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Characterization of tau prion seeding activity and strains from formaldehyde-fixed tissue

Characterization of tau prion seeding activity and strains from formaldehyde-fixed tissue Tauopathies such as Alzheimer’s disease (AD) feature progressive intraneuronal deposition of aggregated tau protein. The cause is unknown, but in experimental systems trans-cellular propagation of tau pathology resembles prion pathogenesis. Tau aggregate inoculation into mice produces transmissible pathology, and tau forms distinct strains, i.e. conformers that faithfully replicate and create predictable patterns of pathology in vivo. The prion model predicts that tau seed formation will anticipate neurofibrillary tau pathology. To test this idea requires simultaneous assessment of seed titer and immunohistochemistry (IHC) of brain tissue, but it is unknown whether tau seed titer can be determined in formaldehyde-fixed tissue. We have previously created a cellular biosensor system that uses flow cytometry to quantify induced tau aggregation and thus determine seed titer. In unfixed tissue from PS19 tauopathy mice that express 1 N,4R tau (P301S), we have measured tau seeding activity that precedes the first observable histopathology by many months. Additionally, in fresh frozen tissue from human AD subjects at early to mid-neurofibrillary tangle stages (NFT I-IV), we have observed tau seeding activity in cortical regions predicted to lack neurofibrillary pathology. However, we could not directly compare the same regions by IHC and seeding activity in either case. We now describe a protocol to extract and measure tau seeding activity from small volumes (.04 mm ) of formaldehyde-fixed tissue immediately adjacent to that used for IHC. We validated this method with the PS19 transgenic mouse model, and easily observed seeding well before the development of phospho-tau pathology. We also accurately isolated two tau strains, DS9 and DS10, from fixed brain tissues in mice. Finally, we have observed robust seeding activity in fixed AD brain, but not controls. The successful coupling of classical IHC with seeding and strain detection should enable detailed study of banked brain tissue in AD and other tauopathies. Introduction distinct disease pathologies by forming strains. Conse- Tauopathies are diverse neurodegenerative diseases char- quently, we use the term “prion” to describe tau, recog- acterized by the deposition of aggregated tau protein nizing that there is no evidence that tau aggregates and progressive neuronal loss [18]. Each tauopathy has spontaneously transmit disease between individuals. We unique patterns of neuropathology, rates of progression, propose that a prion is best understood as a self- and regional involvement. This variability is reminiscent replicating assembly of defined structure that produces a of distinct prionopathies, which are caused by prion pro- specific biological effect, whether for good or ill. This tein (PrP) strains. Strains are unique aggregate structures definition encompasses an enormous literature on func- that faithfully self-replicate, and produce distinct pat- tional yeast prions and other mammalian proteins that terns of neuropathology [8, 20]. Tau resembles a PrP utilize induced, self-amplifying conformational change to prion in experimental systems, as it mediates transmis- functional ends [23]. sible pathology in cells and animals, and transmits Strong evidence indicates that, like PrP, tau forms self- replicating strains that create unique patterns of path- * Correspondence: marc.diamond@utsouthwestern.edu ology in cell and animal models [2, 7, 16, 22]. According Center for Alzheimer’s and Neurodegenerative Diseases, Peter O’Donnell Jr. to the prion model, uniquely structured tau assemblies Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, form in one brain region, where they escape from USA Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 2 of 12 affected cells. These “seeds” act as functional templates on a B6C3 background, and raised with wild-type (WT) to trigger further protein aggregation following internal- littermates. Mice were provided food and water ad libi- ization by adjacent or synaptically connected cells. tum, and housed under a 12-hour light/dark cycle. All It is still unknown whether this mechanism can ac- animal maintenance and experiments adhered to the ani- count for progressive pathology in humans. A central mal care and use protocols of the University of Texas challenge has been to determine the relationship of tau Southwestern Medical Center, and Washington University prion titer and strain composition to classical neuro- in St. Louis. pathological descriptions of phospho-tau accumulation, which have been the gold standard for disease staging Human tissue and discrimination among tauopathies [1, 15, 17]. Meas- The autopsy brains used for this study were obtained urement of tau seeding activity in brain tissue and deter- from five individuals (1 female, 4 males) in compli- mination of strain composition will facilitate ance with Ulm University ethics committee guidelines characterization of neuropathological specimens, and as well as German federal and state law governing may help answer this question. human tissue usage. Informed written permission was We have previously engineered HEK293T cells to de- obtained from all patients and/or their next of kin. tect tau seeding activity in unfixed tissues [13] and to All cases were neuropathologically staged according isolate and characterize tau prion strains present in to published protocols [3–6, 15]. human brain [22]. In transgenic mouse models, the seed biosensor assay detects the emergence of pathological Isolation of mouse brain tau prions far in advance of frank neuropathology [13]. Animals were anesthetized with isoflurane and perfused In selected fresh frozen brain samples from Alzheimer’s with chilled PBS with 0.03% heparin. Whole-brains were disease (AD) patients, these methods indicate that seed- drop-fixed in 4% paraformaldehyde (PFA) in PBS over- ing activity might predict the accumulation of phospho- night at 4 °C. For time course samples, brains were first tau in characteristic neurofibrillary tangles (NFTs) [11]. bisected; the left hemispheres were drop-fixed in 4% However, because it has been impossible to directly PFA, while right hemispheres were stored as fresh frozen compare adjacent, thin sections of brain, we have been tissue at−80 °C until use. unable to use the biosensor assay to simultaneously compare tau pathology with microscopy, seeding activ- Immunohistochemistry of mouse tissue ity, or strain composition. We now describe assessment A freezing microtome was used to collect 50 μm free- of tau seeding activity and strain composition in small floating coronal sections from fixed mouse brains or amounts of fixed brain tissue from mice and humans. 50 μm sections from fresh frozen tissue. For immunohis- tochemistry (IHC) analysis of tau pathology in mice, sec- Methods tions were incubated with biotinylated AT8 antibody Cell culture and cell lines (1:500, Thermo Fisher Scientific) overnight at 4 °C. Sec- Seeding assay experiments were performed with a previ- tions were washed and incubated with VECTASTAIN ously published biosensor cell line that expresses a fusion Elite ABC Kit (Vector Labs) prepared in TBS for 30 mi- between 4R tau repeat domain (RD) containing the nutes at room temperature. Samples were developed disease-associated P301S mutation, and cyan or yellow with 3’3-diaminobenzidine using the DAB Peroxidase fluorescent proteins (tauRD(P301S)-CFP/YFP). These cells Substrate Kit with optional nickel enhancement (Vector are freely available (ATCC CRL-3275) [13]. LM1, DS1, Labs). Sections were imaged using an Olympus DS9, and DS10 are monoclonal HEK cell lines that were Nanozoomer 2.0-HT (Hamamatsu). Tau pathology was previously described [22]. They are derived from a mono- assessed using ImageJ as previously described [13, 26]. clonal HEK cell line that stably expresses tauRD(P301L/ Briefly, the regions of interest (entorhinal cortex/amg- V337M)-YFP. DS9 and DS10 stably propagate distinct dala, dentate gyrus) were outlined, and tau AT8 path- tau aggregate conformations and exhibit unique inclu- ology was analyzed by brightness thresholding. The area sion morphology, biochemistry, and phenotypes in covered with AT8 staining was reported as a percentage culture and in vivo [16, 22]. DS1 is a negative control of total area analyzed. cell line that expresses only monomeric tauRD(P301L/ V337M)-YFP. Isolation of brain regions from mouse and human brain slices Transgenic animals Free-floating mouse brain regions were washed in 1x PS19 transgenic mice that express 1N4R P301S human TBS (50 mM Tris-Cl, pH 7.5 150 mM NaCl) and placed tau under the murine prion promoter [27] were pur- under a dissection microscope. Single brain regions were chased from Jackson Laboratory. They were maintained collected with a 1 mm punch biopsy tool, and placed Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 3 of 12 into 40uL per punch of 1x TBS in PCR tubes. To avoid Mouse tissue samples were assayed in triplicate, and cross-contamination of seeding activity, only one punch human tissue samples were added in sextuplicate. Cells biopsy tool was used for each brain, and then discarded. were kept at 37 °C in a humidified incubator for 24 hours Human brain tissue blocks fixed in 4% formaldehyde for experiments with cell lysate or transgenic mouse were embedded in polyethylene glycol (PEG 1000, brain homogenate, and for 48 hours for all human tissue Merck) and sectioned coronally at 100 μm, as described experiments described here. Cells were subsequently col- elsewhere [3, 25]. Tissue sections with the human trans- lected and prepared for flow cytometry analysis. entorhinal cortex, entorhinal cortex, and CA1–4 with AT8-positive tau pathology and from CA1/3 of the Inoculation of strains into transgenic mice mouse hippocampus (2.5 mm x 2.5 mm) were collected DS1, 9, or 10 cell lines were trypsinized from 3 x 10 cm and placed into 1 x TBS for homogenization. Final con- dishes, pelleted, and washed with 1 x PBS. Cell pellets centration of mouse samples was 1 mm of tissue per were frozen at−80 °C until use. Cell lines were frozen on 1 mL of TBS, while human samples were homogenized ice and resupsended in 1 x PBS with cOmplete mini in 100 μL and then diluted to 10 mm per 1 mL of TBS protease inhibitor tablet (Roche). Cells were sonicated at (v/v). 