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Catechol derivatives inhibit the fibril formation of amyloid-β peptides
Journal of Bioscience and Bioengineering VOL. 109 No. 6, 629 – 634, 2010 www.elsevier.com/locate/jbiosc
Catechol derivatives inhibit the fibril formation of amyloid-β peptides Vu Thi Huong,1 Toshinori Shimanouchi,1 Naoya Shimauchi,1 Hisashi Yagi,2 Hiroshi Umakoshi,1 Yuji Goto,2 and Ryoichi Kuboi1,⁎ Division of Chemical Engineering, Department of Engineering Science, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan 1 and Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan 2 Received 23 July 2009; accepted 13 November 2009 Available online 16 December 2009
The inhibition of fibril formation of amyloid β proteins (Aβ) would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). Dopamine (DA) and other catechol derivatives were used as inhibitory factors for Aβ fibril formation. The fibril formation of Aβ was monitored by Thioflavin T fluorescence, a transmission electron microscopy (TEM) and a total internal reflection fluorescence microscopy (TIRFM). Catechol and its derivatives showed the dose-dependent inhibitory effects on the spontaneous Aβ fibril formation. The inhibitory activity depended on the chemical structure of catechol derivatives both in the presence and absence of the liposome a model of biomembrane. Formation of catechol quinone-conjugated-Aβ adduct by a Schiff-base is a key step for the inhibition effect of Aβ fibril formation. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Liposome; Fibril formation; Inhibitor; Catechol derivatives; Amyloid beta; Alzheimer's disease]
The progressive deposition of amyloid-β protein (Aβ) in Alzheimer's disease is generally considered to be fundamental to the development of neurodegenerative pathology (1). Many researchers have demonstrated that Aβ fibrils promote the neurodegeneration in cell culture systems (2). The soluble monomeric Aβ is found to be non-toxic although its physiological function is not known in detail. The deposition of amyloid Aβ fibrils is believed to be causally linked to Alzheimer's disease (AD) (3). The aggregation of the soluble Aβ monomer into toxic oligomeric or fibrillar species is considered to be a crucial step in the pathology of the disease (4). It was reported that the most neurotoxic species are oligomers acting as intermediates during the formation of fibrils (5). Currently, there is no way to cure Alzheimer's disease or stop its progression. Therefore, preventing the formation of Aβ oligomers and fibrils are promising therapeutic strategies against AD. In previous studies, a number of inhibitors of fibril formation have been studied. It has been shown that the amyloid fibril formation can be inhibited by various compounds, such as pyrroloquinoline quinone (PQQ) (6), nitrophenols (7), salvianolic acid B (8), biocompatible nanogels (9), benzofurans (10), baicalein (11), curcumin, dopamine, L-DOPA, rosmarinic acid, nordihydroguaiaretic acid, selegiline hydrochloride (12, 13). The previous study has demonstrated that oxidative damage plays a central role in AD pathogenesis (14). In the in vitro experiments, many antioxidant compounds, such as vitamin E (15, 16), and nicotine (17, 18), have been demonstrated to protect the brain from the toxicity of Aβ, and clinical trials to test the ability of high dose vitamin E to slow down an AD progression have been ⁎ Corresponding author. Tel./fax: +81 6 6850 6286. E-mail address: [email protected] (R. Kuboi).
carried out. Polyphenol compounds, antioxidant compounds (19, 20), also have been reported as inhibitors for fibril formation of Aβ (21), such as DA and L-DOPA. Polyphenol compounds were also reported to inhibit the fibril formation of α-synuclein (22, 23). Aβ has recently been reported to form the fibrils on biomembrane which plays the role as the seeds for fibril formation (24). The Aβ fibrils formed in buffer and on membranes showed the different βsheet structure (25). It is likely that the biomemrane affects the formation of Aβ fibrils as a modulating factors of Aβ fibril formation. Amyloid fibrils deposit on neuronal cell membrane at which catechol derivatives such as dopamine and L-DOPA are present. It is, therefore, likely that the influence of dopamine towards the Aβ fibril formation occurred on the neuronal cell membrane. There is, however, no report to investigate the effect of catechol derivatives as a neurotransmitter against the inhibition of amyloid fibrils. In this study, we examined the inhibitory effect of representative compounds, shown in Fig. 1A, on fibril formation of Aβ with 40 and 42 amino acid residues. The inhibitory activity of these compounds on the Aβ fibril formation was examined not only in the bulk phase but also on the fatty acid and cholesterol-containing domain-like liposomes mimicking biomembranes. The inhibitory effect of catechol derivatives on Aβ fibril formation was clarified to affect on Aβ nucleation rather than on elongation process. Finally, the molecular mechanism of inhibition of amyloid fibril formation was discussed base on experimental data and previous reports summarized on Table 1. MATERIALS AND METHODS Materials Amyloid-β proteins with 40 and 42 amino acid residues (Aβ(1–40) and Aβ(1–42), respectively) were purchased from the Peptide Institute (Osaka, Japan).
1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.11.010
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FIG. 1. Chemical structure of the compounds used in this study (A). Effect of various compounds on fibril formation of Aβ(1–40) (B) and Aβ(1–42) (C) in the absence and in the presence 1 mM DMPC/SA (10:4) or 1 mM POPC/CH (6:4) liposomes. Aβ fibrils were prepared by incubating 10 μM Aβ monomer in Tris–HCl buffer solution at 37 °C for 24 h. Fibril formation was estimated by ThT assay, and the ThT fluorescence intensity in the absence of any compound was set to 100%.
Thioflavin T (ThT) was obtained from Dojindo (Kumamoto, Japan). Dopamine hydrochloride, L-Tyrosine (Tyr), Catechol (Cat), 3,4-dihydroxyphenylacetic acid (L-DOPA), Norepinephrine (NE), Epinephrine (EP) (Fig. 1A) and Stearic acid (SA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3–phosphocholine (DMPC) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol (Ch) was obtained from Wako Pure Chemical (Osaka, Japan). Other reagents used were of analytical grade. Fibril preparation and inhibitory experiment of Aβ fibril formation Aβ(1– 40) and Aβ(1–42) peptide solutions were prepared from powder by dissolving in 0.1% NH3 solution to 200 μM as stock solution at 4 °C. The stock solutions were stored at − 80 °C until using. Just before the experiment, the Aβ stock solutions were thawed and then diluted 20-fold by Tris-HCl buffer containing 50 mM Tris and 100 mM NaCl, pH 7.4, to a final concentration of 10 μM. The fibrils were prepared by incubating Aβ monomer
solution at 37 °C for at least 24 h. The fibril formation was monitored by measuring the ThT fluorescence intensity carried out at 37 °C using JASSO, PF-6500 fluorescence spectroscopy at an excitation wavelength of 442 nm and an emission wavelength of 485 nm. For the inhibition of Aβ fibril formation, the inhibitors, such as DA or other catechol derivatives, were added at the beginning of incubation time. To minimize potential interactions between the dye and catecholamines, the ThT fluorescence was measured immediately after addition of an aliquot of the incubated sample to the dye. Aβ seed preparation Seed stock solutions were prepared by the following procedure as previously reported (26). In brief, the seed was prepared from 10 μM of already fibrillated sample solution prepared under the conditions as described above. Fibrils were disrupted into seeds by sonication of the fibril solution with a probe sonicator in a 10-ml plastic tube on an ice-bath at 40 W with five-cycle sonication (each 1 min in sonication and 1 min in interval). The seed stocks were kept at 4 °C and briefly vortex mixed before use.
