Phenolic compounds prevent the oligomerization of α‐synuclein and reduce synaptic toxicity

Lewy bodies, mainly composed of α‐synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low‐order αS oligomers are neurotoxic and critical species in the pathogenesis of α‐synucleinopathies. To develop disease modifying therapies for α‐synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo‐induced cross‐linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N‐terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long‐term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers.

aS is a neuronal pre-synaptic protein, and is thought to be involved in vesicular trafficking, neurotransmitter release, and regulation of neurotransmission (Clayton and George 1999;Fujiwara et al. 2002). The monomeric aS is a natively unfolded soluble protein without well-defined secondary or tertiary structures (Weinreb et al. 1996), but it transforms into cross-b-sheet-rich amyloid by self-assembly at physiological conditions via partially folded intermediates and soluble oligomers (Uversky et al. 2001). In a recent study with kinetic assays, it was proposed that aS fibrils grew by monomer and not oligomer addition and were subject to higher order assembly processes that decreased their capacity to grow (Buell et al. 2014). While the origin of aS toxicity is yet to be clear, accumulated evidence suggests that oligomeric forms of aS, rather than the larger intracellular inclusions, may be more bioactive and, possibly, cytotoxic, causing not only neuronal dysfunction but also cell death (Winner et al. 2011;Martin et al. 2012).
Epidemiological studies showed an inverse relationship between green tea consumption and the risk of developing PD (Chan et al. 1998;Ascherio et al. 2001). The major polyphenols present in green tea are catechins, especially epigallo catechin 3-gallate (EGCG), and green tea contains more myricetin (Myr) compared with black tea (Bosetti et al. 2005). Recently, a prospective study showed habitual intake of some polyphenols may reduce PD risk, and the association was more pronounced in men than women (Gao et al. 2012).
Results from in vitro and in vivo studies have indicated the protective effects of polyphenols, such as EGCG in green tea, curcuminoids in curry, baicalein extracted from the root of Scutellaria baicalensis, a traditional Chinese herb, or extracts from grape and blueberry, against neuronal damage in PD (Masuda et al. 2006;Chao et al. 2012). Levites et al. (2001) reported the neuroprotective activity of EGCG on 1methyl-4-phenyl-1,2,3,4-tetrahydropyridine-induced parkinsonism in animal models. It is suggested that the neuroprotective effects of EGCG are mediated by iron-chelating activities and free-radical-scavenging activities possessed by the cathecol group (Weinreb et al. 2009). Polyphenols also have protective effects against aS toxicity (Liu et al. 2011;Jiang et al. 2013). In PD cell model experiments, Curcumin (Cur) reduced aS-induced cytotoxicity by reduction of intracellular reactive oxygen species, mitochondrial depolarization, cytochrome c release, and caspase 9 and caspase 3 activation (Liu et al. 2011), or down-regulation of mTOR/ p70S6K signaling and the recovery of macroautophagy (Jiang et al. 2013).
We showed that phenolic compounds such as the winerelated polyphenol Myr, a major component of curry spice turmeric Cur, rosmarinic acid (RA), nordihydroguaiaretic acid (NDGA), and ferulic acid (FA) inhibited the formation of aS fibrils, as well as destabilizing preformed fibrils (Ono and Yamada 2006). Similarly, it was reported that baicalein (Zhu et al. 2004) and EGCG (Ehrnhoefer et al. 2008;Bieschke et al. 2010) also inhibited aS fibril formation and destabilized preformed aS fibrils.
Given this background, we examined the ability of five phenolic compounds -Myr, FA, NDGA, Cur, and RA ( Fig. 1)to interact with aS and inhibit the oligomerization of aS using well-established biophysical techniques. We assessed the oligomerization of aS with the methods of photo-induced cross-linking of unmodified proteins (PICUP), sodium dodecyl sulfate-polyaclylamide gel electrophoresis (SDS-PAGE), circular dichroism spectroscopy (CD), electron microscope (EM), atomic force microscope, and nuclear magnetic resonance (NMR) (Bitan and Teplow 2004;Ono et al. 2008;. We also evaluated the seeding effect of oligomeric aS using thioflavin S (ThS) assay (LeVine 1993;Naiki and Nakakuki 1996;Ono et al. 2013). Finally, we examined whether the phenolic compounds reduced aS oligomer-induced synaptic dysfunction with electrophysiological assays for long-term potentiation (LTP).

