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• W2051214521 abstract "Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282–11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of Nϵ-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression. Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282–11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of Nϵ-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression. Alzheimer disease (AD) 3The abbreviations used are: ADAlzheimer diseaseAβamyloid β proteinAMDage-related macular degenerationANOVAanalysis of varianceAPPamyloid precursor proteinhAPPhuman APPCMLNϵ-(carboxymethyl) lysineGFAPglial fibrillary acid proteinIba-1ionized calcium binding adaptor molecule 1dGdeoxyguanosine8-OHdG8-hydroxydeoxyguanosineSODsuperoxide dismutaseTgtransgenicTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. is characterized by amyloid deposits in senile plaques mainly consisting of 40- and 42-mer amyloid β proteins (Aβ40 and Aβ42) (1Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4213) Google Scholar, 2Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 4245-4249Crossref PubMed Scopus (3662) Google Scholar). These proteins are produced from amyloid precursor protein (APP) by β- and γ-secretases. Aβ42 plays a more important role in the pathogenesis of AD than Aβ40 because of its greater aggregation propensity and higher neurotoxicity (3Haass C. Selkoe D.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 101-112Crossref PubMed Scopus (3885) Google Scholar). It has been well demonstrated that oxidative stress is a contributing factor to neurodegenerative disease progression (4Sayre L.M. Perry G. Smith M.A. Chem Res. Toxicol. 2008; 21: 172-188Crossref PubMed Scopus (643) Google Scholar, 5Barnham K.J. Masters C.L. Bush A.I. Nat. Rev. Drug Discov. 2004; 3: 205-214Crossref PubMed Scopus (2753) Google Scholar). Aβ-induced neurotoxicity has been linked to oxidative stress via protein radicalization in vitro (6Varadarajan S. Yatin S. Aksenova M. Butterfield D.A. J. Struct. Biol. 2000; 130: 184-208Crossref PubMed Scopus (652) Google Scholar, 7Murakami K. Irie K. Ohigashi H. Hara H. Nagao M. Shimizu T. Shirasawa T. J. Am. Chem. Soc. 2005; 127: 15168-15174Crossref PubMed Scopus (143) Google Scholar). Soluble oligomeric assemblies (50∼60 kDa; e.g. Aβ-derived diffusible ligand, Aβ*56, and globulomer) of Aβ rather than insoluble fibrils are believed to inhibit long term potentiation and induce neuronal loss (8Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Crossref PubMed Scopus (3694) Google Scholar, 9Roychaudhuri R. Yang M. Hoshi M.M. Teplow D.B. J. Biol. Chem. 2009; 284: 4749-4753Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). Alzheimer disease amyloid β protein age-related macular degeneration analysis of variance amyloid precursor protein human APP Nϵ-(carboxymethyl) lysine glial fibrillary acid protein ionized calcium binding adaptor molecule 1 deoxyguanosine 8-hydroxydeoxyguanosine superoxide dismutase transgenic N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Many defensive systems protect mammals from oxidative stress caused by reactive oxygen species, including superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen. Superoxide dismutase (SOD) is one of the major antioxidant enzymes that catalyzes the conversion of superoxide radicals to hydrogen peroxide (10Okado-Matsumoto A. Fridovich I. J. Biol. Chem. 2001; 276: 38388-38393Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar). SOD consists of three isozymes: copper/zinc SOD (CuZn-SOD, SOD1), which is localized in the cytosol, nucleus, and intermembrane space of mitochondria; manganese SOD (Mn-SOD, SOD2), which occurs in the mitochondrial matrix; extracellular SOD (EC-SOD, SOD3), which is also a complex of Cu and Zn. Our previous investigation proposed that Sod1-deficient (Sod1−/−) mice showed increased drusen formation, which is a typical characteristic of age-related macular degeneration (AMD) (11Imamura Y. Noda S. Hashizume K. Shinoda K. Yamaguchi M. Uchiyama S. Shimizu T. Mizushima Y. Shirasawa T. Tsubota K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11282-11287Crossref PubMed Scopus (340) Google Scholar), fatty liver (12Uchiyama S. Shimizu T. Shirasawa T. J. Biol. Chem. 2006; 281: 31713-31719Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), skin thinning (13Murakami K. Inagaki J. Saito M. Ikeda Y. Tsuda C. Noda Y. Kawakami S. Shirasawa T. Shimizu T. Biochem. Biophys. Res. Commun. 2009; 382: 457-461Crossref PubMed Scopus (56) Google Scholar), osteoporosis (14Nojiri H. Saita Y. Morikawa D. Kobayashi K. Tsuda C. Miyazaki T. Saito M. Marumo K. Yonezawa I. Kaneko K. Shirasawa T. Shimizu T. J. Bone Miner. Res. 2011; 26: 2682-2694Crossref PubMed Scopus (129) Google Scholar), and infertility (15Noda Y. Ota K. Shirasawa T. Shimizu T. Biol. Reprod. 2011; (in press)Google Scholar). The suggestion that drusen deposits contain nonfibrillar amyloid oligomers (16Luibl V. Isas J.M. Kayed R. Glabe C.G. Langen R. Chen J. J. Clin. Invest. 2006; 116: 378-385Crossref PubMed Scopus (170) Google Scholar) led us to predict the existence of a common mechanism in AD and AMD involving cytoplasmic oxidative damage. Recently, there have been reports on the involvement of mitochondrial oxidative stress in the pathogenesis of AD; transgenic mouse models of AD were crossed with Sod2+/−, resulting in increased plaque formation (17Li F. Calingasan N.Y. Yu F. Mauck W.M. Toidze M. Almeida C.G. Takahashi R.H. Carlson G.A. Flint Beal M. Lin M.T. Gouras G.K. J. Neurochem. 2004; 89: 1308-1312Crossref PubMed Scopus (236) Google Scholar), accelerated behavioral deficits (18Esposito L. Raber J. Kekonius L. Yan F. Yu G.Q. Bien-Ly N. Puoliväli J. Scearce-Levie K. Masliah E. Mucke L. J. Neurosci. 2006; 26: 5167-5179Crossref PubMed Scopus (197) Google Scholar), and the hyperphosphorylation of Tau (19Melov S. Adlard P.A. Morten K. Johnson F. Golden T.R. Hinerfeld D. Schilling B. Mavros C. Masters C.L. Volitakis I. Li Q.X. Laughton K. Hubbard A. Cherny R.A. Gibson B. Bush A.I. PLoS One. 2007; 2: e536Crossref PubMed Scopus (272) Google Scholar), but no effects on Aβ oligomerization were reported. Information on the contribution of SOD1 to Aβ oligomer formation and the localization is, therefore, required to further elucidate the role of cytoplasmic superoxide in the mechanism of AD. To achieve this we generated an AD model mouse lacking Sod1 and analyzed it for AD-like pathology. This report shows that cytoplasmic SOD reduction induced Aβ oligomerization, causing cognitive impairment, and that neuronal dysfunction is mediated by oxidative damage of brain tissues. Consistent with the animal studies, the levels of SOD1, but not those of SOD2 or SOD3, were significantly decreased in the brains of human AD subjects compared with non-AD individuals, thereby highlighting a potential causative role for SOD1-mediated Aβ oligomerization in the pathogenesis of AD. Tg2576 (20Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3695) Google Scholar) expressing the human APP (hAPP, C57BL6/SJL background) possessing the Swedish mutation shows the “early” (cognitive impairment) and “late” phenotypes (plaque formation) of AD (21Jacobsen J.S. Wu C.C. Redwine J.M. Comery T.A. Arias R. Bowlby M. Martone R. Morrison J.H. Pangalos M.N. Reinhart P.H. Bloom F.E. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 5161-5166Crossref PubMed Scopus (582) Google Scholar). We utilized Tg2576 (Taconic) because it is possible to evaluate each effect of SOD1 deletion on the cognitive function or amyloid depositions. Sod1−/− mice (The Jackson Laboratory) were backcrossed with C57BL/6NCrSlc mice for 5 or 6 generations. To generate hAPP/Sod1+/− mice, we bred hAPP/Sod1+/+ mice with Sod1−/− mice. Studies were conducted on sibling offspring from hAPP/Sod1+/− × Sod1+/− matings, giving hAPP/Sod1−/− mice together with the corresponding littermate controls (hAPP/Sod1+/−, hAPP/Sod1+/+, Sod1−/−, Sod1+/−, and Sod1+/+). All experiments were performed at two age points; a young age (6–8 months old) and old age groups (15–17 months old), in which groups consisted of sex-balanced females and males. Mice were genotyped by PCR using genomic DNA isolated from the tail tip as reported previously (13Murakami K. Inagaki J. Saito M. Ikeda Y. Tsuda C. Noda Y. Kawakami S. Shirasawa T. Shimizu T. Biochem. Biophys. Res. Commun. 2009; 382: 457-461Crossref PubMed Scopus (56) Google Scholar). The animals were housed under a 12-h light/dark cycle and with ad libitum access to food and water. The mice were maintained and studied according to protocols approved by the Animal Care Committee of the Tokyo Metropolitan Institute of Gerontology. All experiments were performed by examiners blinded to the genotypes of the mice. The frontal lobes of the brains of AD (6 female, 4 male) and non-AD (5 female, 5 male) individuals (Table 1) were used in the experiment with written informed consent obtained from the patients' families, and the experiment was approved by the Ethics Committee of Tokyo Metropolitan Institute of Gerontology and Tokyo Metropolitan Geriatric Hospital. The National Institute on Aging-Reagan criteria (modified) were adopted for the diagnosis of AD (22Murayama S. Saito Y. Neuropathology. 2004; 24: 254-260Crossref PubMed Scopus (77) Google Scholar). The normal controls were defined using clinical documentation of unimpaired cognition as well as minimal senile changes consisting of Braak's neurofibrillary tangle stage equal to or less than II, senile plaque stage equal to or less than A, and a lack of any vascular, inflammatory, or traumatic changes or tumors.TABLE 1Summary of neuropathological diagnosis of human materialsCaseAgeSexCDRNFTSPNP diagnosis186F3VCAD283F3VCAD387F3VCAD474M0VCAD582F1VCAD684M3VCAD786F1VCAD881M2VICAD987M3VICAD1091F1VCAD1179M0.5II0Non-AD1281M0I0Non-AD1382M0IANon-AD1478FN/AIANon-AD1583MN/AI0Non-AD1680F0II0Non-AD1777F0I0Non-AD1879FN/AI0Non-AD1982FN/AIIANon-AD2080M0IIANon-AD Open table in a new tab The procedure was based on the method of previous works (23Murakami K. Horikoshi-Sakuraba Y. Murata N. Noda Y. Masuda Y. Kinoshita N. Hatsuta H. Murayama S. Shirasawa T. Shimizu T. Irie K. ACS Chem. Neurosci. 2010; 1: 747-756Crossref PubMed Scopus (43) Google Scholar, 24Murata N. Murakami K. Ozawa Y. Kinoshita N. Irie K. Shirasawa T. Shimizu T. Biosci. Biotechnol. Biochem. 2010; 74: 2299-2306Crossref PubMed Scopus (56) Google Scholar, 25Murakami K. Yokoyama S. Murata N. Ozawa Y. Irie K. Shirasawa T. Shimizu T. Biochem. Biophys. Res. Commun. 2011; 409: 34-39Crossref PubMed Scopus (17) Google Scholar, 26Murakami K. Murata N. Ozawa Y. Kinoshita N. Irie K. Shirasawa T. Shimizu T. J. Alzheimers Dis. 2011; 26: 7-18Crossref PubMed Google Scholar, 27Toda T. Noda Y. Ito G. Maeda M. Shimizu T. J. Biomed. Biotechnol. 