TABLE 1. Summary inhibitory factors and suggested mechanisms of inhibition of amyloid fibril formation. Chemical compounds Pyrroloquinoline (PQQ)
Peptide
Proposed mechanism
Ref.
α-synuclein
PQQ, which contains quinine group, bind to α-synuclein via a Schiff-base to form a PQQ-conjugated-α-synuclein adduct which prevent fibril formation. Nitrophenols bind to Aβ by hydrophobic interaction between its hydrophobic region and the hydrophobic region of Aβ, thus blocking the association between Aβ molecules, therefore lead to the inhibition of the fibril formation. Sal B has many hydroxyl groups that might interact with the peptide side chain to inactivate fibril aggregation. Sal B inhibits elongation process Baicalein quinone (oxidized baicalein) binds to α-synuclein by formation of a Schiff-base with a lysine side chain in α-synuclein to inhibit α-synuclein nucleation. The compact and symmetric structure of Curcumin (Cur) might be suitable for specially binding to free Aβ and subsequently inhibiting polymerization of Aβ into Aβ fibrils. TA and Myr could bind to the ends of short fibrils and then inhibit extension of fibrils. TA and Myr would bind to Aβ monomers and consequently inhibit of polymerization. Oxidized product of DA (DAQ) form coupling with Tyr to form a covalent adduct. The DAQ-α-synuclein covalent adduct formation by nucleophilic attack of DAQ to Lys side-chains (Lys forming a Schiff base with DAQ). DAQ-α-synuclein covalent adduct induced stabilization of protofibrils.
(6)
Nitrophenols
Aβ(1-42)
Salvianolic acid B (Sal B)
Aβ(1–40)
Baicalein
α-synuclein
Curcumin
Aβ(1–40) Aβ(1–42) Aβ(1–40) Aβ(1–42) α-synuclein
Tannic acid (TA), Myricetin (Myr) Dopamine L-DOPA
(7)
(8) (11) (12) (21) (22, 23)
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TABLE 2. The inhibition concentration (IC50) of Catechol, DA, L-DOPA, NE and EP for the Aβ(1–40) and Aβ(1–42) fibril formation. IC50 a/μM
Inhibitor
Catechol DA L-DOPA NE EP
Aβ(1–40)
Aβ(1–42)
5.2 ∼1 1.3 1.4 2.3
26.4 3.4 3.8 7.4 8.1
a IC50 (μM) was defined as the concentration of Catechol, DA, L-DOPA, NE and EP to inhibit the fibril formation to 50% of the control value.
Direct observation of Aβ fibrils The image of Aβ fibrils was obtained by a transmission electron microscope (TEM) according to the previous method (27). In brief, a 5-μl aliquot of diluted solution was placed on a copper grid (400-mesh) covered with a carbon-coated collodion film for 1 min and the excess sample solution was removed by blotting with filter paper. After the residual solution had dried up, the grid was negatively stained with a 2% (wt/vol) uranyl acetate solution. Again, the liquid on the grid was removed with filter paper and dried. TEM images were acquired using a JEOL JEM-1200EX transmission microscope (JEOL, Tokyo, Japan) with an acceleration voltage of 80 kV. The total internal reflection fluorescence microscope (TIRFM) system used to observe amyloid fibril formation was developed based on an inverted microscope (IX70, Olympus, Tokyo, Japan) (27). The ThT molecule was excited at 442 nm by a helium-cadmium laser (IK5552R-F, Kimmon, Tokyo, Japan). The laser power was 40– 80 mW, and the observation period was 3–5 s. The fluorescence image was filtered with a band pass filter (D490/30, Omega Optical, Bratteboro, VT) and visualized using a digital steel camera (DP70, Olympus). CD spectroscopy The secondary structures of Aβ with and without DA were analyzed by using a circular dichroism spectrometer (J-720W, JASCO, Tokyo, Japan). CD spectra were measured by using a quartz cell from 195 to 250 nm with a step interval 0.1 nm bandwidth and a scanning speed of 10 nm min− 1. Liposome preparation The liposomes were prepared by the previous method (28, 29). The lipid mixture was dissolved in chloroform and then dried onto the wall of a round-bottom flask in vacuum and was then left overnight to remove all of the solvent. The dried thin lipid film was hydrated with adequate buffer solution to form multilamellar vesicles (MLVs). Large unilamellar vesicles (LUVs) were formed from the MLVs with five cycles of freeze-thaw treatment. Alternatively, monodisperse phospholipids molecules of DMPC and stearic acid (SA) at molar ratio (10:4), and phospholipids molecules of POPC and cholesterol (Ch) at molar ratio (6:4) were used for making liposomes in diameter of 100 nm.
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RESULTS AND DISCUSSION Catechol derivatives inhibit Aβ fibril formation The inhibition of amyloid fibril formation was believed to be a promising strategy for the treatment of AD (30). In this study, the possibility that catechol and its derivatives inhibit Aβ fibril formation was investigated by incubating Aβ monomers in Tris–HCl buffer at 37 °C in the presence of catechol derivative compounds (Fig. 1B, C). The control experiments were performed without any catechol derivative compound. After 24 h of incubation, the ThT fluorescence intensity in the solution increased in the absence of catechol derivatives, showing the fibril formation. The fluorescence in the absence of catechol derivatives was then set to 100%. In the presence of 100 μM catechol derivative compounds, the value of ThT fluorescence relative to the control experiment was very small, indicating the inhibition of the fibril formation. We also examined the inhibitory effect of Tyr with similar chemical structure to L-DOPA. The result shows that Tyr had no effect on inhibition of fibril formation. This result suggests the requirement of a catechol group for an inhibitory effect on the Aβ fibril formation. Furthermore, we examined the inhibitory activity of these compounds on Aβ fibril formation in the presence of fatty acidand cholesterol-containing domain-like liposomes as a model biomembrane. These liposomes strongly have been reported to interact with Aβ fibrils. The results showed that catechol and its derivatives exhibited the inhibitory effect on Aβ fibril formation not only in the buffer but also in the presence of liposomes as model biomembranes (Fig. 1B, C). The inhibitory effects of catechol derivatives on the conformation of Aβ were investigated in the presence of catechol derivatives at various concentrations. Catechol and its derivatives showed the dosedependent inhibitory effect on Aβ fibril formation (data not shown). The concentration of these compounds necessary for half-maximal effect (IC50) increased in an order of DA b L-DOPA b NE b EP b Catechol for both of Aβ(1–40) and Aβ(1–42) (Table 2). From the experimental results of ThT monitoring, DA was suggested to posses the strongest inhibitory effect against the fibril formation of both Aβ(1–40) and
FIG. 2. The direct observation with two different microscopic techniques. The TIRFM images (scale: 10 μm) of fibrils formed in the absence of DA (A1, A2) and in the presence of 100 μM DA (C1, C2) for Aβ(1–40) and Aβ(1–42), respectively. The TEM images (scale: 200 nm) of fibrils formed in the absence of DA (B1, B2) and in the presence of 100 μM DA (D1, D2) for Aβ(1–40) and Aβ(1–42), respectively. Aβ fibrils were prepared by incubating 10 μM Aβ monomer in Tris–HCl buffer solution at 37 °C for 24 h.
