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Intracellular Accumulation of Insoluble, Newly Synthesized Aβn-42 in Amyloid Precursor Protein-transfected Cells That Have Been Treated with Aβ1–42

1999; Elsevier BV; Volume: 274; Issue: 29 Linguagem: Inglês

10.1074/jbc.274.29.20650

1083-351X

Austin J. Yang, Dhusdee Chandswangbhuvana, Theo Shu, Agnes Henschen, Charles Glabe,

Cholinesterase and Neurodegenerative Diseases

Our early study indicates that intracellular Aβ1–42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Aβ1–42 also leads to the accumulation of newly synthesized intracellular Aβ. Here we describe that newly synthesized Aβ, especially Aβn-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Aβ1–42 treatment. These results suggest that intracellular Aβ may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Aβ1–40, the solid-phase intracellular pathway preferentially produces Aβn-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Aβ also share the same types of Aβ structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway. Our early study indicates that intracellular Aβ1–42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Aβ1–42 also leads to the accumulation of newly synthesized intracellular Aβ. Here we describe that newly synthesized Aβ, especially Aβn-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Aβ1–42 treatment. These results suggest that intracellular Aβ may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Aβ1–40, the solid-phase intracellular pathway preferentially produces Aβn-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Aβ also share the same types of Aβ structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway. The major protein component of amyloid deposits associated with Alzheimer's disease (AD) 1The abbreviations ADAlzheimer's diseaseAPPamyloid precursor proteinHPLChigh performance liquid chromatographyACNacetonitrile is a 39–42-amino acid, self-assembling peptide known as the amyloid Aβ peptide. Although significant progress has been made in our understanding of the proteolytic processing of amyloid precursor protein (APP) and the secretion of soluble amyloid Aβ peptide, the mechanisms for the accumulation of insoluble amyloid deposits and their role in AD pathogenesis remains a matter of speculation. It is clear that at least two pathways for APP processing give rise to fragments bearing Aβ sequences at their amino termini: processing by α-secretase, which cleaves within the Aβ sequence, thereby precluding amyloid accumulation, and β-secretase processing, which generates carboxyl-terminal APP fragments containing the Aβ sequence. Amyloidogenic, β-secretase processing events may take place within several intracellular organelles, including the rough endoplasmic reticulum, trans-Golgi network, and lysosomes (1Wild-Bode C. Yamazaki T. Capell A. Leimer U. Steiner H. Ihara Y. Haass C. J. Biol. Chem. 1997; 272: 16085-16088Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 2Chyung A.S.C. Greenberg B.D. Cook D.G. Doms R.W. Lee V.M. J. Cell Biol. 1997; 138: 671-680Crossref PubMed Scopus (137) Google Scholar, 3Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (430) Google Scholar, 4Xu H. Sweeney D. Wang R. Thinakaran G. Lo A.C. Sisodia S.S. Greengard P. Gandy S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3748-3752Crossref PubMed Scopus (253) Google Scholar, 5Refolo L.M. Sambamurti K. Efthimiopoulos S. Pappolla M.A. Robakis N.K. J. Neurosci. Res. 1995; 40: 694-706Crossref PubMed Scopus (52) Google Scholar, 6Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 7Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar). Further processing of APP within the transmembrane domain by γ-secretase releases soluble 3- and 4-kDa fragments containing all or part of the Aβ sequence (8Selkoe D.J. Annu. Rev. Cell Biol. 1994; 10: 373-403Crossref PubMed Scopus (745) Google Scholar). Recent evidence indicates that the familial AD amino acid substitutions within the APP transmembrane domain and presenilin favor the production of Aβ1–42 form of Aβ, which is preferentially localized within diffuse plaques and senile plaques in AD brain. This suggests that Aβ1–42 is more closely associated with AD pathogenesis than shorter Aβ isoforms (9Suzuki N. Cheung T.T. Cai X.D. Odaka A. Otvos Jr., L. Eckman C. Golde T.E. Younkin S.G. Science. 