Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7437-7441, August 1992

Biochemistry

Intracellular accumulation and resistance to degradation of the Alzheimer 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 constitutes the major protein component of senile plaques in Alzheimer disease. The A4/13 protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, 181-42, corresponding to one of the longer forms of the A4/13 protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The peptide appears to accumulate intracellularly, since it does not accumulate under conditions that prevent endocytosis and accumulation is correlated with the acquisition of resistance to removal by trypsin digestion. This intracellular accumulation is also correlated with the ability of the peptide to aggregate as determined by SDS/polyacrylamide gel electrophoresis. At low concentrations of the 131-42 peptide, which favor the nonaggregated state, no accumulation is observed. Shorter peptide analogs (28 or 39 residues) that are truncated at the C terminus, which lack the ability to aggregate in SDS gels, fail to accumulate. The accumulated intracellular 131-42 peptide is in an aggregated state and is contained in a dense organellar compartment that overlaps the distribution of late endosomes or secondary lysosomes. Immunofluorescence of the internalized peptide in permeabilized cells reveals that it is contained in granular deposits, consistent with localization in late endosomes or secondary lysosomes. Sequence analysis indicates that some of the internalized peptide is subject to N-terminal trimming. These results suggest that the aggregated A4/13 protein may be resistant to degradation and suggest that the A4/1B protein may arise, at least in part, by endosomal or lysosomal processing of APP. Our results also suggest that relatively nonspecific proteolysis may be sufficient to generate the A4/13 protein if this part of APP is selectively resistant to proteolysis.

7). The fact that normal processing precludes A4/13 accumulation suggests that abnormal processing may be the initial step leading to amyloid deposition. Using synthetic peptide analogs of the A4/(3 protein, we have defined some of its intrinsic biochemical and physical properties that are related to its assembly into amyloid-like fibrils and its ability to aggregate (8). We found that assembly into amyloid-like fibrils and aggregation in SDS/polyacrylamide gels are separate and distinct properties of the amyloid peptides. Low pH (pH 3.5-6.5) and high concentrations of peptide are important for promoting assembly of the peptides into amyloid-like fibrils (8). The length of the hydrophobic C terminus (-42 residues) and a high concentration of peptide are critical for the ability of the (31 2 peptide to self-aggregate into multiple discrete bands in SDS/polyacrylamide gels. These intrinsic factors may be important for amyloid deposition in vivo because the acid environment of endosomes and lysosomes and their ability to concentrate solutes would promote amyloid fibril formation and peptide aggregation, which could compromise the ability of the cell to degrade the A4/13 protein. To explore whether cells are able to degrade the A4//3 protein, we examined uptake and degradation of three peptides that corresponded to residues 1-28 (extracellular portion) (p13-28), 1-39 [predominant form of the A4/,8 protein in cerebrovascular amyloid from HCHWA (hereditary cerebral hemorrhage with amyloidosis) Dutch-type patients (9)] (p1-39), and 1-42 [a major form of the A4/,8 protein in senile plaque (10)] (131-42) of the A4/,8 protein in human neonatal foreskin fibroblast (HF cell) cultures. Here we report that the 42-residue senile-plaque form selectively accumulates intracellularly and is largely resistant to degradation for at least 3 days.

Alzheimer disease is a specific form of neuronal degeneration and consequent dementia characterized by the accumulation of intraneuronal neurofibrillary tangles and extracellular amyloid deposits. Amyloid occurs as diffuse aggregates or more dense deposits, which are termed senile plaques. Deposits surrounding brain microvessels also occur, where they are termed cerebrovascular amyloid. The major protein component of the senile plaque is a 42- or 43-amino acid peptide, known as the 13 protein (1) or A4 peptide (2), which is extremely insoluble and has a strong tendency to aggregate. The A4/13 protein is derived from a larger transmembrane protein, the amyloid precursor protein (APP), which is expressed as multiple different mRNA splicing products (3-5). Accumulation of the A4/1B protein is apparently the result of abnormal processing, since the extracellular domain of APP is normally cleaved at residue 16 within the A4/13 region (6,

FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) were synthesized by fluoren-9-ylmethoxycarbonyl chemistry using a continuous-flow semiautomatic instrument, purified by reverse-phase HPLC, and characterized by sequencing and electrospray mass spectrometry (8). Na1251 was obtained from Amersham; chloroglycouracil (lodo-Gen) from Pierce, Bio-Gel P-2 from Bio-Rad, fetal bovine serum from GIBCO, Dulbecco's modified Eagle's medium (DMEM) from Irvine Scientific, and Percoll from Pharmacia. Analytical-grade solvents and other reagents were from various commercial sources (Sigma; Fisher Scientific). Peptide Iodination. Aliquots (10 pg) of each peptide in 40 jul of 1 M Tris (pH 7.4) were radioiodinated to a specific activity of 50,000-150,000 cpm per ng in the presence of 50 u.g of Iodo-Gen at 0C for 20 min (8). Free iodine was separated from peptide by elution over a 5-ml Bio-Gel P-2 column

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

MATERIALS AND METHODS Materials. Peptides 131-28,

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Biochemistry: Knauer et al.