4 °C with an Omni-Ruptor 250 probe sonicator at 30% power for 1-second pulses x 30 cycles. Cell lysate was Paraffin embedding, sectioning, and tissue preparation clarified with a 1000 x g spin and the supernatant was Paraffin sections from mice were collected as 10 x 5 μm stored at−80 °C until use. left hemisphere coronal brain slices and stored in 100% PS19 mice that underwent stereotaxic inoculations ethanol until use. Excess paraffin was removed by per- were anesthetized with isoflurane. A regulated heating forming 2 x 100% ethanol washes at 60 °C. pad was used to maintain core body temperature throughout the procedure. Animals were inoculated in Tissue homogenization the left hippocampus with 20 μg of cell lysate (from Mouse samples were sonicated in a water bath in PCR Bregma: x =−2.0, y =−2.5, Z =−1.8), and kept for 3, 6, or tubes for 30 minutes under 50% power at 4 °C (Qsonica 12 weeks after injection. Q700 power supply, 431MPX microplate horn, with chiller). For fresh frozen tissue homogenization, right Generation of strains in secondary cells hemisphere slices from aged mice were collected by Fixed hippocampal sections from P301S mice injected freezing microtome as described above, and placed into with DS9 or DS10 and incubated for 12 weeks were col- 1 x TBS with protease inhibitors. These samples were lected and sonicated as above. LM1 cells were plated at kept on ice, and sonicated immediately after collection 8000 cells per well of a 96-well plate and allowed to in parallel with fixed tissue slices taken from the left grow overnight. Wells were subsequently treated with hemisphere. These samples were immediately trans- 5uL of pooled, fixed tissue hippocampal sections from duced into biosensor cells as described below. Human DS9 and DS10 mice (n =3–4 mice per condition) and tissue samples were sonicated under identical conditions incubated for 48 hours. Cells were re-plated into a 12- for 60 minutes. well dish, and grown for an additional two days. Single cells were sorted by fluorescence activated cell sorting Transduction into biosensor cell lines (FACS) into five 96-well plates per condition using the Biosensor cells were plated at 25,000 cells per well in Beckman Coulter MoFlo at the Siteman Flow Cytometry 96-well plates. After 18 hours, cells were transduced core facility at the Washington University in St. Louis. with mouse or human homogenates as previously de- Cells were observed for individual colony growth for scribed [13]. Tissue from the mouse aging study and hu- 10 days. Single cell colonies that contained aggregates man samples were used at stock concentrations were isolated as monoclonal secondary cell lines and prepared as described above, while samples from the grown to confluency in 10 cm dishes. Cell pellets were spreading paradigm were diluted 1:5 in TBS. Tissue collected for biochemistry, plated for confocal micros- samples were added to Opti-MEM (Thermo Fisher Sci- copy, and subsequently frozen in 90% FBS/10% DMSO entific) and incubated for 5 minutes (5 μL of mouse tis- for long-term storage. sue lysate with 5 μL of Opti-MEM, or 3.3 μL human with 6.7 μL of Opti-MEM per well). Lipofectamine was Immunocytochemistry of secondary cell lines incubated with Opti-MEM (1.25 uL Lipofectamine with Secondary cell lines were plated at low confluency on 8.75 μL Opti-MEM per well) for five minutes. Lipofecta- glass cover slips (0.09 to 0.12 mm thickness; Carolina mine complexes were then mixed with samples and in- Biologicals) and allowed to grow for 72 hours. Cells were cubated for 20 minutes prior to addition to biosensor fixed with 4% PFA, permeabilized with 0.25% Triton X- cells. 100 and DAPI stained. Cells were imaged with a Zeiss Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 4 of 12 LSM780 inverted confocal microscope. For secondary We used the quantitative cell-based seeding assay to cell line seeding assays, protein content was normalized test whether tau protein also retains its seeding proper- by a Bradford assay to 0.5 μg/μL, and 2 μg of cell lysate ties after fixation [10, 13]. This assay utilizes HEK cells was transduced per well in triplicate. Transduction was that stably express tau four repeat domain (RD) with a performed as described above. P301S mutation fused to cyan or yellow fluorescent pro- teins (termed tau-CFP/YFP for brevity). Transduction of material that contains tau prion seeds into biosensor Flow cytometry and analysis of seeding activity cells triggers aggregation of tau-CFP/YFP (Fig. 1a). This Biosensor cell lines were harvested with 0.05% trypsin, brings CFP and YFP into close association and results in and quenched with media (DMEM + 50% FBS, 1% Pen/ fluorescence resonance energy transfer (FRET) that we Strep, 1% Glutamax). Cells were centrifuged at 500 x g quantify by flow cytometry (Fig. 1b) [10, 13]. and resuspended in 4% PFA in 1x PBS. Cells were subse- Previous work has shown that fixed tissue samples quently centrifuged at 500 x g, resuspended in flow buf- with Aβ pathology can be lysed using a Precellys 24- fer (HBSS + 1% FBS + 1 mM EDTA), and stored for less Dual homogenizer [9]. However, this method yielded than 24 hours prior to performing flow cytometry. All approximately 50% reduction in the level of seeding ob- flow cytometry for biosensor cells treated with mouse served in fresh-frozen tissue. Sonication-based and human-derived tissue was performed using a Milte- homogenization techniques have been utilized to detect nyi VYB flow cytometer. Flow cytometry data was ana- minute amounts of misfolded PrP [21]. Thus, we lyzed as previously described [10]. Integrated FRET adopted an extended water-bath sonication protocol to density was calculated as integrated FRET density homogenize fixed tissue samples. (IFD) = (percentage of FRET-positive cells)*(median PS19 mice that express 1N4R tau(P301S) under the fluorescence intensity). IFD was normalized to nega- prion promoter develop progressive pathology over tive control samples. ~1 year [27]. Tau seeding activity was compared in fresh frozen versus fixed tissue samples from mice at Statistical analysis 10 months, when these animals show robust tau path- All statistical analysis was performed using GraphPad ology (Fig. 1c). In parallel, fixed tissue slices were col- Prism. Tau AT8 and seeding time course data were stan- lected and homogenized from the left hemisphere and dardized from 0 to 100%, and modeled using nonlinear equivalent fresh frozen tissue slices from the right hemi- regression analysis as a log(agonist) vs. normalized re- sphere. Fixed and fresh frozen tissue similarly seeded ag- sponse (variable slope). S and S refer to the log(EC ) 10 50 10 gregation after transduction into biosensor cells and log(EC ) calculated from these curves. Unless expli- (Fig. 1d). In contrast, fixed and fresh frozen slices from citly stated, all statistical analyses used one-way analysis negative control transgenic mice that express α- of variance with Bonferroni’s multiple comparison test. synuclein (A53T) under the prion promoter [12] did not induce aggregation in this biosensor line (Fig. 1d) nor did Results tissue from a wild-type (WT) mouse (Additional file 1: Fixed tissue retains tau seeding activity Figure S1a). Formaldehyde-fixed tissue samples are a stable resource To verify that seeding can be detected in fixed tissue to assess tau pathology by microscopy, but are not suit- processed with other embedding protocols, we tested able for classical biochemistry. Nonetheless, fixed tissue brains from aged P301S mice embedded in paraffin or from Creutzfeld-Jakob disease (CJD) patients retains PEG. Paraffin-embedded sections required 100% ethanol prion infectivity and strain properties [20]. Moreover, washes at 60 °C to remove excess wax, while PEG was PrP aggregates from fixed tissue will amplify natively readily removed with aqueous buffer. We detected seed- folded PrP in the real time quaking-induced conversion ing activity in samples embedded in both PEG and par- assay (RT-QuIC) [14]. The RT-QuIC technique relies affin (Additional file 1: Figure S1b). upon seeded aggregation of recombinant PrP by patho- logical PrP present in samples. Quantification of seeding activity in fixed tissue from Fixed tissue that contains Aβ pathology also retains its PS19 mice seeding activity and conformation upon inoculation into IHC is typically used to assess phosphorylated tau path- transgenic mice [9]. Similarly, fixed samples with α- ology in tauopathy autopsy samples [5, 17]. To directly synuclein pathology can induce synucleinopathy after in- compare seeding activity to IHC, brain slices were taken oculation into transgenic mice that express human from PS19 mice at 1–12 months of age. Aged WT cage- forms of α-synuclein [24]. Thus, several aggregation- mates served as negative controls. We assessed tau AT8 prone proteins implicated in neurodegenerative diseases histopathology in the dentate gyrus (DG) and entorhinal retain seeding activity after fixation. cortex/amygdala (EC/A). In PS19 mice, we observed Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 5 of 12 Fig. 1 Fixed and fresh frozen tissue exhibit similar levels of seeding activity. a Transduction of aggregated tau into biosensor cells induces aggregation of endogenously expressed tauRD(P301S)-YFP fusion proteins. b Flow cytometry of tau biosensor cells can detects tau seeding based on FRET from aggregation of tauRD(P301S)-CFP and tauRD(P301S)-YFP fusion proteins. Note the population of cells that shift to the FRET-positive gate. c AT8 phospho-tau pathology is apparent in aged PS19 mice, whereas WT mice show no tau pathology. d Tau seeding activity is similar between fixed and fresh frozen tissue sections collected from aged PS19 mice. No significant difference was detected between fixed and fresh tissue at each concentration tested. Seeding is not detected in aged mice that express α-synuclein (A53T) [12]. Integrated FRET density is calculated as (Percent FRET-positive cells)*(median fluorescence intensity of positive cells). This value is normalized to a negative control sample. See Additional file 1: Figure S1a for an additional comparison of fixed versus fresh frozen tissue seeding, and Additional file 1: Figure S1b for seeding of fixed tissue embedded in PEG or paraffin. Error bars = S.E.M increasing levels of AT8 tau pathology over time, whereas brains and aged PS19 mouse samples, which facilitated in WT mice no phospho-tau pathology was detected even this quantitative analysis even among samples with rela- at 12 months of age (Fig. 2a, Additional file 2: Figure S2a, tively variable patterns of histopathology. Table 1). To determine the rate at which seeding activity and AT8 pathology in the DG of PS19 mice did not exceed AT8pathology developin the PS19mouse line,we low baseline levels until 5 months of age (Fig. 2b). The standardized data from both metrics from 0 to 100% EC/A exhibited a more apparent, but highly variable and performed nonlinear regression analyses. Seeding AT8 signal from early ages, but this did not rise above reached 10% (S ) and 50% (S )ofmaximum signal 10 50 the baseline until 7 months (Fig. 2b). The low dynamic earlier than AT8 pathology both in the DG and EC/A range of AT8 staining in this mouse model hinders (Additional file 2: Figure S2b). We also plotted seed- quantitative assessment of pathology using this metric. ing activity against the AT8 pathology from individual Next, we measured seeding activity in 1 mm punch bi- mice. Seeding activity increased earlier than AT8 opsies from fixed tissue slices immediately adjacent pathology in individual mice. We observed robust (50 μm) to those stained for AT8 pathology. We homog- phospho-tau pathology above 6 months of age in the enized biopsies from the DG and EC/A, transduced DG and above 3 months in the EC/A, whereas seed- them into the biosensor cell line, and assessed them ing scored positive much earlier (Additional file 2: after 24 hours (Fig. 2c). In PS19 mice, the DG and EC/A Figure S2c). We conclude that tau seeding activity exhibited higher seeding activity at 2 vs. 1 month. As re- measured in fixed tissue is a robust and highly sensi- ported earlier with unfixed samples [13], we observed a tive metric for tau pathology, and anticipates AT8 1000-fold dynamic range between negative control WT staining in this mouse model. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 6 of 12 Fig. 2 Seeding activity increases with age and anticipates AT8 pathology in PS19 mice. a AT8 staining of phospho-tau pathology in the dentate gyrus (DG) and entorhinal cortex/amygdala (EC/A) of a 12 month old WT mouse, and in PS19 mice at 1, 3, 6, 9, and 12 months of age. AT8 staining increases with age in PS19 mice. See Additional file 2: Figure S2a for representative images of whole-brain slices from 12-month-old WT and PS19 mice. b AT8 tau pathology was quantified in the DG and EC/A of WT and PS19 mice. WT mice were collected from various ages. Six PS19 mice at each age were assessed for tau pathology. Threshold analysis was performed to quantify the percentage of area occupied by AT8 staining in the region of interest. DG AT8 pathology did not rise above baseline staining in 1 month mice until 5 months of age. EC/A AT8 pathology did not show significant increases until 7 months of age. See Additional file 2: Figure S2b for nonlinear regression model of time-course AT8 staining data, and Table 1 for details regarding mice used in this study. c. Seeding activity was assessed from adjacent free-floating brain slices of WT and PS19 mice. 1 mm punch biopsies from the DG and EC/A were homogenized and transduced into biosensor cells. Tau seeding activity increased above baseline by 2 months of age for both the DG and EC/A. See Additional file 2: Figure S2b for nonlinear regression model of tau seeding activity time course data, and Additional file 2: Figure S2c for a direct comparison of seeding activity versus AT8 tau pathology. Error bars = S.E.M; * = p < .05; ** = p <.01 Quantification of spreading tau pathology neurofibrillary tangle (NFT)-like pathology in CA1 and Tau pathology progressively accumulates along neuronal CA3, while DS10 induced mossy-fiber pathology networks in AD patient brains [4]. We have developed (Fig. 3c,d). By 6 weeks, we observed spreading of an in vivo model of tau propagation through inoculation phospho-tau pathology into CA1 of the contralateral of distinct tau strains (DS1 (monomer control), DS9, hippocampus of DS9-inoculated mice. At this time point DS10) derived from stable cells into the hippocampus of in mice inoculated with DS10, we observed mossy fiber PS19 mice (Fig. 3a) [22]. To compare the utility of the pathology in the contralateral hippocampus, but limited fixed tissue seeding assay with IHC, we collected brain CA1 pathology (Fig. 3c,d). samples at 3, 6, or 12 weeks after injection and assessed We quantified tau histopathology by determining the pathology by AT8 stain or seeding activity (Fig. 3b, percentage area covered by AT8 staining in the ipsilat- Table 2). As previously described, DS9 produced eral and contralateral hippocampi of inoculated mice. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 7 of 12 Table 1 Aging PS19 mouse study whether tau strains similarly retain their conformations after fixation, we homogenized fixed hippocampi from Age at Collection N (Total) Male Female mice inoculated with DS9 or DS10 12 weeks post- P301S Mice injection. We transduced hippocampal lysate into the 1 month 6 3 3 LM1 biosensor cell line. After four days, we used FACS 2 month 6 3 3 to isolate aggregate-containing monoclonal cell lines in 3 month 6 3 3 96-well plates. 4 month 6 3 3 We examined the wells for cell colony growth at 7– 5 month 6 3 3 10 days post-plating; 8% of DS9 derived wells and 5% of DS10 derived wells contained aggregate-positive 6 month 6 3 3 cell colonies at 7–10 days. As observed previously, 7 month 6 3 3 theinitial passageof isolatedmonoclonalsecondary 8 month 6 3 3 cell lines is toxic. Thus, 24 DS9-derived secondary 9 month 6 3 3 lines and 9 DS10-derived lines were isolated. Three 10 month 6 3 3 secondary cell lines from each DS9 inoculated mouse 11 month 6 3 3 and all DS10 derived cell lines (2–4 lines per mouse) were analyzed for morphology and/or seeding activity 12 month 6 3 3 (Fig. 4a). WT Mice Secondary cell lines derived from mice inoculated 1 month 4 3 1 with DS9 and DS10 displayed cellular morphologies 2 month 1 0 1 identical to the original strains as seen in Fig. 3a 5 month 1 0 1 (e.g., nuclear speckles versus no nuclear speckles and 6 month 1 1 0 a large juxtanuclear aggregate) (Fig. 4b). This is con- sistent with previous results from fresh frozen tissue, 9 month 1 1 0 which demonstrated that tau strains stably template 10 month 1 0 1 onto humantau expressedinPS19miceand then 12 month 1 0 1 back into LM1 cells [22]. Secondary cell lines derived from fixed tissue showed similar seeding activity to DS1-injected mice exhibited limited AT8 pathology at the original DS9 and 10 strains (Fig. 4c). Thus, tau this time point. DS9 and DS10 inoculates induced in- strains retain their conformation and strain-specific creasing levels of pathology in the hippocampus, but properties in young and aged mice even after threshold analysis of AT8 staining was difficult in fixation. DS10 mice owing to the subtle mossy fiber phenotype (Fig. 3e). To assess tau seeding activity, we dissected and ho- Quantification of seeding activity in fixed tissue from the mogenized ipsilateral and contralateral hippocampi from human brain fixed free-floating 50 μm brain sections of inoculated To test the seeding assay in formaldehyde-fixed human mice. We transduced hippocampal tissue into biosensor tissue samples, we examined the transentorhinal cortex cells, and quantified seeding activity by flow cytometry and Ammon’s horn (CA1/3) of five individuals with dif- after 24 hours. Mice inoculated with DS1 exhibited a ferent stages of tau pathology (Table 3) [4]. While we minimal signal possibly due to the endogenous seeding observed robust seeding from PS19 mice, they express activity present at this age (Fig. 2c, Fig. 3f). Mice inocu- an aggregation-prone form of tau at approximately 5x lated with DS9 and DS10 exhibited robust seeding the level of endogenous tau expression in humans [27]. activity that increased over time in the ipsilateral hippo- To increase the sensitivity of the assay, human samples campus. We also detected clear seeding activity in the were added to biosensor cells at 10x concentration and contralateral hippocampus 3 weeks after injection with the incubation period was extended to 48 hours. Sub- DS9 or DS10, in contrast to the AT8 staining. We con- jects with no tau pathology or with very subtle pretangle clude that seeding activity in fixed tissue anticipates the pathology (stages a/1b) did not display detectable seed- spread of tau pathology and is robust prior to AT8 path- ing activity in either region. In contrast, tissue from indi- ology detectable by immunohistochemistry. viduals with NFT stages III and V tau pathology contained seeding activity in both the transentorhinal Fixed tissue retains strain-specific conformations cortex and Ammon’s horn (Fig. 5; Table 3). Thus, tau After fixation, PrP and Aβ retain their ability to serve as seeding activity can be quantified from fixed mouse and distinct conformational templates [9, 19]. To test archival human tissue alike. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 8 of 12 Fig. 3 Seeding activity detects spread of tau pathology. a Tau strains DS1, 9, and 10 have different inclusion morphologies. DS1 does not contain aggregated tau. DS9 cells feature nuclear speckles, while DS10 cells have a large juxtanuclear aggregate and no nuclear speckles. b Cell lysate from DS1, 9, or 10 was inoculated into the hippocampi of young PS19 mice. At 3, 6, or 12 weeks, brains were collected for tau histopathology and seeding analysis. c Inoculation of DS1 did not induce AT8 pathology. Mice inoculated with DS9 developed NFT-like AT8 pathology in CA1 of the ipsilateral hippocampus by three weeks. This pathology spread to the contralateral hippocampus by 6 weeks. DS10 produced limited tau pathology in this region. d DS9 inoculation induced neurofibrillary tangle-like pathology in CA3 of the ipsilateral hippocampus, and limited pathology in the contralateral hippocampus by six weeks. DS10 inoculation primarily induced mossy fiber AT8 pathology that progressed over time, and spread to the contralateral hippocampus by 12 weeks. e The percentage of the hippocampus covered with AT8 tau pathology was assessed in mice inoculated with DS1, 9, and 10 at each time point. Tau AT8 pathology and spread was apparent in DS9 inoculated mice. However, DS10 mossy fiber pathology was difficult to detect with this technique and showed variable pathology among animals (f). Tau seeding activity was detected in ipsilateral and contralateral hippocampi of DS9 and DS10 inoculated mice at 3 weeks. Seeding activity increased by 12 weeks, suggesting tau pathology continues to develop over time. ANOVA analysis was performed by comparing samples within each time point to DS1 inoculated controls. Error bars = S.E.M, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 Discussion from miniscule amounts of fixed tissue (approximately Propagation of tau aggregation along neuronal networks .04 mm )topermitdirectcomparisonwithtissues may mediate the progressive accumulation of pathology stained by IHC. observed in tauopathy patients. To measure tau seeding We first tested this method in PS19 mice that overex- activity in well-characterized human brains, it will be ne- press full-length human tau (1 N,4R) containing the cessary to analyze formaldehyde-fixed tissues. We now P301S mutation. We drop-fixed brain samples that had present a method for extracting tau seeding activity been embedded either in paraffin or PEG and sectioned Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 9 of 12 Table 2 Inoculation model of spread of tau pathology them coronally for microscopy. We analyzed adjacent 50 μm sections using standard IHC to detect phospho-tau Time N (Total) Male Female Average Age at Average Age at Course Surgery (Days) Collection (Days) or 1 mm circular punch biopsies of tissue for seeding as- 3 weeks says. We homogenized punch biopsies by water-bath son- ication in closed tubes, and assayed them in a cellular DS1 2 0 2 94 115 FRET bioassay system as described previously [10, 13]. DS9 4 0 4 90 111 Tau seeding activity tracked the development of path- DS10 4 0 4 91 112 ology more efficiently than IHC, with a lower degree of 6 weeks inter-animal variation, and a higher dynamic range. This DS1 3 0 3 77 119 was perfectly comparable to previously obtained results DS9 4 1 3 80 122 using fresh frozen tissue [13]. In addition, we detected seeding activity relatively early in the course of disease DS10 4 0 4 80 122 (1–2 months) and it steadily increased over time. Next, 12 weeks we tested brain tissues from animals previously inocu- DS1 2 0 2 71 155 lated with two distinct tau prion strains. We recovered DS9 4 2 2 70 154 these strains from fixed mouse brain tissue as accurately DS10 3 1 2 71 155 as we had previously from fresh frozen tissue. Finally, we tested the extraction method in fixed human brain tissue with documented AT8-positive tau pathology, including AD, and readily detected tau seeding activity in cases ar- chived for up to 27 years in formaldehyde. Fig. 4 Strain-specific properties are retained after fixation. a Fixed hippocampi were isolated from PS19 mice at 12 weeks post injection with DS9 or 10. This tissue was homogenized and transduced into the original LM1 cell line. Fluorescence-activated cell sorting was used to isolate monoclonal cells into 96-well plates. Cells that stably propagated aggregates were amplified and characterized. b Confocal images of representative secondary cell strains derived from mice inoculated with DS9 or 10. Secondary strains displayed the same inclusion morphology as the original inoculum (nuclear speckles or a large juxtanuclear aggregate). See Fig. 3a for images of original strain morphology. c. Seeding activity was assessed for DS9 and 10, as well as secondary cell lines. Cell lysate from each line was transduced into biosensor cells and assessed for tau seeding activity after 24 hours (2 μgper