tau oligomers impair artificial membrane integrity and cellular

Tau Oligomers Impair Artificial Membrane Integrity andCellular Viability*□S

Received for publication, June 28, 2012, and in revised form, October 26, 2012 Published, JBC Papers in Press, November 5, 2012, DOI 10.1074/jbc.M112.396176

Katharina Flach‡, Isabel Hilbrich‡, Andrea Schiffmann‡, Ulrich Gärtner‡, Martin Krüger§, Marion Leonhardt‡,Hanka Waschipky¶, Lukas Wick�, Thomas Arendt‡, and Max Holzer‡1

From the ‡Department of Molecular and Cellular Mechanisms of Neurodegeneration, Paul Flechsig Institute of Brain Research,Faculty of Medicine, Jahnallee 59, University of Leipzig, 04109 Leipzig, Germany, the §Institute of Anatomy, 04103 Leipzig,Germany, the ¶Institute for Medical Physics and Biophysics, 04107 Leipzig, Germany, and the �Department of EnvironmentalMicrobiology, Helmholtz Centre for Environmental Research, 04318 Leipzig, Germany

Background:Tau aggregation is amultistep process. The identity of Tau species compromising cell viability remains largelyunknown.Results: Analysis of Tau aggregation dynamic identifies oligomeric Tau aggregates as toxic species that impairviability.Conclusion:Membrane leakage induced by oligomeric Tau is a mechanism for toxicity.Significance: Tau belongs to the class of amyloidogenic proteins that share a common toxicity-mediating mechanism.

The microtubule-associated protein Tau is mainly expressedin neurons, where it binds and stabilizes microtubules. InAlzheimer disease and other tauopathies, Tau protein has areduced affinity toward microtubules. As a consequence, Tauprotein detaches from microtubules and eventually aggregatesinto �-sheet-containing filaments. The fibrillization of mono-meric Tau to filaments is a multistep process that involves theformation of various aggregates, including spherical and proto-fibrillar oligomers. Previous concepts, primarily developed forA� and �-synuclein, propose these oligomeric intermediates asthe primary cytotoxic speciesmediating their deleterious effectsthrough membrane permeabilization. In the present study, wethus analyzed whether this concept can also be applied to Tauprotein. To this end, viability and membrane integrity wereassessed on SH-SY5Y neuroblastoma cells and artificial phos-pholipid vesicles, treated with Tau monomers, Tau aggregationintermediates, or Tau fibrils. Our findings suggest that oligo-meric Tau aggregation intermediates are the most toxic com-pounds of Tau fibrillogenesis, which effectively decrease cellviability and increase phospholipid vesicle leakage. Our dataintegrate Tau protein into the class of amyloidogenic proteinsand enforce the hypothesis of a common toxicity-mediatingmechanism for amyloidogenic proteins.

Alzheimer disease (AD)2 is a neurodegenerative disorder,histopathologically characterized by the formation of extracel-

lular neuritic plaques and intracellular neurofibrillary tangles.Both lesions are made up by fibrillar aggregates of specific pro-teins.Whereas plaques contain theA�-peptide, derived by pro-teolytic cleavage from the amyloid precursor protein, tanglesare composed of the microtubule-associated protein Tau (1).Although plaques and tangles are typical hallmarks of the dis-ease, their precise role in the pathomechanism of the diseaseand how their formation is linked to neurodegeneration are notentirely clear.Under normal conditions, Tau protein is predominantly

localized in axons, where it binds to microtubules and pro-motesmicrotubule assembly (2–5). In AD and some other neu-rodegenerative disorders, such as frontotemporal dementiaand parkinsonism linked to chromosome 17, Tau proteinbecomes hyperphosphorylated and detaches from microtu-bules. This hyperphosphorylated Tau protein, which showsreduced affinity toward microtubules, eventually sequestersinto neurofibrillary tangles (6–7). The distribution of neurofi-brillary tangles correlates well with the loss of neurons and cog-nitive dysfunction in AD (8, 9). The conversion from nativesoluble Tau to aggregatedTau seems to involve conformationalchanges from a random coil to a �-sheet structure and theassembly of �-sheet-containing Tau monomers into filaments(10–12). The increase of �-sheet structures in the Tau mole-cule and the formation of cross-� structures duringTauproteinaggregation classify Tau protein as an amyloidogenic protein(13, 14).Amyloidogenic proteins that can lose their native functional

conformation and convert into�-sheet-containing deposits area characteristic feature of a group of diseases, commonlyreferred to as amyloidosis (15, 16). Although a direct causalrelationship has not unequivocally been proven, a number ofindirect lines of evidence indicate a link between protein aggre-gation and degeneration in these disorders: (i) protein depositsand cell death are often found colocalized (17–19); (ii) in famil-ial forms of amyloidosis,mutations frequently occur in the geneencoding for the amyloidogenic protein (20–23); (iii) these

* This work was supported, in part, by Deutsche ForschungsgemeinschaftGrant HO2368-4/1 and by Alzheimer Forschung Initiative Project 06825.