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Aβ(1–42). Therefore, DA-induced inhibition of fibril formation was investigated in the following. Direct observation of DA inhibit Aβ fibril formation In order to demonstrate the inhibition of Aβ fibril formation by DA, a direct observation of Aβ fibrils was performed with a TIRFM combined with ThT fluorescence (Fig. 2). TIRFM is a useful technique to evaluate a population and a length of fibrils on a quartz slide. In Fig. 2A1, a considerable Aβ(1–40) fibrils with 10.2 μm in average length were observed in the absence of any inhibitor. The fibrils observed by a TEM were confirmed to posse the microscopic structure peculiar to amyloid fibrils (Fig. 2B1), consistent with the previous reports (23). On the contrary, the samples of Aβ(1−40) monomers co-incubated with 100 μM DA (for 24 h) showed no ThT fluorescence intensity in the observation field of TIRFM, indicating the inhibitory effect of DA on the fibril formation of Aβ (Fig. 2C1). At a microscopic observation with TEM, the Aβ fibrils could not be observed and small amorphous aggregates were observed in the samples of 10 μM Aβ monomers coincubated with 100 μM DA for 24 h (Fig. 2D1). The similar results were obtained for Aβ(1–42) as shown in Fig. 2(A2–D2). The samples of Aβ(1–42) monomers co-incubated with 100 μM DA (for 24 h) showed a slight ThT fluorescence intensity in the observation field of TIRFM (Fig. 2C2), few very short fibrils coexisting with small
J. BIOSCI. BIOENG., amorphous aggregates were obtained by TEM (Fig. 2D2) indicating DA also shows inhibitory effect on Aβ(1–42) fibril formation. We thus considered that DA could inhibit the fibril formation of Aβ. Effect of DA on the kinetics of Aβ fibril formation To investigate the effects of DA on the kinetics of Aβ fibril formation, the ThT fluorescence intensity was measured during fibril formation (Fig. 3). The control experiment was performed by incubating 10 μM Aβ monomers without any inhibitor. The time-course of ThT fluorescence intensity showed a characteristic sigmoidal curve. According to the previous report (31), this curve consists of two processes: (i) a nucleation process and (ii) an elongation process. In (i), the oligomer formation or the nucleation occurs while the fluorescence variation was not detected. This duration is defined as “lag phase” and its time is a “lag time.” And in (ii), the fibrils grow by the binding of monomers to the ends of fibrils and the ThT fluorescence intensity increased (Fig. 3A). This duration is called as “elongation phase” and its rate is an “elongation rate”. When a 10-μM Aβ(1–40) monomer solution was incubated in the presence of 5 μM DA, the final equilibrium level of ThT was low in comparison with the control experiment. However, the lag time and elongation time (time of elongation phase) were similar to those of control experiment. Furthermore, the fluorescence intensity did not
FIG. 3. Effect of DA on kinetics of Aβ fibril formation. The time-course of the ThT fluorescence during Aβ(1–40) (A) and Aβ(1–42) (C) fibril formation without DA (filled circles) and with 5 μM DA (open circles), with 100 μM DA (open squares) from Aβ monomer solutions. The time course of the ThT fluorescence during Aβ(1–40) (B) and Aβ(1–42) (D) fibril formation without DA and with 5 μM, 100 μM DA, from 7 μM Aβ monomer and 3 μM Aβ seed solution. Aβ seed + Aβ only (filled circles); Aβ seed + Aβ monomer + 5 μM DA (open circles); Aβ seed + Aβ monomer + 100 μM DA(open squares); Aβ seed + 10 h pre-incubated solution containing 100 μM DA and 7 μM Aβ monomer (filled squares). All the solution were prepared in Tris–HCl buffer solution and incubated at 37 °C.
VOL. 109, 2010 increase in the case of 100 μM DA, indicating fibril elongation process during 24 h. In order to estimate the effect of DA on fibril growth rate, the elongation rate was estimated to be 0.81 h− 1 and 0.78 h− 1 in the absence and the presence of 5 μM DA, respectively. Therefore, it is likely that DA varied the elongation phase slightly. The similar results were obtained for Aβ(1–42) (Fig. 3C). The inhibition of Aβ fibril formation by catechol derivative compounds may involve two broadly different mechanisms, in inhibition of either a nucleation process or an elongation process. In order to clarify if DA could inhibit a nucleation process or elongation process or both of these processes, we investigated the effect of DA on kinetic of Aβ fibril formation started from seeds of Aβ fibrils. Their seeds were added to Aβ monomer solution to make the final concentrations of seeds and monomers at 3 μM and 7 μM, respectively. The results were shown in Fig. 3B and D for Aβ(1–40) and Aβ(1–42), respectively. In the absence of DA, the fluorescence intensity significantly increased without a lag phase and reached to plateau more rapidly in contrast to the sample without seeds. These curves are consistent with the previous first-order kinetic model (32). The elongation rate was estimated to be 0.80 h− 1, which is similar to the case of a spontaneous fibrillation (Fig. 3A). The addition of seeds to the monomer solution in the presence of 5 μM DA also showed the similar increase of the ThT fluorescence intensity to the case in the absence of DA. The elongation of Aβ(1–40) fibrils was induced up to 4 h. 100 μM DA could not inhibit the elongation up to 1 h or so in the presence of seeds (Fig. 3B) although 100 μM DA could completely inhibit the spontaneous Aβ fibril formation (Fig. 3A). The initial rate in ThT fluorescence intensity (apparent elongation rate) was similar in the experiments with 5 and 100 μM of DA. Thus, we considered that the DA did not inhibit the elongation process of Aβ fibril formation and that DA inhibits the nucleation process in the fibril formation of Aβ(1–40). The similar results were obtained for Aβ(1–42) (Fig. 3D). As another reason for the above consideration, we have reported the disaggregation of Aβ fibrils by the same compounds listed in Fig. 1A (Vu, H. T., Shimanouchi, T., Ishikawa, D., Matsumoto, T., Yagi, H., Umakoshi, H., Goto, Y., and Kuboi, R., unpublished data). Our latest study indicated that the decrease in ThT fluorescence intensity resulted from the disaggregation of Aβ fibrils. Both the decrement change in ThT intensity and the transition time from elongation phase to disaggregation phase depends on the concentration of DA, which strongly supports the DA-induced disaggregation of fibrils. From the results, it is considered that DA inhibits the nucleation process, rather than the elongation process. Also, the other catechol derivatives showed the similar trends to DA. Possible mechanism of inhibition of Aβ fibril formation and the effect of liposome The obtained results clearly showed that catechol derivatives could inhibit the Aβ fibril formation. The remaining problem is the mechanism responsible for the inhibition of amyloid fibril formation. Then, the previously proposed mechanisms for the inhibition on amyloid fibril formation by reagents were summarized to elucidate the possible mechanism (Table 1). It has been reported that, for inhibition of α-synuclein fibril formation, the oxidized product of dopamine (dopamine quinone, DAQ) stabilized the protofibrils of α-synuclein, leading to an inhibition of fibril formation, where 5–10% of the α-synuclein molecules formed a covalent adduct with DAQ by a reaction of DAQ with Tyr of α-synuclein (22, 23). Also, in the other chemical compounds, it has been reported that the formation of a Schiff-base between quinone group of inhibitory compounds and Lys-side chain of α-synuclein induced the inhibition of fibril formation (6, 11). On the other hands, a nucleophilic attack of DAQ to Lys (Lys forming a Schiff base with DAQ) is also considered as another possible mechanism to form the complex formation between proteins and compounds (22, 23). Therefore, we supposed that the oxidized product of
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catechol derivatives (catechol quinone and its derivatives) bound to Aβ monomer to form the covalent adduct-like complex. The effect of DA on the secondary structure of Aβ monomer was investigated though the circular dichroism measurement. Fig. 4A shows that the structure of DAQ-Aβ(1–40) complex (incubated for 24 h) was similar to that of Aβ(1–40) monomer, suggesting that DA/DAQ retains the secondary structure of Aβ. Therefore, the catechol ring of catechol derivatives as a common chemical structure might contribute to their interaction with Aβ. The sequence of Aβ(1–40) and Aβ(1–42) involves two Lys residues (Lys16 and Lys28) (33). It is therefore likely that the catechol quinone and its derivatives could form a Schiff-base with Aβ molecule although we have no direct evidence. This conjugated adduct might stabilize the disordered conformation and be advantageous for a prevention of the conversion to a β-sheet-rich structure. The addition of seeds to a 10-h pre-incubated sample of 7 μM Aβ monomer and 100 μM DA inhibited the formation of intermediate of Aβ fibrils (closed square in Fig. 3B, D). No increase in ThT fluorescence intensity was observed, strongly suggesting that the conjugated adduct has the structure disadvantageous for a binding to the seeds for fibril growth. The similar results were obtained for
FIG. 4. CD spectra of Aβ(1–40) (A) and Aβ(1–42) (B) at the beginning (0 h) and the end (24 h) of incubation in the absence and the presence of 100 μM DA. Aβ only at 0 h (curve 1), Aβ only at 24 h (curve 2); Aβ and 100 μM DA at 0 h (curve 3); and Aβ and 100 μM DA at 24 h (curve 4). The solutions were prepared by incubating 10 μM Aβ monomer on PBS buffer pH 7.4 at 37 °C.
