Frozen frontal cortices from autopsied and histopathologicallyconfirmed AD (80 years old, 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−80°C until analysis. The use of postmortem human braintissue was in accordance with the National Institutes of HealthGuidelines and was exempted by the Institutional Review Board 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.”

AD O-tau was isolated from frozen autopsy cerebral cortex of AD patient as described (Kopke et al., 1993). Briefly, the brain tissue was homogenized in ninefold volume of ice-cold lysis buffer containing 20 mM Tris-HCl, pH 8.0, 0.32 M sucrose, 10 mM β-mercaptoethanol, 10 mM glycerophosphate, 5 mM MgSO₄, 50 mM NaF, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), and 10 μg/ml each of aprotinin, leupeptin, and pepstatin. The homogenate was centrifuged at 27,000 × g for 30 min at 4°C. The supernatant was aspirated and further centrifuged at 235,000 × g for 45 min at 4°C. The resulting pellet rich in AD O-tau was collected, washed twice, and resuspended in saline. AD O-Tau was stored at −80°C and probe-sonicated for 2 min (0.5 s on, 3 s off) at 20% power before use.

The supernatant was saved and NaCl was added to a final concentration of 0.75 M. The remaining aggregated tau in the supernatant was removed by boiling for 5 min. After cooling, the samples were centrifuged at 25,000 × g for 30 min. The supernatant was collected, dialyzed against 10 mM Tris-HCl, pH 7.6, and concentrated by five times to obtain HS-tau.

Dp-AD O-tau was obtained by incubating AD O-tau (4 mg/ml protein) with 196 U/ml AP in the reaction buffer (100 mM Tris-HCl, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml pepstatin, and 5 μg/ml leupeptin) at 37°C for 5 h. Dephosphorylated products were analyzed by Western blots developed with Tau-1 antibody to show dephosphorylation efficacy.

Various amounts of AD O-tau were spotted on a nitrocellulosemembrane (Schleicher and Schuell, Keene, NH, United States) at 5 μl/grid of 7 × 7 mm. The membrane was incubated at37°C for 1 h to allow protein binding to the membrane. Proteins on the membrane were dephosphorylated as describedpreviously (Kopke et al., 1993) with slight modification. The membranewas wet by TBS and incubated with 196 U/ml AP in the reaction bufferas described above at 37°C for 5 h. After blocking with 5% milk in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) for 30 min, membrane was then developed with specific antibodies or subjected totau capture assay.

Brain homogenates were prepared in 10% in ice-cold lysis buffer consisting of 50 mM Tris-HCl, pH 7.4, 8.5% sucrose, 2.0 mM EDTA, 10 mM β-mercaptoethanol, 1 mM Na₃VO₄, 50 mM NaF, 1 mM AEBSF, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin. The homogenates were diluted in 2 × Laemmli sample buffer (250 mM Tris-HCl, pH 6.8, 20% glycerol, 20% β-mercaptoethanol, 4% SDS and 0.008% bromophenol blue), followed by boiling for 10 min. Protein concentration of each sample was measured by the Pierce^TM 660 nm Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, United States). Same amounts of protein in brain homogenate were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to a polyvinylidene difluoride (PVDF) membrane.

For dot blots, the cells were lysed with RIPA (radio immunoprecipitation assay) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Non-idet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM Na₃VO₄, 1 mM AEBSF, 5 mM benzamidine, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin) and the protein concentration was determined. Different amounts of protein were spotted on a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, United States) at 5 μl/grid of 7 × 7 mm. The blot was incubated at 37°C for 1 h to allow protein binding to the membrane.

The PVDF or nitrocellulose membrane was blocked with 5% skim milkin TBS buffer (50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 30 minand was subsequently incubated overnight with specific primaryantibodies (Table 1) at room temperature. After washing three times with TBST (TBS containing 0.05% Tween-20), the membrane was incubated with horseradish peroxidase(HRP)-conjugated secondary antibody (1:4,000, JacksonImmunoResearch, West Grove, PA, United States) at room temperaturefor 2 h, washed with TBST, incubated with enhanced chemiluminescence(ECL) kit (Thermo Fisher Scientific, Rockford, IL, United States), and exposed to X-ray films (Denville Science, Holliston, MA, United States). The intensity of bands on Western blots and dotblots was quantified by Multi Gauge software (Fujifilm, Mito, Tokyo, Japan).