□S This article contains supplemental Figs. S1–S5.1 To whom correspondence should be addressed. Tel.: 49-341-9725759; Fax:

49-341-9725729; E-mail: [email protected] The abbreviations used are: AD, Alzheimer disease; bis-ANS, 4,4�-dianilino-

1,1�-binaphthyl-5,5�-disulfonic acid; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; DPX, p-xylene-bis-pyridinium bromide; LDH, lactate dehy-drogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; ThT, thioflavin T; SEC, size exclusion chromatography; RH, hydro-dynamic radius.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 52, pp. 43223–43233, December 21, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

DECEMBER 21, 2012 • VOLUME 287 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 43223

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mutations increase aggregation propensity of recombinantproteins (24–27); and (iv) overexpression of amyloidogenicproteins in animal models can lead to a disease-associated phe-notype (28–30).The pathogenic cascade potentially linking amyloidosis to

cell degeneration appears to be a multistep process. It mightinvolve the formation of various aggregation intermediates,such as spherical oligomers, containing 15–40 monomers aswell as chainlike protofibrils composed of several spherical olig-omers. Protofibrils further convert into amyloid fibrils but inaddition might form circular, porelike species, which couldpuncture the cell membrane (31–34). Thus, prefibrillar aggre-gation intermediates rather than the fibrillar aggregation endproduct more likely represent the cytotoxic species (35–38).Accordingly, it has been demonstrated that oligomeric forms ofA�, �-synuclein, and IAPP are toxic to cells (39–41), findingsthat have provided the basis for a concept referred to as the“toxic oligomer hypothesis” (42, 43).Still, for Tau protein, cytotoxicity of intermediates, gener-

ated in the process of fibrillogenesis, has not been investigatedsystematically. Therefore, in the present study, we analyzedwhether a definedTau species generated in the process of fibril-lar aggregation can be identified, accounting for the deleteriousconsequences of Tau fibrillogenesis. In addition, we addressedthe question of whether the cell membrane, which is generallyconsidered to be a primary target of amyloidogenic com-pounds (44–46), might be involved in the cascade of patho-genic events.To this end, viability and membrane integrity were assessed

on SH-SY5Y neuroblastoma cells and artificial phospholipidvesicles, treatedwithTaumonomers, Tau aggregation interme-diates, or Tau fibrils. Our data suggest that Tau aggregationintermediates are themost toxic compounds of Tau fibrillogen-esis, which effectively decrease cell viability and increase phos-pholipid vesicle leakage. The present findings integrate Tauprotein into the class of amyloidogenic proteins and enforce thehypothesis of a common toxicity-mediating mechanism foramyloidogenic proteins.

EXPERIMENTAL PROCEDURES

Expression and Purification of Recombinant Tau Protein—The prk172-ht40 plasmid, encoding the longest human Tauisoform (Tau; 441 amino acids, including exons 2, 3, and 10)was provided courtesy ofM. Goedert (Cambridge, UK) (1). Theintegrity of the plasmid was confirmed by automated sequenc-ing. Tau purification was performed using a modified proce-dure described by Yoshida and Goedert (47). Briefly, prk172-ht40 transformed E. coli were extracted with extraction buffer(50 mM Tris, pH 7.4, 5 mM EDTA, 0.1 mM DTT, 1% TritonX-100, 1 mM PMSF). Supernatant was cleared using a diethyl-aminoethyl cellulose column and afterward loaded onto a phos-phocellulose column. Tauwas eluted stepwise in 5-ml fractionswith NaCl (0.1–0.5 M). The purest fractions containing Tauwere pooled, and Tau was precipitated by ammonium sulfatewith 60% saturation. Tau precipitate was solubilized in gel fil-tration buffer (50mMMES, pH 6.5, 50mMNaCl, 0.1mMEDTA,and 1 mM PMSF) with 0.5% �-mercaptoethanol, heat-dena-tured, cleared, and loaded onto aHiPrep Sephacryl S300HR gel

filtration column.Tau fractionswere pooled, concentrated, anddialyzed against 5 mM MOPS, pH 7.0, 50 mM NaCl, 0.1 mM

EDTA, and 0.1 mM PMSF).Tau Filament Assembly—Aggregation of 400 �l of recombi-

nant Tau protein (100 �M) was carried out with the aid of hep-arin (�14 kDa; BDH) in a final concentration of 0.4 mg/ml asdescribed byHaase et al. (48). Aggregationwas performed for 0,6, 24, 48, 72, or 144 h.Detection of Tau FilamentAssembly—Aggregation of recom-

binant Tau protein was monitored by SDS-PAGE, 4,4�-diani-lino-1,1�-binaphthyl-5,5�-disulfonic acid (bis-ANS), and thio-flavin T (ThT) fluorescence spectrometry and electronmicroscopy. A 2-�l aliquot of each aggregation samplewas sep-arated on 10% SDS-PAGE and stainedwith Coomassie BrilliantBlue R250 followed by a densitometric analysis. Quantitativeaggregation analysis via bis-ANS fluorescence spectrometry(excitation 360 nm, emission 485 nm) and ThT fluorescencespectrometry (excitation 430 nm, emission 485 nm)was carriedout in a volume of 100 �l containing 27 mMMOPS (pH 7.4), 20�M bis-ANS, or 5 �MThT and 5% (v/v) aggregation sample in ablack 96-well plate (HTS 7000 Bio Assay Reader, PerkinElmerLife Sciences). For electron microscopy, aggregation sampleswere diluted 1:5 in aqua dest (B. Braun Melsungen AG) andplaced on carbon-coated 200-mesh gold grids with a diameterof 3.05 mm (S160A, Plano) for 2 min. Grids were washed withaqua dest for 2 min and stained with 2% (w/v) uranyl acetate inaqua dest for a further 2min. Grids were analyzed in a LEO 912OMEGA electron microscope (Zeiss), and microphotographswere taken. Diluted samples from FPLC purification were con-centrated by centrifugation for 2 h at 200,000 � g before trans-fer of 1.5 �l on Formvar-coated copper grids for ultrastructuralanalysis. After uranyl acetate staining, samples were analyzedwith a Zeiss EM900 transmission electron microscope (ZeissNTS, Oberkochen, Germany).Characterization of Tau Aggregation Intermediates by Size

