©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Aggregation of Secreted Amyloid -Protein into Sodium Dodecyl Sulfate-stable Oligomers in Cell Culture (*)

Marcia B. Podlisny , Beth L. Ostaszewski , Sharon L. Squazzo , Edward H. Koo , Russell E. Rydell (2), David B. Teplow (1), Dennis J. Selkoe

From the (1) Department of Neurology and Program in Neuroscience, Harvard Medical School, the Center for Neurologic Diseases and Biopolymer Laboratory, Brigham and Women's Hospital Boston, Massachusetts 02115, and (2) Athena Neurosciences, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Filamentous aggregates of the 40-42-residue amyloid -protein (A) accumulate progressively in the limbic and cerebral cortex in Alzheimer's disease, where they are intimately associated with neuronal and glial cytopathology. Attempts to model this cytotoxicity in vitro using synthetic peptides have shown that monomeric A is relatively inert, whereas aggregated A reproducibly exerts a variety of neurotoxic effects. The processes that mediate the conversion of monomeric A into a toxic aggregated state are thus of great interest. Previous studies of this conversion have employed high concentrations (10-10 M) of synthetic A peptides under nonbiological conditions. We report here the detection of small amounts (<10 M) of SDS-stable A oligomers in the culture media of Chinese hamster ovary cells expressing endogenous or transfected amyloid -protein precursor genes. The identity of these oligomers (primarily dimers and trimers) was established by immunoprecipitation with a panel of A antibodies, by electrophoretic comigration with synthetic A oligomers, and by amino acid sequencing. The oligomeric A species comprised 10-20% of the total immunoprecipitable A in these cultures. A truncated A species beginning at Arg 5 was enriched in the oligomers, suggesting that amino-terminal heterogeneity can influence A oligomerization in this system. Addition of Congo red (10 µ M) during metabolic labeling of the cells led to increased monomeric and decreased oligomeric A. The ability to detect and quantitate oligomers of secreted A peptides in cell culture should facilitate dynamic studies of the critical process of initial A aggregation under physiological conditions.


INTRODUCTION

Amyloid -protein (A)() is a hydrophobic proteolytic fragment of a ubiquitously expressed integral membrane polypeptide termed the amyloid -protein precursor (PP). Progressive cerebral deposition of A is an invariant feature of Alzheimer's disease (AD) that appears to precede the development of the characteristic neuronal and glial cytopathology of AD (1) . One established molecular cause of familial (autosomal dominant) AD (FAD) is the occurrence of missense mutations within or immediately flanking the A coding region of the PP gene on chromosome 21 (2) . Cerebral deposits of A can exist as dense, filamentous aggregates of the peptide surrounded by dystrophic neurites and glial cells (classical or neuritic amyloid plaques) and as loose, amorphous deposits associated with little or no local cellular alteration (diffuse or preamyloid plaques). Recently, similar A deposits, including some associated with neuritic dystrophy and synaptic loss, have been described in transgenic mice overexpressing a FAD-linked mutant PP, strongly supporting the primacy of PP processing to A in AD (3) .

Although A was originally identified as the protein comprising the insoluble filaments in AD amyloid deposits (4) , it also occurs as a normal, soluble product of cellular metabolism that is constitutively secreted by PP-expressing cells and is found in cerebrospinal fluid and plasma (5, 6, 7, 8) . As a result, the mechanism by which soluble, monomeric A gradually accumulates as insoluble aggregates associated with surrounding cytotoxicity has become a central question in AD pathobiology. Attempts to model the toxicity of A by adding synthetic peptides to neural cultures have provided strong evidence that freshly solubilized, monomeric A is relatively inert, whereas aggregated, oligomeric, and polymeric A that forms after incubating the peptide in vitro ( e.g. at 37° for 3-7 days) is cytotoxic ( e.g. see Refs. 9-12). Many biophysical studies of the aggregation and/or cytotoxicity of synthetic A have been reported, but these have several limitations. These studies employ high doses of synthetic peptides, usually in the 10-10 M range, whereas concentrations of A in physiological fluids are 10-10 M(6, 13) , and normal concentrations in brain are believed to be even lower (14) . Aggregation is generally examined under nonbiological conditions, e.g. using synthetic peptides solubilized in organic solvents and then studied in water or simple aqueous buffers free of other proteins. Peptides of a single, specified length ( e.g. 28, 40, or 42 residues) are generally used in contrast to the rich array of endogenous A peptides having heterogeneous N and C termini that are produced by cultured human cells (5, 13, 15, 16) and found in human cerebrospinal fluid (6, 17) and brain (18, 19, 20) .

