©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Vacuolar H-ATPase Inhibitor Bafilomycin A1 Differentially Affects Proteolytic Processing of Mutant and Wild-type -Amyloid Precursor Protein (*)

(Received for publication, October 27, 1994; and in revised form, December 29, 1994)

Christian Haass Anja Capell Martin Citron David B. Teplow Dennis J. Selkoe

From the Department of Neurology and Program in Neuroscience, Harvard Medical School and Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We analyzed the effect of the vacuolar H-ATPase inhibitor bafilomycin A1 (bafA1) on the processing of beta-amyloid precursor protein (betaAPP). In kidney 293 cells stably transfected with the wild-type betaAPP cDNA, bafA1 caused a stabilization of mature betaAPP and its 10-kDa COOH-terminal fragment. Moreover, it caused a 2-3-fold increase in secretion of soluble APP and amyloid-beta protein (Abeta). Interestingly, bafA1 treatment of cells transfected with a mutant betaAPP isoform that occurs in a Swedish kindred with familial Alzheimer's disease resulted in a decrease of Abeta production and no increase of soluble APP secretion. Identical results were obtained when the effect of bafA1 was analyzed on fibroblasts derived from affected versus unaffected members of the Swedish family. These data demonstrate a differential effect of bafA1 on the production of Abeta derived from wild-type or Swedish mutant betaAPP. Radiosequencing of Abeta derived from bafA1-treated cells expressing wild-type betaAPP revealed a marked increase of Abeta peptides starting at amino acids phenylalanine 4 and valine -3 and a relative decrease of Abeta molecules beginning at the typical NH(2) terminus of aspartate 1. Cells transfected with the Swedish mutation and treated with bafA1 did not produce these alternative Abeta peptides, so that bafA1 treatment resulted in a decrease of Abeta starting at aspartate 1. Our data indicate that multiple proteases are able to cleave Abeta at or near its NH(2) terminus. Inhibition of the protease cleaving at aspartate 1 by bafA1 and perhaps other similar agents can result in an increase of alternatively cleaved peptides.


INTRODUCTION

One of the most characteristic features of Alzheimer's disease (AD) (^1)is the accumulation of extracellular amyloid plaques in the cerebral and limbic cortices and biochemically similar deposits in selected cerebral blood vessels. Amyloid plaques are predominantly composed of the 4-kDa amyloid beta-peptide (Abeta). Abeta is derived from a high molecular weight precursor protein, the beta-amyloid precursor protein (betaAPP) by proteolysis (Kang et al., 1987). betaAPP can apparently be cleaved by multiple proteases to yield a variety of secreted derivatives (reviewed by Haass and Selkoe, 1993). The predominant cleavage is mediated by an unknown protease called alpha-secretase, leading to the secretion of the NH(2)-terminal ectodomain of betaAPP, called soluble APP (APP(s)) (Weidemann et al., 1989) and the retention of a 10-kDa COOH-terminal fragment in the cell membrane (Selkoe et al., 1988). Because this cleavage occurs within the Abeta domain (at position 17 of Abeta) (Esch et al., 1990; Wang et al., 1992), it prevents the production of Abeta. This cleavage occurs in part on the cell surface (Sisodia et al., 1992; Haass et al., 1992a, 1994a), although some studies have also demonstrated apparent intracellular cleavage in an unknown subcellular location (Sambamurti et al., 1992; DeStrooper et al., 1993; Haass et al., 1995).

Besides the alpha-secretory pathway, a second trafficking pathway has been documented for betaAPP. betaAPP has been detected within clathrin-coated vesicles (Nordstedt et al., 1993) and shown to be reinternalized from the cell surface as a full-length uncleaved molecule (Haass et al., 1992a; Koo and Squazzo, 1994). betaAPP can subsequently be detected within endosomes and lysosomes (Cole et al., 1989; Golde et al., 1992; Haass et al., 1992a; Estus et al., 1992; Carporaso et al., 1994). However, it is not clear whether betaAPP reaches endosomes only by reinternalization from the cell-surface or also via a direct trafficking pathway from the trans-Golgi network.