Exclusion Chromatography (SEC)—400 �l of aggregated Tauprotein was centrifuged at 1000� g for 10min and then loadedonto a HiPrep 16/60 Sephacryl S-500 HR and eluted with gelfiltration buffer (50 mM MOPS, pH 7.0, 50 mM NaCl, 0.1 mM

EDTA at 0.5 ml/min). Elution of Tau protein was monitored at280 nm, and 2-ml fractions were collected and stored at 4 °C.Each fraction was analyzed for protein content using Fluram(Fluka) and for �-sheet content using thioflavin S and bis-ANS.Fluram is a sensitive fluorimetric reagent for detecting primaryamines. Using a black microtiter plate, we combined 80 �l of aFPLC fraction with 20 �l of 100 mM phosphate buffer, pH 8.0,and 50�l of 1mM Fluram in acetone. After 5min of incubation,we measured fluorescence using 355-nm excitation and480-nm emission filters. Protein content was calculated usingBSA as a dilution curve because content of the primary aminesin both proteins is similar (arginine content 3.2% versus 3.9%and lysine content 10% versus 10.1% in 441-amino acid (2N-terminal inserts 4 microtubule-binding repeats (2N4R)) Tauprotein and BSA, respectively). �-Sheet content was measuredwith ThT and bis-ANS as described above using 80 �l of eachFPLC fraction.Dynamic Light Scattering—Dynamic light scattering meas-

urements of FPLC fractions were performed with a Zetasizer

Toxicity of Intermediate Tau Aggregates

43224 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 52 • DECEMBER 21, 2012


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Nano ZS (Malvern, Herrenberg, Germany) containing a 5-mil-liwatt helium-neon 633-nm laser at 173° measurement angle.Sample were thermally equilibrated at 25 °C for 2 min. Particlesize was obtained as an average of two measurements with 25runs each and expressed as volume distribution.Detection of Tau Oligomers via Sandwich ELISA—A 96-mi-

crowell plate with MaxiSorb surface and flat bottom cavities(Nunc)was coated overnightwith 5�g/mlAffiniPure goat anti-rabbit IgG (H�L) (Jackson ImmunoResearch Laboratories) incoating buffer (50mMTris, pH 8.0, 1mMEDTA). Furtherwash-ing and incubation steps were carried out using Tris-bufferedsaline containing 0.1% (v/v) Tween 20 (TBS-T). Coated platewas washed and incubated for 1 h with 2 �g/ml rabbit (poly-clonal) anti-oligomer antibody (A11) (Invitrogen). The platewas washed, blocked for 2 h with 2% (w/v) skimmed milk pow-der, and afterward incubated overnight with Tau aggregationsamples diluted 1:1000 in aqua dest. After washing, the platewas incubated for 1 h with 0.25 �g/ml detection antibody anti-TAU1 monoclonal antibody (mouse IgG2a) (Chemicon), fol-lowed by a 1-h incubation with ImmunoPure antibody goatanti-mouse IgG�IgM (H�L), horseradish peroxidase-labeled(Pierce) and diluted 1:3000. Detection was performed with100 �l/well chromogen solution (2.4 mM o-phenylenedi-amine, 0.03% H2O2 in 0.1 M acetate buffer, pH 5.0). Thereaction was stopped after 15 min with 6 M HCl. Absorptionwas analyzed at 492 nm (HT3 microtiter plate reader,Anthos Mikrosysteme).Cell Culture, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltet-

razolium bromide (MTT), and Lactate Dehydrogenase (LDH)Assay—For the incubation of SH-SY5Y cells with aggregatedTau protein, cells weremaintained in a 96-well plate inDMEM/Ham’s F-12 medium supplemented with 15% (v/v) fetal bovineserum (FBS), 2 mM L-glutamine, non-essential amino acids(BiochromAG), 50 �g/ml gentamicin (PAA Laboratories), and15 mM HEPES, pH 7.4, in a 5% CO2 environment at 37 °C for24 h. Afterward, cells were maintained for 24 h in 185 �l of thedescribed medium containing only 2% (v/v) FBS. Then 15�l/well aggregated Tau protein were added to the cells for 48 h.MTT (Sigma) was added in triplicate to cells to a final concen-tration of 1mg/ml, and cells were incubated for 2 h. After incu-bation, cell culture medium with MTT was removed, 200 �l ofDMSO and 25 �l of Soerensen’s glycine buffer (0.1 M glycine,pH 10.5, 0.1 M NaCl) per well were added, and absorbance val-ues were measured at 550 nm (HT3 microtiter plate reader).Absorbance values of SH-SY5Y cells cultures treated with Tauprotein were normalized to heparin-only-treated SH-SY5Y cellcultures at each aggregation time point. The LDH assay wasperformed according to the manufacturer’s recommendations(Roche Applied Science). Absorbance values were measured at492 nm (HT3 microtiter plate reader). The obtained absorb-ance values were normalized as mentioned for the MTT assay.Cellular Tau Protein Uptake—400 �l of 48-h preaggregated