Materials and methods
Chemicals and reagents Chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) and were of the highest purity available. Water was produced using a Milli-Q system (Nihon Millipore K.K., Tokyo, Japan).
Proteins and phenolic compounds aS peptides were obtained from rPeptide (Osaka, Japan), and were > 95% purity. Purified peptides were stored as lyophilizates at À20°C. To prepare peptides for study, aS peptide lyophilizates were dissolved at a nominal concentration of 50 lM in 20 mM Tris HCl buffer, pH 7.4. The peptide solution was centrifuged for 30 min at 16 000 g at 4°C. A stock solution of glutathione S-transferase (GST;~26 kDa) (Sigma-Aldrich) was prepared by dissolving the lyophilizate to a concentration of 250 lM in 60 mM NaOH. Prior to use, aliquots were diluted 10-fold into 20 mM Tris-HCl, pH 7.4. We examined 5-phenolic compounds such as Myr, FA, NDGA, Cur, and RA. They were dissolved in ethanol to a concentration of 25 mM and then diluted to a final concentration of 2.5 mM with 20 mM Tris HCl buffer, pH 7.4, and we produced compounds of concentrations 5, 10, 25, 50, 100, and 500 lM for CD, PICUP, and atomic force microscope (AFM) as described previously (Bitan and Teplow 2004;Ono et al. 2012a).
Circular dichroism spectroscopy CD spectra of aS compound mixtures were acquired immediately after sample preparation or following 1-5 days of incubation. CD measurements were made by removing a 200-lL aliquot from the reaction mixture, adding the aliquot to a 1-mm path length CD cuvette (Hellma, Forest Hills, NY, USA), and acquiring spectra in a J-805 spectropolarimeter (JASCO, Tokyo, Japan). The CD cuvettes were maintained on ice prior to introduction into the spectrometer. Following temperature equilibration, spectra were recorded at 22°C from~190 to 260 nm at 0.2 nm resolution with a scan rate of 100 nm/min. Ten scans were acquired and averaged for each sample. Raw data were manipulated by smoothing and subtraction of buffer spectra according to the manufacturer's instructions.
Chemical cross-linking and determination of oligomer frequency distributions Immediately after their preparation, samples were cross-linked using PICUP, as described (Ono et al. 2012b). Briefly, 1 lL of 4 mM tris (2,2 0 -bipyridyl)dichlororuthenium(II) (Ru(bpy)) and 1 lL of 80 mM ammonium persulfate were added to 18 lL of 50 lM protein solution. The final protein of aS: Ru(bpy): ammonium persulfate molar ratios were 1 : 4 : 80. The mixture was irradiated for 1 s with visible light, and then the reaction was quenched with 2 lL of 1 M dithiothreitol (Invitrogen, Carlsbad, CA, USA) in ultrapure water. Determination of the frequency distribution of monomers and oligomers was accomplished using SDS-PAGE and silver staining as described (Ono et al. 2012b). Briefly, 8 lL of each cross-linked sample was electrophoresed on a 10-20% gradient tricine gel and visualized by silver staining (Invitrogen). Uncrosslinked samples were used as controls in each experiment. Densitometry was performed with a luminescent image analyzer (LAS 4000 mini; Fujifilm, Tokyo, Japan) and image analysis software (Multi gauge, version 3.2; Fujifilm). The intensity of each band in a lane from the SDS gel was normalized to the sum of the intensities of all the bands in that lane according to the formula, where R i is the normalized intensity of band I, and I i is the intensity of each band i. R i varies from 0 to 100. To calculate the oligomer ratio, the sum of oligomers intensities of aS with 2.5, 5, 25, 50, and 250 lM Myr, FA, NDGA, Cur, or RA, respectively, was divided by the sum of oligomer intensities without each compound. The EC 50 was defined as the concentration of phenolic compounds to inhibit a-synuclein oligomerization to 50% of the control value. The EC 50 was calculated by sigmoidal curve fitting, using GraphPad Prism software (version 4.0a; GraphPad Software Inc., San Diego, CA, USA). The effects on cross-linking were examined at different temperatures, pH values, and NaCl or ethanol concentrations.
Size-exclusion chromatography PICUP reagents and phenolic compounds were removed from crosslinked samples by size-exclusion chromatography as described previously (Volles et al. 2001;Ono et al. 2012a). To do so, we used the Superdex 200 10/300 GL column (GE Healthcare, Tokyo, Japan). At first, the column was washed twice with 0.5 M NaOH. Two hundred lL of cross-linked sample was then loaded. The column was eluted with 20 mM Tris HCl buffer at a flow rate of 0.5 mL/min. The first 4 mL of elute was collected. Fractions were lyophilized immediately after collection.
Seeding activity of assemblies of aS For the seeding assay, uncross-linked aS or cross-linked aS with or without Myr and RA at a concentration of 25 lM in 20 mM Tris buffer, pH 7.4 were added as seeds to uncross-linked aS at a ratio of 10% (v/v). The mixtures were incubated at 37°C for 0-7 days. ThS fluorescence were measured as mentioned below at 0, 1, 2, 6, 24. 48, 72, 96, 120, 144, and 168 h. Before the seeding assay, secondary structure of each aS assembly was checked, using CD studies.
Thioflavin S binding Thioflavin binding assays were performed because fluorescence intensities do correlate with aS fibril content (LeVine 1993;Naiki and Nakakuki 1996). The reaction mixture contained 5 lM ThS (MP Biomedicals, Irvine, CA, USA) and 50 mM glycine-NaOH buffer, pH 8.5. After brief vortexing, fluorescence was determined three times at intervals of 10 s using a Hitachi F-7500 fluorometer (Hitachi, Tokyo, Japan). Excitation and emission wavelength of 440 and 521 nm were used for aS assay, respectively. Fluorescence was determined by averaging three readings and subtracting the ThS blank readings.

