Proc. Natl. Acad. Sci. USAVol. 89, pp. 7437-7441, August 1992Biochemistry

Intracellular accumulation and resistance to degradation of theAlzheimer amyloid A4/, protein

(senile plaque/cerebrovascular amyloid/lysosomes/protein catabolism)

MARY F. KNAUER, BRIAN SOREGHAN, DEBRA BURDICK, JOSEPH KOSMOSKI, AND CHARLES G. GLABE*Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717

Communicated by William J. Lennarz, May 11, 1992

ABSTRACT The A4 or 13 protein is a peptide that consti-tutes the major protein component of senile plaques in Alzhei-mer disease. The A4/13 protein is derived from a larger,transmembrane amyloid precursor protein (APP). The puta-tive abnormal processing events leading to amyloid accumu-lation are largely unknown. Here we report that a 42-residuesynthetic peptide, 181-42, corresponding to one of the longerforms of the A4/13 protein, accumulates in cultured human skinfibroblasts and is stable for at least 3 days. The peptide appearsto accumulate intracellularly, since it does not accumulateunder conditions that prevent endocytosis and accumulation iscorrelated with the acquisition of resistance to removal bytrypsin digestion. This intracellular accumulation is also cor-related with the ability of the peptide to aggregate as deter-mined by SDS/polyacrylamide gel electrophoresis. At lowconcentrations of the 131-42 peptide, which favor the nonaggre-gated state, no accumulation is observed. Shorter peptideanalogs (28 or 39 residues) that are truncated at the C terminus,which lack the ability to aggregate in SDS gels, fail to accu-mulate. The accumulated intracellular 131-42 peptide is in anaggregated state and is contained in a dense organellar com-partment that overlaps the distribution of late endosomes orsecondary lysosomes. Immunofluorescence of the internalizedpeptide in permeabilized cells reveals that it is contained ingranular deposits, consistent with localization in late endo-somes or secondary lysosomes. Sequence analysis indicates thatsome of the internalized peptide is subject to N-terminaltrimming. These results suggest that the aggregated A4/13protein may be resistant to degradation and suggest that theA4/1B protein may arise, at least in part, by endosomal orlysosomal processing of APP. Our results also suggest thatrelatively nonspecific proteolysis may be sufficient to generatethe A4/13 protein if this part of APP is selectively resistant toproteolysis.

Alzheimer disease is a specific form ofneuronal degenerationand consequent dementia characterized by the accumulationof intraneuronal neurofibrillary tangles and extracellularamyloid deposits. Amyloid occurs as diffuse aggregates ormore dense deposits, which are termed senile plaques. De-posits surrounding brain microvessels also occur, where theyare termed cerebrovascular amyloid. The major protein com-ponent of the senile plaque is a 42- or 43-amino acid peptide,known as the 13 protein (1) or A4 peptide (2), which isextremely insoluble and has a strong tendency to aggregate.The A4/13 protein is derived from a larger transmembraneprotein, the amyloid precursor protein (APP), which is ex-pressed as multiple different mRNA splicing products (3-5).Accumulation of the A4/1B protein is apparently the result ofabnormal processing, since the extracellular domain of APPis normally cleaved at residue 16 within the A4/13 region (6,

7). The fact that normal processing precludes A4/13 accumu-lation suggests that abnormal processing may be the initialstep leading to amyloid deposition.Using synthetic peptide analogs of the A4/(3 protein, we

have defined some of its intrinsic biochemical and physicalproperties that are related to its assembly into amyloid-likefibrils and its ability to aggregate (8). We found that assemblyinto amyloid-like fibrils and aggregation in SDS/polyacryl-amide gels are separate and distinct properties of the amyloidpeptides. Low pH (pH 3.5-6.5) and high concentrations ofpeptide are important for promoting assembly of the peptidesinto amyloid-like fibrils (8). The length of the hydrophobic Cterminus (-42 residues) and a high concentration of peptideare critical for the ability ofthe (31 2 peptide to self-aggregateinto multiple discrete bands in SDS/polyacrylamide gels.These intrinsic factors may be important for amyloid depo-sition in vivo because the acid environment ofendosomes andlysosomes and their ability to concentrate solutes wouldpromote amyloid fibril formation and peptide aggregation,which could compromise the ability of the cell to degrade theA4/13 protein.To explore whether cells are able to degrade the A4//3

protein, we examined uptake and degradation of three pep-tides that corresponded to residues 1-28 (extracellular portion)(p13-28), 1-39 [predominant form of the A4/,8 protein in cere-brovascular amyloid from HCHWA (hereditary cerebral hem-orrhage with amyloidosis) Dutch-type patients (9)] (p1-39), and1-42 [a major form of the A4/,8 protein in senile plaque (10)](131-42) of the A4/,8 protein in human neonatal foreskin fibro-blast (HF cell) cultures. Here we report that the 42-residuesenile-plaque form selectively accumulates intracellularly andis largely resistant to degradation for at least 3 days.

