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Oxidative processes in Alzheimer's disease: the role of Aβ-metal interactions
T. Lynch et al. / Experimental Gerontology 35 (2000) 445–451
445
Experimental Gerontology 35 (2000) 445–451 www.elsevier.nl/locate/expgero
Review
Oxidative processes in Alzheimer’s disease: the role of Ab-metal interactions T. Lynch a, R.A. Cherny a, A.I. Bush a,b,* a
Department of Pathology, The University of Melbourne, and Neuropathology Laboratory, The Mental Health Research Institute of Victoria, Parkville, Vic. 3052, Australia b Laboratory for Oxidation Biology, Genetics and Aging Unit, and Department of Psychiatry, Harvard Medical School, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA Received 14 February 2000; accepted 11 April 2000
Abstract Alzheimer’s disease is characterized by signs of a major oxidative stress in the neocortex and the concomitant deposition of Amyloid beta (Ab). Ab is a metalloprotein that binds copper, and is electrochemically active. Ab converts molecular oxygen into hydrogen peroxide by reducing copper or iron, and this may lead to Fenton chemistry. Hydrogen peroxide is a freely permeable prooxidant that may be responsible for many of the oxidative adducts that form in the Alzheimer-affected brain. The electrochemical activity of various Ab species correlates with the peptides’ neurotoxicity in cell culture, and participation in the neuropathology of Alzheimer’s disease. These reactions present a novel target for Alzheimer therapeutics. 䉷 2000 Elsevier Science Inc. All rights reserved. Keywords: Amyloid beta; Metals; Oxidative stress; Hydrogen peroxide; Neurodegeneration; Antioxidants; Superoxide dismutase
1. Introduction The term ‘oxidative stress’ is used when the body’s natural defense mechanisms are exceeded by the production of deleterious reactive oxygen species, resulting in damage to susceptible cell components such as DNA, proteins, and lipids. Features of the brain that cause it to be particularly sensitive to oxidative stress include a high rate of oxidative metabolic activity, relatively low levels of antioxidant enzymes (e.g. catalase and glutathione peroxidase), a high concentration of unsaturated fatty acids, large iron and copper stores, and a low mitotic index. * Corresponding author. Tel.: ⫹1-617-726-8244; fax: ⫹1-617-724-9610. E-mail address: [email protected] (A.I. Bush). 0531-5565/00/$ - see front matter 䉷 2000 Elsevier Science Inc. All rights reserved. PII: S0531-556 5(00)00112-1
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Oxidative stress associated with amyloid beta (Ab) accumulation in the neocortex is characteristic of Alzheimer’s disease (AD) and potentially related to the pathogenesis of neuronal degeneration in this disorder (Atwood et al., 1999). Similarly, markers of oxidative stress and damage response, including elevated levels of redox-active iron, (Smith et al., 1997a) directly correlate with the presence of Ab deposits in the neocortex of transgenic mice overexpressing amyloid precursor protein (APP) (Smith et al., 1998), thereby providing evidence that Ab and oxidative damage are linked in vivo. Increased oxidation of brain lipids, carbohydrates, proteins, and DNA, as well as systemic signs of oxidative stress, altered levels of antioxidants, and metabolic signs of excess oxidative stress in the neocortex of patients, have all been observed in AD. Frontal lobe autopsy specimens from AD brains show an increase in the oxidative modifications of proteins (i.e. carbonylation) with significant reductions in the activities of the oxidation-sensitive enzymes, creatine kinase and glutamine synthetase, effects of which may contribute directly to the symptoms of frontal dysfunction observed in AD (Aksenova et al., 1999). Several oxidative stress products are detected in the vicinity of Ab amyloid plaques and neurofibrillary tangles (the other major neuropathological feature of AD brain), as well as in the cerebrospinal fluid of AD patients. These include the highly reactive breakdown products of lipid peroxidation such as 4-hydroxynonenal (Sayre et al., 1997), Maillard end products pyrraline and pentosidine (Smith et al., 1994a), nitrotyrosine (Smith et al., 1997b), advanced glycation endproducts, and neurofilament-related protein carbonyls (Smith et al., 1994a). As expected, there is an up-regulation of antioxidants such as copper/zinc-superoxide dismutase (Cu/Zn-SOD) (Pappolla et al., 1992), catalase (Pappolla et al., 1992), and heme oxygenase-1 (HO-1) (Smith et al., 1994b). There are also increased markers for lipid and protein oxidation in Down’s syndrome fetal brain cortex (Odetti et al., 1998) which is important since individuals with Trisomy 21 possess an extra copy of the APP gene, develop an accelerated rate of cerebral Ab deposition, and express premature AD neuropathology. Paradoxically, there appears to be no spatial association between histological amyloid plaque deposits and histological markers of oxidation (Nunomura et al., 1999). Moreover, we recently found that total Ab load in AD neocortex is not related to disease severity or numbers of amyloid plaques (McLean et al., 1999). Instead, the concentration of soluble Ab, most likely in the form of soluble oligomers, is an accurate determinant of the severity of neurodegeneration in AD brain. It is therefore not surprising that plaque load does not correlate well with other properties of neocortical damage in general.
