Biochimica et Biophysica Acta 1814 (2011) 388–395

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Biochimica et Biophysica Acta

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Pseudophosphorylation of tau protein directly modulates its aggregation kinetics

Edward Chang, Sohee Kim, Kelsey N. Schafer, Jeff Kuret ⁎Center for Molecular Neurobiology, Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine, 1060 Carmack Rd., Columbus, OH 43210, USA

Abbreviations: ODS, Sodium octadecyl sulfate⁎ Corresponding author. Tel.: +1 614 688 5899; fax:

E-mail address: kuret.3@osu.edu (J. Kuret).

1570-9639/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbapap.2010.10.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 September 2010Received in revised form 11 October 2010Accepted 15 October 2010Available online 23 October 2010

Keywords:AggregationAlzheimer's diseaseTau proteinPhosphorylationKinetics

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's diseaseand other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentrationof free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify hownegative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudopho-sphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as afunction of time and submicromolar tau concentrations using electron microscopy assay methods. Kineticconstants for nucleation and extension phases of aggregation were then estimated by direct measurementand mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tauaggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensityby stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative chargeinto the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.

+1 614 292 5379.

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The neurofibrillary lesions of Alzheimer's disease develop intra-cellular aggregates of the microtubule-associated protein tau [1].Although certain familial tauopathies result frommutations in the taugene (MAPT), the pathogenesis of Alzheimer's disease is notassociated with changes in tau amino acid sequence. Rather, lesionformation is accompanied by a 3- to 4-fold increase in tauphosphorylation stoichiometry [2,3]. The covalently bound phosphateis distributed among ~40 sites within and adjacent to the taumicrotubule-binding domain [4–6]. Occupancy of these sites mayinfluence tau aggregation in twoways. First, occupancy of certain sitesmodulates tau-tubulin affinity [7], fostering an increase in the levels offree cytoplasmic tau available to nucleate and support the aggregationreaction [8–11]. Second, hyperphosphorylation can increase tauaggregation propensity directly [12,13]. However, the precise mech-anism of these direct effects has been difficult to establish. Challengesto overcome include the difficulties of recapitulating the complexphosphorylation patterns observed in disease tissue and of quantify-ing the aggregation reaction under controlled conditions. Thechallenge of site occupancy has been addressed through phosphor-ylation mimicry, where phosphorylatable hydroxy-amino acids areconverted to negatively charged Asp or Glu residues. The approachfosters site-specific incorporation of negative charge at full occupancy.Resultant pseudophosphorylation mutants have been shown tomimic phosphorylation-induced changes in tau structure and func-

tion [14–16] and to be recognized by phosphorylation-sensitive anti-tau antibodies [17]. The challenge of aggregation kinetics has beenaddressed by the development of agents that drive efficientaggregation in vitro over tractable time periods and near physiologicalconcentrations of tau protein [18].

Despite these advances, aggregation kinetics in the presence ofexogenous inducers can be difficult to analyze with explicit models.For example, the effects of some inducers, such as heparin, depend onthe concentration ratio between inducer and tau protein [19]. Otherinducers, such as anionic surfactants, micellize on contact with tau[20]. When aggregation reactions are initiated with sodium octadecylsulfate (ODS), for example, the rate of micellization is slow relative toaggregation, and so the early stages of aggregation may be obscured[21,22].

Recently, we found that aggregation of full-length tau atsubmicromolar concentrations can be achieved with Thiazine red[23]. Thiazine red-mediated aggregation can be explicitly modeled asa homogeneous nucleation scheme involving the formation of anunstable dimeric nucleus followed by monomer addition to growingfilament ends [24]. Under these conditions, the nucleation andextension phases of aggregation can be assessed and quantified.Thus, the inherent aggregation propensity of pseudophosphorylatedtau can be quantified and compared to that of wild-type tau.

Here, we examine the aggregation propensity of a tau mutantpseudophosphorylated at residue T212 in a full-length four-repeat taubackground. This site composespart of theAT100epitope [25,26],whichis recognized by multiple protein kinases [27–31], and is selectivelyoccupied in disease [32]. The results show that the introduction ofnegative charge at this position directly promotes tau fibrillization byacting at multiple points along the aggregation pathway.