Tau was mixed with 1 mg of Alexa488-SDP ester (Invitrogen)after adjusting pH to 7.6 and allowed to react for 1 h. The reac-tion was terminated with hydroxylamine, and labeled Taumonomer and oligomer fractions were purified by gel filtrationusing Sephacryl S-500 HR matrix. Equal quantities ofAlexa488-labeled Tau monomer or oligomer were applied to

SH-SY5Y cells for 24 h. Cellular localization of labeled Tau wasanalyzed by optical sectioning with a Zeiss Apotome fluores-cence microscope after washing cells with 0.1 M acetate buffer,pH 5.0.Vesicle Leakage Assay with Preaggregated Tau—The vesicle

leakage assay was performed as described by Zschörnig et al.(49). Large unilamellar vesicles filled with a 20 mM NaCl solu-tion containing a 12.5 mM concentration of the fluorescencedye 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and a45 mM concentration of the quencher p-xylene-bis-pyridiniumbromide (DPX) were used. For the measurement, 30 �l of thelarge unilamellar vesicle suspension were transferred to 2.5 mlof 10 mM HEPES (pH 7.4) and 150 �l of Tau protein preaggre-gated for 0, 24, or 144 hwas added. Dialysis buffer containing allingredients needed for Tau protein aggregation but withoutTau protein incubated for 24 h served as a control. ANTS fluo-rescence was measured every 15 s over a time of 220 min withan excitation wavelength of 360 nm and an emission wave-length of 530 nm. After 220 min, 100% leakage was determinedby adding 0.1% (v/v) Triton X-100.

RESULTS

Characterization of Tau Aggregation Intermediates—Theinnate ability of recombinant Tau to aggregate in vitro into highmolecular weight adducts is low. Thus, aggregation of recom-binant human 441 amino acid Tau isoform (Tau) was triggeredusing the polyanionic aggregation inducer heparin (50). Tauaggregation was allowed to proceed for up to 144 h at 37 °C.Aggregation of Tau over time in the presence and absence ofheparin was monitored by SDS-PAGE, SEC on SephacrylS-500, ThT and bis-ANS fluorescence spectroscopy, electronmicroscopy, and ELISA with oligomer-specific antibodies.On SDS-PAGE, monomeric Tau runs as a single band at

around 63 kDa. After a 24-h incubation of Tau with heparin,additional bands occurred in the range of 130–200 kDa andincreased with further incubation time (Fig. 1A). Most likely,they represent SDS-stable aggregation products. Alzheimer-like Tau filaments arising during Tau aggregation do not enterthe stacking gel and therefore cannot be quantified via SDS-PAGE. It is noteworthy that monomeric Tau decreases withincreasing incubation time due to the formation of aggregated,high molecular weight Tau species. In contrast, Tau incubatedwithout heparin exhibits a low aggregation tendency. Besidesvisualization of Tau aggregation via SDS-PAGE and proteinstaining, it could also be confirmed that Tau remains stableduring incubation time and is not degraded. The densitometricanalysis of shifted Tau (smear and bands) above the monomerband, representing high molecular weight Tau species, affirmsthe increase of Tau aggregation in the presence of heparin withincreasing incubation time. Aggregated Tau above the mono-mer band occurs only marginally with proceeding incubationtime in the absence of heparin (Fig. 1C).Tau aggregation was also monitored using the fluorescence

dyes ThT and bis-ANS, detecting �-sheet content and exposedhydrophobic patches, respectively (Fig. 1, D and E). Both ThTand bis-ANS fluorescence intensity gradually increased withtime, when Tau was incubated with heparin. ThT fluorescenceand bis-ANS fluorescence intensity reached a steady-state level




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at 48 h (Fig. 1D) and between 72 and 144 h (Fig. 1E), respec-tively. Fluorescence intensity of both dyes did not increase withtime for Tau incubated without heparin (Fig. 1, D and E).To obtain more precise information on the size distribution

and kinetics in the formation of Tau aggregation intermediates,aggregates were sampled after different periods of time (24, 48,72, and 120 h) and subjected to SEC on Sephacryl S-500. Eachchromatographic fraction was characterized with respect toprotein content, �-sheet content, and exposed hydrophobicpatches by fluorescence spectroscopy (Fig. 2). The hydrody-namic radius of peak fractionswas determined by dynamic lightscattering, which allowed us to discriminate between Taumonomers (hydrodynamic radius (RH) � 4.7 nm), oligomers(RH � 14 nm), and higher aggregates (RH � 92 nm) (Fig. 3).Integrating the protein peaks obtained by SEC allowed us toquantify the relative amount of each of these aggregation forms(Table 1). Oligomeric Tau showed its highest amount after 48 hof aggregation and decreased again thereafter. 72 h of aggrega-tion led to the formation of Tau fibrils eluting in the void vol-ume of the SEC column. At the same time point, monomericTau had almost completely disappeared. However, Tau mono-mers reappeared again after 120 h, although with a higher bis-ANS signal than unaggregated Tau, which most likely is due toan altered secondary structure. A prominent finding of Tauaggregate size exclusion chromatography is the presence of asingle, defined oligomeric Tau species with a uniform hydrody-namic radius of 14 nm, which can be calculated to consist of 40Tau protein molecules.

