©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Enhanced Release of Amyloid -Protein from Codon 670/671 Swedish Mutant -Amyloid Precursor Protein Occurs in Both Secretory and Endocytic Pathways (*)

(Received for publication, February 2, 1995; and in revised form, January 26, 1996)

Ruth G. Perez (1) (2) Sharon L. Squazzo (1) Edward H. Koo (1) (3)(§)

From the  (1)Center for Neurologic Diseases and the Departments of (2)Neurology and (3)Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mutation at codons 670/671 of beta-amyloid precursor protein (betaPP) dramatically elevates amyloid beta-protein (Abeta) production. Since increased Abeta may be responsible for the disease phenotype identified from a Swedish kindred with familial Alzheimer's disease, evaluation of the cellular mechanism(s) responsible for the enhanced Abeta release may suggest potential therapies for Alzheimer's disease. In this study, we analyzed Chinese hamster ovary cells stably transfected with either wild type betaPP (betaPP-wt) or ``Swedish'' mutant betaPP (betaPP-sw) for potential differences in betaPP processing. We confirmed that increased amounts of Abeta and a beta-secretase-cleaved COOH-terminally truncated soluble betaPP (betaPP(s)) were secreted from betaPP-sw cells. As shown previously for betaPP-wt cells, Abeta was released more slowly than the secretion of betaPP(s) from surface-labeled betaPP-sw cells, indicating that endocytosis of cell surface betaPP is one source of Abeta production. In contrast, by [S]methionine metabolic labeling, the rates of Abeta and betaPP(s) release were virtually identical for both cell lines. In addition, the identification of intracellular betaPP(s) and Abeta shortly after pulse labeling suggests that Abeta is produced in the secretory pathway. Interestingly, more Abeta was present in medium from betaPP-sw cells than betaPP-wt cells after either cell surface iodination or [S]methionine labeling, indicating that betaPP-sw cells have enhanced Abeta release in both the endocytic and secretory pathways. Furthermore, a variety of drug treatments known to affect protein processing similarly reduced Abeta release from both betaPP-wt and betaPP-sw cells. Taken together, the data suggest that the processing pathway for betaPP is similar for both betaPP-wt and betaPP-sw cells and that increased Abeta production by betaPP-sw cells arises from enhanced cleavage of mutant betaPP by beta-secretase, the as-yet unidentified enzyme(s) that cleaves at the NH(2) terminus of Abeta.


INTRODUCTION

In Alzheimer's disease a characteristic pathological finding in the brains of affected individuals is the deposition of amyloid beta-protein (Abeta) (^1)in senile plaques(1) . Abeta is the 39-43-amino acid proteolytic cleavage product of the type I integral membrane protein beta-amyloid precursor protein (betaPP). The betaPP gene is encoded on chromosome 21, and alternative exon splicing produces three major isoforms of 695, 751, or 770 amino acids(2) . During constitutive secretion some full-length betaPP molecules are proteolytically cleaved between lysine and leucine residues at positions 16 and 17 of Abeta (Fig. 1) by an enzyme termed alpha-secretase(3, 4) . Cleavage of betaPP at this position creates a soluble 100-120-kDa NH(2)-terminal fragment (betaPP(s)) (5) and a COOH-terminal membrane-retained fragment of 10 kDa(6) . Generation of these fragments by alpha-secretase precludes formation of an intact Abeta sequence from full-length betaPP.


Figure 1: betaPP structure, enzymatic cleavage sites, COOH-terminal fragments, and antibody epitopes. Schematic diagram of betaPP. The vertical cross-hatched box represents the plasma membrane. The white box labeled Abeta represents the Abeta peptide (also shown enlarged with the amino acid sequence listed). The horizontally striped box labeled KPI represents the Kunitz protease inhibitor domain alongside the adjacent exon indicated by the small open box; the NH(2)-terminal black box represents the signal sequence. alpha, beta, and mark the sites of the enzymatic cleavages by alpha-, beta-, and -secretases, respectively. Also indicated are the 10-kDa fragment (including the p3 region, transmembrane region, and COOH terminus) and the 12-kDa fragment (including the Abeta region, transmembrane region, and COOH terminus). -NPTY- indicates the putative clathrin internalization signal. Horizontal black bars indicate the approximate epitopes of antibodies B5, C7, 6E10, MMAb, R1280 and R1282, and R1736.



Abeta, however, is known to be released during normal cellular metabolism both in vivo(7, 8) and in a number of cell culture systems(9, 10) . Cleavage of betaPP at the NH(2) terminus of the Abeta sequence by an enzyme designated beta-secretase creates a shortened form of betaPP(s) and the 12-kDa COOH-terminal fragment(11, 12) . An additional enzymatic cleavage at the COOH terminus of the Abeta sequence by the as yet unidentified enzyme designated -secretase generates the 4-kDa Abeta peptide. The -secretase enzyme is also hypothesized to generate p3, the 3-kDa NH(2)-terminal piece of the membrane-retained 10-kDa COOH-terminal fragment of betaPP produced by alpha-secretase cleavage (7, 8, 9, 13) . In addition to the secretory cleavage, betaPP can also be processed in an endosomal/lysosomal pathway(14, 15, 16, 17) . Although Abeta-containing COOH-terminal fragments are generated in lysosomes, evidence suggests that these are not an important source of Abeta(18) . Recently, it was shown that cell surface betaPP molecules can be processed in the endocytic pathway and may be the direct precursors of Abeta, presumably by recycling internalized molecules from the cell surface(19) .