2011; 2011617974 Crossref PubMed Scopus (38) Google Scholar). In brief, human brain tissue (0.1–0.2 g) was homogenized in 10 volumes (w/v) of 50 mm TBS (Tris-HCl buffer (pH 7.6) containing 150 mm NaCl, a mixture of protease and phosphatase inhibitors (CompleteTM; Roche Diagnostics) supplemented with 0.7 μg/ml pepstatin A and 1 mm phenylmethylsulfonyl fluoride). The animal brain was then removed, split into two hemispheres, and one-half was frozen on liquid nitrogen. The other hemisphere was fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemical study. Indeed, there are the differences of distribution for plaque depositions in several hAPP transgenic mice. In preliminary experiments, we investigated the variance of plaque distribution using several lines (Tg2576 (20Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3695) Google Scholar), J20 (28Mucke L. Masliah E. Yu G.Q. Mallory M. Rockenstein E.M. Tatsuno G. Hu K. Kholodenko D. Johnson-Wood K. McConlogue L. J. Neurosci. 2000; 20: 4050-4058Crossref PubMed Google Scholar), and PS2Tg2576 (27Toda T. Noda Y. Ito G. Maeda M. Shimizu T. J. Biomed. Biotechnol. 2011; 2011617974 Crossref PubMed Scopus (38) Google Scholar)); Tg2576 and PS2Tg2576 had almost no preference in the plaque areas, whereas the plaque deposits was frequently found in the hippocampus of J20, as Mucke et al. (28Mucke L. Masliah E. Yu G.Q. Mallory M. Rockenstein E.M. Tatsuno G. Hu K. Kholodenko D. Johnson-Wood K. McConlogue L. J. Neurosci. 2000; 20: 4050-4058Crossref PubMed Google Scholar) also previously described. Therefore, we carried out the biochemical analysis using the whole brain lysates of mice crossed with Tg2576. The mouse brain (∼250 mg) was homogenized in 3 volumes (w/v) of TBS buffer as described above, and homogenates were centrifuged at 186,000 × g for 30 min at 4 °C using an Optima TL ultracentrifuge and a TLA100.4 rotor to give the supernatant (TBS-soluble) and pellet (TBS-insoluble) fractions. The pellet was then dissolved by sonication in 70% formic acid containing a mixture of protease inhibitors. The solubilized pellet was centrifuged at 186,000 × g for 30 min at 4 °C, after which the supernatant was neutralized with 1 m Tris base of pH 11 (1:20, v:v) as an insoluble fraction (formic acid-soluble). The total protein concentration of the brain was determined using the DC protein assay (Bio-Rad). The fractions (2 μg/μl) were subjected to Western blotting using 10–20% Tricine gel (for Aβ, Invitrogen) or 10% Bis-Tris gel (for other proteins, Invitrogen) and transferred to a PVDF membrane (0.2 μm pore size, Bio-Rad). The membranes were blocked in TBS-T (TBS containing 0.01% Tween 20 and 2.5% skimmed milk) and incubated with the primary antibody (anti-Aβ antibody (6E10) 1:1000 (Signet); anti-N-terminal Aβ (82E1), 1:100 (Immuno-Biological Laboratories (IBL), Gunma, Japan); anti-synaptophysin, 1:200 (Sigma); anti-total Tau, 1:200 (Dako); anti-phosphorylated Tau at Ser-396, 1:5000 (Epitomics); anti-β-actin, 1:2000 (Sigma); anti-SOD1, SOD2, SOD3, 1:1000 (Stressgen); anti-Nϵ-(carboxymethyl) lysine (CML), 1:400 (Cosmo Bio)) overnight at 4 °C, before being washed with TBS-T and treated with the secondary antibody (1 h). Development was performed with enhanced chemiluminescence and quantified using LAS-3000 (Fujifilm). Two microliters of TBS-soluble fractions (2 μg/μl) were applied to a nitrocellulose membrane (0.2-μm pore size, Bio-Rad) basically according to the protocol developed by Kayed et al. (29Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3443) Google Scholar). After being blocked, the membrane was incubated with anti-Aβ oligomer (A11, 0.1 μg/ml, Invitrogen) overnight at 4 °C before being incubated with the secondary antibody (1 h). As implied previously (29Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3443) Google Scholar), TBS containing 0.01% Tween 20 was used as a washing buffer to reduce the interference with the detection of oligomers by higher concentrations of detergent. Development was performed with enhanced chemiluminescence and quantified using LAS-3000 (Fujifilm). Memory impairment was assessed using the Morris water maze test, as described previously (25Murakami K. Yokoyama S. Murata N. Ozawa Y. Irie K. Shirasawa T. Shimizu T. Biochem. Biophys. Res. Commun. 