2. Metal-mediated aggregation of Ab in vitro and in vivo There is convincing evidence that the toxic effects of Ab are generated during, or following, the process of polymerization and oligomerization of this normally soluble peptide, therefore strategies aimed at preventing Ab aggregation or, alternatively, dissolving Ab deposits, may be of therapeutic value. The length of the Ab species (40–42 amino acids), following cleavage from APP, is considered to be an important factor in AD pathogenesis. Despite existing as a minor free soluble species in biological fluids, Ab 1-42 is enriched in cerebral amyloid deposits in AD.
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Ab interacts with Zn(II) and Cu(II) at low micromolar and submicromolar concentrations (Bush et al., 1993, 1994; Huang et al., 1997; Atwood et al., 1998). Ab is rapidly precipitated by Zn 2⫹ at pH ⬎ 7.0, whereas Cu 2⫹ and Fe 3⫹ induce greater Ab aggregation under mildly acidic conditions (Atwood et al., 1998), comparable to those reported in AD brain (Yates et al., 1990). In contrast, the solubility of rat/mouse Ab 1-40 is unaffected by Zn(II) or Cu(II) at low micromolar concentrations (Bush et al., 1994; Atwood et al., 1998), perhaps contributing to the inability of these animals to form cerebral Ab amyloid. Significantly, Ab 1-42 is more readily precipitated by Cu 2⫹ than Ab 1-40, but both peptide species are equally destabilized by Zn 2⫹ (Atwood et al., 1998). Moir et al. (1999) reported that apolipoprotein E (apoE) isoforms differentially modulate the aggregation of Ab by Cu 2⫹ and Zn 2⫹. Consistent with the increased risk of late-onset AD attached to inheriting apoE4, they discovered that this isoform (compared with apoE2 and apoE3) is the least effective chaperone for Ab solubility, whether the precipitating stress is Cu 2⫹ or Zn 2⫹. The affinity of the Zn 2⫹ binding sites on Ab 1-40 were measured as 100 nM and 5 mM, indicating that they may be occupied under physiological conditions (Bush et al., 1994). Ab species also possess an unusually strong affinity for Cu 2⫹ (Ka ⬃ 10 ⫺10 M and 10 ⫺16 M for Ab1-40 and Ab1-42, respectively (Atwood et al., 2000), and are therefore likely to bind Cu 2⫹ in vivo. Indeed, we have recently reported that zinc and copper-selective chelators markedly enhance the solubilization of Ab deposits from post-mortem AD brain tissue (Cherny et al., 1999), supporting the possibility that zinc and copper ions are intrinsic to the architecture of these deposits.
3. Reactive oxygen species formation and neurotoxicity of Ab Since Ab binds redox active metals Cu 2⫹ and Fe 3⫹, we investigated whether potentially adverse electrochemical reactions between the peptide and these metals could result. Huang et al. (1999a) reported that human Ab is redox-active and can directly produce H2O2 in a cell-free manner dependent upon oxygen and the reduction of Fe(III) or Cu(II) to Fe(II) and Cu(I), respectively, setting up conditions that could promote Fenton-type chemistry. The metal reducing ability and H2O2 production of Ab species is greatest when generated by Ab42human ⬎ Ab40human Ⰷ Ab40rat/mouse, an order that correlates with the neurotoxicity of the respective Ab species in cell culture (Huang et al., 1999b) and its association with AD neuropathology. Ab cytotoxicity was also shown to be mediated largely by the H2O2 produced directly by the Ab species, since the toxicity of the peptide augmented by Cu 2⫹ was abolished by catalase (Huang et al., 1999b). The effects of Cu(II)-mediated auto-oxidation and hydroxyl radical production cause multiple modifications in Ab (carbonyl adduct formation, histidine loss, and dityrosine cross-linking) that may consequently increase the protease resistance of the peptide. These Cu(II)-induced oxidative modifications can be mimicked by direct hydroxyl radical attack (again greatest for Ab1-42human ⬎ Ab1-40human Ⰷ Ab1-40rat, corresponding to the participation of the respective peptide in AD pathophysiology) (Atwood et al., unpublished results). Our data indicate that when human Ab is abnormally metallated with Cu(II), it produces H2O2 which then reacts with reduced Cu(I) and may produce the hydroxyl radical
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(Fenton-type chemistry). This highly reactive oxygen species then oxidizes Ab. Apart from being a substrate for Fenton-mediated hydroxyl radical generation, H2O2 is also cytotoxic, pro-apoptotic, and is freely permeable across membranes. Hence, an elevation in H2O2 will not just cause Ab oxidation and accumulation, but may also contribute to oxidative damage extending beyond the site of generation. This theory correlates well with the global increase in oxidative damage observed in AD. Another neurochemical feature of the cerebral microenvironment in AD that may contribute to excessive H2O2 production is the mild acidosis that promotes Cu(II)-binding to Ab (reviewed in Atwood et al., 1999). The redox activity of Ab is compatible with reports that Ab peptides can initiate synaptosomal lipoperoxidation (Butterfield et al., 1994), and can raise cellular hydrogen peroxide (H2O2) (Behl et al., 1994), although it was not appreciated in these previous reports that the source of H2O2 could be the peptide itself. Both in vitro and in vivo data emphasize the importance of the single methionine 35 residue of Ab in mediating this free radical production and neurotoxicity (Varadarajan et al., 1999), and we suspect that this residue is critical in allowing Ab the electrochemical property that allows it to produce H2O2 from molecular oxygen. SOD and synthetic catalytic scavengers of superoxide and H2O2 (Thomas et al., 1996; Bruce et al., 1996) can abrogate Ab toxicity in vitro. The free radical dependence of Ab-associated toxicity is further supported by the ability of Vitamin E, a free radical scavenger, to protect against the toxic effects of Ab (Subramaniam et al., 1998). Moreover, clinical data shows that AD patients benefit from treatment with antioxidants, Vitamin E and/or selegiline hydrochloride (Sano et al., 1997). In this regard, it is interesting that a relationship between oxidative damage in AD brain and apoE genotype has been recently reported (Ramassamy et al., 1999). These workers described elevated levels of lipid oxidation in tissues from AD cases homozygous for apoE4 and increased catalase and glutathione peroxidase activities in AD cases with at least one apoE4 allele.
