©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracellular A142 Aggregates Stimulate the Accumulation of Stable, Insoluble Amyloidogenic Fragments of the Amyloid Precursor Protein in Transfected Cells (*)

Austin J. Yang , Mary Knauer (§) , Debra A. Burdick (1), Charles Glabe (¶)

From the (1)Department of Molecular Biology and Biochemistry and Department of Developmental and Cell Biology, University of California, Irvine, California 92717

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have analyzed the effect of internalized amyloid -protein (A) 1-42 aggregates on the metabolism of the amyloid precursor protein (APP) in stably transfected 293 cells. The amount of potentially amyloidogenic fragments of APP immunoprecipitated by anti-carboxyl-terminal APP and anti-A antibodies is dramatically enhanced by the treatment of the cells with A1-42, which is resistant to degradation, but not A1-28, which does not accumulate in cells. This accumulation of amyloidogenic carboxyl-terminal fragments is specific, since there is relatively little effect of A1-42 on the amount of the nonamyloidogenic -secretase carboxyl-terminal fragment. The amyloidogenic fragments accumulate in the same nonionic detergent-insoluble fraction of the cell that contains the internalized A1-42. Western analysis indicates that a subset of the amyloidogenic fragments react with antibodies that recognize a conformation of A that is specifically associated with aggregated forms of A, suggesting that the adoption of this aggregation-related conformation may be an early event which precedes the final processing that produces A. Pulse-chase analysis of the [S]Met-labeled 16-kDa amyloidogenic fragment indicates that it is relatively stable in A1-42-treated cells, with a half-life of approximately 50 h. This fragment is degraded with a half-life of 30 min in control cells treated with A1-28. In contrast, the turnover of the nonamyloidogenic -secretase product is not significantly altered by the presence of A1-42. The continuous uptake of A1-42 from the medium is not required for the stimulation of amyloidogenic fragment accumulation, suggesting that the presence of intracellular A1-42 aggregates establishes a new pathway for APP catabolism in cells which leads to the long term stability of the fragments. If these amyloidogenic fragments of APP ultimately give rise to A, then the production of A may be an autocatalytic, ``runaway'' process in cells containing A1-42 nuclei. It is conceivable that the accumulation of insoluble APP and amyloidogenic fragments of APP in response to A1-42 aggregates may mimic the pathophysiology of dystrophic neurites, where the accumulation of intracellular APP and APP fragments has been documented by immunohistochemistry.


INTRODUCTION

The major protein component of amyloid deposits associated with Alzheimer's disease (AD)()is a 39-42-amino acid, self-assembling peptide, known as the amyloid A peptide. Although remarkable progress has been made in our understanding of the proteolytic processing of APP and the secretion of soluble amyloid A peptide(1) , the mechanisms for the accumulation of insoluble amyloid deposits and their role in Alzheimer's disease pathogenesis remains a matter of speculation. It is clear that at least two pathways exist for APP processing which give rise to fragments bearing A sequences at their amino termini: processing by -secretase, which cleaves within the A sequence thereby precluding amyloid accumulation(2, 3, 4) , and -secretase processing, which generates carboxyl-terminal APP fragments containing the entire A sequence(2, 5, 6, 7) . Amyloidogenic, -secretase-processing events may occur within several intracellular organelles, including the rough endoplasmic reticulum(8) , trans-Golgi network(9) , and lysosomes (10-12). Further processing of APP within the transmembrane domain by -secretase releases soluble 3- and 4-kDa fragments containing all or part of the A sequence(13, 14, 15) . Recent evidence indicates that the familial AD amino acid substitutions within the transmembrane domain favor the production of the longer A1-42 form of A (16) which is preferentially localized with diffuse and senile plaque amyloid deposits in AD brain(17) . This suggests that A1-42 is more closely associated with AD pathogenesis than shorter A isoforms.

Our previous studies have demonstrated that internalized A1-42 is largely resistant to degradation and accumulates as insoluble aggregates in lysosomes. In contrast, A1-39 and shorter peptides fail to accumulate, although they are also internalized by endocytosis(18) . Since lysosomes are a site of APP processing and catabolism, we examined the effect of internalized A1-42 peptide on the proteolytic process of the APP in APP-overexpressing cells. We found that the intracellular A1-42 causes a dramatic increase in the amounts of amyloidogenic carboxyl-terminal fragments of APP. The accumulation of these fragments is due to an increase in their stability. These results suggest that the intracellular A aggregates may stimulate the accumulation of more A by providing a stable nucleus on which newly synthesized amyloidogenic fragments and A can accrete, thereby acquiring resistance to degradation.