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inhibition of Aβ(1–42) fibril formation but the effect of catechol derivatives on Aβ(1–42) fibril formation was less than that of Aβ(1– 40) (Table 2 and Fig. 4B). Aβ(1–42) was reported to show the higher propensity to form fibrils by showing the shorter lag time compare to Aβ(1–40). Therefore, these data also confirmed the inhibitory effect of catechol derivatives on nucleation process rather than elongation process. In the presence of liposome membranes, hydrophobic catechol and its derivatives, such as dopamine, prefer to partition into the membrane surface (data not shown). Aβ(1–40) and Aβ(1–42) could also interact with the liposome membranes in which the liposome could play a promotion factor for fibril formation (24). These will effectively enhance the formation of the conjugated adduct on liposome membrane. Therefore, the environment of the neuronal cell membrane with coexisting catecholamine would be advantageous to inhibit the amyloid fibril formation although the detailed experiments are needed. Under normal conditions, the concentration of Aβ is approximately 50 nM. The dopamine present at the neuronal cell is in micromolar levels enough to delay the fibril formation of Aβ if the inhibitory effect by dopamine could play a dominant role in the inhibition of fibril formation. In conclusion, our data suggest that the coexistence of catechol and its derivatives at micromolar concentrations is sufficient to significantly inhibit the spontaneous fibril formation. The catecholamine quinone formed an adduct disadvantageous for a nucleation process and for its binding to formed nuclei, leading to the inhibition of the spontaneous amyloid fibril formation. Catechol derivatives would be useful for inhibiting the formation of Aβ fibrils in its early stages. ACKNOWLEDGMENTS The authors also thank the Grant-in-Aid for Scientific Research (No: 19566203, 19656220, 20760539, 20360350, 21246121) from the Ministry of Education, Science, Sports, and Culture of Japan, a grant from the 21st Century COE program “Creation of Integrated EcoChemistry” and the Global COE program “Bio-Environmental Chemistry” of JSPS. This work was also partly supported by the Core University Program between JSPS and Vietnamese Academy of Science and Technology (VAST). The authors are grateful to the Research Center for Solar Energy Chemistry of Osaka University and the Gas hydrate Analyzing System (Osaka University, Japan). A part of the present experiments was carried out by using a facility in the Research Center for Ultrahigh Voltage Electron Microscopy (Osaka University). References 1. Mattson, M. P.: Pathways towards and away from Alzheimer's disease, Nature, 430, 631–639 (2004). 2. Iversen, L. L., Mortishiresmith, R. J., Pollack, S. J., and Shearman, M. S.: The toxicity in vitro of β-amyloid protein, Biochem. J., 311, 1–16 (1995). 3. Yankner, B. A.: Mechanisms of neuronal degeneration in Alzheimer's disease, Neuron, 16, 921–932 (1996). 4. Resende, R., Ferreiro, E., Pereira, C., and Resende, D. O. C.: Neurotoxic effect of oligomeric and fibrillar species of amyloid-beta peptide 1-42: involvement of endoplasmic reticulum calcium release in oligomer-induced cell death, Neuroscience, 155, 725–737 (2008). 5. Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W., and Glabe, C. G.: Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis, Science, 300, 486–489 (2003). 6. Kobayashi, M., Kim, J., Kobayashi, N., Han, S., Nakamura, C., Ikebukuro, K., and Sode, K.: Pyrroloquinoline quinone (PQQ) prevents fibril formation of α-synuclein, Biochem. Biophys. Res. Com., 349, 1139–1144 (2006). 7. Felice, F. G. D., Houzel, J. C., Garcia-Abreu, J., Louzada Jr., P. R. F., Jr., Afonso, R. C., Meirelles, M. N. L., Lent, R., Neto, V. M., and Ferreira, S. T.: Inhibition of Alzheimer's disease β-amyloid aggregation, neurotoxicity, and in vivo deposition by nitrophenols: implications for Alzheimer's therapy, FASEB J., 15, 1297–1299 (2001).