Besides the alpha-secretase cleavage of betaAPP, an alternative proteolytic mechanism has been observed which leads to the physiologic production and secretion of Abeta by cells in culture (Haass et al., 1992b; Seubert et al., 1992; Shoji et al., 1992; Busciglio et al., 1993). Two enzyme activities have been postulated for Abeta generation (for review, see Haass and Selkoe, 1993). Enzymes referred to as beta- and -secretases cleave at the NH(2) terminus (at a methionine-aspartate bond) and the COOH terminus of Abeta (around residues 40-42), respectively. These cleavages result in the release of Abeta into the culture medium. An acidic intracellular environment appears necessary for Abeta production (Shoji et al., 1992; Haass et al., 1993; Koo and Squazzo, 1994). Inhibition of betaAPP reinternalization leads to decreased Abeta generation (Koo and Squazzo, 1994). Such a mechanism is consistent with the detection of COOH-terminal fragments of betaAPP that contain the complete Abeta domain within isolated endosomes/lysosomes (Golde et al., 1992; Haass et al., 1992b; Nordstedt et al., 1993).

Interestingly, missense mutations associated with familial AD (FAD) have been mapped to the betaAPP gene (for review, see Hardy, 1992). The mutations are localized at the NH(2) terminus (Mullan et al., 1992), in the middle (Hendriks et al., 1992) or at the COOH terminus (Goate et al., 1991) of the Abeta domain, close to the beta, alpha, and -secretase sites, respectively. Each of these mutations has now been shown to strongly influence Abeta production. The double mutation at the Abeta NH(2) terminus causes a 3-6-fold increase in Abeta production (Citron et al., 1992, 1994; Cai et al., 1993). The mutations at the Abeta COOH terminus result in alternative -secretase cleavages leading to COOH terminally elongated peptides (Suzuki et al., 1994) that are believed to aggregate faster than do wild-type peptides (Jarrett and Lansbury, 1993). The mutation in the middle of Abeta results in a relative increase of Abeta peptides beginning at aspartate 1 and the increased production of NH(2) terminally truncated Abeta-like peptides (Haass et al., 1994b).

Little is known about the proteases involved in Abeta generation. beta-Secretase cleaving at the methionine-aspartate bond has been shown to be highly sequence specific (Citron et al., 1995). Alternative NH(2)-terminal cleavages of Abeta (Busciglio et al., 1993; Haass et al., 1992b, 1993, 1994a, 1994b; Shoji et al., 1992) result in the secretion of Abeta-like peptides not beginning at the principal NH(2) terminus (aspartate). This finding raises the question of whether the other peptides are generated by one and the same beta-secretase due to some degree of miscleavage or by multiple beta-secretase-like enzymes with different sequence specificities and subcellular localizations. To examine the latter possibility, we attempted to find pharmacological agents that inhibit the predominant methionine-aspartate cleavage activity but not the alternative Abeta-like cleavages. Based on the knowledge that agents which interfere with intracellular pH, such as NH(4)Cl and chloroquine, inhibit Abeta production (Shoji et al., 1992; Haass et al., 1993; Koo and Squazzo, 1994), we studied the effects of bafilomycin A1 (bafA1), a more specific compound that does not perturb the formation of intracellular organelles like Golgi vesicles and endosomes. BafA1 is a macrolide antibiotic shown to be a specific inhibitor of the vacuolar class of H-ATPases in vitro (Bowman et al., 1988; Hanada et al., 1990). In vivo, bafA1 can inhibit acidification of endosomes, lysosomes, and phagosomes, resulting in the inhibition of lysosomal protein degradation (Umata et al., 1990; Lukacs et al., 1992; Yoshimori et al., 1991). BafA1 also inhibits acidification of the trans-Golgi network, which can result in the inhibition of prohormone processing (Xu and Shields, 1994). In addition it cannot be excluded that bafA1 might also inhibit acidification of secretory vesicles (Xu and Shields, 1994). However, bafA1 does not alter the morphology of vacuolar compartments, as do weak bases and ionophores (Umata et al., 1990; Yoshimori et al., 1991; Johnson et al., 1993). Moreover, it has been shown that bafA1 slows recycling of the transferrin receptor without influencing its reinternalization (Johnson et al., 1993).


MATERIALS AND METHODS

Cell Lines

Human kidney 293 cells were stably transfected with wild-type cytomegalovirus betaAPP cDNA (Selkoe et al., 1988) and with the cytomegalovirus Swedish betaAPP cDNA construct bearing a double mutation (K595N/M596L) (Citron et al., 1992). Primary skin fibroblasts derived from affected and unaffected members of a Swedish family with familial AD were described by Citron et al. (1994).