We have observed that immunoprecipitation of the conditioned media of certain PP-transfected cells with A-specific antibodies reveals, in addition to monomeric 4-kDa A and a related 3-kDa fragment (p3), peptides migrating at 6, 8, and 12 kDa (21) . We report here the characterization of these peptides and show that they represent stable oligomers that form from A peptides secreted by cells at high picomolar to low nanomolar levels. The ability to detect these oligomers and to quantify their abundance among different PP-expressing cell types will facilitate dynamic analyses of the factors influencing the critical process of initiation of A aggregation under physiological conditions.


MATERIALS AND METHODS

PP-expressing Cell Lines

Chinese hamster ovary (CHO) cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal clone II (Hyclone). CHO cell lines expressing wild-type PPwere generated with the expression vector pCMV751 (22) using the Lipofectin (Life Technologies, Inc.) transfection method and selected by G418 resistance. Additional stable cell lines expressing certain PP missense mutations (for review see Ref. 2) were generated by oligonucleotide-directed mutagenesis of pCMV751 and transfection into CHO cells. These included the Val Phe or Val Ile mutations at codon 717 (PPnumbering) and the Lys Asn and Met Leu double mutation at codons 670 and 671 that are linked to early onset familial AD, and the Glu Gln mutation at codon 693 that causes hereditary cerebral hemorrhage with amyloidosis, Dutch type. Multiple stable single clones were established from each of these constructs.

Antibodies

The epitopes for many of the PP antibodies used in this study are diagrammed (see Fig. 2). Rabbit antisera to synthetic A peptides include R1280 raised against A1-40 (5, 23) , antiserum Y to A1-38 (24) , and R1963 to A21-37 (5) . Antiserum R1282 to synthetic A1-40 was produced as described for antiserum Y (24) characterized immunocytochemically on AD brain sections and by immunoprecipitation of culture media and found to be highly similar to R1280. Monoclonal antibodies 266 to A13-28 (6) and 4G8 to A17-24 (25) were also used. Polyclonal antisera to synthetic peptides of PP outside the A region included those raised to PP602-615 (26) , PP652-671, PP724-739 (27) , PP751-770 (C7) (28) , and a bacterial recombinant protein of human PP519-667 (5) (29) (all numbered according to the PPisoform). Antiserum PP652-671 was generated similarly to C7 (28) and characterized on immunoblots of PP-transfected cells and by immunoprecipitation of cell lysates. Antibodies 266 and 5 were generously provided by Athena Neurosciences, Inc.


Figure 2: The 6-14-kDa proteins are specifically immunoprecipitated by several A antibodies but not by antibodies to PP epitopes flanking A. The positions of the epitopes for the seven antibodies are indicated on the PP schematic. Antiserum dilution was 1:300 except for R1282 (1:150) and R1963 (1:100). PP antibodies flanking A failed to precipitate A, p3, and the higher Mbands, while four different A antisera precipitated all of these bands.



Immunoprecipitation, Gel Fluorography, and Radiosequencing

Confluent 100-mm dishes of CHO cells were incubated for 30-45 min in Met-free medium and then labeled overnight with 300 µCi of [S]Met (75 µCi/ml; DuPont NEN) in 10% dialyzed fetal bovine serum (FBS) (Hyclone). The conditioned media (CM) were spun at 2100 g for 30 min, precleared with protein A-Sepharose, and immunoprecipitated overnight with the relevant antiserum (1:300 or as noted), as described previously (26) , except that all washes were centrifuged at 5000 g. Immunoprecipitated samples were then boiled for 3 min in SDS/-mercaptoethanol (ME) sample buffer (Novex, San Diego, CA), electrophoresed on 10-20 or 16% Tris-tricine gels (Novex) and subjected to autofluorography. In some control experiments, instead of eluting the immunoprecipitates from protein A-Sepharose by boiling in SDS sample buffer, 0.2 M glycine, pH 2.5, was used, after which the samples were neutralized with Tris base, mixed with SDS sample buffer lacking ME, and directly electrophoresed without heating. Synthetic A peptides used as positive controls for comigration studies were A1-42 (Bachem) and A1-43 (generously provided by S. Little, Lilly Research Labs). Antibodies were adsorbed at a concentration of 10-15 µg of synthetic peptide/µl of undiluted antiserum in 1% bovine serum albumin/phosphate-buffered saline for 2-4 h at 4 °C and cleared at 16,000 g for 10 min. Gel bands were quantitated using a Molecular Dynamics PhosphorImager 400A and ImageQuant software.