Evidence that Abeta and betaPP contribute to the pathogenesis of Alzheimer's disease comes from the findings of missense mutations within and adjacent to the Abeta region of the betaPP gene in families with autosomal dominant forms of Alzheimer's disease(20) . The concurrence of the mutations with the disease phenotype suggests that altered betaPP function or processing may be pathogenic. A double mutation at amino acids 670 and 671 (betaPP numbering) changing Lys to Asn and Met to Leu (K670N/M671L) was identified in a Swedish pedigree with familial Alzheimer's disease(21) . In vitro analyses of transfected cells expressing the Swedish form of betaPP (12, 22) and primary cell cultures of fibroblasts obtained from affected individuals (23) reveal a dramatic increase in Abeta production. However, the mechanism by which Abeta generation is increased has not been elucidated. Furthermore, a detailed analysis of cellular processing of betaPP with this mutation has not been reported. Because recent studies have implicated the endocytic pathway in Abeta production(19) , we speculated that Abeta production may be similarly enhanced in this pathway in cells expressing the K670N/M671L betaPP mutation.

In this report, biosynthetic analyses confirmed the increase in Abeta production and the abundant secretion of a shorter betaPP(s) species by Chinese hamster ovary (CHO) cells stably transfected with the betaPP K670N/M671L mutation. Furthermore, Abeta generation was increased in both the secretory and endocytic pathways. We postulate that this increase in Abeta production is the result of enhanced proteolytic cleavage of the mutant betaPP by the beta-secretase enzyme.


EXPERIMENTAL PROCEDURES

Cell Culture

Stably transfected CHO cell lines were generated with wild type betaPP(19) or with 670/671 betaPP ``Swedish'' mutation cloned into pcDNA3 (Invitrogen, San Diego) by CaPO(4) transfection and selection by G418 resistance. The mutant betaPP construct was obtained by subcloning the 500-base pair BamHI-EcoRI fragment containing the mutation from betaPP K670N/M671L (22, generously provided by Dr. Martin Citron) into the wild type betaPP expression vector. Cells were grown in Dulbecco's modified Eagle's medium (DMEM, BioWhitaker, Walkersville, MD) with 10% Fetal Clone II (HyClone Laboratories, Logan, UT) at 37 °C, with 5% CO(2).

Antibodies

Several anti-betaPP antibodies were used (see Fig. 1). The monoclonal antibodies 5A3 and 1G7 (referred to as MMAb when used together), recognize a midregion extracellular betaPP domain (19) . betaPP monoclonal antibody 6E10 (from K. S. Kim and H. Wisniewski) recognizes Abeta(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) and alpha-secretase-cleaved betaPP(s)(24) . Five previously described polyclonal antibodies were also used: R1280 and R1282 raised against synthetic Abeta(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) precipitate Abeta and p3 fragments(25, 26) . R1282 (1:100) gave consistent recovery of both Abeta and the p3 fragment by immunoprecipitation. C7 was raised against betaPP(751-770) and recognizes full-length betaPP and COOH-terminal fragments of betaPP(27) . B5 raised against the midregion of betaPP(519-667) precipitates both alpha-secretase- and beta-secretase-cleaved betaPP(s)(28) , and R1736 raised against betaPP(670-686) recognizes alpha-secretase-cleaved betaPP(s)(15) .

Metabolic Labeling

Confluent cultures of betaPP-transfected CHO cells were incubated in methionine-free DMEM for 15 min followed by incubation with methionine-free DMEM supplemented with 200 µCi/ml [S]methionine for 10 min (pulse labeling) or for 2-4 h with 50-100 µCi/ml [S]methionine (long labeling). Cells were lysed immediately or incubated with 2-fold unlabeled methionine (chase) in DMEM from 10 min to 4 h. For some experiments, single dishes of confluent cells were pulse labeled and chased at multiple time points with repeated collection of medium to evaluate the incremental secretion of betaPP products at each time point of the chase period. betaPPs were immunoprecipitated using betaPP-specific antibodies and separated by SDS-polyacrylamide gel electrophoresis (using 6-10% Tris-glycine gels for high molecular weight proteins and 16.5% Tris-Tricine gels for low molecular weight proteins). Gels were either fluorographically enhanced and exposed to x-ray film or dried and exposed on a Phosphor screen (Molecular Dynamics). All experiments reported herein were performed two to six times, and a representative example of each is shown. Where applicable, average values ± S.E. are given.

Assessment of Total betaPP and betaPP(s)

betaPP expression and betaPP(s) quantity were determined using parallel triplicate cultures of stably transfected CHO cells expressing wild type betaPP or ``Swedish'' betaPP. One set of cultures was lysed immediately after a 10-min pulse labeling and immunoprecipitated with C7 to determine total betaPP. The other set was chased for 4 h, and media were collected and immunoprecipitated with MMAb to determine betaPP(s) secretion. Samples were separated by SDS-polyacrylamide gel electrophoresis and analyzed by Phosphorimaging. Comparison of betaPP(s) levels was made after normalization for total betaPP expression.