MATERIALS AND METHODSMaterials. Peptides 131-28, P11-39, and P1l-42 (131-42, DAE-

FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG-GVVIA) were synthesized by fluoren-9-ylmethoxycarbonylchemistry using a continuous-flow semiautomatic instru-ment, purified by reverse-phase HPLC, and characterized bysequencing and electrospray mass spectrometry (8). Na1251was obtained from Amersham; chloroglycouracil (lodo-Gen)from Pierce, Bio-Gel P-2 from Bio-Rad, fetal bovine serumfrom GIBCO, Dulbecco's modified Eagle's medium(DMEM) from Irvine Scientific, and Percoll from Pharmacia.Analytical-grade solvents and other reagents were from var-ious commercial sources (Sigma; Fisher Scientific).

Peptide Iodination. Aliquots (10 pg) ofeach peptide in 40 julof 1 M Tris (pH 7.4) were radioiodinated to a specific activityof 50,000-150,000 cpm per ng in the presence of 50 u.g ofIodo-Gen at 0C for 20 min (8). Free iodine was separatedfrom peptide by elution over a 5-ml Bio-Gel P-2 column

Abbreviations: APP, amyloid precursor protein; HF cells, humanneonatal foreskin fibroblasts.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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equilibrated in 0.1 M Tris (pH 7.4) with polyvinylpyrrolidone(Mr 40,000) at 2 mg/ml. Peptide was eluted in the void volumeand these fractions were pooled, frozen, and lyophilizedovernight. Specific activity was determined by trichloroace-tic acid precipitation of an aliquot of the radioiodinatedpeptide sample prior to elution.

Cell Culture. When HF cells reached confluence (4-8days), they were passaged at a 1:4 dilution in DMEM with 10mM Hepes, 10o fetal bovine serum, 4 mM glutamine,penicillin (200 units/ml) and streptomycin (200 ug/ml) (5%C02, 370C). Test cultures were plated in 100-mm (12 ml) or35-mm (2 ml) dishes and used when they reached confluence.Uptake/Degradation. Cultures were grown in 35-mm

plates, rinsed in 1 ml of binding medium (DMEM/10 mMHepes/2% bovine serum albumin), and then returned toincubation in binding medium containing the indicated con-centrations of 125I-labeled peptide and unlabeled peptide. Atthe indicated times, aliquots of medium from each culturedish were frozen for later TLC analysis, and the cells wererinsed with ice-cold phosphate-buffered saline and treatedwith trypsin (2.5 mg/ml) at 0C for 15 min. Cells were pelletedby microcentrifugation and radioactivity in the trypsin su-pernatant was quantitated in a Beckman Gamma 5500 ycounter. The cell pellet was resuspended in phosphate-buffered saline and the radioactivity was quantitated asabove. Each data point is the mean of three independentdeterminations. Cell pellets were frozen for subsequent anal-ysis by tricine SDS/PAGE (11). Cell surface adsorption wasmeasured in parallel cultures incubated at 4°C. lodotyrosinewas quantitated by y counting following TLC separation ofintact peptide in a 1-butanol/acetic acid/water (100:10:10,vol/vol) solvent system (12).

Characterization of Internalized P1-42. The subcellular dis-tribution of internalized 3142 and the lysosomal markershexosaminidase and acid phosphatase was determined afterfractionation of cell homogenates on 12.5% Percoll gradients(13-15). The internalized 125SI438142 was extracted from tryp-sin-treated cell pellets by addition of 88% formic acid fol-lowing freezing and thawing. The solution was centrifuged at14,000 x g for 10 min. The supernatant was dried, suspendedin water containing 0.1% trifluoroacetic acid and 1 mg ofunlabeled P142 as a carrier, and fractionated by reverse-phase HPLC (8). An aliquot (8000 cpm) of the purifiedinternalized peptide was subjected to automated Edmandegradation and the sequence products were quantified by ycounting.For immunolocalization, cells that had been incubated with

(31-42 (100 jkg/ml) for 24 hr were trypsin-treated, plated oncoverslips, incubated in the absence of peptide for 24 hr, andfixed in cold methanol (0°C, 10 min). After fixation, thecoverslips were treated with formic acid (88%, 5 min),washed, and incubated with affiity-purified rabbit anti-P3l12IgG (2 pg, 12 hr). The coverslips were washed and incubatedwith fluorescein-conjugated goat anti-rabbit IgG and fluores-cence micrographs were taken with an Olympus epifluores-cence microscope using x40 and x20 objectives.