To confirm the structural identity of A and related oligomers, cells were labeled with [H]Phe, and radiosequencing was performed as described previously (5, 30) .

Congo Red Experiments

Cells were labeled as above, except in serum-free Dulbecco's modified Eagle's medium. Congo red (Aldrich, Milwaukee, WI) was prepared as a stock solution of 10 m M in MeSO and added to tissue culture medium at a 1:1000 dilution during metabolic labeling.


RESULTS

We initially asked whether secreted A peptides underwent polymerization in cell culture, analogous to the aggregation, precipitation, and seeding experiments reported for synthetic A peptides in vitro ( e.g. see Refs. 10-12 and 31-35). Conditioned media from [S]methionine-labeled CHO cells stably transfected with PPcDNA (CHO) were immunoprecipitated with R1280, a high-titer polyclonal antibody to synthetic human A, and electrophoresed in Tris-tricine gels. Autofluorographs revealed bands of 6, 8, and 12 kDa, in addition to the characteristic 4- (A) and 3-kDa (p3) peptides (Fig. 1 A, lane 3). Absorption of R1280 with synthetic Aabolished the precipitation of A and p3 as well as the three larger proteins (Fig. 1 A, lane 4). As in numerous previous reports (31, 33) , an A synthetic peptide spontaneously formed SDS-stable oligomers upon storage; comparison of an R1280 immunoblot of this peptide preparation (Fig. 1 A, lane 2) with the fluorograph of the R1280 immunoprecipitate of CHOmedium run in the adjacent lane (Fig. 1 A, lane 3) revealed that the 4-, 8-, and 12-kDa CHO-derived peptides comigrated with bands of these sizes in the synthetic peptide sample, strongly suggesting that the bands of molecular mass greater than 4 kDa represent A oligomers. Examination of several CHO lines stably expressing mutant (PPV717I, PPV717F, and PPE693Q) or wild-type PPalways revealed the 6, 8, and 12 kDa R1280-reactive bands in all lines (Fig. 1 B). Quantitation of these A-specific bands by PhosphorImaging demonstrated that differences among the cell lines examined to date were not consistently dependent on the mutation expressed (data not shown). The average percent of each A-specific band as a function of the total signal for all four A bands was calculated for all of the above cell lines. The mean percents ± S.D. ( n = 32) were: 4 kDa, 65 ± 11%; 6 kDa, 19 ± 7%; 8 kDa, 11 ± 4%; and 12 kDa, 4 ± 4%. Nontransfected CHO cells, which endogenously express low levels of PP, showed substantially smaller amounts of these bands, as expected (Fig. 1 C). In the experiments that follow, high PP expressing stable cell lines were utilized to enhance detection of the oligomers. On longer fluorographic exposures of our highest PP expressing cell lines, faint A-specific bands of 10, 14, and sometimes 16 kDa were detected (Fig. 1 D).


