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Oxidative stress has long been linked to the pathogenesis of neurodegenerative diseases; however, whether it is a cause or merely a consequence of the degenerative process is still unknown. We show that mice deficient in Cu, Zn-superoxide dismutase (SOD1) have features typical of age-related macular degeneration in humans. Investigations of senescent Sod1 ؊/؊ mice of different ages showed that the older animals had drusen, thickened Bruch's membrane, and choroidal neovascularization. The number of drusen increased with age, and exposure of young Sod1 ؊/؊ mice to excess light induced drusen. The retinal pigment epithelial cells of Sod1 ؊/؊ mice showed oxidative damage, and their ␤-cateninmediated cellular integrity was disrupted, suggesting that oxidative stress may affect the junctional proteins necessary for the barrier integrity of the retinal pigment epithelium. These observations strongly suggest that oxidative stress may play a causative role in age-related retinal degeneration, and our findings provide evidence for the free radical theory of aging. In addition, these results demonstrate that the Sod1 ؊/؊ mouse is a valuable animal model to study human age-related macular degeneration.animal model ͉ superoxide dismutase A ge-related macular degeneration (AMD) is the leading cause of legal blindness in humans in developed countries (1-5). AMD is characterized by a progressive degeneration of the macula, usually bilateral, leading to a severe decrease in vision and a central scotoma. The decrease in vision results either from retinal degeneration, called geographic atrophy (dry or nonexudative AMD), or from the secondary effects of choroidal neovascularization (CNV; wet or exudative AMD). An early sign of AMD is the appearance of drusen, which are extracellular deposits that accumulate below the retinal pigment epithelium (RPE) and are known to be risk factors for developing CNV (6-8).Mouse models of AMD that manifest some of the features of human AMD have recently begun to appear (9-14); however, most of these mice have only some of the characteristics of human AMD (15). The severity of AMD in humans progresses with increasing age, finally resulting in extensive degeneration of the retina. Therefore, animal models that mimic the complex and progressive characteristics of AMD are needed to investigate the pathogenesis of AMD.Oxidative stress, which refers to cellular or molecular damage caused by reactive oxygen species (ROS), has been implicated in many age-related diseases and aging itself (16,17). ROS include free radicals, hydrogen peroxide, and singlet oxygen and are often the by-products of oxygen metabolism. The retina is particularly susceptible to oxidative stress because of its high consumption of oxygen, high concentration of polyunsaturated fatty acids, and exposure to light (18). A growing body of evidence suggests that cumulative oxidative damage may be responsible for AMD (18, 19); however, a causative link has not been definitively demonstrated (18).To determine whether there is a causative ro...

Amyloid fibrils in Alzheimer's disease mainly consist of 40-and 42-mer ␤-amyloid peptides (A␤40 and A␤42) that exhibit aggregative ability and neurotoxicity. Although the aggregates of A␤ peptides are rich in intermolecular ␤-sheet, the precise secondary structure of A␤ in the aggregates remains unclear. To identify the amino acid residues involved in the ␤-sheet formation, 34 proline-substituted mutants of A␤42 were synthesized and their aggregative ability and neurotoxicity on PC12 cells were examined. Prolines are rarely present in ␤-sheet, whereas they are easily accommodated in ␤-turn as a Pro-X corner. Among the mutants at positions 15-32, only E22P-A␤42 extensively aggregated with stronger neurotoxicity than wild-type A␤42, suggesting that the residues at positions 15-21 and 24 -32 are involved in the ␤-sheet and that the turn at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of A␤42. The C-terminal proline mutants (A42P-, I41P-, and V40P-A␤42) hardly aggregated with extremely weak cytotoxicity, whereas the C-terminal threonine mutants (A42T-and I41T-A␤42) aggregated potently with significant cytotoxicity. These results indicate that the hydrophobicity of the C-terminal two residues of A␤42 is not related to its aggregative ability and neurotoxicity, rather the C-terminal three residues adopt the ␤-sheet. These results demonstrate well the large difference in aggregative ability and neurotoxicity between A␤42 and A␤40. In contrast, the proline mutants at the N-terminal 13 residues showed potent aggregative ability and neurotoxicity similar to those of wild-type A␤42. The identification of the ␤-sheet region of A␤42 is a basis for designing new aggregation inhibitors of A␤ peptides. Alzheimer's disease (AD)1 is neuropathologically characterized by the progressive deposition of amyloid fibrils in the brain parenchyma and cortical blood vessels (1). This deposition mainly consists of 40-and 42-mer peptides (A␤40 and A␤42) generated from amyloid precursor protein by two proteases, ␤-and ␥-secretase (2, 3). A␤42 plays a pivotal role in the pathogenesis of AD, because the aggregative ability and neurotoxicity of A␤42 are considerably higher than those of A␤40 (4). Because the aggregative ability of A␤ peptides is closely related to the neurotoxicity, precise structural information for amyloid fibrils is indispensable for understanding the molecular mechanisms of AD and related folding diseases and for developing new medicinal leads using the inhibitory activity of amyloid fibril formation.Previous studies on A␤ fibrils showed that A␤ aggregates mainly consist of intermolecular parallel ␤-sheet (5-10