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2. Materials and methods

2.1. Materials

Recombinant polyhistidine-tagged 2N4R tau and pseudopho-sphorylation mutant 2N4R–T212E were prepared as describedpreviously [21,33]. Aggregation inducer Thiazine red (ChemicalAbstract Service registry number 2150-33-6) was obtained from TCIAmerica (Portland, OR, USA). Formvar/carbon-coated copper grids,glutaraldehyde, and uranyl acetate were obtained from ElectronMicroscopy Sciences (Fort Washington, PA, USA). Primary mousemonoclonal Tau5 [34] was the gift of L. I. Binder (NorthwesternUniversity), whereas HRP-linked goat anti-mouse IgG was fromKirkegaard and Perry (Gaithersburg, MD). Nitrocellulose membranes(0.45 μm) were from Bio-Rad Laboratories (Hercules, CA).

2.2. Tau fibrillization assay

Tau filaments were formed from purified tau incubated withoutagitation in assembly buffer (10 mM HEPES, pH 7.4, 100 mM NaCl,and 5 mM dithiothreitol) for up to 24 h at 37 °C. Aggregation wasinitiated with Thiazine red (100 μM final concentration). For samplesanalyzed by electron microscopy, reactions were terminated with 2%glutaraldehyde, adsorbed to Formvar/carbon-coated copper grids,stained with 2% uranyl acetate, and viewed in a Tecnai G2 SpiritBioTWIN transmission electron microscope (FEI, Hillsboro, OR, USA)operated at 80 kV and 23,000–49,000× magnification. At least threeviewing fields were captured for each reaction condition in whichfilaments N10 nm in length were counted and quantified with ImageJsoftware (National Institutes of Health, Bethesda, MD, USA). Totalfilament length is defined as the sum of the lengths of all resolvedfilaments per field and is reported as±SD.

For quantification by immuno-dot blot, reactionswere centrifuged at200,000 g for 1 h at 16 °C, after which time aliquots of the resultantsupernatants were spotted onto nitrocellulose membranes. Membraneswereblocked in4%nonfatdrymilkdissolved inblockingbuffer (100 mMTris–HCl, pH 7.4, 150 mM NaCl, and 0.5% Tween 20) for 2 h, and thenincubated with mouse monoclonal antibody Tau5 at 1:1000 dilution for2 h. The membrane was washed three times in blocking buffer andincubated with HRP-linked goat anti-mouse IgG for 2 h. The membranewas thenwashed three times in blocking buffer and developed with theEnhanced Chemiluminescence Western Blotting Analysis System (GEHealthcare, Buckinghamshire, UK). Chemiluminescence was recordedon an Omega 12iC Molecular Imaging System and quantified usingUltraQuant software (UltraLum, Claremont, CA, USA).

2.3. Critical concentration

Critical concentrations (Kcrit) were determined by inverse predictionof the abscissa intercept on a plot of the concentration dependence oftau aggregation, as described previously [24]. The accompanyingstandard error of the estimate (Sx) was calculated as:

Sx =CI

2 t0:975;n−2

� � ð1Þ

where CI is the Fieller 95% confidence interval of each regression, andt0.975,n–2 is the Student's t distribution percentage at 1–α=0.975 andn–2 degrees of freedom.

2.4. Dissociation kinetics

Assembled tau filaments prepared as described abovewere diluted10-fold into assembly buffer containing 100 μM Thiazine red andincubated at 37 °C. Aliquots were removed as a function of time up to

5 h post-dilution and assayed for total filament length. The disaggre-gation time course was fit to an exponential decay function:

y = y0e−kappt ð2Þ

where y is the filament length at time t, y0 is filament length at timezero, and kapp is the pseudo-first order rate constant for the process.After solving for kapp, the initial velocity of disaggregation (dy/dt) wasdetermined from the first derivative of the exponential decay function[35,36] at time t=0:

dy= dt = −y0 kapp ð3Þ

Dissociation rate constant ke− was then extracted from initialvelocities by converting length into tau protomer units (assuming3.85 taumolecules/nanometer filament [24]) and then dividing by thenumber of filaments measured at time zero (i.e., it was assumed thatdissociation proceeded from only one end of each filament). Theassociation rate constant ke+ was then determined from therelationship [24]:

Kcrit = ke− = ke+ ð4Þ

assuming a two state model (i.e., all tau was either monomeric orincorporated into filaments).

