Serial Review: Oxidatively Modified Proteins in Aging and DiseaseGuest Editor: Earl Stadtman

OXIDATIVELY MODIFIED PROTEINS IN AGING AND DISEASE

M. FLINT BEAL

Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York Presbyterian Hospital,New York, NY, USA

(Received 28 December 2001;Accepted 8 February 2002)

Abstract—There is a large body of evidence implicating oxidative damage in the pathogenesis of both normal agingand neurodegenerative diseases. Oxidative damage to proteins has been well established. Although there are a largenumber of potential oxidative modifications only a few have been systematically studied. The most frequently studiedmarker of oxidative damage to proteins is protein carbonyl groups. 3-Nitrotyrosine is thought to be a relatively specificmarker of oxidative damage mediated by peroxynitrite. Increased concentrations of both protein carbonyls and3-nitrotyrosine have been documented in both normal aging as well as in Alzheimer’s disease (AD), Parkinson’s disease(PD), and amyotrophic lateral sclerosis (ALS). These findings help to provide a rationale for trials of antioxidants inneurodegenerative diseases. © 2002 Elsevier Science Inc.

Keywords—3-Nitrotyrosine, Carbonyls, Dityrosine, Alzheimer’s, Parkinson’s, Huntington’s, Amyotrophic lateralsclerosis, Free radical

INTRODUCTION

Oxidative modification of proteins by reactive oxygenspecies (ROS) or reactive nitrogen species (RNS) isimplicated in the pathogenesis of both normal aging andneurodegenerative diseases. The generation of ROS andRNS may occur by a large number of physiological andnonphysiological processes. These include the genera-

tion of superoxide anion O2•� and H2O2 by inadvertent

autooxidation of reduced forms of electron carriers. Thegeneration of nitric oxide (NO•) from arginine by nitricoxide synthase is a process which is involved in neuro-transmission, regulation of vascular relaxation, and ininflammatory processes. The generation of NO• is cata-lyzed by three isoforms of nitric oxide synthase, neuronalnitric oxide synthase (nNOS), endothelial nitric oxidesynthase (eNOS), and inducible nitric oxide synthase(iNOS). Under pathological circumstances O2

•� can di-rectly react with NO• to generate peroxynitrite(ONOO�). The latter compound, although strictly not afree radical, has the capacity to act in a hydroxyl (OH•)radical-like manner to induce lipid and protein oxidation,and it can also nitrate tyrosines, generating 3-nitroty-rosine. The latter reaction is markedly enhanced in thepresence of CO2 [1]. Peroxynitrite plays a key role inneuronal damage associated with excitotoxicity [2].

The most widely studied marker of protein oxidationis protein carbonyl groups. Protein carbonyl groups areformed by oxidation of the side chains of lysine, proline,arginine, and threonine residues [3]. In addition to thedirect oxidation of protein side chains, carbonyl groupscan be introduced into proteins by Michael additionreactions of 4-hydroxynonenal, a product of lipid peroxi-

This article is part of a series of reviews on “Oxidatively ModifiedProteins in Aging and Disease.” The full list of papers may be found onthe homepage of the journal.

Dr. M. Flint Beal is the Anne Parrish Titzell Professor and Chairmanof the Department of Neurology and Neuroscience at the Weill MedicalCollege of Cornell University and Director of the Neurology service atthe New York Presbyterian Cornell Campus. Dr. Beal received hismedical degree from the University of Virginia in 1976 and did hisinternship and first year residency in Medicine at New York-Cornellbefore completing his residency in Neurology at The MassachusettsGeneral Hospital. He joined the neurology faculty at Harvard in 1983.Dr. Beal was Professor of Neurology at the Harvard Medical Schooland Chief of the Neurochemistry laboratory at Massachusetts GeneralHospital before moving to Cornell. Dr. Beal’s research has focused onthe mechanism of neuronal degeneration in Alzheimer’s Disease, Hun-tington’s Disease, Parkinson’s Disease, and amyotrophic lateral scle-rosis (ALS).

Address correspondence to: M. Flint Beal, M.D., Chairman, Neurol-ogy Department, New York Hospital-Cornell Medical Center, 525 East68th Street, New York, NY 10021, USA; Tel: (212) 746-6575; Fax:(212) 746-8532; E-Mail: fbeal@mail.med.cornell.edu.

Free Radical Biology & Medicine, Vol. 32, No. 9, pp. 797–803, 2002Copyright © 2002 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/02/$–see front matter

PII S0891-5849(02)00780-3

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dation. The measurement of protein carbonyls followingtheir reaction with 2,4-dinitrophenylhydrazine was pio-neered by Levine and Stadtman, and has become themost widely utilized measure of protein oxidation. Theassay can be performed either biochemically or by West-ern blot. In addition, methods have been developed todetect protein carbonyls using immunohistochemistry[4].