4. Does Ab in AD, like mutant SOD1 in familial ALS, represent a corrupted antioxidant? A mutation in the ubiquitous antioxidant, SOD1, confers a toxic gain of function that results in the degenerative disorder, FALS. Associated with this mutation are oxidative damage and the formation of SOD1 aggregates in affected motor neurons and glia (Bruijn et al., 1998). The biochemistry behind this toxic gain of function by SOD1 may provide an insight into other neurodegenerative disorders also characterized by oxidative stress, including AD. We had previously noted that the Cu 2⫹ –Ab1-42 complex has a notably strong reduction potential (⫹550 mM vs Ag/AgCl) which is likely to denote a biological purpose (Huang et al., 1999b). This feature, together with the observation that Ab binds Cu 2⫹ and Zn 2⫹ simultaneously (Atwood et al., in press) and is released upon oxidative stress of cells, lead us to speculate that Ab may also function as a superoxide dismutasemimetic. Using pulse radiolysis and cell culture techniques, we recently reported that Cu/ Zn-loaded Ab (at nanomolar concentrations) exhibits catalytic SOD-like activity, greatest for Ab1-42human ⬎ Ab1-40human Ⰷ rat/mouse homologues (Bush et al., 1999). We are currently investigating the possibility that copper and zinc appear to play complementary roles when bound to Ab in a fashion similar to mutant SOD1 (Estevez et al., 1999); in
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addition to promoting structural integrity, zinc may modulate the activity of copper which has the potential to be abnormally redox-reactive, generating unwanted reactive oxygen species. 5. Conclusions There is growing evidence of significant interaction between Ab and biometals (copper, zinc and iron) as playing a major role in the pathogenesis of AD. These metals are normally enriched in the neocortical areas most affected by Alzheimer neuropathology, but are even further enriched in AD, especially in amyloid deposits. The interactions between these metals and Ab modulate the precipitation of the peptide, its electrochemical activity, its toxicity and its oxidative modification. Perhaps most importantly, Ab may itself be a metalloprotein with normal electrochemical activity, like CuZnSOD. Interdiction of the abnormal metallation of Ab by small molecules that complex to the redox active site on the protein may have therapeutic promise in AD. References Aksenova, M.V., Aksenov, M.Y., Payne, R.M., Trojanowski, J.Q., Schmidt, M.L., Carney, J.M., Butterfield, D.A., Markesbery, W.R., 1999. Oxidation of cytosolic proteins and expression of creatine kinase BB in frontal lobe in different neurodegenerative disorders. Dement. Geriatr. Cogn. Disord. 10, 158–165. Atwood, C.S., Moir, R.D., Huang, X., Bacarra, N.M.E., Scarpa, R.C., Romano, D.M., Hartshorn, M.A., Tanzi, R.E., Bush, A.I., 1998. Dramatic aggregation of Alzheimer Ab by Cu(II) is induced by conditions representing physiological acidosis. J. Biol. Chem. 273, 12 817–12 826. Atwood, C.S., Huang, X., Moir, R.D., Tanzi, R.E., Bush, A.I., 1999. Role of free radicals and metal ions in the pathogenesis of Alzheimer’s disease. Met. Ions Biol. Syst. 36, 309–364. Atwood, C.S., Scarpa, R.C., Huang, X., Moir, R.D., Jones, W.D., Fairlie, D.P., Tanzi, R.E., Bush, A.I., 2000. Characterization of copper interactions with Alzheimer Ab peptides-identification of an attomolar affinity copper binding site on Ab1-42. J. Neurochem. (in press). Behl, C., Davis, J.B., Lesley, R., Schubert, D., 1994. Hydrogen peroxide mediates amyloid b protein toxicity. Cell 77, 817–827. Bruce, A.J., Malfroy, B., Baudry, M., 1996. Beta-Amyloid toxicity in organotypic hippocampal cultures: protection by Euk-8, a synthetic catalytic free radical scavenger. Proc. Natl. Acad. Sci. USA 93, 2312–2316. Bruijn, L.I., Houseweart, M.K., Kato, S., Anderson, K.L., Anderson, S.D., Ohama, E., Reaume, A.G., Scott, R.W., Cleveland, D.W., 1998. Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science 281, 1851–1854. Bush, A.I., Multhaup, G., Moir, R.D., Williamson, T.G., Small, D.H., Rumble, B., Pollwein, P., Beyreuther, K., Masters, C.L., 1993. A novel zinc(II) binding site modulates the function of the bA4 amyloid protein precursor of Alzheimer’s disease. J. Biol. Chem. 268, 16109–16112. Bush, A.I., Pettingell, W.H., Multhaup, G., Paradis, Md., Vonsattel, J.P., Gusella, J.F., Beyreuther, K., Masters, C.L., Tanzi, R.E., 1994. Rapid induction of Alzheimer Ab amyloid formation by zinc. Science 265, 1464–1467. Bush, A.I., Lynch, T., Cherny, R.C., Atwood, C.S., Goldstein, L.E., Moir, R.D., Li, Q-X., Cabelli, D.E., Multhaup, G., Masters, C.L., Tanzi, R.E., Huang, X., 1999. Alzheimer Ab functions as superoxide antioxidant in vitro and in vivo. Soc. Neurosci. Abstr. 25, 14. Butterfield, D.A., Hensley, K., Harris, M., Mattson, M., Carney, J., 1994. Beta-amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer’s disease. Biochem. Biophys. Res. Commun. 200, 710–715. Cherny, R.A., Legg, J.T., McLean, C.A., Fairlie, D., Huang, X., Atwood, C.S., Beyreuther, K., Tanzi, R.E.,