RESULTSWe examined the uptake and degradation of A4/( proteinanalogs in HF cells because the endosomal and lysosomalpathways are ubiquitous and they have been well character-ized in HF cells (16-18). In addition, human skin fibroblastcultures are available from Alzheimer disease patients. Allthree peptides adsorbed to the cell surface at 4°C (Fig. 1A).No evidence was obtained for the existence of a specificreceptor for these peptides in HF cells. The cell surface-adsorbed peptide was effectively removed (>95%) by trypsindigestion (Fig. 1A). Upon incubation at 370C, a significantamount of (1-42 accumulated that was resistant to removal by

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FIG. 1. Intracellular accumulation and stability of internalizedA4,B peptides. (A) Adsorption of peptides to the cell layer at 40C. HFcells (500,000 per 35-mm plate) were rinsed with 1 ml of bindingmedium and then incubated for 2 hr at 4C in 1 ml of mediumcontaining 25 ng of radioiodinated 81-28 (A, A), 181-39 (O, e), or 13-42

(0, m) peptide (30,000-45,000 cpm per ng) and 0-150 Mg of thecorresponding unlabeled peptide. After incubation, the cells were

washed with cold phosphate-buffered saline and trated with trypsin.Cell-associated counts before (open symbols) and after (filled sym-bols) trypsin treatment are shown. No significant decrease in cell-associated counts is observed over the range of 10-150 Mg ofunlabeled peptide added. Trypsin treatment removed >95% of thecell-associated peptide. (B) Concentration dependence of the accu-

mulation of 1-42. Cells were incubated for 6 hr at 370C in 1 ml ofmedium containing 0.75 Mg of radioiodinated P1_2s (A), 131-39 (0), or

131-42 (n) peptide (30,000-45,000 cpm per ng) and 0-200 Mg of thecorresponding unlabeled peptide. After incubation, the cells werewashed and trypsin-treated and the amount ofcell-associated peptidewas determined. At high concentrations of peptide (100-200 pg/ml)significant amounts of trypsin-resistant 11-42 remained associatedwith the cells in comparison to the shorter 1-protein analogs. (C)Intracellular retention of 13-42 peptide. HF cells (900,000 per plate)were incubated in binding medium with 0.375 Mg of radioiodinated13142 (42,500 cpm/ng) plus unlabeled peptide (100 Mg/ml). At 24 hr,medium was aspirated, cells were rinsed, and fresh medium without(v) or with (-) unlabeled 11-42 (100 Mg/ml) was added. At varioustimes after the initial incubation with radiolabeled peptide, cells wereremoved from incubation and intracellular peptide was determined.

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trypsin (Fig. 1B). This accumulated peptide appeared torepresent internalized material, since it was resistant totrypsin under conditions where the surface-adsorbed peptidewas sensitive. The amount of trypsin-resistant, internalizedpeptide accumulated after a 24-hr incubation at 100 ug/ml(19.4 pg per cell) was higher than the amount of adsorbedpeptide released by trypsin digestion (7.9 pg per cell). Theamount of intracellular peptide accumulated depended on theconcentration of the peptide in the culture medium. At lowconcentrations of (1-42 (0.75 ug/ml), HF cells accumulated<1 fg per cell. Accumulation also depended on the length ofthe hydrophobic C terminus. At higher concentrations of(31-42 (10-200 ug/ml), the amount of intracellular peptideaccumulated was significantly greater than for cell culturesincubated with the same concentrations of the shorter, f1-28or (1-39 analogs; >22 pg of the peptide accumulated per cellwhen cultures were incubated with (81-42 at 200 ug/ml,compared to <0.7 and <0.05 pg per cell when cultures wereincubated with the same concentration of 81-39 and (31-2sanalogs, respectively (Fig. 1B). The differences in the accu-mulation of 81-42 and the shorter peptides cannot be ex-plained by differences in the rates of internalization. Theinitial rates of internalization during a 10-min incubation at370C were 0.18, 0.07 and 0.053 pg per cell per min for 81-28,(31-39 and (31-42, respectively.The majority ofthe internalized (1-42 was stable for at least