EXPERIMENTAL PROCEDURES

Tissue Culture

Two cDNA clones, which transcribe the full-length human APP751 and APP695 cDNAs under the direction of the strong cytomegalovirus promoter, were kindly provided by Dr. T. Oltersdorf of Athena Neurosciences. The constructs were used to co-transfect the human kidney 293 cell line, along with the selection marker pSVneo, by CaPO co-precipitation. Cells were then maintained in DMEM, 10% fetal bovine serum with G418. The stably transfected cells were then screened for APP expression by Western blot analysis.

Antibody Production and Purification

Rabbits were immunized with high performance liquid chromatography-purified synthetic peptides A1-28 or A1-42 containing an extra cysteine residue at the carboxyl terminus conjugated to ovalbumin lysine residues using the cross-linker, N-succinimidyl 3-(2-pyridyldithio)propionate (Pierce) and emulsified in Freud's complete adjuvant. Subsequent injections utilized unconjugated synthetic A1-42 emulsified in Freud's incomplete adjuvant. After at least three immunizations, serum samples were collected and affinity-purified. To immobilize the immunizing peptide, 10 mg of the cysteine-containing A1-28 or A1-42 were dissolved in 5 ml of 20 mM TES buffer (Sigma) and allowed to react with 8 ml of Bio-Rad Affi-Gel 401 for 16 h at room temperature. Preparation and washing of the gel was done according to the manufacturer's recommendations. Serum samples (20-30 ml) were diluted 1:1 in PBS, and the IgG fraction was eluted from a protein G-Sepharose column using 0.2 M glycine, pH 2.7. The IgG fraction was neutralized, dialyzed in PBS, recirculated over the A1-42 affinity resin, and eluted with 0.2 M glycine, pH 2.7. The purified antibody was neutralized and dialyzed against PBS, and aliquots were stored at -80 °C. Once thawed, antibody samples were kept at 4 °C for use. Typically, 30 ml of serum yielded 2 mg of affinity-purified antibody. Monoclonal antibody 13G8, which recognizes the carboxyl terminus of APP, and anti-BX5 polyclonal antibody, which recognizes an epitope in the extracellular domain of APP, were generous gifts of Athena Neurosciences.

Metabolic Labeling and Immunoprecipitation

Transfected cell cultures (1 10 cells in a 10-cm plate) were preincubated with methionine-deficient DMEM for 2 h prior to labeling. The cells were then incubated in 2 ml of methionine-deficient DMEM, containing 25 µM amyloid peptide and 1% bovine serum albumin, and labeled with 100 µCi/ml of [S]methionine/cysteine (1000 Ci/mmol; TranS-label, ICN) for 4-16 h. At the end of the labeling period, the conditioned medium was collected, and cells were washed twice with cold PBS and lysed either in RIPA (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 2 µg/ml leupeptin, 0.2 unit/ml soybean trypsin inhibitor, 1 µg/ml aprotinin) or Nonidet P-40 lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 2 µg/ml leupeptin, 0.2 unit/ml soybean trypsin inhibitor, 1 µg/ml aprotinin). The cell lysate was then clarified at 10,000 g for 10 min, and the supernatant was transferred to a fresh tube. The pellet fraction was then resuspended in 88% formic acid, centrifuged at 10,000 g for 10 min, and lyophilized. After lyophilization, the dry sample was resolublized with 2 RIPA, sonicated until clarified, diluted to 1 RIPA, and centrifuged at 10,000 g for 10 min. The supernatant of these samples was then subjected to immunoprecipitation analysis. The immunoprecipitation of soluble APP with anti-BX5 antibody from conditioned medium was carried out by the method described by Oltersdorf et al. (3). The immunoprecipitated products were then subjected to either Tris-Tricine SDS (45) or Tris-glycine (46) PAGE analysis, the gel was treated with ENHANCE (DuPont NEN), and the dry gel was exposed to x-ray film.

