Nevertheless, systemic administration of AP is still proposed as a potential therapeutic intervention against the pathology of AD, since sufficient anti-inflammatory AP in circulation might leave endogenous TNAP to perform its normal functions in the brain (Pike et al., 2015). N-terminal truncated and C-terminalhyperphosphorylated tau species were enriched in AD O-tau. Dephosphorylation of AD O-tau by alkaline phosphatasediminished its activity in capturing tau and ininducing insoluble aggregates in cultured cells. BSc5371 Our resultssuggested that dephosphorylation passivated the seeding activity ofAD O-tau. Inhibition BSc5371 of phosphorylation may be a potentstrategy to prevent the distributing of tau patho3logy. by binding tothe interface between tubulin heterodimers with itsmicrotubule-binding repeats (Kadavath et al., 2015). As a phosphoprotein, tau contains more than 80 residues that can potentially bephosphorylated, and at least 18 of these sites are abnormallyhyperphosphorylated in the brains of Alzheimers patients(Chu and Liu, 2018; Iqbal et al., 2018). Hyperphosphorylated tau detachesfrom microtubules, resulting in microtubule loss in neurons(Austin et al., 2017). Accumulation of intracellular neurofibrillarytangles (NFTs), which mainly consist of hyperphosphorylated andtruncated tau, is correlated directly with the degree of cognitivedecline in AD patients and is considered as one of the predominanthallmarks of AD. The NFT pathology in AD brains initiates in the locus coeruleus and transentorhinal area, and sequentially progresses to the limbic system and further to the isocortex, as explained in the Braak stages (Braak and Braak, 1991). Intrahippocampal injection of tau aggregates isolated from AD patients or produced successfully induced tau hyperphosphorylation and NFT formation at the injection sites and anatomically related regions in rodent brains, showing a similar stereotypical propagation of tau pathology as observed in AD brain (Clavaguera et al., 2009; Boluda et al., 2015; Takeda et al., 2015; Hu et al., 2016; Miao et al., 2019). Emerging evidence suggests that the prion-like seeding activity of pathological tau in AD brain is crucial for its propagation. Due to the induction of molecules with strong unfavorable charges (such as heparin and RNA) or even proteins, the inert tau monomer could switch its conformation to form -sheet structures that are prone to oligomerization (Goedert et al., 1996; Mudher et al., 2017; Wischik et al., 2018). The oligomeric tau aggregates, acting like seeds, capture normal tau proteins and template their conformational switch in a prion-like mechanism, and eventually assembles the paired helical filaments (PHFs) and NFTs in neurons (von Bergen et al., 2005; Lasagna-Reeves et al., 2012b). Oligomeric tau isolated from Alzheimers brain (AD O-tau) has been reported to capture tau protein (Alonso et al., 1996), andreduced the number of NFTs in mouse brain (Hu et al., 2016). However, the mechanism that dephosphorylation inhibits the prion-likeactivity of harmful tau seeds remains unclear. In the present study, we isolated oligomeric tau from AD brains and analyzed AD O-tau by Western blots using antibodies against different epitopes of tau protein. We found that AD O-tau was mainly N-terminal truncated and C-terminal hyperphosphorylated. AP treatment successfully reduced the phosphorylation of AD O-tau. Tau capture assay revealed that compared with AD O-tau, the ability of dephosphorylated AD O-tau (Dp-AD O-tau) to capture free tau is usually decreased. Immunofluorescence showed that Dp-AD O-tau templated less aggregates formation in HeLa cells. Seeded tau aggregation assay in HEK-293FT cells BSc5371 revealed that Dp-AD O-tau induced less accumulation of total and phosphorylated tau in the insoluble fractions from cell lysates. Our results suggested that dephosphorylation could be an effective way to passivate the prion-like seeding activity of AD O-tau. Materials and Methods Human Brain Samples Frozen frontal cortices from autopsied and histopathologicallyconfirmed AD (80 years aged, female, 2.9 h post mortem interval, Harvard Brain Tissue Resource Center McLean Hospital) andage-matched normal human (84 years old, female, 4.25 h post morteminterval, De Nederlandse Hersenbank) brains were obtained withoutidentification of donors. Brain samples were frozen at?80C until analysis. The use of postmortem human braintissue was in accordance with the National Institutes of HealthGuidelines and was exempted by the Institutional Review Table ofNew York State Institute for Basic Research in DevelopmentalDisabilities because the research does not involve intervention orinteraction with the individuals, nor is the informationindividually identifiable. Preparation of AD O-Tau, Dephosphorylated-AD O-Tau (Dp-AD O-Tau), and Heat-Stable Tau (HS-Tau) AD O-tau was isolated from frozen autopsy cerebral cortex of AD patient as Mouse monoclonal to CD15 explained (Kopke BSc5371 et al., 1993). Briefly, the brain tissue was homogenized in ninefold volume of ice-cold lysis buffer made up of 20 mM Tris-HCl, pH 8.0, 0.32 M sucrose, 10 mM -mercaptoethanol, 10 mM glycerophosphate, 5 mM MgSO4, 50 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 1 mM.