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Masters, C.L., Bush, A.I., 1999. Aqueous dissolution of Alzheimer’s disease Ab amyloid deposits by biometal depletion. J. Biol. Chem. 274, 23 223–23 228. Estevez, A.G., Crow, J.P., Sampson, J.B., Reiter, C., Zhuang, Y., Richardson, G.J., Tarpey, M.M., Barbeito, L., Beckman, J.S., 1999. Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficient superoxide dismutase. Science 286, 2498–2500. Huang, X., Atwood, C.S., Moir, R.D., Hartshorn, M.A., Vonsattel, J.-P., Tanzi, R.E., Bush, A.I., 1997. Zinc-induced Alzheimer’s Ab1-40 aggregation is mediated by conformational factors. J. Biol. Chem. 272, 26 464–26 470. Huang, X., Atwood, C.S., Hartshorn, M.A., Multhaup, G., Goldstein, L.E., Scarpa, R.C., Cuajungco, M.P., Gray, D.N., Lim, J., Moir, R.D., Tanzi, R.E., Bush, A.I., 1999. The Ab peptide of Alzheimer’s disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry 38, 7609–7616. Huang, X., Atwood, C.S., Cuajungco, M.P., Hartshorn, M.A., Tyndall, J., Hanson, G.R., Stokes, K.C., Leopold, M., Multhaup, G., Goldstein, L.E., Scarpa, R.C., Saunders, A.J., Lim, J., Moir, R.D., Glabe, C., Bowden, E.F., Masters, C.L., Fairlie, D.P., Tanzi, R.E., Bush, A.I., 1999. Cu(II) potentiation of Alzheimer Ab neurotoxicity: correlation with cell-free hydrogen peroxide production and metal reduction. J. Biol. Chem. 274, 37111– 37116. McLean, C.A., Cherny, R.A., Fraser, F.W., Fuller, S.J., Smith, M.J., Beyreuther, K., Bush, A.I., Masters, C.L., 1999. Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer’s Disease. Ann. Neurol. 46, 860–866. Moir, R.D., Atwood, C.S., Romano, D.M., Laurans, M.H., Huang, X., Bush, A.I., Smith, J.D., Tanzi, R.E., 1999. Differential effects of apolipoprotein E isoforms on metal-induced aggregation of A-beta using physiological concentrations. Biochemistry 38, 4595–4603. Nunomura, A., Perry, G., Pappolla, M.A., Wade, R., Hirai, K., Chiba, S., Smith, M.A., 1999. RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer’s disease. J. Neurosci. 19, 1959–1964. Odetti, P., Angelini, G., Dapino, D., Zaccheo, D., Garibaldi, S., Dagna-Bricarelli, F., Piombo, G., Perry, G., Smith, M., Traverso, N., Tabaton, M., 1998. Early glycoxidation damage in brains from Down’s syndrome. Biochem. Biophys. Res. Commun. 243, 849–851. Pappolla, M.A., Omar, R.A., Kim, K.S., Robakis, N.K., 1992. Immunohistochemical evidence of antioxidant stress in Alzheimer’s disease. Am. J. Pathol. 140, 621–628. Ramassamy, C., Averill, D., Beffert, U., Bastianetto, S., Theroux, L., Lussier-Cacan, S., Cohn, J.S., Christen, Y., Davignon, J., Quirion, R., Poirier, J., 1999. Oxidative damage and protection by antioxidants in the frontal cortex of Alzheimer’s disease is related to the apoliprotein E genotype. Free Rad. Biol. Med. 27, 544–553. Sano, M., Ernesto, C., Thomas, R.G., Klauber, M.R., Schafer, K., Grundman, M., Woodbury, P., Growdon, J., Cotman, C.W., Pfeiffer, E., Schneider, L.S., Thal, L.J., 1997. A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer’s disease. The Alzheimer’s disease cooperative study. N. Engl. J. Med. 336, 1216–1222. Sayre, L.M., Zelasko, D.A., Harris, P.L., Perry, G., Salomon, R.G., Smith, M.A., 1997. 4-Hydroxynonenalderived advanced lipid peroxidation end products are increased in Alzheimer’s disease. J. Neurochem. 68, 2092–2097. Smith, M.A., Harris, P.L.R., Sayre, L.M., Perry, G., 1997. Iron accumulation in Alzheimer’s disease is a source of redox-generated free radicals. Proc. Natl. Acad. Sci. USA 94, 9866–9868. Smith, M.A., Richey, P.L., Taneda, S., Kutty, R.K., Sayre, L.M., Monnier, V.M., Perry, G., 1994. Advanced Maillard reaction end products, free radicals, and protein oxidation in Alzheimer’s disease. Ann. NY Acad. Sci. 738, 447–454. Smith, M.A., Kutty, R.K., Richey, P.L., Yan, S.D., Stern, D., Chader, G.J., Wiggert, B., Petersen, R.B., Perry, G., 1994. Heme oxygenase-1 is associated with the neurofibrillary pathology of Alzheimer’s disease. Am. J. Pathol. 145, 42–47. Smith, M.A., Richey, P.L., Harris, P.L., Sayre, L.M., Beckman, J.S., Perry, G., 1997. Widespread peroxynitritemediated damage in Alzheimer’s disease. J. Neurosci. 17, 2653–2657. Smith, M.A., Hirai, K., Hsiao, K., Pappolla, M.A., Harris, P., Siedlak, S., Tabaton, M., Perry, G., 1998. Amyloidbeta deposition in Alzheimer transgenic mice is associated with oxidative stress. J. Neurochem. 70, 2212–2215. Subramaniam, R., Koppal, T., Green, M., Yatin, S., Jordan, B., Drake, J., Butterfield, D.A., 1998. The free radical antioxidant vitamin E protects cortical synaptosomal membranes from amyloid beta-peptide(25–35) toxicity