In the pretreatment experiments, some of the cell cultures exposed to 25 µM A1-42 for 6 h were treated with trypsin and resuspended in fresh medium lacking A1-42 prior to metabolic labeling. Cells treated with A peptide for 6-12 h were incubated in 2 ml of methionine-free DMEM containing 1% bovine serum albumin for 2 h and then pulse-labeled with 250 mCi/ml [S]Met for 30 min. The [S]Met-labeled cells were then washed twice with PBS and chased in the methionine-deficient DMEM supplemented with 10 µg/ml methionine for 2-12 h. Immunoprecipitation of APP and its products was then performed as described above.

Immunoblotting

Cells treated with A peptide were lysed in 1 ml of RIPA as described above and centrifuged at 10,000 g for 15 min. The RIPA-insoluble pellet was then solubilized in 88% formic acid, and the supernatant was then collected by centrifugation at 10,000 g for 10 min. The formic acid-soluble cell extract was then lyophilized and redissolved in 4 SDS sample buffer with sonication. About 30 µg of protein were subjected to SDS-PAGE and blotted onto nitrocellulose. Nitrocellulose filters were blocked with 5% dried milk and incubated with primary antibody that was specific to either A peptide or APP COOH-terminal fragments. The bound primary antibody was then detected by a horseradish peroxide-coupled secondary antibody and an ECL detection system (Amersham Corp.).


RESULTS

We examined whether the presence of A1-42 affects the catabolism of APP and APP fragments in stably transfected 293 cell lines. We chose this cell culture model because the nonamyloidogenic -secretase and amyloidogenic - and -secretase pathways for APP processing have been demonstrated in this system(2, 3, 11, 13, 19) . The ability to compare the products of nontransfected cells is an important control for immunoprecipitation and Western blot identification of fragments, since the endogenous levels of APP expression in nontransfected cells is relatively low(3) . We first confirmed that A1-42 is internalized by APP751-transfected 293 cells and accumulates as nonionic detergent-insoluble aggregates that are resistant to degradation as described previously in human fibroblasts(18) . Five percent of the total I-labeled A1-42 in the medium accumulates intracellularly over a 6-h incubation period in comparison to 0.02% of the total A1-28 as determined by the amount of cell-associated A that is resistant to removal by trypsin treatment. After lysis of the cells in the nonionic detergent-containing RIPA, two-thirds of this internalized, I-labeled A1-42 is sedimentable at 10,000 g. These results closely parallel those previously described for human fibroblasts(18) .

We metabolically labeled APP with [S]Met in cell cultures treated with A1-42 or nonaccumulating A1-28 and compared the amount of S-labeled APP or amyloidogenic fragments of APP associated with the cell by immunoprecipitation with 13G8 monoclonal antibody directed against the carboxyl terminus of APP. Since most of the intracellular A1-42 is contained in the low speed sedimentable fraction after nonionic detergent lysis, we examined both the insoluble and soluble fractions for APP fragments. The amounts of APP and amyloidogenic APP fragments are greatly increased in the insoluble fraction of A1-42-treated cells (Fig. 1A). In particular, the accumulation of a band migrating at an apparent mass of 16 kDa is dramatically stimulated by A1-42. Although the amount of this band is much lower in control cells, it can be visualized at longer exposures (see below). The stimulatory effect is specific for A1-42, since the amounts of APP and APP fragments in cells treated with nonaccumulating A1-28 are indistinguishable from that in cells incubated in the absence of peptide. The response to A1-42 treatment is also specific for potentially amyloidogenic fragments of APP, since the amounts of the nonamyloidogenic 12-kDa -secretase product are not significantly affected by the presence of A1-42. Similar increases in the amounts of APP and amyloidogenic fragments of APP were observed in cells transfected with APP695 (data not shown). Control experiments with nontransfected cells indicate that the immunoprecipitated bands arise from the transfected APP751 gene and are not an artifact of immunoprecipitation. In contrast to the results observed in the insoluble fraction of the cells, no significant effect of A1-42 on the amounts of APP and APP fragments was observed in the RIPA-soluble fraction of cells (Fig. 1B). The stimulation of the accumulation of fragments of APP does not appear to be due to an increase in the rate of APP synthesis or a decrease in the -secretase processing of APP, since the amounts of soluble APP immunoprecipitated from the culture medium are the same in A1-42-treated and control cells (Fig. 1C).


