Sulfated Polysaccharides Promote the Assembly of Amyloid β1–42 Peptide into Stable Fibrils of Reduced Cytotoxicity

Ramona Bravo,Muriel Arimon,Juan José Valle-Delgado,Raquel García,Núria Durany,Susanna Castel,Montserrat Cruz,Salvador Ventura,Xavier Fernàndez-Busquets

doi:10.1074/jbc.m709870200

Abstract

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (Abeta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, Abeta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for Abeta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different Abeta(1-42) species on Ca(2+) homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca(2+) that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca(2+) increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

Highlights

Mulation of A␤ that in turn triggers a transmembrane signal having as ultimate effect the formation of neurofibrillary tangles by the microtubule-associated protein Tau [1,2,3], followed by collapse of the microtubular cytoskeleton
Chondroitin sulfate proteoglycans have been found to be associated with the lesions of AD [37], and heparan and chondroitin sulfate GAGs attenuate the neurotoxic effect of A␤ in primary neuronal cultures [38] and in neuron-like cell lines [39]
During the first stages of aggregation, amyloid ␤ peptide (A␤)1–42 can adopt either a globular or a fibrillar structure depending on the method used to prepare the sample (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Preparation of A␤1–42 and Tau—Chemicals and reagents were purchased from Sigma, except where otherwise indicated. After 6 h, the medium was removed and cells were incubated in Ham’s F-12 containing 15% fetal calf serum for 4 – 6 days. Controls included the incubation of cells in HCl-containing medium, which in the absence of A␤1–42 did not induce any Ca2ϩ response. After incubation for the specified times in the absence or presence of A␤1–42 HCl fibrils, cells were rinsed with PBS and fixed for 15 min with a solution of 3% paraformaldehyde in PBS containing 60 mM saccharose. After 24 h at 37 °C in 5% CO2 atmosphere the medium was substituted by 100 ␮l of peptide-containing solution prepared by dissolving 10 ␮l of concentrated A␤1–42 in 90 ␮l of Ham’s F-12 with 0.5% fetal calf serum, to reach the desired final A␤ concentration. All samples were observed in a JEM-1010 electron microscope (Jeol, Japan) operated at 80 kV

RESULTS

Methods

Peptide into