Measurements of 3-nitrotyrosine have been made us-ing HPLC with the electrochemical detection and bymass spectroscopy [5]. It can also be detected usingimmunohistochemistry. A number of other markers ofoxidative damage to proteins are used less commonly.These include dityrosine, 2-oxohistidine, methione sul-foxide, and cysteine disulfide [3].

OXIDATIVE DAMAGE AND AGING

A number of studies have shown increases in theintracellular concentrations of oxidized proteins as afunction of age. Increases in protein carbonyls occur inrat hepatocytes, drosophila, brain, and kidney of miceand in brain tissue of gerbils [6–8]. In humans proteincarbonyls increase with age in brain, muscle, and humaneye lens [9–12]. The carbonyl content of human fibro-blasts also increases as a function of age of the donor[13]. The rate of oxidation of proteins increases dramat-ically in the last third of life such that an average of onein three proteins is affected. This is likely to have phys-iologic relevance since oxidative modification can inac-tivate catalytic function [14,15]. In drosophila, restrictingflying increases life span, and this correlates with re-duced protein carbonyls [16]. Recent work showed thattransgenic mice with a knockout of methionine sulfoxidereductase, which repairs oxidized methione, have a re-duced life span and show increased protein carbonyls[17].

Interestingly, not all proteins are uniformly suscepti-ble to oxidative damage. Using an immunochemicalprobe for oxidative damage it was demonstrated thatmitochondrial aconitase was particularly vulnerable tooxidative damage accompanying aging in drosophila[18]. Similarly, the mitochondrial adenine nucleotidetranslocase was shown to be particularly vulnerable [19].Prior studies showed that glutamine synthetase and cre-atine kinase are particularly vulnerable to oxidative dam-age [12,20]. These proteins can also be inactivated bynitration.

Only a limited number of proteins undergo nitrationas well. A proteomic method identified about 40 nitratedproteins out of 1000 during an inflammatory challenge[21]. These included a large number of mitochondrialproteins, which regulate cellular energy metabolism. Oneprotein was manganese superoxide dismutase, which was

previously shown to be selectively nitrated at tyr-34, andinactivated [22]. Prostacyclin synthase (PGI2) is anotherheme protein that is nitrated and inactivated by very lowlevels of peroxynitrite [23]. Nitration of tyrosine residuesin glutamine synthetase by peroxynitrite inhibits oxida-tion of methione residues, and leads to inactivation of theenzyme [3]. This compromises its regulation by adeny-lylation. Peroxynitrite-mediated nitration of lymphocyte-specific tyrosine kinase inhibits its ability to phosphory-late tyrosine residues [24]. This is a signal transductionmechanism involved in a myriad of pathways, includingthose mediated by neurotrophins. Creatine kinase is an-other key intracellular enzyme regulating energy metab-olism that is nitrated and inactivated by peroxynitrite[25].

ALZHEIMER’S DISEASE

Alzheimer’s disease is the most common neurodegen-erative disease. Clinically, it leads to progressive mem-ory loss and dementia. The neuropathologic hallmarksare senile plaques containing �-amyloid and neurofibril-lary tangles, which occur in pyramidal neurons of thecerebral cortex and hippocampus. In Alzheimer’s disease(AD) there is a large body of evidence implicating oxi-dative damage. Furthermore, administration of vitamin Eleads to a slowing of disease progression [26]. There isalso epidemiologic evidence that patients taking antiox-idant vitamins and anti-inflammatory compounds have alower incidence of AD. Several biochemical studiesshowed increased concentrations of protein carbonyls inAD patients using both biochemical assays and spinlabeling assays [27]. Protein carbonyls were significantlyincreased in both hippocampus and the inferior parietallobule, but unchanged in the cerebellum, consistent withthe regional pattern of histopathology in AD (Table 1).There were also significant decreases in glutamine syn-thetase and creatine kinase activity, and alterations inspin-labeled synaptosomes consistent with oxidativedamage. Oxidative damage to the glial glutamate trans-porter was recently reported in AD [28]. We developedimmunochemical methods to examine protein carbonylsat the cellular level. These studies showed that there wereincreases in protein carbonyls both in neurofibrillarytangles as well as in the cytoplasm of tangle free neurons[29,30]. Neurofibrillary tangles are protein aggregates,which are largely made up of the microtubule-associatedprotein tau. The immunocytochemical staining was ab-sent in age-matched controls and was blocked by priorreduction with NaBH4. Carbonyl related posttransla-tional modification of neurofilament proteins was alsodemonstrated [31]. Histochemical methods showed 4-hy-droxynonenal staining of amyloid deposits [32]. Acroleinis a highly reactive product of lipid peroxidation that is

798 M. F. BEAL

readily incorporated into proteins, generating a carbonylderivative. Immunohistochemistry showed strong reac-tivity in neurofibrillary tangle-bearing neurons, and a fewtangle-free neurons in AD [33]. Abnormal glycosylationof proteins in AD has also been found in several studies[34].

