Exploring the multimeric tau interactome and its convergence with human tauopathy

Background Neurofibrillary tangles (NFTs) comprised of the phosphorylated Tau protein are a core pathological feature of Alzheimer’s disease (AD) and several other neurodegenerative diseases collectively termed tauopathies include progressive supranuclear palsy, corticobasal degeneration, and subtypes of frontotemporal dementia. Physiological Tau is involved in many processes essential for cell function via protein‐protein interactions. However, under pathological conditions, the gain or loss of Tau protein interactions is an important consequence of disease pathophysiology and may result in the ‘rewiring’ of Tau protein‐protein interaction (PPI) networks. Therefore, modeling and identifying Tau interacting partners could reveal signaling mechanisms and therapeutic targets in tauopathies. Method To gain an unbiased understanding of the aggregated Tau interactors, we generated a split Turbo Tau (sTurbo Tau) proximity labeling system. We chose to express the isolated Tau repeat domain (RD) containing a pro‐aggregation P301L familial tauopathy substitution. Immunocytochemistry (ICC) and Western blot (WB) were performed in sTurbo Tau expressing cells following biotin labeling. Biotinylated proteins were affinity purified and analyzed in triplicate via mass spectrometry (MS) to identify these Tau interacting partners. Differential expression and Gene Ontology (GO) analyses were performed. We overlapped the sTurbo Tau interactors in cell culture and Tau interacting partners in AD brain lysates to further validate our model. Result ICC displayed speckle‐like Tau‐positive aggregates in the cytoplasm and nucleus that colocalized with biotin. WB with streptavidin dye also revealed high and low molecular weight biotinylated proteins in sTurbo Tau cells. MS identified nearly 1,900 proteins enriched in sTurbo Tau lysates compared to controls. GO analysis of these putative Tau interacting proteins confirmed various pathways, including cellular structural components, RNA binding, and translation. This overlap revealed 176 shared partners with roles in translation, synaptic processing, RNA‐binding, and proteasome function. Candidate spliceosome proteins, U1‐70K, U1A, and SNRPD2, have previously been shown to aggregate and colocalize with Tau in AD. Additionally, HSP90 family members were enriched in the overlapped datasets and are the target of mechanistic studies. Conclusion Collectively, we established a cell‐based Tau proximity labeling approach to identify Tau co‐aggr