HEK-293FT and HeLa cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, United States) at 37°C in a humidified atmosphere of 5% CO₂/95% air.

pCI/HA (hemagglutinin)-tau_1–441 andpCI/HA-tau_151–391 were generated as previouslydescribed (Gu et al., 2020). The DNA sequence of the plasmids wasconfirmed by Sanger sequencing. PCI-neo plasmid was used as anegative control. pCI/HA-tau_1–441 orpCI/HA-tau_151–391 was transfected intoHEK-293FT or HeLa cells by FuGENE^®, HD TransfectionReagent (Promega, Madison, WI, United States) following themanufacturer’s instructions.

HEK-293FT cells were transfected with pCI/HA-tau_1–441 or pCI/HA-tau_151–391. AD O-Tau or Dp-AD O-tau was sonicated at 20% power for 2 min (0.5 s on, 3 s off) before use. Six hours after transfection, AD O-Tau or Dp-AD O-tau was mixed with 3% Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States) in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, United States) for 20 min at room temperature and then added into the cell culture at a final concentration of 6.6 μg/ml. Forty-eight hours after transfection, the cells were harvested and analyzed for tau aggregation by Western blots or immunofluorescence.

To examine the accumulation of insoluble tau, the cells were lysed in RIPA buffer for 20 min on ice and centrifuged at 130,000 × g for 45 min. The supernatants were collected as RIPA-soluble fraction and diluted in 4 × Laemmli sample buffer. The resulting pellets containing the insoluble fraction were washed twice with RIPA buffer and resuspended in 1 × Laemmli buffer by sonicating at 80% power for 8 min (5 s on, 20 s off) with sonicator equipped with cup horn (Thermo Fisher Scientific, Waltham, MA, United States). The samples were boiled for 5 min. Levels of RIPA-insoluble and -soluble tau were determined by Western blots developed with anti-HA antibody (Table 1). Levels of phosphorylated tau at each specific site were analyzed by Western blots developed with site-specific and phosphorylation-dependent tau antibodies.

HeLa cells were transfected with pCI/HA-tau_1–441 orpCI/HA-tau_151–391 by FuGENE^®, HDand treated with 6.6 μg/ml AD O-tau or Dp-ADO-tau as described above. The cells were harvested, fixed for15 min with 4% paraformaldehyde in phosphate-buffered saline (PBS), and treated with 0.3% Triton in PBS for 15 min. After blocking with5% newborn goat serum, 0.05% Tween-20, and 0.1% Triton X-100 inPBS for 30 min, the cells were incubated with 77G7 (1:500) oranti-HA (1:1,000) antibody in the blocking solution overnight at4°C. After washing three times with PBS, the cells wereincubated with Alexa Fluor 555-conjugated or 488-conjugated IgG(1:1,000, Thermo Fisher Scientific, Waltham, MA, United States) for2 h and TO-PRO-3 (5 mg/ml, Thermo Fisher Scientific, Waltham, MA, United States) for 15 min at room temperature. After washing withPBS, the cells were mounted using ProLong^TM GoldAntifade Mountant (Thermo Fisher Scientific, Waltham, MA, United States). The images were captured with a Nikon EZ-C1 confocalmicroscope (Nikon Instruments, Melville, NY, United States).

The number of HA-tau_151–391-expressing cells and the number of cells with tau aggregates were counted under the microscope. The percentage of cells with tau aggregates was calculated by dividing the number of cells with aggregates by the total number of HA-tau_151–391-expressing cells on each slide.

Tau capture assay was carried out as described (Kremer et al., 1988; Alonso et al., 1994; Li et al., 2019). HEK-293FT cells overexpressingpCI/HA-tau_151–391 were lysed in PBS containing50 mM NaF, 1 mM Na₃VO₄, 1 mM AEBSF, 5 mM benzamidine, and 10 μg/ml each of aprotinin, leuprotinin, and pepstatin. The cell lysate was sonicated at 20% power for 2 min (0.5 s on, 3 soff) and centrifuged at 10,000 × g for 10 min toextract the supernatant containingHA-tau_151–391. Different amounts of ADO-tau or HS-tau were spotted on the nitrocellulose membraneand dried at 37°C for 1 h. The membrane was then blockedwith 5% skim milk in TBS for 1 h, and incubated overnight with thecell extract containing HA-tau_151–391. Afterwashing three times with TBST, the membrane was examined by anti-HAantibody using dot blot method.

Data were presented as mean ± standard deviation (SD). The statistical significance was analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, United States). P < 0.05 was considered statistically significant.