Electron microscope
A 10 lL aliquot of each sample was spotted onto a glowdischarged, carbon-coated formvar grid (Okenshoji, Co, Ltd, Tokyo, Japan) and incubated for 20 min. The droplet then was displaced with an equal volume of 2.5% (v/v) glutaraldehyde in water and incubated for an additional 5 min. Finally, the peptide was stained with 8 lL of 1% (vol/vol) filtered (0.2 lm) uranyl acetate in water (Wako Pure Chemical Industries, Ltd, Osaka, Japan). This solution was wicked off and then the grid was air-dried. The samples were examined using a JEM-1210 transmission EM (JEOL Ltd., Tokyo, Japan).

Atomic force microscope
Peptide solutions were characterized using a Nanoscope IIIa controller (Veeco Digital Instruments, Santa Barbara, CA, USA) with a multimode scanning probe microscope equipped with a JV (Jtype vertical) scanner. All measurements were carried out in the tapping mode under ambient conditions using single-beam silicon cantilever probes. A 10-lL aliquot of each sample was spotted onto freshly cleaved mica (Ted Pella, Inc., Redding, CA, USA), incubated at 25°C for 5 min, rinsed with water, and then blown dry with air. At least four regions of the mica surface were examined to confirm the homogeneity of the structures throughout the sample. Mean particle heights were analyzed by averaging the measured values of eight individual cross-sectional line scans from each image only when the particle structure was confirmed.

Electrophysiology
The field excitatory post-synaptic potentials (fEPSPs) were recorded from the CA1 region of acute hippocampal slices derived from C57BL/6 mice (male, 4-5 weeks of age). The procedures for slice preparation and electrophysiological recording were described previously (Takamura et al. 2014). Briefly, 300-lm-thick transverse hippocampal slices were placed in a physiological chamber perfused with artificial cerebrospinal fluid (125 mM NaCl, 3.5 mM KCl, 1.25 mM NaH 2 PO 4 , 25 mM NaHCO 3 , 2.0 mM MgSO 4 , 2.0 mM CaCl 2 , and 20 mM glucose and aerated with a mixture of 95% O 2 and 5% CO 2 ) at a rate of 1 mL/min at 30°C. Shaffer collaterals/ commissural bundle in the CA3 hippocampal subfield were stimulated using a bipolar stainless steel wire electrode at 20-s intervals throughout the experiment. The fEPSPs were recorded from the stratum radiatum in the CA1 hippocampal subfield using a sharp glass electrode (2-6 Mohms, filled with 2 M NaCl). After fEPSP baseline became stabilized, slice was incubated with circulation of 10 mL aS sample (1 lM) for 90 min on the experimental chamber without electric stimulation. fEPSP recording was restarted after incubation and confirmed baseline stability at least 20 min. LTP was induced by two train of tetanic stimulation delivered at 100 Hz for 1 s. The evoked potential was amplified (91000), filtered (0.1-1000 Hz), digitized (20 kHz), and stored in a computer for off-line analysis using the PowerLab system (AD Instruments, Colorado Springs, CO, USA). LTP values were presented as the percentage of average fEPSPs slope relative to the mean value of the base line before tetanic stimulation.

