Modulation of Tau Protein Fibrillization by OleocanthalMaria Chiara Monti, Luigi Margarucci, Raffaele Riccio, and Agostino Casapullo*

Dipartimento di Scienze Farmaceutiche e Biomediche, Universita degli Studi di Salerno, Via Ponte don Melillo, 84084 Fisciano, Italy

*S Supporting Information

ABSTRACT: Among the phenolic compounds extractedfrom extra virgin olive oil, oleocanthal (1) has attractedconsiderable attention in the modulation of many humandiseases, such as inflammation and Alzheimer’s disease (AD).Indeed, 1 is capable of altering the fibrillization of tau protein,which is one of the key factors at the basis of neuro-degenerative diseases, and of covalently reacting with lysine ε-amino groups of the tau fragment K18 in an unspecific fashion.In the present study, an investigation of the recognitionprocess and the reaction profile between 1 and the wild-typetau protein has been conducted by a circular dichroism, surface plasmon resonance, fluorescence, and mass spectrometrycombined approach. As a result, 1 has been found to interact with tau-441, inducing stable conformational modifications of theprotein secondary structure and also interfering with tau aggregation. These findings provide experimental support for thepotential reduced risk of AD and related neurodegenerative diseases associated with olive oil consumption and may offer a newchemical scaffold for the development of AD-modulating agents.

Extra virgin olive oil, one of the principal constituents of theMediterranean diet, has been associated for a long time

with health benefits.1,2 Indeed, the occurrence of breast andcolon cancers and cardiovascular diseases3−5 is remarkably lowin the Mediterranean area compared to other geographicalregions of the world. In this context, the biological properties ofphenolic compounds present in extra virgin olive oil from Oleaeuropea L. (Oleaceae) and their involvement in somepathogenic processes have attracted wide attention since theirdiscovery.6−10

Among the phenolic olive oil constituents, (−)-oleocanthal(1), the dialdehydic form of (−)-deacetoxyligstroside aglycone,found mainly in freshly pressed extra virgin olive oil, has shownan anti-inflammatory profile similar to the nonsteroidal anti-inflammatory drug ibuprofen.11 Indeed, after the completion ofthe total synthesis, 1 was subjected to extensive biologicalinvestigations.12 In some of these studies, 1 has been suggestedto reduce the polymerization of tau protein through a possiblecovalent mechanism.13,14 Since the aggregation of tau correlateswith clinical progression of Alzheimer’s disease (AD), it seemslikely that inhibition or reversal of tau aggregation could protectthe affected neurons.15,16

Tau protein, found in six isoforms from 352 to 441 aminoacids in length, is involved in the stabilization of the

microtubules (MTs) by direct interaction through a micro-tubule-binding domain (MBD),17,18 thereby modulating theplasticity of the cytoskeleton. It has been reported recently thattwo VQIXXK motifs in the MT binding region, named PHF6(from V306 to K311) and PHF6* (from V275 to K280), areresponsible for β-sheet structural development and theinitiation of tau fibrils formation.19 Indeed, tau is a highlysoluble protein with a random conformation in aqueoussolution and hardly shows any tendency to assemble underphysiological conditions.20 In the brains of AD patients,however, it dissociates from axonal microtubules andabnormally aggregates to form insoluble paired helical filaments(PHFs), which are implicated in neurodegeneration.19 Sincethe amount of tau aggregates has been correlated with neuronloss and the severity of dementia, the analysis of its self-assembly mechanism and the discovery of lead compounds ableto reduce the PHF formation21−24 could provide essentialinformation to develop an effective way to slow theneurodegenerative process.With this background, compound 1 has been studied herein

to potentially better understand its interaction profile with wild-type tau protein (tau-441). Recently, its chemical reactivityprofile with K18, a tau-441 fragment containing all four MT-binding domains responsible for the fibrillization process, hasbeen analyzed thoroughly, demonstrating that this peptide isprone to covalent modification by 1 in an unspecific fashion,due to its unstructured conformation.14 Starting with thisinformation, the ability of oleocanthal to interfere with the taufibrillization process has been analyzed along with an

Received: May 31, 2012

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investigation of the recognition process between thecomponent units, by a combination of circular dichroism(CD), surface plasmon resonance (SPR), fluorescence, and MS.Several pieces of evidence have established that 1 is able tocovalently interact with tau-441 and to induce α-helix formationin the secondary structure of the protein, interfering also withthe tau aggregation process.

