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Keywords:

  • Alzheimer's disease;
  • β-amyloid fibrils;
  • cytotoxicity;
  • electron microscopy;
  • polyphenols;
  • thioflavin T

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

Cerebral deposition of amyloid β-peptide (Aβ) in the brain is an invariant feature of Alzheimer's disease (AD). A consistent protective effect of wine consumption on AD has been documented by epidemiological studies. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of wine-related polyphenols (myricetin, morin, quercetin, kaempferol (+)-catechin and (–)-epicatechin) on the formation, extension, and destabilization of β-amyloid fibrils (fAβ) at pH 7.5 at 37°C in vitro. All examined polyphenols dose-dependently inhibited formation of fAβ from fresh Aβ(1–40) and Aβ(1–42), as well as their extension. Moreover, these polyphenols dose-dependently destabilized preformed fAβs. The overall activity of the molecules examined was in the order of: myricetin = morin = quercetin > kaempferol > (+)-catechin = (–)-epicatechin. The effective concentrations (EC50) of myricetin, morin and quercetin for the formation, extension and destabilization of fAβs were in the order of 0.1–1 µm. In cell culture experiments, myricetin-treated fAβ were suggested to be less toxic than intact fAβ, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Although the mechanisms by which these polyphenols inhibit fAβ formation from Aβ, and destabilize pre-formed fAβin vitro are still unclear, polyphenols could be a key molecule for the development of preventives and therapeutics for AD.

Abbreviations used

amyloid β-peptide

AD

Alzheimer's disease

apoE

apolipoprotein E

BBB

blood–brain barrier

Cat

(+)-catechin

DMEM

Dulbecco's modified Eagle's medium

DMSO

dimethyl sulfoxide

EC50

effective concentrations at 50% value

epi-Cat

(–)-epicatechin

fAβ

β-amyloid fibrils

HEK

human embryonic kidney

Kmp

kaempferol

Mor

morin

MTT

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

Myr

myricetin

NDGA

nordihydroguaiaretic acid

Qur

quercetin

RIF

rifampicin

TC

tetracycline

ThT

thioflavin T

Alzheimer's disease (AD) is characterized by the abundance of intraneuronal neurofibrillary tangles and the extracellular deposition of the amyloid β-peptide (Aβ) as amyloid plaques and vascular amyloid (Selkoe 2001). Despite recent progress in the symptomatic therapy with cholinergic drugs (Doody et al. 2001), an effective therapeutic approach that interferes directly with the neurodegenerative process in AD, especially the accumulation of Aβ in the CNS is eagerly awaited. Recently, immunization with Aβ (Schenk et al. 1999) and treatment with a copper-zinc chelator (Cherny et al. 2001) were reported to attenuate the accumulation of Aβ in AD transgenic mice.

The notion that red wine may have potential health benefits initially received a great deal of attention following the report that moderate wine consumption was linked to a lower incidence of coronary heart disease, an effect known as the French paradox (Renaud and de Lorgeril 1992). In addition to the purported cardioprotective effects of red wine, French and Danish epidemiological studies have suggested that moderate wine drinking may protect against AD (Dartigues and Orgogozo 1993; Dorozynski 1997; Orgogozo et al. 1997; Truelsen et al. 2002). The role of ethanol in the protective properties of red wine is however, uncertain (Soleas et al. 1997; van Golde et al. 1999). In addition to ethanol, red wine contains a broad range of polyphenols that are present in the skin and seeds of grapes (Hertog et al. 1993; Celotti et al. 1996; Goldberg et al. 1996; Sato et al. 1997; Soleas et al. 1997). Recently, several studies have suggested that many kinds of natural polyphenols may have neuroprotective effects both in vivo and in vitro, possibly due to their abilities to scavenge reactive oxygen species (Inanami et al. 1998; Shutenko et al. 1999; Bastianetto et al. 2000; Virgili and Contestabile 2000; Choi et al. 2001; Levites et al. 2001). Choi et al. (2001) demonstrated that green tea polyphenol (–)-epigallocatechin gallate attenuates β-amyloid-induced neurotoxicity in cultured hippocampal neurons through scavenging reactive oxygen species. However, few reports that describe the effects of polyphenols on the formation and destabilization of Alzheimer's β-amyloid fibrils (fAβ) in vitro have thus far been available.

