Abstract
Post-translational modifications have emerged as a key biomolecular process in the onset of neurodegenerative disorders, such as the hyperphosphorylation of tau protein in Alzheimer's disease (AD). High levels of phosphorylation are related to tau malfunction, self-assembly into amyloids forming neurofibrillary tangles in the brain, and cellular toxicity. These molecular processes are also reflected in human biofluids, allowing us to use tau phosphorylation as a biomarker of the disease onset and progression. However, it is not yet clear what structural changes the tau protein undergoes upon phosphorylation and what the early self-assembly steps are that lead to the formation of the final amyloid species. This knowledge gap is related in large part to the experimental challenge in achieving a multiparametric physical-chemical characterization of nanoscale size and heterogeneous amyloid at the single-molecule level. Here, we employ high-resolution and advanced atomic force microscopy methods to study the effect of phosphorylation on the tau pathway of self-assembly and on the physical-chemical properties of the heterogeneous amyloid species formed, down to the single-oligomer level. We report the correlative analysis of single-oligomer structural and chemical properties and achieve, for the first time, the quantitative determination of their phosphorylation state. Our findings reveal that hyperphosphorylation results in the formation of smaller, stiffer, and more adhesive oligomers, which might be critical for their pathological role in AD. This nanoresolved information might be, in turn, useful to understand the early molecular mechanisms of disease, as well as to improve the detection of pathological tau species in biofluids as diagnostic biomarkers.