well). Secondary strains showed similar seeding activity to the original inoculum. See Table 2 for additional information regarding the mice used in this study Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 10 of 12 Table 3 Human tauopathy and control subjects Patient Case NFT/Aβ/α-synuclein Stage M/F Age Year of formalin Notes fixation 1 a/0/0 M 31 1989 Control 2 1b/0/0 M 42 1989 Control 3 III/0/0 F 90 1992 No clinical AD diagnosis 4 III/2/0 M 65 1989 No clinical AD diagnosis 5 V/3/0 M 88 1990 Clinical AD Seeding activity Moreover, we detected seeding activity in a small sam- Our laboratory previously detected tau seeding activity ple of human tauopathy cases that were collected and in fresh frozen brain tissue from mouse tauopathy stored in formalin for over 20 years prior to this study. models and human AD cases[11, 13]. However, fresh We observed lower seeding activity in these human sam- frozen samples are much more difficult to obtain than ples than in PS19 mice, probably because of the overex- fixed tissue sections, must be carefully stored at−80 °C, pression of an aggregation-prone form of tau in this and are very challenging to dissect precisely to isolate mouse model. However, the length of fixation may affect specific brain regions. The assay described here accur- the level of seeding observed in samples. Further, differ- ately quantifies tau seeding from fixed tissue sections ences in seeding activity observed between patients at over three log orders of signal. Remarkably, in a mouse Braak stage III and V likely reflect differences in the model from which we sampled tissue at different time level of tau aggregate burden between these patients, cell points, fixed tissue seeding proved comparable to seed- loss, or ghost-tangle formation at later disease stages. ing activity detected in fresh frozen tissue. Thus, we ex- Given the early detection of seeding activity relative to pect that this assay will enable assessment of tau seeding AT8 staining in PS19 mice, we anticipate that this activity in a range of fixed tissues at a similar level of assay could represent a more sensitive metric of tau sensitivity to fresh frozen samples. pathology. Additional studies in a large number of Fig. 5 Fixed human brain samples with tau pathology exhibit seeding activity. AT8-immunostained (hyperphosphorylated tau, DAB) 100 μm sections from cases 3 (a) and 4 (b) in Table 3. In NFT stage III, the tau pathology in the hippocampal formation increases. a,b. The entorhinal layers pre-α and, in addition, pri-α become heavily involved. Tau pathology extends through the transentorhinal region into the adjoining high order sensory association areas of the temporal neocortex but not yet into the superior temporal gyrus. c The NFT in the late stage V case shown here is not identical to case 5 in Table 1 but is from another Alzheimer’s disease patient in her eighties. During NFT stage V, tau lesions develop in the superior temporal gyrus and progress into first order sensory association and premotor areas of the neocortex. d Fixed tissue was isolated from the transentorhinal cortex and the hippocampus (CA1/3) of 100 μm human brain sections that were blinded prior to collection (Table 3). Samples were homogenized and transduced into tau biosensor cells. The integrated FRET density was normalized to a negative control treated only with Lipofectamine. Error bars = S.E.M, ** = p < 0.01, **** = p < 0.0001 Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 11 of 12 well-characterized human tissue samples will help ad- tau pathology based on seeding activity and is also sensi- dress these important questions, and provide additional tive to strain composition. We anticipate that punch biop- insight into the progression of seeding activity in human sies taken from tissue sections will be useful to measure tauopathies. strain identity with high anatomical precision. By carefully Earlier work described a dose-dependent increase in comparing seeding activity and strain composition with tau seeding activity in the PS19 mouse tauopathy model standard neuropathology, it should be possible to add new [13]. However, the regional specificity possible with fresh dimensions to analyses of tissue samples from a range of frozen tissue was limited to gross dissection. We now neurodegenerative diseases. In turn, this will facilitate have reliably isolated and characterized punch biopsies more widespread testing of the putative role of tau prion as small as 1 mm diameter x 50 μm (or ~ .04 mm ). activity in human tauopathies. When we quantified the level of seeding activity at in- creasing ages vs. the tau pathology observed in adjacent Additional files tissue slices using anti-tau AT8 staining, we easily de- tected tau seeding activity, even in fixed tissue sections Additional file 1: Figure S1. Fixed tissue reliably seeds tau aggregation. a. Comparison of fixed and fresh tissue seeding from aged PS19 mice. WT with a minimal AT8 signal. For example, when PS19 mouse tissue did not induce seeding. Seeding displays a dose-response, mice were inoculated with tau strains, we induced strong and no significant difference was detected at each concentration. b. Tau AT8 pathology with DS9, whereas DS10 produced a seeding was equivalently detected from aged PS19 brain tissue embedded in either paraffin or polyethylene glycol. Paraffin embedded tissue requires weak signal. In both cases, the pathology spread from heated ethanol washes to remove excess wax prior to homogenization for the site of inoculation to connected regions, as described robust seeding. A sham sample (Lipo) was used as a negative control. elsewhere [22]. The fixed tissue seeding assay more read- (PDF 97 kb) ily detected the spread of tau pathology in this propaga- Additional file 2: Figure S2. Seeding activity precedes AT8 pathology in PS19 mice. a. Representative images of 12 month WT and PS19 mouse tion model. Furthermore, we readily detected seeding hemi-brain slices stained with AT8. No AT8 staining was detected in WT activity in DS10 inoculated mice despite the relatively mice, whereas PS19 mice exhibited robust phospho-tau pathology subtle AT8 staining phenotype induced by this strain throughout the brain. b. Schematic of punch biopsy, transduction, and seeding assay workflow. c. Seeding and AT8 pathology time course data (mossy fiber dots). Consequently seeding activity can were modeled with nonlinear regression analysis using log (agonist) serve as an important measure of tau pathology when versus normalized response (variable slope). S and S refer to the 10 50 routine AT8 staining reports otherwise minimal path- time point at which seeding or AT8 pathology reaches 10% or 50% of maximal signal, and is represented in months. Seeding preceded ology. The combination of precise quantification of AT8 pathology in both the DG and EC/A. d. Scatter plot analysis of seeding activity with the ability to sample brain tissue to tau seeding activity versus AT8 pathology for each animal. Seeding 1 mm resolution indicates that this method could help activity increases before robust AT8 pathology is observed in the DG and EC/A. (PDF 440 kb) define the seeding activity in human brain with remark- ably high accuracy. Acknowledgements The authors SKK and MID thank Bill Eades for expert advice and guidance. Detection of tau strains in formaldehyde-fixed tissue The authors (HB, KDT) thank the Hans and Ilse Breuer Foundation (Frankfurt Prior experimental work indicates that distinct tau ag- am Main, Germany) for generously supporting their research and Mr. David gregate conformations may underlie different patterns of Ewert (University of Ulm) for technical assistance with the graphics (Fig. 5a- c). This work was supported by the Siteman Flow Cytometry core and Hope pathology, rates of progression, and disease phenotypes Center Alafi Neuroimaging Laboratory at Washington University in St. Louis, observed in distinct tauopathies [2, 7, 22]. Distinct tau and the Neuro-Models Facility and Whole Brain Microscopy Facility at University strains are associated with different tauopathies [22], and of Texas Southwestern Medical Center. inoculation of unique tau strains produces different pat- Funding terns and tau pathology rates of progression [16]. We This work was funded by NIH/NIA grant F30AG048653 (S.K.K); NIH/ observed that fixed tissue from mice inoculated with NIAR01AG048678, NIH/NINDSR01NS071835, the Tau Consortium, and the DS9 and DS10 produced strain phenotypes identical to Cure Alzheimer’s Fund (M.I.D.); Hans and Ilse Breuer Foundation (Frankfurt am Main, Germany) (KDT and HB). the original strains upon inoculation into LM1 biosensor cells. Thus, tau strains are stable upon fixation. We an- Availability of data and materials ticipate that formaldehyde-fixed tissues will serve as an The datasets used and/or analyzed during the current study are available invaluable resource to examine the role of strain com- from the corresponding author by reasonable request. position in tauopathies. Authors’ contributions Studies that use traditional IHC techniques to detect SKK designed and performed all animal, cell culture, and flow cytometry tau pathology have provided important insights into the experiments. TLT assisted with tissue collection and immunohistochemistry progression and anatomy of macromolecular accumula- of animal experiments. KDT and HB performed all human tissue collection, IHC and neuropathological staging. MID assisted with the design and tions of tau assemblies. However, these methods cannot interpretation of all animal and flow cytometry experiments. All authors discriminate among distinct strains, nor can they detect assisted in the writing and figure preparation for this manuscript. All authors submicroscopic tau assemblies. The present assay measures read and approved the final manuscript. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 12 of 12 Competing interests 13. Holmes BB, Furman JL, Mahan TE, Yamasaki TR, Mirbaha H, Eades WC, MID is co-developer of an anti-tau antibody currently in clinical trials (C2N Belaygorod L, Cairns NJ, Holtzman DM, Diamond MI (2014) Proteopathic tau 8E12 [NCT02494024]). The remaining authors declare that they have no seeding predicts tauopathy in vivo. Proc Natl Acad Sci 111:E4376–E4385. competing interests. doi:10.1073/pnas.1411649111 14. Hoover CE, Davenport KA, Henderson DM, Pulscher LA, Mathiason CK, Zabel Consent for publication MD, Hoover EA (2016) Detection and Quantification of CWD Prions in Fixed Not applicable. Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion. Sci Rep 1–10. doi: 10.1038/srep25098 Ethics approval and consent to participate 15. Hyman BT, Phelps CH, Beach TG, Bigio EH, Cairns NJ, Carrillo MC, Dickson All animal maintenance and experiments adhered to the animal care and DW, Duyckaerts C, Frosch MP, Masliah E, Mirra SS, Nelson PT, Schneider JA, use protocols of the University of Texas Southwestern Medical Center, and Thal DR, Thies B, Trojanowski JQ, Vinters HV, Montine TJ (2012) National Washington University in St. Louis. The autopsy brains used for this study Institute on Aging-Alzheimer“s Association guidelines for the were obtained in compliance with Ulm University ethics committee neuropathologic assessment of Alzheimer”s disease. 8:1–13. doi: 10.1016/j. guidelines as well as German federal and state law governing human tissue jalz.2011.10.007 usage. 16. 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Characterization of tau prion seeding activity and strains from formaldehyde-fixed tissue

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Copyright © 2017 by The Author(s).