equilibrated in 0.1 M Tris (pH 7.4) with polyvinylpyrrolidone (Mr 40,000) at 2 mg/ml. Peptide was eluted in the void volume and these fractions were pooled, frozen, and lyophilized overnight. Specific activity was determined by trichloroacetic acid precipitation of an aliquot of the radioiodinated peptide sample prior to elution. Cell Culture. When HF cells reached confluence (4-8 days), they were passaged at a 1:4 dilution in DMEM with 10 mM 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) or 35-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 mM Hepes/2% bovine serum albumin), and then returned to incubation in binding medium containing the indicated concentrations of 125I-labeled peptide and unlabeled peptide. At the indicated times, aliquots of medium from each culture dish were frozen for later TLC analysis, and the cells were rinsed with ice-cold phosphate-buffered saline and treated with trypsin (2.5 mg/ml) at 0C for 15 min. Cells were pelleted by microcentrifugation and radioactivity in the trypsin supernatant was quantitated in a Beckman Gamma 5500 y counter. The cell pellet was resuspended in phosphatebuffered saline and the radioactivity was quantitated as above. Each data point is the mean of three independent determinations. Cell pellets were frozen for subsequent analysis by tricine SDS/PAGE (11). Cell surface adsorption was measured in parallel cultures incubated at 4°C. lodotyrosine was quantitated by y counting following TLC separation of intact peptide in a 1-butanol/acetic acid/water (100:10:10, vol/vol) solvent system (12). Characterization of Internalized P1-42. The subcellular distribution of internalized 3142 and the lysosomal markers hexosaminidase and acid phosphatase was determined after fractionation of cell homogenates on 12.5% Percoll gradients (13-15). The internalized 125SI438142 was extracted from trypsin-treated cell pellets by addition of 88% formic acid following freezing and thawing. The solution was centrifuged at 14,000 x g for 10 min. The supernatant was dried, suspended in water containing 0.1% trifluoroacetic acid and 1 mg of unlabeled P142 as a carrier, and fractionated by reversephase HPLC (8). An aliquot (8000 cpm) of the purified internalized peptide was subjected to automated Edman degradation and the sequence products were quantified by y counting. For immunolocalization, cells that had been incubated with (31-42 (100 jkg/ml) for 24 hr were trypsin-treated, plated on coverslips, incubated in the absence of peptide for 24 hr, and fixed in cold methanol (0°C, 10 min). After fixation, the coverslips were treated with formic acid (88%, 5 min), washed, and incubated with affiity-purified rabbit anti-P3l12 IgG (2 pg, 12 hr). The coverslips were washed and incubated with fluorescein-conjugated goat anti-rabbit IgG and fluorescence micrographs were taken with an Olympus epifluorescence microscope using x40 and x20 objectives.

RESULTS We examined the uptake and degradation of A4/( protein analogs in HF cells because the endosomal and lysosomal pathways are ubiquitous and they have been well characterized in HF cells (16-18). In addition, human skin fibroblast cultures are available from Alzheimer disease patients. All three peptides adsorbed to the cell surface at 4°C (Fig. 1A). No evidence was obtained for the existence of a specific receptor for these peptides in HF cells. The cell surfaceadsorbed peptide was effectively removed (>95%) by trypsin digestion (Fig. 1A). Upon incubation at 370C, a significant amount of (1-42 accumulated that was resistant to removal by

Proc. NatL Acad Sci. USA 89 (1992)

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FIG. 1. Intracellular accumulation and stability of internalized A4,B peptides. (A) Adsorption of peptides to the cell layer at 40C. HF cells (500,000 per 35-mm plate) were rinsed with 1 ml of binding medium and then incubated for 2 hr at 4C in 1 ml of medium containing 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 the corresponding 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 symbols) trypsin treatment are shown. No significant decrease in cellassociated counts is observed over the range of 10-150 Mg of unlabeled peptide added. Trypsin treatment removed >95% of the cell-associated peptide. (B) Concentration dependence of the accumulation of 1-42. Cells were incubated for 6 hr at 370C in 1 ml of medium 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 the corresponding unlabeled peptide. After incubation, the cells were washed and trypsin-treated and the amount ofcell-associated peptide was determined. At high concentrations of peptide (100-200 pg/ml) significant amounts of trypsin-resistant 11-42 remained associated with 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 radioiodinated 13142 (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 various times after the initial incubation with radiolabeled peptide, cells were removed from incubation and intracellular peptide was determined.

Proc. Nat. Acad. Sci. USA 89 (1992)

Biochemistry: Knauer et al. trypsin (Fig. 1B). This accumulated peptide appeared to represent internalized material, since it was resistant to trypsin under conditions where the surface-adsorbed peptide was sensitive. The amount of trypsin-resistant, internalized peptide accumulated after a 24-hr incubation at 100 ug/ml (19.4 pg per cell) was higher than the amount of adsorbed peptide released by trypsin digestion (7.9 pg per cell). The amount of intracellular peptide accumulated depended on the concentration of the peptide in the culture medium. At low concentrations of (1-42 (0.75 ug/ml), HF cells accumulated 22 pg of the peptide accumulated per cell when cultures were incubated with (81-42 at 200 ug/ml, compared to