Gold nanoparticle is becoming a promising platform for the delivery of drugs to treat neurodegenerative diseases. Parkinson’s disease, associated with the aggregation of α-synuclein, is a condition that results in...

Aggregates of amyloid-β (Aβ) are characteristic of Alzheimer's disease, but there is no consensus as to either the nature of the toxic molecular complex or the mechanism by which toxic aggregates are produced. We report on a novel feature of amyloid-lipid interactions where discontinuities in the lipid continuum can serve as catalytic centers for a previously unseen microscale aggregation phenomenon. We show that specific lipid membrane conditions rapidly produce long contours of lipidbound peptide, even at sub-physiological concentrations of Aβ. Using single molecule fluorescence, time-lapse TIRF microscopy and AFM imaging we characterize this phenomenon and identify some exceptional properties of the aggregation pathway which make it a likely contributor to early oligomer and fibril formation, and thus a potential critical mechanism in the etiology of AD. We infer that these amyloidogenic events occur only at areas of high membrane curvature, which suggests a range of possible mechanisms by which accumulated physiological changes may lead to their inception. The speed of the formation is in hours to days, even at 1 nM peptide concentrations. Lipid features of this type may act like an assembly line for monomeric and small oligomeric subunits of Aβ to increase their aggregation states. We conclude that under lipid environmental conditions, where catalytic centers of the observed type are common, key pathological features of AD may arise on a very short timescale under physiological concentration. A widely adopted framework to understand the biochemical underpinnings of AD is based on the amyloid cascade hypothesis, which assumes that aggregation of the Aβ peptide into oligomeric or fibrous structures triggers a cascade of toxic events that cause functional deterioration of the human brain 1. The aggregation of Aβ in cell-free solution is well studied and explained by the primary nucleation of monomers, secondary nucleation of monomers on the fibrils and the elongation of fibrils by monomer addition 2,3. For these molecular events to occur, a critical concentration of monomers of ~90 nM in solution is required 4 , far above the concentration found in vivo. Furthermore, for fibril formation in cell free systems, µM concentration of Aβ is required 5 ; while in the brain, the peptide concentration reaches only up to 4 nM 6. Given these numbers it is unlikely that the currently established in vitro mechanisms can account for the formation of plaques and tangles in the brain of AD patients 7,8. Converse to the aggregation mechanism in the absence of cells, it is well documented that interactions of Aβ with cell membranes can induce Aβ aggregation. Aβ-membrane interactions occur via prion 9 , cholesterol 10 and gangliosides 11 or non-specifically with the lipid membranes themselves 12-20. Previous studies found changes in the lipid composition of the brain in AD patients during disease progression affecting the carbon chain length, linkages, and degree of unsaturation 21. This brings to the idea that phospholipid metab...

Aggregation kinetics of proteins and peptides have been studied extensively due to their significance in many human diseases, including neurodegenerative disorders, and the roles they play in some key physiological processes. However, most of these studies have been performed as bulk measurements using Thioflavin T or other fluorescence turn-on reagents as indicators of fibrillization. Such techniques are highly successful in making inferences about the nucleation and growth mechanism of fibrils, yet cannot directly measure assembly reactions at low protein concentrations which is the case for amyloid-β (Aβ) peptide under physiological conditions. In particular, the evolution from monomer to low-order oligomer in early stages of aggregation cannot be detected. Single-molecule methods allow direct access to such fundamental information. We developed a high-throughput protocol for single-molecule photobleaching experiments using an automated fluorescence microscope. Stepwise photobleaching analysis of the time profiles of individual foci allowed us to determine stoichiometry of protein oligomers and probe protein aggregation kinetics. Furthermore, we investigated the potential application of supervised machine learning with support vector machines (SVMs) as well as multilayer perceptron (MLP) artificial neural networks to classify bleaching traces into stoichiometric categories based on an ensemble of measurable quantities derivable from individual traces. Both SVM and MLP models achieved a comparable accuracy of more than 80% against simulated traces up to 19-mer, although MLP offered considerable speed advantages, thus making it suitable for application to high-throughput experimental data. We used our high-throughput method to study the aggregation of Aβ40 in the presence of metal ions and the aggregation of α-synuclein in the presence of gold nanoparticles.