©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell-type and Amyloid Precursor Protein-type Specific Inhibition of A Release by Bafilomycin A1, a Selective Inhibitor of Vacuolar ATPases (*)

(Received for publication, November 4, 1994; and in revised form, December 15, 1994)

Jeroen Knops Susanna Suomensaari Michael Lee Lisa McConlogue Peter Seubert Sukanto Sinha (§)

From the From Athena Neurosciences, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Treatment of human 293 cells transfected with amyloid precursor protein (APP) (the ``Swedish'' mutation) with a specific inhibitor of the vacuolar H-ATPases, bafilomycin A1 (baf A), leads to a potent inhibition of the release of the Abeta peptide. This is accompanied by a selective inhibition of beta-secretase activity. Surprisingly, baf A did not inhibit the production of Abeta from either wild-type APP (WT APP) or from APP (the ``Hardy'' mutation), expressed in the same cell type. In contrast, the robust production of Abeta from a human neuroglioma-derived cell line (HS683) transfected with WT APP, or from primary human mixed brain cultures (HMBC) expressing genomic WT APP, were also effectively inhibited by baf A. The inhibition of Abeta production from the HMBC was also accompanied by the inhibition of beta-s-APP release. No inhibition of alpha-s-APP release was seen in any of the cell types tested. These results indicate that intracellular acidic processes are rate-limiting for beta-secretase cleavage and Abeta production from SW APP, but not WT APP, in the peripheral 293 cell line. Furthermore, such acidic processes also play a rate-limiting role in Abeta release from human central nervous system-derived cells, including HMBC. Differential trafficking of the SW APP into an acidic compartment conducive to beta-secretase cleavage and Abeta release could be one explanation for the increased production of Abeta observed on expression of this mutation.


INTRODUCTION

The pathogenesis of Alzheimer's disease involves the deposition of the Abeta peptide as an early, and perhaps causative, event in the subsequent maturation of such deposits into senile or neuritic plaques (1) . The secretion of Abeta by human mixed brain cultures (HMBC)(^1)(2) , as well as a number of APP-transfected cell lines in culture(3, 4) , suggests that Abeta is produced by a normal metabolic pathway of the cell. However, the cellular mechanisms that lead to the production of Abeta from APP are not well understood. One metabolic fate of newly synthesized APP appears to be constitutive secretion of the bulk of its ectodomain as alpha-s-APP(5) , after an endoproteolytic non-amyloidogenic cleavage by an unidentified enzyme, ``alpha-secretase''(6) . However, other cellular processing pathways must also contribute to APP metabolism, since a substantial proportion of APP metabolism results in lysosomal degradation of the mature, full-length protein(7, 8, 9) . Although intermediates of APP degradation containing the entire Abeta peptide region accumulate in the cell upon treatment with lysosomal cysteine protease inhibitors(8) , such inhibitors do not have any effect on Abeta production(10) . Considerable doubt is thus cast upon the relevance of such ``potentially amyloidogenic'' intermediates to the formation of Abeta. More relevant to Abeta formation, however, are observations on the analysis of a truncated secreted form of APP that appears to terminate at Met-596 (beta-s-APP)(11) . These forms, produced at a relatively low level by the 293 cell line transfected with WT APP, form a larger proportion of the total s-APP produced by HMBC, which correlates well with the higher level of Abeta production in the primary cultures. This suggests that an alternate enzymatic activity, dubbed ``beta-secretase,'' is responsible for the production of the truncated secreted form, and, by inference, a cell-associated carboxyl-terminal fragment starting at Asp-597. Subsequent cleavage at Val-637 or Ala-639 in the transmembrane domain of such a fragment by a second protease would then release Abeta into the extracellular medium.

