First-order kinetic model of Alzheimer's β-amyloid fibril extension in vitro

Recently, several studies have proposed models describing the mechanisms of Alzheimer's β-amyloid fibril formation in vitro. However, these models are somewhat controversial and no exact kinetic analyses measuring the polymerization velocity as an indicator of the reaction, have thus far been available. We first formed β-amyloid fibrils from a synthetic peptide, β-amyloid(1-40), and determined the optimum conditions for quantitative fluorometry of these β-amyloid fibrils with thioflavine T. Optimum fluorescence measurements of β-amyloid fibrils were obtained at the excitation and emission wavelengths of 446 and 490 nm, respectively, with the reaction mixture containing 5 μM thioflavine T and 50 mM of glycine-NaOH buffer, pH 8.5. We then focused our study on the extension phase of β-amyloid fibril formation in vitro. When β-amyloid fibrils were incubated with monomeric β-amyloid(1-40) in conditions where de novo seed formation does not occur, the extension of β-amyloid fibrils was observed with electron microscopy. Quantitative fluorometry revealed that : (a) extension of amyloid fibrils proceeded by a pseudo-first-order exponential increase as measured by the fluorescence of thioflavine T ; (b) the rate of extension was maximum around pH 7.5, and was dependent on the incubation temperature. Between 20 and 37° C, good linearity was observed between the common logarithm of the initial rate and the reciprocal of the absolute temperature ; (c) the rate of polymerization was found to be proportional to the product of β-amyloid fibrils number concentration and the β-amyloid(1-40) concentration ; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization. These results show that β-amyloid fibril formation can be explained by a first-order kinetic model : i.e., the extension of β-amyloid fibrils proceeds via the consecutive association of β-amyloid(1-40) onto the ends of existing fibrils.