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Biomedicine; Neurosciences; Pathology; Neurology
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Abstract

Tauopathies such as Alzheimer’s disease (AD) feature progressive intraneuronal deposition of aggregated tau protein. The cause is unknown, but in experimental systems trans-cellular propagation of tau pathology resembles prion pathogenesis. Tau aggregate inoculation into mice produces transmissible pathology, and tau forms distinct strains, i.e. conformers that faithfully replicate and create predictable patterns of pathology in vivo. The prion model predicts that tau seed formation will anticipate neurofibrillary tau pathology. To test this idea requires simultaneous assessment of seed titer and immunohistochemistry (IHC) of brain tissue, but it is unknown whether tau seed titer can be determined in formaldehyde-fixed tissue. We have previously created a cellular biosensor system that uses flow cytometry to quantify induced tau aggregation and thus determine seed titer. In unfixed tissue from PS19 tauopathy mice that express 1 N,4R tau (P301S), we have measured tau seeding activity that precedes the first observable histopathology by many months. Additionally, in fresh frozen tissue from human AD subjects at early to mid-neurofibrillary tangle stages (NFT I-IV), we have observed tau seeding activity in cortical regions predicted to lack neurofibrillary pathology. However, we could not directly compare the same regions by IHC and seeding activity in either case. We now describe a protocol to extract and measure tau seeding activity from small volumes (.04 mm ) of formaldehyde-fixed tissue immediately adjacent to that used for IHC. We validated this method with the PS19 transgenic mouse model, and easily observed seeding well before the development of phospho-tau pathology. We also accurately isolated two tau strains, DS9 and DS10, from fixed brain tissues in mice. Finally, we have observed robust seeding activity in fixed AD brain, but not controls. The successful coupling of classical IHC with seeding and strain detection should enable detailed study of banked brain tissue in AD and other tauopathies. Introduction distinct disease pathologies by forming strains. Conse- Tauopathies are diverse neurodegenerative diseases char- quently, we use the term “prion” to describe tau, recog- acterized by the deposition of aggregated tau protein nizing that there is no evidence that tau aggregates and progressive neuronal loss [18]. Each tauopathy has spontaneously transmit disease between individuals. We unique patterns of neuropathology, rates of progression, propose that a prion is best understood as a self- and regional involvement. This variability is reminiscent replicating assembly of defined structure that produces a of distinct prionopathies, which are caused by prion pro- specific biological effect, whether for good or ill. This tein (PrP) strains. Strains are unique aggregate structures definition encompasses an enormous literature on func- that faithfully self-replicate, and produce distinct pat- tional yeast prions and other mammalian proteins that terns of neuropathology [8, 20]. Tau resembles a PrP utilize induced, self-amplifying conformational change to prion in experimental systems, as it mediates transmis- functional ends [23]. sible pathology in cells and animals, and transmits Strong evidence indicates that, like PrP, tau forms self- replicating strains that create unique patterns of path- * Correspondence: marc.diamond@utsouthwestern.edu ology in cell and animal models [2, 7, 16, 22]. According Center for Alzheimer’s and Neurodegenerative Diseases, Peter O’Donnell Jr. to the prion model, uniquely structured tau assemblies Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, form in one brain region, where they escape from USA Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 2 of 12 affected cells. These “seeds” act as functional templates on a B6C3 background, and raised with wild-type (WT) to trigger further protein aggregation following internal- littermates. Mice were provided food and water ad libi- ization by adjacent or synaptically connected cells. tum, and housed under a 12-hour light/dark cycle. All It is still unknown whether this mechanism can ac- animal maintenance and experiments adhered to the ani- count for progressive pathology in humans. A central mal care and use protocols of the University of Texas challenge has been to determine the relationship of tau Southwestern Medical Center, and Washington University prion titer and strain composition to classical neuro- in St. Louis. pathological descriptions of phospho-tau accumulation, which have been the gold standard for disease staging Human tissue and discrimination among tauopathies [1, 15, 17]. Meas- The autopsy brains used for this study were obtained urement of tau seeding activity in brain tissue and deter- from five individuals (1 female, 4 males) in compli- mination of strain composition will facilitate ance with Ulm University ethics committee guidelines characterization of neuropathological specimens, and as well as German federal and state law governing may help answer this question. human tissue usage. Informed written permission was We have previously engineered HEK293T cells to de- obtained from all patients and/or their next of kin. tect tau seeding activity in unfixed tissues [13] and to All cases were neuropathologically staged according isolate and characterize tau prion strains present in to published protocols [3–6, 15]. human brain [22]. In transgenic mouse models, the seed biosensor assay detects the emergence of pathological Isolation of mouse brain tau prions far in advance of frank neuropathology [13]. Animals were anesthetized with isoflurane and perfused In selected fresh frozen brain samples from Alzheimer’s with chilled PBS with 0.03% heparin. Whole-brains were disease (AD) patients, these methods indicate that seed- drop-fixed in 4% paraformaldehyde (PFA) in PBS over- ing activity might predict the accumulation of phospho- night at 4 °C. For time course samples, brains were first tau in characteristic neurofibrillary tangles (NFTs) [11]. bisected; the left hemispheres were drop-fixed in 4% However, because it has been impossible to directly PFA, while right hemispheres were stored as fresh frozen compare adjacent, thin sections of brain, we have been tissue at−80 °C until use. unable to use the biosensor assay to simultaneously compare tau pathology with microscopy, seeding activ- Immunohistochemistry of mouse tissue ity, or strain composition. We now describe assessment A freezing microtome was used to collect 50 μm free- of tau seeding activity and strain composition in small floating coronal sections from fixed mouse brains or amounts of fixed brain tissue from mice and humans. 50 μm sections from fresh frozen tissue. For immunohis- tochemistry (IHC) analysis of tau pathology in mice, sec- Methods tions were incubated with biotinylated AT8 antibody Cell culture and cell lines (1:500, Thermo Fisher Scientific) overnight at 4 °C. Sec- Seeding assay experiments were performed with a previ- tions were washed and incubated with VECTASTAIN ously published biosensor cell line that expresses a fusion Elite ABC Kit (Vector Labs) prepared in TBS for 30 mi- between 4R tau repeat domain (RD) containing the nutes at room temperature. Samples were developed disease-associated P301S mutation, and cyan or yellow with 3’3-diaminobenzidine using the DAB Peroxidase fluorescent proteins (tauRD(P301S)-CFP/YFP). These cells Substrate Kit with optional nickel enhancement (Vector are freely available (ATCC CRL-3275) [13]. LM1, DS1, Labs). Sections were imaged using an Olympus DS9, and DS10 are monoclonal HEK cell lines that were Nanozoomer 2.0-HT (Hamamatsu). Tau pathology was previously described [22]. They are derived from a mono- assessed using ImageJ as previously described [13, 26]. clonal HEK cell line that stably expresses tauRD(P301L/ Briefly, the regions of interest (entorhinal cortex/amg- V337M)-YFP. DS9 and DS10 stably propagate distinct dala, dentate gyrus) were outlined, and tau AT8 path- tau aggregate conformations and exhibit unique inclu- ology was analyzed by brightness thresholding. The area sion morphology, biochemistry, and phenotypes in covered with AT8 staining was reported as a percentage culture and in vivo [16, 22]. DS1 is a negative control of total area analyzed. cell line that expresses only monomeric tauRD(P301L/ V337M)-YFP. Isolation of brain regions from mouse and human brain slices Transgenic animals Free-floating mouse brain regions were washed in 1x PS19 transgenic mice that express 1N4R P301S human TBS (50 mM Tris-Cl, pH 7.5 150 mM NaCl) and placed tau under the murine prion promoter [27] were pur- under a dissection microscope. Single brain regions were chased from Jackson Laboratory. They were maintained collected with a 1 mm punch biopsy tool, and placed Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 3 of 12 into 40uL per punch of 1x TBS in PCR tubes. To avoid Mouse tissue samples were assayed in triplicate, and cross-contamination of seeding activity, only one punch human tissue samples were added in sextuplicate. Cells biopsy tool was used for each brain, and then discarded. were kept at 37 °C in a humidified incubator for 24 hours Human brain tissue blocks fixed in 4% formaldehyde for experiments with cell lysate or transgenic mouse were embedded in polyethylene glycol (PEG 1000, brain homogenate, and for 48 hours for all human tissue Merck) and sectioned coronally at 100 μm, as described experiments described here. Cells were subsequently col- elsewhere [3, 25]. Tissue sections with the human trans- lected and prepared for flow cytometry analysis. entorhinal cortex, entorhinal cortex, and CA1–4 with AT8-positive tau pathology and from CA1/3 of the Inoculation of strains into transgenic mice mouse hippocampus (2.5 mm x 2.5 mm) were collected DS1, 9, or 10 cell lines were trypsinized from 3 x 10 cm and placed into 1 x TBS for homogenization. Final con- dishes, pelleted, and washed with 1 x PBS. Cell pellets centration of mouse samples was 1 mm of tissue per were frozen at−80 °C until use. Cell lines were frozen on 1 mL of TBS, while human samples were homogenized ice and resupsended in 1 x PBS with cOmplete mini in 100 μL and then diluted to 10 mm per 1 mL of TBS protease inhibitor tablet (Roche). Cells were sonicated at (v/v). 