1994; 264: 1336-1340Crossref PubMed Scopus (1354) Google Scholar, 10Younkin S.G. Tohoku J. Exp. Med. 1994; 174: 217-223Crossref PubMed Scopus (22) Google Scholar). Alzheimer's disease amyloid precursor protein high performance liquid chromatography acetonitrile Biochemical studies of synthetic amyloid peptides have elucidated several important properties regarding their ability to assemble into the amyloid fibrils that characteristically accumulate in AD. Peptides that end at residue 42 aggregate much more rapidly than those ending at residue 39 or 40 (11Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Abstract Full Text PDF PubMed Google Scholar, 12Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1760) Google Scholar). The pH optimum for β-sheet formation and aggregation is between pH 4.0 and 5.5 (11Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Abstract Full Text PDF PubMed Google Scholar, 13Barrow C.J. Yasuda A. Kenny P.T. Zagorski M.G. J. Mol. Biol. 1992; 225: 1075-1093Crossref PubMed Scopus (609) Google Scholar, 14Fraser P.E. Nguyen J.T. Surewicz W.K. Kirschner D.A. Biophys. J. 1991; 60: 1190-1201Abstract Full Text PDF PubMed Scopus (355) Google Scholar). Perhaps because of the fact that it aggregates much more rapidly, Aβ1–42 is resistant to degradation once it has been internalized by endocytosis. It accumulates as an insoluble residue in late endosomes or secondary lysosomes, whereas Aβ1–40 and shorter peptides are degraded and eliminated (15Knauer M.F. Soreghan B. Burdick D. Kosmoski J. Glabe C.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7437-7441Crossref PubMed Scopus (205) Google Scholar, 16Burdick D. Kosmoski J. Knauer M.F. Glabe C.G. Brain Res. 1997; 746: 275-284Crossref PubMed Scopus (101) Google Scholar). The amyloid that accumulates in AD is structurally heterogeneous with ragged amino termini, but most of the Aβ peptides end at residue 42 (17Miller D.L. Papayannopoulos I.A. Styles J. Bobin S.A. Lin Y.Y. Biemann K. Iqbal K. Arch. Biochem. Biophys. 1993; 301: 41-52Crossref PubMed Scopus (424) Google Scholar, 18Roher A.E. Lowenson J.D. Clarke S. Woods A.S. Cotter R.J. Gowing E. Ball M.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10836-10840Crossref PubMed Scopus (622) Google Scholar). The amyloid that accumulates in AD brain has the hallmarks of a long-lived protein, such as d-amino acids and isopeptide bonds (19Roher A.E. Lowenson J.D. Clarke S. Wolkow C. Wang R. Cotter R.J. Reardon I.M. Zurcher-Neely H.A. Heinrikson R.L. Ball M.J. Greenberg B.D. J. Biol. Chem. 1993; 268: 3072-3083Abstract Full Text PDF PubMed Google Scholar). The selective resistance of aggregated Aβ1–42 to degradation provides a simple and direct mechanism for why Aβ1–42 preferentially accumulates in the brain (17Miller D.L. Papayannopoulos I.A. Styles J. Bobin S.A. Lin Y.Y. Biemann K. Iqbal K. Arch. Biochem. Biophys. 1993; 301: 41-52Crossref PubMed Scopus (424) Google Scholar, 18Roher A.E. Lowenson J.D. Clarke S. Woods A.S. Cotter R.J. Gowing E. Ball M.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10836-10840Crossref PubMed Scopus (622) Google Scholar). The accumulation of insoluble Aβ1–42 in lysosomes also alters the catabolism of APP and causes the accumulation of APP and a series of potentially APP amyloidogenic fragments (20Yang A.J. Knauer M. Burdick D.A. Glabe C. J. Biol. Chem. 1995; 270: 14786-14792Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 21Davis-Salinas J. Saporito-Irwin S.M. Cotman C.W. Van Nostrand W.E. J. Neurochem. 1995; 65: 931-934Crossref PubMed Scopus (159) Google Scholar). Like the internalized Aβ1–42, these fragments accumulate in the insoluble fraction of the cell and display very long half-lives (20Yang A.J. Knauer M. Burdick D.A. Glabe C. J. Biol. Chem. 1995; 270: 14786-14792Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Several features of this accumulation are analogous to prion replication (22Prusiner S.B. DeArmond S.J. Lab. Invest. 1987; 56: 349-363PubMed Google Scholar). The prion model postulates a conformation change in the precursor protein preceding their proteolytic conversion to more prions. The APP amyloidogenic fragments appear to undergo such a conformation change, since they display an epitope that is specifically associated with Aβ aggregates (20Yang A.J. Knauer M. Burdick D.A. Glabe C. J. Biol. Chem. 1995; 270: 14786-14792Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This suggests that they may have the same shape as aggregated Aβ and are therefore capable of adding on to the fibril lattice established by the internalized exogenous Aβ1–42. The fact that they co-purify in the insoluble fraction of the cell is consistent with the suggestion that they co-aggregate (20Yang A.J. Knauer M. Burdick D.A. Glabe C. J. Biol. Chem. 