Using a nucleation-dependent polymerization model to explain the mechanism of fAβ formation in vitro (Jarrett and Lansbury 1993; Lomakin et al. 1997; Naiki et al. 1997; Naiki and Gejyo 1999), we previously reported that nordihydroguaiaretic acid (NDGA) and rifampicin (RIF) inhibit fAβ formation from Aβ and fAβ extension dose-dependently in vitro (Naiki et al. 1998). Moreover, we reported that NDGA also destabilizes fAβ(1–40) and fAβ(1–42) in a concentration-dependent manner within a few hours at pH 7.5 at 37°C, based on fluorescence spectroscopic analysis with thioflavin T (ThT) and electron microscopic studies (Ono et al. 2002b). The activity of NDGA to destabilize fAβ(1–40) and fAβ(1–42) in comparison with other molecules reported to inhibit fAβ formation from Aβ and/or to destabilize pre-formed fAβ both in vivo and in vitro was in the order of: NDGA > > RIF = tetracycline (TC) > poly(vinylsulfonic acid, sodium salt) = 1,3-propanedisulfonic acid, disodium salt > β-sheet breaker peptide (iAβ5) > nicotine (Ono et al. 2002a; Ono et al. 2002b). Moreover, in cell culture experiments, fAβ treated by NDGA was significantly less toxic than intact fAβ (Ono et al. 2002b).

Using fluorescence spectroscopy with ThT and electron microscopy, we examined the effects of the major natural polyphenols on the formation, extension, and destabilization of fAβ(1–40) and fAβ(1–42) at pH 7.5 and 37°C in vitro. We also compared the cytotoxicity of myricetin (Myr)-treated fAβ with that of intact fAβ, by measuring the reducing rate of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT).

Preparation of Aβ and fAβ solutions

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

Aβ(1–40) (a trifluoroacetate salt, lot number 520130, Peptide Institute Inc., Osaka, Japan) and Aβ(1–42) (a trifluoroacetate salt, lot number 520625, Peptide Institute Inc., Osaka, Japan) were dissolved by brief vortexing in 0.02% ammonia solution at a concentration of 500 µm (2.2 mg/mL) and 250 µm, respectively, in a 4°C room and stored at − 80°C before assaying (fresh Aβ(1–40) and Aβ(1–42) solutions). fAβ(1–40) and fAβ(1–42) were formed from the fresh Aβ(1–40) and Aβ(1–42) solutions, respectively, sonicated, and stored at 4°C as described in Hasegawa et al. (1999).

Fresh, non-aggregated fAβ(1–40) and fAβ(1–42) were obtained by extending sonicated fAβ(1–40) or fAβ(1–42) with fresh Aβ(1–40) or Aβ(1–42) solutions, respectively, just before the destabilization reaction (Ono et al. 2002a; Ono et al. 2002b). The reaction mixture was 600 µL and contained 10 µg/mL (2.3 µm) fAβ(1–40) or fAβ(1–42), 50 µm Aβ(1–40) or Aβ(1–42), 50 mm phosphate buffer, pH 7.5, and 100 mm NaCl. After incubation at 37°C for 3–6 h under non-agitated conditions, the extension reaction proceeded to equilibrium as measured by the fluorescence of ThT. In the following experiment, the concentration of fAβ(1–40) and fAβ(1–42) in the final reaction mixture was regarded as 50 µm.

Fluorescence spectroscopy, electron microscopy, and polarized light microscopy

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

A fluorescence spectroscopic study was performed as described by Naiki and Nakakuki (1996) on a Hitachi F-2500 fluorescence spectrophotometer (Tokyo, Japan). Optimum fluorescence measurements of fAβ(1–40) and fAβ(1–42) were obtained at the excitation and emission wavelengths of 445 and 490 nm, respectively, with the reaction mixture containing 5 µm ThT (Wako Pure Chemical Industries Ltd, Osaka, Japan) and 50 mm of glycine-NaOH buffer, pH 8.5. An electron microscopic study and polarized light microscopic study of the reaction mixtures were performed as described elsewhere (Hasegawa et al. 1999).

Polymerization assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

Polymerization assay was performed as described in Naiki et al. (1998). The reaction mixture contained 50 µm Aβ(1–40), or 25 or 50 µm Aβ(1–42), 0 or 10 µg/mL fAβ(1–40) or fAβ(1–42), 0, 0.01, 0.1, 1, 10, or 50 µm NDGA or polyphenols (Myr, morin (Mor), quercetin (Qur), kaempferol (Kmp) (+)-catechin (Cat) (–)-epicatechin (epi-Cat) [Sigma Chemical Co., St. Louis, MO, USA], 1% dimethyl sulfoxide (DMSO) (Nacalai Tesque, Inc., Kyoto, Japan), 50 mm phosphate buffer, pH 7.5, and 100 mm NaCl. NDGA and polyphenols were first dissolved in DMSO at concentrations of 1, 10, 100 µm, 1 and 5 mm, then added to the reaction mixture to make the final concentrations 0.01, 0.1, 1, 10 and 50 µm, respectively.