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Neurochem., 87, 172–181 (2003). 21. Ono, K., Hasegawa, K., Naiki, H., and Yamada, M.: Anti-amyloidogenic activity of tannic acid and its activity to destabilize Alzheimer's β-amyloid fibrils in vitro, Biochim. Biophys. Acta, 1690, 193–202 (2004). 22. Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T.: Kinetic stabilization of the α-synuclein protofibril by a dopamine-α-synuclein adduct, Science, 294, 1346–1349 (2001). 23. Li, J., Zhu, M., Manning-Bog, A. B., Monte, D. A. D., and Fink, A. L.: Dopamine and L-DOPA disaggregate amyloid fibrils: implications for Parkinson's and Alzheimer's disease, FASEB J., 18, 962–964 (2004). 24. Matsuzaki, K., Noguch, T., Wakabayashi, M., Ikeda, K., Okada, T., Ohashi, Y., Hoshino, M., and Naiki, H.: Inhibitors of amyloid β-protein aggregation mediated by GM1-containing raft-like membranes, Biochim. Biophys. Acta, 1768, 122–130 (2007). 25. Kakio, A., Yano, Y., Takai, D., Kuroda, Y., Matsumoto, O., Kozutsumi, Y., and Matsuzaki, K.: Interaction between amyloid β-protein aggregates and membranes, J. Peptide Sci., 10, 612–621 (2004). 26. Andersen, C. B., Yagi, H., Manno, M., Martorana, V., Ban, T., Christiansen, G., Otzen, D. E., Goto, Y., and Rischel, C.: Branching in amyloid fibril growth, Biophys. J., 96, 1529–1536 (2009). 27. Yagi, H., Ban, T., Morigaki, K., Naiki, H., and Goto, Y.: Visualization and classification of amyloid β supuramolecular assemblies, Biochemistry, 46, 15009–15017 (2007). 28. Shimanouchi, T., Ishii, H., Yoshimoto, N., Umakoshi, H., and Kuboi, R.: Calcein permeation across phosphatidylcholine bilayer membrane: effects of membrane fluidity, liposome size, and immobilization, Coll. Surf. B, 73, 156–160 (2009). 29. Kuboi, R., Shimanouchi, T., Yoshimoto, M., and Umakoshi, H.: Detection of protein conformation under stress condition using liposomes as sensor materials, Sens. Mater., 16, (5) 241–254 (2004). 30. Soto, C., Sigurdsson, E. M., Morelli, L., Kumar, A., Castano, E. M., and Frangione, B.: β-Sheet breaker pepides inhibit fibrillogenesis in a rat brain model of amyloidosis: implication for Alzheimer's therapy, Nat. Med., 4, 822–826 (1998). 31. Nuria, B. C., Mercedes, C., and Josep, C.: Conversion of non-fibrillar β-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation, Biochem. Biophys. Res. Com., 361, 916–921 (2007). 32. Naiki, H. and Nakakuki, K.: First-order kinetic model of Alzheimer's beta-amyloid fibril extension in vitro, Lab. Invest., 74, 374–383 (1996). 33. Petkova, A. T., Ishii, Y., Balbach, J. J., Antzukin, O. N., Leapman, R. D., Delaglio, F., and Tycko, R.: A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR, Proc. Natl. Acad. Sci. U. S. A., 99, 16742–16747 (2002).
Catechol derivatives inhibit the fibril formation of amyloid-β peptides Vu Thi Huong,1 Toshinori Shimanouchi,1 Naoya Shimauchi,1 Hisashi Yagi,2 Hiroshi Umakoshi,1 Yuji Goto,2 and Ryoichi Kuboi1,⁎ Division of Chemical Engineering, Department of Engineering Science, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan 1 and Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan 2 Received 23 July 2009; accepted 13 November 2009 Available online 16 December 2009
The inhibition of fibril formation of amyloid β proteins (Aβ) would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). Dopamine (DA) and other catechol derivatives were used as inhibitory factors for Aβ fibril formation. The fibril formation of Aβ was monitored by Thioflavin T fluorescence, a transmission electron microscopy (TEM) and a total internal reflection fluorescence microscopy (TIRFM). Catechol and its derivatives showed the dose-dependent inhibitory effects on the spontaneous Aβ fibril formation. The inhibitory activity depended on the chemical structure of catechol derivatives both in the presence and absence of the liposome a model of biomembrane. Formation of catechol quinone-conjugated-Aβ adduct by a Schiff-base is a key step for the inhibition effect of Aβ fibril formation. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Liposome; Fibril formation; Inhibitor; Catechol derivatives; Amyloid beta; Alzheimer's disease]
The progressive deposition of amyloid-β protein (Aβ) in Alzheimer's disease is generally considered to be fundamental to the development of neurodegenerative pathology (1). Many researchers have demonstrated that Aβ fibrils promote the neurodegeneration in cell culture systems (2). The soluble monomeric Aβ is found to be non-toxic although its physiological function is not known in detail. The deposition of amyloid Aβ fibrils is believed to be causally linked to Alzheimer's disease (AD) (3). The aggregation of the soluble Aβ monomer into toxic oligomeric or fibrillar species is considered to be a crucial step in the pathology of the disease (4). It was reported that the most neurotoxic species are oligomers acting as intermediates during the formation of fibrils (5). Currently, there is no way to cure Alzheimer's disease or stop its progression. Therefore, preventing the formation of Aβ oligomers and fibrils are promising therapeutic strategies against AD. In previous studies, a number of inhibitors of fibril formation have been studied. It has been shown that the amyloid fibril formation can be inhibited by various compounds, such as pyrroloquinoline quinone (PQQ) (6), nitrophenols (7), salvianolic acid B (8), biocompatible nanogels (9), benzofurans (10), baicalein (11), curcumin, dopamine, L-DOPA, rosmarinic acid, nordihydroguaiaretic acid, selegiline hydrochloride (12, 13). The previous study has demonstrated that oxidative damage plays a central role in AD pathogenesis (14). In the in vitro experiments, many antioxidant compounds, such as vitamin E (15, 16), and nicotine (17, 18), have been demonstrated to protect the brain from the toxicity of Aβ, and clinical trials to test the ability of high dose vitamin E to slow down an AD progression have been ⁎ Corresponding author. Tel./fax: +81 6 6850 6286. E-mail address: [email protected] (R. Kuboi).
carried out. Polyphenol compounds, antioxidant compounds (19, 20), also have been reported as inhibitors for fibril formation of Aβ (21), such as DA and L-DOPA. Polyphenol compounds were also reported to inhibit the fibril formation of α-synuclein (22, 23). Aβ has recently been reported to form the fibrils on biomembrane which plays the role as the seeds for fibril formation (24). The Aβ fibrils formed in buffer and on membranes showed the different βsheet structure (25). It is likely that the biomemrane affects the formation of Aβ fibrils as a modulating factors of Aβ fibril formation. Amyloid fibrils deposit on neuronal cell membrane at which catechol derivatives such as dopamine and L-DOPA are present. It is, therefore, likely that the influence of dopamine towards the Aβ fibril formation occurred on the neuronal cell membrane. There is, however, no report to investigate the effect of catechol derivatives as a neurotransmitter against the inhibition of amyloid fibrils. In this study, we examined the inhibitory effect of representative compounds, shown in Fig. 1A, on fibril formation of Aβ with 40 and 42 amino acid residues. The inhibitory activity of these compounds on the Aβ fibril formation was examined not only in the bulk phase but also on the fatty acid and cholesterol-containing domain-like liposomes mimicking biomembranes. The inhibitory effect of catechol derivatives on Aβ fibril formation was clarified to affect on Aβ nucleation rather than on elongation process. Finally, the molecular mechanism of inhibition of amyloid fibril formation was discussed base on experimental data and previous reports summarized on Table 1. MATERIALS AND METHODS Materials Amyloid-β proteins with 40 and 42 amino acid residues (Aβ(1–40) and Aβ(1–42), respectively) were purchased from the Peptide Institute (Osaka, Japan).
1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.11.010
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FIG. 1. Chemical structure of the compounds used in this study (A). Effect of various compounds on fibril formation of Aβ(1–40) (B) and Aβ(1–42) (C) in the absence and in the presence 1 mM DMPC/SA (10:4) or 1 mM POPC/CH (6:4) liposomes. Aβ fibrils were prepared by incubating 10 μM Aβ monomer in Tris–HCl buffer solution at 37 °C for 24 h. Fibril formation was estimated by ThT assay, and the ThT fluorescence intensity in the absence of any compound was set to 100%.