Treatment with bafA1 in 2-h Pulse-Chase Experiments

To analyze the effect of bafA1 on the processing of betaAPP, 293 cells were pulse-labeled with 300 µCi of [S]methionine in serum-free medium, as described (Haass et al., 1993). Cells were than chased with excess unlabeled methionine and the indicated concentrations of either bafA1 (Waco Inc.) or Me(2)SO (as a control). Conditioned media and cell lysates were analyzed by immunoprecipitation, as described (Haass et al., 1991, 1992a). The following polyclonal antibodies were used: R1280, raised to Abeta 1-40 (Tamaoka et al., 1992); C7, raised to the COOH-terminal 20 amino acids of betaAPP (Podlisny et al., 1991); and B5, raised to recombinant betaAPP 444-592 (Oltersdorf et al., 1991). Abeta immunoprecipitates were separated on 10-20% Tris-Tricine gels (Novex), while APP(s) immunoprecipitates were separated on 10% SDS-polyacrylamide Tris glycine gels. Abeta and APP(s) quantitations were performed with a PhosphorImager 400A using the Image-Quant software (Molecular Dynamics).

Treatment with bafA1 in 15-min Pulse-chase Experiments

For these experiments, cells were labeled with [S]methionine in serum-free medium for 15 min. The cells were chased with medium containing 10% fetal bovine serum and an excess of unlabeled methionine for 10 sequential 15-min time intervals (2.5 h total). After every 15-min interval, media were removed and replaced with fresh medium. Two-thirds of the collected sample of conditioned media were immunoprecipitated with antibody R1280 (for Abeta) and one-third with antibody B5 (for APP(s)).

Radiosequencing

For radiosequencing, 293 cells were metabolically labeled with 2.5 mCi of L-[2,3,4,5,6-^3H]phenylalanine (Amersham Corp.) during a 2-h pulse in phenylalanine-free Dulbecco's modified Eagle's medium containing dialyzed 10% fetal calf serum. After this pulse, the cells were chased with medium containing excess unlabeled phenylalanine and 0.25 µM bafA1. Abeta was immunoprecipitated, electrophoresed, transferred to polyvinylidene difluoride membrane and radiosequenced as described (Haass et al., 1992b, 1994b).

Immunocytochemistry

293 cells stably transfected with BAPP were plated onto glass coverslips coated with 0.5 mg/ml poly-L-lysine. After 48 h, cells were incubated for 2 h in 0.25 µM bafA1, or in Me(2)SO as a control. Cells were fixed in 4% paraformaldehyde/phosphate-buffered saline for 20 min at room temperature. Cell surface betaAPP was detected with a mixture of the monoclonal antibodies 5A3 and 1G7 (Koo and Squazzo, 1994) used at a dilution of 1:200 in 1% bovine serum albumin/phosphate-buffered saline. Fluorescein isothiocyante-labeled goat anti-mouse secondary antibodies (Boehringer Mannheim) were used to visualize betaAPP. Cells were photographed with a Zeiss Axioplan microscope.


RESULTS

Bafilomycin A1 Increases Abeta Production in Human 293 Cells Transfected with the Wild-type betaAPP cDNA

Kidney 293 cells stably transfected with a betaAPP 695 cDNA were metabolically labeled for 2 h and chased in the presence of 0.25 µM bafA1 for 2 h. Media were then immunoprecipitated with a polyclonal antibody (R1280) raised to Abeta 1-40. As a control, identical numbers of cells were treated with the Me(2)SO carrier during a 2 h-cold chase. A 3-fold increase of Abeta production was observed in cells treated with bafA1 (Fig. 1, A and B). In parallel, a similar increase in the release of p3 was observed (Fig. 1A). Aliquots of the same media were precipitated with antibody B5 to the betaAPP ectodomain. Fig. 1, C and D, show a 2-fold increase of APP(s) secretion upon bafA1 treatment. To assess whether cell-associated full-length betaAPP is also affected by bafA1, the cell lysates were immunoprecipitated after a 2-h pulse-chase experiment with antibody C7 to the betaAPP COOH terminus. Stabilization of mature N`- and O`-glycosylated betaAPP and its 10-kDa COOH-terminal fragment occurred upon bafA1 treatment, whereas little mature betaAPP and 10-kDa COOH-terminal fragment were detected after the 2-h cold chase in the presence of the Me(2)SO carrier alone (Fig. 1E). These data suggest that bafA1 inhibits betaAPP degradation within an acidic compartment, presumably endosomes and lysosomes, resulting in the stabilization of holobetaAPP and the 10-kDa fragment. This finding is in agreement with the knowledge that inhibition of vacuolar H ATPases by bafA1 prevents proteolytic activity within endosomes and lysosomes (Yoshimori et al., 1991).