1995; 270: 14786-14792Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The accumulation of amyloid is autocatalytic as predicted for prion replication, and once amyloid core is seeded, the continued presence of exogenous Aβ1–42 is not required for further accumulation of APP and amyloidogenic fragments (20Yang A.J. Knauer M. Burdick D.A. Glabe C. J. Biol. Chem. 1995; 270: 14786-14792Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). To complete the prion-like cycle, the APP amyloidogenic fragments would need to be further proteolytically processed to Aβ. In this report, we examined whether the addition of exogenous Aβ1–42 causes the accumulation of newly synthesized intracellular Aβ. Here we describe that 4-kDa Aβ, especially Aβn-42, is produced from metabolically labeled APP molecules and accumulates in the detergent-insoluble fraction of cells that have been incubated with synthetic Aβ1–42. Most of the newly synthesized Aβ peptides that accumulate have ragged amino termini and end at residue 42. The structure of these peptides is remarkably similar to the structure described for amyloid Aβ isolated from Alzheimer's brain tissue (17Miller D.L. Papayannopoulos I.A. Styles J. Bobin S.A. Lin Y.Y. Biemann K. Iqbal K. Arch. Biochem. Biophys. 1993; 301: 41-52Crossref PubMed Scopus (424) Google Scholar,18Roher A.E. Lowenson J.D. Clarke S. Woods A.S. Cotter R.J. Gowing E. Ball M.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10836-10840Crossref PubMed Scopus (622) Google Scholar), suggesting that much of the brain amyloid may be derived from this solid-phase, intracellular pathway. Transfected cell cultures (1 × 107 cells in a 10-cm plate) were incubated with methionine-deficient Dulbecco's modified Eagle's medium for 2 h before labeling. The cells were then incubated in 2 ml of methionine-deficient Dulbecco's modified Eagle's medium containing 25 μm amyloid peptide and 1% bovine serum albumin and labeled with 100 μCi/ml [35S]methionine/cysteine (1000 Ci/mmol, Tran35S-label, ICN) for 16 h. At the end of incubation time, the cells were washed twice with cold phosphate-buffered saline and lysed with Nonidet P-40 lysis buffer (50 mm Tris (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride, 5 mm EDTA, 2 mg/ml leupeptin, 0.2 unit/ml soybean trypsin inhibitor, 1 mg/ml aprotinin). The insoluble cell lysate was collected by centrifugation at 10,000 × g for 10 min, solubilized in 88% formic acid (v/w), and lyophilized. After lyophilization, the dried sample was resolubilized with 2× radioimmune precipitation buffer, sonicated until clarified, diluted to 1× radioimmune precipitation buffer, and centrifuged at 10,000 × g for 10 min. The supernatant of sample was then subjected to immunoprecipitation analysis with various antibodies and analyzed by SDS-polyacrylamide gel electrophoresis. APP751-overexpressing cells were preincubated with methionine-deficient Dulbecco's modified Eagle's medium for 2 h before labeling. The cells were then incubated in 2 ml of methionine-deficient Dulbecco's modified Eagle's medium containing 25 μm Aβ1–42 and 1% bovine serum albumin and labeled with 100 μCi/ml [35S]methionine/cysteine (1000 Ci/mmol; Tran35S-label, ICN) for 16 h. At the end of labeling period, the cells were then washed twice with cold phosphate-buffered saline and lysed in Nonidet P-40 lysis buffer. The insoluble cell pellet was then collected by centrifugation at 10,000 ×g for 10 min and solublized in 88% formic acid. The formic acid-solublized cell fraction was then injected over a Superdex 75 gel filtration column that had been previously equilibrated in 60% formic acid. The sample was eluted from the gel filtration column in 60% formic acid at 1 ml/min. The fractions containing Aβ were then pooled and lyophilized. The lyophilized material was then redissolved in 60% formic acid and subjected to a second round of reverse-phase HPLC chromatography in a Vydac C-4 column. The sample was eluted in a 5–95% ACN gradient, and fractions coeluted with synthetic Aβ1–42 were collected for further analysis. APP751-transfected cells were metabolically labeled with either [35S]Met or [3H]Phe, and the secreted or intracellular Aβ were then purified as described above. The amyloid peptides were then subjected to automated Edman degradation amino acid sequencing analysis, and the amount of [3H]Phe radioactivity eluted from each sequencing cycle was then determined by liquid scintillation counting. In the case of [35S]Met-labeled amyloid peptide sequencing reaction, 4,000 cpm of purified labeled Aβ was