Phenolic Compounds Prevent Alzheimer’s Pathology through Different Effects on the Amyloid-β Aggregation Pathway
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.090417
ISSN1525-2191
AutoresTsuyoshi Hamaguchi, Kenjiro Ono, Atsushi Murase, Masahito Yamada,
Tópico(s)Aluminum toxicity and tolerance in plants and animals
ResumoInhibition of amyloid-β (Aβ) aggregation is an attractive therapeutic strategy for Alzheimer’s disease (AD). Certain phenolic compounds have been reported to have anti-Aβ aggregation effects in vitro. This study systematically investigated the effects of phenolic compounds on AD model transgenic mice (Tg2576). Mice were fed five phenolic compounds (curcumin, ferulic acid, myricetin, nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA)) for 10 months from the age of 5 months. Immunohistochemically, in both the NDGA- and RA-treated groups, Aβ deposition was significantly decreased in the brain (P < 0.05). In the RA-treated group, the level of Tris-buffered saline (TBS)-soluble Aβ monomers was increased (P < 0.01), whereas that of oligomers, as probed with the A11 antibody (A11-positive oligomers), was decreased (P < 0.001). However, in the NDGA-treated group, the abundance of A11-positive oligomers was increased (P < 0.05) without any change in the levels of TBS-soluble or TBS-insoluble Aβ. In the curcumin- and myricetin-treated groups, changes in the Aβ profile were similar to those in the RA-treated group, but Aβ plaque deposition was not significantly decreased. In the ferulic acid-treated group, there was no significant difference in the Aβ profile. These results showed that oral administration of phenolic compounds prevented the development of AD pathology by affecting different Aβ aggregation pathways in vivo. Clinical trials with these compounds are necessary to confirm the anti-AD effects and safety in humans. Inhibition of amyloid-β (Aβ) aggregation is an attractive therapeutic strategy for Alzheimer’s disease (AD). Certain phenolic compounds have been reported to have anti-Aβ aggregation effects in vitro. This study systematically investigated the effects of phenolic compounds on AD model transgenic mice (Tg2576). Mice were fed five phenolic compounds (curcumin, ferulic acid, myricetin, nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA)) for 10 months from the age of 5 months. Immunohistochemically, in both the NDGA- and RA-treated groups, Aβ deposition was significantly decreased in the brain (P < 0.05). In the RA-treated group, the level of Tris-buffered saline (TBS)-soluble Aβ monomers was increased (P < 0.01), whereas that of oligomers, as probed with the A11 antibody (A11-positive oligomers), was decreased (P < 0.001). However, in the NDGA-treated group, the abundance of A11-positive oligomers was increased (P < 0.05) without any change in the levels of TBS-soluble or TBS-insoluble Aβ. In the curcumin- and myricetin-treated groups, changes in the Aβ profile were similar to those in the RA-treated group, but Aβ plaque deposition was not significantly decreased. In the ferulic acid-treated group, there was no significant difference in the Aβ profile. These results showed that oral administration of phenolic compounds prevented the development of AD pathology by affecting different Aβ aggregation pathways in vivo. Clinical trials with these compounds are necessary to confirm the anti-AD effects and safety in humans. 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This indicated that the effects of these phenolic compounds on Aβ aggregation remain controversial. These different results may reflect different experimental conditions in these studies. To resolve this problem, a systematic in vivo study is required; however, few reports on the effects of phenolic compounds on Aβ aggregation in vivo have been published so far, except for reports about Cur.39Yang F Lim GP Begum AN Ubeda OJ Simmons MR Ambegaokar SS Chen PP Kayed R Glabe CG Frautschy SA Cole GM Curcumin inhibits formation of amyloid-β oligomers and fibrils, binds plaques, and reduces amyloid in vivo.J Biol Chem. 2005; 280: 5892-5901Crossref PubMed Scopus (2014) Google Scholar, 40Garcia-Alloza M Borrelli LA Rozkalne A Hyman BT Bacskai BJ Curcumin labels amyloid pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an Alzheimer mouse model.J Neurochem. 2007; 102: 1095-1104Crossref PubMed Scopus (566) Google Scholar, 41Lim GP Chu T Yang F Beech W Frautschy SA Cole GM The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse.J Neurosci. 2001; 21: 8370-8377Crossref PubMed Google Scholar, 42Begum AN Jones MR Lim GP Morihara T Kim P Heath DD Rock CL Pruitt MA Yang F Hudspeth B Hu S Faull KF Teter B Cole GM Frautschy SA Curcumin structure-function, bioavailability, and efficacy in models of neuroinflammation and Alzheimer's disease.J Pharmacol Exp Ther. 2008; 326: 196-208Crossref PubMed Scopus (505) Google Scholar, 44Frautschy SA Hu W Kim P Miller SA Chu T Harris-White ME Cole GM Phenolic anti-inflammatory antioxidant reversal of Aβ-induced cognitive deficits and neuropathology.Neurobiol Aging. 