Figure 1: A1-42 stimulates the accumulation of amyloidogenic fragments of APP. A, [S]Methionine-labeled APP and carboxyl-terminal fragments from APP were immunoprecipitated with the monoclonal antibody, 13G8, from the insoluble fraction of A1-42-treated and control cell cultures. The amounts of APP and amyloidogenic fragments of APP are greatly increased in cells treated with A1-42 (lane 6) in comparison to controls treated with A1-28 (lane 5) or cells labeled in the absence of A peptide (lane 4). In particular, the amount of a 16-kDa amyloidogenic fragment (indicated by the arrowhead) is dramatically increased. In contrast, the amount of the nonamyloidogenic 12-kDa -secretase band is not significantly altered by A1-42. The amount of immunoprecipitable fragments is greatly reduced in nontransfected cells which have low levels of endogenous APP expression, indicating that the labeled bands are derived from the transfected APP gene and are not an artifact of immunoprecipitation. B, A1-42 has no significant effect on the amounts of APP or amyloidogenic APP carboxyl-terminal fragments in the soluble fraction of the cells. APP and its carboxyl-terminal fragments were immunoprecipitated from the RIPA-soluble cell extracts of cells incubated with A1-42 for 6 h (695 cells-A1-42; 751 cells-A1-42) or control cells (695 cells only; 751 cells only) with the monoclonal antibody 13G8. No significant and reproducible changes in the amounts of APP or amyloidogenic fragments were observed (compare lanes 1 and 2; 3 and 4). A small (2-fold) increase in the amount of the -secretase product (indicated by the arrowhead) is observed in A1-42-treated APP751-transfected cells, but this small increase has not proven to be reproducible in subsequent experiments. C, the secretion of soluble APP is not affected by the treatment of cells with A1-42. Conditioned medium from cells treated with A1-42 for 6 h (751 cells, A1-42) or untreated control cells (751 cells, no peptide and 293 cells) were immunoprecipitated with antisera directed against APP residues 444-592 of APP695 (polyclonal anti-Bx5). No difference in the amount of immunoprecipitated APP is observed in the medium from A1-42-treated and control APP751 cells. D, immunoprecipitation of APP COOH-terminal fragments with antisera directed against residues 1-28 of A. APP751-transfected 293 cells were metabolically labeled with [S]Met in the presence (+) or absence (-) of 25 µM A1-42 for 6 h. The APP COOH-terminal fragments were then immunoprecipitated by anti-A1-28 antibody and resolved on a 15% Tris Tricine gel. The levels of a 14.4-kDa APP COOH-terminal fragment (arrowhead) are greatly increased in A1-42-treated cells (lane 2) in comparison to control cultures (lane 1). Bands with a higher electrophoretic mobility also accumulate in response to A treatment. The A1-42-induced APP COOH-terminal fragments can be eliminated by preadsorbing the anti-A1-28 antibody with 10 µg/ml of excess A peptide before immunoprecipitation (lanes 3 and 4).



The accumulation of amyloidogenic fragments of APP in the insoluble fraction of cells treated with A1-42 was also observed by immunoprecipitation with antibodies directedagainst residues 1-28 of A, providing further evidence that these fragments contain A epitopes (Fig. 1D). The amount of APP and amyloidogenic APP fragments immunoprecipitated by affinity-purified anti-A1-28 antisera is observed to increase dramatically in the cells incubated with A1-42. The amount of amyloidogenic fragments is greatly reduced in control, nontransfected cells, indicating that the labeled bands are products of the transfected APP gene (data not shown). The immunoprecipitation of the amyloidogenic fragments is blocked by preadsorption of the antibody with excess A1-28 peptide. On Tris-Tricine gels (Fig. 1D), the apparent molecular weight of the APP fragments is slightly lower than that observed on Tris-glycine gels (Fig. 1A) and the carboxyl-terminal fragments are further resolved into a series of closely spaced bands as has been previously described(6) .