72 hr after uptake, in either the presence or the absence ofunlabeled peptide in the culture medium (Fig. 1C). Seventy-two hours after medium containing the radioactive peptidewas replaced by fresh incubation medium, 70-80%o of theinternalized 125I-labeled peptide was still retained intracellu-larly. These results indicate that the majority of the accumu-lated intracellular (1-42 peptide is stable over this incubationperiod and is not degraded via the lysosomal pathway.The intracellular accumulation of the P1-42 peptide and its

resistance to degradation correlate with the potential of thepeptide to aggregate and with conditions that promote bothamyloid fibril formation and aggregation of the peptide (8).The (3-28 and (1-39 peptides lack the ability to aggregate asdefined by the appearance of discrete higher molecular massforms in SDS/polyacrylamide gels (8), and these peptides donot accumulate to significant levels intracellularly (Fig. 1A).Aggregation in SDS may reflect the ability ofthe hydrophobicC termini of 81-42 peptides to interact. This interaction isnoncovalent, as the higher molecular mass forms are inequilibrium with the peptide monomer in SDS (8). Thesehigher molecular mass aggregates are also observed by nativegel electrophoresis and gel filtration, indicating that they arenot an artifact of SDS gel electrophoresis (data not shown).The critical concentration for aggregation ofthe (1-42 peptideis between 200 and 500 ,ug/ml in DMEM (Fig. 2A). We alsoanalyzed the aggregation state of the radioiodinated peptideinside the cell (Fig. 2B). Upon incubation in the presence ofP1-42 at 100 ug/ml, the intracellular fraction displayed mul-tiple discrete, higher molecular mass forms at the earliesttime point in addition to the monomeric form of the peptide.Since no aggregated forms were observed at this concentra-tion in the absence of cells (Fig. 2A), this suggests that eitherthe internalized peptide was concentrated or the conditions inthe intracellular compartment promoted aggregation of thepeptide. Although multiple discrete, higher molecular massbands were formed both intracellularly and in the incubationmedium, there were some apparent differences in the intra-cellular aggregated forms compared with the aggregatesformed in medium alone. The amount of radiolabeled peptidethat remained at the top of the stacking gel was much greaterin the intracellular fraction, and the higher molecular massband within the separating gel was broader (9-18 kDa). Thepattern of discrete bands formed by (1-42 incubated at acidpH was similar to the one observed intracellularly (8). Since

A ug / ml100 200 500 1,000 2,000

B hours0 1 2 24 48 72

- - w

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432918.4 -4

14.3 -4

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FIG. 2. (A) Concentration dependence of 81-42 aggregation inmedium. Lyophilized (1-42 was diluted with serum-free medium (pH7.4) to concentrations indicated. Each sample contained 0.25 ng of125I-.1142 (12,000 cpm). The samples were incubated for 6 hr at 37TC.After addition of 2x SDS/PAGE sample buffer, the samples wereboiled for 3 min and then run in a tricine SDS gel. The gel wasimmediately dried and then exposed to film at -70'C. (B) Kinetics ofintracellular (31-42 aggregation. HF cells were incubated in mediumcontaining 125I-438142 (0.25 j.g/ml, 65,000 cpm/ng) plus unlabeledpeptide (100 pg/ml). At various times cells were rinsed, trypsin-treated and pelleted as described in Fig. 1.

(31-42 forms higher molecular mass bands in the absence ofany other macromolecules, the simplest interpretation is that*the intracellular peptide is aggregated, but we have not ruledout the possibility that the accumulated (1-42 aggregates withother cellular macromolecules.We characterized the intracellular fraction of the (1-42

peptide by TLC, reverse-phase HPLC, and automated Ed-man degradation. TLC indicated that essentially all of theintracellular radioiodinated material was macromolecularand was not degraded to iodotyrosine. In contrast, 4-7% ofthe total radioactivity in the culture medium was in the formof iodotyrosine over a 6-hr incubation at 370C for all threepeptides. The intracellular (31-42 was extracted with formicacid and analyzed by reverse-phase HPLC (Fig. 3). Theintracellular fraction of the 1251-381-42 was coeluted withradioiodinated peptide that had not been incubated with cellsand with noniodinated peptide. The broad elution profile is acharacteristic feature of the P1-42 peptide and is not a reflec-tion of heterogeneity (8). Reverse-phase HPLC can resolve(31-42 from (1-39 and shorter peptides, suggesting that the Cterminus of the internalized peptide is not substantiallydegraded. Radiochemical sequencing of the extracted intra-cellular fraction by automated Edman degradation revealed

a.