NMR spectroscopy
The synthetic DNA encoding human aS was inserted into pOPTH plasmid. Sequencing of the inserted DNA was performed on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster city, CA, USA). The aS protein with an N-terminal His-tag (MAH-HHHHH) was expressed in E. coli BL21(DE3) harboring the pOPTH plasmid. The cells were grown in M9 minimal medium supplemented with 15 NH 4 Cl and 13 C-glucose. Purification of aS was carried out with a Ni-NTA agarose resin (Qiagen, Valencia, CA, USA), followed by further purification by gel-filtration on a Superdex 75 16/60 column (GE Healthcare Bio-Sciences). Matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed that His-tagged aS has no methionine at the N-terminus. MALDI-TOF MS analysis was performed on a Bruker Daltonics Autoflex-T1 mass spectrometer (Billerica, MA, USA). The three-repeat domain of tau with an Nterminal His-tag (AHHHHHH) was obtained in the same manner as the His-tagged aS.
Stock solutions of Myr and RA (54 mM) were prepared by dissolution in dimethylsulfoxide. Aliquots of the phenolic solutions were mixed with a solution containing 41 lM 13 C/ 15 N-synuclein, 10 mM HEPES (pH 7.4), 50 mM NaCl, 90 lM NaN 3 , 90 lM 2,2dimethyl-2-silapentane-5-sulfonate sodium salt and 10% D 2 O. The polyphenol concentration was 0.41 mM so that the final aS: polyphenol concentration ratio was 1 : 10. An NMR sample of the aS alone was also prepared in the same manner without polyphenol. The NMR sample was incubated at 15°C for 6 days before NMR measurements. NMR spectra were obtained with a Bruker Avance 800 MHz spectrometer equipped with a cryoprobe (Bruker BioSpin, Rheinstetten, Germany). NMR data were processed with NMRPipe (Delaglio et al. 1995) and analyzed with NMRView (Johnson 2004). The published backbone resonance assignments of aS (BMRB Entry 16300) were transferred to the data of His-tagged aS without polyphenol (Rao et al. 2009). The transferred assignments were confirmed by analyzing CBCANH, CBCA(CO)NH, HNCO, and HN(CA)CO (Clubb et al. 1992;Bax 1992a,b, 1993).

NMR-based molecular modeling
The model of aS was obtained with the use of CS-ROSETTA and the chemical shift assignment data (Shen et al. 2008(Shen et al. , 2009Rao et al. 2009). CS-ROSETTA generated the ensemble of the disordered aS, and one of them was used to show the regions affected by the polyphenol binding. Figure depicting the aS model were prepared with the program PyMOL (DeLano Scientific, San Carlos, CA, USA).

Statistical analysis
Dunnett multiple comparisons were used to determine statistical significance between cross-linked aS group versus other groups in LTP analysis. These tests were implemented within GraphPad Prism software (MDF, Tokyo, Japan). Significance was defined as p < 0.05.