The molecular weight of the amyloidogenic fragments was further characterized by Tris-Tricine SDS-gel electrophoresis in comparison to a series of carboxyl-terminal fragments of known structure (Fig. 2). The major amyloidogenic fragment migrates at a position that is significantly larger than the authentic -secretase products from transfected CHO cells (lane 1),()or the carboxyl-terminal 100 residues of APP (20) expressed in a reticulocyte lysate (lane 4), which have an apparent molecular mass of approximately 12 kDa. These results indicate that the fragments which accumulate in response to A1-42 are sufficiently large to contain the entire A sequence and suggest that the amino terminus of the major fragment may extend approximately 20 amino acids amino-terminal to the beginning of the A domain.


Figure 2: Characterization of the sizes of the amyloidogenic fragments by Tris-Tricine gel electrophoresis. The electrophoretic mobility of the amyloidogenic carboxyl-terminal fragments of APP were compared to APP fragments of known structure. The amyloidogenic fragments were immunoprecipitated with 13G8 monoclonal antibody from APP-751-transfected cells and resolved on 15% Tris-Tricine gels as described previously. The major carboxyl-terminal fragment of APP from A1-42-treated cells migrates with an apparent molecular mass of 14 kDa on Tris-Tricine gels (lane 2) which is substantially larger than the smallest amyloidogenic fragment from transfected CHO cells (lane 1) that was verified by protein sequencing. It also migrates more slowly than an in vitro translation product of the carboxyl-terminal 100 residues of APP expressed in a rabbit reticulocyte lysate (lane 4).



The accumulation of the amyloidogenic fragments of APP in A1-42-treated cells is also observed by Western analysis (Fig. 3A). The 16-kDa band and several additional higher molecular mass bands are stained by affinity-purified anti-A1-42 antibodies in A1-42-treated transfected cells (lanes 2 and 3), but not in transfected cells incubated in the absence of A (lane 1) nor in nontransfected control cells treated with A1-42 (lane 5). These results indicate that the immunoreactive bands arise from the transfected APP751 gene and not the added A1-42 and confirm that the accumulation of these bands in the preceding immunoprecipitation experiments is not an artifact. The reactivity of these bands with this antibody also reveals that some of the amyloidogenic fragments display a conformation that is resistant to denaturation in SDS and found only in A aggregates. The specificity of this antibody for an epitope found in SDS-resistant A aggregates is demonstrated in Fig. 3B, which compares the staining of synthetic A1-42 and A1-40 standards. Previously published work has demonstrated that A1-42 forms aggregates that are not disrupted by heating at 100 °C in SDS sample buffer, while aggregates formed by A1-40 are disrupted by this treatment(21, 22) . Although bands corresponding to 4-kDa A monomer are prominently revealed by Amido Black staining of the nitrocellulose membrane for both A1-40 and A1-42 (lanes 4-6), only the bands corresponding to aggregated forms of A1-42 (at approximately 18 kDa and at the top of the gel) strongly react with the anti-A1-42 antibodies (lane 3). The 4-kDa band corresponding to the A1-40 monomer is not stained by this antibody, and the 4-kDa band corresponding to A1-42 reacts only weakly (lanes 1-3). The fact that the amyloidogenic fragments are recognized by the same antibody suggests that they display the same SDS-resistant epitope found in A aggregates.


Figure 3: The amyloidogenic fragments of APP that accumulate in response to A1-42 treatment display a conformation-dependent epitope associated with aggregated forms of A. A, Western analysis of A1-42-treated and control cells with an affinity-purified antibody which reacts with a conformation of A found specifically in A aggregates. This panel shows the fluorogram of a blot transferred from a 15% Tris-glycine gel. The prominent 16-kDa amyloidogenic and minor bands at molecular masses of 30 and 28 kDa are labeled by the antibody (lane 2). No bands are detected in control 293 cells (lane 4) or APP751-transfected cells incubated in the absence of A1-42 (lane 1) nor in nontransfected 293 cells incubated with A1-42 (lane 5). The steady-state amounts of these fragments does not decrease rapidly after the A1-42-containing medium is removed and replaced with fresh medium lacking A and incubated for an additional 6 h (lane 3), suggesting that the fragments are relatively stable. B, the specificity of the antibody for a conformation of A associated with A aggregates is demonstrated by Western analysis of synthetic A peptides. At concentrations above 25 µM, A1-42 forms higher molecular weight aggregates that are not disrupted by heating at 100 °C in SDS sample buffer (21, 22), while the aggregates formed by A1-40 and shorter A peptides are disrupted by this treatment. Lanes 1-3 are anti-A1-42-stained, and lanes 4-6 are Amido Black-stained nitrocellulose strips after the electrophoretic transfer of peptide from A1-40 (lanes 1 and 4), A1-41 (lanes 2 and 5) or A1-42 (lanes 3 and 6). The aggregated forms of A1-42 migrating with an apparent molecular mass of 18 kDa and material that remains at the top of the gel are intensely stained by the antibody, even though there is little Amido Black-staining peptide at these positions. The 4-kDa band corresponding to the monomeric A reacts weakly or not detectably with the antibody, even though this band is prominently stained by Amido Black.