Intracellular betaPP(s)

Separate dishes of stably transfected CHO cells, one dish for each chase time, were pulse labeled for 10 min and chased for either 10, 20, 30, or 60 min. Chase media were collected, and cells were washed with ice-cold DPBS and rapidly chilled to 4 °C. After washing, DPBS was replaced with DPBS containing 0.1% saponin plus protease inhibitors (leupeptin and Pefabloc, Boehringer Mannheim). Cells were treated with saponin buffer for 40 min at 4 °C as described previously(29) , to allow the release of intracellular betaPP(s). Saponin buffer was then collected, and both the chase media and saponin buffers were immunoprecipitated using antibody B5 for total betaPP(s) and antibody C7 for full-length betaPP.

Cell Surface Iodination

Derivatized Bolton-Hunter reagent, sulfosuccinmidyl-3-(4-hydroxyphenyl)propionate (sulfo-SHPP, Pierce) was labeled with NaI in the presence of IODO-GEN (Pierce) at room temperature essentially as described(30) . After 5 min, the iodination reaction was quenched with p-hydroxyphenylacetic acid (Sigma), diluted with DPBS, and immediately added to chilled washed cells for 40 min at 4 °C. After iodination, the cells were extensively washed in DPBS containing 1 mg/ml lysine followed by incubation in prewarmed CHO medium at 37 °C. Three independent experiments were performed.

Surface Antibody Binding

To determine the amount of cell surface and total betaPP in stably transfected CHO cells, 5A3 monoclonal antibody Fab fragments were radioiodinated with IODO-GEN to 2-4 µCi/µg(19) . Confluent cell cultures were chilled and washed. One set of cultures was treated with 0.1% saponin in DPBS for 30 min at 4 °C to permeabilize cells gently and permit the labeling of both cell surface and intracellular betaPP. Parallel cultures for each cell line were treated for 30 min at 4 °C with DPBS for evaluation of cell surface betaPP. Both sets were incubated with radioiodinated antibody at 10 nM in binding medium (RPMI 1640 medium supplemented with 0.2% bovine serum albumin) at 4 °C for 1 h, followed by two washes with binding medium and two washes with DPBS. The cells were then lysed with 0.2 M NaOH, and radioactivity was determined by -counting. To calculate specific binding, background levels of radioactivity were determined from parallel cultures of untransfected CHO cells to subtract from the counts obtained from betaPP-transfected CHO cells. Four separate experiments were performed using triplicate cultures.

Drug Studies

Confluent CHO cells were metabolically labeled with 50-100 µCi/ml [S]methionine for 2 h and then chased for 2 h in media containing either chloroquine (100 µM), bafilomycin A1 (0.25 µM, Wako BioProducts, Richmond, VA), or brefeldin A (35 µM, Epicentre Technologies, Madison, WI). These drugs are known to affect protein processing as described below. Some bafilomycin experiments were performed using a 10-min pulse with [S]methionine followed by a 20-min chase in 0.25 µM bafilomycin A1. betaPP products were immunoprecipitated and visualized as described previously. Six separate experiments were performed.


RESULTS

betaPP Processing by beta-Secretase Is Enhanced in CHO Cells Stably Expressing ``Swedish'' Mutant betaPP

Multiple stably transfected betaPP-sw CHO cell lines were selected based on their equivalent betaPP expression levels to betaPP-wt cell lines. Consistent with earlier reports (12, 22, 31, 32, 33) all of the betaPP-sw cell lines released severalfold more of the 4-kDa Abeta peptide than wild type cells, which had comparable levels of betaPP expression (not shown). Pulse-chase experiments also showed that the timing of appearance and disappearance of labeled betaPP was essentially identical in betaPP-wt and betaPP-sw cells (Fig. 2A). Both betaPP-wt and betaPP-sw cells produced abundant N-glycosylated betaPP during the 10-min labeling reaction (time 0, Fig. 2A). At 1 h of the chase, higher molecular weight N+O-glycosylated betaPP molecules were seen in addition to the N-glycosylated forms. Full-length betaPP decreased by 2 h; and by 4 h, little full-length betaPP remained (Fig. 2A).


Figure 2: betaPP turnover, betaPP(s) species, and precursor product relationship of 12-kDa fragments and Abeta in CHO cells stably transfected with betaPP. Panel A, turnover of full-length betaPP immunoprecipitated with antibody C7 from betaPP-wt and betaPP-sw cells pulse-labeled for 10 min with [S]methionine and chased for 0, 1, 2, or 4 h. Panel B, immunoprecipitation of betaPP(s) from conditioned medium with antibodies B5, R1736, and 6E10 from betaPP-wt and betaPP-sw cells labeled for 4 h with [S]methionine. R1736 and 6E10 are specific for alpha-secretase-cleaved betaPP(s). Panel C, COOH-terminal fragments and Abeta release from betaPP-sw cells following a 10-min pulse with [S]methionine and 10- or 20-min chase. The COOH-terminal fragments were immunoprecipitated with antibody C7 from cell lysates after the 10- or 20-min chase. The 12-kDa fragments (at arrowhead), clearly apparent by 10 min, increased by 20 min. Media from parallel cultures immunoprecipitated with antibody R1282 show an Abeta (at arrowhead) signal by 20 min. No Abeta signal is observed at 10 min even when the signal is intentionally amplified as in the lanes on the right. For comparison, the unamplified Abeta image is presented to the left of the darkened image. Molecular weights determined from prestained standards are indicated. wt = betaPP-wt cells; sw = betaPP-sw cells.



