate in secretory transport vesicles leaving the late Golgi compartment and heading for the cell surface (Busciglio et al. 1993; Haass et al. 1993; Caporaso et al. 1994).

The intracellular localization and trafficking of APP may be crucially important for its metabolism. This has been studied mostly in neuronal cells (Yamazaki et al. 1995,1996; Koo et al. 1996; Marquez-Sterling et al. 1997). APP in neurons has been demonstrated in Rab5-positive endosomes (Ikin et al. 1996), late endosomes, and axon membranes (Ferreira et al. 1993) and in the Golgi complex (Caporaso et al. 1994). Putatively, neurons play an important role in the pathogenesis of senile plaques and possibly also of cerebral amyloid angiopathy (CAA) (Weller et al. 1998). In addition, cerebrovascular cells may also play an important role in the genesis of cerebrovascular amyloid (Shoji et al. 1990; Tamaoka et al. 1992; Frackowiak et al. 1994; Verbeek et al. 2000). We performed a detailed study of the intracellular distribution of APP in cerebrovascular cells and investigated whether a change in intracellular localization of APP is related to alterations in its processing. Cultured human cerebrovascular cells were used in this study because at present these are the only primary cells related to a specific lesion in AD brains (i.e., CAA) that can be isolated and cultured in sufficient amounts from human adult brains.

Materials and Methods

Cells

Human brain pericytes (HBPs) were isolated and cultured from human autopsy brain tissue from both DAT and control patients as described previously (Verbeek et al. 1994). Human leptomeningeal arterial smooth muscle cells (HLSMCs) were isolated by collagenase digestion (1 mg/ml, 30 min, 37C) of dissected leptomeningeal vessels from human autopsy brain tissue from both DAT and control patients and subsequent seeding of the vessel fragments on fibronectin-coated culture plates. Isolated cells were characterized by positive staining for α-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 (Verbeek et al. 1994). Human microvascular endothelial cells (HMEC-1) were a kind gift of Dr. E. Ades (Centers for Disease Control and Prevention; Atlanta, GA). This cell line has retained the morphological, phenotypical, and functional characteristics of normal human microvascular endothelial cells (Ades et al. 1992). Cultured cells were grown in Eagle's modification of essential medium (EMEM) containing 10% human serum (Red Cross Blood Bank; Nijmegen, The Netherlands), 20% newborn calf serum (Flow Laboratories; Irvine, Scotland), endothelial cell growth factor isolated from bovine brain (150 μg/ml), heparin (5 U/ml; Organon Teknika, Boxtel, The Netherlands), glutamine (2 mM; Flow Laboratories), and antibiotics. In experiments, triplicate wells with cultured cells were incubated with EMEM containing 0.1% bovine serum albumin, 1 μg/ml hydrocortisone, 1 ng/ml basic fibroblast growth factor, and antibiotics (serum-free medium) after two washes with EMEM. Interferon-γ (IFN-γ), 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-α (TNF-α) was a kind gift of Boehringer Ingelheim (Ingelheim, Germany). Cells were treated with IFN-γ at 200 U/ml for the indicated time periods, with chloroquine at 10–25 μM overnight or 50–100 μM for 1–4 hr, with IL-1 and IL-6 at 50 U/ml or with TNF-α at 50 ng/ml. Levels of cellular and secreted APP were analyzed by Western blotting as described previously (Verbeek et al. 1997). Equal protein samples of cell lysates of treated or control cells, or aliquots of supernatants normalized to cell protein, were loaded on the gels. APP expression and secretion were similar in cultures derived from control or DAT patients. Cellular and secreted APP levels in the human serum-containing medium were similar to those in the serum-free medium.