Since the amount of soluble APP secretion is not significantly altered by the presence of A1-42 peptide, the accumulation of the insoluble amyloidogenic APP fragments may be due to a decrease in their rate of turnover. Western analysis suggests that the 16-kDa amyloidogenic fragment is relatively stable after the removal of A1-42 from the medium (Fig. 3A, lane 3). The intensity of the immunoreactivity of the 16-kDa band is only slightly reduced when the cells are removed from the A1-42-containing medium and incubated in peptide-free medium for an additional 6 h (lane 3) as compared to cells incubated continuously with A1-42 for 12 h (lane 2). This suggests that the steady-state amount of the fragment does not change rapidly when the cells are no longer exposed to A1-42. To quantify the stability of the 16-kDa fragment, we determined its turnover rate by pulse-chase analysis. As shown in Fig. 4A, the 16-kDa fragment turns over at a much slower rate in cells treated with A1-42 as compared to the control cells exposed to A1-28. Although the amount of this 16-kDa fragment is greatly reduced in control cells, the turnover of this fragment can be quantified by longer exposures of the PhosphorImager plate. In the presence of A1-42, the half-life is approximately 50 h, whereas in the presence of A1-28 the half-life is approximately 30 min. The increased stability of the amyloidogenic APP fragments does not appear to be due to a generalized inhibition of degradative enzymes. The half-life of the nonamyloidogenic -secretase carboxyl-terminal fragment of APP is not detectably stabilized by A1-42 treatment, suggesting that the stability is specific for amyloidogenic fragments (Fig. 4B). This is consistent with previous results that demonstrate that intracellular A1-42 aggregates do not interfere with the degradation of control peptides and proteins internalized from the culture medium(18) .


Figure 4: The turnover of the 16-kDa amyloidogenic fragment is specifically stabilized by A1-42. A, pulse-chase analysis of the turnover of the 16-kDa carboxyl-terminal fragment. The 16-kDa fragment is much more stable in A1-42-treated cells (--) in comparison to the lifetime of the fragment in control cells treated with A1-28 (--). The half-life of the 16-kDa fragment is approximately 50 h in A1-42-treated cells and approximately 30 min in control cells. Although the amount of the 16-kDa fragment in control cells is much lower than that in A1-42-treated cells, this fragment can be detected and quantified accurately using a PhosphorImager. B, pulse-chase analysis of the turnover of the 12-kDa nonamyloidogenic -secretase product. The lifetime of the nonamyloidogenic fragment is that same in A1-42-treated cells (⊡--⊡) and cells treated with A1-28 (--). This indicates that A1-42 treatment does not result in a generalized inhibition in the degradation of membrane proteins, but rather suggests that the effect is specific for amyloidogenic fragments of APP.



To probe the mechanism for the accumulation of amyloidogenic fragments of APP in response to A1-42 treatment, we asked whether the continued internalization of A1-42 from the medium is required for the stimulation of amyloidogenic fragment accumulation. APP751-transfected cells were incubated in the presence of 25 µM A1-42 for 6 h. One set of cultures was immediately labeled with [S]Met in the presence of A and prepared for immunoprecipitation, whereas another set of cultures was trypsin-treated to remove surface-adsorbed A(18) , cultured for an additional 6 h in the absence of A, and then labeled and prepared for immunoprecipitation. Both cultures labeled in the continuous presence of A and cultures pretreated with A before labeling show an increase in amyloidogenic carboxyl-terminal fragments of APP ranging from 8 to 14 kDa (Fig. 5) on a Tris Tricine gel. These results demonstrate that the continued uptake of A from the culture medium is not required to stimulate the accumulation of amyloidogenic fragments and suggest that, once stable A1-42 aggregates have nucleated within the cell, the pathways for APP catabolism are altered to favor the accumulation of amyloidogenic fragments and potentially more A.