Secretion of a shortened betaPP(s) species has been reported from a betaPP chimeric molecule expressing the ``Swedish'' mutation (31) . To confirm this finding with authentic betaPP molecules, betaPP(s) was immunoprecipitated from conditioned media using B5 antibody, which recognizes both alpha- and beta-secretase species of betaPP(s), and two antibodies that recognize only alpha-secretase-cleaved betaPP(s) (R1736 and 6E10). As observed for untransfected CHO cells (not shown), betaPP(s) from transfected CHO cells migrates as a doublet of bands on low percentage polyacrylamide gels. As a result, the higher molecular weight betaPP(s) cleaved by alpha-secretase and the slightly lower molecular weight betaPP(s) cut by beta-secretase can best be compared by observing the lower of the two bands of each doublet (Fig. 2B). The betaPP(s) from betaPP-sw cells migrated at an M(r) consistently lower than that of betaPP-wt cells, indicating the secretion of a shorter betaPP(s) species. Although both cell lines secreted comparable levels of total betaPP(s) by B5 antibody immunoprecipitation (Fig. 2B), betaPP-sw cells had dramatically reduced levels of alpha-secretase-cleaved betaPP(s) (6 ± 1.3-fold less) than betaPP-wt cells using antibodies R1736 and 6E10 (Fig. 2B). Consistent with this finding, and as reported by others(12, 31, 32, 33) , betaPP-sw cells also had correspondingly higher levels of 12-kDa COOH-terminal betaPP fragments (see below).

Timing of Secretion of betaPP(s), Abeta, and p3 Is Identical in betaPP-wt and betaPP-sw Cells

Since the turnover rate of full-length betaPP was essentially the same in the betaPP-wt and betaPP-sw cells, we next examined the biosynthetic rate for the generation of betaPP-secreted products (betaPP(s), Abeta, and p3). Following a 10-min pulse labeling, media were collected from a single dish each from betaPP-wt and betaPP-sw cells and reapplied at 10-min intervals to define the incremental release of betaPP secretion products during the 1st h of the chase period.

The onset of secretion of total betaPP(s) was first detectable at 10 min as determined with B5 immunoprecipitation (Fig. 3B). However, at this first time interval only minute amounts of betaPP(s) were secreted from both betaPP-wt and betaPP-sw cells because the signal could be seen in the 10-min lane only after prolonged autoradiographic exposures (Fig. 3B). betaPP(s) became pronounced at 20 min for both cell lines with peak secretion at approximately 30 min (Fig. 3A and Fig. 4). The profile of betaPP(s) secretion as a function of time was essentially identical for the two cell lines (Fig. 4). In the experiment shown, although betaPP(s) secretion by betaPP-sw cells was lower because of diminished expression of full-length betaPP, the profile of secretion is essentially identical to that of betaPP-wt cells. This profile of betaPP(s) secretion did not depend on the level of betaPP expression because other wild type and Swedish cell lines exhibited the same patterns of release (not shown). Furthermore, comparison of betaPP-wt and betaPP-sw cells that expressed equivalent levels of betaPP confirmed that betaPP(s) secretion by both cell lines was essentially the same (within 10% of each other as determined by Phosphorimage analysis of media from triplicate cultures from each cell line, Student's t test, p = 0.49).


Figure 3: Incremental release of betaPP(s), Abeta, and p3 from CHO cells transfected with wild type or K670N/M671L mutant betaPP. Immunoprecipitations of betaPP(s), Abeta, and p3 from conditioned chase media from single cultures of betaPP-wt and betaPP-sw cells following a 10-min [S]methionine pulse label were collected at 10-min intervals. The level of betaPP holoprotein expression was somewhat lower in betaPP-sw cells in this experiment. On low percentage polyacrylamide gels, betaPP(s) from CHO cells migrates as a doublet. Panel A, total betaPP(s) was immunoprecipitated with antibody B5. Note the lower molecular weight species of betaPP(s), indicated by the arrowhead, from betaPP-sw cell media. Panel B, the presence of betaPP(s) at 10 min is confirmed by this long exposure of the gel shown in panel A. The shortened betaPP(s) form, at the arrowhead, is apparent at the earliest time point. Panel C, Abeta and p3 immunoprecipitated by R1282 from the same media as panels A and B. Positions of Abeta and p3 are indicated. Molecular weights determined from prestained standards are indicated on the right. wt = betaPP-wt cells; sw = betaPP-sw cells.




Figure 4: Profiles of betaPP(s), Abeta, and p3 release from CHO cells transfected with wild type or K670N/M671L mutant betaPP. Data from Phosphorimage analysis of gels in Fig. 3represent the percent secretion for each time point relative to the cumulative (100%) secretion during the entire 60-min chase. The top panel shows the betaPP(s) release from betaPP-wt (designated by the solid line and circles in all graphs) and betaPP-sw cells (designated by the dotted line and triangles in all graphs) from antibody B5 immunoprecipitation. Abeta (middle panel) and p3 (bottom panel) release from betaPP-wt and betaPP-sw cells, immunoprecipitated with R1282 antibody, are also shown. wt = betaPP-wt cells; sw = betaPP-sw cells.