An increase in 3-nitrotyrosine immunostaining in ADpostmortem brain tissue has been documented by twogroups. Increased 3-nitrotyrosine was found in both neu-rons containing neurofibrillary tangles and in those inwhich they were absent [35–37]. Dimethylargininase, anitric oxide regulatory protein, was increased in neuronswith neurofibrillary tangles in AD [30]. The source of thenitric oxide is possibly from upregulation of an induciblenitric oxide synthase in neurofibrillary tangle-bearingneurons or from nitric oxide synthase in astrocytes andmicroglia adjacent to senile plaques [38,39]. Increases in3-nitrotyrosine and dityrosine were found in hydrolyzedproteins from AD postmortem brain tissue and ventric-ular cerebrospinal fluid, reaching concentrations 5–8-fold greater than those seen in age-matched controls [40].A 6-fold increase in 3-nitrotyrosine concentrations wasdetected in AD cerebrospinal fluid as compared to age-matched controls [41], and an increase in nitrated man-ganese superoxide dismutase was also reported [42].

PARKINSON’S DISEASE

Parkinson’s disease is the second most common neu-rodegenerative disease. It causes a progressive move-ment disorder. There is a loss of substantia nigra dopa-minergic neurons. The histopathologic hallmark iseosinophilic cytoplasmic inclusions in the substantianigra neurons known as Lewy bodies. In Parkinson’s

disease (PD) increases in protein carbonyls were foundin all brain regions examined including the substantianigra, basal ganglia, globus pallidus, substantia innomi-nata, frontal cortex, and cerebellum [43]. This findingwas unexpected since the neuropathology is much morerestricted. No changes, however, were found in patientswith incidental Lewy bodies, although the substantianigra was not examined. It was therefore speculated thatthe changes could be a consequence of L-DOPA treat-ment. An alternative explanation, however, would be awidely expressed genetic defect in the brain leading tooxidative damage. In patients with dementia with Lewybodies there was a trend for protein carbonyls to beincreased in cerebral cortex, and in the same cases oxi-dative damage to DNA was increased [44].

There is substantial evidence implicating peroxyni-trite-induced protein damage in PD and in animal modelsof PD. We and others showed that inhibitors of neuronalnitric oxide synthase blocked MPTP induced dopaminer-gic toxicity in mice, and that MPTP neurotoxicity wasattenuated in mice deficient in neuronal nitric oxidesynthase [45,46]. We subsequently showed that neuronalnitric oxide synthase inhibitors blocked MPTP neurotox-icity in baboons, and this was accompanied by an inhi-bition of 3-nitrotyrosine staining [47]. MPTP neurotox-icity and 3-nitrotyrosine generation are also attenuated inmice deficient in inducible NOS [48].

Increased 3-nitrotyrosine immunostaining was shownin Lewy bodies and in amorphous deposits in intact anddegenerating neurons in PD substantia nigra [49]. Otherstudies showed increased cerebrospinal fluid nitrate con-centrations and nitrosyl adducts in brains of PD patients[50]. An increase in nitrated manganese superoxide dis-mutase was found in cerebrospinal fluid [42].

Table 1. Protein Oxidation in Neurodegeneration

Disease Oxidative modification Reference

Alzheimer’s disease Protein carbonyls [27,28]Immunocytochemistry of carbonyls [29–31]4-hydroxynonenal immunostaining [32]Acrolein immunostaining [33]3-nitrotyrosine immunostaining [35–37]Protein 3-nitrotyrosine and dityrosine [40]Cerebrospinal fluid 3-nitrotyrosine [41]Cerebrospinal fluid nitrated manganese superoxide dismutase [42]

Parkinson’s disease Protein carbonyls [43]3-nitrotyrosine immunostaining [49]Cerebrospinal fluid nitrated manganese superoxide dismutase [42]Nitrated �-synuclein immunostaining [51]

ALS Protein carbonyls [57–59]Hydroynonenal modified protein [61]3-nitrotyrosine concentrations [5]Cerebrospinal fluid 3-nitrotyrosine [62]Cerebrospinal fluid nitrated manganese superoxide dismutase [42]3-nitrotyrosine immunostaining [64–66]

Huntington’s disease 3-nitrotyrosine immunostaining in mouse model [74]

799Protein oxidation and neurodegeneration

A major finding in PD pathogenesis was the geneticlinkage of autosomal dominant PD to mutations in theprotein �-synuclein. This protein was subsequentlyfound to be a major component of Lewy bodies. Anti-bodies to specific nitrated residues of �-synuclein weretherefore raised, and demonstrated to extensively labelLewy bodies in idiopathic PD, dementia with Lewybodies, and in multiple system atrophy brains, in whichLewy body inclusions occur in oligodendrocytes [51].Recent work showed that intracellular formation of per-oxynitrite in cells stably expressing mutant or wild-type�-synuclein leads to the formation of perinuclear�-synuclein aggregates that resemble Lewy bodies [52].This finding supports a direct role of peroxynitrite-in-duced modification of �-synuclein in Lewy body gener-ation. Other work showed that dityrosine cross-linkingpromotes the formation of stable �-synuclein polymers[53]. The over expression of �-synuclein in neuronalcells can also promote mitochondrial dysfunction andoxidative stress [54]. Other evidence for oxidative dam-age to proteins in PD is increased expression of neuralheme oxygenase-1 and increased immunostaining of gly-cosylated proteins [55,56].