Thioflavin T (ThT) was obtained from Dojindo (Kumamoto, Japan). Dopamine hydrochloride, L-Tyrosine (Tyr), Catechol (Cat), 3,4-dihydroxyphenylacetic acid (L-DOPA), Norepinephrine (NE), Epinephrine (EP) (Fig. 1A) and Stearic acid (SA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3–phosphocholine (DMPC) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol (Ch) was obtained from Wako Pure Chemical (Osaka, Japan). Other reagents used were of analytical grade. Fibril preparation and inhibitory experiment of Aβ fibril formation Aβ(1– 40) and Aβ(1–42) peptide solutions were prepared from powder by dissolving in 0.1% NH3 solution to 200 μM as stock solution at 4 °C. The stock solutions were stored at − 80 °C until using. Just before the experiment, the Aβ stock solutions were thawed and then diluted 20-fold by Tris-HCl buffer containing 50 mM Tris and 100 mM NaCl, pH 7.4, to a final concentration of 10 μM. The fibrils were prepared by incubating Aβ monomer
solution at 37 °C for at least 24 h. The fibril formation was monitored by measuring the ThT fluorescence intensity carried out at 37 °C using JASSO, PF-6500 fluorescence spectroscopy at an excitation wavelength of 442 nm and an emission wavelength of 485 nm. For the inhibition of Aβ fibril formation, the inhibitors, such as DA or other catechol derivatives, were added at the beginning of incubation time. To minimize potential interactions between the dye and catecholamines, the ThT fluorescence was measured immediately after addition of an aliquot of the incubated sample to the dye. Aβ seed preparation Seed stock solutions were prepared by the following procedure as previously reported (26). In brief, the seed was prepared from 10 μM of already fibrillated sample solution prepared under the conditions as described above. Fibrils were disrupted into seeds by sonication of the fibril solution with a probe sonicator in a 10-ml plastic tube on an ice-bath at 40 W with five-cycle sonication (each 1 min in sonication and 1 min in interval). The seed stocks were kept at 4 °C and briefly vortex mixed before use.
TABLE 1. Summary inhibitory factors and suggested mechanisms of inhibition of amyloid fibril formation. Chemical compounds Pyrroloquinoline (PQQ)
Peptide
Proposed mechanism
Ref.
α-synuclein
PQQ, which contains quinine group, bind to α-synuclein via a Schiff-base to form a PQQ-conjugated-α-synuclein adduct which prevent fibril formation. Nitrophenols bind to Aβ by hydrophobic interaction between its hydrophobic region and the hydrophobic region of Aβ, thus blocking the association between Aβ molecules, therefore lead to the inhibition of the fibril formation. Sal B has many hydroxyl groups that might interact with the peptide side chain to inactivate fibril aggregation. Sal B inhibits elongation process Baicalein quinone (oxidized baicalein) binds to α-synuclein by formation of a Schiff-base with a lysine side chain in α-synuclein to inhibit α-synuclein nucleation. The compact and symmetric structure of Curcumin (Cur) might be suitable for specially binding to free Aβ and subsequently inhibiting polymerization of Aβ into Aβ fibrils. TA and Myr could bind to the ends of short fibrils and then inhibit extension of fibrils. TA and Myr would bind to Aβ monomers and consequently inhibit of polymerization. Oxidized product of DA (DAQ) form coupling with Tyr to form a covalent adduct. The DAQ-α-synuclein covalent adduct formation by nucleophilic attack of DAQ to Lys side-chains (Lys forming a Schiff base with DAQ). DAQ-α-synuclein covalent adduct induced stabilization of protofibrils.
(6)
Nitrophenols
Aβ(1-42)
Salvianolic acid B (Sal B)
Aβ(1–40)
Baicalein
α-synuclein
Curcumin
Aβ(1–40) Aβ(1–42) Aβ(1–40) Aβ(1–42) α-synuclein
Tannic acid (TA), Myricetin (Myr) Dopamine L-DOPA
(7)
(8) (11) (12) (21) (22, 23)
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TABLE 2. The inhibition concentration (IC50) of Catechol, DA, L-DOPA, NE and EP for the Aβ(1–40) and Aβ(1–42) fibril formation. IC50 a/μM
Inhibitor
Catechol DA L-DOPA NE EP
Aβ(1–40)
Aβ(1–42)
5.2 ∼1 1.3 1.4 2.3
26.4 3.4 3.8 7.4 8.1
a IC50 (μM) was defined as the concentration of Catechol, DA, L-DOPA, NE and EP to inhibit the fibril formation to 50% of the control value.
Direct observation of Aβ fibrils The image of Aβ fibrils was obtained by a transmission electron microscope (TEM) according to the previous method (27). In brief, a 5-μl aliquot of diluted solution was placed on a copper grid (400-mesh) covered with a carbon-coated collodion film for 1 min and the excess sample solution was removed by blotting with filter paper. After the residual solution had dried up, the grid was negatively stained with a 2% (wt/vol) uranyl acetate solution. Again, the liquid on the grid was removed with filter paper and dried. TEM images were acquired using a JEOL JEM-1200EX transmission microscope (JEOL, Tokyo, Japan) with an acceleration voltage of 80 kV. The total internal reflection fluorescence microscope (TIRFM) system used to observe amyloid fibril formation was developed based on an inverted microscope (IX70, Olympus, Tokyo, Japan) (27). The ThT molecule was excited at 442 nm by a helium-cadmium laser (IK5552R-F, Kimmon, Tokyo, Japan). The laser power was 40– 80 mW, and the observation period was 3–5 s. The fluorescence image was filtered with a band pass filter (D490/30, Omega Optical, Bratteboro, VT) and visualized using a digital steel camera (DP70, Olympus). CD spectroscopy The secondary structures of Aβ with and without DA were analyzed by using a circular dichroism spectrometer (J-720W, JASCO, Tokyo, Japan). CD spectra were measured by using a quartz cell from 195 to 250 nm with a step interval 0.1 nm bandwidth and a scanning speed of 10 nm min− 1. Liposome preparation The liposomes were prepared by the previous method (28, 29). The lipid mixture was dissolved in chloroform and then dried onto the wall of a round-bottom flask in vacuum and was then left overnight to remove all of the solvent. The dried thin lipid film was hydrated with adequate buffer solution to form multilamellar vesicles (MLVs). Large unilamellar vesicles (LUVs) were formed from the MLVs with five cycles of freeze-thaw treatment. Alternatively, monodisperse phospholipids molecules of DMPC and stearic acid (SA) at molar ratio (10:4), and phospholipids molecules of POPC and cholesterol (Ch) at molar ratio (6:4) were used for making liposomes in diameter of 100 nm.