Regarding Abeta release, the timing of Abeta secretion during the 1st h from betaPP-wt and betaPP-sw cells was also identical (Fig. 3C and Fig. 4). The Abeta signal was first apparent at the 20-min collection time by autoradiography (Fig. 3C) and reached a peak at 30-40 min. Although no discernible Abeta signal was ever seen on either autoradiograms or Phosphorimages at the 10-min chase time, after long exposures a few Phosphorimage counts higher than background were detected in the 10-min lane (Fig. 4). At each chase time, betaPP-sw cells consistently released more Abeta than betaPP-wt cells. The timing of p3 secretion mirrored that of Abeta in both betaPP-wt and betaPP-sw cells throughout the chase period (Fig. 3C), although betaPP-wt cells consistently released more p3 relative to Abeta than did betaPP-sw cells. Authentication of Abeta (beginning at Asp^1) and p3 (beginning at Lys) was obtained by radiosequencing (not shown), as reported previously(15, 19) . Thus, a difference in the ratios of alpha-secretase- and beta-secretase-generated molecules was also reflected by the levels of p3 and Abeta released by these cell lines. Finally, the formation of the beta-secretase-generated 12-kDa betaPP COOH-terminal fragments preceded the release of Abeta from pulse-labeled betaPP-sw cells (Fig. 2C). After a 10-min labeling with [S]methionine, the 12-kDa fragment was apparent by the 10-min chase time in betaPP-sw cells and increased at 20 min (Fig. 2C). Consistent with the above results, Abeta was not apparent in the corresponding media until 20 min of the chase period (Fig. 2C). This earlier generation of the 12-kDa fragment prior to Abeta release, consistently seen in three experiments, indicates a precursor-product relationship between the two molecules.

betaPP(s) and Abeta Appear to Be Present Intracellularly in betaPP-wt and betaPP-sw Cells

The release of betaPP(s) at very early chase times suggested that betaPP may be cleaved by beta-secretase in the secretory pathway. To determine if soluble betaPP(s) was present intracellularly, metabolically labeled cells were treated with 0.1% saponin in buffer. Saponin is a mild detergent that permeabilizes cells but does not solubilize the lipid bilayer (29) and therefore allowed the intracellular betaPP(s) to diffuse into the buffer. Essentially no full-length betaPP was detected in the saponin buffer of treated cells. However, soluble intracellular betaPP(s) was recovered from the saponin buffer from both betaPP-wt and betaPP-sw cells (Fig. 5, A and B). Intracellular betaPP(s) species from betaPP-wt cells (a finding previously reported by others; see (34, 35, 36) ) migrated with an M(r) consistent with alpha-secretase-cleaved molecules (Fig. 5A). A shorter betaPP(s) species with an M(r) identical to secreted betaPP(s) and consistent with beta-secretase-cleaved molecules was observed from betaPP-sw cells (Fig. 5A; (32) ). Furthermore, in pulse-chase experiments a precursor-product relationship could be demonstrated between intracellular betaPP(s) from the saponin-treated cells and betaPP(s) secreted into the medium (Fig. 5B).


Figure 5: Generation of intracellular betaPP(s) and Abeta from CHO cells transfected with wild type or K670N/M671L mutant betaPP. Panel A, B5 antibody immunoprecipitation of betaPP(s) from chase media and saponin buffers of betaPP-wt and betaPP-sw cells pulse-labeled for 10 min with [S]methionine and chased for 20 min. Since betaPP(s) from CHO cells migrates as a doublet, the higher molecular weight betaPP(s) cleaved by alpha-secretase (alpha at arrow) and the slightly lower molecular weight betaPP(s) cut by beta-secretase (beta at arrow) can best be appreciated by observing the lower of the two bands. The shorter betaPP(s) species is observed both intracellularly (intra) and secreted into the medium (sec) of betaPP-sw cells. The faint bands that run below 97 kDa in the saponin lanes are degradation products. Molecular weights determined from prestained standards are indicated on the right. Panel B, antibody B5 immunoprecipitations of intracellular and secreted betaPP(s) from betaPP-wt and betaPP-sw cells following a 10-min pulse with [S]methionine and 10-60-min chase. Intracellular betaPP(s) was immunoprecipitated from saponin buffers; secreted betaPP(s) was obtained from chase media. Panel C, immunoprecipitation of betaPP-wt and betaPP-sw control (cont) cell lysates after 4 h [S]methionine labeling with antibody R1280 or R1280 that had been preabsorbed (abs) with the Abeta 1-40 peptide. The positions of the 12-kDa COOH-terminal fragments and Abeta are indicated at arrows on the left. wt = betaPP-wt cells; sw = betaPP-sw cells.



These results suggested that Abeta can be formed within the secretory pathway. Indeed, intracellular Abeta appeared to be present in both betaPP-wt and betaPP-sw cell lysates labeled for 4 h (Fig. 5C). Preabsorption of R1280 antibody with the Abeta 1-40 peptide totally eliminated immunoprecipitation of Abeta from the cell lysates by R1280 antibody (Fig. 5C), and no 4-kDa band was observed from the same lysate using antibody C7. Treatment with trypsin prior to immunoprecipitation did not diminish the Abeta signal (not shown), indicating that Abeta was present inside the cells. In addition, cells pulse labeled with [S]methionine followed by a 20-min or 30-min chase had both Abeta and p3 isolated from cell lysates (not shown). Thus, the immunoprecipitated Abeta had not been derived from secreted molecules present on the extracellular plasma membrane at the time of cell lysis. Furthermore, the appearance of these intracellular Abeta and p3 molecules after short pulse-chase intervals provides indirect evidence of their production in the secretory and not the endosomal/lysosomal pathway. Nevertheless, Abeta and p3 bands were visualized only after 8-10 weeks of autoradiographic exposure, suggesting that only very low levels of Abeta were ever present intracellularly. The minute amounts of intracellular Abeta precluded definitive identification by amino acid radiosequencing.