■ RESULTS AND DISCUSSIONAnalysis of the Tau-441−Olecanthal Recognition

Process. In order to assess the interactions between 1 andtau-441, SPR analysis was performed.25,26 The binding eventswere monitored by injecting 1, at a concentration rangebetween 0.1 and 25 μM, on the protein immobilized over thesensor surface, giving rise to the sensogram curves shown inFigure 1.These results confirmed the occurrence of the interaction

between tau-441 and 1, although the baseline response unitswere not recovered, even after more than 400 s, symptomatic ofa potential covalent binding between 1 and tau. To address thishypothesis, the structural analysis of the tau−oleocanthalcomplex was investigated by mass spectrometry. In particular,MALDI spectra of the incubation mixture between the proteinand 1, after treatment with NaBH4, were measured andcompared with that of the free tau-441 (Figure 2). The ΔMw of

around 350 amu, calculated on the singly and doubly chargedspecies present in the spectra of the two samples, wasconsistent with the formation of a covalent protein−ligandcomplex, in which the stoichiometry was evaluated as 1:4(protein:ligand), on the basis of a previous study reporting amass increment of 92 Da for each ligand bound.14 Then, thetau lysine residues involved in the covalent bond were identifiedby submitting the free tau-441 and the tau-441−1 mixture totreatment with NaBH4, trypsin digestion, and MALDI massspectrometric analysis. More than 90% of the protein sequencewas covered by peptide identification, and as expected for anintrinsically disordered polypeptide, several lysine residues weremodified by 1 (see Supporting Information, Figure S1). It isnoteworthy that, despite the presence of the likely reactivedialdehyde moiety, 1 showed a low reactivity towardnucleophilic amino acids, such as lysine and arginine, and nobinding with several proteins (see Supporting Information,Figures S2 and S3).The binding selectivity of 1 with tau-441 points to a

significant role of the metabolite scaffold in the molecularrecognition process, even if a facile approach of the ligandtoward the protein lysine side chain can be postulated, due tothe random coil structure of tau.

Effect of Oleocanthal (1) on Tau Protein Conforma-tion and Fibrillization. In order to estimate the effect of 1 on

Figure 1. Sensograms obtained from the binding of 1 (0.1−25 μM) to tau-441.

Figure 2. MALDI mass spectra of tau-441 incubated in the absence (upper panel) and presence (lower panel) of a 20-fold molar excess of 1 for 45min at 37 °C. The spectra clearly show a tau-441 mass increment of around 350 Da upon incubation with 1. All the other spectra collected withdifferent molar excesses of 1 and a different incubation time showed the same ΔMw..

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tau protein conformational dynamics, the amide hydrogen/deuterium (H/D) exchange coupled with a mass spectrometricapproach was applied.27 Using this method, the different amidehydrogen atoms undergo isotopic exchange at different ratesdue to solvent accessibility, hydrogen-bonding strength, andprotein secondary structure elements. The results indicate thereplacement of about 320 hydrogen atoms at the plateau for thefree tau-441, whereas, after incubation with 1, the protein isable to exchange approximately 200 hydrogen atoms,suggesting a consistent conformational rearrangement in thetau-441 structure (Figure 3).