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but not from hydroxynonenal toxicity: relevance to the free radical hypothesis of Alzheimer’s disease. Neurochem. Res. 23, 1403–1410. Thomas, T., Thomas, G., McLendon, C., Sutton, T., Mullan, M., 1996. Beta-Amyloid-mediated vasoactivity and vascular endothelial damage. Nature 380, 168–171. Varadarajan, S., Yatin, S., Kanski, J., Jahanshahi, F., Butterfield, D.A., 1999. Methionine residue 35 is important in amyloid beta-peptide-associated free radical oxidative stress. Brain Res. Bull 50, 133–141. Yates, C.M., Butterworth, J., Tennant, M.C., Gordon, A., 1990. Enzyme activities in relation to pH and lactate in postmortem brain in Alzheimer-type and other dementia. J. Neurochem. 55, 1624–1630.
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Experimental Gerontology 35 (2000) 445–451 www.elsevier.nl/locate/expgero
Review
Oxidative processes in Alzheimer’s disease: the role of Ab-metal interactions T. Lynch a, R.A. Cherny a, A.I. Bush a,b,* a
Department of Pathology, The University of Melbourne, and Neuropathology Laboratory, The Mental Health Research Institute of Victoria, Parkville, Vic. 3052, Australia b Laboratory for Oxidation Biology, Genetics and Aging Unit, and Department of Psychiatry, Harvard Medical School, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA Received 14 February 2000; accepted 11 April 2000
Abstract Alzheimer’s disease is characterized by signs of a major oxidative stress in the neocortex and the concomitant deposition of Amyloid beta (Ab). Ab is a metalloprotein that binds copper, and is electrochemically active. Ab converts molecular oxygen into hydrogen peroxide by reducing copper or iron, and this may lead to Fenton chemistry. Hydrogen peroxide is a freely permeable prooxidant that may be responsible for many of the oxidative adducts that form in the Alzheimer-affected brain. The electrochemical activity of various Ab species correlates with the peptides’ neurotoxicity in cell culture, and participation in the neuropathology of Alzheimer’s disease. These reactions present a novel target for Alzheimer therapeutics. 䉷 2000 Elsevier Science Inc. All rights reserved. Keywords: Amyloid beta; Metals; Oxidative stress; Hydrogen peroxide; Neurodegeneration; Antioxidants; Superoxide dismutase
1. Introduction The term ‘oxidative stress’ is used when the body’s natural defense mechanisms are exceeded by the production of deleterious reactive oxygen species, resulting in damage to susceptible cell components such as DNA, proteins, and lipids. Features of the brain that cause it to be particularly sensitive to oxidative stress include a high rate of oxidative metabolic activity, relatively low levels of antioxidant enzymes (e.g. catalase and glutathione peroxidase), a high concentration of unsaturated fatty acids, large iron and copper stores, and a low mitotic index. * Corresponding author. Tel.: ⫹1-617-726-8244; fax: ⫹1-617-724-9610. E-mail address: [email protected] (A.I. Bush). 0531-5565/00/$ - see front matter 䉷 2000 Elsevier Science Inc. All rights reserved. PII: S0531-556 5(00)00112-1
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Oxidative stress associated with amyloid beta (Ab) accumulation in the neocortex is characteristic of Alzheimer’s disease (AD) and potentially related to the pathogenesis of neuronal degeneration in this disorder (Atwood et al., 1999). Similarly, markers of oxidative stress and damage response, including elevated levels of redox-active iron, (Smith et al., 1997a) directly correlate with the presence of Ab deposits in the neocortex of transgenic mice overexpressing amyloid precursor protein (APP) (Smith et al., 1998), thereby providing evidence that Ab and oxidative damage are linked in vivo. Increased oxidation of brain lipids, carbohydrates, proteins, and DNA, as well as systemic signs of oxidative stress, altered levels of antioxidants, and metabolic signs of excess oxidative stress in the neocortex of patients, have all been observed