Figure 1: The influence of bafA1 on the processing of betaAPP. Kidney 293 cells expressing the wild-type betaAPP cDNA were metabolically labeled for 2 h and chased for 2 h in the presence of 0.25 µM bafA1 or the Me(2)SO vehicle alone as a control. A, immunoprecipitation of Abeta from conditioned media with antibody 1280. B, quantitation of the observed increase of Abeta upon bafA1 treatment. Data are expressed as percent release (untreated control = 100%) plus standard errors of the means (± S.E.); n = 4. C, immunoprecipitation of APP(s) from conditioned media with antibody B5. The lower band is the transfected betaAPP isoform of betaAPP (arrowhead). The upper band is the endogenously expressed betaAPP isoform. D, quantitation of the observed increase of APP(s) upon bafA1 treatment. Data are expressed as percent release (untreated control = 100%) ± S.E.; n = 4. E, immunoprecipitation of membrane-associated betaAPP from cell lysates with antibody C7. The upper band is mature N`- and O`-glycosylated betaAPP (N`/O`), and the lower band is the immature N`-glycosylated betaAPP (N`). Arrow indicates the characteristic COOH-terminal 10-kDa fragment generated by alpha-secretase cleavage. Asterisk indicates nonspecifically immunoprecipitated protein.



The increased secretion of Abeta and APP(s) is dose-dependent. Elevated Abeta release was observed at bafA1 concentrations as low as 25 nM (Fig. 2A). The effect of bafA1 on APP(s) secretion was also found to be dose-dependent. However, we observed an increase of APP(s) secretion at a concentration of 2.5 nM of bafA1 (Fig. 2B). The observed difference in the dose-dependent secretion of APP(s) and Abeta might indicate that these proteolytic products of betaAPP are generated within distinct subcellular compartments which are differentially affected by bafA1. In agreement with published data on the action of bafA1 (Bowman et al., 1988; Hanada et al., 1990), our results suggest a highly specific dose effect of bafA1 on the processing of betaAPP.


Figure 2: The effect of bafA1 in enhancing secretion of Abeta and APP(s) is dose-dependent. Kidney 293 cells expressing the wild-type betaAPP 695 cDNA were metabolically labeled for 2 h and chased in the presence of the indicated concentrations of bafA1. A, Abeta and p3 were immunoprecipitated from conditioned media with antibody 1280. B, APP(s) was immunoprecipitated with antibody B5.



bafA1 Slows the Kinetics of Abeta and APPs Secretion

It has been shown that bafA1 slows externalization of the transferrin receptor but has no effect on receptor internalization (Johnson et al., 1993). In parallel, endosomal/lysosomal degradation of proteins seems to be inhibited (Yoshimori et al., 1991). To determine if such a kinetic effect is also observed during betaAPP processing, we performed pulse-chase experiments on cells treated with bafA1 or the Me(2)SO vehicle alone. Under the Me(2)SO control conditions, APP(s) was secreted very rapidly, with a major peak occurring in the first 30-60 min (Fig. 3A). After bafA1 treatment, APP(s) secretion was reduced at 30-45 min, a time point at which the major secretion of APP(s) in untreated cells occurred (Fig. 3A). Instead, APP(s) secretion dramatically increased after 90 min and continued at high levels. Similar results were observed for the secretion of Abeta. Under control conditions, Abeta was predominantly secreted around 45-75 min (Fig. 3B). After exposing the cells to bafA1, Abeta release was dramatically slowed; however, a strong increase of Abeta secretion was observed after 60 min. Therafter, Abeta secretion continued at high levels (Fig. 3B). These data clearly demonstrate that bafA1 has an effect on the kinetics of betaAPP processing, resulting in a slower but more prolonged release of the proteolytic products of betaAPP.


Figure 3: The kinetics of APP(s) and Abeta release in pulse-chase experiments. Kidney 293 cells expressing the betaAPP wild-type cDNA were metabolically labeled for 15 min and chased for 2.5 h in the presence of bafA1 or Me(2)SO as a control. The media were completely exchanged after every 15-min time point. A, APP(s) was immunoprecipitated with antibody B5. B, Abeta and p3 were immunoprecipitated with antibody 1280.



