Quantitative proteomics of tau and Aβ in detergent fractions from Alzheimer's disease brains

The two hallmarks of Alzheimer's disease (AD) are amyloid-β (Aβ) plaques and neurofibrillary tangles marked by phosphorylated tau. Increasing evidence suggests that aggregating Aβ drives tau accumulation, a process that involves synaptic degeneration leading to cognitive impairment. Conversely, there is a realization that non-fibrillar (oligomeric) forms of Aβ mediate toxicity in AD. Fibrillar (filamentous) aggregates of proteins across the spectrum of the primary and secondary tauopathies were the focus of recent structural studies with a filament structure-based nosologic classification, but less emphasis was given to non-filamentous co-aggregates of insoluble proteins in the fractions derived from post-mortem human brains. Here, we revisited sarkosyl-soluble and -insoluble extracts to characterize tau and Aβ species by quantitative targeted mass spectrometric proteomics, biochemical assays, and electron microscopy. AD brain sarkosyl-insoluble pellets were greatly enriched with Aβ₄₂ at almost equimolar levels to N-terminal truncated microtubule-binding region (MTBR) isoforms of tau with multiple site-specific post-translational modifications (PTMs). MTBR R3 and R4 tau peptides were most abundant in the sarkosyl-insoluble materials with a 10-fold higher concentration than N-terminal tau peptides. This indicates that the major proportion of the enriched tau was the aggregation-prone N-terminal and proline-rich region (PRR) of truncated mixed 4R and 3R tau with more 4R than 3R isoforms. High concentration and occupancies of site-specific phosphorylation pT₁₈₁ (~22%) and pT₂₁₇ (~16%) (key biomarkers of AD) along with other PTMs in the PRR and MTBR indicated a regional susceptibility of PTMs in aggregated tau. Immunogold labelling revealed that tau may exist in globular non-filamentous form (N-terminal intact tau) co-localized with Aβ in the sarkosyl-insoluble pellets along with tau filaments (N-truncated MTBR tau). Our results suggest a model that Aβ and tau interact forming globular aggregates, from which filamentous tau and Aβ emerge. These characterizations contribute towards unravelling the sequence of events which lead to end-stage AD changes.