Tau-proximity ligation assay reveals extensive previously undetected pathology prior to neurofibrillary tangles in preclinical Alzheimer’s disease

Abstract

Background Multimerization is a key process in prion-like disorders such as Alzheimer’s disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau–tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers. Methods Here, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-proximity ligation assay (tau-PLA). We then analyzed MAPT KO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold. Results Our novel tau-proximity ligation assay (tau-PLA) directly visualized tau–tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal in MAPT KO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large lesions. Conclusions Tau-PLA is the first method to directly visualize tau multimers both in vitro and in situ with high specificity. We find that tau multimerization appears extensively from the earliest presymptomatic Braak stages as a previously unreported type of diffuse pathology. Importantly, in our study multimerization is the earliest detectable molecular event of AD tau pathology. Our findings open a new window to the study of early tau pathology, with potential implications in early diagnosis and the design of therapeutic strategies.

Tau aggregation and its relation to selected forms of neuronal cell death

“…Indeed it has been hypothesised from a kinetic analysis that the formation of sarkosyl-insoluble tau species must also include a fragmentation process [ 23 ], which could give rise to secondary oligomers. A recent study using a tau-proximity ligation assay has suggested that tau–tau interactions occur before detection of formation of tau aggregates in AD (at Braak stage I/II ), with positive structures occuring in both the neuropile and in neuronal cell bodies [ 24 ] but the sizes of the multimers and their purity remains to be determined.…”

SETD7-mediated monomethylation is enriched on soluble Tau in Alzheimer’s disease

“…While our antibodies were not suited for immunofluorescence applications, we took advantage of an antibody recognizing monomethylated lysines (meK) and combined it with a total Tau antibody in a proximity ligation assay (PLA) in hiPSC-derived neurons. The PLA method relies on the spatial proximity of two antibodies, and can be used to detect PTMs, protein-protein interactions as well as protein oligomerization [ 39 ]. With the antibody combination meK and Tau 12, we observed a predominantly somatic and nuclear distribution of the PLA signal (Fig.…”

Role of Environmental Toxicants on Neurodegenerative Disorders

“…In Parkinson’s disease, insoluble, misfolded proteins aggregate to form Lewy bodies, which are abnormal protein aggregations that develop inside the nerve cells severely affected by Parkinson’s disease ( Breydo et al, 2012 ). While neurofibrillary tangles (NFTs) are hyperphosphorylated tau protein aggregates that are widely recognized as a basic biomarker of Alzheimer’s disease ( Bengoa-Vergniory et al, 2021 ). Pesticide exposure can be minimized in school premises, workplaces, and residences by changing dietary habits and using sustainable pest management strategies ( McBurney, 2017 ).…”