aS oligomerization
To determine whether the five phenolic compounds blocked formation of low n-order aS oligomers, we used PICUP, a photochemical cross-linking method that is rapid, efficient, requires no structural modification of aS, and accurately reveals the oligomerization state of aS (Li et al. 2006). Following cross-linking as reported previously (Ono et al. 2012b), aS existed predominately as a mixture of monomers and oligomers of order 2-4, and higher order oligomers appear as smear bands (Fig. 2a). When 25 lM Myr was mixed with aS at a peptide: compound ratio of 1 : 1, oligomerization was blocked, bands of tetramer and higher order oligomers disappeared, and intensity of trimer band was decreased (Fig. 2a). When 250 lL of Myr was mixed with aS at a peptide: compound ratio of 1 : 10, oligomerization was blocked almost completely (Fig. 2a). When 25 (1 : 1) or 250 lM (1 : 10) RA was mixed with aS at the same ratios as used above, comparable effects were observed on aS oligomerization (Fig. 2a).
With aS: FA at a 1 : 1 ratio, intensities of bands of tetramer and higher order oligomers were decreased (Fig. 2a). At a higher concentration of FA (aS: FA, 1 : 10), oligomerization was blocked almost completely. With aS: NDGA at a 1 : 1 ratio and 1 : 10 ratio, similar effects was observed on aS oligomerization (Fig. 2a). With aS: Cur at a 1 : 1 ratio, no inhibition of aS oligomerization was observed. With aS: Cur at a 1 : 10 ratio, tetramer band was disappeared, and intensity of trimer band was decreased.
The results indicated that Myr and RA had the stronger inhibitory effects on aS oligomerization, compared to FA, NDGA, and Cur. We confirmed dose dependence of inhibitions by Myr and RA ( Fig. 2c and d). The EC 50 of Myr and RA for the aS oligomerization were 23.9 and 22.7 lM, respectively.
A potential problem relates to the possibility that the inhibition of aS oligomerization could have resulted from an alternative compound, which may form from a possible side reaction of the inhibitor and the PICUP sensitizer. To evaluate this possibility, cross-linking reactions also were performed on GST, a positive control for the cross-linking chemistry (Fancy and Kodadek 1999  exhibited an intense monomer band and a relatively faint dimer band (Fig. 2b). Cross-linking produced an intense dimer band, which was expected because GST exists normally as a homodimer, as well as higher order crosslinked species. No alterations in GST cross-linking were observed in the presence of Myr, FA, NDGA, Cur, or RA at either of the two protein: compound ratios tested, 1 : 1, 1 : 10. Thus, the significant inhibition of aS oligomerization is from a direct interaction with the phenolic compounds.
aS is a dipolar molecule with its basic N-terminal and acidic C-terminal, and weak protein-protein interactions could be the origin of the observed species upon chemical cross-linking rather than actual oligomer formation. To show that the cross-linking is not because of weak protein-protein interactions, we also performed PICUP experiments of aS at different temperatures, pH values, and NaCl concentrations, and did not detect any difference on aS oligomerization ( Figure S1a-c). The final ethanol concentrations in different samples ranged from 0 to 1%, and ethanol at these concentrations did not affect aS oligomerization in PICUP experiments ( Figure S1d).

Size-exclusion chromatography
The chromatograms were shown in Figure S2a. Reconstitution of the lyophilizates to a nominal concentration of 25 lM in 20 mM Tris HCl buffer, pH 7.4, followed by SDS-PAGE analysis, showed that removal of reagents and phenolic compounds, lyophilization and reconstitution did not alter the oligomer composition of any of the peptide populations under study ( Figure S2b).
aS assembly morphology We used AFM and EM to determine the morphology of the small assemblies present following PICUP of aS with or without phenolic compounds. The height of uncross-linked aS was 0.53 AE 0.05 nm ( Fig. 3 and Table 1) from AFM analysis. Following PICUP, the height of aS oligomers became 1.60 AE 0.24 nm. These morphologies are consistent with our previous findings (Ono et al. , 2012b. When aS was cross-linked with Myr at a compound: peptide ratio of 1 : 2, the height of treated aS decreased to 0.58 AE 0.06 nm. When aS was cross-linked with RA at a compound: peptide ratio of 1 : 2, the height of treated aS was decreased to 0.57 AE 0.06 nm (Fig. 3 and Table 1). Similarly, when aS was cross-linked with NDGA, FA, or Cur at a compound: peptide ratio of 1 : 2, the height of treated aS decreased to 0.54 AE 0.06 nm, 0.57 AE 0.07 nm, or 0.60 AE 0.07 nm, respectively. Similar data were obtained from EM analysis. The diameter of uncross-linked aS was 2.32 AE 0.17 nm. Following PICUP, the diameter of aS oligomers became 11.94 AE 1.51 nm. These morphologies are also consistent with our previous findings (Ono et al. 2012b). When aS was cross-linked with Myr, NDGA, FA, Cur, or RA at a compound: peptide ratio of 1 : 2, the diameters of treated aS decreased to 2.80 AE 0.34 nm, 2.70 AE 0.27 nm, 2.99 AE 0.38 nm, 3.15 AE 0.38 nm, or 2.67 AE 0.35 nm, respectively (Table 1).
aS secondary structure dynamics The above oligomerization studies revealed effects of the phenolic compounds at the initial stages of peptide selfassociation. To examine whether the phenolic compounds altered the secondary structure of the aS, we undertook CD studies (Fig. 4). aS, incubated alone, produced initial spectra characteristic of statistical coils (Fig. 4a). The major feature of these spectra was a large magnitude minimum centered at 198 nm. aS displayed substantial secondary structure changes between days 2-3 that were consistent with the previous study (Ono et al. 2012b). When aS were incubated with Myr and RA at a compound: peptide ratio of 1 : 2, no such transitions were observed ( Fig. 4b and c).