Increased Cell Surface Expression of betaAPP upon bafA1 Treatment

As described above, bafA1 stabilizes membrane-associated holobetaAPP and slows down the release of its proteolytic products. To determine whether increased levels of betaAPP can be detected at distinct subcellular sites, kidney 293 cells stably transfected with betaAPP 695 were analyzed by immunofluorescence upon treatment with bafA1. The only substantial change in betaAPP localization following bafA1 treatment was observed when the cell surface expression of membrane-bound betaAPP was examined. Under control conditions, very little betaAPP was detected on the cell surface (Fig. 4A), a finding consistent with published data (Haass et al., 1991; Caporaso et al., 1994). However, after bafA1 treatment, we observed a punctate cell surface expression of betaAPP (Fig. 4B). These data suggest that stabilized holobetaAPP molecules are targeted in increased amounts to the cell surface. However, we did not detect an inhibition of the reinternalization of betaAPP from the cell surface (data not shown), consistent with the observation that bafA1 does not inhibit the re-uptake of the transferrin receptor (Johnson et al., 1993).


Figure 4: Detection of cell surface betaAPP upon bafA1 treatment. Kidney 293 cells expressing the wild-type betaAPP cDNA were plated on poly-L-lysine coated glass coverslips. After a 2-h treatment with either Me(2)SO (DMSO) as a control (A) or 0.25 µM bafA1 (B), cells were fixed with paraformaldehyde. Cell surface betaAPP was detected with a mixture of the ectodomain monoclonal antibodies 5A3 and 1G7, followed by a FITC-labeled goat anti-mouse secondary antibody. Note the punctate cell surface expression of betaAPP upon bafA1 treatment. Magnification times 400.



bafA1 Decreases Abeta Production in Cells Expressing the Swedish Mutant Form of betaAPP

A variety of missense mutations in the betaAPP gene have been found in patients affected by certain early-onset forms of familial AD (reviewed in Mullan and Crawford, 1993). To assess whether such mutations influence the effects of bafA1 on betaAPP processing, we treated 293 cells stably transfected with a cDNA encoding the valine to isoleucine mutation at position 717 of betaAPP (Goate et al., 1991). BafA1 resulted in increased secretion of Abeta and APP(s) indistinguishable from that of wild-type transfectants (data not shown). Next, we analyzed a double mutation at the NH(2) terminus of the Abeta region which is found in a Swedish FAD family and has been shown to cause a 3-6-fold increase of Abeta production (Citron et al., 1992, 1994; Cai et al., 1993; Felsenstein et al., 1994). Kidney cells transfected with this cDNA construct were treated with bafA1 in a 2-h pulse-chase experiment. As shown in Fig. 5, A and B, this treatment results in a 30-40% reduction of Abeta secretion. This result is in striking contrast to the observed 2-3-fold increase in Abeta production in cells transfected with wild-type betaAPP (Fig. 1, A and B). Moreover, the increase in APP(s) secretion observed after bafA1 treatment of wild-type transfectants was not seen in cells transfected with the Swedish mutation (Fig. 5, C and D). However, we still found the marked stabilization of the cell-associated N`- and O`-glycosylated betaAPP and its 10-kDa COOH-terminal fragment (Fig. 5E).


Figure 5: Treatment of kidney 293 cells expressing the Swedish betaAPP mutation with bafA1. Cells were metabolically labeled for 2 h and chased for 2 h in the presence of 0.25 µM bafA1 or Me(2)SO as a control. A, conditioned media were immunoprecipitated with antibody 1280. B, quantitation of the immunoprecipitation shown in A. BafA1 causes a 30-40% reduction of Abeta secretion in cells expressing the Swedish mutation, compared to the marked increase of Abeta secretion bafA1 causes in cells transfected with the wild-type betaAPP cDNA. Data are expressed as percent increase/decrease over the untreated control (± S.E.); n = 4. C, APPs was immunoprecipitated with antibody B5. D, quantitation of the immunoprecipitation shown in C (± S.E.); n = 4. BafA1 has no effect on the amount of APP(s) secretion. E, BafA1 stabilizes holoAPP and the 10-kDa fragment in cells transfected with the Swedish mutation, as seen with wild-type betaAPP (compare to Fig. 1E).