For the qualitative analysis of Tau aggregates, electronmicrographs were taken at different time points (Fig. 4). Smallspherical structures, most likely representing Tau oligomers,were visible after 6 h of Tau aggregation in the presence ofheparin. Short filaments with straight appearance were clearlyvisible after 48 h of Tau aggregation.With proceeding aggrega-tion time up to 144 h, an elongation of the filaments could beobserved. Similar results were obtained using gel filtration-pu-rified aggregation products, which, however, had to be centri-fuged before coating grids (supplemental Fig. S5). In these elec-tron micrographs, oligomeric Tau appears also as sphericalstructures but less complex and with a smaller diameter of 12nm. This value is very close to the hydrodynamic radiusobtained by light scattering.To validate the kinetics of oligomer formation with an

independent method, we quantified the relative amounts ofTau oligomers after different periods of aggregation by asandwich ELISA (Fig. 5). The oligomer-specific antibodydescribed by Kayed et al. (46) was used for capturing Tauoligomers. Preincubated Tau samples were allowed to aggre-gate in the presence or absence of heparin for up to 144 h andwere subsequently diluted 1:1000 to stop aggregation. Tauoligomer content in the presence of heparin increased after6 h and reached its maximum at 48 h. It decreased thereafterand remained stable at an intermediate level. This decreaseof Tau oligomer content at an aggregation time of 72 and144 h might be due to the progressive formation of fibrils,which depletes Tau oligomers. The fact that Tau oligomers

FIGURE 1. Monitoring Tau aggregation. Tau was aggregated for different periods of time (0, 6, 24, 48, 72, and 144 h) with (A) and without (B) heparin. Tauaggregation was visualized by separating aggregation samples by 10% SDS-PAGE, followed by protein staining with Coomassie Brilliant Blue R250. A,increasing aggregation time leads to an increase of high molecular weight Tau aggregates above the Tau monomer band at 63 kDa. B, incubation of Tauwithout heparin does not lead to formation of high molecular weight products. Please note shifted Tau bands in the range of 65–72 kDa in A and B, whichprobably represent conformationally changed monomeric Tau. C, densitometric analysis also reveals an increase of high molecular weight Tau aggregateswith increasing incubation time if Tau fibrillogenesis is induced by heparin. Tau aggregation with and without heparin was also measured by ThT (D) andbis-ANS fluorescence spectrometry (E). Both fluorescence dyes indicate Tau protein aggregation in the presence of heparin, whereas Tau protein aggregationdoes not occur in the absence of heparin. Data are expressed as mean values � S.D. (error bars).




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after 72 h decrease toward a level that is well above the initiallevel at 0 h might indicate an equilibrium reaction main-tained by the still ongoing conversion from monomers tooligomers and its further incorporation into fibrils.Tau Oligomers Decrease SH-SY5Y Cell Viability—SH-SY5Y

cells were incubated for 48 h with Tau samples preaggregatedfor different periods of time. The stability of Tau protein duringincubation of SH-SY5Y cell culture has been confirmed (sup-plemental Fig. S1). The neurotoxicity of preaggregated Tauwasdetermined by theMTT assay, measuringmitochondrial dehy-drogenase activity of treated cells (Fig. 6A). Controls such asTau protein only and heparin only were included for each timepoint to account for the trophic effect of heparin (supplementalFig. S2). SH-SY5Y cells incubated with non-aggregated Tau (0h) exhibit the highest MTT turnover. With increasingpreaggregation time of Tau, MTT turnover by SH-SY5Y cellsdecreased and reached its minimum for a Tau preaggregationtime of 24 h. With further increasing preaggregation time ofTau, MTT turnover increased again slightly without reachingthe initial level at 0 h. This time curve of decreased cell viabilitymatches the formation of oligomeric Tau (Table 1), whichshows its highest amount after 48 h of aggregation, in contrasttomonomericTau (0 h) orTau filaments (144 h).We comparedthe size distribution of 6-h preaggregated Tau protein after a

48-h incubation of SH-SY5Y cells with non-incubated Tau pro-tein and can confirm that Tau aggregation does not progress incell culture medium (supplemental Fig. S3).The membrane integrity of SH-SY5Y cells treated with

preaggregatedTauwas assayed by LDH leakage (Fig. 6B). Therewas only a low LDH leakage for cells incubated with Taupreaggregated for 0 and 6 h, which increased with increasingpreaggregation time. It reached its maximum for a Taupreaggregation time of 48 h and decreased again thereafterslightly (Fig. 6B). This overall pattern of LDH leakage matchesthe pattern of cell viability obtained by the MTT assay (see Fig.6A). Thus, the LDH assay and MTT assay, both indicators ofcell viability, show a highly significant correlation with contentof oligomeric Tau (Fig. 7). To analyze the mode of action, weinvestigated whether oligomeric Tau gains access to the cellmembrane. Alexa488-labeled Tau monomer and oligomerwere applied to SH-SY5Y cells for 24 h. Fluorescence micros-copy revealed more intracellular residence of oligomeric Tauprotein compared with monomeric Tau protein (supplementalFig. S4). Monomeric Tau protein is concentrated in the perinu-clear region, where endocytic vesicles are located.We find olig-omeric Tau protein more evenly distributed with some associ-ation with the cell membrane.