2011; 409: 34-39Crossref PubMed Scopus (17) Google Scholar, 26Murakami K. Murata N. Ozawa Y. Kinoshita N. Irie K. Shirasawa T. Shimizu T. J. Alzheimers Dis. 2011; 26: 7-18Crossref PubMed Google Scholar, 27Toda T. Noda Y. Ito G. Maeda M. Shimizu T. J. Biomed. Biotechnol. 2011; 2011617974 Crossref PubMed Scopus (38) Google Scholar). The water maze pool (Muromachi Kikai, Tokyo), diameter 120 cm, contained opaque water (20 °C) with a platform (10 cm in diameter) submerged 2 cm below the surface. The hidden platform task took 4 (6–8-month group) or 7 (15–17-month group) days (2 sessions per day, 3 h apart) during which 2 trials were performed each day (15 min apart). The platform location remained constant, and the entry points were changed semi-randomly between trials. Twenty-four hours after the last day of the hidden platform task, a 1-min probe trial was carried out without the platform. The entry point for the probe trials was in the quadrant opposite the target quadrant. Memory retention was evaluated by the amount of time spent in the correct quadrant where the escape platform was located in the hidden platform trial. Performance was monitored with the CompACT VAS/DV video-tracking system. Exploratory behavior was tested using the Y maze test, as described previously (24Murata N. Murakami K. Ozawa Y. Kinoshita N. Irie K. Shirasawa T. Shimizu T. Biosci. Biotechnol. Biochem. 2010; 74: 2299-2306Crossref PubMed Scopus (56) Google Scholar, 25Murakami K. Yokoyama S. Murata N. Ozawa Y. Irie K. Shirasawa T. Shimizu T. Biochem. Biophys. Res. Commun. 2011; 409: 34-39Crossref PubMed Scopus (17) Google Scholar, 26Murakami K. Murata N. Ozawa Y. Kinoshita N. Irie K. Shirasawa T. Shimizu T. J. Alzheimers Dis. 2011; 26: 7-18Crossref PubMed Google Scholar). The Y maze apparatus (Muromachi Kikai) was made of gray plastic walls, 12 cm high consisting of three compartments (40 × 2 cm) connected with 2 × 2-cm passages. The mice were placed into one of the three arms of the maze and allowed to explore the two open arms for 5 min, during which the third arm remained closed (training trial). After a 1-h interval, the closed arm was opened, and the mice were allowed to explore all three arms for 5 min (test trial). An arm entry was recorded when all four paws entered the compartment. After testing each mouse, the maze was thoroughly cleaned to standardize odors. Performance was monitored with the CompACT VAS/DV video-tracking system. The procedure was basically adopted based on the method of the previous works (23Murakami K. Horikoshi-Sakuraba Y. Murata N. Noda Y. Masuda Y. Kinoshita N. Hatsuta H. Murayama S. Shirasawa T. Shimizu T. Irie K. ACS Chem. Neurosci. 2010; 1: 747-756Crossref PubMed Scopus (43) Google Scholar, 24Murata N. Murakami K. Ozawa Y. Kinoshita N. Irie K. Shirasawa T. Shimizu T. Biosci. Biotechnol. Biochem. 2010; 74: 2299-2306Crossref PubMed Scopus (56) Google Scholar, 25Murakami K. Yokoyama S. Murata N. Ozawa Y. Irie K. Shirasawa T. Shimizu T. Biochem. Biophys. Res. Commun. 2011; 409: 34-39Crossref PubMed Scopus (17) Google Scholar, 26Murakami K. Murata N. Ozawa Y. Kinoshita N. Irie K. Shirasawa T. Shimizu T. J. Alzheimers Dis. 2011; 26: 7-18Crossref PubMed Google Scholar, 27Toda T. Noda Y. Ito G. Maeda M. Shimizu T. J. Biomed. Biotechnol. 