In a subsequent experiment, the analysis was extended to theconformational transition of tau-441 from its native unfoldedstate to filament formation induced by arachidonic acid (AA),28

by measuring CD spectra of the protein in the presence orabsence of 1 at different incubation times. Indeed, it is well-known that different inducers, such as AA, heparin, and otherpolyanionic compounds, are used to drive rapid self-associationof tau into fibrillar structures in vitro.29 Although taupolymerization in AD seems to be strongly related to itsabnormal phosphorylation,30 AA has been used to generate invitro filaments structurally similar to those found in the disease,and therefore they represent a good model for mimickingpathological tau fibrillization.A sample of the free tau-441 and the mixture tau-441−1 were

separately treated with AA to start the fibrillization process, andCD spectra were acquired for each sample at different timeintervals up to 300 min. Analysis of the CD spectra by theSOMCD31 software gave the variation of the secondarystructural elements of the tau protein in the presence of a37-fold excess of AA (Figure 4, panel A). Tau protein, as alsoreported in the literature, showed an unfolded characterconsistent with more than 95% random coil structure that,after 60 min of incubation with AA, gave a transition towardboth α-helix and β-sheet elements, a sign of conformationalconversion during the fibrillization process. When tau-441 was

preincubated with 1, the CD spectrum of the sample (Figure 4,panel B) revealed a high percentage of α-helical elements,which confirmed the protein structural changes induced by 1.This new conformational arrangement was stable even after AAaddition, suggesting a protecting effect of 1 against thefibrillization process.To give a more clear picture of tau aggregation, an in vitro

assay, based on the fluorescence of thioflavin S (ThS),32 wasperformed to analyze the effect of 1 on this event. ThSundergoes a relevant increase in fluorescence quantum yieldupon binding to fibrils, as a result of the ability of the fibrils tohamper the ThS free rotation.33−35 As reported in Figure 5A,the fluorescence intensity of ThS, in the presence of the free tauexposed to AA, increased in the first five hours. Uponpreincubation of tau with a different molar excess of 1, asimilar trend in ThS fluorescence was monitored (Figure 5B),even if the intensity of the emission signal was sufficiently low ifcompared with the free tau (Figure 5A). This result confirmedthe ability of 1 to prevent AA-induced tau fibrillization in aconcentration-dependent manner.In conclusion, oleocanthal (1) exhibited nonspecific covalent

interactions with tau-441, inducing a conformational rearrange-ment as a consequence of a fast transition of the tau-441secondary structure from a random coil to an α-helix, whichcould explain the antifibrillogenic ability of 1, and this couldaccount for a downregulation of the pathological phosphor-ylation of tau. However, since there is a lack of data on theadsorption or biotransformation of 1 after olive oil ingestion, aswell as on its ability to cross the blood−brain barrier, furtherbiological investigations are needed to define the potential

Figure 3. Bars show the number of hydrogens exchanged by tau-441in the presence or absence of a 20 molar excess of 1.

Figure 4. Variation of the percentage of secondary structure of tau-441protein in the presence of a 37-fold excess of AA. The incubation oftau-441 with AA (panel A) induces a decrease in the percentage of tau-441 random coil and a concurrent increase of both α-helix and β-sheets. Panel B shows that 1 rapidly induces tau-441 α-helical moietiesand indicates a structural change induced by 1 that prevents AAfibrillization.

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application of 1 for the treatment of neurodegenerativedisorders.

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. The samples of extra virgin

olive oil were kindly provided by Dr. C. Prattico from TenutaAcquamara, Rocca d’Evandro, Caserta, Italy. The extraction andpurification procedure of 1 was carried out essentially as reported byMonti et al.14 The purity of 1 was estimated by reversed-phase HPLCon a HP 1100 binary pump system (Agilent Technologies, Palo Alto,CA, USA) using a Jupiter C18 Phenomenex (250 × 4.6 mm) column,by means of an isocratic gradient at 25% aqueous CH3CN. Thechromatographic profile was monitored at 264 nm, showing a purity of1 greater than 95%. Escherichia coli-expressed recombinant tau-441(Sigma Aldrich, Saint Louis, MO, USA), used in all reportedexperiments, is an isoform of tau (variant 2N4R) having fourmicrotubule binding repeats and two amino terminal inserts.Surface plasmon resonance sensograms were determined using a