Figure 1: CM from labeled CHO cells immunoprecipitated with A antibodies contains proteins of 6-16 kDa in addition to A and p3. A, R1280-immunoprecipitated CM from [S]Met-labeled CHOcells ( wt-1 line; see B below) was electrophoresed on a 16% Tris-tricine gel ( lanes 3 and 4); purified synthetic A peptides were loaded directly in adjacent lanes ( 1 and 2). The gel was transferred to polyvinylidine difluoride membrane and either immunoblotted with R1280 ( lanes 1 and 2) or subjected to autoradiography ( lanes 3 and 4). Lane 1, synthetic A(100 ng); lane 2, synthetic A(100 ng). The extent of oligomerization of the two peptides is not directly comparable, because the preparations had different histories of in vitro handling and storage. Lane 3, CHOCM precipitated by R1280; lane 4, CHOCM precipitated by R1280 preabsorbed with synthetic A. B, comparison of wild-type and mutant PPstably transfected CHO lines reveals the 6, 8, and 12 kDa bands in all lines. Cell lysates (above) and CM (below) were immunoprecipitated with anti-PP( C7) or R1280, respectively, and electrophoresed on a 10-20% Tris-tricine gel. Arrowhead indicates position of the full-length PP in the lysates. Quantitation of the A-specific bands by PhosphorImaging ( n = 4) demonstrated that differences among the cell lines in these experiments were not consistently dependent on the mutation (data not shown). wt, wild-type; E693Q, Glu to Gln at PP(PPnumbering); V717I ( V717F), Val to Ile (or Phe) at PP. C, R1280 immunoprecipitation of CM from nontransfected CHO cells reveals smaller amounts of A-related bands. Lane 1, nontransfected cells; lane 2, CHOwt-1 cells. D, longer exposures of R1280-precipitated CM from the highest expressing cell line ( V717F- 2; see B) reveal additional bands of 10, 14, and 16 kDa.



To determine whether the bands migrating between 6 and 14 kDa represented A oligomers or larger fragments of PP containing the A region, we precipitated CHO-conditioned media with antibodies directed at PP epitopes flanking the A sequence (residues 672-713 of PP). Anti-PP(5), anti-PP, anti-PP, anti-PP, and anti-PP(C7) all failed to precipitate A, p3, and the higher Mpeptides ( Fig. 2and data not shown). In contrast, six additional A antisera specific for either synthetic A, A, or Apeptides (Fig. 2) or to native A purified from AD amyloid filament fractions (36) (not shown), precipitated each of the 3-5 higher Mbands, A, and in some cases, p3. Adsorption of each of the synthetic A antisera with its respective peptide immunogen abolished the reaction (not shown).

Confirmation of the identity of the 3 most abundant bands as A oligomers was obtained by sequencing them after metabolically labeling the cells with [H]phenylalanine (5, 30) . Radiosequence analysis of the 4-kDa band demonstrated peaks of [H]Phe in cycles 4, 19, and 20, consistent with an A peptide beginning at Asp(Fig. 3 A), as has been found by radiosequencing in other cell types (5, 30) . Analysis of the 6 kDa band revealed a profile similar to that of the 4 kDa band, as well as a peak centered at cycle 15, consistent with an A peptide beginning at Arg(as described previously in other cells (30, 37) ) (Fig. 3 B). Minor [H]Phe peaks centered at cycles 10 and 24-25 consistent with A species beginning at Ileand/or Gluwere also seen (5, 30) . This result suggests that the 6 kDa band represents an A monomer or dimer with N-terminal heterogeneity that migrates anomalously in this gel system. Radiosequence analysis of the 8 kDa band revealed major peaks of [H]Phe at cycles 4, 19, and 20, consistent with A beginning at Asp, and at cycles 15 and 16, consistent with an A peptide beginning at Arg. Minor peaks at cycles 9 and 10 are consistent with an Ileand/or a Gluspecies (Fig. 3 C). This result suggests that the 8 kDa band represents a dimeric species composed in large part of the A Asp 1 peptide but including dimers that are N-terminally heterogeneous. In this regard, the 8 kDa gel band can sometimes be seen to comprise a tightly spaced doublet ( e.g. Fig. 2 ), presumably representing structurally different A dimers. Radiosequencing of the 12 kDa band revealed [H]Phe at cycles 4, 19, and 20, again consistent with A beginning at Asp, as well as at cycles 15 and 16, consistent with A beginning at Arg. In addition, a major peak at cycle 3 is consistent with an A species starting at Ala(Fig. 3 D). These results suggest that the 12 kDa band comprises A trimers having N-terminal heterogeneity.


Figure 3: Radiosequencing of 6-, 8-, and 12-kDa A-reactive proteins confirms their identity as oligomers. [H]Phenylalanine radioactivity obtained at each cycle of Edman degradation is graphed for the 4 (A), 6 ( B), 8 ( C), and 12 kDa ( D) bands precipitated by R1280 from CHO-conditioned medium.