betaPP from the Cell Surface Contributes to Abeta Production in Both betaPP-wt and betaPP-sw Cells

To determine if the endocytic pathway contributed to Abeta production in betaPP-sw cells, release of Abeta was analyzed after selective cell surface radioiodination. Consistent with an earlier report(19) , little radiolabeled Abeta was secreted within the first 10 min by either cell line, but considerable Abeta was released by 2 h from both betaPP-wt and betaPP-sw cells (Fig. 6A), with betaPP-sw cells releasing greater than 2-fold more Abeta than betaPP-wt cells at the 2-h collection time (2.4 ± 0.4). The timing of Abeta release following labeling of cell surface betaPP was essentially the same in the two cell lines (Fig. 6A). However, in sharp contrast to the timing of Abeta secretion, the majority of the iodinated betaPP(s) was released within the first 5 min of incubation at 37 °C from both betaPP-wt and betaPP-sw cells (Fig. 6B). In addition, these profiles of both Abeta and betaPP(s) release are distinctly different from the timing observed for Abeta and betaPP(s) release observed using [S]methionine labeling ( Fig. 3and Fig. 4).


Figure 6: Release of Abeta and betaPP(s) from cell surface-iodinated betaPP molecules. Panel A, immunoprecipitation of Abeta with antibody R1280 from chase media of betaPP-wt and betaPP-sw cells following iodination of cell surface betaPP. The timing of release of Abeta was the same from both cell lines. Molecular weights determined from prestained standards are indicated. Panel B, rapid release of betaPP(s) was observed from both betaPP-wt and betaPP-sw cells after surface iodination and immunoprecipitation by antibody B5. Panel C, immunoprecipitation of cell lysates with antibody C7 after iodination revealed more full-length betaPP on the surface of betaPP-wt cells than betaPP-sw cells. betaPP-sw cells, however, had more iodinated 12-kDa COOH-terminal fragments and fewer 10-kDa fragments than betaPP-wt cells. wt = betaPP-wt cells; sw = betaPP-sw cells.



Two additional observations are noteworthy from these experiments. First, betaPP(s) derived from cell surface betaPP by betaPP-sw cells had an M(r) compatible with alpha-secretase-cleaved betaPP(s) (Fig. 6B). A lower M(r) beta-secretase-cleaved betaPP(s) species was not readily apparent after surface labeling. However, resolution of the labeled bands is significantly less distinct from an iodine signal because of radiographic intensification, and minor differences may be undetectable. Second, we consistently observed more full-length betaPP on the surface of betaPP-wt cells than betaPP-sw cells (Fig. 6C) expressing the same amount of betaPP. To confirm and quantitate this difference, the levels of cell surface and total betaPP were measured by an antibody binding assay using radioiodinated antibody 5A3 Fab fragments, which bind to an extracellular betaPP epitope(19) . Treatment with 0.1% saponin permitted labeling of both cell surface and intracellular betaPP. Multiple repetitions of this experiment showed that betaPP-sw cells had approximately 50% less cell surface betaPP than betaPP-wt cells (49.8% ± 0.7, p < 0.0001). Interestingly, betaPP-sw cells showed more of the COOH-terminal 12-kDa fragment and less of the 10-kDa fragment than betaPP-wt cells (Fig. 6C) present on the cell surface.

Drug Treatments Affect betaPP-wt and betaPP-sw Cells Similarly

To assess further whether the processing of betaPP molecules is similar in betaPP-wt and betaPP-sw cells, both cell lines were treated with a variety of compounds known to affect protein processing. Control lanes reveal the higher production of both Abeta and 12-kDa COOH-terminal betaPP fragments from betaPP-sw cells (Fig. 7A). Treatment with brefeldin A, a drug that blocks the maturation of proteins by collapsing the Golgi into the endoplasmic reticulum, inhibited the production of both Abeta and the 12-kDa fragment in both betaPP-wt and betaPP-sw cells (Fig. 7A). Treatments that alkalinize intracellular vesicles were also used because Abeta generation in cultured cells appears to require an acidic compartment(8, 13) . To determine if beta-secretase cleavage of both betaPP-wt and betaPP-sw cells occurs in an acidic intracellular compartment, cells were exposed to chloroquine or bafilomycin A1(37) . In response to chloroquine presented during the 2-h chase, both betaPP-wt and betaPP-sw cells released 30%-60% less Abeta than untreated controls (Fig. 7A). As anticipated(10, 13) , chloroquine also dramatically elevated the level of COOH-terminal fragments in both cell lines (Fig. 7A). Exposure of cells to bafilomycin A1, a drug that specifically inhibits vacuolar H-ATPases and thus prevents vesicular acidification(37) , produced a 60% decrease in Abeta release from both betaPP-wt and betaPP-sw cells (as measured by Phosphorimaging, Fig. 7A). A corresponding increase in 10-kDa COOH-terminal fragments was observed following exposure to bafilomycin A1 (Fig. 7A), suggesting that less betaPP was cleaved by beta-secretase in the presence of the drug. Consistent with this interpretation is the finding of a dramatic decrease in the amount of 12-kDa fragments in betaPP-sw cells when bafilomycin was presented using a short pulse-chase paradigm (Fig. 7B). This reduction of the 12-kDa fragment (Fig. 7B) was seen at a time when abundant 12-kDa COOH-terminal fragments were normally observed from betaPP-sw cell lysates following a 10-min pulse-labeling (Fig. 2C and Fig. 7B). In sum, the processing of betaPP and release of Abeta by CHO cells expressing either wild type or mutant betaPP were similarly affected by these drug treatments.