mixed with 10 μg of synthetic Aβ1–42 in 0.2 m ammonium bicarbonate buffer (pH 8.0) and digested with 0.1 μg of l-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (Sigma) at 37 °C for 16 h. The [35S]Met-containing tryptic fragments (Aβ29–42) were then collected by centrifugation on a tabletop centrifuge at 14,000 rpm for 30 min. The pellet was then washed two times with water and dissolved in 40% ACN, 60% formic acid right before the radiochemical sequence analysis. Our previous work indicates that Aβ1–42 preferentially accumulates in late endosomes and lysosomes of both cultured human fibroblast and PC12 cells and is resistant to degradation. We examined whether the presence of intracellular Aβ affects the catabolism of APP and Aβ in APP-overexpressing human embryonic kidney 293 cells since both nonamyloidogenic and amyloidogenic APP-processing pathways have been demonstrated in this cell line. Previous studies on the uptake of125I-labeled Aβ1–42 demonstrated that most of the internalized 125I-labeled Aβ is sedimentable at 10,000 × g (15Knauer M.F. Soreghan B. Burdick D. Kosmoski J. Glabe C.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7437-7441Crossref PubMed Scopus (205) Google Scholar, 16Burdick D. Kosmoski J. Knauer M.F. Glabe C.G. Brain Res. 1997; 746: 275-284Crossref PubMed Scopus (101) Google Scholar). Because the amyloidogenic fragments of APP also accumulate in the nonionic detergent-insoluble fraction of cells treated with Aβ1–42 (20Yang A.J. Knauer M. Burdick D.A. Glabe C. J. Biol. Chem. 1995; 270: 14786-14792Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), we investigated whether some of these fragments are ultimately converted to 4-kDa Aβ. APP-overexpressing human embryonic kidney 293 cells were treated with Aβ1–42, metabolically labeled with [35S]Met for 16 h, and then lysed with Nonidet P-40 lysis buffer as described under "Materials and Methods." The Nonidet P-40-insoluble fraction was then dissolved in 88% formic acid, lyophilized, and resuspended in radioimmune precipitation buffer, and the 100,000 ×g-soluble supernatant was immunoprecipitated with antibodies raised against Aβ1–42, Aβ1–28, or the carboxyl terminus of APP (13G8). All three antibodies immunoprecipitate a broad size range of labeled products from Aβ1–42-treated cells that are absent in untreated cells (Fig. 1 A). Because of the low efficiency of immunoprecipitation with anti-Aβ antibodies due to the presence of the exogenously supplied, unlabeled Aβ1–42, we devised a more specific method of immunoprecipitating Aβ using cells that were treated with synthetic Aβ4–42 instead of Aβ1–42. We verified that Aβ4–42 is not recognized by an anti-Aβ monoclonal antibody, 3D6, that recognizes the first 5 residues of Aβ (Fig. 1 B). When the detergent-insoluble fraction of cells treated with Aβ4–42 is immunoprecipitated with this antibody, a small amount of 4-kDa Aβ is detected (lane 5, Fig.1 A indicated by arrow). To further demonstrate that the accumulation of intracellular newly synthesized Aβ is because of the Aβ treatment, a time course analysis was performed. APP-overexpressing cells were treated with 25 μm Aβ, and the amount of 35S-labeled Aβ was then detected by immunoprecipitation at indicated times. Our previously published data (20Yang A.J. Knauer M. Burdick D.A. Glabe C. J. Biol. Chem. 1995; 270: 14786-14792Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and the results from this study (Fig. 1 A) indicate that there is a large amount of APP carboxyl-terminal fragments in this insoluble fraction after Aβ treatment. To improve the sensitivity of Aβ detection by eliminating the cross-reactivity between the APP carboxyl-terminal fragments and the anti-Aβ antibody, cell extracts were preabsorbed with anti-APP carboxyl-terminal antibodies (13G8) before immunoprecipitation with the anti-Aβ antibody, 3D6. As shown in Fig. 1 C, there is no detectable newly synthesized [35S]Met-labeled Aβ in the first 4 h of Aβ incubation. Newly synthesized Aβ is first observed after 6 h of Aβ treatment, suggesting that the 35S-labeled Aβ is derived from the amyloidogenic APP fragments that accumulate in the insoluble fraction of cells that have internalized Aβ1–42. This observation is consistent with the recent findings that Aβ1–42 can be produced intracellularly in the detergent-insoluble fraction of both cultured human NT2 neurons and APP-overexpressing human embryonic kidney cells (1Wild-Bode C. Yamazaki T. Capell A. Leimer U. Steiner H. Ihara Y. Haass C. J. Biol. Chem. 1997; 272: 16085-16088Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 