Secondary structures of aS assembly
We measured secondary structure of seeds, such as uncrosslinked aS, cross-linked aS with or without Myr or RA, using CD studies. The seed of cross-linked aS without phenolic compounds produced spectrum characteristic of b-sheet, on the other hand, the seeds of uncross-linked aS and crosslinked aS with Myr or RA produced spectrum characteristic of statistical coils ( Figure S3a).
Seeding activities of aS assemblies aS fibril assembly proceeds along a nucleation-dependent polymerization process (Wood et al. 1999). To monitor the abilities of cross-linked aS with or without phenolic compounds to exert fibril formation as seeds, we measured the time dependence of ThS fluorescence in seeded fibril formation experiments ( Figure S3b). Uncross-linked aS displayed a quasisigmoidal process curve characterized by an~6 h lag time, an~96 h period of increasing ThS binding, and a binding plateau occurring after~120 h ( Figure S3b). The unseeded reaction did not display initial fluorescence increase, within experimental error. Adding 10% cross-linked aS oligomers eliminated the lag period and produced a quasihyperbolic increase in fluorescence that reached maximal levels at~48 h, suggesting that crosslinked aS oligomers functioned as seeds. However, this seeding activity had disappeared in the cross-linked aS with Myr or RA ( Figure S3b). Maximal ThS levels for the aS oligomers seeded reaction was reached in 72 h, whereas those of seeded with uncross-linked aS, and cross-linked aS with Myr or RA reached in 120 h.

Electrophysiology
To obtain an index of cross-linked aS-induced functional alteration of synaptic transmission, we analyzed LTP in the CA1 region of mouse hippocampal slices. Synaptic current strength was estimated from fEPSP slope (Fig. 5). The vehicle group indicated LTP by tetanus stimulation (166 AE 7.6%). Uncross-linked aS did not affect LTP (177 AE 15.5%). Cross-linked aS completely inhibited induction of LTP (87 AE 19.4%). In contrast, cross-linked aS treated with Myr-and RA-induced LTP comparable with that in the vehicle (150 AE 15.6% and 155 AE 18.1%, respectively). Figure 5 shows differences in LTP induction among the five treatment groups. There was a significant group effect on %fEPSP slope in the cross-linked aS group was significantly lower than those in the other four groups, indicating that cross-linked aS-induced LTP suppression, but cross-linked aS treated with Myr and RA did not.

NMR studies
To study the interaction between the phenolic compounds and aS, we utilized NMR spectroscopy, a widely accepted method to obtain atomic level aspects of protein structure and ligand binding. Figure 6 shows the overlaid 1 H-15 N HSQC spectra of the aS alone and the aS containing the phenolic compounds at 1 : 10 molar ratio (aS: polyphenol). The 1 H-15 N HSQC spectra of aS are typical of an unstructured protein with limited resonance dispersion in the proton dimension (Wu et al. 2008).
Myr causes the reduction in the signal intensities owing to the broadening of NMR resonances (Fig. 6a). The broadening results from the interactions between Myr and aS. Severe broadening was observed only within the first nine residues of aS, indicating that the N-terminal region was involved in the Myr binding (Fig. 6a). On the other hand, neither chemical shift changes nor line broadening was observed upon RA addition, suggesting that RA did not bind to monomeric aS (Fig. 6b). The N-terminal His-tag on aS was not affecting the interaction between aS with Myr because we did not detect any significant binding by NMR of Myr to the three-repeat domain of tau expressed with an N-terminal His-tag ( Figure S4).