Figure 7: Effects of various treatments on Abeta production and COOH-terminal fragments from CHO cells transfected with wild type or K670N/M671L mutant betaPP. Panel A, immunoprecipitation of betaPP-wt and betaPP-sw conditioned media with antibody R1282, and cell lysates with antibody C7 from a 2-h [S]methionine label followed by a 2-h chase containing either no drug (Cont), brefeldin A (Bref), chloroquine (Cq), or bafilomycin A1 (Balfilo). Less Abeta was released in the presence of drugs compared with the control condition for both cell lines. Panel B, antibody C7 immunoprecipitation of betaPP-sw cell lysates after a 10-min [S]methionine pulse followed by a 20-min chase in the absence (0) or presence (.25) of bafilomycin A1. Note the presence of the 12-kDa COOH-terminal fragment generated by beta-secretase cleavage at 20 min in the control lane (0) and its near absence after bafilomycin A1 treatment. wt = betaPP-wt cells; sw = betaPP-sw.




DISCUSSION

A double mutation in the betaPP gene from a Swedish kindred with familial Alzheimer's disease is invariably linked with Alzheimer's disease(21) . All cells reported to date which express the betaPP mutation produce dramatically more Abeta peptide than do cells expressing wild type betaPP(12, 22, 23, 31, 32, 33) . Since excess Abeta production may be causally related to the Alzheimer's phenotype in individuals affected with the ``Swedish'' mutation(21) , it is important to evaluate the mechanism by which Abeta is produced from betaPP with this alteration. In this study we performed a detailed analysis of the biosynthetic processing of betaPP in betaPP-wt and betaPP-sw CHO cells.

Our results showed that, as anticipated, betaPP-sw cells released substantially more Abeta than betaPP-wt cells. Interestingly, the timing of onset and the duration of Abeta secretion during the 1st h following a short pulse labeling were coincident with p3 release for both cell lines. Only the amounts of Abeta and p3 varied between betaPP-wt and betaPP-sw cells. Furthermore, treatments known to decrease Abeta in betaPP-wt cells (8, 13, 38) also affected betaPP-sw cells. We interpret our data to suggest that the pathway of Abeta production is similar for betaPP-wt and betaPP-sw cells. In contrast, however, the timing of Abeta release differed substantially depending on whether cells were [S]methionine-labeled or surface-iodinated. In both cell lines Abeta was released with a shorter time course from [S]methionine-labeled cells than from cells that were surface-iodinated. This difference in the timing of Abeta secretion leads us to propose that Abeta is generated in both the secretory and endocytic pathways from both betaPP-wt and betaPP-sw cells.

A number of observations suggest that Abeta is generated in the secretory pathway(10, 39) . First, the timing of secretion of betaPP(s), Abeta, and p3 was essentially identical at early chase times in both cell lines. Specifically, within the first 30 min in a short pulse-chase experiment, the profiles of betaPP(s), Abeta, and p3 secretion were remarkably similar. This chase paradigm was chosen specifically to reveal the incremental release of these early secretory products. Second, permeabilization of [S]methionine pulse-labeled cells followed by immunoprecipitation with a betaPP midregion antibody (B5) showed that intracellular soluble betaPP(s) was present in both betaPP-wt (34, 35, 36) and betaPP-sw cells as reported previously(32) . The major intracellular species of soluble betaPP(s) from betaPP-sw cells had a lower M(r) than betaPP(s) from betaPP-wt cells, consistent with production by beta-secretase cleavage. Significantly, intracellular betaPP(s) was present before abundant betaPP(s) was secreted into the culture medium, thus demonstrating a precursor-product relationship. Third, the 12-kDa COOH-terminal fragment of betaPP and Abeta showed a precursor-product relationship, with the 12-kDa molecules apparent 10 min prior to the appearance of Abeta. Moreover, consistent with a recent report(12) , this COOH-terminal 12-kDa fragment was specifically increased in betaPP-sw cells compared with betaPP-wt cells. Fourth, our data suggest that intracellular Abeta is present in both betaPP-wt and betaPP-sw cells. Based on results from trypsin digestion using a short pulse-chase paradigm, Abeta in cell lysates did not appear to represent extracellular Abeta attached to the cell surface or to be derived from the lysosomal pathway. However, the exceedingly small amount of intracellular Abeta suggests that Abeta turnover and secretion are rapid. This is consistent with the earlier postulation that Abeta is released from cells soon after it is formed and suggests that -secretase cleavage occurs at or near the cell surface(19) . Previously, intracellular Abeta has only been detected in neurons(40) . Thus our preliminary findings suggest that the pathways of Abeta production in neurons and non-neuronal cells may be more similar than was previously thought.