To show that this differential inhibition of Abeta production is not only observed in transfected 293 cells, we repeated the same experiment with primary skin fibroblasts obtained directly from affected and unaffected members of the Swedish FAD family. It was recently shown by Citron et al.(1994) that primary fibroblasts derived from these patients carrying the betaAPP mutation show a striking overproduction of Abeta, similar to that observed in transfected cell lines. Treating fibroblasts from unaffected members of the Swedish pedigree with bafA1 again led to an increase of Abeta secretion (Fig. 6A). In contrast, fibroblasts cultured from patients with the betaAPP mutation secreted lower amounts of Abeta in response to bafA1 treatment (Fig. 6A). Again, we observed a marked stabilization of cell-associated betaAPP and the 10-kDa COOH-terminal fragment in fibroblasts derived from both control subjects and patients with the mutation (Fig. 6B).


Figure 6: Treatment of primary skin fibroblasts cultured from affected and unaffected members of the Swedish FAD family with bafA1 or Me(2)SO. A, Abeta was immunoprecipitated from the media with antibody 1280. B, membrane-associated betaAPP was immunoprecipitated from the cell lysates with antibody C7 after the 2-h chase. Asterisk indicates nonspecifically precipitated protein.



Alternative NH(2)-terminal Cleavages of Abeta Are Enhanced after bafA1 Treatment in Cells Expressing Wild-type But Not Swedish Mutant betaAPP

Previously, we reported that Abeta produced normally by some cultured cells results not only from cleavage at the usual methionine-aspartate peptide bond but also from cleavages at positions valine -3, phenylalanine 4, arginine 5, glutamate 11, and others (Haass et al., 1992b, 1993, 1994a, 1994b). To establish whether the increased production of Abeta from wild-type betaAPP caused by bafA1 is due to the production of peptides starting at the characteristic aspartate 1 NH(2) terminus or at alternative cleavage sites, we sequenced the NH(2) termini of Abeta peptides isolated from cells metabolically labeled with [^3H]phenylalanine. Relative to untreated wild-type BAPP transfectants, radiosequencing of Abeta peptides from bafA1 treated wild-type betaAPP transfectants revealed a decrease of Abeta starting at aspartate 1 and a strong increase of peptides beginning at the alternative cleavage sites isoleucine -6, valine -3, and phenylalanine 4 (Fig. 7, A and B). The major peptide produced by 293 cells expressing wild-type betaAPP during bafA1 treatment had its NH(2) terminus at valine -3, represented by [^3H]phenylalanine peaks in cycles 7 and 22/23. Therefore, our data show that the increase of Abeta secretion observed upon bafA1 treatment in cells expressing wild-type betaAPP is due to the dramatically enhanced production of Abeta-like peptides with alternative NH(2) termini and a concomitant inhibition of Abeta starting at Asp^1. In contrast, radiosequencing of Abeta secreted from cells transfected with the Swedish mutation during bafA1 treatment revealed predominantly peptides begining at the characteristic NH(2) terminus of Asp^1 (Fig. 7A). 293 cells expressing the Swedish mutation do not compensate for the bafA1-induced decrease in production of Abeta peptides starting at aspartate 1 by undergoing increased generation of alternative Abeta peptides; therefore, a reduction in overall secretion of Abeta is observed. This finding is consistent with data reported previously showing that 293 cells expressing the Swedish form of betaAPP produce Abeta molecules exclusively starting at aspartate 1 (Citron et al., 1994).


Figure 7: A, radiosequencing of Abeta produced from kidney 293 cells expressing the betaAPP wild-type cDNA (open circles) and the betaAPP cDNA containing the Swedish mutation (closed circles). Cells were labeled for 2 h with 2.5 mCi [^3H] phenylalanine and chased for 2 h in the presence of 0.25 µM bafA1. Data are expressed as % counts per min (% CPM) per sequencing run (a total of 30 cycles is shown). B, all of the observed NH(2)-termini of Abeta peptides seen in both cell lines are shown as bars in reference to the betaAPP amino acid sequence of the region -6 to 40 of Abeta.




DISCUSSION

Very little is known about the identity and heterogeneity of the secretases involved in the proteolytic processing of betaAPP. Sisodia (1992) suggested that alpha-secretase might be a membrane-bound protease which cleaves its substrate in a sequence independent manner. The cleavage specificity of alpha-secretase seemed to be mediated by its cleaving at a fixed distance from the membrane and by the recognition of an alpha-helical structure around the alpha-secretase site. This model was supported by the observation that some mutations close to the alpha-secretase cleavage site, which might diminish alpha-helical structure, inhibit cleavage by this enzyme, resulting in the secretion of truncated APP(s) molecules (Haass et al., 1994b).