Elderly people insidiously manifest the symptoms of heart failure, such as dyspnea and/or physical disabilities in an age-dependent manner. Although previous studies suggested that oxidative stress plays a pathological role in the development of heart failure, no direct evidence has been documented so far. In order to investigate the pathological significance of oxidative stress in the heart, we generated heart/muscle-specific manganese superoxide dismutase-deficient mice. The mutant mice developed progressive congestive heart failure with specific molecular defects in mitochondrial respiration. In this paper, we showed for the first time that the oxidative stress caused specific morphological changes of mitochondria, excess formation of superoxide (O 2 . ), reduction of ATP, and transcriptional alterations of genes associated with heart failure in respect to cardiac contractility. Accordingly, administration of a superoxide dismutase mimetic significantly ameliorated the symptoms. These results implied that O 2 . generated in mitochondria played a pivotal role in the development and progression of heart failure. We here present a bona fide model for human cardiac failure with oxidative stress valuable for therapeutic interventions.

Cerebral amyloid angiopathy (CAA) due to ␤-amyloid (A␤) is one of the specific pathological features of familial Alzheimer's disease. A␤ mainly consisting of 40-and 42-mer peptides (A␤40 and A␤42) exhibits neurotoxicity and aggregative abilities. All of the variants of A␤40 and A␤42 found in CAA were synthesized in a highly pure form and examined for neurotoxicity in PC12 cells and aggregative ability. All of the A␤40 mutants at positions 22 and 23 showed stronger neurotoxicity than wild-type A␤40. Similar tendency was observed for A␤42 mutants at positions 22 and 23 whose neurotoxicity was 50 -200 times stronger than that of the corresponding A␤40 mutants, suggesting that these A␤42 mutants are mainly involved in the pathogenesis of CAA. Although the aggregation of E22G-A␤42 and D23N-A␤42 was similar to that of wild-type A␤42, E22Q-A␤42 and E22K-A␤42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis. In contrast, A21G mutation needs alternative explanation with the exception of physicochemical properties of A␤ mutants. Attenuated total reflection-Fourier transform infrared spectroscopy spectra suggested that ␤-sheet content of the A␤ mutants correlates with their aggregation. However, ␤-turn is also a critical secondary structure because residues at positions 22 and 23 that preferably form two-residue ␤-turn significantly enhanced the aggregative ability. Alzheimer's disease (AD)1 is neuropathologically characterized by the progressive deposition of amyloid in the brain parenchyma and cortical blood vessels (1). This deposition mainly consists of 40-and 42-mer ␤-amyloid peptides (A␤40 and A␤42) generated from amyloid precursor protein by two proteases, ␤-and ␥-secretases (2, 3). Cerebral amyloid angiopathy (CAA) in familial Alzheimer's disease is linked to missense mutations inside the A␤-coding region in the amyloid precursor protein. The mutations of A␤ sequence are concentrated at positions 21-23 and are called Flemish (A21G) (4), Arctic (E22G) (5, 6), Dutch (E22Q) (7), Italian (E22K) (8), and Iowa (D23N) (9) mutations. These A␤ mutant peptides may play a pathological role in the CAAs because wild-type A␤ peptides induce neuronal death in vitro (10). Neurotoxicity and formation of amyloid fibrils of some CAA-related A␤40 mutants have been independently reported by several groups (11-15). However, there are no reports on the neurotoxicity and aggregation of the CAA-related A␤42 mutants with the exception of Dutch mutation (E22Q) (11), the investigation of which is essential to reveal the mechanism of CAA because wild-type A␤42 shows considerably stronger neurotoxicity and aggregative ability than wild-type A␤40 (11). Moreover, it is indispensable to simultaneously compare neurotoxicity and aggregative ability of all of the CAA-related A␤40 and A␤42 mutants in the same conditions such as pH, peptide concentration, reaction buffer, and temperature.It is difficult to synthesize A␤42 with 14 hydrophobic and/or ...