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) (Van Nostrand et al. 1989), MAb 12–10 (anti-MHC class II; Dr. W. Tax, Nijmegen, The Netherlands), MAb F12 (anti-APP, C-terminal; Dr. J.E. Gardella, Stony Brook, NY), MAb Rab5 (anti-Rab5; Dr. J. Buxbaum, NY) (Ikin et al. 1996), polyclonal antibodies (PAbs) directed against MHC class II molecules (Dr. H. Ploegh; Amsterdam, The Netherlands), PAb R57 (anti-APP, C-terminal; Dr. D.L. Miller, New York), PAbs directed against lamp-1 and lamp-2 (lysosome-associated membrane proteins; Dr. S. Carlsson, Umeå, Sweden) (Carlsson et al. 1988). Anti-Rab4 and anti-Rab5A PAbs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

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 periodate-lysine-paraformaldehyde (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 2–4 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 Danscher (1981) for 18 min, after which the cells were extensively washed in distilled water, dehydrated, and embedded in Epon 812. Filters were dissected from the Transwells with a diamond knife (gift of Drukker; Cuijk, The Netherlands). Ultrathin sections were stained with uranyl acetate and lead nitrate and examined with a JEOL 1200 EX/II electron microscope (Tokyo, Japan) at 50 kV.

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.

Results

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 (Figures 1A and 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 (Figure 1E). In addition, few intracellular organelles were stained. Staining for APP with MAb F12 was similar to P2–1 staining (not shown).

Intracellular Localization of APP is Altered by IFN-γ and Chloroquine

After 1 day of exposure of the cells to the cytokine IFN-γ (200 U/ml, 3 days) large APP-positive intracellular organelles were observed in addition to the staining described above (Figures 1B, 1D, and 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-γ (25–400 U/ml). Incubation with 200 U/ml IFN-γ resulted in a consistently robust response of the cells. In cells treated for 3 days with IFN-γ and subsequently incubated in medium without the cytokine, the large APP-positive organelles gradually disappeared. In contrast, incubation for 1–72 hr with the cytokine IL-1, IL-6, or TNF-α, 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.

Figure 1

Immunofluorescence staining of untreated (A, C,E) and IFN-γ treated (200 U/ml, 3 days; B, D,F) cultures of HBP (A, B), HLSMC (C, D), and HMEC-1 (E, F) with the anti-APP MAb P2–1. Large APP-positive intracellular organelles are formed in all cell types after exposure to IFN-γ. Bar = 3 μm.

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-γ- 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 (Westphal et al. 1993; Verbeek et al. 1995).

APP Immunoreactivity in Multivesicular Body-like Organelles after Exposure to IFN-γ or Chloroquine as Demonstrated by IEM

In resting HBPs and HLSMCs, small organelles (diameter 250–300 nm) were stained for APP (Figure 2A). In HMEC-1, the ER/Golgi-like staining could not be observed. In IFN-γ-treated cells (200 U/ml, 3 days), especially in HBPs and HLSMCs and to a minor extent in HMEC-1, large organelles (diameter 500–1000 nm) were immunogold-labeled with P2–1 (Figures 2B and 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 (Figure 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 10–20% 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-γ.

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 (Figure 2F), but not in HMEC-1. Large intracellular MVB-like organelles were found in chloroquine-treated cells as well and were immunostained for APP (Figure 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 (Figure 2A). The number of coated pits and coated vesicles in IFN-γ-treated cells was markedly increased compared to untreated cells, but all these coated structures remained unstained for APP (Figure 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 (Figure 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).

Treatment with IFN-γ 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-γ or chloroquine treatment of HBPs, HLSMCs, or HMEC-1 remained unstained for lamp-1 or lamp-2 (Figures 3B and 3C).

Exposure to IFN-γ 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 (Figure 3D). P2–1 staining only partially overlapped with intracellular MHC class II staining in IFN-γ-treated HBP and HLSMC cultures, similar to that observed for P2–1/lamp-1 double staining. The APP-positive MVB-like organelles in IFN-γ-treated cells remained unstained for MHC class II (not shown) and Rab5A (Figure 3E). Rab4 staining of HBPs or HLSMCs was unaffected by IFN-γ treatment and did not co-localize with P2–1 staining (not shown).