0 10 20 304Fraction number

1.6

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FIG. 3. Reverse-phase HPLC analysis of intracellular (31-42. HFcells were incubated for 24 hr in binding medium containing 0.3 ,ugof 125I-,1l-42 (10,000 cpm/ng) and unlabeled peptide (100 Ag/ml). Thesurface-adsorbed fraction was removed by trypsin digestion and thecell pellet was solubilized in 88% formic acid, mixed with 1 mg ofunlabeled peptide, and subjected to reverse-phase HPLC. *, Intra-cellular 125I-01(42 (cpm); o, unincubated control 125I-91-42 peptide(cpm); m, unlabeled peptide (A214).

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that radioiodine was released at cycles 5-9, in addition to theexpected position at cycle 10 (Fig. 4A). No radioiodine wasreleased until cycle 10 in control, unincubated peptide,indicating that the His-6 was not detectably iodinated. Ra-dioactivity released at cycles 11 and 12 was most likely dueto sequencing "lag," since it was also observed in theunincubated control. These results suggest that the N termi-nus of the intracellular P1-42 was "ragged" and may havebeen subjected to limited proteolysis (Fig. 4B). Sequenceanalysis of the A4/fB protein isolated from Alzheimer diseasebrain tissue indicates that the N terminus of the naturalproduct is also ragged, with a major product beginning atPhe-4 (2).The intracellular distribution of the radiolabeled peptide

accumulated during a 24-hr incubation with medium contain-ing P1-42 at 100 pg/ml was determined by fractionation of thecell organellar homogenate on Percoll gradients. The distri-butions of both radioactivity and the lysosomal markers(3-hexosaminidase and acid phosphatase are shown in Fig. 5.The intracellular radioactivity was associated primarily withfractions 17-20 and had a peak density of 1.048 g/ml. Thelysosomal markers peaked in fractions 18-21. Thus, themajority of the intracellular peptide was associated with adense organelle that overlapped the distribution of the lateendosome/secondary lysosomal fractions.The internalized (1-42 was also localized by immunofluo-

rescence microscopy, which revealed numerous apparentlycytoplasmic deposits (Fig. 6 A and B). No specific fluores-cence staining was observed in control samples in which theprimary antibody was omitted (Fig. 6 C and D) or in cellsincubated in the absence of (81-42 (Fig. 6 D and E). Many ofthe anti-131-42-reactive deposits were in a perinuclear loca-tion, consistent with localization in late endosomes or sec-ondary lysosomes. The putative localization of the peptidewithin an acidic compartment is consistent with previousstudies which demonstrated that the rate and extent of 131-42aggregation and assembly into an insoluble form were greatlyenhanced at the pH range typically found within late endo-somes and secondary lysosomes (8).

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FIG. 4. Radiochemical sequencing of intracellular 125I-Pl-42.Fractions 29-40 of the intracellular peptide from the reverse-phaseseparation in Fig. 3 were pooled and lyophilized, and an aliquot wasused for sequence analysis by automated Edman degradation. (A)w-5I radioactivity released at each cycle. (B) Interpretation of thesequence data, assuming that the peptide is iodinated at Tyr-10 andthat the counts released at cycles 5-9 are due to the population ofN-terminal truncation products. Arrows and corresponding numbersshow distribution of shorter products in the population.

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FIG. 5. Subcellular distribution of internalized #1.42. HF cellswere incubated in binding medium containing 0.3 pg of 1"I-81-4e2(10,000 cpm/ng) and unlabeled peptide (100 pg/ml). After 24 hr. themedium containing the radioiodinated peptide was removed, the celllayer was rinsed with phosphate-buffered saline, and fresh mediumcontaining unlabeled peptide (100 ,ug/ml) was added and allowed toincubate for an additional 24 hr. The cells were homogenized andfractionated on 12.5% Percoll gradients (0.25 M sucrose). Radioac-tivity (n) and lysosomal marker enzymes acid phosphatase (o) and,B-hexosaminidase (A) were determined for each fiaction. Duplicategradients were loaded with density marker beads and fractionated todetermine density (shown above the graph, in g/ml).