Thirty µL aliquots of the mixture were put into oil-free PCR tubes (size: 0.5 mL, code number: 9046; Takara Shuzo Co. Ltd, Otsu, Japan). The reaction tubes were then put into a DNA thermal cycler (PJ480; Perkin Elmer Cetus, Emeryville, CA, USA). The plate temperature was elevated at maximal speed, starting at 4°C, to 37°C. Incubation times ranged from between 0 and 8 days (as indicated in each figure), and the reaction was stopped by placing the tubes on ice. The reaction tubes were not agitated. From each reaction tube, triplicate 5-µL aliquots were removed, then subjected to fluorescence spectroscopy and the mean of each triplicate determined. In the ThT solution, the concentration of NDGA and polyphenols examined in this study was diluted up to 1/200 of that in the reaction mixture. We confirmed that these compounds did not quench ThT fluorescence at the diluted concentration (data not shown).

Destabilization assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

Destabilization assay was performed as described by Ono et al. (2002b). Briefly, the reaction mixture contained 25 µm fresh fAβ(1–40) or fAβ(1–42), 0, 0.01, 0.1, 1, 10, or 50 µm NDGA or polyphenols, 1% DMSO, 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 1% (wt/vol) polyvinyl alcohol (Wako Pure Chemical Industries Ltd) to avoid the aggregation of fAβ and the adsorption of fAβ onto the inner wall of the reaction tube during the reaction.

Triplicate 5 µL aliquots were mixed by pipette then subjected to fluorescence spectroscopy, 30 µL aliquots were put into PCR tubes. The reaction tubes were then transferred into a DNA thermal cycler. The plate temperature was elevated to maximal speed, starting at 4°C, and raised to 37°C. Incubation times ranged between 0 and 72 h (as indicated in each figure), and the reaction was stopped by placing the tubes on ice. The reaction tubes were not agitated during the reaction. From each reaction tube, 5 µL aliquots in triplicate were subjected to fluorescence spectroscopy and the mean of the three measurements was determined.

Cell Cultures and MTT assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

Cell cultures and MTT assay were performed as described by Yoshiike et al. (2001). Human embryonic kidney (HEK) 293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma, St Louis, MO, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA) and incubated in a humidified chamber (85% humidity) containing 5% CO2 at 37°C. A day prior to the fAβ treatment, the cell culture medium was replaced with serum-free DMEM, and the cells were plated onto a 96-well coated plate (Corning, NY, USA) at a final cell count of 20 000 cells/well.

Ten µM fAβ(1–40) were incubated with 0 or 0.2% DMSO, or 10 µm Myr + 0.2% DMSO at pH 7.5 at 37°C. ThT fluorescence was regularly monitored as described in Yoshiike et al. (2001). After 6 h, the incubation mixtures were added immediately to HEK 293 cell cultures. Cells were treated with a final concentration of 0 or 1 µm intact, DMSO-treated or Myr-treated fAβ(1–40) containing 0 or 0.02% DMSO, or 1 µm Myr + 0.02% DMSO, respectively, in serum-free DMEM for 2 h. MTT was then added to each well and the plate was kept in a CO2 incubator for an additional 2 h. The cells were then lysed by the addition of a lysis solution (50% dimethylformamide, 20% SDS, pH 4.7) and were incubated overnight. The degree of MTT reduction in each sample was subsequently assessed by measuring absorption at 570 nm and at 37°C using a Bio Kinetics EL340 reader (Bio-Tek Instruments, Winooski, VT, USA). Background absorbance values, as assessed from cell-free wells, were subtracted from the absorption values of each test sample. Percentage of MTT reduction was calculated by taking the average from a condition with neither fAβ, Myr, nor DMSO as 100% in each experiment.