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RESULTS AND DISCUSSION Catechol derivatives inhibit Aβ fibril formation The inhibition of amyloid fibril formation was believed to be a promising strategy for the treatment of AD (30). In this study, the possibility that catechol and its derivatives inhibit Aβ fibril formation was investigated by incubating Aβ monomers in Tris–HCl buffer at 37 °C in the presence of catechol derivative compounds (Fig. 1B, C). The control experiments were performed without any catechol derivative compound. After 24 h of incubation, the ThT fluorescence intensity in the solution increased in the absence of catechol derivatives, showing the fibril formation. The fluorescence in the absence of catechol derivatives was then set to 100%. In the presence of 100 μM catechol derivative compounds, the value of ThT fluorescence relative to the control experiment was very small, indicating the inhibition of the fibril formation. We also examined the inhibitory effect of Tyr with similar chemical structure to L-DOPA. The result shows that Tyr had no effect on inhibition of fibril formation. This result suggests the requirement of a catechol group for an inhibitory effect on the Aβ fibril formation. Furthermore, we examined the inhibitory activity of these compounds on Aβ fibril formation in the presence of fatty acidand cholesterol-containing domain-like liposomes as a model biomembrane. These liposomes strongly have been reported to interact with Aβ fibrils. The results showed that catechol and its derivatives exhibited the inhibitory effect on Aβ fibril formation not only in the buffer but also in the presence of liposomes as model biomembranes (Fig. 1B, C). The inhibitory effects of catechol derivatives on the conformation of Aβ were investigated in the presence of catechol derivatives at various concentrations. Catechol and its derivatives showed the dosedependent inhibitory effect on Aβ fibril formation (data not shown). The concentration of these compounds necessary for half-maximal effect (IC50) increased in an order of DA b L-DOPA b NE b EP b Catechol for both of Aβ(1–40) and Aβ(1–42) (Table 2). From the experimental results of ThT monitoring, DA was suggested to posses the strongest inhibitory effect against the fibril formation of both Aβ(1–40) and
FIG. 2. The direct observation with two different microscopic techniques. The TIRFM images (scale: 10 μm) of fibrils formed in the absence of DA (A1, A2) and in the presence of 100 μM DA (C1, C2) for Aβ(1–40) and Aβ(1–42), respectively. The TEM images (scale: 200 nm) of fibrils formed in the absence of DA (B1, B2) and in the presence of 100 μM DA (D1, D2) for Aβ(1–40) and Aβ(1–42), respectively. Aβ fibrils were prepared by incubating 10 μM Aβ monomer in Tris–HCl buffer solution at 37 °C for 24 h.
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Aβ(1–42). Therefore, DA-induced inhibition of fibril formation was investigated in the following. Direct observation of DA inhibit Aβ fibril formation In order to demonstrate the inhibition of Aβ fibril formation by DA, a direct observation of Aβ fibrils was performed with a TIRFM combined with ThT fluorescence (Fig. 2). TIRFM is a useful technique to evaluate a population and a length of fibrils on a quartz slide. In Fig. 2A1, a considerable Aβ(1–40) fibrils with 10.2 μm in average length were observed in the absence of any inhibitor. The fibrils observed by a TEM were confirmed to posse the microscopic structure peculiar to amyloid fibrils (Fig. 2B1), consistent with the previous reports (23). On the contrary, the samples of Aβ(1−40) monomers co-incubated with 100 μM DA (for 24 h) showed no ThT fluorescence intensity in the observation field of TIRFM, indicating the inhibitory effect of DA on the fibril formation of Aβ (Fig. 2C1). At a microscopic observation with TEM, the Aβ fibrils could not be observed and small amorphous aggregates were observed in the samples of 10 μM Aβ monomers coincubated with 100 μM DA for 24 h (Fig. 2D1). The similar results were obtained for Aβ(1–42) as shown in Fig. 2(A2–D2). The samples of Aβ(1–42) monomers co-incubated with 100 μM DA (for 24 h) showed a slight ThT fluorescence intensity in the observation field of TIRFM (Fig. 2C2), few very short fibrils coexisting with small
J. BIOSCI. BIOENG., amorphous aggregates were obtained by TEM (Fig. 2D2) indicating DA also shows inhibitory effect on Aβ(1–42) fibril formation. We thus considered that DA could inhibit the fibril formation of Aβ. Effect of DA on the kinetics of Aβ fibril formation To investigate the effects of DA on the kinetics of Aβ fibril formation, the ThT fluorescence intensity was measured during fibril formation (Fig. 3). The control experiment was performed by incubating 10 μM Aβ monomers without any inhibitor. The time-course of ThT fluorescence intensity showed a characteristic sigmoidal curve. According to the previous report (31), this curve consists of two processes: (i) a nucleation process and (ii) an elongation process. In (i), the oligomer formation or the nucleation occurs while the fluorescence variation was not detected. This duration is defined as “lag phase” and its time is a “lag time.” And in (ii), the fibrils grow by the binding of monomers to the ends of fibrils and the ThT fluorescence intensity increased (Fig. 3A). This duration is called as “elongation phase” and its rate is an “elongation rate”. When a 10-μM Aβ(1–40) monomer solution was incubated in the presence of 5 μM DA, the final equilibrium level of ThT was low in comparison with the control experiment. However, the lag time and elongation time (time of elongation phase) were similar to those of control experiment. Furthermore, the fluorescence intensity did not
FIG. 3. Effect of DA on kinetics of Aβ fibril formation. The time-course of the ThT fluorescence during Aβ(1–40) (A) and Aβ(1–42) (C) fibril formation without DA (filled circles) and with 5 μM DA (open circles), with 100 μM DA (open squares) from Aβ monomer solutions. The time course of the ThT fluorescence during Aβ(1–40) (B) and Aβ(1–42) (D) fibril formation without DA and with 5 μM, 100 μM DA, from 7 μM Aβ monomer and 3 μM Aβ seed solution. Aβ seed + Aβ only (filled circles); Aβ seed + Aβ monomer + 5 μM DA (open circles); Aβ seed + Aβ monomer + 100 μM DA(open squares); Aβ seed + 10 h pre-incubated solution containing 100 μM DA and 7 μM Aβ monomer (filled squares). All the solution were prepared in Tris–HCl buffer solution and incubated at 37 °C.