Figure 5: The accumulation of amyloidogenic APP fragments does not require the continued uptake of A1-42 from the medium. APP751-transfected 293 cells were incubated with A1-42 for 6 h, after which half of the cultures were treated with trypsin to remove any surface-adsorbed A and resuspended in fresh medium lacking A. The A-treated cultures and control cells not exposed to peptide were then labeled for 6 h with [S]Met, and samples were immunoprecipitated with monoclonal antibody 13G8. The amounts of amyloidogenic fragments immunoprecipitated is not significantly different in the cell cultures labeled in the continuous presence of A1-42 (lane 2) and the cultures pretreated with A1-42 and labeled in the absence of A in the medium (lane 3).




DISCUSSION

In this report, we have extended our previous studies of A internalization and turnover to examine the effects of internalized A1-42 aggregates on the catabolism of APP and its proteolytic processing products. Our results suggest that intracellular A1-42 aggregates alter the pathways which normally degrade APP and its amyloidogenic fragments to stabilize them and favor their accumulation. The accumulation of amyloidogenic fragments is not due to an increase in the rate of APP synthesis or an inhibition of the -secretase processing pathway, since the amount of soluble APP secreted into the medium is not altered by A1-42 treatment. Instead, the mechanism for the accumulation of the amyloidogenic fragments appears to be derived from a specific enhancement in their stability. The half-life of the 16-kDa amyloidogenic fragment is approximately 100-fold longer in cells treated with A1-42 than in control cells treated with nonaccumulating A1-28.

Several reports have suggested that a series of carboxyl-terminal APP fragments may arise from the endosomal/lysosomal processing of APP(5, 6, 10, 11, 19) . Under normal conditions, these fragments are rapidly degraded by lysosomal cysteine proteases(5, 19) . Sequence analysis of these APP fragments has indicated that some of these carboxyl-terminal fragments contain the entire A peptide sequence and are potentially amyloidogenic(7) . A similar series of carboxyl-terminal fragments are detected in both AD and aged control brains(5) , and some reports indicate that there may be a direct correlation between the extent of neuronal degeneration and accumulation of APP carboxyl-terminal fragments(23, 24) . These results indicate that APP fragments of the same size that we have observed accumulating in A1-42-treated cultured cells are also produced by brain cells in vivo and accumulate in AD and aged brain tissue.

Although it is not yet clear what significance the accumulation and increased stability of the amyloidogenic fragments is for the mechanisms of amyloid accumulation and AD pathogenesis, there are some intriguing possibilities that will serve as the basis for further analysis of this phenomenon. It is possible that the accumulation of insoluble A, APP, and amyloidogenic APP fragments may mimic the pathophysiology of dystrophic neurites(25, 26, 27, 28, 29, 30, 31, 32, 33) and vascular smooth muscle cells in amyloid angiopathy(34, 35) , where A and carboxyl-terminal fragments of APP have been demonstrated to accumulate intracellularly by immunohistochemical staining. The amounts of potentially amyloidogenic, carboxyl-terminal fragments of APP and 4-kDa A were also found to be significantly enriched in lysates of leptomeningeal vessels from AD cases with amyloid angiopathy but not controls(35) . The increased stability and accumulation of amyloidogenic, carboxyl-terminal fragments of APP has also been reported in lymphoblastoid cells from patients with familial forms of AD(36, 37) . The accumulation of carboxyl-terminal fragments of APP may also contribute directly to pathogenesis. In some cell culture and transgenic animal models, overexpression of carboxyl-terminal fragments bearing the entire A sequence has been reported to result in cell death and neurotoxicity(20) -(41) . Determining whether these possibilities are valid or not will require further analysis.