2.5. Aggregation time series

Aggregation lag times, defined as the time when the tangent to thepoint of maximum aggregation rate intersects the abscissa of thesigmoidal curve [37], were obtained ±SE from each time series byGompertz regression as described in Ref. [22]. To determine thenucleation dissociation equilibrium constant, Kn, filament length datawere converted to protomer concentration (cp*) assuming that allprotein above the critical concentration formed filaments [24] andthat the resultant filaments contained two tau protomers per β-sheetspacing [24]. Data were then fitted to the simplified homogeneousnucleation scheme of Wegner and Engel [38] assuming a dimericnucleus [24]:

c1 = ctotal–cp* ð5Þ

dcpdt

=kn+ ke+ c1−ke−

� �c21

kn− + ke+ c1−ke−ð6Þ

dcp*dt

= ke+ c1−ke−� �

cp ð7Þ

where ctotal, c1, and cp represent bulk tau, taumonomer, and taufilamentconcentrations, respectively. Parameter estimates were obtained byfitting experimentally determined values of ctotal, cp*, ke−, and ke+to Eqs. 5–7 in JACOBIAN™ modeling software (Numerica Technol-ogy, LLC, Cambridge, MA). The simulation yielded estimates offorward and reverse nucleation rate constants kn+ and kn−, withthe ratio kn−/kn+ recorded as Kn.

2.6. Statistical analysis

The probability of differences between kinetic parameters wasassessed by z-test:

z =x1−x2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

Sx1ð Þ2 + Sx2ð Þ2q ð8Þ

where x1±Sx1 and x2±Sx2 are the pair of estimates ±SE beingcompared, and z is the 1–α point of the standard normal distribution.

390 E. Chang et al. / Biochimica et Biophysica Acta 1814 (2011) 388–395

All statistical analyses were carried out using SigmaPlot 10.0 (SystatSoftware, Chicago, IL) and JMP 7.0 (SAS Institute, Cary, NC).

3. Results

3.1. Effect of pseudophosphorylation on critical concentration

Approximately thirty phosphorylation sites have been mapped tothe microtubule-binding repeat region of filamentous tau isolatedfrom AD brain (Fig. 1A). Using recombinant tau preparations, wepreviously showed that incorporation of negatively charged Gluresides at some of these sites modulated aggregation propensityrelative to unmodified tau in the presence of anionic surfactantinducers [21]. Among these missense mutants, T212E showed thegreatest effect and so was selected for detailed analysis in thepresence of Thiazine red aggregation inducer. To facilitate comparisonwith previous work [21], recombinant T212E was prepared in a full-length four-repeat background corresponding to the 2N4R tauisoform. After purification from a bacterial expression system,recombinant 2N4R–T212E and wild-type 2N4R tau migrated identi-cally on SDS-polyacrylamide gel electrophoresis, confirming that theywere prepared with consistent concentration and purity (Fig. 1B).When wild-type 2N4R tau (≤1 μM) was incubated with Thiazinered at near-physiological conditions of pH, ionic strength, andreducing environment, filaments with twisted ribbon morphologyformed (Fig. 1C). These filaments were previously reported to havea mass-per-unit length similar to authentic brain-derived pairedhelical filaments [24]. When incubated under the same conditions,2N4R–T212E produced filaments that were morphologically iden-tical to those formed by wild-type 2N4R tau (Fig. 1D). In contrast,incubation of either tau preparation in the absence of Thiazine red

Fig. 1. Comparison of 2N4R and 2N4R–T212E tau preparations. (A) Distribution of ~30 hydisoform contains alternatively spliced exons 2 and 3 (E2 and E3), each of which encodes an abinding repeat. Pseudophosphorylation mutant T212E is distinguished graphically by a rai(right lane) protein preparations by SDS-polyacrylamide gel electrophoresis (10% acrylamideproteins [33], whereas the band below 22 kDa corresponds to the dye front. The preparationssynthetic tau filaments. Full-length wild-type 2N4R tau (C) andmutant 2N4R–T212E (D) wered (24 h at 37 °C) and viewed by transmission electronmicroscopy. 2N4R–T212E produced ulength distribution from wild-type 2N4R. Scale bar=100 nm.

for up to 24 h did not yield any detectable filaments (data notshown), suggesting that spontaneous aggregation was minimalunder these conditions. These data indicate that 2N4R–T212Eshares the fundamental aggregation characteristics of wild-type2N4R tau and can be studied at physiological bulk tau concentra-tions in the presence of Thiazine red inducer.