Biacore 3000 from GE Healthcare equipped with a CM5 sensor chip(GE Healthcare). MALDI mass spectra were acquired on a MALDIMicro (Waters Co., Manchester, UK) using α-cyano-4-hydroxycin-namic acid (Sigma-Aldrich) as matrix. Circular dichroism measure-ments were performed using a JASCO J-810 spectrometer equippedwith a cell holder thermostatically controlled by a circulating waterbath. Fluorescence intensity was measured using a Perkin-Elmer LS55luminescence spectrometer equipped with a 96-well plate (blackmicrotiter 96 round well plate).Tau−Oleocanthal Binding by Surface Plasmon Resonance.

Tau-441 was immobilized onto a CM5 sensor chip using standardamine coupling procedures. Phosphate-buffered saline, which con-

sisted of 10 mM Na2HPO4 and 150 mM NaCl at pH 7.4, was used asrunning buffer. The carboxymethyl dextran surface was activated witha 5 min injection of a 1:1 ratio of 100 mM EDC and 100 mM NHS at5 μL min−1. Tau protein was diluted to a final concentration of 30 ngmL−1 in 10 mM sodium acetate, pH 4.5, before its injection onto theactivated chip surface at a flow rate of 5 μL min−1. Proteinconcentration was adjusted to give an optimal response (around9000 RU). The remaining active groups were blocked with a 7 mininjection of 1.0 M ethanolamine−HCl, pH 8.5, at 5 μL min−1.Solutions of 1 (0.1−25 μM), used in the biosensor experiments, werediluted in 10 mM phosphate saline buffer (pH 7.4) containing 2%DMSO. Each concentration was tested at least three times. Theinteraction experiments were carried out at a flow rate of 10 μL min−1,employing a 3 min injection time. The dissociation time was set at 350s.

Analysis of the Tau−Oleocanthal (1) Complex by MassSpectrometry. Compound 1 was dissolved in CH3CN (1 mg/mL),and then aliquots of this stock solution were added to a solution oftau-441 (150 nM in 50 mM MES buffer, 100 mM NaCl, and 0.5 mMEGTA, pH 6.8) for different times (from 5 min to 3 h) at 37 °C, withmolar excesses of inhibitor ranging from 10:1 to 40:1. The reactionmixture was diluted with the same volume of NaBH4 (molar ratioNaBH4:Tau-441, 150:1) in NaOH (15 mM) for 10 min at 4 °C, andthe reaction was quenched by adding 3 μL of a 6 M HCl solution.

The final concentration of CH3CN in the reaction mixture wasalways kept lower than 6% (v/v). The mixture was loaded on aMALDI plate, and MALDI mass spectra were performed using α-cyano-4-hydroxycinnamic acid dissolved in 1:1 CH3CN−H2O−0.1%TFA as matrix. Then, the mixture was incubated with trypsin solution(E/S ratio of 1:100) at 37 °C for 4 h and analyzed by MALDI massspectrometry.

H/D Exchange Mass Spectrometry to Monitor Tau-441Conformational Changes Induced by Oleocanthal (1). Tauprotein 441 (1 mg/mL) was diluted 1:20 in D2O and incubated witheither 1 (10 mM) or solvent (CH3CN) at 25 °C. The reaction wasmonitored using a MALDITOF mass spectrometer for 60 min. Briefly,1 μL of the samples was spotted directly on the target under a nitrogenflow and mixed 1:1 with a solution of 10 mg/mL α-cyano-4-hydroxycinnamic acid in H2O−CH3CN−0.1% TFA. After crystal-lization (60 s), the target was transferred directly to the massspectrometer. The spectra were acquired in positive linear ion mode(5000−50 000 m/z) using tau-441 or tau-441−1 (1:20 in H2O) aslock mass.