AMYOTROPHIC LATERAL SCLEROSIS

ALS is a rapidly progressive neurodegenerative dis-ease leading to progressive motor weakness and death.Neuropathologically, there is a loss of motor neurons inboth the motor cortex and the spinal cord. In amyotro-phic lateral sclerosis (ALS) there is a large amount ofevidence implicating oxidative damage in disease patho-genesis. We found a large increase in protein carbonylsin frontal cortex (Broadman area 6) and in motor cortex(Broadman area 4) [57,58]. In one study, protein carbon-yls were increased by 119% in spinal cord tissue ofsporadic ALS patients [59]. In another study, however,there was no change in levels of protein carbonyls inALS motor cortex [60]. A study of hydroxynonenalmodified protein by immunohistochemistry showed anincrease in ALS spinal cord motor neurons, and immu-noprecipitation showed that one of the modified proteinswas the glial glutamate transporter [61].

There is substantial evidence for increased proteinnitration in ALS. We found increased immunocytochem-ical staining for 3-nitrotyrosine in spinal cord motorneurons of both sporadic and familial ALS patients [5].Furthermore, biochemical measurements of 3-nitroty-rosine and 3-nitro-4-hydroxyphenylacetic acid showedsignificant increases in the lumbar and thoracic spinalcord of ALS patients. Other groups reported markedincreases of both free 3-nitrotyrosine and nitrated man-ganese superoxide dismutase in the cerebrospinal fluid ofsporadic ALS patients [42,62]. In transgenic mouse mod-

els of ALS, protein carbonyls are significantly increasedin the spinal cord and one of the most heavily oxidizedproteins in Cu/Zn superoxide dismutase. We found sig-nificant decreases in creatine kinase activity in the trans-genic ALS mice that were mimicked by exposure ofbrain extracts of peroxynitrite [63].

A number of other immunocytochemical studiesshowed increased 3-nitrotyrosine staining in spinal cordmotor neurons of ALS patients [64–66]. An increase inneuronal nitric oxide synthase (nNOS) was found inmotor neurons in one study [65–67]. Other recent studiesshowed no alteration in nNOS in motor neurons, butshowed an increase in nNOS in reactive astrocytes inALS spinal cord and subcortical white matter [68,69]. Apotential source of O2

•� in ALS is the proinflammatoryenzyme cyclooxygenase-2. Its activity is dramaticallyincreased in spinal cord tissue of sporadic ALS patients,and immunochemistry shows increased staining in glialcells [70].

HUNTINGTON’S DISEASE

Huntington’s disease is an autosomal dominant inher-ited neurodegenerative disease in which there is both amovement disorder and dementia. Neuropathologically,the damage predominates in the basal ganglia. In Hun-tington’s disease (HD) evidence of increased oxidativedamage is much more limited. One study found noincreased protein carbonyl or oxidative damage to lipidsor DNA [71]. We, however, found increased oxidativedamage to DNA in both HD postmortem tissue and atransgenic mouse model [72,73]. Increased 3-nitroty-rosine immunostaining has also been reported in a trans-genic mouse model of HD [74].

CONCLUSION

There is a large body of evidence implicating oxida-tive damage to proteins in the pathogenesis of bothnormal aging and neurodegenerative illnesses. Oxidativedamage is selective in inactivating particular proteinspreferentially. This is true of both protein carbonyls andprotein nitration. This leads to inactivation of enzymaticactivity and kinase signaling pathways. The finding ofoxidative damage in neurodegenerative diseases raisesthe therapeutic possibilities that antioxidants might beuseful in slowing the progression of these illnesses.

Acknowledgements — The secretarial assistance of Sharon Melansonand Greta Strong is gratefully acknowledged. This work was supportedby the NIH, the Department of Defense, the ALS Association, and theHuntington’s Disease Society of America.

800 M. F. BEAL

REFERENCES

[1] Uppu, R. M.; Squadrito, G. L.; Pryor, W. A. Acceleration ofperoxynitrite oxidations by carbon dioxide. Arch. Biochem. Bio-phys. 327:335–343; 1996.

[2] Baranano, D. E.; Snyder, S. H. Neural roles for heme oxygenase:contrasts to nitric oxide synthase. Proc. Natl. Acad. Sci. USA98:10996–11002; 2001.