2001; 22: 993-1005Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar To elucidate the inhibitory effects of phenolic compounds on Aβ aggregation in vivo, several phenolic compounds, including Cur, FA, Myr, NDGA, and RA, were fed to AD model mice, and the cerebral plaque burden and formation of Aβ oligomers were compared systematically. 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At the age of 14 months, the mice were sacrificed. The mice were perfused before brain dissection with 0.9% normal saline, followed by HEPES buffer containing protease inhibitor mixture (Nacalai Tesque, Kyoto, Japan). Brains were harvested and hemidissected. One hemisphere was fixed in 4% paraformaldehyde for 24 hours for histological studies, and the opposite hemisphere was frozen rapidly in liquid nitrogen and stored at −80°C for biochemical studies. All animal studies were approved by the Institutional Animal Experiment Committee of Kanazawa University. For the assessment of brain Aβ deposition in Tg2576 mice brain, 4% paraformaldehyde-fixed, paraffin-embedded left hemi-brains were sectioned in the coronal plane using a microtome at a thickness of 5 μm. Sections were deparaffinized and hydrated in a graded series of ethanol, pretreated with 99% formic acid for 5 minutes, and immersed in 0.3% hydrogen peroxide and methanol for 30 minutes to block endogenous peroxidase before preblocking at ambient temperature with serum-free protein block (Dako, Glostrup, Denmark). Aβ immunohistochemical staining was performed using anti-human amyloid-β antibody (4G8, 1/2000; Chemicon International, Temecula, CA) in conjunction with the Liquid Diaminobenzidine Substrate Chromogen System (Dako). 4G8-positive Aβ deposits were examined under bright field using an Olympus BX-51 microscope, Olympus DP71 digital camera, and custom-designed software WinROOF (Mitani, Fukui, Japan). The percentage of 4G8-positive deposits area (Aβ plaque burden) and numbers of 4G8-positive blood vessels per 1 mm2 (CAA counts) were investigated. In total, seven coronal sections were assessed by a scientist (A.M.) who was blinded to the treatment profile of each section. Frozen brains were homogenized in 10 volumes of Tris-buffered saline (TBS) (20 mmol/L Tris (pH 7.3) and 140 mmol/L NaCl) containing protease inhibitors mixture (Nacalai Tesque) (Figure 2). Samples were sonicated briefly (2 × 10 s) and centrifuged at 100,000 × g for 60 minutes at 4°C to generate a TBS-soluble fraction. The TBS-insoluble pellet was sonicated in 8 volumes of 5 M guanidine and 50 mmol/L Tris-HCl and solubilized by agitation at room temperature for 4 hours (TBS-insoluble fraction) (Figure 2). 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After binding of a biotinylated reporter antibody, quantification was made by binding of a third fluorochrome coupled to streptavidin. In TBS-soluble fractions, Aβ1-40 (Aβ40), Aβ1-42 (Aβ42), and aggregated Aβ were analyzed using human Aβ40, Aβ42, and aggregated Aβ antibody bead kit (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions (Figure 2). TBS-insoluble fractions were diluted 1/10,000 with TBS containing 5% bovine serum albumin with protease inhibitor mixture (Nacalai Tesque) and then analyzed for Aβ40 and Aβ42 (Figure 2). Aβ40 and Aβ42 antibody bead kits recognized monomeric forms of Aβ40 and Aβ42, and they have no cross-reactivity with each other (information sheet of the kit from Invitrogen). The aggregated Aβ antibody bead kit recognized aggregated Aβ, and it had no cross-reactivity with Aβ40 but slight reactivity with Aβ42 (2.2%) (information sheet of the kit from Invitrogen). To investigate TBS-soluble Aβ oligomers in the brains, dot blot assays were performed as described previously.38Wang J Ho L Zhao W Ono K Rosensweig C Chen L Humala N Teplow DB Pasinetti GM Grape-derived polyphenolics prevent Aβ oligomerization and attenuate cognitive deterioration in a mouse model of Alzheimer's disease.J Neurosci. 2008; 28: 6388-6392Crossref PubMed Scopus (331) Google Scholar, 51Wang J Ho L Chen L Zhao Z Zhao W Qian X Humala N Seror I Bartholomew S Rosendorff C Pasinetti GM Valsartan lowers brain β-amyloid protein levels and improves spatial learning in a mouse model of Alzheimer disease.J Clin Invest. 2007; 117: 3393-3402Crossref PubMed Scopus (281) Google Scholar Five micrograms of protein from the TBS-soluble fractions were applied directly to a nitrocellulose membrane, air-dried, and blocked with 5% nonfat dry milk. 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