Characterization of aggregation propensity began with estimationof the minimal tau concentration required to support aggregation of2N4R and 2N4R–T212E. In nucleation-dependent reactions, theminimal concentration is termed the critical concentration (Kcrit)and approximates the dissociation equilibrium constant for elonga-tion (Ke) [24]. The minimal concentration of 2N4R estimated from theabscissa intercept of a plot of aggregation versus tau concentrationwas 186±25 nM (Fig. 2; Table 1). In contrast, the minimalconcentration of 2N4R–T212E was 84±28 nM (Fig. 2; Table 1),differing from wild-type tau by 2.2±0.4-fold (pb0.01).

Critical behavior also is reflected in the levels of protein monomerat reaction plateau, which remain constant as bulk concentrations riseabove Kcrit [39]. To confirm that tau aggregation in the presence ofThiazine red displayed critical behavior, the levels of soluble tau atreaction plateau were estimated after ultracentrifugation (200,000×gfor 1 h). In the absence of Thiazine red, soluble 2N4R and 2N4R–T212Eincreased linearly with bulk tau concentration up to at least 1 μM(Fig. 3), consistent with the absence of detectable filament formationin these samples. In contrast, the presence of Thiazine red aggregationinducer yielded a nearly constant amount of 2N4R and 2N4R–T212E inthe supernatant regardless of bulk tau concentration (Fig. 3). Underthese conditions, average plateau levels of soluble 2N4R were 2.1±0.2-fold higher (pb0.01) than soluble 2N4R–T212E (Fig. 3). Thesedata were consistent with critical behavior and indicated thatphosphorylation mimicry at residue 212 increased aggregation

roxyamino acid residues affected by phosphorylation depicted on isoform 2N4R. Thiscidic 29-residue segment, and exon 10 (E10), which encodes an additional microtubulesed hollow symbol. (B) Comparison of recombinant 2N4R (left lane) and 2N4R–T212E) and Coomassie blue staining. The bands migrating at ~66 kDa correspond to these tauwere consistent with respect to protein concentration and purity. (C, D) Morphology ofre incubated (1 μM concentration) without agitation in the presence of 100 μM Thiazinenbranched filaments ~16 nm in diameter with no obvious differences inmorphology or

Fig. 2. Pseudophosphorylation lowers critical concentration. Wild-type 2N4R tau (●)and mutant 2N4R–T212E (○) were incubated at varying bulk concentrations in thepresence of Thiazine red inducer for 24 h at 37 °C, then assayed for filament formationby electron microscopy. Each data point represents total filament length as a function ofbulk protein concentration (triplicate determination), whereas the solid lines representbest fit of the data points to linear regression. The abscissa intercept, which wasobtained by extrapolation (dotted lines), was used to estimate critical concentration(Kcrit; Table 1). 2N4R–T212E aggregated with a lower Kcrit than did wild-type 2N4R.

391E. Chang et al. / Biochimica et Biophysica Acta 1814 (2011) 388–395

propensity by depressing the minimum concentration of tau neededto support fibril formation.

To confirm that the decrease in Kcrit was a result of increasedability to aggregate and not a change in sensitivity to Thiazine redinduction, 2N4R and 2N4R–T212E were incubated with differentconcentrations of Thiazine red at constant tau supersaturation (i.e., ata constant tau concentration above Kcrit) (Fig. 4). The concentration–effect relationship was similar for both tau forms with maximalefficacy near 100 μM, indicating that the response of 2N4R–T212E toThiazine red was not perturbed. Together these data indicate that thedepression of Kcrit observed with pseudophosphorylation reflectsdifferences in aggregation propensity and not differential sensitivityto Thiazine red inducer.

3.2. Effect of pseudophosphorylation on the extension reaction

From Eq. 4, Kcrit approximates the ratio of the rate of monomerdissociation from filament ends (ke−) to the rate of monomer additionto filament ends (ke+). A decrease in Kcrit could result from astabilization of filaments (i.e., a decrease in ke−), enhanced monomeraddition (an increase in ke+), or a combination of both. To distinguishbetween these possibilities, ke− was estimated from the disaggregationrate of preassembledfilaments composed of 2N4R and2N4R–T212E tau.Rate constant ke+was then calculated from estimates of ke− and Kcrit foreach mutant through Eq. 4. Results show that disaggregation followed

Table 1Summary of aggregation parameters.

