Amyloid beta aggregation kinetics : The role of intrinsic and extrinsic factors.

Cerebral senile plaque is one of the main pathologies of Alzheimer's disease (AD). The amyloid cascade hypothesis suggests that the aggregation of amyloid beta (Abeta) peptide is involved in the pathogenesis of AD, which is supported by the fact that Abeta overexpression or production of more aggregation-prone variants lead to early-onset dementia. In this work, we mainly studied the in vitro Abeta aggregation kinetics, to investigate the mechanistic shift as a result of intrinsic factors and extrinsic factors. We employ a fluorescence probe Thioflavin T (ThT) to follow the aggregation kinetics. The aggregation half-time is then extracted and plotted against monomer concentration. By fitting the curve with a power function, a scaling exponent is extracted and reflects the aggregation monomer dependence. The ThT data can be globally fitted using master equations to determine the dominant aggregation reaction step at the microscopic level. Circular dichroism spectroscopy and cryogenic transmission electron microscopy are used to study the fibril structure transition and morphology. Surface plasmon resonance and mass spectrometry provide information of molecular interaction and the latter one is also used to identify peptide segments of the soluble and insoluble Abeta species. Our results show a two-step saturated secondary nucleation dominated mechanism in several cases: Abeta mutants E22K, E22Q, E22G, D23N and A2V, which link to an early-onset of AD, Abeta40 aggregation at high monomer concentration (> 30 uM, pH 7.4), and Abeta42 aggregation at high ionic strength (> 92 mM) and at pH 7.4. The mechanistic shift in these cases is mainly attributed to a reduced repulsion between monomers and other aggregating species due to decreased absolute charges from point mutation or pH shifting to a value close to the isoelectric point, or due to increased ionic strength by adding salt. This effect is combined with additional hydrophobic effect or other side chain properties in some cases to reach a more enhanced secondary nucleation. The secondary nucleation that produces enormous amount of toxic oligomers is found to be severely inhibited by a chaperone protein, Brichos, through specifically binding to the fibril surface and blocking the catalytic cycle. In our co-aggregation work, the most abundant Abeta variants, Abeta40 and Abeta42 that differ in length at C-terminus, do not co-aggregate and do not form mixed fibrils. The result implies that Abeta40 and Abeta42 interact exclusively at primary nucleation level and Abeta aggregation is a highly selective process that tolerates a low level of sequence mismatch in C-terminus