[3] Berlett, B. S.; Levine, R. L.; Stadtman, E. R. Carbon dioxidestimulates peroxynitrite-mediated nitration of tyrosine residuesand inhibits oxidation of methionine residues of glutamine syn-thetase: both modifications mimic effects of adenylylation. Proc.Natl. Acad. Sci. USA 95:2784–2789; 1998.

[4] Smith, M. A.; Sayre, L. M.; Anderson, V. E.; Harris, P. L.; Beal,M. F.; Kowall, N.; Perry, G. Cytochemical demonstration ofoxidative damage in Alzheimer disease by immunochemical en-hancement of the carbonyl reaction with 2,4-dinitrophenylhy-drazine. J. Histochem. Cytochem. 46:731–735; 1998.

[5] Beal, M. F.; Ferrante, R. J.; Browne, S. E.; Matthews, R. T.;Kowall, N. W.; Brown, R. H. Jr. Increased 3-nitrotyrosine in bothsporadic and familial amyotrophic lateral sclerosis. Ann. Neurol.42:646–654; 1997.

[6] Sohal, R. S.; Agarwal, S.; Dubey, A.; Orr, W. C. Protein oxidativedamage is associated with life expectancy of houseflies. Proc.Natl. Acad. Sci. USA 90:7255–7259; 1993.

[7] Sohal, R. S.; Ku, H.-H.; Agarwal, S.; Forster, M. J.; Lal, H.Oxidative damage, mitochondrial oxidant generation and antiox-idant defenses during aging and in response to food restriction inthe mouse. Mech. Ageing Dev. 74:121–133; 1994.

[8] Sohal, R. S.; Agarwal, S.; Sohal, B. H. Oxidative stress and agingin the mongolian gerbil (Meriones unguiculatus). Mech. AgeingDev. 81:15–25; 1995.

[9] Russell, P.; Garland, D.; Zigler, J. S. Jr.; Meakin, S. O.; Tsui,L. C.; Breitman, M. L. Aging effects of vitamin C on a humanlens protein produced in vitro. FASEB J. 1:32–35; 1987.

[10] Pansarasa, O.; Bertorelli, L.; Vecchiet, J.; Felzani, G.; Marzatico,F. Age-dependent changes of antioxidant activities and markers offree radical damage in human skeletal muscle. Free Radic. Biol.Med. 27:617–622; 1999.

[11] Mecocci, P.; Fano, G.; Fulle, S.; MacGarvey, U.; Shinobu, L.;Polidori, M. C.; Cherubini, A.; Vecchiet, J.; Senin, U.; Beal, M. F.Age-dependent increases in oxidative damage to DNA, lipids, andproteins in human skeletal muscle. Free Radic. Biol. Med. 26:303–308; 1999.

[12] Smith, C. D.; Carney, J. M.; Starke-Reed, P. E.; Oliver, C. N.;Stadtman, E. R.; Floyd, R. A.; Markesbery, W. R. Excess brainprotein oxidation and enzyme of dysfunction in normal aging andAlzheimer disease. Proc. Natl. Acad. Sci. USA 88:10540–10543;1991.

[13] Oliver, C. N.; Ahn, B.-W.; Moerman, E. J.; Goldstein, S.; Stadt-man, E. R. Age-related changes in oxidized proteins. J. Biol.Chem. 262:5488–5491; 1987.

[14] Starke, P. E.; Oliver, C. N.; Stadtman, E. R. Modification ofhepatic proteins in rats exposed to high oxygen concentration.FASEB J. 1:36–39; 1987.

[15] Starke-Reed, P. E.; Oliver, C. N. Protein oxidation and proteolysisduring aging and oxidative stress. Arch. Biochem. Biophys. 275:559–567; 1989.

[16] Yan, L. J.; Sohal, R. S. Prevention of flight activity prolongs thelife span of the housefly, Musca domestica, and attenuates theage-associated oxidative damage to specific mitochondrial pro-teins. Free Radic. Biol. Med. 29:1143–1150; 2000.

[17] Moskovitz, J.; Bar-Noy, S.; Williams, W. M.; Requena, J.; Ber-lett, B. S.; Stadtman, E. R. Methionine sulfoxide reductase(MsrA) is a regulator of antioxidant defense and lifespan inmammals. Proc. Natl. Acad. Sci. USA 98:12920–12925; 2001.

[18] Yan, L.-J.; Levine, R. L.; Sohal, R. S. Oxidative damage duringaging targets mitochondrial aconitase. Proc. Natl. Acad. Sci. USA94:11168–11172; 1997.

[19] Yan, L. J.; Sohal, R. S. Mitochondrial adenine nucleotide trans-locase is modified oxidatively during aging. Proc. Natl. Acad. Sci.USA 95:12896–12901; 1998.

