The progressive neurodegeneration of Alzheimer's disease has been hypothesized to be mediated, at least in part, by beta-amyloid protein. A relationship between the aggregation state of beta-amyloid protein and its ability to promote degeneration in vitro has been previously suggested. To evaluate this hypothesis and to define a structure-activity relationship for beta-amyloid, aggregation properties of an overlapping series of synthetic beta-amyloid peptides (beta APs) were investigated and compared with beta AP neurotoxic properties in vitro. Using light microscopy, electrophoresis, and ultracentrifugation assays, we found that few beta APs assembled into aggregates immediately after solubilization, but that over time peptides containing the highly hydrophobic beta 29-35 region formed stable aggregations. In short-term neuronal cultures, toxicity was associated specifically with those beta APs that also exhibited significant aggregation. Further, upon the partial reversal of beta 1-42 aggregation, a concomitant loss of toxicity was observed. A synthetic peptide derived from a different amyloidogenic protein, islet amyloid polypeptide, exhibited aggregation but not toxicity, suggesting that beta AP-induced neurotoxicity in vitro is not a nonspecific reaction to aggregated protein. The correlation between beta AP aggregation and neurotoxicity was also observed in long-term neuronal cultures but not in astrocyte cultures. These data are consistent with the hypothesis that beta-amyloid protein contributes to neurodegeneration in Alzheimer's disease.

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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 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, 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), FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG-GVVIA) 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 ...

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