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid ␤ (A␤) 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 agerelated 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 A␤ 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 N ⑀ -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 agematched 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)3 is characterized by amyloid deposits in senile plaques mainly consisting of 40-and 42-mer amyloid ␤ proteins (A␤40 and A␤42) (1, 2). These proteins are produced from amyloid precursor protein (APP) by ␤-and ␥-secretases. A␤42 plays a more important role in the pathogenesis of AD than A␤40 because of its greater aggregation propensity and higher neurotoxicity (3). It has been well demonstrated that oxidative stress is a contributing factor to neurodegenerative disease progression (4, 5). A␤-induced neurotoxicity has been linked to oxidative stress via protein radicalization in vitro (6, 7). Soluble oligomeric assemblies (50ϳ60 kDa; e.g. A␤-derived diffusible ligand, A␤ * 56, and globulomer) of A␤ rather than insoluble fibrils are believed to inhibit long term potentiation and induce neuronal loss (8, 9).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 (10). SOD consists of three isozymes: copper/zinc SOD (CuZn-SOD, SOD1), which is localized in the cytosol, nucle...

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Amyloid fibrils mainly consist of 40-mer and 42-mer peptides (Abeta40, Abeta42). Abeta42 is believed to play a crucial role in the pathogenesis of Alzheimer's disease because its aggregative ability and neurotoxicity are considerably greater than those of Abeta40. The neurotoxicity of Abeta peptides involving the generation of free radicals is closely related to the S-oxidized radical cation of Met-35. However, the cation's origin and mechanism of stabilization remain unclear. Recently, structural models of fibrillar Abeta42 and Abeta40 based on systematic proline replacement have been proposed by our group [Morimoto, A.; et al. J. Biol. Chem. 2004, 279, 52781] and Wetzel's group [Williams, A. D.; et al. J. Mol. Biol. 2004, 335, 833], respectively. A major difference between these models is that our model of Abeta42 has a C-terminal beta-sheet region. Our biophysical study on Abeta42 using electron spin resonance (ESR) suggests that the S-oxidized radical cation of Met-35 could be generated by the reduction of the tyrosyl radical at Tyr-10 through a turn structure at positions 22 and 23, and stabilized by a C-terminal carboxylate anion through an intramolecular beta-sheet at positions 35-37 and 40-42 to form a C-terminal core that would lead to aggregation. A time-course analysis of the generation of radicals using ESR suggests that stabilization of the radicals by aggregation might be a main reason for the long-lasting oxidative stress of Abeta42. In contrast, the S-oxidized radical cation of Abeta40 is too short-lived to induce potent neurotoxicity because no such stabilization of radicals occurs in Abeta40.