2Chyung A.S.C. Greenberg B.D. Cook D.G. Doms R.W. Lee V.M. J. Cell Biol. 1997; 138: 671-680Crossref PubMed Scopus (137) Google Scholar, 3Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (430) Google Scholar), suggesting that a common amyloidogenic pathway exists in these cells. To further characterize the newly synthesized 4-kDa Aβ generated by cells treated with Aβ, we size-selected the 4-kDa fraction using gel filtration methods that were developed to isolate and characterize 4-kDa Aβ from insoluble brain amyloid deposits (19Roher A.E. Lowenson J.D. Clarke S. Wolkow C. Wang R. Cotter R.J. Reardon I.M. Zurcher-Neely H.A. Heinrikson R.L. Ball M.J. Greenberg B.D. J. Biol. Chem. 1993; 268: 3072-3083Abstract Full Text PDF PubMed Google Scholar). APP-overexpressing cells were treated with Aβ1–42 and metabolically labeled with [35S]Met for 6–12 h and then lysed with Nonidet P-40 lysis buffer as described under "Materials and Methods." The Nonidet P-40-insoluble fraction was then dissolved in 60% formic acid and then loaded onto a Superdex 75HR size exclusion column and eluted in 60% formic acid as described (19Roher A.E. Lowenson J.D. Clarke S. Wolkow C. Wang R. Cotter R.J. Reardon I.M. Zurcher-Neely H.A. Heinrikson R.L. Ball M.J. Greenberg B.D. J. Biol. Chem. 1993; 268: 3072-3083Abstract Full Text PDF PubMed Google Scholar, 24Naslund J. Karlstrom A.R. Tjernberg L.O. Schierhorn A. Terenius L. Nordstedt C. J. Neurochem. 1996; 67: 294-301Crossref PubMed Scopus (30) Google Scholar). A peak elutes between fractions 27 and 30 that co-migrates with authentic Aβ1–42 and is absent from control cells that were not treated with Aβ1–42. (Fig.2 A). The Aβ42-containing fractions (fractions 27–30, as indicated on Fig. 2 A) were pooled and further purified by a semi-preparative reverse-phase HPLC. As shown in Fig. 2 B, the majority of the35S-labeled material in the 4-kDa peak from the gel filtration column elutes as a broad peak from fractions 31–51, and the peak is coincident with the elution profile of synthetic Aβ1–42. Therefore, most of the newly synthesized, [35S]Met-labeled Aβ elutes with a profile that is identical to authentic Aβ1–42. As we reported earlier and shown in Fig. 2 B (inset), Aβ1–40 elutes as a sharp peak on an analytical reverse-phase HPLC that precedes the broad elution profile of Aβ1–42 (11Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Abstract Full Text PDF PubMed Google Scholar). The sharp peak of [35S]Met-labeled material elutes at fraction 31, and this elution position is identical to synthetic Aβ1–40. This suggests that the [35S]Met-labeled insoluble fraction contains a mixture of Aβ1–42 and Aβ1–40, and this question is addressed in greater detail below. We radiochemically sequenced the purified 4-kDa newly synthesized Aβ from Aβ1–42-treated cells to determine the amino terminus of newly synthesized intracellular Aβ. APP-overexpressing cells were metabolically labeled with [3H]Phe overnight in the presence of 25 μmAβ1–42, and the [3H]Phe-labeled peptides were then purified as described above and subjected to automated Edman degradation amino acid sequence analysis. Phenylalanine residues occur in the Aβ sequence at positions 4, 19, and 20. As indicated on Fig.3 A, the [3H]Phe radioactivity from peptide isolated from the insoluble fraction of cell lysate is detected in two major peak fractions (fractions 2–10 and fractions 18–22). This result indicates that the [3H]Phe-labeled Aβ is "ragged" at its amino terminus, containing both longer and shorter peptides than those beginning at residue 1. To confirm that the radiochemical sequence is derived from amyloid Aβ peptides, we radiochemically sequenced [35S]Met-labeled peptides after cleaving them with trypsin to create homogeneous ends. The Aβ sequence contains one methionine residue at position 35 that would occur at cycle 7 after trypsin cleavage at lysine 28. The purified intracellular [35S]Met-labeled Aβ was subjected to tryptic fragmentation, and the hydrophobic carboxyl-terminal fragment from residues 28–42 was purified as described previously (19Roher A.E. Lowenson J.D. Clarke S. Wolkow C. Wang R. Cotter R.J. Reardon I.M. Zurcher-Neely H.A. Heinrikson R.L. Ball M.J. Greenberg B.D. J. Biol. Chem. 1993; 268: 3072-3083Abstract Full Text PDF PubMed Google Scholar) and subjected to radiochemical Edman sequence analysis. Sixty percent of the total starting 35S label was recovered in the purified carboxyl-terminal fragment. As indicated in Fig. 3 B, the [35S]Met radioactivity begins in cycle 7, which