Other analytical procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

Protein concentrations of the supernatants of the reaction mixtures after centrifugation were determined by the method of Bradford (1976) with a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). The Aβ1–40 solution quantified by amino acid analysis was used as the standard. Paired Student's t-test and linear least squares fit were used for statistical analysis. The effective concentrations (EC50) were defined as the concentrations of NDGA or polyphenols to inhibit the formation or extension of fAβs to 50% of the control value, or the concentrations to destabilize fAβs to 50% of the control value. EC50 were calculated by the sigmoidal curve fitting of the data as shown in Figs 1(e), 2(f) and 4(e), using Igor Pro ver.4 (WaveMetrics, Inc., Lake Oswego, OR, USA).

image

Figure 1. (a- d) Effects of Myr (a, b), Kmp (c), and Cat (d) on the kinetics of fAβ(1–40) (a, c, d) and fAβ(1–42) (b) formation from fresh Aβ(1–40) and Aβ(1–42), respectively. The reaction mixtures containing 50 µm Aβ(1–40) (a, c, d) or 25 µm Aβ(1–42) (b), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0 (•), 10 (^), or 50 µm (^) of Myr (a, b), Kmp (c), or Cat (d), were incubated at 37°C for the indicated times. Each figure is a representative pattern of 3 independent experiments. (e) Dose-dependent inhibition of fAβ(1–40) formation from fresh Aβ(1–40). The reaction mixtures containing 50 µm Aβ(1–40), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0, 0.01, 0.1, 1, 10 and 50 µm NDGA (•), Myr (^), Kmp (▪), or Cat (□) were incubated at 37°C for 7 days. Points represent means of three independent experiments. In all points, standard errors were within symbols. The average without compounds was regarded as 100%.

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image

Figure 2. (a- d) Effects of Myr (a, b), Kmp (c), and Cat (d) on the kinetics of fAβ(1–40) (a, c, d) and fAβ(1–42) (b) extension. The reaction mixtures containing 10 µg/mL (2.3 µm) sonicated fAβ(1–40) (a, c, d) or fAβ(1–42) (b), 50 µm Aβ(1–40) (a, c, d) or Aβ(1–42) (b), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0 (•), 10 (^), or 50 µm (□) of Myr (a, b), Kmp (c), or Cat (d), were incubated at 37°C for the indicated times. Each figure is a representative pattern of 3 independent experiments. (e) Effect of Aβ(1–40) concentration on the initial rate of fAβ(1–40) extension in the presence (^) and absence (•) of Myr. The reaction mixtures containing 10 µg/mL (2.3 µm) sonicated fAβ(1–40), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, 0 (•) or 10 µm (^) Myr, and 0, 10, 20, 30, 40, and 50 µm Aβ(1–40), were incubated at 37°C for 1 h. Points represent means of three independent experiments. In all points, standard errors were within symbols. Liner least-square fit was performed for each straight line (R2 = 0.998 and 0.994 for ^and •, respectively). (f) Dose-dependent inhibition of fAβ(1–40) extension. The reaction mixtures containing 10 µg/mL (2.3 µm) sonicated fAβ(1–40), 50 µm Aβ(1–40), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0, 0.01, 0.1, 1, 10 and 50 µm NDGA (•), Myr (^), Kmp (▪), or Cat (□) were incubated at 37°C for 1 h. Points represent means of three independent experiments. In all points, standard errors were within symbols. The average without compounds was regarded as 100%.

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image

Figure 4. (a- d) Effects of Myr (a, b), Kmp (c), and Cat (d) on the kinetics of fAβ(1–40) (a, c, d) and fAβ(1–42) (b) destabilization. The reaction mixtures containing 25 µm fAβ(1–40) (a, c, d) or fAβ(1–42) (b), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0 (•), 10 (^), or 50 µm (□) of Myr (a, b), Kmp (c), or Cat (d), were incubated at 37°C for the indicated times. Each figure is a representative pattern of 3 independent experiments. (e) Dose-dependent destabilization of fAβ(1–40). The reaction mixtures containing 25 µm fAβ(1–40), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0, 0.01, 0.1, 1, 10 and 50 µm NDGA (•), Myr (^), Kmp (▪), or Cat (□) were incubated at 37°C for 6 h. Points represent means of three independent experiments. In all points, standard errors are within symbols. The average without compounds was regarded as 100%.

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Effect of polyphenols on the kinetics of fAβ polymerization

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

As shown in Fig. 1(a-d), when fresh Aβ(1–40) or Aβ(1–42) was incubated at 37°C, the fluorescence of ThT followed a characteristic sigmoidal curve. This curve is consistent with a nucleation-dependent polymerization model (Jarrett and Lansbury 1993; Naiki et al. 1997). fAβ(1–40) and fAβ(1–42) stained with Congo red showed typical orange-green birefringence under polarized light (data not shown). When Aβ(1–40) was incubated with 10 and 50 µm Myr, Mor or Qur, no increase in the fluorescence was observed throughout the reaction (Fig. 1(a) and data not shown). A similar effect of Myr, Mor and Qur was observed with Aβ(1–42) (Fig. 1(b) and data not shown). When Aβ(1–40) was incubated with 10 and 50 µm Kmp, Cat or epi-Cat, the final equilibrium level decreased dose-dependently (Fig. 1 (c) and (d) and data not shown). A similar effect of Kmp, Cat and epi-Cat was observed with Aβ(1–42) (data not shown).