Circular Dichroism Experiments on Tau−Oleocanthal (1)Complex. A Solution of tau-441 at 2.5 μM was prepared using a 50mM MES buffer, 100 mM NaCl, and 0.5 mM EGTA (pH 6.8), and92.5 μM arachidonic acid was added to induce the fibrillization of tau-441. CD spectra were collected from 1 to 28 h of incubation. The tausolution was mixed with 1 (125 μM) in a molar excess of 50 times, andthe solution was kept at 37 °C for 1 h before adding AA at 92.5 μM.Measurements were recorded at 25 °C, in a cuvette with a 1 mm pathlength with a 4 s time response, at a rate of 100 nm/min, and wereaveraged for 16 acquisitions. Then, molar ellipticity was determinedafter normalizing protein concentration. The same measurement wasrepeated at least three times using newly prepared samples, and thereproducibility of the results was confirmed. Data are expressed interms of mean residue ellipticity [θ] in deg cm2 dmol−1. Thesecondary structure contents (i.e., random, α-helix, and β-sheetstructures) were estimated using the program SOMCD (http://geneura.ugr.es).

Use of Anionic Inducers of Tau Aggregation and ThSFluorescence Experiments. Solutions of tau-441 at 0.5 μM wereprepared using 2 mM MES buffer, 10 mM NaCl, and 50 μM EGTA(pH 6.8), with arachidonic acid in a molar excess of 37 times over tauconcentration (18.5 μM) added to induce fibrillization. The mixturewas kept at 37 °C for 5 min, and 1 μL of ThS diluted in H2O at a finalconcentration of 2.5 μM was added. The kinetics of tau aggregationwas analyzed by recording the time-course of fluorescence intensityover 24 h with excitation at 440 nm and emission at 521 nm. Theexcitation and emission slit widths were set at 10 nm. The background

Figure 5. Panel A shows the variation of ThS emission at 521 nm (as apercentage) when exposed to tau-441 alone and tau-441−1. Thehistograms reported in panel B shows the percent of tau-441−AA-induced fibrillization in the presence or absence of different molarexcess values of 1.

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fluorescence of the sample was subtracted when necessary, andcontrols without protein or without AA or ThS were included inaddition.Evaluation of Tau Aggregation in the Presence of

Oleocanthal (1) Using a ThS Assay. Tau-441 at 0.5 μMconcentration was incubated with 1 at a concentration range from 5to 200 μM for 1 h at 37 °C. Compound 1 was diluted in CH3CN; thevolume of the organic solvent was kept lower than 5% in theincubation solution. Then, tau-441 was mixed for 1 h at 37 °C, andarachidonic acid in a molar excess of 37 times over the tauconcentration (18.5 μM) was added to induce fibrillization. Themixture was kept at 37 °C for 5 min, and 1 μL of ThS, diluted in H2Oat a final concentration of 2.5 μM, was added. Fluorescence intensitywas measured as described above. All of the measurements werecarried out in triplicate. Control reactions were normalized forCH3CN vehicle [5% (v/v) final concentration].

■ ASSOCIATED CONTENT

*S Supporting InformationTau-441-modified peptides after treatment with 1 are includedalong with the reaction profile of 1 with basic amino acids andwith myoglobin, lysozyme, and human synovial PLA2. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Tel: +39 089 969243. Fax: +39 089 969602. E-mail:casapullo@unisa.it.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSFinancial support by the University of Salerno is gratefullyacknowledged. The authors also acknowledge the use of theinstrumental facilities of the Center of Competence inDiagnostics and Molecular Pharmaceutics supported byRegione Campania (Italy) through POR funds.

■ DEDICATION

In memory of Professor Ernesto Fattorusso.

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