FIGURE 2. Characterization of Tau aggregation intermediates. Tau samples aggregated for different periods of time were separated by SEC on Sephacryl-S500, and fractions were analyzed for protein content (Fluram), �-sheet content (thioflavin S), and disordered structure (bis-ANS). Tau aggregation for 48 hleads to the highest relative amount of oligomeric Tau. After 72-h aggregation, fibrillar Tau appears eluting in the void volume of the SEC column, whereasmonomeric Tau disappears. After 120 h, disaggregation of Tau adducts leads to a reappearance of Tau monomers, which confer a higher bis-ANS signal thanunaggregated Tau protein, probably due to altered secondary structure.




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Tau Oligomers Disturb Membrane Integrity of ArtificialPhospholipid Vesicles—To further specify the effects of Tauaggregates onmembrane integrity, we analyzed its influence onleakage of artificial phospholipid vesicles (Fig. 8). Phospholipidvesicles were filled with the fluorescence dye ANTS and thefluorescence quencher DPX. Upon vesicle leakage by pore for-mation, ANTS and DPX became separated, and the ANTS flu-orescence yield increases. Phospholipid vesicles were treatedwith Tau preaggregated for 0, 24, or 144 h, and the increase offluorescence dye intensity was monitored over a time of 220min. In Fig. 8, the leakage of phospholipid vesicles is plotted asa function of time after applying preaggregatedTau. The vesicleleakage caused by preaggregated Tau appears to be a slow proc-ess. After 50–60min of application of Tau preaggregated for 0,24, and 144 h, a difference in vesicle leakage becomes visible.Tau preaggregated for 24 h causes the most dramatic vesicleleakage, which after 220 min reaches almost 100%. Taupreaggregated for 144 h and non-aggregated Tau (0 h) lead to avesicle leakage of about 70 and 20%, respectively. This probablyindicates that oligomeric Tau, which represents the major con-stituent of theTau sample aggregated for 24 h, evokes the great-est disturbance of membrane integrity.

DISCUSSION

The cytotoxic effects of amyloidogenic proteins have beenexplained by the concept that oligomers formed during theprocess of amyloidogenesis lead to a disturbance of membraneintegrity by a non-channelmechanism or by forming pores intothe membrane (39, 51).The aim of the present study was to analyze whether this

concept can also be applied to the Tau protein, representingan integral part of Alzheimer pathology. We thus analyzedwhether, during Tau aggregation, a toxic species is gener-ated, which can attack cellular membranes. To this end, weincubated the longest isoform of recombinant human Tau inthe presence of heparin for different periods of time andanalyzed the amount of oligomeric Tau that had formed afterthis time. Subsequently, we used these probes and testedtheir effects on cell viability and phospholipid vesicle leak-age. As a result, we obtained a clear cut relationship betweenthe content of oligomeric Tau contained in the probe and itsdeleterious effect on cell viability and phospholipid vesicleleakage.Tau Aggregation and Oligomer Detection—For our experi-

ments, we preaggregated Tau for different time periods in thepresence of heparin. Subsequently, we characterized aggrega-tion intermediates obtained after defined aggregation periodsby (i) SDS-PAGE, (ii) bis-ANS andThT fluorescence spectrom-etry; (iii) electron microscopy, (iv) quantification of Tau oligo-mers with an ELISA using an anti-oligomer antibody, and (v)size exclusion chromatography, followed by an analysis of pro-tein content, �-sheet content, and exposed hydrophobicpatches of each fraction. The hydrodynamic radius of peak frac-tions was measured by dynamic light scattering, which allowed

FIGURE 3. RH of peak fractions was determined by dynamic light scattering, which allowed identification of the peak for monomeric Tau (4.7 nm),oligomeric Tau (14 nm), and higher molecular aggregates (mean value, 92 nm). Monomeric and oligomeric Tau peaks each consist of molecules of similarmolecular size, whereas the high molecular weight peak represents a more heterogeneous mixture of different sized aggregates.

TABLE 1Determination of relative protein content of eluted protein fractionsafter size exclusion chromatography

Aggregationtime

Relative amount of molecular Tau speciesMonomer Oligomer Filament

h % % %0 10024 28.2 66.5 5.348 9.0 87.5 3.572 1.8 56.3 41.9120 25.4 46.7 27.9




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the allocation of protein peaks to monomeric Tau, oligomericTau, and higher molecular Tau aggregates.The biochemicalmechanisms that lead to aggregation of Tau

into paired helical filaments remain not fully understood. It hasbeen shown that sulfated glycosaminoglycans like heparin orheparan sulfate can trigger Tau aggregation in vitro and thatheparan sulfate is coexistent with hyperphosphorylated Tau inpretangle neurons (50). We thus used heparin as an effectivetrigger to accelerate Tau aggregation in vitro. Our SDS-PAGEdata indicate that in the presence of heparin, Tau first under-goes conformational changes and then forms high molecularsize aggregates in a time-dependent manner.Bis-ANS and ThT fluorescence spectrometry enabled us to