Intracellular accumulation did not appear to be due to ageneralized inhibition of lysosomal proteolysis at high con-centrations of (81-42. High concentrations of unlabeled (81-42

FIG. 6. Immunofluorescence microscopy of HF cells incubatedwith unlabeled #91-42 (100 pg/ml). Cells were immunostained andviewed under either phase-contrast (A, C, and E) or fluorescence (B,D, and F) illumination. (A and B) f1-42-treated HF cells stained withaffinity-purified anti-P#k42 rabbit Igp. (x400.) (CandD) P1-42-treatedHF control cells stained without the primary antibody. (x130.) (Eand F) Control HF cells incubated in the absence of P1-42 peptide andstained with anti-#1_42 antibody. (x130.)

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Table 1. Effect of unlabeled A4/3 peptides on the intracellularaccumulation of 1251-labeled peptides

Unlabeled % of total radioactivity internalizedpeptide l25I-Pl_42 125I.pl_39 125i s

None 0.100 + 0.038 0.046 ± 0.002 0.018 ± 0.00181-42 1.12 ± 0.134 0.548 + 0.036 0.035 ± 0.001P1-39 0.082 ± 0.006 0.050 ± 0.002 NDpi-2 0.078 ± 0.004 ND 0.022 ± 0.002HF cells were incubated for 6 hr with unlabeled peptides (200

Ag/ml) plus radioiodinated peptide (0.50 Ag, 40,000-45,000 cpm perng). ND, not done.

did not significantly inhibit the degradation of low concen-trations of radioiodinated Pf-2 (Table 1). Conversely, highconcentrations ofthe nonaggregating peptides (1-28 and 13-39failed to stimulate the accumulation of low concentrations ofradioiodinated 131-42. However, high concentrations of unla-beled 131-42 did cause a significant increase in the amount of125I-,81-39 that accumulated intracellularly. Although the ex-planation for this is not yet clear, preliminary experimentsindicate that the presence of 131-42 can significantly increasethe extent of assembly of 1-39 into insoluble amyloid-likefilaments at pH 7.4.

DISCUSSIONAlthough 131-42 appears to accumulate in an intracellularcompartment with a similar density and cytoplasmic distri-bution as late endosomes and secondary lysosomes, themechanism for its accumulation is not clear. The high mo-lecular mass, aggregated forms of 81-42 may be intrinsicallyresistant to lysosomal proteolysis, but this remains to beestablished. The resistance to degradation could also beexplained by a failure to transport the 1P-42 peptide containedwithin late endosomes to secondary lysosomes. At lowconcentrations, which favor the nonaggregated state, thepeptide does not accumulate. The 1-28 and 11-39 analogs,which do not form SDS-resistant high molecular weightaggregates, also fail to accumulate even though they areinternalized at rates comparable to that of 131-42. The shorterpeptides may be degraded, which is the fate of most proteinsinternalized by pinocytosis, but we cannot rule out thepossibility that they may be released intact by exocytosis. Itis also conceivable that 13-42 might accumulate in a trypsin-resistant form in an extracellular location. However, thisinterpretation (i) cannot readily account for the observationthat trypsin-resistant peptide does not accumulate at 4°C (atemperature that prevents endocytosis) and (ii) does notexplain the subcellular distribution of the peptide deposits.

Intracellular accumulation of the A4/,1 protein and itsresistance to degradation may be relevant to amyloid depo-sition in vivo. Previous work has implicated secondary ly-sosomes or late endosomes as sites of accumulation of APP,"amyloidogenic" fragments of APP, and the A4/,1 protein(19-22). Lysosomal enzymes have also been localized withinextracellular senile-plaque cores (23, 24). Together with thisprevious work, our results suggest that the A4/13 protein maybe produced by late endosomal or lysosomal proteolysis ofthe intact APP, or a truncated "amyloidogenic" form ofAPPcontaining the entire A4/,8 protein, to the protease-resistantA4/13 protein. Abnormal processing ofAPP at a site preceding

the A4/,1 peptide (21, 22) or improper folding of APP mayinitiate events resulting in the aggregation ofthe A4/,1 peptideand its sorting to secondary lysosomes. Thus, production ofthe A4/13 protein in Alzheimer disease may be due to theaggregation of abnormally processed or incorrectly foldedAPP and its degradation to the protease-resistant A4/,1 pep-tide within the lysosome. The intracellular accumulation ofthe A4/,8 protein may be directly related to the inability ofneuronal cells to degrade the aggregated A4/13 protein overthe normal human life-span, thereby contributing to thecellular pathology of Alzheimer disease.

We thank Dr. Carl Cotman and Mr. Fritz Bieth for helpfuldiscussions and suggestions. This work was supported by grantsfrom the National Institutes of Health (AG07918 and HD21379).

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Biochemistry: Knauer et al.

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