Because aggregation of synthetic A peptides may be modified by several serum proteins, including apolipoprotein E, apolipoprotein J, and transthyretin (38, 39, 40, 41) , we investigated the effect of serum on A oligomer formation in our system. CHO cells labeled overnight in the absence of fetal bovine serum showed markedly reduced levels of the 4 and 6 kDa A bands and an increase in the 8-12 kDa bands when compared with cells labeled conventionally in 10% serum (Fig. 4 A). Importantly, the addition of serum to labeled cell-free conditioned medium just prior to the immunoprecipitation reaction resulted in no change in the monomer/oligomer pattern (Fig. 4 B), indicating that serum does not alter the immunoprecipitation. These results suggest that proteins in serum can stabilize the amount of A monomer and decrease the formation of A oligomers in our cultures.


Figure 4: Conditions affecting the relative amounts of A monomer and oligomers. A, the omission of FBS during overnight labeling resulted in marked decreases in the A and 6 kDa bands, and a concurrent increase in the amounts of the 8 and 12 kDa oligomeric bands. Preabsorption of R1280 with synthetic A( lanes marked -) shows the specificity of all bands. B, the addition of FBS to conditioned medium just prior to the immunoprecipitation reaction had no effect. Lanes 1 and 2, overnight labeling in the presence ( lane 1) or absence ( lane 2) of 10% FBS. Lane 3, 10% FBS added to serum-free conditioned medium prior to immunoprecipitation. C, the addition of 10 µ M Congo red ( lane 2) during overnight labeling in serum-free medium resulted in substantial increases in the A and 6 kDa bands, with a slight decrease in the amount of the 8 and 12 kDa bands when compared with the addition of the MeSO vehicle alone ( lane 1). Lane 3, overnight labeling in serum-free medium alone; lane 4, overnight labeling in serum-free medium plus MeSO at 1:1000 caused no change in A and the oligomers; lane 5, overnight labeling in 10 µ M Congo red; lane 6, conditioned serum-free medium plus 10 µ M Congo red added just prior to immunoprecipitation also caused no change in the A/oligomer pattern. D, elution of R1280 immunoprecipitates from protein A-Sepharose with 0.2 M glycine, pH 2.5 ( lane 2) gave the same A monomer and oligomer pattern as conventional elution by boiling in SDS/ME sample buffer ( lane 1). V717F-2 cells used in A-D.



To assess whether the extent of A oligomerization in culture can be altered pharmacologically, we chose to investigate the effect of the dye Congo red. Congo red has been shown to decrease the accumulation of the protease-resistant form of the prion protein and scrapie infectivity in vitro, resulting in the inhibition of amyloid fibril formation in this model (42) . Addition of 10 µ M Congo red during serum-free metabolic labeling of our cells overnight produced a dramatic increase of the 4 and 6 kDa A bands and a concurrent slight decrease of the 8 and 12 kDa oligomeric bands (Fig. 4 C, lanes 1 and 2). At lower doses (2.5 and 5 µ M), a dose-dependent increase of the 4 and 6 kDa bands occurred (data not shown). Addition of the Congo red vehicle, MeSO (1:1000), to the labeling medium had no effect on the A pattern (Fig. 4 C, lanes 3 and 4). Addition of 10 µ M Congo red to the labeled conditioned medium just prior to immunoprecipitation also had no effect on the A pattern (Fig. 4 C, lane 6). Experiments are underway to determine whether the consistent A-raising/oligomer-lowering effect of Congo red is due to inhibition of the conversion of monomeric A to oligomers, stabilization of the A monomer from degradation, altered A production, or some combination of these.