VOL. 109, 2010 increase in the case of 100 μM DA, indicating fibril elongation process during 24 h. In order to estimate the effect of DA on fibril growth rate, the elongation rate was estimated to be 0.81 h− 1 and 0.78 h− 1 in the absence and the presence of 5 μM DA, respectively. Therefore, it is likely that DA varied the elongation phase slightly. The similar results were obtained for Aβ(1–42) (Fig. 3C). The inhibition of Aβ fibril formation by catechol derivative compounds may involve two broadly different mechanisms, in inhibition of either a nucleation process or an elongation process. In order to clarify if DA could inhibit a nucleation process or elongation process or both of these processes, we investigated the effect of DA on kinetic of Aβ fibril formation started from seeds of Aβ fibrils. Their seeds were added to Aβ monomer solution to make the final concentrations of seeds and monomers at 3 μM and 7 μM, respectively. The results were shown in Fig. 3B and D for Aβ(1–40) and Aβ(1–42), respectively. In the absence of DA, the fluorescence intensity significantly increased without a lag phase and reached to plateau more rapidly in contrast to the sample without seeds. These curves are consistent with the previous first-order kinetic model (32). The elongation rate was estimated to be 0.80 h− 1, which is similar to the case of a spontaneous fibrillation (Fig. 3A). The addition of seeds to the monomer solution in the presence of 5 μM DA also showed the similar increase of the ThT fluorescence intensity to the case in the absence of DA. The elongation of Aβ(1–40) fibrils was induced up to 4 h. 100 μM DA could not inhibit the elongation up to 1 h or so in the presence of seeds (Fig. 3B) although 100 μM DA could completely inhibit the spontaneous Aβ fibril formation (Fig. 3A). The initial rate in ThT fluorescence intensity (apparent elongation rate) was similar in the experiments with 5 and 100 μM of DA. Thus, we considered that the DA did not inhibit the elongation process of Aβ fibril formation and that DA inhibits the nucleation process in the fibril formation of Aβ(1–40). The similar results were obtained for Aβ(1–42) (Fig. 3D). As another reason for the above consideration, we have reported the disaggregation of Aβ fibrils by the same compounds listed in Fig. 1A (Vu, H. T., Shimanouchi, T., Ishikawa, D., Matsumoto, T., Yagi, H., Umakoshi, H., Goto, Y., and Kuboi, R., unpublished data). Our latest study indicated that the decrease in ThT fluorescence intensity resulted from the disaggregation of Aβ fibrils. Both the decrement change in ThT intensity and the transition time from elongation phase to disaggregation phase depends on the concentration of DA, which strongly supports the DA-induced disaggregation of fibrils. From the results, it is considered that DA inhibits the nucleation process, rather than the elongation process. Also, the other catechol derivatives showed the similar trends to DA. Possible mechanism of inhibition of Aβ fibril formation and the effect of liposome The obtained results clearly showed that catechol derivatives could inhibit the Aβ fibril formation. The remaining problem is the mechanism responsible for the inhibition of amyloid fibril formation. Then, the previously proposed mechanisms for the inhibition on amyloid fibril formation by reagents were summarized to elucidate the possible mechanism (Table 1). It has been reported that, for inhibition of α-synuclein fibril formation, the oxidized product of dopamine (dopamine quinone, DAQ) stabilized the protofibrils of α-synuclein, leading to an inhibition of fibril formation, where 5–10% of the α-synuclein molecules formed a covalent adduct with DAQ by a reaction of DAQ with Tyr of α-synuclein (22, 23). Also, in the other chemical compounds, it has been reported that the formation of a Schiff-base between quinone group of inhibitory compounds and Lys-side chain of α-synuclein induced the inhibition of fibril formation (6, 11). On the other hands, a nucleophilic attack of DAQ to Lys (Lys forming a Schiff base with DAQ) is also considered as another possible mechanism to form the complex formation between proteins and compounds (22, 23). Therefore, we supposed that the oxidized product of
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catechol derivatives (catechol quinone and its derivatives) bound to Aβ monomer to form the covalent adduct-like complex. The effect of DA on the secondary structure of Aβ monomer was investigated though the circular dichroism measurement. Fig. 4A shows that the structure of DAQ-Aβ(1–40) complex (incubated for 24 h) was similar to that of Aβ(1–40) monomer, suggesting that DA/DAQ retains the secondary structure of Aβ. Therefore, the catechol ring of catechol derivatives as a common chemical structure might contribute to their interaction with Aβ. The sequence of Aβ(1–40) and Aβ(1–42) involves two Lys residues (Lys16 and Lys28) (33). It is therefore likely that the catechol quinone and its derivatives could form a Schiff-base with Aβ molecule although we have no direct evidence. This conjugated adduct might stabilize the disordered conformation and be advantageous for a prevention of the conversion to a β-sheet-rich structure. The addition of seeds to a 10-h pre-incubated sample of 7 μM Aβ monomer and 100 μM DA inhibited the formation of intermediate of Aβ fibrils (closed square in Fig. 3B, D). No increase in ThT fluorescence intensity was observed, strongly suggesting that the conjugated adduct has the structure disadvantageous for a binding to the seeds for fibril growth. The similar results were obtained for
FIG. 4. CD spectra of Aβ(1–40) (A) and Aβ(1–42) (B) at the beginning (0 h) and the end (24 h) of incubation in the absence and the presence of 100 μM DA. Aβ only at 0 h (curve 1), Aβ only at 24 h (curve 2); Aβ and 100 μM DA at 0 h (curve 3); and Aβ and 100 μM DA at 24 h (curve 4). The solutions were prepared by incubating 10 μM Aβ monomer on PBS buffer pH 7.4 at 37 °C.
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inhibition of Aβ(1–42) fibril formation but the effect of catechol derivatives on Aβ(1–42) fibril formation was less than that of Aβ(1– 40) (Table 2 and Fig. 4B). Aβ(1–42) was reported to show the higher propensity to form fibrils by showing the shorter lag time compare to Aβ(1–40). Therefore, these data also confirmed the inhibitory effect of catechol derivatives on nucleation process rather than elongation process. In the presence of liposome membranes, hydrophobic catechol and its derivatives, such as dopamine, prefer to partition into the membrane surface (data not shown). Aβ(1–40) and Aβ(1–42) could also interact with the liposome membranes in which the liposome could play a promotion factor for fibril formation (24). These will effectively enhance the formation of the conjugated adduct on liposome membrane. Therefore, the environment of the neuronal cell membrane with coexisting catecholamine would be advantageous to inhibit the amyloid fibril formation although the detailed experiments are needed. Under normal conditions, the concentration of Aβ is approximately 50 nM. The dopamine present at the neuronal cell is in micromolar levels enough to delay the fibril formation of Aβ if the inhibitory effect by dopamine could play a dominant role in the inhibition of fibril formation. In conclusion, our data suggest that the coexistence of catechol and its derivatives at micromolar concentrations is sufficient to significantly inhibit the spontaneous fibril formation. The catecholamine quinone formed an adduct disadvantageous for a nucleation process and for its binding to formed nuclei, leading to the inhibition of the spontaneous amyloid fibril formation. Catechol derivatives would be useful for inhibiting the formation of Aβ fibrils in its early stages. ACKNOWLEDGMENTS The authors also thank the Grant-in-Aid for Scientific Research (No: 19566203, 19656220, 20760539, 20360350, 21246121) from the Ministry of Education, Science, Sports, and Culture of Japan, a grant from the 21st Century COE program “Creation of Integrated EcoChemistry” and the Global COE program “Bio-Environmental Chemistry” of JSPS. This work was also partly supported by the Core University Program between JSPS and Vietnamese Academy of Science and Technology (VAST). The authors are grateful to the Research Center for Solar Energy Chemistry of Osaka University and the Gas hydrate Analyzing System (Osaka University, Japan). A part of the present experiments was carried out by using a facility in the Research Center for Ultrahigh Voltage Electron Microscopy (Osaka University). References 1. Mattson, M. P.: Pathways towards and away from Alzheimer's disease, Nature, 430, 631–639 (2004). 2. Iversen, L. L., Mortishiresmith, R. J., Pollack, S. J., and Shearman, M. S.: The toxicity in vitro of β-amyloid protein, Biochem. J., 311, 1–16 (1995). 3. Yankner, B. A.: Mechanisms of neuronal degeneration in Alzheimer's disease, Neuron, 16, 921–932 (1996). 4. Resende, R., Ferreiro, E., Pereira, C., and Resende, D. O. C.: Neurotoxic effect of oligomeric and fibrillar species of amyloid-beta peptide 1-42: involvement of endoplasmic reticulum calcium release in oligomer-induced cell death, Neuroscience, 155, 725–737 (2008). 5. Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W., and Glabe, C. G.: Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis, Science, 300, 486–489 (2003). 6. Kobayashi, M., Kim, J., Kobayashi, N., Han, S., Nakamura, C., Ikebukuro, K., and Sode, K.: Pyrroloquinoline quinone (PQQ) prevents fibril formation of α-synuclein, Biochem. Biophys. Res. Com., 349, 1139–1144 (2006). 7. Felice, F. G. D., Houzel, J. C., Garcia-Abreu, J., Louzada Jr., P. R. F., Jr., Afonso, R. C., Meirelles, M. N. L., Lent, R., Neto, V. M., and Ferreira, S. T.: Inhibition of Alzheimer's disease β-amyloid aggregation, neurotoxicity, and in vivo deposition by nitrophenols: implications for Alzheimer's therapy, FASEB J., 15, 1297–1299 (2001).
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