[20] Oliver, C. N.; Starke-Reed, P. E.; Stadtman, E. R.; Liu, G. J.;Carney, J. M.; Floyd, R. A. Oxidative damage to brain proteins,loss of glutamine synthetase activity, and production of freeradicals during ischemia/reperfusion-induced injury to gerbilbrain. Proc. Natl. Acad. Sci. USA 87:5144–5147; 1990.

[21] Aulak, K. S.; Miyagi, M.; Yan, L.; West, K. A.; Massillon, D.;Crabb, J. W.; Stuehr, D. J. Proteomic method identifies proteinsnitrated in vivo during inflammatory challenge. Proc. Natl. Acad.Sci. USA 98:12056–12061; 2001.

[22] MacMillan-Crow, L. A.; Thompson, J. A. Tyrosine modificationsand inactivation of active site manganese superoxide dismutasemutant (Y34F) by peroxynitrite. Arch. Biochem. Biophys. 366:82–88; 1999.

[23] Zou, M. H.; Daiber, A.; Peterson, J. A.; Shoun, H.; Ullrich, V.Rapid reactions of peroxynitrite with heme-thiolate proteins as thebasis for protection of prostacyclin synthase from inactivation bynitration. Arch. Biochem. Biophys. 376:149–155; 2000.

[24] Kong, S.-K.; Yim, M. B.; Stadtman, E. R.; Chock, P. B. Per-oxynitrite disables the tyrosine phosphorylation regulatory mech-anism: lymphocyte-specific tyrosine kinase fails to phosphorylatenitrated cdc2(6-20)NH2 peptide. Proc. Natl. Acad. Sci. USA 93:3377–3382; 1996.

[25] Stachowiak, O.; Dolder, M.; Wallimann, T.; Richter, C. Mito-chondrial creatine kinase is a prime target of peroxynitrite-in-duced modification and inactivation. J. Biol. Chem. 273:16694–16699; 1998.

[26] 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. A controlled trial ofselegiline, alpha-tocopherol, or both as treatment for Alzheimer’sdisease. N. Engl. J. Med. 336:1216–1222; 1997.

[27] Hensley, K.; Hall, N.; Subramaniam, R.; Cole, P.; Harris, M.;Aksenov, M.; Aksenova, M.; Gabbita, S. P.; Wu, J. F.; Carney,J. M.; Lovell, M.; Markesbery, W. R.; Butterfield, D. A. Brainregional correspondence between Alzheimer’s disease histopa-thology and biomarkers of protein oxidation. J. Neurochem. 65:2146–2156; 1995.

[28] Lauderback, C. M.; Hackett, J. M.; Huang, F. F.; Keller, J. N.;Szweda, L. I.; Markesbery, W. R.; Butterfield, D. A. The glialglutamate transporter, GLT-1, is oxidatively modified by 4-hy-droxy-2-nonenal in the Alzheimer’s disease brain: the role ofAbeta1- 42. J. Neurochem. 78:413–416; 2001.

[29] Smith, M. A.; Perry, G.; Richey, P. L.; Sayre, L. M.; Anderson,V. E.; Beal, M. F.; Kowall, N. Oxidative damage in Alzheimer’s.Nature 382:120–121; 1996.

[30] Smith, M. A.; Vasak, M.; Knipp, M.; Castellani, R. J.; Perry, G.Dimethylargininase, a nitric oxide regulatory protein, in Alzhei-mer disease. Free Radic. Biol. Med. 25:898–902; 1998.

[31] Smith, M. A.; Rudnicka-Nawrot, M.; Richey, P. L.; Praprotnik,D.; Mulvihill, P.; Miller, C. A.; Sayre, L. M.; Perry, G. Carbonyl-related posttranslational modification of neurofilament protein inthe neurofibrillary pathology of Alzheimer’s disease. J. Neuro-chem. 64:2660–2666; 1995.

[32] Ando, Y.; Brannstrom, T.; Uchida, K.; Nyhlin, N.; Nasman, B.;Suhr, O.; Yamashita, T.; Olsson, T.; El Salhy, M.; Uchino, M.;Ando, M. Histochemical detection of 4-hydroxynonenal proteinin Alzheimer amyloid. J. Neurol. Sci. 156:172–176; 1998.

[33] Calingasan, N. Y.; Uchida, K.; Gibson, G. E. Protein-boundacrolein: a novel marker of oxidative stress in Alzheimer’s dis-ease. J. Neurochem. 72:751–756; 1999.

[34] Guevara, J.; Espinosa, B.; Zenteno, E.; Vazguez, L.; Luna, J.;Perry, G.; Mena, R. Altered glycosylation pattern of proteins inAlzheimer disease. J. Neuropathol. Exp. Neurol. 57:905–914;1998.

[35] Good, P. F.; Werner, P.; Hsu, A.; Olanow, C. W.; Perl, D. P.Evidence for neuronal oxidative damage in Alzheimer’s disease.Am. J. Pathol. 149:21–28; 1996.