The data presented so far indicate that SDS-stable low oligomers of secreted A can be detected in CHO-conditioned medium by immunoprecipitation. To exclude the possibility that these oligomers formed only during the immunoprecipitation reaction, several control experiments were performed. Synthetic Awas added to either fresh Dulbecco's modified Eagle's medium with 10% FBS or to this medium conditioned overnight by CHOcells at concentrations of the peptide that matched or exceeded those of secreted A (5 n M). Incubation at 37 °C for 4 h followed by R1280 precipitation under conditions identical to those that demonstrated A oligomers in cell culture revealed only the A monomer and no oligomeric bands on immunoblots (data not shown), indicating that our immunoprecipitation procedure does not induce oligomerization of synthetic A. To exclude the possibility that boiling the samples in SDS sample buffer prior to electrophoresis induced the aggregation, immunoprecipitated samples were eluted from protein A-Sepharose with glycine, pH 2.5, neutralized and loaded without heating in a sample buffer devoid of ME; gel fluorography revealed the usual pattern of monomeric and oligomeric A bands (Fig. 4 D). Furthermore, addition of either FBS or Congo red to serum-free labeled conditioned medium just prior to the immunoprecipitation reaction had no effect, whereas their presence during cell labeling consistently resulted in dramatically altered patterns of A monomer and oligomers (Fig. 4, B and C).


DISCUSSION

Experiments conducted in numerous laboratories on the neurotoxic effects of synthetic A peptides in hippocampal cortical cultures ( e.g. see Refs. 43-45) and in brain-injected animals ( e.g. see Refs. 46-48) have led to widely divergent results. In some studies, neuronal toxicity and loss were reproducibly seen when synthetic Awas compared with control peptides administered in the same vehicle. Other studies using putatively highly similar or identical peptides showed little or no induction of specific toxicity (for review, see Ref. 49). Subsequent analyses of different lots of peptides synthesized by the same or different facilities have revealed that A peptides of identical sequence can adopt varying degrees of random coil, -helical, and -pleated sheet secondary structure as well as varying states of aggregation ( e.g. see Refs. 10-12). Freshly prepared A peptides can exist in a largely random coil monomeric state that is unassociated with significant cellular effects, but they can be converted into a principally -pleated sheet conformation and a partially aggregated state that induces neurotoxicity by incubating (``aging'') the peptide in buffer for several days (10, 11, 12) . There is evidence that a conformational transition to increasing -sheet structure and the subsequent polymerization of the peptide to fibrils underlies its conversion to a toxic moiety (11, 12, 32, 50) . It is possible that an analogous slow aggregation/polymerization process may explain in part the differences between the apparently pathologically inert, largely nonfibrillar A found in diffuse plaques (51) , and the fibrillar deposits found in plaques that are intimately associated with neuritic and glial dystrophy (52) .

It is important to characterize and model accurately the process of aggregation of A peptides that occurs under physiological conditions in cerebral tissue. To this end, we have searched for evidence of spontaneous aggregation of the native A peptides that are produced by cells. Several lines of evidence support the conclusion that the 8-16-kDa R1280-reactive proteins we have detected in conditioned medium are oligomers of A: ( a) their specific, adsorbable immunochemical reaction with all 6 A antibodies examined but not with various antibodies to flanking PP regions; ( b) their electrophoretic co-migration with oligomers of synthetic A peptide; ( c) the substantially higher amounts of these proteins in PP-transfected than untransfected cells; and ( d) the direct confirmation of the A composition of the 6-, 8-, and 12-kDa proteins by radiosequencing.

We have also conducted several control experiments to exclude the possibility that A aggregation arises only during the immunoprecipitation reaction. First, synthetic A, which is the same length as the principal A peptide secreted by cultured cells (15) , does not undergo oligomer formation during immunoprecipitation when added to CHO-conditioned medium at endogenous or higher concentrations. Second, elimination of boiling and ME in the immunoprecipitation protocol does not change the appearance of the oligomers. Third, and most importantly, we describe two manipulations (omission of serum and addition of Congo red) that reproducibly alter the relative amounts of A oligomers versus monomers in the CHO medium when present during metabolic labeling ( i.e. during putative formation of the oligomers) but have no effects when performed just prior to the immunoprecipitation ( i.e. after the oligomers have formed). This finding clearly indicates that the oligomers exist in the medium prior to the immunoprecipitation reaction rather than only after it. However, because there is no method to assay unconcentrated conditioned medium directly for these very low abundance (picomolar level) oligomers, we cannot exclude the possibility that some additional A oligomerization could occur during concentration. We are currently examining the effects of Congo red and serum proteins on the A oligomer to monomer ratio; these mechanisms are likely to be heterogeneous and complex.