Truncation and hyperphosphorylation of tau are commonly found in ADbrains (Zhou et al., 2018; Li et al., 2019). Brain-derived tau oligomersfrom the individuals with AD and related tauopathies can capturenormal tau and trigger the propagation of tau pathology as cytotoxicseeds (Hu et al., 2016; Chu and Liu, 2018). To determine whetheroligomeric tau from AD brain is also hyperphosphorylated and/ortruncated, AD O-tau was analyzed by Western blots developedwith antibodies raised against specific epitopes of tau (Figure 1A and Table 1). R134d, a polyclonal antibodyraised against the longest human tau isoform, tau441 (Tatebayashi et al., 1999), detected smear bands in AD O-tau and ADbrain lysate but not in control brain lysates, confirming thetruncation and SDS- and β-mercaptoethanol-resistanthigh-molecular weight of AD O-tau (Figure 1B). Monoclonal antibodies 43D andHT7, which recognize the N-terminal 6–18 a.a. and 159–163 a.a. of tau441, respectively, detected tau bands in both control andAD brain lysates, but not in AD O-tau. Similar results werefound when using the polyclonal antibody 113e (Li et al., 2019) thatrecognizes 19–32 a.a. of tau, indicating that AD O-tau wasmostly N-terminal truncated (Figure 1B). 77G7 (244–368 a.a.) and Tau5 (210–230 a.a.) that target the epitopes in or close to themicrotubule binding repeats of tau both showed stronger affinity toAD O-tau than to AD or control brain lysates. The level oftau detected by Tau46 (404–421 a.a.) was decreased in ADO-tau, implying that AD O-tau could be partiallyC-terminal truncated. These results suggested that truncatedtau species were accumulated in AD O-tau.

Tau is abnormally hyperphosphorylated in AD brain (Zhou et al., 2018). Therefore, we used a series of phosphorylation site-specific antibodies of tau to examine the phosphorylation pattern of AD O-tau (Figure 1C). Phosphorylation of all the epitopes tested, pS199, pT217, pS262, pS396/404, and pS422, were increased in AD compared to control lysate, consistent with previous reports (Zhou et al., 2018). However, the changes of site-specific phosphorylation in AD O-tau were not the same as that in AD lysate. In AD O-tau, pS199 phosphorylation was distinctly eliminated, which might be because of tau cleavage at the N-terminus. pT217 showed comparable phosphorylation levels in AD O-tau and AD lysate. Phosphorylation of pS262, pS396/404, and pS422 was dramatically elevated in AD O-tau than in AD and control lysate. The site-selective phosphorylation of AD O-tau implied that C-terminal hyperphosphorylated tau species were prone to aggregate in AD O-tau.

Recently, we constructed 11 truncated forms offull-length tau and analyzed the pathological activities of them(Gu et al., 2020). We found that deletion of the first 150 or230 a.a. and the last 50 a.a. of tau enhanced its site-specificphosphorylation and seeded aggregation by AD O-Tau. Amongthese truncated proteins, tau_151–391 exhibitedthe highest pathological activities but did not show significantdifference in cytotoxicity compared with full-length tau(tau_1–441) (Gu et al., 2020). In the present study, HeLa cells were transfected with HA-tau_1–441 orHA-tau_151–391. Six hours later, the cells weretreated with AD O-tau for 42 h and then subjected toimmunofluorescence. The immunofluorescence staining with 77G7 antibodies revealed that both HA-tau_1–441 andHA-tau_151–391 were expressed in HeLa cells. ADO-tau induced remarkable aggregates in the cytoplasm andnucleus of HA-tau_151–391-expressing cells, butonly diffusion distribution of tau inHA-tau_1–441-transfectedcells (Figure 2A).

For biochemical analyses, HEK-293FT cells were transfected with HA-tau_1–441 or HA-tau_151–391, treated with AD O-tau for 42 h and then lysed with RIPA buffer and centrifuged at 130,000 × g for 45 min to separate RIPA-insoluble pellets and RIPA-soluble supernatants. Serial dilutions of the RIPA-soluble or -insoluble samples were spotted on nitrocellulose membranes and subjected to dot blots using anti-HA antibody (Figure 2B). The results showed higher level of RIPA-soluble tau in HEK-293FT/HA-tau_1–441 cells than in HEK-293FT/HA-tau_151–391 cells. O-tau treatment induced intensive insoluble aggregates of HA-tau_151–391, but not that of HA-tau_1–441. Next, RIPA-soluble or -insoluble fraction from HA-tau_1–441- or HA-tau_151–391-expressing cells treated with or without AD O-tau were examined by Western blots developed with tau antibodies (Figure 2C). AD O-tau induced dramatic accumulation of tau_151–391 in RIPA-insoluble fraction, whereas less tau_151–391 in RIPA-soluble fraction, compared with tau_1–441. These results further support that tau_151–391 could be more susceptible to AD O-tau seeded aggregation. Thus, HA-tau_151–391, instead of HA-tau_1–441, was used in the following analyses for tau seeding activity.