We have utilized 293 cells stably transfected with either WT APP751 or APP-NL, incorporating the so-called ``Swedish'' mutation (APP), as a system for investigating the role of such an alternative secretory cleavage in the cellular production of Abeta peptide. Tissue culture expression of this double mutation, identified as pathogenetic for Alzheimer's disease in the affected kindred(12) , results in a substantial stimulation of Abeta production (13) , without any significant effect on total s-APP released into the CM. Direct sequencing of the Abeta produced by these cells shows that the primary product is Abeta 1-40(14) , indicating that the presumptive site of beta-secretase cleavage has not been altered by the presence of the double mutation. Preliminary data indeed suggested that in comparison to wild-type APP751, transient transfections incorporating the Swedish mutation produce more of a truncated s-APP (data not shown). This form was not immunoprecipitable with antibodies that recognize epitopes specific to the amino-terminal region of the Abeta peptide, consistent with the previously described truncated cleavage. The relatively high levels of Abeta produced by stable transfectants incorporating the Swedish mutation allowed the design of experiments that tested the ability of specific compounds to alter or affect the relative balance between normal secretase cleavage and beta-secretase cleavage. We were especially interested in identifying whether alpha-secretase and beta-secretase activity are separate manifestations of the same enzymatic process, or whether they occur in different intracellular compartments. Although acidotropic amines have previously been shown to have an inhibitory effect on Abeta production(10) , the lack of specificity of such compounds precluded a clear identification of cellular compartments that contributed to Abeta production. Since the vacuolar-type H-ATPase(s) are thought to be ubiquitously involved in the maintenance of the acidic milieu in diverse intracellular acidic organelles(16) , we decided to test bafilomycin A1 (baf A), a potent and selective inhibitor of the vacuolar-type H-ATPase(s) (17) as to its effect on APP metabolism, including Abeta secretion.


EXPERIMENTAL PROCEDURES

293 cells (transformed human embryonic kidney cells) overexpressing either APP751 wild-type (APP-WT) or APP751 (APP-NL) were grown to semiconfluence and subsequently metabolically labeled in the absence or presence of 1 µM baf A. Human fetal cortical cultures were prepared as described previously(2) . Cultures were grown at least 30 days in vitro and were subsequently metabolically labeled for 16 h in the absence or presence of 1 µM baf A. Bafilomycin A1 was obtained from Dr. K. Altendorf (Osnabrück, Germany). Abeta was immunoprecipitated from aliquots of conditioned medium (CM) using R1282 antiserum (obtained from Dr. D. Selkoe (Brigham and Women's Hospital, Boston, MA)). Total s-APP was similarly determined by immunoprecipitating aliquots of the CM with alpha-5(5) . alpha-s-APP was separately immunoprecipitated with 6C6(11) , and beta-s-APP immunoprecipitated with 92 (wild-type APP) (10) or SW192 (APP-NL) antibodies. The SW192 polyclonal was produced essentially according to the method described for the wild-type 92 antibody(11) , with the exception that the synthetic pentapeptide antigen ended with NL rather than KM. Cell-associated forms of APP were immunoprecipitated from aliquots of the cell lysate protein with alpha-6(5) .

Metabolic labeling and immunoprecipitations were carried out as described before(5) . Labeling was carried out for the indicated period of time with 100 µCi/ml L-[S]methionine in methionine-free medium containing 10% heat-inactivated, dialyzed fetal bovine serum. After labeling, CM were gently removed and the cells lysed in 1 ml of lysis buffer (50 mM Tris pH 8.0, 0.15 M NaCl, 20 mM Na-EDTA, 1% sodium deoxycholate, 1% Triton X-100, and 0.1% SDS) and the crude lysates were cleared of debris by centrifugation at 100,000 times g and 4 °C for 5 min. Approximately 100 µg of total cell lysate or the equivalent amount of supernatant were immunoprecipitated with 5 µl of antiserum or 5 µg of antibody and 10 µl of protein A-Sepharose beads (Pharmacia Biotech Inc.). Immunoprecipitated protein was solubilized from the beads after boiling in reducing Laemmli buffer. Solubilized protein was separated by SDS-polyacrylamide gel electrophoresis on 10-20% Tricine gradient gels or 6% Tris-glycine gels. The gels were fixed, soaked in Amplify (Amersham), dried, and exposed to Kodak X-AR autoradiography film.