As shown in Fig. 2(a-d), when fresh Aβ(1–40) or Aβ(1–42) was incubated with fAβ(1–40) or fAβ(1–42), respectively, at 37°C, the fluorescence increased hyperbolically without a lag phase and proceeded to equilibrium much more rapidly than without seeds (compare Figs 1 and 2). This curve is consistent with a first-order kinetic model (Naiki and Nakakuki 1996). When Aβ(1–40) and fAβ(1–40) were incubated with polyphenols, the final equilibrium level decreased (Fig. 2a, 2c,d). A similar effect of polyphenols was observed for the extension of fAβ(1–42) (Fig. 2(b) and data not shown). At a constant fAβ(1–40) concentration, a perfect linearity was observed between the Aβ(1–40) concentration and the initial rate of fAβ(1–40) extension both in the presence and absence of Myr (Fig. 2e). This linearity is again consistent with a first-order kinetic model and indicates that at each Aβ(1–40) concentration, the net rate of fAβ(1–40) extension is the sum of the rates of polymerization and depolymerization (Naiki and Nakakuki 1996; Hasegawa et al. 2002). In the presence of 10 µm Myr, the slope of the straight line decreased to about 3/10. The interpretation of this figure implicating the mechanism of the anti-amyloidogenic effect of Myr will be discussed later.

When fresh Aβ(1–40) was incubated with fAβ(1–40) at 37°C, clear fibril extension was observed electron-microscopically (Fig. 3b). However, 50 µm Myr completely inhibited the extension of sonicated fAβ(1–40) (Fig. 3a,c). Myr inhibited the extension of fAβ(1–42) (data not shown). Similarly, Mor, Qur and Kmp also inhibited the extension of fAβ(1–40) and fAβ(1–42) (data not shown).

image

Figure 3. Electron micrographs of extended fAβ(1–40). The reaction mixtures containing 10 µg/mL (2.3 µm) fAβ(1–40), 50 µm Aβ(1–40), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0 (b) or 50 µm Myr (a, c), were incubated at 37°C for 0 (a), or 6 h (b, c). Scale bars indicate a length of 250 nm.

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Destabilization assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

As shown in Fig. 4(a-d), the fluorescence of ThT was almost unchanged during the incubation of fresh fAβ(1–40) or fAβ(1–42) at 37°C without additional molecules. On the other hand, the ThT fluorescence decreased immediately after addition of polyphenols to the reaction mixture. After incubation of 25 µm fresh fAβ(1–40) with 50 µm Myr for 1 h, many short, sheared fibrils were observed (Fig. 5b). At 6 h, the number of fibrils was reduced markedly, and small amorphous aggregates were occasionally observed (Fig. 5c). Similar morphology was observed when 25 µm fresh fAβ(1–42) were incubated with 50 µm Myr (data not shown). Other polyphenols also destabilized preformed fAβ(1–40) and fAβ(1–42) (data not shown).

image

Figure 5. Electron micrographs of destabilized fAβ(1–40). The reaction mixture containing 25 µm fAβ(1–40), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 50 µm Myr was incubated at 37°C for 0 (a), 1 (b), or 6 h (c). Scale bars indicate a length of 250 nm.

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After incubation with 50 µm Myr, Mor, Qur or Kmp for 6 h, or 50 µm Cat or epi-Cat for 72 h, fAβ(1–40) and fAβ(1–42) were stained with Congo red to a much lesser degree than fresh fAβ(1–40) and fAβ(1–42). However, they all showed orange-green birefringence under polarized light. This means that a significant amount of intact fAβ(1–40) and fAβ(1–42) still remains in the mixture after the reaction. When the protein concentration of the supernatant after centrifugation at 4°C for 2 h at 1.6 × 104g was measured by the Bradford assay, no proteins were detected in the supernatant in any case. This implies that although polyphenols could destabilize fAβ(1–40) and fAβ(1–42) to visible aggregates (Fig. 5c), they could not depolymerize fAβ(1–40) and fAβ(1–42) to monomers or oligomers of Aβ(1–40) and Aβ(1–42).