detect the partial structural transition from a random coil to amore hydrophobic cross-� structure in our preaggregated Tausamples. The hydrophobic fluorescent probe bis-ANS binds tosolvent-accessible hydrophobic protein structures, resulting inan increase of bis-ANS fluorescence intensity (52). ThT bindsto �-sheet structures, whereupon ThT fluorescence intensityincreases (53, 54). Up to 48 h of Tau preaggregation, weobserved a strong increase of fluorescence intensity withincreasing preaggregation time for both dyes. From 48 to 144 hof Tau preaggregation, we found a slight increase of fluores-

cence intensity with increasing preaggregation time for bothdyes. This result might indicate an equilibrium of Tau fibrillo-genesis that is reached after about 48 h of preaggregation. Still,our SDS-PAGE data indicate a progression of fibrillogenesis upto 144 h. Here we observed a continuous depletion of mono-meric Tau and an increase of high molecular smear up to 144 hof preaggregation. These findings might suggest that fluores-cence dyes have a higher affinity to oligomers and small fibrils,which are more easily accessible than the long and compactfibrils. To quantify the content of oligomers in preaggregatedTau samples, we used two independentmethods, which yieldedconsistent results (i.e. SEC combined with bis-ANS and ThTfluorescence spectrometry and dynamic light scattering of peakfractions and ELISA with an anti-oligomer antibody, A11).The A11 antibody is known to recognize oligomers of amy-

loidogenic proteins independent of the amino acid sequence(46). With a sandwich ELISA, using the A11 antibody for cap-turing oligomers, we detected the highest oligomer content inthe Tau samples aggregated for 24 and 48 h. Tau samplespreaggregated for more than 48 h showed a reduced oligomercontent, probably due to increasing filament formation, whichconsumes Tau oligomers. Tau protein samples preaggregatedfor less than 24 h exhibited the lowest oligomer content, indi-cating that a certain period of time needs to elapse before olig-omers are formed to a detectable amount. These results are inagreement with the findings of Maeda et al. (55), who obtainedthe highest level of granular Tau oligomers consisting of 40� 3Tau molecules after 21 h. The peak formation of oligomericTau after 24–48 h of aggregation time was confirmed throughdynamic light scattering analysis of protein fractions separatedby SEC. The oligomeric Tau species possesses an RH of 14 nm,which amounts to 40 monomers (55). Formation of Tau oligo-mers continuously increased until this time point, when oligo-mers accounted for about two-thirds of total Tau, and slightlydecreased thereafter.Influence of Preaggregated Tau on Cell Viability and Artifi-

cial PhospholipidVesicles—Effects of Tau aggregation interme-diates on cell viability and membrane integrity were analyzedon SH-SY5Y neuroblastoma cells and artificial phospholipidvesicles. SH-SY5Y cells were treated for 48 h with Taupreaggregated for up to 144h.To characterize the effects on cellviability, we performed two assays. We measured MTT reduc-tion and LDH release. MTT turnover of SH-SY5Y cells treatedwith Tau that were preaggregated for at least 6 h was signifi-cantly reduced comparedwith SH-SY5Y cells treatedwith non-

FIGURE 4. Visualization of Tau aggregation intermediates by electron microscopy. For qualitative analysis, Tau aggregation samples were applied tocarbon-coated gold grids, stained with 2% uranyl acetate, and analyzed using a LEO 912 OMEGA transmission electron microscope. After 6 h of aggregation(left), spherical oligomeric aggregates predominate, which after longer aggregation time progressively convert to fibrillar aggregates (48 and 144 h) (middleand right). Magnification was �50,000; scale bar, 242 nm.

FIGURE 5. Quantification of Tau oligomers by ELISA. The oligomer contentin Tau protein samples aggregated for different periods of time (0, 6, 24, 48,72, and 144 h) with or without heparin was quantified by a sandwich ELISA.Tau oligomers were captured with the anti-oligomer antibody A11 anddetected with anti-TAU1 antibody. In the presence of heparin, the Tau oli-gomer content rapidly increased up to 48 h of aggregation and decreasedthereafter toward an intermediate level. Formation of Tau oligomers in theabsence of heparin remains marginal. Error bars, S.D.




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aggregatedTau.Of note, effectsweremost pronounced for cellstreated with probes that had been preaggregated for 24 or 48 h(i.e. which contained the highest amount of Tau oligomers).

Similar results were obtained with the LDH assay, where theoligomer content of probes highly significantly correlated withits deleterious effects on membranes, leading to LDH release.Permeabilizing effects of Tau oligomers on membranes werecorroborated further in the vesicle permeabilization assay,where we measured the leakage of the fluorescence dye ANTSout of phospholipid vesicles. In agreementwith results from theLDH assay, effects were most pronounced for preaggregatedTau with the highest content of oligomers.The present results suggest that Tau oligomers are the cyto-

toxic species ofTau fibrillogenesis that lead to an impairment ofcell viability. Furthermore, our results identify the cellularmembrane as a primary target for Tau oligomers.These findings support previous concepts that amyloido-

genic proteins are produced as soluble proteins that can pro-gressively convert into lower molecular weight oligomers thatrepresent the primary toxic species that contribute to diseaseprogression (43, 51, 56). Amyloid-related diseases might thusbe characterized by a common mechanism of aggregation,cytotoxicity and propagation of pathology, which criticallyinvolves permeabilization of membranes (57, 58). Several mod-els have been proposed on how oligomers might lead to a dis-ruption of cellular membranes, including transmembrane olig-omeric pore structures reminiscent of those of core-formingtoxins (57, 58), nonspecific binding of amyloid oligomers to themembrane surface (51, 56), and detergent-like membrane dis-solution by amyloid fibrils growing on the membrane surface(59). Oligomers of amyloidogenic proteins, for example, aresuspected to form circular porelike structures, which couldpuncture the cell membrane (31–34). Since the original findingbyArispe et al. (39) that A� exhibits ion channel activity in lipidbilayers, channel activity has been described for other amy-loidogenic proteins, such as IAPP (60), �-synuclein (61–63),polyglutamine (64), and prion-derived peptides (65).If one assumes that Tau oligomersmight formadiscrete pore