RESULTS AND DISCUSSION

Treatment of cells stably transfected with SW-APP with baf A at 1 µM leads to a virtually complete inhibition of Abeta release (-82%) from the transfected cells (Fig. 1; Table 1) as determined by the loss of the 4-kDa Abeta band, immunoprecipitable by R1282. In contrast, there appears to be a stimulation of secretion of the 3-kDa APP-derived fragment (+234%), previously determined to start at either Leu-17 or Val-18(18) . This fragment is presumed to derive from the cell-associated 9-10-kDa APP carboxyl-terminal fragment generated as a result of alpha-secretory cleavage.


Figure 1: Immunoprecipitation of Abeta from conditioned medium of bafilomycin A1-treated 293 cells. 293 cells (transformed human embryonic kidney cells) overexpressing APP751 wild-type (WT-APP) or SW-APP751 (APP-NL) were grown to semiconfluence and subsequently metabolically labeled with [S]methionine for 4 h in the absence or presence of 1 µM bafilomycin A1. Aliquots of supernatants equivalent to 100 µg of cell lysate protein were immunoprecipitated with 5 µl of R1282 antiserum.





Immunoprecipitation of s-APP from the CM with two separate APP antibodies revealed that although total s-APP production was somewhat diminished (-9%) from the SW APP-transfected cells (Fig. 2A; Table 1), there was actually a modest increase in alpha-s-APP secretion (+11%) (Fig. 2B; Table 1). In contrast, there is a dramatic inhibition of beta-s-APP release (-55%) (Fig. 2C; Table 1) in the presence of baf A. The less dramatic inhibition of total s-APP appears to result as a consequence of this as well, since, on careful examination of the immunoprecipitated total s-APP, which resolves into a closely spaced doublet, it appears that bafilomycin treatment abolishes the shorter (beta-s-APP) component, accompanied by an increase in intensity of the longer (alpha-s-APP) species (data not shown). Treatment with baf A thus does not inhibit alpha-s-APP, but selectively inhibits beta-s-APP secretion, along with Abeta secretion.


Figure 2: A, immunoprecipitation of total s-APP from conditioned medium of baf A-treated 293 cells. 293 cells overexpressing APP-WT or APP-NL were metabolically labeled for 4 h in the presence or absence of 1 µM bafilomycin A1. Aliquots of CM equivalent to 100 µg of cell lysate protein were immunoprecipitated with 5 µg of alpha5 antibody, which specifically recognizes total secreted s-APP. B, immunoprecipitation of alpha-s-APP from CM of baf A-treated 293 cells. CM aliquots as in A were immunoprecipitated with 5 µg of 6C6 antibody, which specifically recognizes alpha-secretase-cleaved s-APP (alpha-s-APP). C, immunoprecipitation of beta-s-APP from CM of baf A-treated 293 cells. CM aliquots as in A from 293 cells overexpressing APP-WT were immunoprecipitated with 5 µg of 92 antibody, which specifically recognizes beta-secretase-cleaved s-APP wild-type (beta-s-APP-WT). In parallel, supernatants of 293 cells overexpressing APP-NL were immunoprecipitated with 5 µg of SW192 antibody, which specifically recognizes beta-secretase-cleaved s-APP NL (beta-s-APP-NL).



Investigation of the fate of full-length APP in the cell lysates revealed that there is a selective increase in the mature APP full-length protein, but no detectable change in the immature holoprotein (Fig. 3). The mobilities of either species are not altered on SDS-polyacrylamide gel electrophoresis, suggesting that the effects observed with baf A are probably not owing to effects on protein glycosylation, for example.


Figure 3: Immunoprecipitation of cell associated full-length forms of APP from baf A-treated cells. 293 cells overexpressing APP WT or APP751 NL were metabolically labeled for 4 h as described before, and 100 µg of cell lysate protein were immunoprecipitated with 5 µg of alpha6 antibody, which recognizes both full-length mature APP (m-APP) and immature APP (i-APP).