Normal tau is heat-stable (Weingarten et al., 1975). Heat treatment iscommonly used to remove tau aggregates from the soluble fraction ofbrain lysate (Planel et al., 2007; Miao et al., 2019). Various amounts of ADO-tau or heat-stable tau (HS-tau) were dotted onnitrocellulose membranes, which were further incubated with celllysate containing HA-tau_151–391 to allow thecapture of HA-tau_151–391 by AD O-tau orHS-tau. Captured HA-tau_151–391 was detected byanti-HA antibody (Figure 2D). The resultsindicated that AD O-tau, but not HS-tau, capturedHA-tau_151–391 (Figure 2D).

To determine the role of phosphorylation in modulating the captureability of AD O-tau, we applied various amount of ADO-tau onto nitrocellulose membranes parallelly. One set ofmembranes was treated with AP to dephosphorylate AD O-tau onit. The control membranes were treated with the reaction bufferparallelly. Dephosphorylation of AD O-tau by AP wasidentified by reduced PHF-1 (recognizing phosphorylated tau atSer396/404) and enhanced Tau-1 (recognizing tau unphosphorylatedSer195/198/199/202) immunoreactivity(Figures 3A,B). Similar R134d(Tatebayashi et al., 1999) and 92e (Grundke-Iqbal et al., 1988) immunoreactivity indicatedthat AP treatment did not affect total tau levels on the membrane(Figure 3A). The control and AP-treatedmembranes were further incubated with cell lysate containingHA-tau_151–391 to allow the binding ofHA-tau_151–391 to AD O-tau. CapturedHA-tau_151–391 developed with anti-HA wasmarkedly decreased in AP-treated membrane, suggesting thatdephosphorylation of AD O-tau inhibited its ability tocapture tau (Figures 3C,D).

To assess the ability of dephosphorylated AD O-tau (Dp-ADO-tau) in triggering tau aggregation, we used AP todephosphorylate AD O-tau and added Dp-AD O-tau intoHeLa cells expressing HA-tau_151–391 toinvestigate the formation of aggregates. Western blots developedwith Tau-1 antibody (Binder et al., 1985) confirmed the up-regulation ofdephosphorylation levels in Dp-AD O-tau after AP treatment(Figure 4A). Overexpression ofHA-tau_151–391 in HeLa cells usually leads toaggregation in less than 10% of the transfected cells (HA positive) after 48 h transfection when examined by immunofluorescence(Figure 4C). Incubation with AD O-taufor 42 h significantly increased the proportion of aggregate-bearingcells (Figures 4B,C; Li et al., 2019; Gu et al., 2020). Unlike AD O-tau, Dp-AD O-tau did notpromote the formation of tau aggregates(Figures 4B,C). In another set ofexperiments, 0.26 U AP was added into the culture medium ofHA-tau_151–391-expressing cells together with ADO-tau and also inhibited AD O-tau-induced tauaggregation (Figures 4B,C). These resultssuggested that dephosphorylation of AD O-tau and/or itsrecruiting targets could help to reduce the formationof tau aggregates.

Next, HEK-293FT cells were transfected with HA-tau_151–391 and treated with AD O-tau or Dp-AD O-tau. The cell lysates were separated into RIPA-soluble and RIPA-insoluble fractions. Total and phosphorylated tau levels in each fraction were analyzed by Western blot (Figure 5). AD O-tau induced dramatic accumulation of total tau (Figures 5A,B) and tau phosphorylated at T181, T212, S214, and T217 (Figures 5C,D) in RIPA-insoluble fractions. However, compared with AD O-tau, Dp-AD O-tau treatment significantly decreased both total and phosphorylated tau deposit in RIPA-insoluble fractions (Figure 5). In both groups, the levels of RIPA-soluble tau were not grossly affected. These data further supported that dephosphorylation of AD O-tau could suppress its ability in templating tau aggregation.