The extent to which secreted A peptides of various lengths participate in A oligomerization in vivo and in cell culture remains to be determined. It has been proposed that A peptides longer than 40 residues, particularly the Apeptide believed to be a major constituent of the compacted plaque cores (19, 20, 53, 54) , could serve as a nidus for the aggregation of shorter species, including the much more abundantly secreted Apeptide (34, 55) . Small amounts of A peptides ending at residue 42 have been detected in the media of PP-transfected cells (13, 15) and in human cerebrospinal fluid (17) . Moreover, FAD-linked PP missense mutations located near the carboxyl end of the A region have been shown to cause enhanced secretion of the longer (42 residue) A species (13) . Synthetic Ahas been shown to aggregate preferentially into fibrillar amyloid (34) . The role of Ain the formation of the 6-16-kDa A oligomers in cell culture needs to be elucidated using carboxyl-terminal specific antibodies for immunoprecipitation and enzyme-linked immunosorbent analysis. We have not yet observed a consistant effect of these mutations on oligomer levels in our transfected cells. This could be due to the use of antibodies that do not preferentially detect longer A peptides or to the short period of time over which we assayed in vitro A aggregation. In vivo, although A1-42 is made throughout life, it takes at least several decades before one can detect any aggregated A deposits in human brain.

Relatively more hydrophobic A peptides, such as species beginning at Argor Glu, have also been detected in the medium of PP-expressing cells and hypothesized to be important in enhanced A aggregation (30, 56) . In this regard, we could detect virtually no ArgA in the 4 kDa monomeric band from our cultures, and yet we found small amounts in the SDS-stable multimers we obtained from the same cells (Fig. 3), supporting the hypothesis of enhanced participation of the heterogeneous A monomeric species in oligomerization. That A N-terminal heterogeneity influences the aggregation of A into amyloid in vivo has recently been shown in AD brain tissue by the use of N-terminal specific A antibodies (57) .

In addition to peptide length, a large number of variables that may influence A oligomerization could be assessed under physiological conditions and concentrations in the cell culture system we describe. These variables include the effects of amyloid-associated proteins (``pathological chaperones'') ( e.g. heparan sulfate proteoglycan (58) and apolipoproteins E and J (38, 39) ), metal ions (35, 59) , and oxidizing and anti-oxidizing agents (60, 61, 62) . The addition of these molecules to the cultures, the use of doubly transfected CHO cell lines expressing PP and a chaperone protein of interest, and the study of cell lines genetically deficient in a particular chaperone could lead to new insights about how such molecules influence the earliest stages of A aggregation. It is also likely that certain cell types and certain culture conditions may be found that allow much more extensive aggregation of endogenous A than we have observed in this initial study. We have recently detected small amounts of A oligomers after long autofluorographic exposures of the R1280-precipitated media of PP-transfected human kidney 293 cells.() The levels of the putative oligomers were far lower than those in CHO transfectants secreting similar amounts of A monomer. This result suggests that in vitro oligomerization of A can occur in different cell types and that cell-specific factors are likely to promote or retard the extent of A aggregation.

Finally, the ability to detect and quantitate dimers, trimers, and tetramers of native A at submicromolar concentrations similar to those likely to exist within early A deposits could allow such cultures to be used to screen and characterize small molecules that inhibit the formation, or proteases that enhance the clearance, of neurotoxic A aggregates. We have observed a consistent change in A monomer to oligomer ratios after treatment with Congo red, providing an example of such an amyloid binding compound. Further characterization of the cellular and extracellular effects of this and similar compounds on the production, initial aggregation, and clearance of A can now be conducted under physiological conditions.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant AG07911 (LEAD Award) and by Bristol-Myers Squibb unrestricted neuroscience grant (to D. J. S.). 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.

The abbreviations used are: A, amyloid -protein; PP, -protein precursor; AD, Alzheimer's disease; FAD, familial (autosomal dominant) Alzheimer's disease; CHO, Chinese hamster ovary; FBS, fetal bovine serum; CM, conditioned medium; ME, -mercaptoethanol.

D. J. Watson, M. B. Podlisny, and D. J. Selkoe, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Christian Haass and Wei Qiao Qiu for helpful discussions.


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