Surprisingly, baf A failed to inhibit Abeta production from the WT APP (Fig. 1), expressed in the same cell type. In fact, increased Abeta production (+48%) was actually observed (Table 1). Treatment with baf A, however, stimulated alpha-s-APP production (+125%) (Fig. 2, A and B; Table 1) and increased levels of mature holoprotein (Fig. 3) detected in the cell lysates. These results were similar to that observed with the SW APP. The lack of inhibition of Abeta was paralleled by an undiminished secretion of WT beta-s-APP (+100%) (Fig. 2C). A slightly decreased electrophoretic mobility of the beta-s-APP also indicated baf A-induced alterations in post-translational processing events.

The differential effect of baf A on Abeta production from the mutated APP in comparison to the WT APP prompted us to investigate the effect of the drug on a separate pathogenetic APP mutation, that at codon 717 (19) . As with WT APP, treatment of 293 cells stably transfected with APP695 with baf A did not lead to any detectable inhibition of Abeta production (Fig. 4); again, as with the WT APP, a small increase was seen. Unlike the Swedish mutation, the cellular expression of this mutation does not lead to an overproduction of the Abeta peptide. It has been reported, instead, that the expression of this mutation leads to a selective increase in the longer, 1-42 form of the Abeta peptide(20) , which is known to be more prone to aggregation than the shorter 1-40 peptide. It thus appears that the baf A inhibitory effect observed with the SW APP in these cells is specific to the SW mutation and is not shared by the Hardy mutation.


Figure 4: Immunoprecipitation of Abeta from CM of baf A-treated 293 cells and a human neuroglioma cell line. 293 cells overexpressing APP751-WT, APP751-NL, APP695 V717I, and the human neuroglioma cell line HS683 overexpressing APP695 were metabolically labeled for 4 h in the absence or presence of 1 µM baf A. CM aliquots equivalent to 100 µg of cell lysate protein were immunoprecipitated with 5 µl of R1282 antiserum.



We were interested in knowing whether the insensitivity to baf A treatment observed with Abeta produced from WT APP in 293 cells represented an universal phenomenon. To this end, we evaluated a human neuroglioma-derived cell line, HS683, stably transfected with APP695. Immunoprecipitation from the CM of this cell line revealed very robust production of a 4-kDa Abeta band, comparable to the level of secretion from 293 cells transfected with the SW APP (Fig. 4). Treatment with baf A resulted in a significant inhibition of production of the Abeta fragment, sparing the 3-kDa fragment. Thus, the cell type apparently determines whether or not Abeta is produced from APP in an acidic milieu.

To investigate these puzzling observations further, HMBC, which also secrete Abeta as a result of endogenous APP processing, were treated with 1 µM baf A. CM was collected overnight and evaluated using an enzyme-linked immunosorbent assay that specifically detects Abeta in the CM from these cells(2) . Greater than 75% inhibition of Abeta production was observed in these cultures (Table 2). As in the 293 cells expressing the Swedish mutation, this was also accompanied by a dramatic inhibition of the beta-s-APP, whereas no significant effect was observed with alpha-s-APP. At the levels of baf A tested, no cytotoxicity was evident. Removal of the drug, in fact, led to a reversal of the inhibitory effect over a 48-h recovery period (data not shown). Immunoprecipitation with R1282 from the CM of HMBC treated with baf A for 24 h confirmed the selective inhibition of release of the 4-kDa Abeta peptide (Fig. 5), without loss of the 3-kDa fragment. Therefore, as in the 293 cells expressing the SW APP, and the HS683 cells expressing WT APP, Abeta production from the primary cultures expressing genomic WT APP is thus effectively inhibited by baf A as well.




Figure 5: Immunoprecipitation of Abeta from CM of human mixed brain cultures in the absence or presence of baf A. Human fetal cortical cultures were grown 30 days in vitro and were subsequently metabolically labeled for 16 h in the absence or presence of 1 µM baf A. Aliquots of CM equivalent to 100 µg of cell lysate protein were immunoprecipitated with 5 µl of R1282 antiserum.