Protein Kcrita

(nM)ke−

a

(s−1)ke+

a

(mM−1s−1)Lag time(h)

Kn

(mM)

2N4R 186±25 0.020±0.001 105±15 0.81±0.06 21.7T212E 84±28⁎ 0.011±0.001⁎ 133±46 0.28±0.05⁎ 5.1

a Overall constants reflecting events at both filament ends.⁎ pb0.01 versus 2N4R tau.

first-order kinetics as predicted for a Poisson-like distribution offilament lengths undergoing endwise depolymerization [35] (Fig. 5),and that ke− for 2N4R–T212E is decreased nearly 2-fold relative towild-type 2N4R tau (Table 1). In contrast, there was no significant differencein calculated values of ke+ (Table 1). These data indicate that filamentsformed by 2N4R–T212E are inherently more stable and less prone todisaggregate than filaments formed by wild-type 2N4R tau. Further-more, these observations were consistent for two different filamentmorphologies formed from two distinct inducers (Thiazine red, herein;octadecyl sulfate, [21,22]).

3.3. Effect of pseudophosphorylation on the nucleation reaction

In the presence of Thiazine red inducer, the aggregationreaction of tau is driven by the rapid equilibration of assembly-competent monomers with a thermodynamic nucleus, defined asthe least stable species reversibly interconverted with monomer[40]. Since elongation can proceed efficiently only after thenucleus has formed, the rate of aggregation depends on nucleationrate as well as protein concentration and the rate of elongation. Todetermine whether pseudophosphorylation affected nucleationrate, tau aggregation time course was quantified for both wild-type 2N4R and 2N4R–T212E at constant supersaturation in thepresence of Thiazine red. Under these conditions, differences inreaction rates primarily reflect differences in rates of nucleationand of protein concentrations [41]. Both reaction progress curvesdisplayed lag, exponential growth, and equilibrium phases (Fig. 6).However,fittingeachcurve to a 3-parameterGompertz growth functionrevealed that 2N4R–T212E aggregated with a significantly shorter lagtime than wild-type 2N4R tau despite being present at lower bulkconcentrations (Table 1). These data suggest that pseudophosphoryla-tion accelerated the nucleation phase of the tau aggregation reaction.

To test this hypothesis, each time series was fit to an explicitnucleation–extensionmodel using Eqs. 5–7 and experimental values forrate constants ke+ and ke− as constraints. The nucleation dissociationequilibrium constant, Kn, was then estimated from the model. Thecalculations revealed that pseuphosphorylation mutant 2N4R–T212Eincreased the efficiency of nucleation by decreasingKn ~4-fold (Table 1).Together these data indicate that incorporation of negative charge atresidue 212 directly increases aggregation propensity and that it does soat the nucleation step by decreasing Kn and at the extension step bydecreasing ke−.

4. Discussion

These results confirm that incorporation of negative charge intotau protein can directly modulate aggregation propensity irrespectiveof reported indirect effects on tau turnover [42] or prolineisomerization [43]. Two mechanisms have been proposed to accountfor direct effects. The first posits that charge neutralization decreasesthe isoelectric point of tau protein resulting in lower solubility atphysiological pH [44,45]. The second mechanism predicts thatconformational changes induced by phosphorylation promote orstabilize self-association [16]. These mechanisms need not bemutually exclusive. In fact, phosphorylation sites on tau may exerttheir effects in tandem, with some modifications enhancing fibrilliza-tion ([17,21,46–48] and herein) and others having neutral orinhibitory effects [49,50].