in AD. Frontal lobe autopsy specimens from AD brains show an increase in the oxidative modifications of proteins (i.e. carbonylation) with significant reductions in the activities of the oxidation-sensitive enzymes, creatine kinase and glutamine synthetase, effects of which may contribute directly to the symptoms of frontal dysfunction observed in AD (Aksenova et al., 1999). Several oxidative stress products are detected in the vicinity of Ab amyloid plaques and neurofibrillary tangles (the other major neuropathological feature of AD brain), as well as in the cerebrospinal fluid of AD patients. These include the highly reactive breakdown products of lipid peroxidation such as 4-hydroxynonenal (Sayre et al., 1997), Maillard end products pyrraline and pentosidine (Smith et al., 1994a), nitrotyrosine (Smith et al., 1997b), advanced glycation endproducts, and neurofilament-related protein carbonyls (Smith et al., 1994a). As expected, there is an up-regulation of antioxidants such as copper/zinc-superoxide dismutase (Cu/Zn-SOD) (Pappolla et al., 1992), catalase (Pappolla et al., 1992), and heme oxygenase-1 (HO-1) (Smith et al., 1994b). There are also increased markers for lipid and protein oxidation in Down’s syndrome fetal brain cortex (Odetti et al., 1998) which is important since individuals with Trisomy 21 possess an extra copy of the APP gene, develop an accelerated rate of cerebral Ab deposition, and express premature AD neuropathology. Paradoxically, there appears to be no spatial association between histological amyloid plaque deposits and histological markers of oxidation (Nunomura et al., 1999). Moreover, we recently found that total Ab load in AD neocortex is not related to disease severity or numbers of amyloid plaques (McLean et al., 1999). Instead, the concentration of soluble Ab, most likely in the form of soluble oligomers, is an accurate determinant of the severity of neurodegeneration in AD brain. It is therefore not surprising that plaque load does not correlate well with other properties of neocortical damage in general.
2. Metal-mediated aggregation of Ab in vitro and in vivo There is convincing evidence that the toxic effects of Ab are generated during, or following, the process of polymerization and oligomerization of this normally soluble peptide, therefore strategies aimed at preventing Ab aggregation or, alternatively, dissolving Ab deposits, may be of therapeutic value. The length of the Ab species (40–42 amino acids), following cleavage from APP, is considered to be an important factor in AD pathogenesis. Despite existing as a minor free soluble species in biological fluids, Ab 1-42 is enriched in cerebral amyloid deposits in AD.
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Ab interacts with Zn(II) and Cu(II) at low micromolar and submicromolar concentrations (Bush et al., 1993, 1994; Huang et al., 1997; Atwood et al., 1998). Ab is rapidly precipitated by Zn 2⫹ at pH ⬎ 7.0, whereas Cu 2⫹ and Fe 3⫹ induce greater Ab aggregation under mildly acidic conditions (Atwood et al., 1998), comparable to those reported in AD brain (Yates et al., 1990). In contrast, the solubility of rat/mouse Ab 1-40 is unaffected by Zn(II) or Cu(II) at low micromolar concentrations (Bush et al., 1994; Atwood et al., 1998), perhaps contributing to the inability of these animals to form cerebral Ab amyloid. Significantly, Ab 1-42 is more readily precipitated by Cu 2⫹ than Ab 1-40, but both peptide species are equally destabilized by Zn 2⫹ (Atwood et al., 1998). Moir et al. (1999) reported that apolipoprotein E (apoE) isoforms differentially modulate the aggregation of Ab by Cu 2⫹ and Zn 2⫹. Consistent with the increased risk of late-onset AD attached to inheriting apoE4, they discovered that this isoform (compared with apoE2 and apoE3) is the least effective chaperone for Ab solubility, whether the precipitating stress is Cu 2⫹ or Zn 2⫹. The affinity of the Zn 2⫹ binding sites on Ab 1-40 were measured as 100 nM and 5 mM, indicating that they may be occupied under physiological conditions (Bush et al., 1994). Ab species also possess an unusually strong affinity for Cu 2⫹ (Ka ⬃ 10 ⫺10 M and 10 ⫺16 M for Ab1-40 and Ab1-42, respectively (Atwood et al., 2000), and are therefore likely to bind Cu 2⫹ in vivo. Indeed, we have recently reported that zinc and copper-selective chelators markedly enhance the solubilization of Ab deposits from post-mortem AD brain tissue (Cherny et al., 1999), supporting the possibility that zinc and copper ions are intrinsic to the architecture of these deposits.