[36] Smith, M. A.; Richey Harris, P. L.; Sayre, L. M.; Beckman, J. S.;Perry, G. Widespread peroxynitrite-mediated damage in Alzhei-mer’s disease. J. Neurosci. 17:2653–2657; 1997.

[37] Su, J. H.; Deng, G.; Cotman, C. W. Neuronal DNA damage

801Protein oxidation and neurodegeneration

precedes tangle formation and is associated with up-regulation ofnitrotyrosine in Alzheimer’s disease brain. Brain Res. 774:193–199; 1997.

[38] Vodovotz, Y.; Lucia, M. S.; Flanders, K. C.; Chesler, L.; Xie,Q.-W.; Smith, T. W.; Weidner, J.; Mumford, R.; Webber, R.;Nathan, C.; Roberts, A. B.; Lippa, C. E.; Sporn, M. B. Induciblenitric oxide synthase in tangle-bearing neurons of patients withAlzheimer’s disease. J. Exp. Med. 184:1425–1433; 1996.

[39] Wallace, M. N.; Geddes, J. G.; Farquhar, D. A.; Masson, M. R.Nitric oxide synthase in reactive astrocytes adjacent to beta-amyloid plaques. Exp. Neurol. 144:266–272; 1997.

[40] Hensley, K.; Maidt, M. L.; Yu, Z.; Sang, H.; Markesbery, W. R.;Floyd, R. A. Electrochemical analysis of protein nitrotyrosine anddityrosine in the Alzheimer brain indicates region-specific accu-mulation. J. Neurosci. 18:8126–8132; 1998.

[41] Tohgi, H.; Abe, T.; Yamazaki, K.; Murata, T.; Ishizaki, E.; Isobe,C. Alterations of 3-nitrotyrosine concentration in the cerebrospi-nal fluid during aging and in patients with Alzheimer’s disease.Neurosci. Lett. 269:52–54; 1999.

[42] Aoyama, K.; Matsubara, K.; Fujikawa, Y.; Nagahiro, Y.;Shimizu, K.; Umegae, N.; Hayase, N.; Shiono, H.; Kobayashi, S.Nitration of manganese superoxide dismutase in cerebrospinalfluids is a marker for peroxynitrite-mediated oxidative stress inneurodegenerative diseases [In Process Citation]. Ann. Neurol.47:524–527; 2000.

[43] Alam, Z. I.; Daniel, S. E.; Lees, A. J.; Marsden, D. C.; Jenner, P.;Halliwell, B. A generalised increase in protein carbonyls in thebrain in Parkinson’s but not incidental Lewy body disease. J. Neu-rochem. 69:1326–1329; 1997.

[44] Lyras, L.; Perry, R. H.; Perry, E. K.; Ince, P. G.; Jenner, A.;Jenner, P.; Halliwell, B. Oxidative damage to proteins, lipids, andDNA in cortical brain regions from patients with dementia withLewy bodies. J. Neurochem. 71:302–312; 1998.

[45] Przedborski, S.; Jackon-Lewis, V.; Yokoyama, R.; Shibata, T.;Dawson, V. L.; Dawson, T. M. Role of neuronal nitric oxide in1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induceddopaminergic neurotoxicity. Proc. Natl. Acad. Sci. USA 93:4565–4571; 1996.

[46] Schulz, J. B.; Matthews, R. T.; Muqit, M. M. K.; Browne, S. E.;Beal, M. F. Inhibition of neuronal nitric oxide synthase by 7-ni-troindazole protects against MPTP-induced neurotoxicity in mice.J. Neurochem. 64:936–939; 1995.

[47] Hantraye, P.; Brouillet, E.; Ferrante, R.; Palfi, S.; Dolan, R.;Matthews, R. T.; Beal, M. F. Inhibition of neuronal nitric oxidesynthase prevents MPTP-induced parkinsonism in baboons. Nat.Med. 2:1017–1021; 1996.

[48] Liberatore, G. T.; Jackson-Lewis, V.; Vukosavic, S.; Mandir,A. S.; Vila, M.; McAuliffe, W. G.; Dawson, V. L.; Dawson,T. M.; Przedborski, S. Inducible nitric oxide synthase stimulatesdopaminergic neurodegeneration in the MPTP model of Parkin-son disease. Nat. Med. 5:1403–1409; 1999.

[49] Good, P. F.; Hsu, A.; Werner, P.; Perl, D. P.; Olanow, C. W.Protein nitration in Parkinson’s disease. J. Neuropathol. Exp.Neurol. 57:338–339; 1998.

[50] Shergill, J. K.; Cammack, R.; Cooper, C. E.; Cooper, J. M.; Mann,V. M.; Schapira, A. H. V. Detection of nitrosyl complexes inhuman substantia nigra, in relation to Parkinson’s disease. Bio-chem. Biophys. Res. Commun. 228:298–305; 1996.

