Acceleration of Amyloid β-Peptide Aggregation by Physiological Concentrations of Calcium

James P. Shine,David B. Senn,Menglan Yuan,Adrian M. Isaacs,Bruce A. Yankner

doi:10.1074/jbc.m602061200

James P. Shine, David B. Senn

Open Access

https://doi.org/10.1074/jbc.m602061200

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Abstract

Alzheimer disease is characterized by the accumulation of aggregated amyloid beta-peptide (Abeta) in the brain. The physiological mechanisms and factors that predispose to Abeta aggregation and deposition are not well understood. In this report, we show that calcium can predispose to Abeta aggregation and fibril formation. Calcium increased the aggregation of early forming protofibrillar structures and markedly increased conversion of protofibrils to mature amyloid fibrils. This occurred at levels 20-fold below the calcium concentration in the extracellular space of the brain, the site at which amyloid plaque deposition occurs. In the absence of calcium, protofibrils can remain stable in vitro for several days. Using this approach, we directly compared the neurotoxicity of protofibrils and mature amyloid fibrils and demonstrate that both species are inherently toxic to neurons in culture. Thus, calcium may be an important predisposing factor for Abeta aggregation and toxicity. The high extracellular concentration of calcium in the brain, together with impaired intraneuronal calcium regulation in the aging brain and Alzheimer disease, may play an important role in the onset of amyloid-related pathology.

Highlights

Amyloid ␤-peptide (A␤)2 is the primary constituent of amyloid plaques in Alzheimer disease (AD)
We show that calcium can predispose to A␤ aggregation and fibril formation
In this report we show that calcium can accelerate aggregation of A␤1–42, with marked effects on the formation of early protofibrillar structures, as well as the protofibril to fibril conversion event

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Reagents—All cell culture reagents were purchased from Invitrogen, and chemicals were from SigmaAldrich, unless stated otherwise. For experiments in which A␤ was aggregated in the presence of 150 mM NaCl or in Dulbecco’s modified Eagle’s medium (DMEM), A␤ was resuspended at 200 ␮M in reverse osmosis-purified water (MP Biomedicals) and taken to pH 11.5 with 1 N NaOH to solubilize the peptide. 2 mM A␤ in Me2SO was an Optima TLX desktop ultracentrifuge (Beckman Coulter) to diluted to 100 ␮M in 75 mM MOPS pH 7.4 buffer, and metal ions remove pre-existing aggregates. For preformed fibrils and protofibrils that had been added to culture medium for 24 h at 20 ␮M, the medium was centrifuged as above, and the pellet was resuspended at 100 ␮M for use in the. Electron microscopy of early forming A␤ species no spin was performed and samples were diluted 1:1 with reverse osmosispurified water. The grids were examined using a JEOL 1200EX transmission electron microscope

Inductively Coupled Plasma Mass

RESULTS

DISCUSSION