3. Reactive oxygen species formation and neurotoxicity of Ab Since Ab binds redox active metals Cu 2⫹ and Fe 3⫹, we investigated whether potentially adverse electrochemical reactions between the peptide and these metals could result. Huang et al. (1999a) reported that human Ab is redox-active and can directly produce H2O2 in a cell-free manner dependent upon oxygen and the reduction of Fe(III) or Cu(II) to Fe(II) and Cu(I), respectively, setting up conditions that could promote Fenton-type chemistry. The metal reducing ability and H2O2 production of Ab species is greatest when generated by Ab42human ⬎ Ab40human Ⰷ Ab40rat/mouse, an order that correlates with the neurotoxicity of the respective Ab species in cell culture (Huang et al., 1999b) and its association with AD neuropathology. Ab cytotoxicity was also shown to be mediated largely by the H2O2 produced directly by the Ab species, since the toxicity of the peptide augmented by Cu 2⫹ was abolished by catalase (Huang et al., 1999b). The effects of Cu(II)-mediated auto-oxidation and hydroxyl radical production cause multiple modifications in Ab (carbonyl adduct formation, histidine loss, and dityrosine cross-linking) that may consequently increase the protease resistance of the peptide. These Cu(II)-induced oxidative modifications can be mimicked by direct hydroxyl radical attack (again greatest for Ab1-42human ⬎ Ab1-40human Ⰷ Ab1-40rat, corresponding to the participation of the respective peptide in AD pathophysiology) (Atwood et al., unpublished results). Our data indicate that when human Ab is abnormally metallated with Cu(II), it produces H2O2 which then reacts with reduced Cu(I) and may produce the hydroxyl radical
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(Fenton-type chemistry). This highly reactive oxygen species then oxidizes Ab. Apart from being a substrate for Fenton-mediated hydroxyl radical generation, H2O2 is also cytotoxic, pro-apoptotic, and is freely permeable across membranes. Hence, an elevation in H2O2 will not just cause Ab oxidation and accumulation, but may also contribute to oxidative damage extending beyond the site of generation. This theory correlates well with the global increase in oxidative damage observed in AD. Another neurochemical feature of the cerebral microenvironment in AD that may contribute to excessive H2O2 production is the mild acidosis that promotes Cu(II)-binding to Ab (reviewed in Atwood et al., 1999). The redox activity of Ab is compatible with reports that Ab peptides can initiate synaptosomal lipoperoxidation (Butterfield et al., 1994), and can raise cellular hydrogen peroxide (H2O2) (Behl et al., 1994), although it was not appreciated in these previous reports that the source of H2O2 could be the peptide itself. Both in vitro and in vivo data emphasize the importance of the single methionine 35 residue of Ab in mediating this free radical production and neurotoxicity (Varadarajan et al., 1999), and we suspect that this residue is critical in allowing Ab the electrochemical property that allows it to produce H2O2 from molecular oxygen. SOD and synthetic catalytic scavengers of superoxide and H2O2 (Thomas et al., 1996; Bruce et al., 1996) can abrogate Ab toxicity in vitro. The free radical dependence of Ab-associated toxicity is further supported by the ability of Vitamin E, a free radical scavenger, to protect against the toxic effects of Ab (Subramaniam et al., 1998). Moreover, clinical data shows that AD patients benefit from treatment with antioxidants, Vitamin E and/or selegiline hydrochloride (Sano et al., 1997). In this regard, it is interesting that a relationship between oxidative damage in AD brain and apoE genotype has been recently reported (Ramassamy et al., 1999). These workers described elevated levels of lipid oxidation in tissues from AD cases homozygous for apoE4 and increased catalase and glutathione peroxidase activities in AD cases with at least one apoE4 allele.