Figure 2

Ultrastructural analysis of APP expression in cultured cells. Small organelles are stained by P2–1 (anti-APP) in untreated HBP (A). Large intracellular, MVB-like organelles in IFN-γ (200 U/ml, 3 days)-treated HLSMCs (B) and HBPs (C) and in HBPs treated with chloroquine (10 μM overnight; D) are stained with P2–1. Morphological examination of IFN-γ- (E) or chloroquine - (F) treated HBP reveals the presence of MVB-like organelles (large arrows in E, F). Coated pits and coated vesicles (arrowheads in E, F), non-coated vesicles (small arrows in E, F), and irregularly shaped organelles are visible. Note that in A and B coated pits and coated vesicles (arrowheads) remain unstained in untreated and IFN-γ-treated cells, respectively. Bar = 0.5 μm.

Figure 3

(A-C) Double immunostaining of untreated (A), IFN-γ-treated (B), and chloroquine-treated (C) HBPs for APP (red) and lamp-1 (green). (A) P2–1 staining partially overlaps with lamp-1 staining, but many organelles stained only with P2–1 for APP are visible in red. (B, C) Large intracellular MVB-like organelles stained for APP remain unstained for lamp-1. (D) Double immunostaining of IFN-γ-treated HBPs for MHC class II (red) and lamp-1 (green). MHC class II is expressed both at the cell surface and in intracellular organelles that are also stained for lamp-1 (yellow). (E) Double immunostaining of IFN-γ-treated HBPs for APP (red) and Rab5A (green). The APP-positive MVB-like organelles remain unstained for Rab5A. Bar = 0.85 μm.

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-γ- and chloroquine-treated cells, contained the FITC label (Figures 4A and 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-γ-treated cells always contained 6-nm gold particles (Figures 4C and 4D). Furthermore, organelles containing the FITC label but lacking APP reactivity could be observed (long arrows in Figure 4B). These organelles are probably represented by the gold-containing organelles in Figures 4E and 4F. Occasionally, the fluorescent marker and the 6-nm gold particles were found in lamp-1-positive lysosomes (not shown).

IFN-γ 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 ~105–125 kD. Cellular APP expression in HBPs and HLSMCs was elevated approximately twofold after exposure to IFN-γ (Figure 5A). Identical results were obtained with the anti-C-terminal antibodies R57 and F12 (not shown). Chloroquine treatment (10–25 μM, overnight) resulted in a two- to fourfold increase in the APP content of HBP and HLSMC cultures. Secreted APP (APP_s) appeared as two bands with relative molecular weights of approximately 115 kD in supernatants of the cell cultures. Both IFN-γ and chloroquine treatment slightly, but reproducibly, reduced the secretion of APP (Figure 5B). No effect on either APP production or secretion was observed after treatment with IL-1, IL-6, or TNF-α.

Discussion

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-γ 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-γ treatment because induction of MHC class II expression in the three cell types by IFN-γ resulted in intracellular accumulation of MHC class II molecules in lysosomes but not in the MVB-like organelles. Unlike “true” MVBs described elsewhere (Carlsson et al. 1988), the MVB-like organelles we observed were not labeled by the lysosomal marker lamp-1, but they do have a comparable size (van Deurs et al. 1993). Because they lack lamp-1-reactivity the MVB-like organelles we identified may be an immature form (van Deurs et al. 1993). The effects of chloroquine appear to be in partial agreement with other studies (Caporaso et al. 1992,1994) because we observed an accumulation of APP in lysosomes after chloroquine treatment, together with a slight reduction in the secretion of APP. However, accumulation in MVB-like organelles after treatment with IFN-γ 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 (Verbeek et al. 1995). Furthermore, although our IEM procedures did not preserve the APP epitopes in the ER and Golgi compartments in HLSMCs treated with Aβ_1–42 (25 μM, 6–12 days), which resulted in increased APP expression (Verbeek et al. 1997), APP immunoreactivity was readily and predominantly observed by us at the cell surface by IEM and immunofluorescence analysis (unpublished observations), confirming that the absence of APP membrane expression observed in the present study was not an artifact. This indicates either that only a small fraction of full-length APP is expressed at the cell surface, in line with previous reports (Weidemann et al. 1989; Haass et al. 1991) or that the residence time of APP at the cell surface is very short due to rapid internalization or secretion (Yamazaki et al. 1995; Koo et al. 1996).