4 °C with an Omni-Ruptor 250 probe sonicator at 30% power for 1-second pulses x 30 cycles. Cell lysate was Paraffin embedding, sectioning, and tissue preparation clarified with a 1000 x g spin and the supernatant was Paraffin sections from mice were collected as 10 x 5 μm stored at−80 °C until use. left hemisphere coronal brain slices and stored in 100% PS19 mice that underwent stereotaxic inoculations ethanol until use. Excess paraffin was removed by per- were anesthetized with isoflurane. A regulated heating forming 2 x 100% ethanol washes at 60 °C. pad was used to maintain core body temperature throughout the procedure. Animals were inoculated in Tissue homogenization the left hippocampus with 20 μg of cell lysate (from Mouse samples were sonicated in a water bath in PCR Bregma: x =−2.0, y =−2.5, Z =−1.8), and kept for 3, 6, or tubes for 30 minutes under 50% power at 4 °C (Qsonica 12 weeks after injection. Q700 power supply, 431MPX microplate horn, with chiller). For fresh frozen tissue homogenization, right Generation of strains in secondary cells hemisphere slices from aged mice were collected by Fixed hippocampal sections from P301S mice injected freezing microtome as described above, and placed into with DS9 or DS10 and incubated for 12 weeks were col- 1 x TBS with protease inhibitors. These samples were lected and sonicated as above. LM1 cells were plated at kept on ice, and sonicated immediately after collection 8000 cells per well of a 96-well plate and allowed to in parallel with fixed tissue slices taken from the left grow overnight. Wells were subsequently treated with hemisphere. These samples were immediately trans- 5uL of pooled, fixed tissue hippocampal sections from duced into biosensor cells as described below. Human DS9 and DS10 mice (n =3–4 mice per condition) and tissue samples were sonicated under identical conditions incubated for 48 hours. Cells were re-plated into a 12- for 60 minutes. well dish, and grown for an additional two days. Single cells were sorted by fluorescence activated cell sorting Transduction into biosensor cell lines (FACS) into five 96-well plates per condition using the Biosensor cells were plated at 25,000 cells per well in Beckman Coulter MoFlo at the Siteman Flow Cytometry 96-well plates. After 18 hours, cells were transduced core facility at the Washington University in St. Louis. with mouse or human homogenates as previously de- Cells were observed for individual colony growth for scribed [13]. Tissue from the mouse aging study and hu- 10 days. Single cell colonies that contained aggregates man samples were used at stock concentrations were isolated as monoclonal secondary cell lines and prepared as described above, while samples from the grown to confluency in 10 cm dishes. Cell pellets were spreading paradigm were diluted 1:5 in TBS. Tissue collected for biochemistry, plated for confocal micros- samples were added to Opti-MEM (Thermo Fisher Sci- copy, and subsequently frozen in 90% FBS/10% DMSO entific) and incubated for 5 minutes (5 μL of mouse tis- for long-term storage. sue lysate with 5 μL of Opti-MEM, or 3.3 μL human with 6.7 μL of Opti-MEM per well). Lipofectamine was Immunocytochemistry of secondary cell lines incubated with Opti-MEM (1.25 uL Lipofectamine with Secondary cell lines were plated at low confluency on 8.75 μL Opti-MEM per well) for five minutes. Lipofecta- glass cover slips (0.09 to 0.12 mm thickness; Carolina mine complexes were then mixed with samples and in- Biologicals) and allowed to grow for 72 hours. Cells were cubated for 20 minutes prior to addition to biosensor fixed with 4% PFA, permeabilized with 0.25% Triton X- cells. 100 and DAPI stained. Cells were imaged with a Zeiss Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 4 of 12 LSM780 inverted confocal microscope. For secondary We used the quantitative cell-based seeding assay to cell line seeding assays, protein content was normalized test whether tau protein also retains its seeding proper- by a Bradford assay to 0.5 μg/μL, and 2 μg of cell lysate ties after fixation [10, 13]. This assay utilizes HEK cells was transduced per well in triplicate. Transduction was that stably express tau four repeat domain (RD) with a performed as described above. P301S mutation fused to cyan or yellow fluorescent pro- teins (termed tau-CFP/YFP for brevity). Transduction of material that contains tau prion seeds into biosensor Flow cytometry and analysis of seeding activity cells triggers aggregation of tau-CFP/YFP (Fig. 1a). This Biosensor cell lines were harvested with 0.05% trypsin, brings CFP and YFP into close association and results in and quenched with media (DMEM + 50% FBS, 1% Pen/ fluorescence resonance energy transfer (FRET) that we Strep, 1% Glutamax). Cells were centrifuged at 500 x g quantify by flow cytometry (Fig. 1b) [10, 13]. and resuspended in 4% PFA in 1x PBS. Cells were subse- Previous work has shown that fixed tissue samples quently centrifuged at 500 x g, resuspended in flow buf- with Aβ pathology can be lysed using a Precellys 24- fer (HBSS + 1% FBS + 1 mM EDTA), and stored for less Dual homogenizer [9]. However, this method yielded than 24 hours prior to performing flow cytometry. All approximately 50% reduction in the level of seeding ob- flow cytometry for biosensor cells treated with mouse served in fresh-frozen tissue. Sonication-based and human-derived tissue was performed using a Milte- homogenization techniques have been utilized to detect nyi VYB flow cytometer. Flow cytometry data was ana- minute amounts of misfolded PrP [21]. Thus, we lyzed as previously described [10]. Integrated FRET adopted an extended water-bath sonication protocol to density was calculated as integrated FRET density homogenize fixed tissue samples. (IFD) = (percentage of FRET-positive cells)*(median PS19 mice that express 1N4R tau(P301S) under the fluorescence intensity). IFD was normalized to nega- prion promoter develop progressive pathology over tive control samples. ~1 year [27]. Tau seeding activity was compared in fresh frozen versus fixed tissue samples from mice at Statistical analysis 10 months, when these animals show robust tau path- All statistical analysis was performed using GraphPad ology (Fig. 1c). In parallel, fixed tissue slices were col- Prism. Tau AT8 and seeding time course data were stan- lected and homogenized from the left hemisphere and dardized from 0 to 100%, and modeled using nonlinear equivalent fresh frozen tissue slices from the right hemi- regression analysis as a log(agonist) vs. normalized re- sphere. Fixed and fresh frozen tissue similarly seeded ag- sponse (variable slope). S and S refer to the log(EC ) 10 50 10 gregation after transduction into biosensor cells and log(EC ) calculated from these curves. Unless expli- (Fig. 1d). In contrast, fixed and fresh frozen slices from citly stated, all statistical analyses used one-way analysis negative control transgenic mice that express α- of variance with Bonferroni’s multiple comparison test. synuclein (A53T) under the prion promoter [12] did not induce aggregation in this biosensor line (Fig. 1d) nor did Results tissue from a wild-type (WT) mouse (Additional file 1: Fixed tissue retains tau seeding activity Figure S1a). Formaldehyde-fixed tissue samples are a stable resource To verify that seeding can be detected in fixed tissue to assess tau pathology by microscopy, but are not suit- processed with other embedding protocols, we tested able for classical biochemistry. Nonetheless, fixed tissue brains from aged P301S mice embedded in paraffin or from Creutzfeld-Jakob disease (CJD) patients retains PEG. Paraffin-embedded sections required 100% ethanol prion infectivity and strain properties [20]. Moreover, washes at 60 °C to remove excess wax, while PEG was PrP aggregates from fixed tissue will amplify natively readily removed with aqueous buffer. We detected seed- folded PrP in the real time quaking-induced conversion ing activity in samples embedded in both PEG and par- assay (RT-QuIC) [14]. The RT-QuIC technique relies affin (Additional file 1: Figure S1b). upon seeded aggregation of recombinant PrP by patho- logical PrP present in samples. Quantification of seeding activity in fixed tissue from Fixed tissue that contains Aβ pathology also retains its PS19 mice seeding activity and conformation upon inoculation into IHC is typically used to assess phosphorylated tau path- transgenic mice [9]. Similarly, fixed samples with α- ology in tauopathy autopsy samples [5, 17]. To directly synuclein pathology can induce synucleinopathy after in- compare seeding activity to IHC, brain slices were taken oculation into transgenic mice that express human from PS19 mice at 1–12 months of age. Aged WT cage- forms of α-synuclein [24]. Thus, several aggregation- mates served as negative controls. We assessed tau AT8 prone proteins implicated in neurodegenerative diseases histopathology in the dentate gyrus (DG) and entorhinal retain seeding activity after fixation. cortex/amygdala (EC/A). In PS19 mice, we observed Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 5 of 12 Fig. 1 Fixed and fresh frozen tissue exhibit similar levels of seeding activity. a Transduction of aggregated tau into biosensor cells induces aggregation of endogenously expressed tauRD(P301S)-YFP fusion proteins. b Flow cytometry of tau biosensor cells can detects tau seeding based on FRET from aggregation of tauRD(P301S)-CFP and tauRD(P301S)-YFP fusion proteins. Note the population of cells that shift to the FRET-positive gate. c AT8 phospho-tau pathology is apparent in aged PS19 mice, whereas WT mice show no tau pathology. d Tau seeding activity is similar between fixed and fresh frozen tissue sections collected from aged PS19 mice. No significant difference was detected between fixed and fresh tissue at each concentration tested. Seeding is not detected in aged mice that express α-synuclein (A53T) [12]. Integrated FRET density is calculated as (Percent FRET-positive cells)*(median fluorescence intensity of positive cells). This value is normalized to a negative control sample. See Additional file 1: Figure S1a for an additional comparison of fixed versus fresh frozen tissue seeding, and Additional file 1: Figure S1b for seeding of fixed tissue embedded in PEG or paraffin. Error bars = S.E.M increasing levels of AT8 tau pathology over time, whereas brains and aged PS19 mouse samples, which facilitated in WT mice no phospho-tau pathology was detected even this quantitative analysis even among samples with rela- at 12 months of age (Fig. 2a, Additional file 2: Figure S2a, tively variable patterns of histopathology. Table 1). To determine the rate at which seeding activity and AT8 pathology in the DG of PS19 mice did not exceed AT8pathology developin the PS19mouse line,we low baseline levels until 5 months of age (Fig. 2b). The standardized data from both metrics from 0 to 100% EC/A exhibited a more apparent, but highly variable and performed nonlinear regression analyses. Seeding AT8 signal from early ages, but this did not rise above reached 10% (S ) and 50% (S )ofmaximum signal 10 50 the baseline until 7 months (Fig. 2b). The low dynamic earlier than AT8 pathology both in the DG and EC/A range of AT8 staining in this mouse model hinders (Additional file 2: Figure S2b). We also plotted seed- quantitative assessment of pathology using this metric. ing activity against the AT8 pathology from individual Next, we measured seeding activity in 1 mm punch bi- mice. Seeding activity increased earlier than AT8 opsies from fixed tissue slices immediately adjacent pathology in individual mice. We observed robust (50 μm) to those stained for AT8 pathology. We homog- phospho-tau pathology above 6 months of age in the enized biopsies from the DG and EC/A, transduced DG and above 3 months in the EC/A, whereas seed- them into the biosensor cell line, and assessed them ing scored positive much earlier (Additional file 2: after 24 hours (Fig. 2c). In PS19 mice, the DG and EC/A Figure S2c). We conclude that tau seeding activity exhibited higher seeding activity at 2 vs. 1 month. As re- measured in fixed tissue is a robust and highly sensi- ported earlier with unfixed samples [13], we observed a tive metric for tau pathology, and anticipates AT8 1000-fold dynamic range between negative control WT staining in this mouse model. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 6 of 12 Fig. 2 Seeding activity increases with age and anticipates AT8 pathology in PS19 mice. a AT8 staining of phospho-tau pathology in the dentate gyrus (DG) and entorhinal cortex/amygdala (EC/A) of a 12 month old WT mouse, and in PS19 mice at 1, 3, 6, 9, and 12 months of age. AT8 staining increases with age in PS19 mice. See Additional file 2: Figure S2a for representative images of whole-brain slices from 12-month-old WT and PS19 mice. b AT8 tau pathology was quantified in the DG and EC/A of WT and PS19 mice. WT mice were collected from various ages. Six PS19 mice at each age were assessed for tau pathology. Threshold analysis was performed to quantify the percentage of area occupied by AT8 staining in the region of interest. DG AT8 pathology did not rise above baseline staining in 1 month mice until 5 months of age. EC/A AT8 pathology did not show significant increases until 7 months of age. See Additional file 2: Figure S2b for nonlinear regression model of time-course AT8 staining data, and Table 1 for details regarding mice used in this study. c. Seeding activity was assessed from adjacent free-floating brain slices of WT and PS19 mice. 1 mm punch biopsies from the DG and EC/A were homogenized and transduced into biosensor cells. Tau seeding activity increased above baseline by 2 months of age for both the DG and EC/A. See Additional file 2: Figure S2b for nonlinear regression model of tau seeding activity time course data, and Additional file 2: Figure S2c for a direct comparison of seeding activity versus AT8 tau pathology. Error bars = S.E.M; * = p < .05; ** = p <.01 Quantification of spreading tau pathology neurofibrillary tangle (NFT)-like pathology in CA1 and Tau pathology progressively accumulates along neuronal CA3, while DS10 induced mossy-fiber pathology networks in AD patient brains [4]. We have developed (Fig. 3c,d). By 6 weeks, we observed spreading of an in vivo model of tau propagation through inoculation phospho-tau pathology into CA1 of the contralateral of distinct tau strains (DS1 (monomer control), DS9, hippocampus of DS9-inoculated mice. At this time point DS10) derived from stable cells into the hippocampus of in mice inoculated with DS10, we observed mossy fiber PS19 mice (Fig. 3a) [22]. To compare the utility of the pathology in the contralateral hippocampus, but limited fixed tissue seeding assay with IHC, we collected brain CA1 pathology (Fig. 3c,d). samples at 3, 6, or 12 weeks after injection and assessed We quantified tau histopathology by determining the pathology by AT8 stain or seeding activity (Fig. 3b, percentage area covered by AT8 staining in the ipsilat- Table 2). As previously described, DS9 produced eral and contralateral hippocampi of inoculated mice. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 7 of 12 Table 1 Aging PS19 mouse study whether tau strains similarly retain their conformations after fixation, we homogenized fixed hippocampi from Age at Collection N (Total) Male Female mice inoculated with DS9 or DS10 12 weeks post- P301S Mice injection. We transduced hippocampal lysate into the 1 month 6 3 3 LM1 biosensor cell line. After four days, we used FACS 2 month 6 3 3 to isolate aggregate-containing monoclonal cell lines in 3 month 6 3 3 96-well plates. 4 month 6 3 3 We examined the wells for cell colony growth at 7– 5 month 6 3 3 10 days post-plating; 8% of DS9 derived wells and 5% of DS10 derived wells contained aggregate-positive 6 month 6 3 3 cell colonies at 7–10 days. As observed previously, 7 month 6 3 3 theinitial passageof isolatedmonoclonalsecondary 8 month 6 3 3 cell lines is toxic. Thus, 24 DS9-derived secondary 9 month 6 3 3 lines and 9 DS10-derived lines were isolated. Three 10 month 6 3 3 secondary cell lines from each DS9 inoculated mouse 11 month 6 3 3 and all DS10 derived cell lines (2–4 lines per mouse) were analyzed for morphology and/or seeding activity 12 month 6 3 3 (Fig. 4a). WT Mice Secondary cell lines derived from mice inoculated 1 month 4 3 1 with DS9 and DS10 displayed cellular morphologies 2 month 1 0 1 identical to the original strains as seen in Fig. 3a 5 month 1 0 1 (e.g., nuclear speckles versus no nuclear speckles and 6 month 1 1 0 a large juxtanuclear aggregate) (Fig. 4b). This is con- sistent with previous results from fresh frozen tissue, 9 month 1 1 0 which demonstrated that tau strains stably template 10 month 1 0 1 onto humantau expressedinPS19miceand then 12 month 1 0 1 back into LM1 cells [22]. Secondary cell lines derived from fixed tissue showed similar seeding activity to DS1-injected mice exhibited limited AT8 pathology at the original DS9 and 10 strains (Fig. 4c). Thus, tau this time point. DS9 and DS10 inoculates induced in- strains retain their conformation and strain-specific creasing levels of pathology in the hippocampus, but properties in young and aged mice even after threshold analysis of AT8 staining was difficult in fixation. DS10 mice owing to the subtle mossy fiber phenotype (Fig. 3e). To assess tau seeding activity, we dissected and ho- Quantification of seeding activity in fixed tissue from the mogenized ipsilateral and contralateral hippocampi from human brain fixed free-floating 50 μm brain sections of inoculated To test the seeding assay in formaldehyde-fixed human mice. We transduced hippocampal tissue into biosensor tissue samples, we examined the transentorhinal cortex cells, and quantified seeding activity by flow cytometry and Ammon’s horn (CA1/3) of five individuals with dif- after 24 hours. Mice inoculated with DS1 exhibited a ferent stages of tau pathology (Table 3) [4]. While we minimal signal possibly due to the endogenous seeding observed robust seeding from PS19 mice, they express activity present at this age (Fig. 2c, Fig. 3f). Mice inocu- an aggregation-prone form of tau at approximately 5x lated with DS9 and DS10 exhibited robust seeding the level of endogenous tau expression in humans [27]. activity that increased over time in the ipsilateral hippo- To increase the sensitivity of the assay, human samples campus. We also detected clear seeding activity in the were added to biosensor cells at 10x concentration and contralateral hippocampus 3 weeks after injection with the incubation period was extended to 48 hours. Sub- DS9 or DS10, in contrast to the AT8 staining. We con- jects with no tau pathology or with very subtle pretangle clude that seeding activity in fixed tissue anticipates the pathology (stages a/1b) did not display detectable seed- spread of tau pathology and is robust prior to AT8 path- ing activity in either region. In contrast, tissue from indi- ology detectable by immunohistochemistry. viduals with NFT stages III and V tau pathology contained seeding activity in both the transentorhinal Fixed tissue retains strain-specific conformations cortex and Ammon’s horn (Fig. 5; Table 3). Thus, tau After fixation, PrP and Aβ retain their ability to serve as seeding activity can be quantified from fixed mouse and distinct conformational templates [9, 19]. To test archival human tissue alike. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 8 of 12 Fig. 3 Seeding activity detects spread of tau pathology. a Tau strains DS1, 9, and 10 have different inclusion morphologies. DS1 does not contain aggregated tau. DS9 cells feature nuclear speckles, while DS10 cells have a large juxtanuclear aggregate and no nuclear speckles. b Cell lysate from DS1, 9, or 10 was inoculated into the hippocampi of young PS19 mice. At 3, 6, or 12 weeks, brains were collected for tau histopathology and seeding analysis. c Inoculation of DS1 did not induce AT8 pathology. Mice inoculated with DS9 developed NFT-like AT8 pathology in CA1 of the ipsilateral hippocampus by three weeks. This pathology spread to the contralateral hippocampus by 6 weeks. DS10 produced limited tau pathology in this region. d DS9 inoculation induced neurofibrillary tangle-like pathology in CA3 of the ipsilateral hippocampus, and limited pathology in the contralateral hippocampus by six weeks. DS10 inoculation primarily induced mossy fiber AT8 pathology that progressed over time, and spread to the contralateral hippocampus by 12 weeks. e The percentage of the hippocampus covered with AT8 tau pathology was assessed in mice inoculated with DS1, 9, and 10 at each time point. Tau AT8 pathology and spread was apparent in DS9 inoculated mice. However, DS10 mossy fiber pathology was difficult to detect with this technique and showed variable pathology among animals (f). Tau seeding activity was detected in ipsilateral and contralateral hippocampi of DS9 and DS10 inoculated mice at 3 weeks. Seeding activity increased by 12 weeks, suggesting tau pathology continues to develop over time. ANOVA analysis was performed by comparing samples within each time point to DS1 inoculated controls. Error bars = S.E.M, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 Discussion from miniscule amounts of fixed tissue (approximately Propagation of tau aggregation along neuronal networks .04 mm )topermitdirectcomparisonwithtissues may mediate the progressive accumulation of pathology stained by IHC. observed in tauopathy patients. To measure tau seeding We first tested this method in PS19 mice that overex- activity in well-characterized human brains, it will be ne- press full-length human tau (1 N,4R) containing the cessary to analyze formaldehyde-fixed tissues. We now P301S mutation. We drop-fixed brain samples that had present a method for extracting tau seeding activity been embedded either in paraffin or PEG and sectioned Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 9 of 12 Table 2 Inoculation model of spread of tau pathology them coronally for microscopy. We analyzed adjacent 50 μm sections using standard IHC to detect phospho-tau Time N (Total) Male Female Average Age at Average Age at Course Surgery (Days) Collection (Days) or 1 mm circular punch biopsies of tissue for seeding as- 3 weeks says. We homogenized punch biopsies by water-bath son- ication in closed tubes, and assayed them in a cellular DS1 2 0 2 94 115 FRET bioassay system as described previously [10, 13]. DS9 4 0 4 90 111 Tau seeding activity tracked the development of path- DS10 4 0 4 91 112 ology more efficiently than IHC, with a lower degree of 6 weeks inter-animal variation, and a higher dynamic range. This DS1 3 0 3 77 119 was perfectly comparable to previously obtained results DS9 4 1 3 80 122 using fresh frozen tissue [13]. In addition, we detected seeding activity relatively early in the course of disease DS10 4 0 4 80 122 (1–2 months) and it steadily increased over time. Next, 12 weeks we tested brain tissues from animals previously inocu- DS1 2 0 2 71 155 lated with two distinct tau prion strains. We recovered DS9 4 2 2 70 154 these strains from fixed mouse brain tissue as accurately DS10 3 1 2 71 155 as we had previously from fresh frozen tissue. Finally, we tested the extraction method in fixed human brain tissue with documented AT8-positive tau pathology, including AD, and readily detected tau seeding activity in cases ar- chived for up to 27 years in formaldehyde. Fig. 4 Strain-specific properties are retained after fixation. a Fixed hippocampi were isolated from PS19 mice at 12 weeks post injection with DS9 or 10. This tissue was homogenized and transduced into the original LM1 cell line. Fluorescence-activated cell sorting was used to isolate monoclonal cells into 96-well plates. Cells that stably propagated aggregates were amplified and characterized. b Confocal images of representative secondary cell strains derived from mice inoculated with DS9 or 10. Secondary strains displayed the same inclusion morphology as the original inoculum (nuclear speckles or a large juxtanuclear aggregate). See Fig. 3a for images of original strain morphology. c. Seeding activity was assessed for DS9 and 10, as well as secondary cell lines. Cell lysate from each line was transduced into biosensor cells and assessed for tau