In striking contrast to these properties of alpha-secretase, beta-secretase cleaves at the methionine-aspartate peptide bond in a highly sequence-dependent manner. In addition, although beta-secretase cleavage requires a membrane-bound substrate, it does not require a fixed distance of the cleavage site from the membrane (Citron et al., 1995). beta-Secretase seems to be localized in an acidic environment such as late Golgi vesicles or endosomes, since alkalinizing agents such as NH(4)Cl, monensin, and chloroquine reduce beta-secretase activity (Shoji et al., 1992; Haass et al., 1993; Koo and Squazzo, 1994).

As regards -secretase, it seems that its activity occurs independently of a specific amino acid sequence at its cleavage site. Substitution of various hydrophobic amino acids at position 42 of Abeta does not noticeably change Abeta secretion. (^2)However, FAD mutations at position 717 of betaAPP (position 46 by Abeta numbering) cause an increased cleavage activity of -secretase at position 42 of Abeta (Suzuki et al., 1994). Taken together, these data indicate that these secretases are different types of enzymes with distinct biochemical properties and perhaps different subcellular localizations.

In this paper, we further characterized the biochemical properties of beta-secretase. We focused on alternative NH(2)-terminal cleavages of Abeta resulting in the secretion of elongated or truncated Abeta peptides (Busciglio et al., 1993; Haass et al., 1992b, 1993, 1994a, 1994b; Shoji et al., 1992). It is important to note that these heterogenous peptides occur in the human brain and cerebrospinal fluid (Gowing et al., 1994; Miller et al., 1993; Seubert et al., 1992; Shoji et al., 1992). Based on the knowledge that beta-secretase is a highly sequence-specific peptidase (Citron et al., 1995), we hypothesized that different proteases are involved in the cleavage of Abeta at the predominant methionine-aspartate peptide bond or at alternative NH(2)-terminal cleavage sites. Here, we show that bafA1 selectively reduces the formation of Abeta cleaved at the methionine-aspartate peptide bond. In parallel, it causes a pronounced increase of Abeta-like peptides starting at alternative NH(2) termini such as valine -3, phenylalanine 4, and isoleucine -6. Interestingly, Abeta-like peptides with these alternative NH(2) termini have also been observed without pharmacological treatment. Madin-Darby canine kidney cells stably transfected with the wild-type betaAPP cDNA secrete Abeta-like peptides beginning predominantly at arginine 5 and valine -3 (Haass et al., 1994a). Kidney 293 cells stably transfected with betaAPP cDNA constructs containing COOH-terminal deletions or substitutions also show a reduced secretion of peptides beginning at the methionine-aspartate bond and produce increased amounts of Abeta beginning at valine -3, arginine 5, and phenylalanine 4 (Haass et al., 1993). (^3)These data suggest that the alternative cleavages of Abeta observed during bafA1 treatment may not simply result from miscleavage by beta-secretase due to a change in its pH optimum. Rather, our current findings suggest that these cleavages are mediated by different proteases with distinct biochemical properties and/or different subcellular localizations. The latter possibility is supported by the observation that bafA1 treatment causes an increase of cell surface betaAPP (Fig. 4). Such an increase of surface precursor is also observed when kidney 293 cells are transfected with a variety of COOH-terminal deletion constructs. For example, deleting the complete COOH-terminal cytoplasmic domain of betaAPP or mutating the cytoplasmic tyrosines at position 687 (within the NPTY clathrin-coated pit motif) or at 653 cause an increase of cell surface expression of betaAPP which is paralleled by the secretion of increased amounts of Abeta peptides beginning at alternative NH(2) termini (Haass et al., 1993),^3 a result analogous to that reported here after bafA1 treatment. However, this hypothesis does not entirely rule out the possibility that beta-secretase might have multiple cleavage activities at different pH optima. Moreover, it should be noted that the the increased secretion of Abeta after bafA1 treatment might only be observed in cell types which are able to produce alternative NH(2) termini of Abeta. In Chinese hamster ovary cells, which almost exclusively produce Abeta starting at aspartate 1, (^4)bafA1 treatment does not result in an increase of Abeta production. (^5)