Discussion
We previously reported that several antioxidants including the phenolic compounds Myr, FA, NDGA, Cur, and RA had inhibitory effects on aS fibrillization and aS fibrildestabilizing effect (Ono et al. 2003). In this study, we revealed that all five phenolic compounds had dose-dependent inhibitory effects on aS oligomerization, using PICUP studies. Using EM and AFM analysis, the diameters and the heights of cross-linked aS treated with phenolic compounds were found to be smaller than cross-linked aS oligomers. The CD studies unraveled that Myr and RA stabilized aS populations comprising mostly random coil and inhibited statistical coils ? b-sheet conversion. In ThS assay, cross-linked aS treated with Myr and RA lost seeding activities. Taken together, these observations confirm the abilities of Myr and RA to inhibit aS oligomerization and secondary structure conversion. To unravel the chemical and neurophysiological basis of these effects, we performed LTP experiment and NMR analysis. Myr and RA decreased synaptic toxicities induced by aS oligomers on LTP assay of hippocampal slices. NMR showed direct binding of Myr to the first nine residues of the N-terminal region of the monomeric aS protein, whereas no direct binding of RA to the aS monomer was detected.
What is the mechanism underlying the inhibitory effects of the phenolic compounds on aS oligomerization? The binding of Myr to the first nine residues of the N-terminus of aS in the NMR experiment might contribute to inhibition of aS oligomerization. The sequence of aS can be divided into three domains: the N-terminal domain, the central fragment, also known as NAC (non-amyloid b component) region, and the C-terminal region. NAC region (residues 61-95) was reported to represent the critical determinant of oligomerization and the fibrillation process of aS (Hejjaoui et al. 2012). The C-terminal region (residues 96-140) is highly disordered and negatively charged, and it was shown that negatively charged side chains located in C-terminal region of aS acted to retard fibril formation by thioflavin T (ThT) binding assay (Izawa et al. 2012). Similar to Myr, a small  molecular tweezer, CLR01, was recently reported to bind selectively to Lys side chains at the N-terminal region of aS and prevent its aggregation by electron-capture dissociation mass spectrometry and PICUP study (Acharya et al. 2014).
Using ThT fluorescence and EM, we observed that in the presence of CLR01, aS did not form amyloid fibrils, and that CLR01 inhibited aS-mediated toxicity in cell cultures and zebrafish embryos (Prabhudesai et al. 2012). The data of fluorescence and mass-spectrometric analysis suggested that CLR01 kept aS monomeric by increasing its reconfiguration rate (Prabhudesai et al. 2012;Acharya et al. 2014). The polyphenol 3,4-dihydroxyphenylacetic acid bound to the Nterminal region of aS and inhibited fibrillation of aS binding non-covalently (Zhou et al. 2009). By deleting residues 2-11 in the N-terminal, aS aggregation was delayed and cellular membrane permeabilization of aS monomer and oligomer could be abolished (Lorenzen et al. 2014a). Also, N-terminal deletions in aS dramatically reduced toxicity toward yeast (Vamvaca et al. 2009). On the other hand, an NMR analysis revealed that EGCG non-covalently bound to the C-terminal region of the monomeric aS (D119, S129, E130, and D135), and redirected aS into unstructured, off-pathway aS oligomers (Ehrnhoefer et al. 2008). EGCG did not affect oligomer size distribution or secondary structure, and rather, immobilized the C-terminal region and moderately reduced the degree of binding of oligomers to membrane (Lorenzen et al. 2014b). Based on the results of these studies (Vamvaca et al. 2009;Zhou et al. 2009;Prabhudesai et al. 2012;Acharya et al. 2014;Lorenzen et al. 2014a) and our NMR study, it may be assumed that the binding region of aS with phenolic compounds might make equilibrium shift of aS aggregation capacities among the N-terminal, NAC, and the C-terminal regions toward the suppression of aggregation. Interestingly, the anti-oligomerization effects of phenolic compounds on aS are similar to those on Ab in our previous study (Ono et al. 2012a). In our previous NMR study with Ab, Myr was seen to bind to monomeric Ab at Arg-5, Ser-8, Gly-9, His-13, Lys-16, Asp-23, and Ile-31 (Ono et al. 2012a). There were no common amino acid sequences in Myr binding sites between Ab and aS. Unlike the result of Myr, we found no direct binding of RA to monomeric aS in the NMR study. In contrast, a previous NMR study reported the interaction between aS and RA, indicating that residues 3-18 and 38-51 of aS acted as non-covalent binding sites for RA (Rao et al. 2008). The discrepancy between the previous study (Rao et al. 2008) and the present one may be related to the lower concentration of aS used in this study. The ability of Myr and RA to block formation of low-order aS oligomers in our study is valuable, because low-order aS oligomers were thought to be the proximate neurotoxins in asynucleinopathies from the results of several in vitro and in vivo studies (Outeiro et al. 2008;Tsigelny et al. 2008;Paleologou et al. 2009). Visualization of aS oligomerization in living cells using bimolecular fluorescence