Comparison of the activity of polyphenols

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

As shown in Figs 1(e), 2(f) and 4(e), NDGA and polyphenols dose-dependently inhibited the formation and extension of fAβs, as well as dose-dependently destabilized pre-formed fAβs. We calculated EC50, the concentrations of NDGA or polyphenols to inhibit the formation or extension of fAβs to 50% of the control value, or the concentrations to destabilize fAβs to 50% of the control value, by the sigmoidal curve fitting of the data as shown in Figs 1(e), 2(f) and 4(e)(Table 1). EC50 of NDGA, Myr, Mor, Qur and Kmp to inhibit the formation or extension of fAβs were similar to EC50 to destabilize fAβs, respectively. On the other hand, EC50 of Cat and epi-Cat to destabilize fAβs were one-order higher than EC50 to inhibit the formation or extension of fAβs. All data presented in Table 1 may indicate that the anti-amyloidogenic and fibril-destabilizing activity of the molecules examined in this study may be in the order of: NDGA = Myr = Mor = Qur > Kmp > Cat = epi-Cat.

Table 1.  The effective concentrations (EC50)a of NDGA and polyphenols for the formation, extension and destabilization of fAβ(1–40) and fAβ(1–42)
CompoundsFormationb Extensionc Destabilizationd 
  1. a EC50 (µM) were defined as the concentrations of NDGA or polyphenols to inhibit the formation or extension of fAbs to 50% of the control value, or the concentrations to destabilize fAbs to 50% of the control value. EC50 were calculated by the sigmoidal curve fitting of the data as shown in Figs 1(e), 2(f) and 4(e), using Igor Pro ver.4 (WaveMetrics Inc., Lake Oswego, OR, USA).

  2. b The reaction mixtures containing 50 µm Ab(1–40) or 25 µm Ab(1–42), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0, 0.01, 0.1, 1, 10 and 50 µm NDGA, Myr, Mor, Qur, Kmp, Cat, or epi-Cat were incubated at 37°C for 7 days and 24 h, respectively.

  3. c The reaction mixtures containing 10 µg/mL (2.3 µm) sonicated fAb(1–40) or fAb(1–42), 50 µm Ab(1–40) or Ab(1–42), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0, 0.01, 0.1, 1, 10 and 50 µm NDGA, Myr, Mor, Qur, Kmp, Cat, or epi-Cat were incubated at 37°C for 1 h.

  4. d The reaction mixtures containing 25 µm fAb(1–40) or fAb(1–42), 50 mm phosphate buffer, pH 7.5, 100 mm NaCl, and 0, 0.01, 0.1, 1, 10 and 50 µm NDGA, Myr, Mor, Qur, Kmp, Cat, or epi-Cat were incubated at 37°C for 6 h.

 fAβ(1–40)fAβ(1–42)fAβ(1–40)fAβ(1–42)fAβ(1–40)fAβ(1–42)
NDGA0.14 µm0.86 µm0.23 µm0.09 µm1.3 µm0.87 µm
Myr0.290.400.220.131.80.58
Mor0.240.670.200.141.90.68
Qur0.240.720.250.122.10.73
Kmp1.73.20.830.453.72.9
Cat2.95.32.41.42824
epi-Cat2.85.62.41.72324

Cell Cultures and MTT assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

Finally, we examined whether Myr-treated fAβ were toxic to cells. The effects on cell viability were assessed in a simplified model using HEK 293 cell cultures. Cell viability was indirectly measured as a function of the percentage of MTT reduced. Since the dye MTT is known to be converted into a purple formazan by mitochondrial redox activity, MTT reduction assay has been widely used to measure cellular redox activity (Abe and Saito 1998). By measuring the reducing rate of MTT, we recently demonstrated that the cytotoxic effect of Aβ aggregates to HEK 293 cells was similar to that to primary rat hippocampal neurons (Yoshiike et al. 2001). As shown in Fig. 6, the cytotoxicity of Myr-treated fAβ(1–40) was significantly lower than that of fAβ(1–40) incubated with 0 (none) or 0.2% DMSO. This suggests that Myr-treated fAβ might be less toxic than intact fAβ. However, the difference in the rate of MTT reduction was not drastic between Myr-treated and intact or DMSO-treated fAβ(1–40). Moreover, since the cultures treated with Myr-treated fAβ(1–40) contained 1 µm Myr, we cannot rule out the possibility that the difference in the rate of MTT reduction may come from the protective effect of Myr itself, for example, through scavenging reactive oxygen species (Choi et al. 2001).