is consistent with the predicted Aβ sequence. The lag in [35S]Met radioactivity eluting at cycles 8 and 9 was also observed in the Met yield obtained chemically in the same cycles. The [35S]Met is quantitatively released during sequencing, and no labeled material remained associated with the filter. These results provide independent confirmation that the radiochemical sequence obtained is derived from Aβ rather than from contaminating peptides. The presence of ragged amino termini suggests that the ends of the Aβ peptides accumulating in the insoluble fraction of the cell may be generated by relatively nonspecific proteolysis. The proteolytic trimming of exogenously supplied synthetic Aβ1–42 has been previously observed for exogenously added peptide after internalization in human fibroblasts (15Knauer M.F. Soreghan B. Burdick D. Kosmoski J. Glabe C.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7437-7441Crossref PubMed Scopus (205) Google Scholar). In contrast, the radiochemical sequence of Aβ isolated from cultured media is much more homogeneous, and [3H]Phe radioactivity can only be detected in fractions 4, 19, and 20, suggesting the amino terminus of soluble Aβ is generated by a more specific mechanism (25Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1763) Google Scholar). Interestingly, Aβ isolated from the plaques of the brains of Alzheimer's patients also contained such amino-terminal truncation as we describe here (18Roher A.E. Lowenson J.D. Clarke S. Woods A.S. Cotter R.J. Gowing E. Ball M.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10836-10840Crossref PubMed Scopus (622) Google Scholar, 24Naslund J. Karlstrom A.R. Tjernberg L.O. Schierhorn A. Terenius L. Nordstedt C. J. Neurochem. 1996; 67: 294-301Crossref PubMed Scopus (30) Google Scholar,26Naslund J. Schierhorn A. Hellman U. Lannfelt L. Roses A.D. Tjernberg L.O. Silberring J. Gandy S.E. Winblad B. Greengard P. Nordstedt C. Terenius L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8378-8382Crossref PubMed Scopus (370) Google Scholar, 27Saido T.C. Yamao-Harigaya W. Iwatsubo T. Kawashima S. Neurosci. Lett. 1996; 215: 173-176Crossref PubMed Scopus (238) Google Scholar). Our early study from the biochemical properties of synthetic Aβ indicated that Aβ1–42 rather than Aβ1–40 is able to form SDS-resistant aggregates and migrates as a 16-kDa band on SDS-polyacrylamide gel electrophoresis at high concentrations (11Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Abstract Full Text PDF PubMed Google Scholar, 28Soreghan B. Kosmoski J. Glabe C. J. Biol. Chem. 1994; 269: 28551-28554Abstract Full Text PDF PubMed Google Scholar). The specificity of SDS-resistant Aβ aggregates is demonstrated in Fig.4 A, where trace amounts of14C-labeled Aβ1–42 are mixed with increasing amounts of unlabeled Aβ1–40 or Aβ1–42 individually. A 16-kDa SDS-resistant aggregate is only observed at higher concentrations of radiolabeled Aβ1–42 with unlabeled Aβ1–42, whereas the radiolabeled Aβ1–42 does not co-assemble into 16-kDa SDS-resistant aggregates with unlabeled Aβ1–40. Therefore, this property can be used to distinguish between the metabolically labeled Aβ1–42 and Aβ1–40 and shorter Aβ peptides. The HPLC-purified, [35S]Met-labeled intracellular Aβ was mixed with synthetic Aβ1–28, Aβ1–42, and Aβ1–40 at a concentration of 500 μg/ml at pH 5.0 for 24 h. The labeled peptide forms 16-kDa SDS-resistant aggregates only with synthetic Aβ1–42 but not Aβ1–40 (indicated by the arrowhead, Fig. 4 B), suggesting that a fraction of the newly synthesized [35S]Met Aβ from the insoluble fraction of cell lysate is Aβ1–42 or closely related products that are capable of co-assembling with synthetic Aβ1–42 into SDS-resistant aggregates. Recently, an analytical, chromatographic method for separating Aβ1–42 from Aβ1–40 was described that employed reverse-phase separation at high temperature (29Chloupek R.C. Hancock W.S. Marchylo B.A. Kirkland J.J. Boyes B.E. Snyder L.R. J. Chromatogr. A. 1994; 686: 45-59Crossref PubMed Scopus (89) Google Scholar). As indicated in Fig.5 A, Aβ1–42 can be resolved as a single peak on a Zorbax C-18 reverse-phase column at 65 °C and is well separated from Aβ1–40 by almost 2 min. The purified 4-kDa radiolabeled peptides were dissolved in 1 ml of 70% formic acid and injected onto a Zorbax C-18 reverse-phase column. The radioactive sample was then eluted by a 20–45% ACN gradient at 65 °C, and the fractions that coeluted with synthetic Aβ1–42 peptides were collected for further analysis (Fig. 5 B). The radiolabeled peptides elute as two