2011; 2011617974 Crossref PubMed Scopus (38) Google Scholar). In brief, the sections (5 μm) were deparaffinized, rehydrated, and washed in phosphate-buffered saline (PBS) before being treated briefly with formic acid in the case of Aβ staining. After incubation in 3% hydrogen peroxide in methanol to prevent endogenous peroxidation, the sections were blocked with 10% normal goat serum in PBS before being incubated with an anti-Aβ antibody (6E10 and 4G8) (1 μg/ml; Signet), an anti-ionized calcium binding adaptor molecule 1 (Iba-1) antibody (1 μg/ml; Wako), to detect activated microglia or an anti-glial fibrillary acid protein (GFAP) antibody (1 μg/ml; Sigma) to detect activated astrocytes overnight at 4 °C. The sections were incubated with biotinylated secondary antibody for 30 min. Immunoreactivity was visualized using an ABC Elite kit according to the manufacturer's protocol. The sections were counterstained with hematoxylin, and densitometric quantification of the percentage area was measured using Leica QWin V3 image software. After deparaffinization of the sections and washing with PBS, the slides were immersed for 5 min in 0.25% potassium permanganate solution followed by 5 min in 1% potassium metabisulfate, 1% oxalic acid solution. The treated slides were placed in a filtered 0.02% thioflavin-S solution for 8 min before being subjected to florescence photomicrography in aqueous mounting medium. Nuclear DNA was isolated, and 8-hydroxydeoxyguanosine (8-OHdG) was measured using an electrochemical detection-HPLC system as described previously (30Kaneko T. Tahara S. Takabayashi F. Harada N. Free Radic. Res. 2004; 38: 839-846Crossref PubMed Scopus (16) Google Scholar). The 8-OHdG content was expressed as the molar ratio of 8-OHdG to 107 of dG. The amount of dG was calculated from the absorption at 260 nm in the same sample. The glutathione (GSH) content was determined using a total glutathione quantification kit (Dojindo) according to the manufacturer's instructions. The concentration of GSH in the sample was measured from absorbance at 412 nm. 20-μl samples (2 μg/μl) of the TBS-soluble fraction were employed. The amounts of Aβ42, Aβ40, and Aβ oligomers in the TBS-soluble and TBS-insoluble fractions were determined by sandwich ELISA with a human β amyloid ELISA kit (Aβ40: catalog #27714, human amyloid β (1–40)(N); Aβ42: catalog #27712, human amyloid β (1–42)(N); oligomer Aβ: catalog # 27725, human amyloid β oligomers (82E1-specific)) (IBL) according to the manufacturer's instructions. Twenty microliters of fresh blood obtained from the tail vein were subjected to automatic hematological analysis (Celltac, MEK-6358, Nihon Kohden, Tokyo, Japan) after a 100 times dilution with isotonic buffer (Isotonac, MEK-510, Nihon Kohden). Human blood (MEK-3DN, Nihon Kohden) was used as the standard. All data are presented as the means ± S.E., and the differences were analyzed with one-way analysis of variance (ANOVA) followed by Bonferroni's test. p values <0.05 were considered significant. To determine whether SOD1 contributes to the Aβ-dependent pathology of AD in vivo, we generated hAPP/Sod1−/− by crossing hAPP/Sod1+/− mice with Sod1+/− mice, giving six types of littermates: hAPP/Sod1+/+, hAPP/Sod1+/−, hAPP/Sod1−/−, Sod1+/+, Sod1+/−, and Sod1−/−. These groups did not differ with regard to the background strain. We confirmed the approximate half-reduction of SOD1 levels in hAPP/Sod1+/− mice at 6–8 and 15–17 months old in a gene dose-dependent manner using immunoblot analysis (Fig. 1, A–C). The overexpression of hAPP also did not significantly affect the amounts of SOD1 in hAPP/Sod1+/+. To examine the effect of hAPP overexpression on the anemia phenotype of the Sod1−/− mice (31Iuchi Y. Okada F. Onuma K. Onoda T. Asao H. Kobayashi M. Fujii J. Biochem. J. 2007; 402: 219-227Crossref PubMed Scopus (135) Google Scholar), the red blood cells of these mice were counted, and no significant change was observed in either age group by overexpression of hAPP (Fig. 1 D and E). During the acquisition period of Morris maze, mice were trained to search for the hidden platform for 4 days. At 6–8 months of age, all mice quickly achieved