seeding activity after 24 hours (2 μgper well). Secondary strains showed similar seeding activity to the original inoculum. See Table 2 for additional information regarding the mice used in this study Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 10 of 12 Table 3 Human tauopathy and control subjects Patient Case NFT/Aβ/α-synuclein Stage M/F Age Year of formalin Notes fixation 1 a/0/0 M 31 1989 Control 2 1b/0/0 M 42 1989 Control 3 III/0/0 F 90 1992 No clinical AD diagnosis 4 III/2/0 M 65 1989 No clinical AD diagnosis 5 V/3/0 M 88 1990 Clinical AD Seeding activity Moreover, we detected seeding activity in a small sam- Our laboratory previously detected tau seeding activity ple of human tauopathy cases that were collected and in fresh frozen brain tissue from mouse tauopathy stored in formalin for over 20 years prior to this study. models and human AD cases[11, 13]. However, fresh We observed lower seeding activity in these human sam- frozen samples are much more difficult to obtain than ples than in PS19 mice, probably because of the overex- fixed tissue sections, must be carefully stored at−80 °C, pression of an aggregation-prone form of tau in this and are very challenging to dissect precisely to isolate mouse model. However, the length of fixation may affect specific brain regions. The assay described here accur- the level of seeding observed in samples. Further, differ- ately quantifies tau seeding from fixed tissue sections ences in seeding activity observed between patients at over three log orders of signal. Remarkably, in a mouse Braak stage III and V likely reflect differences in the model from which we sampled tissue at different time level of tau aggregate burden between these patients, cell points, fixed tissue seeding proved comparable to seed- loss, or ghost-tangle formation at later disease stages. ing activity detected in fresh frozen tissue. Thus, we ex- Given the early detection of seeding activity relative to pect that this assay will enable assessment of tau seeding AT8 staining in PS19 mice, we anticipate that this activity in a range of fixed tissues at a similar level of assay could represent a more sensitive metric of tau sensitivity to fresh frozen samples. pathology. Additional studies in a large number of Fig. 5 Fixed human brain samples with tau pathology exhibit seeding activity. AT8-immunostained (hyperphosphorylated tau, DAB) 100 μm sections from cases 3 (a) and 4 (b) in Table 3. In NFT stage III, the tau pathology in the hippocampal formation increases. a,b. The entorhinal layers pre-α and, in addition, pri-α become heavily involved. Tau pathology extends through the transentorhinal region into the adjoining high order sensory association areas of the temporal neocortex but not yet into the superior temporal gyrus. c The NFT in the late stage V case shown here is not identical to case 5 in Table 1 but is from another Alzheimer’s disease patient in her eighties. During NFT stage V, tau lesions develop in the superior temporal gyrus and progress into first order sensory association and premotor areas of the neocortex. d Fixed tissue was isolated from the transentorhinal cortex and the hippocampus (CA1/3) of 100 μm human brain sections that were blinded prior to collection (Table 3). Samples were homogenized and transduced into tau biosensor cells. The integrated FRET density was normalized to a negative control treated only with Lipofectamine. Error bars = S.E.M, ** = p < 0.01, **** = p < 0.0001 Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 11 of 12 well-characterized human tissue samples will help ad- tau pathology based on seeding activity and is also sensi- dress these important questions, and provide additional tive to strain composition. We anticipate that punch biop- insight into the progression of seeding activity in human sies taken from tissue sections will be useful to measure tauopathies. strain identity with high anatomical precision. By carefully Earlier work described a dose-dependent increase in comparing seeding activity and strain composition with tau seeding activity in the PS19 mouse tauopathy model standard neuropathology, it should be possible to add new [13]. However, the regional specificity possible with fresh dimensions to analyses of tissue samples from a range of frozen tissue was limited to gross dissection. We now neurodegenerative diseases. In turn, this will facilitate have reliably isolated and characterized punch biopsies more widespread testing of the putative role of tau prion as small as 1 mm diameter x 50 μm (or ~ .04 mm ). activity in human tauopathies. When we quantified the level of seeding activity at in- creasing ages vs. the tau pathology observed in adjacent Additional files tissue slices using anti-tau AT8 staining, we easily de- tected tau seeding activity, even in fixed tissue sections Additional file 1: Figure S1. Fixed tissue reliably seeds tau aggregation. a. Comparison of fixed and fresh tissue seeding from aged PS19 mice. WT with a minimal AT8 signal. For example, when PS19 mouse tissue did not induce seeding. Seeding displays a dose-response, mice were inoculated with tau strains, we induced strong and no significant difference was detected at each concentration. b. Tau AT8 pathology with DS9, whereas DS10 produced a seeding was equivalently detected from aged PS19 brain tissue embedded in either paraffin or polyethylene glycol. Paraffin embedded tissue requires weak signal. In both cases, the pathology spread from heated ethanol washes to remove excess wax prior to homogenization for the site of inoculation to connected regions, as described robust seeding. A sham sample (Lipo) was used as a negative control. elsewhere [22]. The fixed tissue seeding assay more read- (PDF 97 kb) ily detected the spread of tau pathology in this propaga- Additional file 2: Figure S2. Seeding activity precedes AT8 pathology in PS19 mice. a. Representative images of 12 month WT and PS19 mouse tion model. Furthermore, we readily detected seeding hemi-brain slices stained with AT8. No AT8 staining was detected in WT activity in DS10 inoculated mice despite the relatively mice, whereas PS19 mice exhibited robust phospho-tau pathology subtle AT8 staining phenotype induced by this strain throughout the brain. b. Schematic of punch biopsy, transduction, and seeding assay workflow. c. Seeding and AT8 pathology time course data (mossy fiber dots). Consequently seeding activity can were modeled with nonlinear regression analysis using log (agonist) serve as an important measure of tau pathology when versus normalized response (variable slope). S and S refer to the 10 50 routine AT8 staining reports otherwise minimal path- time point at which seeding or AT8 pathology reaches 10% or 50% of maximal signal, and is represented in months. Seeding preceded ology. The combination of precise quantification of AT8 pathology in both the DG and EC/A. d. Scatter plot analysis of seeding activity with the ability to sample brain tissue to tau seeding activity versus AT8 pathology for each animal. Seeding 1 mm resolution indicates that this method could help activity increases before robust AT8 pathology is observed in the DG and EC/A. (PDF 440 kb) define the seeding activity in human brain with remark- ably high accuracy. Acknowledgements The authors SKK and MID thank Bill Eades for expert advice and guidance. Detection of tau strains in formaldehyde-fixed tissue The authors (HB, KDT) thank the Hans and Ilse Breuer Foundation (Frankfurt Prior experimental work indicates that distinct tau ag- am Main, Germany) for generously supporting their research and Mr. David gregate conformations may underlie different patterns of Ewert (University of Ulm) for technical assistance with the graphics (Fig. 5a- c). This work was supported by the Siteman Flow Cytometry core and Hope pathology, rates of progression, and disease phenotypes Center Alafi Neuroimaging Laboratory at Washington University in St. Louis, observed in distinct tauopathies [2, 7, 22]. Distinct tau and the Neuro-Models Facility and Whole Brain Microscopy Facility at University strains are associated with different tauopathies [22], and of Texas Southwestern Medical Center. inoculation of unique tau strains produces different pat- Funding terns and tau pathology rates of progression [16]. We This work was funded by NIH/NIA grant F30AG048653 (S.K.K); NIH/ observed that fixed tissue from mice inoculated with NIAR01AG048678, NIH/NINDSR01NS071835, the Tau Consortium, and the DS9 and DS10 produced strain phenotypes identical to Cure Alzheimer’s Fund (M.I.D.); Hans and Ilse Breuer Foundation (Frankfurt am Main, Germany) (KDT and HB). the original strains upon inoculation into LM1 biosensor cells. Thus, tau strains are stable upon fixation. We an- Availability of data and materials ticipate that formaldehyde-fixed tissues will serve as an The datasets used and/or analyzed during the current study are available invaluable resource to examine the role of strain com- from the corresponding author by reasonable request. position in tauopathies. Authors’ contributions Studies that use traditional IHC techniques to detect SKK designed and performed all animal, cell culture, and flow cytometry tau pathology have provided important insights into the experiments. TLT assisted with tissue collection and immunohistochemistry progression and anatomy of macromolecular accumula- of animal experiments. KDT and HB performed all human tissue collection, IHC and neuropathological staging. MID assisted with the design and tions of tau assemblies. However, these methods cannot interpretation of all animal and flow cytometry experiments. All authors discriminate among distinct strains, nor can they detect assisted in the writing and figure preparation for this manuscript. All authors submicroscopic tau assemblies. The present assay measures read and approved the final manuscript. Kaufman et al. Acta Neuropathologica Communications (2017) 5:41 Page 12 of 12 Competing interests 13. Holmes BB, Furman JL, Mahan TE, Yamasaki TR, Mirbaha H, Eades WC, MID is co-developer of an anti-tau antibody currently in clinical trials (C2N Belaygorod L, Cairns NJ, Holtzman DM, Diamond MI (2014) Proteopathic tau 8E12 [NCT02494024]). The remaining authors declare that they have no seeding predicts tauopathy in vivo. Proc Natl Acad Sci 111:E4376–E4385. competing interests. doi:10.1073/pnas.1411649111 14. Hoover CE, Davenport KA, Henderson DM, Pulscher LA, Mathiason CK, Zabel Consent for publication MD, Hoover EA (2016) Detection and Quantification of CWD Prions in Fixed Not applicable. Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion. Sci Rep 1–10. doi: 10.1038/srep25098 Ethics approval and consent to participate 15. Hyman BT, Phelps CH, Beach TG, Bigio EH, Cairns NJ, Carrillo MC, Dickson All animal maintenance and experiments adhered to the animal care and DW, Duyckaerts C, Frosch MP, Masliah E, Mirra SS, Nelson PT, Schneider JA, use protocols of the University of Texas Southwestern Medical Center, and Thal DR, Thies B, Trojanowski JQ, Vinters HV, Montine TJ (2012) National Washington University in St. Louis. The autopsy brains used for this study Institute on Aging-Alzheimer“s Association guidelines for the were obtained in compliance with Ulm University ethics committee neuropathologic assessment of Alzheimer”s disease. 8:1–13. doi: 10.1016/j. guidelines as well as German federal and state law governing human tissue jalz.2011.10.007 usage. 16. 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Published: Jun 7, 2017

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