major peaks at the positions of both Aβ1–40 and Aβ1–42, even though the amino termini of the peptides were determined to be ragged by radiochemical sequencing. Our experience is that the carboxyl terminus of Aβ has a dominant influence on their chromatographic behavior (11Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Abstract Full Text PDF PubMed Google Scholar), and synthetic amino-terminal deletions of Aβ1–42 and Aβ1–40 tend to co-migrate with the peptides beginning at position 1 (data not shown). Taken together, these results indicate that a mixture of Aβn-42 and Aβn-40 are produced from APP and accumulate in cells treated with Aβ1–42. Our results suggest that a solid-phase pathway for the simultaneous production and accumulation of amyloid Aβ exists within late endosomes or secondary lysosomes of cells that contain degradation-resistant Aβ1–42 aggregates. We have previously shown that the presence of intracellular Aβ1–42 aggregates alters the normal catabolism of APP to cause the accumulation of APP and potentially amyloidogenic APP fragments in lysosomes of cultured cells. In this report, we have demonstrated that newly synthesized 4-kDa Aβ also accumulates in the same insoluble fraction as the amyloidogenic APP fragments. Because of the requirement for exogenously added synthetic Aβ1–42 to initiate the accumulation of insoluble fragments of APP, we must rely on radiochemical methods to unambiguously distinguish the newly synthesized Aβ that is derived from the transfected APP gene from the synthetic Aβ1–42 added to the cell cultures. A [35S]Met-labeled 4-kDa band is immunoprecipitated from the formic acid-soluble fraction with a variety of anti-Aβ antibodies. The efficiency of immunoprecipitation of 4-kDa Aβ is enhanced by using Aβ4–42 instead of Aβ1–42 to prime the cells and immunoprecipitating with 3D6, a monoclonal antibody specific for the first 5 residues of Aβ. Similarly, the efficiency of immunoprecipitation of 4-kDa Aβ is improved by immunodepletion of cross-reacting carboxyl-terminal fragments of APP. Like the amyloid deposits from AD brain, the detergent-insoluble fraction of Aβ1–42-treated cells is largely solubilized in formic acid; therefore, we have used the purification methods employed for purifying Aβ from AD brain to purify and characterize the 4-kDa peptides in the insoluble fraction of Aβ1–42-treated cells. The fractions that elute in formic acid from a Superdex HR75 column at the position of denatured 4-kDa Aβ1–42 were pooled and further purified by reverse-phase HPLC. Most of the radiolabeled material elutes from the reverse-phase HPLC at the same position as Aβ1–42. Radiochemical sequencing of this purified material indicates that the amino termini of these peptides is heterogeneous, with peptide ends both longer and shorter than those beginning at residue 1. The heterogeneity may also be because of non-Aβ contaminants, but after trypsin cleavage to create homogeneous cleavage sites, most of the [35-S]Met label is observed at cycle 7 as expected for Aβ. The carboxyl terminus of the newly synthesized 4-kDa product was characterized by analytical reverse-phase HPLC under conditions that resolve Aβ1–42 and Aβ1–40. Although labeled material is recovered in both peaks, most of the radioactivity is associated with Aβ1–42. Perhaps the most convincing evidence that the newly synthesized 4-kDa peptides are Aβ1–42 and closely related structures is the ability to co-assemble into SDS-resistant aggregates with authentic Aβ1–42 at concentrations above the critical concentration for aggregation. This property is specific to Aβ1–42 and Aβ1–43, since no SDS-resistant aggregates are observed for Aβ1–40 even in the presence of a vast excess of Aβ1–42. Thus, the intracellular Aβ displays a number of similarities to the amyloid that accumulates in AD brain tissue. The intracellular amyloid is insoluble in nonionic detergents, but like amyloid isolated from brain, a substantial fraction of the amyloid is soluble in formic acid (17Miller D.L. Papayannopoulos I.A. Styles J. Bobin S.A. Lin Y.Y. Biemann K. Iqbal K. Arch. Biochem. Biophys. 1993; 301: 41-52Crossref PubMed Scopus (424) Google Scholar, 18Roher A.E. Lowenson J.D. Clarke S. Woods A.S. Cotter R.J. Gowing E. Ball M.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10836-10840Crossref PubMed Scopus (622) Google Scholar). Both the intracellular amyloid and brain amyloid display considerable amino-terminal heterogeneity, and the majority of the amyloid peptides end at residue 42 (17Miller D.L. Papayannopoulos I.A. Styles J. Bobin S.A. Lin Y.Y. Biemann K. Iqbal K. Arch. Biochem. Biophys. 