image

Figure 6. Cytotoxicity of intact and Myr-treated fAβ(1–40). (a) ThT fluorescence of 10 µm fAβ incubated with 10 µm Myr + 0.2% DMSO (Myr + DMSO), 0 (none) or 0.2% DMSO (DMSO) for 6 h at 37°C was measured right before adding to HEK 293 cell cultures. (b) Cells were treated with a final concentration of 0 (□) or 1 µm (▪) Myr-treated (Myr + DMSO), intact (none) or DMSO-treated (DMSO) fAβ1–40 containing 1 µm Myr + 0.02% DMSO, 0 or 0.02% DMSO, respectively, in serum-free DMEM for 2 h and then lysed. Each column represents means + S.E. **p < 0.001 (a), *p < 0.02 (b) (paired Student's t-test).

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

Recently, our systematic in vitro study indicated that the overall activity of the anti-amyloidogenic molecules may be in the order of: NDGA > > RIF = TC > poly(vinylsulfonic acid, sodium salt) = 1,3-propanedisulfonic acid, disodium salt > β-sheet breaker peptide (iAβ5) > nicotine (Ono et al. 2002a; Ono et al. 2002b). NDGA is smaller than RIF, and has two ortho-dihydroxyphenyl rings symmetrically bound by a short carbohydrate chain. This compact and symmetric structure might be quite suitable for specifically binding to free Aβ and subsequently inhibiting the polymerization of Aβ into fAβ (Ono et al. 2002b). Alternatively, this structure might be suitable for a specific binding to fAβ and subsequent destabilization of the β-sheet rich conformation of Aβ molecules in fAβ (Ono et al. 2002b). The anti-amyloidogenic and fibril-destabilizing activity of NDGA and polyphenols examined in this study may be in the order of: NDGA = Myr = Mor = Qur > Kmp > Cat = epi-Cat (see Table 1). Some interesting structure-activity relationships of polyphenols could be considered. First, Myr, Mor, Qur and Kmp have no chirality and the hydroxyphenyl and benzopyran rings of these molecules are able to be located on the same plane by the rotation of the hydroxyphenyl ring (Fig. 7). On the other hand, Cat and epi-Cat have a chirality and the two rings can not be located on the same plane (Fig. 7). This difference in the three-dimensional structure of polyphenols would greatly affect the anti-amyloidogenic and fibril-destabilizing activity. Secondly, the numbers of hydroxyl groups in Myr, Mor, Qur and Kmp are 6, 5, 5 and 4, respectively (Fig. 7). These numbers would also affect the activity, i.e. the more hydroxyl groups in the molecule, the higher the activity. Tomiyama et al. (1994, 1996) suggested that RIF binds to Aβ by hydrophobic interactions between its lipophilic ansa chain and the hydrophobic region of Aβ, thus blocking associations between Aβ molecules leading to fAβ formation. The anti-amyloidogenic and fibril-destabilizing activity of TCs, small-molecule anionic sulfonates or sulfates, melatonin, β-sheet breaker peptides and nicotine may also be related to the propensity to bind to the specific sites of Aβ (Kisilevsky et al. 1995; Soto et al. 1996; Pappolla et al. 1998; Forloni et al. 2001; Zeng et al. 2001). Interestingly, polyphenols, RIF, melatonin, NDGA and nicotine have all been reported to have antioxidant activity (Goodman et al. 1994; Tomiyama et al. 1996; Pappolla et al. 1998; Linert et al. 1999; Bastianetto et al. 2000; Choi et al. 2001). Thus, it may be reasonable to consider that polyphenols and other organic compounds with antioxidant motifs could bind specifically to Aβ and/or fAβ, inhibit fAβ formation and/or destabilize pre-formed fAβ.

image

Figure 7. Structure of Myr, Mor, Qur, Kmp, Cat, and epi-Cat.