4. Does Ab in AD, like mutant SOD1 in familial ALS, represent a corrupted antioxidant? A mutation in the ubiquitous antioxidant, SOD1, confers a toxic gain of function that results in the degenerative disorder, FALS. Associated with this mutation are oxidative damage and the formation of SOD1 aggregates in affected motor neurons and glia (Bruijn et al., 1998). The biochemistry behind this toxic gain of function by SOD1 may provide an insight into other neurodegenerative disorders also characterized by oxidative stress, including AD. We had previously noted that the Cu 2⫹ –Ab1-42 complex has a notably strong reduction potential (⫹550 mM vs Ag/AgCl) which is likely to denote a biological purpose (Huang et al., 1999b). This feature, together with the observation that Ab binds Cu 2⫹ and Zn 2⫹ simultaneously (Atwood et al., in press) and is released upon oxidative stress of cells, lead us to speculate that Ab may also function as a superoxide dismutasemimetic. Using pulse radiolysis and cell culture techniques, we recently reported that Cu/ Zn-loaded Ab (at nanomolar concentrations) exhibits catalytic SOD-like activity, greatest for Ab1-42human ⬎ Ab1-40human Ⰷ rat/mouse homologues (Bush et al., 1999). We are currently investigating the possibility that copper and zinc appear to play complementary roles when bound to Ab in a fashion similar to mutant SOD1 (Estevez et al., 1999); in
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addition to promoting structural integrity, zinc may modulate the activity of copper which has the potential to be abnormally redox-reactive, generating unwanted reactive oxygen species. 5. Conclusions There is growing evidence of significant interaction between Ab and biometals (copper, zinc and iron) as playing a major role in the pathogenesis of AD. These metals are normally enriched in the neocortical areas most affected by Alzheimer neuropathology, but are even further enriched in AD, especially in amyloid deposits. The interactions between these metals and Ab modulate the precipitation of the peptide, its electrochemical activity, its toxicity and its oxidative modification. Perhaps most importantly, Ab may itself be a metalloprotein with normal electrochemical activity, like CuZnSOD. Interdiction of the abnormal metallation of Ab by small molecules that complex to the redox active site on the protein may have therapeutic promise in AD. References Aksenova, M.V., Aksenov, M.Y., Payne, R.M., Trojanowski, J.Q., Schmidt, M.L., Carney, J.M., Butterfield, D.A., Markesbery, W.R., 1999. Oxidation of cytosolic proteins and expression of creatine kinase BB in frontal lobe in different neurodegenerative disorders. Dement. Geriatr. Cogn. Disord. 10, 158–165. Atwood, C.S., Moir, R.D., Huang, X., Bacarra, N.M.E., Scarpa, R.C., Romano, D.M., Hartshorn, M.A., Tanzi, R.E., Bush, A.I., 1998. Dramatic aggregation of Alzheimer Ab by Cu(II) is induced by conditions representing physiological acidosis. J. Biol. Chem. 273, 12 817–12 826. Atwood, C.S., Huang, X., Moir, R.D., Tanzi, R.E., Bush, A.I., 1999. Role of free radicals and metal ions in the pathogenesis of Alzheimer’s disease. Met. Ions Biol. Syst. 36, 309–364. Atwood, C.S., Scarpa, R.C., Huang, X., Moir, R.D., Jones, W.D., Fairlie, D.P., Tanzi, R.E., Bush, A.I., 2000. Characterization of copper interactions with Alzheimer Ab peptides-identification of an attomolar affinity copper binding site on Ab1-42. J. Neurochem. (in press). Behl, C., Davis, J.B., Lesley, R., Schubert, D., 1994. Hydrogen peroxide mediates amyloid b protein toxicity. Cell 77, 817–827. Bruce, A.J., Malfroy, B., Baudry, M., 1996. Beta-Amyloid toxicity in organotypic hippocampal cultures: protection by Euk-8, a synthetic catalytic free radical scavenger. Proc. Natl. Acad. Sci. USA 93, 2312–2316. Bruijn, L.I., Houseweart, M.K., Kato, S., Anderson, K.L., Anderson, S.D., Ohama, E., Reaume, A.G., Scott, R.W., Cleveland, D.W., 1998. Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science 281, 1851–1854. Bush, A.I., Multhaup, G., Moir, R.D., Williamson, T.G., Small, D.H., Rumble, B., Pollwein, P., Beyreuther, K., Masters, C.L., 1993. A novel zinc(II) binding site modulates the function of the bA4 amyloid protein precursor of Alzheimer’s disease. J. Biol. Chem. 268, 16109–16112. Bush, A.I., Pettingell, W.H., Multhaup, G., Paradis, Md., Vonsattel, J.P., Gusella, J.F., Beyreuther, K., Masters, C.L., Tanzi, R.E., 1994. Rapid induction of Alzheimer Ab amyloid formation by zinc. Science 265, 1464–1467. Bush, A.I., Lynch, T., Cherny, R.C., Atwood, C.S., Goldstein, L.E., Moir, R.D., Li, Q-X., Cabelli, D.E., Multhaup, G., Masters, C.L., Tanzi, R.E., Huang, X., 1999. Alzheimer Ab functions as superoxide antioxidant in vitro and in vivo. Soc. Neurosci. Abstr. 25, 14. Butterfield, D.A., Hensley, K., Harris, M., Mattson, M., Carney, J., 1994. Beta-amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer’s disease. Biochem. Biophys. Res. Commun. 200, 710–715. Cherny, R.A., Legg, J.T., McLean, C.A., Fairlie, D., Huang, X., Atwood, C.S., Beyreuther, K., Tanzi, R.E.,
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