Previously, we reported that pseudophosphorylation mutants,including 2N4R–T212E, did not modulate filament nucleation rate[21]. The discrepancy results in part from the use of sodium octadecylsulfate (ODS) as inducer in the previous report. ODS is a surfactantthat functions in micellar form by presenting a negatively chargedsurface for filament nucleation [23]. As a result, the rate of filamentinduction by ODS is limited by its rate of micellization, which is only80% complete after ~45 min [22]. Because lag times for 2N4R and

Fig. 3. Pseudophosphorylation lowers tau solubility. (A)Wild-type 2N4R and (B) 2N4R–T212Ewere incubated at varying bulk concentrations in the presence (○,□) or absence (●,■) ofThiazine red inducer until reaction plateau (24 h at37 °C), then subjected to centrifugation (200,000×g for 1 h). The amount of tau remaining in the soluble fractionwas thenquantifiedonimmuno-dot blots using monoclonal antibody Tau5 and enhanced chemiluminescence detection. Each data point represents tau immunoreactivity as a function of bulk proteinconcentration (triplicate determination), whereas the lines represent best fit of the data points to linear regression (solid line regressions were constrained to pass through the origin). Inthe absence of Thiazine red, soluble 2N4R or 2N4R–T212E increased linearlywith bulk tau concentration (solid lines). In contrast, the presence of Thiazine red greatly reduced solubility ofboth 2N4R–T212E and wild-type 2N4R, both ofwhich remained constant over the 0.4–1 μMbulk tau concentration range (dashed lines). Averaged over this range, levels of soluble 2N4Rwere 2.1±0.2-fold higher than soluble 2N4R–T212E. *pb0.05; **pb0.01, presence compared to absence of Thiazine red inducer.

392 E. Chang et al. / Biochimica et Biophysica Acta 1814 (2011) 388–395

2N4R–T212E ranged from 42 to 59 min [21], the rate of filamentnucleation cannot be distinguished from the rate of ODSmicellization,and potential differences among tau species are masked. In contrast,the depressing effect of pseudophosphorylation on minimal concen-tration is consistent between arachidonic acid, ODS, and Thiazine redinducers ([21] and herein). Absolute values of minimal concentrationvary among these inducers, however, in part because non-fibrillar tau

Fig. 4. Tau mutants share a common sensitivity to aggregation inducer Thiazine red.Wild-type 2N4R (●) and 2N4R–T212E (○) were incubated (24 h at 37 °C) at constantsupersaturation (i.e., 0.5 μM above Kcrit) in the presence of varying concentrations ofThiazine red and then assayed for filament formation by electron microscopy. Each datapoint represents total filament length per field±SD from triplicate determinations.Under these conditions, the concentration effect relationship for Thiazine red wassimilar for both tau species.

binds and coats the surface of anionic inducers, adding a thirdcomponent to the equilibrium between filaments and monomer [23].As a result, minimum concentrations for filament formation are lowmicromolar in the presence of anionic surfactants, but submicromolar

Fig. 5. Dissociation rate constants for filament extension. Tau filaments prepared fromwild-type 2N4R (●) and 2N4R–T212E (○) in the presence of 100 μMThiazine red at 37 °Cwere diluted 10-fold into assembly buffer containing Thiazine red, and the resultantdisaggregationwas followed as a function of time by electronmicroscopy. Each data pointrepresents total filament length per field±SD (triplicate observations), whereas the solidlines represent best fit of data points to linear regression. The first-order decay constantkapp was estimated from each regression and used in conjunction with filament length(shown in the figure) and number (2N4R=127±18; 2N4R–T212E=298±30) at timet=0 to calculate dissociation rate constant ke− (see Table 1). Despite similar decayconstants for 2N4R and 2N4R–T212E filaments in this experiment, estimates of ke−differed owing to the differences in numbers of filaments at time t=0.

Fig. 6. Effects of tau mutations on aggregation time course. Wild-type 2N4R (●) and2N4R–T212E (○) were incubated (37 °C) at constant supersaturation (i.e., 0.2 μMaboveKcrit) in the presence of 100 μM Thiazine red and then assayed for filament formation asa function of time. Each data point represents mean total filament lengths/field(expressed as %plateau length) calculated from triplicate electron microscopy images,whereas each normalized curve represents best fit of the data points to a three-parameter Gompertz growth function. The fits were used to calculate lag time (Table 1).2N4R–T212E aggregated significantly faster than wild-type 2N4R when compared atconstant supersaturation.

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in the presence of Thiazine red. A second source of discrepancypertains to filament morphology. Filaments induced under reducingconditions by anionic surfactants such as arachidonic acid contain ~1tau molecule per β-sheet spacing [51], whereas those induced byThiazine red contain ~2 tau molecules per β-sheet spacing [24].Aggregation kinetics may vary with filament structure.