In this regard, it is interesting to note that bafA1 causes a delayed increase of Abeta secretion (Fig. 3B). Together with our data on the changes in Abeta NH(2) termini with bafA1, this finding leads to the concept that Abeta starting at Asp^1 and the Abeta-like peptides with alternative NH(2) termini are secreted at different time points of the pulse-chase experiment. This would lead to the hypothesis that alternative beta-secretase-like proteases are present in different subcellular environments at or close to the cell surface. Inhibition of the beta-secretase cleavage at the ``classical'' NH(2) terminus of Abeta (aspartate 1) by bafA1 could result in an increased availability of substrate for alternative beta-secretases that are not inhibited by this agent. The latter effect seems to be enhanced by the inhibition of intracellular betaAPP degradation by bafA1, thus leading to more betaAPP at or close to the cell surface. The classical and alternative beta-secretases seem to be expressed in varying amounts in different cell types. For example, Madin-Darby canine kidney cells appear to express lower amounts of the classical beta-secretase, leading to the secretion of peptides starting predominantly at alternative NH(2) termini; the aspartate 1 species represents only 10% of the total secreted Abeta peptides in these cells (Haass et al., 1994).

Citron et al.(1994) have shown that kidney 293 cells transfected with the Swedish mutation exclusively secrete Abeta peptides beginning at the characteristic aspartate 1 position. The same cells showed a decrease of Abeta production upon bafA1 treatment (Fig. 5), whereas 293 cells expressing the wild-type betaAPP cDNA secrete increased amounts of Abeta peptides. It is likely that the Swedish mutation provides a preferred substrate for the classical beta-secretase, in view of the fact that this mutation results in 3-6-fold increase of Abeta secretion in several cell types (Citron et al., 1992, 1994; Cai et al., 1993) and increases levels of the COOH terminally truncated form of APP(s) (Felsenstein et al., 1994). The fact that cells transfected with the Swedish mutation produce less Abeta upon bafA1 treatment might therefore be explained by a sequence specific cleavage by the alternative beta-secretases. In the mutant Swedish betaAPP molecule, the double mutation at the positions -1 and -2 of Abeta might inhibit the recognition of the precursor by the alternative beta-secretases as well as enhancing recognition by the classical beta-secretase. Interestingly, bafA1 causes a preferential increase of an Abeta-like peptide beginning at valine -3 in wild-type betaAPP expressing cells, only 1 residue NH(2)-terminal to the site of the Swedish mutations. Therefore, these mutations might inhibit substrate recognition by the protease which cleaves at valine -3, leading to an overall reduced secretion of Abeta. In addition, Abeta derived from either the mutant or wild-type genes may be generated in different subcellular compartments, thus resulting in a differential effect of bafA1 on Abeta generation. Such an interpretation would be supported by the analysis of betaAPP trafficking in polarized cell types. In Madin-Darby canine kidney cells, proteolytic products of wild-type betaAPP including APP(s), Abeta, and p3 are secreted into the basolateral compartment (Haass et al., 1994a; Haass et al., 1995). However, in the case of the Swedish mutation, truncated APP(s) generated by beta-secretase is secreted into the apical compartment (Lo et al., 1994). (^6)This result is consistent with the hypothesis that the cleavage of the Swedish betaAPP substrate by beta-secretase occurs in a different compartment than the production of Abeta cleaved from wild-type betaAPP (Lo et al., 1994). Taken together, our data indicate that a variety of biochemically distinct beta-secretase-type proteases are able to create the various NH(2) termini of Abeta peptides. These enzymes seem to be localized in different subcellular compartments, with some alternative beta-secretase enzymes close to or at the cell surface, since bafA1 simultaneously increases cell surface betaAPP and enhances alternative beta-secretase cleavage.

These observations have important consequences for the generation of pharmacological inhibitors of beta-secretases as potential drugs to slow amyloid-plaque formation. For example, inhibition of the classical beta-secretase (cleaving at the methionine-aspartate peptide bond) may not result in a corresponding decrease of Abeta secretion. In at least some cell types, Abeta-like peptides with alternative NH(2) termini may be secreted in elevated amounts. Some of these peptides could have an enhanced ability to self-aggregate (Jarret and Lansbury, 1993), resulting in no change or even an increase in amyloid plaque formation.


FOOTNOTES

*
This work was supported by Grants AG 06173 and AG 05134 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: AD, Alzheimer's disease; Abeta, amyloid beta-peptide; APP(s), soluble betaAPP; bafA1, bafilomycin A1; betaAPP; beta-amyloid precursor protein; p3, 3 kDa peptide.

(^2)
C. Haass, D. Watson, and D. Selkoe, unpublished observation.

(^3)
C. Haass and D. Selkoe, manuscript in preparation.

(^4)
E. H. Koo and D. B. Teplow, unpublished observation.

(^5)
C. Haass and D. J. Selkoe, unpublished observation.

(^6)
B. DeStrooper, personal communication.


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