The increase in the half-life of the amyloidogenic fragments in response to A1-42 treatment is not due to a generalized inhibition of the activity of the degradative machinery of the cell. Our results also indicate that the accumulation is very specific for amyloidogenic fragments of APP, since the amount of the nonamyloidogenic -secretase product is not substantially altered by the presence of A1-42, and its rate of turnover is similarly unaffected. The simplest model to account for these results is to postulate that the amyloidogenic fragments interact with the degradation-resistant A aggregates and that this interaction allows the fragments to evade the normal degradation pathways. A prediction of this model is that the rate of amyloid accumulation would depend on the flux of APP and amyloidogenic fragments down the catabolic pathway and the balance between their rates of degradation and accretion onto existing A aggregates. The fact that the amyloidogenic fragments of APP and the A aggregates end up in the same insoluble fraction of the cell support the notion of their interaction, but experiments designed to demonstrate a direct interaction between A and amyloidogenic fragments of APP in vitro have so far been equivocal. The finding that the amyloidogenic fragments display a conformation-dependent epitope found in A aggregates provides a conceptual basis for their potential interaction. If the fragments have the same conformation of A that is required for amyloid assembly, they may be capable of interacting with A aggregates prior to completion of their proteolytic processing to A.

If some of the fragments that accumulate in response to A1-42 treatment are further processed to A, then as a consequence of the nucleation of stable A aggregates intracellularly, amyloid accumulation would be a self-stimulating, ``runaway'' process. A major caveat to this interpretation is that it must await the unambiguous demonstration that these fragments ultimately give rise to more A. This model for amyloid accumulation is mechanistically related to models proposed for the replication of the scrapie prion (42, 43), and the finding that A1-42 stimulates the accumulation of long-lived fragments which are potential precursors to A is consistent with a key feature of this model, which postulates that the scrapie prion catalyzes its own production from its precursor protein. Another prediction of this model is that the scrapie precursor protein undergoes a conformation change leading to the acquisition of resistance to degradation. The observation that aggregation-specific epitopes are displayed by amyloidogenic fragments of APP is also consistent with this prediction. Our working model for A accumulation differs in some details from the model originally proposed for scrapie prion replication. In this model, it is the aggregates of A, rather than the monomer which interacts with the precursor protein or its fragments. It is not clear whether aggregation induces a conformation change in the fragments or whether the conformation change precedes aggregation and merely allows the fragment to interact with the A aggregates. It seems simpler to propose that the resistance to degradation arises from the interaction of the fragments with the stable A aggregates rather than from the conformation change per se.

A prediction of this model is that at the normal, physiological concentrations of A1-42, the formation of A aggregates would be a rare event, and the turnover of APP fragments would proceed normally. The predicted stochastic nature of stable A aggregate formation (44) at the low, physiological concentrations of A1-42 can explain a peculiar feature of A accumulation and AD pathogenesis. Amyloid deposits are focal lesions and even within the regions of the brain which contain large numbers of amyloid deposits, a large number of neurons remain apparently unaffected. The neurons at risk for AD pathogenesis may represent ones which have had the misfortune of containing a stable A aggregate over their lifetime. The remnant of these cells may form the focal nucleus of an amyloid deposit and initiate a cascade of events, including the growth and maturation of the deposits by the addition of soluble, extracellular A to eventually yield senile plaque. This hypothesis remains to be tested by further experimentation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AG00538 and NS31230. 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.

Present address: Dept. of Cell Biology and Anatomy, Cornell University Medical College, 1300 York Ave., New York, NY 10021.

To whom correspondence should be addressed. Tel.: 714-824-6081; Fax: 714-824-8551; E-mail: CGLABE@UCI.EDU.

The abbreviations used are: AD, Alzheimer's disease; A, amyloid -protein; APP, amyloid precursor protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DMEM, Dulbecco's modified Eagle's medium; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; RIPA, radioimmune precipitation buffer; CHO, Chinese hamster ovary. We use the term ``amyloidogenic'' to refer to fragments of APP that are sufficiently large to contain the entire A sequence. These are actually potentially amyloidogenic and it remains to be established that they actually give rise to A.

Z. Zhang and B. Cordell, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Tilman Oltersdorf and Athena Neurosciences for providing the expression constructs and antibodies. We also thank Drs. Barbara Cordell and Ziyang Zhong for supplying the carboxyl-terminal APP fragments of known sequence.


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