the goal except for hAPP/Sod1−/− mice, which took significantly longer to master this task (Fig. 2A). Probe trials, in which the platform was removed and mice were given 1 min to find the missing platform, were performed 1 day after the hidden platform task. As shown in Fig. 2B, only hAPP/Sod1−/− mice failed to find the target location. Estimation of time spent in the target quadrant in the probe trial showed no apparent recognition by hAPP/Sod1−/− mice of the target zone (Fig. 2C), consistent with the results of the hidden trial test (Fig. 2A). There were no differences in swimming speed among all mice investigated (data not shown), indicating that motor functions were normal. Furthermore, no drusen deposits were observed in 6–8-month-old mice knocking out Sod1 (data not shown), as described previously (11Imamura Y. Noda S. Hashizume K. Shinoda K. Yamaguchi M. Uchiyama S. Shimizu T. Mizushima Y. Shirasawa T. Tsubota K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11282-11287Crossref PubMed Scopus (340) Google Scholar), meaning that visual functions were also normal in young mice. At 15–17 months of age, hAPP/Sod1+/+, hAPP/Sod1+/−, and hAPP/Sod1−/− mice took longer to reach the goal than control Sod1+/+ mice (supplemental Fig. S1A). In the probe trial all mice possessing the hAPP transgene showed poor memory of the goal zone (supplemental Fig. S1B), and no preference for the target area was found in these mice (supplemental Fig. S1C). All mice tested in this age group swam at a similar speed in the probe trial (supplemental Fig. S1D). A visible platform trial was carried out before the hidden trial to ensure that the significant difference was not due to visual or motor dysfunctions (data not shown). Although we confirmed the presence of drusens in 15–17-month-old mice without Sod1 (hAPP/Sod1−/− and Sod1−/−, supplemental Fig. S2), consistent with our previous studies (11Imamura Y. Noda S. Hashizume K. Shinoda K. Yamaguchi M. Uchiyama S. Shimizu T. Mizushima Y. Shirasawa T. Tsubota K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11282-11287Crossref PubMed Scopus (340) Google Scholar), hAPP/Sod1+/− showed the significant memory loss compared with Sod1+/+. These results suggest that cytoplasmic SOD deletion significantly accelerates the Aβ-dependent learning and memory deficits of hAPP/Sod1+/+ mice in an age-dependent manner. The Y maze depends on the natural tendency of rodents to explore new environments. As a preference for the novel arm reflects locomotor activity, novel arm entries and the time spent in the novel arm by hAPP mice in the test trial were recorded after the training trial. Young hAPP/Sod1+/+ mice (age 6–8 months) visited the novel arm as often as non-Tg mice, whereas both hAPP/Sod1−/− and hAPP/Sod1+/− mice entered the novel arm less frequently than non-Tg mice (Fig. 2D). hAPP/Sod1−/− and hAPP/Sod1+/− mice also showed poor recognition of the novel arm as determined by the percentage of time spent there (Fig. 2E). Although haplo-deficiency of Sod1 induced behavioral impairment in hAPP/Sod1+/+ mice in the Y maze, these alterations were generally coincident with the learning and memory deficits observed in the Morris water maze test (Fig. 2, A–C), indicating that cytoplasmic SOD plays a role in behavioral function. Neuronal degeneration in animal models is closely associated with memory and behavioral impairments. We examined the levels of synaptophysin as a neuronal marker that is expressed on p" @default.
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• W2051214521 title "SOD1 (Copper/Zinc Superoxide Dismutase) Deficiency Drives Amyloid β Protein Oligomerization and Memory Loss in Mouse Model of Alzheimer Disease" @default.
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