Early synaptic pathology is associated with small tau aggregates in Alzheimer's disease.

Alzheimer's disease (AD) is phenotypically characterised by progressive memory loss, which has been linked to tau aggregation and synaptic dysfunction. Here we characterised the nanoscopic tau aggregates in individual synaptosomes from AD cases and controls, measuring their number and size using SynPull with direct stochastic optical reconstruction microscopy (dSTORM). A total of 7888 synaptosomes from pre-frontal cortex samples were studied, showing the presence of AT8-positive tau aggregates in a small fraction of synaptosomes (~ 3%) from control brains, reaching ~ 20% by Braak stage 6. These key findings of the intra-synaptic localisation of aggregates and existence of synaptic tau pathology at Braak stage 3-preceding tangle formation in this region, were confirmed using aggregate-specific single-molecule array (SIMOA) with proteinase K digestion, three-dimensional super-resolution microscopy, stimulated emission depletion microscopy (STED), and immunohistochemistry. The aggregates also grew in size with AD progression with an average length of 117 nm at stage 0, 154 nm at stage 3 and 182 nm at stage 6, however they mostly remained non-elongated (circular) with average eccentricity values remaining below 0.8. We then investigated the multi-phosphorylation of synaptic tau aggregates for AT8 and T181 and quantified their co-localisation with phosphatidylserine and CD47, synaptic "eat me" and "don't eat me" signals respectively, along with synaptogyrin-3, which contributes to tau-mediated synaptic dysfunction. T181, phosphatidylserine, and synaptogyrin-3 co-localisation with AT8-positive tau were higher during stage 3 and CD47 was lower, indicating early synaptic pathology is associated with the formation of small tau aggregates, contributing to microglia-driven synaptic loss.