As selective and potent inhibitors of the V-ATPases, the bafilomycins are capable of inhibiting the acidification of diverse intracellular vesicles. Indeed, baf A has been shown in a number of different studies to have dramatic effects on specific cellular processes, such as phagosomal acidification, lysosomal acidification and intracellular protein degradation, endosomal carrier vesicle formation, and synaptic vesicle catecholamine uptake(21, 22, 23, 24) . The susceptibility to baf A has been used as diagnostic for the rate-limiting involvement of the V-ATPases in these various processes. The experimental observations in this report therefore strongly suggest that an intracellular acidic compartment plays a critical role in the generation and secretion of Abeta from SW APP, but not WT APP, in 293 cells. The observed increased production of Abeta with the Swedish APP may, at least in part, be due to the mutant protein being differentially trafficked into an acidic intracellular compartment in the 293 cells, much more conducive to both beta-secretase cleavage and release of Abeta. Differential intracellular trafficking of the SW APP metabolites has been recently suggested to occur in polarized cells(25) . By this analogy, increased trafficking of APP into more acidic compartments in HMBC and the neuroglioma cell line may explain the sensitivity to baf A inhibition of Abeta release. An alternative explanation might be that the SW-APP is much more prone to beta-secretase cleavage in an acidic compartment, and the increase in pH as a consequence of baf A treatment thus has a more dramatic inhibitory effect on this mutation, but not on WT. However, one would then have to additionally posit that the beta-secretase protease has different pH optima toward SW as compared to the WT APP. In the absence of an isolated beta-secretase enzymatic activity, it may not be possible to choose between these separate explanations.

These observations, additionally, also directly link Abeta production to the occurrence of the beta-secretase cleavage, reflected in the release of beta-s-APP. In both the SW APP-transfected 293 cells, as well as in the HMBC, the inhibition of Abeta release by baf A also strongly inhibited the release of the beta-s-APP as well. The lack of any negative effect of the drug on either alpha-s-APP secretion or the production of the 3-kDa APP fragment in all of the cell types tested also suggests that alpha-secretase activity, in contrast to beta-secretase, does not require an acidic environment. In one study (26) , treatment with 500 nM baf A led to the inhibition of maturation and targeting of two separate lysosomal enzymes, processes known to involve acidic compartments such as the endosome and lysosome; yet, no effect on the concurrent processing and secretion of either proalbumin, or proC3 was detected. The latter events, mediated presumably by the constitutive secretory pathway involving the trans-Golgi network, appear to be insensitive to alterations of intravesicular pH. The insensitivity of alpha-secretase activity toward baf A also confirms previous conclusions (15) regarding the likely cellular locale for this constitutive secretory cleavage.

In both SW and WT APP-transfected 293 cells, treatment with baf A is accompanied by a significant increase in alpha-s-APP (Fig. 2), as well as an increase in the level of cell-associated mature holoprotein (Fig. 3). Lysosomal degradation of APP has been suggested previously to be an important component of total APP metabolism in 293 cells expressing WT APP(8) . The inhibition of such degradative processes by baf A may lead to the recycling of both WT and SW APP holoprotein into the constitutive secretory pathway. Contrary to its effect on the 293 cells, baf A effected only relatively modest changes on either mature holoprotein levels or on alpha-secretase cleavage in either HMBC or the HS683 cells (data not shown).

The identities of the processes sensitive to bafilomycin treatment, apparently crucial to Abeta/beta-s-APP formation, are not clear from these experiments. It is also unclear whether the presumed elevation of intravesicular pH that leads to the beta-s-APP and Abeta release inhibition is owing to a direct effect on the enzyme(s) involved, or owing to indirect effects, mediated perhaps by inhibition of vesicle fusion events, for example. In any case, the availability of compounds such as baf A stimulate further experimental work aimed at pinpointing the cellular compartments which mediate Abeta production in different cells. Knowledge gained from such studies would lead to increased understanding of the cellular enzymology involved in the formation of Abeta, which would be in the critical path to designing effective therapeutic agents for Alzheimer's disease.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed. Tel.: 415-877-0900; Fax: 415-877-8370.

(^1)
The abbreviations used are: HMBC, human mixed brain culture(s); baf A, bafilomycin A; APP, amyloid precursor protein; WT, wild-type; CM, conditioned medium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.