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Polyphenols and NDGA did not extend the length of a lag phase in the formation fAβs from Aβs (Fig. 1 and Naiki et al. 1998). Moreover, they did not extend the time to proceed to equilibrium in the extension reaction (Fig. 2 and Naiki et al. 1998). These results are in sharp contrast to those of apolipoprotein E (apoE), in which apoE extend both the length of a lag phase and the time to proceed to equilibrium in a dose-dependent manner (Naiki et al. 1998). Although apoE was suggested to inhibit the formation of fAβs in vitro, by making a complex with Aβs, thus eliminating free Aβs from the reaction mixture (Naiki et al. 1997, 1998), polyphenols and NDGA would possibly inhibit the formation of fAβs by different mechanisms. As shown in Fig. 2(e), the extension of fAβ(1–40) followed a first-order kinetic model even in the presence of Myr. The net rate of fAβ(1–40) extension is the sum of the rates of polymerization and depolymerization (Naiki and Nakakuki 1996; Hasegawa et al. 2002). Thus, one possible explanation for the finding in Fig. 2(e) may be that Myr could bind to the ends of extending fAβ(1–40) and increase the rate of depolymerization by destabilizing the conformation of Aβ(1–40) which has just been incorporated into the fibril ends. Alternatively, Myr would bind to Aβ(1–40) and consequently decrease the rate of polymerization. Further studies are essential to clarify the mechanisms by which polyphenols inhibit fAβ formation in vitro.

Recent epidemiological studies have revealed the existence of a negative correlation between moderate red wine drinking and the occurrence of AD (Dartigues and Orgogozo 1993; Dorozynski 1997; Orgogozo et al. 1997; Truelsen et al. 2002). Our study and several reports on the effects of polyphenols may well explain this correlation. First, Bastianetto et al. (2000) showed that major red wine–derived polyphenols, such as Qur, resveratrol, and Cat are capable of both protecting and rescuing cultured rat hippocampal cells against nitric oxide-induced toxicity. These effects are probably mediated by antioxidant activities and do not appear to involve purported inhibitory effects on intracellular enzymes such as cyclo-oxygenase, lipoxygenase, nitric oxide synthase and, with the exception of Qur, protein kinase C. Similarly, Choi et al. (2001) reported that the green tea polyphenol (–)-epigallocatechin gallate attenuates Aβ-induced neurotoxicity through scavenging reactive oxygen species in cultured hippocampal neurons. Virgili and Contestabile (2000) showed that chronic administration of resveratrol to young-adult rats significantly protects from the damage caused by systemic injection of the excitotoxin kainic acid, in the olfactory cortex and the hippocampus. Similarly, Inanami et al. (1998) reported that oral administration of Cat dose-dependently protects against ischemia-reperfusion-induced cell death of hippocampal CA1 in the gerbil. They also showed that superoxide scavenging activities of the brains obtained from Cat-treated gerbils were significantly higher than those of Cat-untreated animals, suggesting that orally administered Cat was absorbed, passed through the blood–brain barrier (BBB) and protected against neuronal death due to its antioxidant activities. Suganuma et al. (1998) also demonstrated that the green tea polyphenol (–)-epigallocatechin gallate administered per os could pass BBB and reach brain parenchyma in mice. It is therefore conceivable that moderate, daily consumption of red wine could provide sufficient amount of active phenolic compounds to the brain to offer neuroprotection. Finally, as shown in this paper, polyphenols dose-dependently inhibit fAβ formation from fresh Aβ, as well as destabilize pre-formed fAβin vitro. Moreover, cell culture experiments with HEK 293 cells suggested that fAβ treated by Myr might be less toxic than intact fAβ. Thus, it may be reasonable to speculate that red wine-derived polyphenols could prevent the development of AD, not only through scavenging reactive oxygen species, but also through directly inhibiting the deposition of fAβ in the brain. Although the exact mechanisms of anti-amyloidogenic and fibril-destabilizing activity of polyphenols are unclear, polyphenols could be a key molecule for the development of therapeutics for AD and other human amyloidoses.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References

The authors thank Dr S. Okino and Dr K. Iwasa (Kanazawa University) for co-operation in the experiments, and Mrs H. Okada and Mr N. Takimoto (Fukui Medical University) for excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (M.Y and H.N), a grant to the Amyloidosis Research Committee from the Ministry of Health, Labour, and Welfare, Japan (M.Y and H.N), and a Grant-in-Aid for Scientific Research on Priority Areas (C) -Advanced Brain Science Project- from the Ministry of Education, Culture, Sports, Science and Technology, Japan (H.N).

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  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Preparation of Aβ and fAβ solutions
  5. Fluorescence spectroscopy, electron microscopy, and polarized light microscopy
  6. Polymerization assay
  7. Destabilization assay
  8. Cell Cultures and MTT assay
  9. Other analytical procedures
  10. Results
  11. Effect of polyphenols on the kinetics of fAβ polymerization
  12. Destabilization assay
  13. Comparison of the activity of polyphenols
  14. Cell Cultures and MTT assay
  15. Discussion
  16. Acknowledgements
  17. References
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