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 (Haass et al. 1992; Caporaso et al. 1994), but we did not observe such expression in HBPs or HLSMCs. Second, we were unable to confirm findings in both the rat pheochromocytoma cell line PC12 and in isolated nerve terminal vesicles on co-localization of APP with Rab5A (Ikin et al. 1996; Marquez-Sterling et al. 1997), a marker of early endosomes. Third, in contrast with our data on HBPs and HLSMCs, APP was identified in cultured rat hippocampal neurons in clathrin-coated vesicles (Ferreira et al. 1993; Caporaso et al. 1994; Yamazaki et al. 1995), which may mediate re-internalization of cell-surface APP (Haass et al. 1992; Nordstedt et al. 1993; Koo and Squazzo 1994). Although coated pits and coated vesicles were frequently observed by IEM in our cells, especially after IFN-γ 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 (Yamazaki et al. 1995; Koo et al 1996), resulting in failure to detect this molecule on the cell surface. Together, these data indicate that the intracellular localization of APP in HBPs and HLSMCs is cell-specific and is different from that in a number of other cell types and from neuronal cells in particular.

Figure 4

Immunofluorescent (A, B) and immunoelectron microscopic (C-F) examination of tracer molecules ingested by IFN-γ-treated HBP (B-F) and comparison with a double-staining for APP (A). (A) Staining for APP; (B) intracellular accumulation of FITC-labeled dextran in the same cells as in A. Large APP-positive, MVB-like organelles contain FITC-labeld dextran (short arrows). Organelles either stained for APP (arrowheads in A) or containing FITC-labeled dextran (long arrows in B) can be identified. (C, E) Electron microscopic micrograph of IFN-γ-treated HBPs that were allowed to ingest 6-nm gold particles coupled to BSA. An MVB-like organelle in C and an endosome in E, both containing 6-nm gold, are shown at higher magnification in D and F, respectively. Bars: A, B = 1 μm; C, E = 500 nm; D, F = 200 nm.

Figure 5

Western blotting analysis of cellular and secreted APP. (A) Cellular APP levels in HBP are increased after IFN-γ (Lane 2) or chloroquine (Lane 3) treatment of cultured HBPs compared to untreated cells (Lane 1). (B) Both IFN-γ and chloroquine treatment slightly reduce the secretion of APP during a 6-day period in cultured HBPs. Molecular weights are indicated at right in kilodaltons.

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 (Parton et al. 1992), and they may play a role in the retrograde transport of APP from axonal terminals to the cell body (Caporaso et al. 1994; Yamazaki et al. 1995; Ikin et al. 1996; Marquez-Sterling et al. 1997). Accordingly, the MVB-like organelles of HBPs or HLSMCs may be a part of the endocytic pathway. These organelles also contained marker molecules taken up by the cells via fluid-phase endocytosis. Therefore, this MVB-like organelle, in addition to endosomes and lysosomes, may be involved in the trafficking of APP from the cell surface into the interior. As pointed out above, however, it is more likely that APP is delivered to the MVB-like organelles via the biosynthetic pathway from the Golgi compartment directly to lysosomes, as has been demonstrated for the mannose-6-phosphate receptor (Kornfeld and Mellman 1989; Dell'Angelica and Payne 2001). Therefore, the MVB-like organelles are at the intersection of these pathways (Hopkins 1992) and their formation could be the result of a blockade in either pathway or of a reduced breakdown of APP after IFN-γ or chloroquine treatment (Caporaso et al. 1992). An intriguing aspect of MVBs is their putative functioning as a sorting compartment, in which internalized molecules, such as APP, may be released from the MVB membrane, thus being exposed to proteolysis, whereas other proteins remain associated with the membrane and can be recycled to the cell surface. This may provide a means by which APP is processed to Aβ in acidic compartments of the cell. However, we did not observe any significant effect of IFN-γ or chloroquine treatment on Aβ 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 Aβ 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-γ, 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.