The results presented herein also disagree with equilibriumaggregation measurements made with pseudophosphorylationmutants including T212E and heparin inducer [26]. The discrepancycould result from differences in protein concentration, which were

Fig. 7. Effect of T212E mutation on the tau fibrillization pathway. Normal tau binds tightly tonatively disordered, assembly incompetent monomer (Ux). A conformational change to anspecies form, the rate-limiting step in tau fibrillization is formation of dimer, which represfurther addition of assembly-competent monomers to the filament (F) ends. Introduction ofphosphorylation, affects multiple points in the pathway. See text for details.

submicromolar range in the present study and ~60 μM in theheparin-induced study. In nucleation-dependent reactions, theamount of aggregation at equilibrium is proportional to the netconcentration of protein above the critical concentration rather thanbulk protein concentration. Thus, when bulk concentrations are highrelative to critical concentrations, net concentrations may not differsubstantially among tau constructs, thereby masking potentialdifferences in aggregation propensity. Heparin-mediated tau aggre-gation reportedly is nucleation dependent under non-reducingconditions [52].

4.1. Implications of tau aggregation mechanism

On the basis of tau aggregation kinetics in the presence ofexogenous inducer Thiazine red [24], we have proposed that fourkey steps must be overcome for tau to aggregate in disease (Fig. 7).First, the concentration of free tau in the cytoplasm must be sufficientto support filament formation. This can be accomplished throughincreased MAPT expression, decreased tau degradation, or decreasesin tau-microtubule binding affinity. The role of tau phosphorylation inmodulating tau-microtubule affinity is well established [8–10]. Forexample, T212E reportedly has diminished ability to promotemicrotubule assembly [26], consistent with an impaired ability tobind tubulin. In addition, tau phosphorylation has been reported todecrease proteasome-mediated tau turnover in a neuronal cell model[53]. Thus, occupancy of certain tau phosphorylation sites may raisefree cytoplasmic tau concentrations through multiple mechanisms.

The second step involves the transition of dissociated taumonomers to an assembly-competent conformation (Fig. 7). Thisstep is proposed to be a barrier to aggregation because highconcentrations (i.e., up to 100 μM) of free tau alone are insufficientto support aggregation or seeding reactions in vitro [54]. Phosphor-ylation of tau at multiple sites within the proline-rich region of tau(including T212) can induce local polyproline II helix conformation[16]. Adoption of such conformations, which are associated withprotein–protein interfaces [55], may help overcome the resistance ofmonomeric unmodified tau proteins to aggregation.

Once aggregation-competent conformations are adopted, the rate-limiting step in filament formation becomes dimerization [24], which

microtubules but dissociates upon phosphorylation to form free tau, which exists as aassembly-competent state accelerates polymerization (Uc). Once assembly-competentents the thermodynamic nucleus (N). Following nucleation, extension occurs throughnegative charge at residue T212 in the form of pseudophosphorylation, and potentially

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is energetically disfavored at physiological tau concentrations, andtherefore a third key point of control (Fig. 7). In the case of 2N4R–T212E, increased aggregation propensity included acceleration offilament nucleation rate. Consistent with this observation, taudimerization can be promoted in vitro by NCLK/cdk5 [56], whichincludes T212 as a target [31].

The final step in fibrillization is mediated by an extensionreaction. Although not rate limiting, equilibria at filament endsdictate the minimal concentration of tau required to supportaggregation. Pseudophosphorylation at T212 enhanced filamentelongation by decreasing the rate at which monomers dissociatedfrom filament ends. These effects are not unique to the twistedribbon morphology induced by Thiazine red, also having beenobserved with the filamentous morphologies induced by anionicsurfactants [36]. These results indicate that the effects of 2N4R–T212E on filament stability are not inducer-specific. However, itseffects on filament elongation differ from that of certain fronto-temporal dementia linked missense mutations, some of which actto increase the rate of monomer addition to filament ends withoutaffecting filament stability [57].

4.2. Conclusions

Together, these data suggest that occupancy of specific tauphosphorylation sites could potentially modulate key rate-limitingsteps along the fibrillization pathway. This reinforces the contributionof tau hyperphosphorylation to neurological disease and providesfurther support for hyperphosphorylation as a target for pharmaco-logical efforts in treatment of tauopathies.

Acknowledgments

This work was supported by the National Institutes of Health grantAG14452.

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