1993; 301: 41-52Crossref PubMed Scopus (424) Google Scholar, 18Roher A.E. Lowenson J.D. Clarke S. Woods A.S. Cotter R.J. Gowing E. Ball M.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10836-10840Crossref PubMed Scopus (622) Google Scholar). The finding that Aβn-42 accumulates inside cells treated with amyloid Aβ1–42 provides further support for the hypothesis that amyloid accumulation is an autocatalytic process mechanistically related to prion replication. Like PrPsc, the core structure of aggregated Aβn-42 is resistant to proteolysis, both inside cells andin vitro (15Knauer M.F. Soreghan B. Burdick D. Kosmoski J. Glabe C.G. Proc. Natl. Acad. Sci. U. 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Misfolded APP molecules and amyloidogenic fragments appear to be intermediates in amyloid accumulation because they also accumulate in the nonionic detergent-insoluble fraction of the cell where they turn over very slowly (20Yang A.J. Knauer M. Burdick D.A. Glabe C. J. Biol. Chem. 1995; 270: 14786-14792Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). At least a subset of these amyloidogenic fragments appear to have adopted the same conformation as aggregated Aβ because they display an unique conformation-dependent epitope that is only detected in aggregated Aβ (20Yang A.J. Knauer M. Burdick D.A. Glabe C. J. Biol. Chem. 1995; 270: 14786-14792Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This suggests that the misfolded amyloidogenic APP fragments may be capable of binding to aggregated Aβn-42 and extending the amyloid lattice. Because both amyloidogenic carboxyl-terminal APP fragment precursors and the 4-kDa Aβ product accumulate in the insoluble fraction, the simplest hypothesis is that this conversion takes place in the solid phase. Co-aggregation of poorly metabolized Aβ and amyloidogenic fragments of Aβ may explain why the intracellular Aβ immunoreactivity is not morphologically recognizable as amyloid fibrils. Because the insoluble aggregates are a heterogeneous collection of fragments, perhaps their underlying fibrillar lattice is not revealed until the fragments have been digested to their protease-resistant amyloid core. The fact that the intracellular amyloidogenic fragments themselves are poorly degraded suggests that this conversion process may be quite slow and may even occur extracellularly, after the insoluble residue has been externalized, either by exocytosis or the death of the cell. This accumulation appears to mimic the "granulovacuolar" pathophysiology of degenerating neurons and dystrophic neurites, where Aβ and APP immunoreactivity have been localized to granular or globular deposits (31Cummings B.J. Su J.H. Geddes J.W. Van Nostrand W.E. Wagner S.L. Cunningham D.D. Cotman C.W. Neuroscience. 1992; 48: 763-777Crossref PubMed Scopus (103) Google Scholar, 32Joachim C. Games D. Morris J. Ward P. Frenkel D. Selkoe D. Am. J. Pathol. 1991; 138: 373-384PubMed Google Scholar, 33Perry G. Siedlak S. Mulvihill P. Kancherla M. Mijares M. Kawai M. Gambetti P. Sharma S. Maggiora L. Cornette J. Kalaria R. Prog. Clin. Biol. Res. 1989; 317: 1021-1025PubMed Google Scholar) that are also positive for ubiquitin immunoreactivity (34Gregori L. Bhasin R. Goldgaber D. Biochem. Biophys. Res. Commun. 1994; 203: 1731-1738Crossref PubMed Scopus (14) Google Scholar,35Tabaton M. Cammarata S. Mancardi G. Manetto V. Autilio-Gambetti L. Perry G. Gambetti P. Proc. Natl. Acad. Sci. U. S. 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Frenkel D. Selkoe D. Am. J. Pathol. 1991; 138: 373-384PubMed Google Scholar, 33Perry G. Siedlak S. Mulvihill P. Kancherla M. Mijares M. Kawai M. Gambetti P. Sharma S. Maggiora L. Cornette J. Kalaria R. Prog. Clin. Biol. Res. 1989; 317: 1021-1025PubMed Google Scholar), these may represent regions of the amyloid deposits where the conversion process is complete. Although the significance of intracellular Aβ immunoreactivity and the origin of extracellular Aβ deposits is still controversial, a recent report by Cataldo et al. (37Cataldo A.M. Barnett J.L. Mann D.M. Nixon R.A. J. Neuropathol. Exp. Neurol. 1996; 55: 704-715Crossref PubMed Scopus (70) Google Scholar) indicates that lysosomal hydrolases can be detected in extracellular amyloid deposits of AD and Down syndrome brains. This is consistent with the lysosomal origin of these deposits under pathological conditions (37Cataldo A.M. Barnett J.L. Mann D.M. Nixon R.A. J. Neuropathol. Exp. 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