into the cell membrane that would allow for the permeation ofLDH (121 kDa; estimated radius � 4.6 nm), an inner diameterof at least 4.6 nm would be required. Lashuel et al. (34) showedthat aggregation of 20–26monomers of�-synuclein can form apore with an inner diameter of 2.0–2.5 nm. Because granular

FIGURE 6. Effect of aggregated Tau protein on SH-SY5Y cell viability. Tau aggregation triggered by heparin was allowed to proceed for different periods oftime up to 144 h. SH-SY5Y cells were incubated for 48 h with the different preaggregated Tau samples or preincubated with heparin only as a control. Cellviability was assessed with MTT and LDH assays. Data were normalized to heparin-only-treated SH-SY5Y cell cultures at each preaggregation time point. A, MTTassay. Tau protein preaggregated for 6 h or more decreases cell viability significantly compared with non-aggregated Tau protein (0 h). B, LDH assay. Tauprotein preaggregated for 24 h or more increases LDH leakage significantly compared with non-aggregated Tau protein (0 h). MTT and LDH assays wereperformed in triplicate. Data are mean values � S.D. (error bars) (S.D. includes error propagation in consequence of normalization); differences are significantat p � 0.05 (*) and p � 0.01 (**) (Student’s t test).

FIGURE 7. High Tau oligomer content correlates with decreased cell via-bility. Oligomer content of Tau samples preaggregated for different periodsof time and membrane integrity of treated SH-SY5Y measured by LDH releasecells are highly correlated (r � 0.956, T � 5.61, p � 0.01).

FIGURE 8. Leakage of phospholipid vesicles treated with preaggregatedTau protein. Large unilamellar vesicles filled with the fluorescence dye ANTSand the quencher DPX were treated for 220 min with Tau protein preaggre-gated for 0, 24, or 144 h. During the time of treatment, the fluorescence yield ofANTS was measured. ANTS fluorescence increases upon separation from DPX,which is caused by vesicle leakage. Tau aggregated for 24 h induces the greatestvesicle leakage compared with long-aggregated and non-aggregated Tau.




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Tau oligomers are proposed to consist of �40 monomers (55),formation of an inner pore with diameter of around 4.6 nm isnot unlikely.The effects of Tau aggregates on cell viability and vesicle

leakage in the present experiments were assessed after externalapplication of Tau samples. This external application was cho-sen because (i) recombinant overexpression of Tau in SH-SY5Ycells would implicate a time lag in expression, which could beavoided with an external treatment; (ii) the aggregation state ofTau can be determined precisely prior to external application;(iii) external application more likely attacks the membrane,thereby generating effects that can be related to Tau and itsaggregation state; and (iv) effects are independent of posttrans-lational modifications, whichmightmodulate intracellular Tauprotein. Testing potential cytotoxic effects of Tau as an intra-cellular protein by external application might be counterintui-tive. Still, recent results on dynamic spreading of Tau pathologyin mouse models of Alzheimer disease (66, 67) indicate a tran-scellular propagation of Tau aggregates, which might requirean inside-out and outside-in passage through the cell mem-brane. Because of phospholipid asymmetry in the plasmamem-brane, it remains unclear whether these two passages rely onthe same mechanism. Accordingly, cell culture models haveshown that extracellular Tau aggregates can be internalized,transmitting Tau misfolding from the outside to the inside ofthe cell (68). The present experimental paradigm of externalapplication of oligomeric Tau aggregates, thus, very likely,might play a pathophysiological role in progression of AD. Cur-rent models on spreading of Tau pathology suggest a prion-likemechanism, where misfolded Tau might act as seed propagat-ing further aggregation of Tau. Transmembrane spreading ofTau oligomers might well be involved in this initiation of wide-spread formation of fibrillar Tau pathology inAD. In support ofthis, formation of the MC1 epitope, as one of the earliest path-ological alterations of Tau in AD (69), has been identified asoccurring during oligomerization of Tau (70).In summary, our results suggest Tau aggregation intermedi-

ates to be the toxic species of Tau fibrillogenesis. Tau aggrega-tion intermediates disturb the integrity of biological mem-branes, probably by a pore-forming mechanism.

Acknowledgments—We thank M. Goedert for providing plasmidprk172ht40. We also thank B. Würz for providing help with the Zeta-sizer measurements.

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Marion Leonhardt, Hanka Waschipky, Lukas Wick, Thomas Arendt and Max HolzerKatharina Flach, Isabel Hilbrich, Andrea Schiffmann, Ulrich Gärtner, Martin Krüger,

Tau Oligomers Impair Artificial Membrane Integrity and Cellular Viability

doi: 10.1074/jbc.M112.396176 originally published online November 5, 20122012, 287:43223-43233.J. Biol. Chem.

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tau oligomers impair artificial membrane integrity and cellular

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