Regarding the endocytic processing of betaPP, cells labeled by selective cell surface iodination confirmed that betaPP-sw cells produced more Abeta from cell surface precursors than did betaPP-wt cells. However, the timing of Abeta release after surface labeling was essentially identical for both betaPP-wt and betaPP-sw cells. As shown previously for cells expressing wild type betaPP(19) , Abeta generated from surface-labeled molecules was released more slowly than betaPP(s) by betaPP-sw cells. These profiles of Abeta and betaPP(s) release from surface-labeled molecules are dramatically different from the [S]methionine labeling experiments in which Abeta and betaPP(s) were released simultaneously. Interestingly, betaPP(s) and Abeta release from [S]methionine pulse-chase experiments showed that betaPP(s) secretion peaked at 30 min followed by a sharp decrease, whereas Abeta release continued at the same level until later chase times. We interpret the sustained Abeta release into the medium at a time when betaPP(s) secretion decreased (40-50 min) to represent the addition of newly generated Abeta, derived from the endocytic pool, after the contribution of the secretory pool of Abeta has peaked. Therefore, our data indicate that Abeta can be derived from both the secretory and endocytic pathways and that more Abeta is formed within each pathway by betaPP-sw cells.

Our studies have defined a number of similarities in betaPP processing between betaPP-wt and betaPP-sw cells. First, the timing of secretion of betaPP(s), Abeta, and p3 is essentially the same for both cell lines within the 1st hour following a 10-min pulse label. Second, various drug treatments decrease Abeta in both betaPP-wt and betaPP-sw CHO cells. Third, intracellular betaPP(s) species and Abeta appear to be present in both cell lines. Fourth, both secretory and endocytic pathways appear to contribute to Abeta generation and release. Fifth, both betaPP-wt and betaPP-sw cells secrete primarily alpha-secretase-cleaved betaPP(s) from surface-labeled betaPP. Thus, within the limits and sensitivity of our experimental system, the timing and the pathway of Abeta secretion appear to be identical in betaPP-wt and betaPP-sw cells. Only the amounts of Abeta and beta-secretase-cleaved precursors differed in betaPP-wt and betaPP-sw cells. Our data and interpretation are therefore consistent with the results of previous investigators who have suggested that the ``Swedish'' mutation at the NH(2) terminus of Abeta enhances beta-secretase cleavage(12, 22, 23) . This altered beta-secretase cleavage produces abundant beta-secretase-cleaved betaPP(s) in the secretory pathway in betaPP-sw cells, leading to excess Abeta production. However, it remains unclear at present which pathway, secretory or endocytic, plays the greater role in Abeta production.

betaPP-sw cells did show some differences in betaPP processing from betaPP-wt cells. In addition to the increase in beta-secretase-cleaved products described above, there was a 50% reduction in the amount of cell surface betaPP in betaPP-sw cells. Concomitantly, there was an increase in the 12-kDa membrane-retained betaPP fragments present on the cell surface of betaPP-sw cells. Whether this increase in 12-kDa fragments is sufficient to account for the decrease in full-length betaPP molecules at the cell surface of betaPP-sw cells is unclear. Because secreted betaPP(s) levels are similar between betaPP-wt and betaPP-sw cells, the reduction in full-length betaPP at the surface of betaPP-sw cells suggests that the amount of betaPP targeted to the cell surface may represent a minor fraction of the total betaPP processed in the secretory pathway. Otherwise, one would expect to see a substantial increase in betaPP(s) released into the medium from betaPP-sw cells, which was not detected. Furthermore, this interpretation is also consistent with reports of other cell types that express little or no betaPP on the cell surface(34, 35, 36) .

In summary, our data suggest that there is a similar mechanism for Abeta generation in both betaPP-wt and betaPP-sw cells. The increased Abeta production from betaPP-sw cells appears to result from enhanced beta-secretase cleavage of the mutant betaPP in both the secretory and endocytic pathways. A recent report has demonstrated altered betaPP processing in mutant betaPP molecules with natural or designed mutations in codon 692(41) , whereas another report demonstrated an increased percentage of longer Abeta peptides from betaPP with codon 717 mutations (42) . Taken together, it appears that FAD betaPP mutations lead to pleiotropic effects on betaPP and Abeta metabolism. The Alzheimer phenotype associated with these dominant mutations may therefore result from different cellular perturbations that specifically modify betaPP processing.


FOOTNOTES

*
This work was supported in part by Alzheimer's Association Grant ZEN-94-011 (to E. H. K.) and National Institute of Aging Grants AG12376 (to E. H. K.) and 5T32AG00222 (to R. G. P.). 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: Center for Neurologic Diseases, Brigham and Women's Hospital, 221 Longwood Ave., LMRC 114, Boston, MA 02115. Tel.: 617-278-0344; Fax: 617-732-7787.

(^1)
The abbreviations used are: Abeta, amyloid beta-protein; betaPP, beta-amyloid precursor protein; betaPP(s), soluble betaPP; betaPP-sw, ``Swedish'' mutant betaPP; betaPP-wt, wild type betaPP; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; MMAb, monoclonal antibodies 5A3 and 1G7 used together; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DPBS, Dulbecco's phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Dr. Martin Citron for the betaPP K670N/M671L cDNA and for helpful discussions, Dr. Margaret Kruse and Deborah Watson for critical reading of the manuscript, and Dr. Dennis Selkoe for the generous contribution of various betaPP antibodies.

Note Added in Proof-Similar findings of intracellular Abeta recently have been reported by Martin et al. (Martin, B. L., Schrader-Fischer, G., Busciglio, J., Duke, M., Paganetti, P., and Yankner, B. A.(1995) J. Biol. Chem.270, 26727-26730) in cells transfected with Swedish mutant [Abstract/Full Text] betaPP.


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