complementation revealed that formation of oligomeric aS species was a central step toward cytotoxicity, which can be targeted through the activity of molecular chaperones, such as heatshock protein 70 (Outeiro et al. 2008). Consistent with this result, toxicity was seen without heavily aggregated aS in the experiment of dopaminergic and non-dopaminergic neurons, and it has been suggested that soluble species mediate toxicity (Xu et al. 2002). Taking this body of evidence into consideration, we performed an LTP assay of hippocampal slices to evaluate synaptic toxicities induced by aS oligomers. LTP has been widely used as a neurophysiological model of activity-dependent synaptic plasticity, and is considered an important neurophysiological model of learning and memory (Martin et al. 2000). It has been reported that human Ab oligomers inhibit hippocampal LTP in vitro and in vivo in rats (Townsend et al. 2006). However, knowledge about the effects of aS oligomers on synaptic plasticity is currently limited. It was reported that the exposure to aS oligomers impaired LTP through NMDA receptor activation, triggering enhanced contribution of calcium-permeable a-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid receptors in rat hippocampus slices (Di ogenes et al. 2012). The application of extracellular aS oligomers was reported to induce LTP suppression in hippocampal neurons via a calcineurin-dependent mechanism (Martin et al. 2012). Our work was consistent with previous studies (Di ogenes et al. 2012;Martin et al. 2012), where aS oligomers, not monomers, suppressed LTP in the hippocampal CA1 subfield, suggesting that memory formation is disturbed by aS oligomers. In contrast, cross-linked aS treated with Myr or RA partly cured LTP suppression. This result suggests that Myr and RA have preventive effects on aS oligomer-induced synaptic dysfunction by interfering with aS oligomerization. In recent studies, Cur showed effectiveness on motor activity, lifespan, oxidative stress, and apoptosis in the transgenic Drosophila model of PD (Siddique et al. 2013(Siddique et al. , 2014. In vivo experiments with animal models of a-synucleinopathies should be conducted to elucidate the effectiveness of phenolic compounds on asynucleinopathies. To develop the therapies for a-synucleinopathies by phenolic compounds, we need to overcome several issues. The first issue is low bioavailability of phenolic compounds. Cur displayed low oral bioavailability, poor water-solubility, short biological half-life, and lower permeability through the brain-blood barrier (Anand et al. 2007). According to the data of clinical trial of Cur, bioavailability of Cur was not high; the range for serum concentration was between 0.51 AE 0.11 lM at a dose of 4000 mg/day and 1.77 AE 1.87 lM at a dose of 8000 mg/day (Cheng et al. 2001). A dose escalation study showed the safety of Cur intake in healthy volunteers, thus they took oral Cur ranged from 500 to 12000 mg and serious adverse events were not reported (Lao et al. 2006). To the best of our knowledge, there have been no reports about the orally ingested Cur concentrations in cerebrospinal fluid. We have insufficient knowledge about bioavailability of Myr and RA, and we need to obtain further information about bioavailability of phenolic compounds. The next issue is the possibility that phenolic compounds may affect physiological functions of aS at synapses. Further experiments are necessary to elucidate whether extracellular phenolic compounds or intracellular phenolic compounds taken into the cell alter synaptic functions. Specific targeting of extracellular aS has an additional advantage as a therapeutic strategy, as this approach will not interfere with the normal function of intracellular aS. Although the most of aS, a neuronal presynaptic protein, exists intracellularly, the secreted extracellular aS, particularly in oligomerized forms, was reported to play important roles in major pathological changes of asynucleinopathies: deposition and spreading of aggregates, neuroinflammation, and neurodegeneration (Lee et al. 2014). We speculate that anti-oligomerization effects of phenolic compounds, such as Myr and RA, may reduce the formation of extracellular aS oligomers as well as intracellular aS oligomers, resulting in favorable effects on a-synucleinopathies.
In conclusion, our data establish that phenolic compounds inhibit oligomerization and statistical coils ? b-sheet conversion of aS through different aS binding, and reduce aS oligomer-induced synaptic toxicity. Although the exact in vivo mechanisms underlying the benefits of polyphenols remain to be established, the present data, coupled with previously reported antioxidant and neuroprotective effects, suggest that phenolic compounds may prove to be formidable candidates as disease modifying therapies for a-synucleinopathies.

Supporting information
Additional supporting information may be found in the online version of this article at the publisher's web-site: Figure S1. The conditions of PICUP experiments. Figure S2. Size exclusion chromatography (SEC) of aS assemblies. Figure S3. The secondary structures and seeding activities of aS assemblies. Figure S4. Analysis of interaction between tau and Myr.