[51] Giasson, B. I.; Duda, J. E.; Murray, I. V.; Chen, Q.; Souza, J. M.;Hurtig, H. I.; Ischiropoulos, H.; Trojanowski, J. Q.; Lee, V. M.Oxidative damage linked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions. Science 290:985–989; 2000.

[52] Paxinou, E.; Chen, Q.; Weisse, M.; Giasson, B. I.; Norris, E. H.;Rueter, S. M.; Trojanowski, J. Q.; Lee, V. M.; Ischiropoulos, H.Induction of a-synuclein aggregation by intracellular nitrativeinsult. J. Neurosci. 21:8053–8061; 2001.

[53] Souza, J. M.; Giasson, B. I.; Chen, Q.; Lee, V. M.; Ischiropoulos,H. Dityrosine cross-linking promotes formation of stable alpha-synuclein polymers. Implication of nitrative and oxidative stress

in the pathogenesis of neurodegenerative synucleinopathies.J. Biol. Chem. 275:18344–18349; 2000.

[54] Hsu, L. J.; Sagara, Y.; Arroyo, A.; Rockenstein, E.; Sisk, A.;Mallory, M.; Wong, J.; Takenouchi, T.; Hashimoto, M.; Masliah,E. a-synuclein promotes mitochondrial deficit and oxidativestress. Am. J. Pathol. 157:401–410; 2000.

[55] Castellani, R.; Smith, M. A.; Richey, P. L.; Perry, G. Glycoxida-tion and oxidative stress in Parkinson disease and diffuse Lewybody disease. Brain Res. 737:195–200; 1996.

[56] Schipper, H. M.; Liberman, A.; Stopa, E. G. Neural heme oxy-genase-1 expression in idiopathic Parkinson’s disease. Exp. Neu-rol. 150:60–68; 1998.

[57] Bowling, A. C.; Barkowski, E. E.; McKenna-Yasek, D.; Sapp, P.;Horvitz, H. R.; Beal, M. F.; Brown, R. H. Jr. Superoxide dis-mutase concentration and activity in familial amyotrophic lateralsclerosis. J. Neurochem. 64:2366–2369; 1995.

[58] Ferrante, R. J.; Browne, S. E.; Shinobu, L. A.; Bowling, A. C.;Baik, M. J.; MacGarvey, U.; Kowall, N. W.; Brown, R. H. Jr.;Beal, M. F. Evidence of increased oxidative damage in bothsporadic and familial amyotrophic lateral sclerosis. J. Neurochem.69:2064–2074; 1997.

[59] Shaw, P. J.; Ince, P. G.; Falkous, G.; Mantle, D. Oxidativedamage to protein in sporadic motor neuron disease spinal cord.Ann. Neurol. 38:691–695; 1995.

[60] Lyras, L.; Evans, P. J.; Shaw, P. J.; Ince, P. G.; Halliwell, B.Oxidative damage and motor neurone disease difficulties in themeasurement of protein carbonyls in human brain tissue. FreeRadic. Res. 24:397–406; 1996.

[61] Pedersen, W. A.; Fu, W.; Keller, J. N.; Markesbery, W. R.; Appel,S.; Smith, G.; Kasarskis, E.; Mattson, M. P. Protein modificationby the lipid peroxidation product 4-hydroxynonenal in the spinalcords of amyotrophic lateral sclerosis patients. Ann. Neurol. 44:819–824; 1998.

[62] Tohgi, H.; Abe, T.; Yamazaki, K.; Murata, T.; Ishizaki, E.; Isobe,C. Remarkable increase in cerebrospinal fluid 3-nitrotyrosine inpatients with sporadic amyotrophic lateral sclerosis. Ann. Neurol.46:129–131; 1999.

[63] Wendt, S.; Dedeoglu, A.; Speer, O.; Wallimann, T.; Beal, M. F.Reduced creatine kinase activity in transgenic amyotrophic lateralsclerosis mice. Free Radic. Biol. Med. In press.

[64] Abe, K.; Pan, L.-H.; Watanabe, M.; Kato, T.; Itoyama, Y. Induc-tion of nitrotyrosine-like immunoreactivity in the lower motorneuron of amyotrophic lateral sclerosis. Neurosci. Lett. 199:152–154; 1995.

[65] Abe, K.; Pan, L. H.; Watanabe, M.; Konno, H.; Kato, T.; Itoyama,Y. Upregulation of protein-tyrosine nitration in the anterior horncells of amyotrophic lateral sclerosis. Neurol. Res. 19:124–128;1997.

[66] Chou, S. M.; Wang, H. S.; Komai, K. Colocalization of NOS andSOD1 in neurofilament accumulation within motor neurons ofamyotrophic lateral sclerosis: an immunohistochemical study.J. Chem. Neuroanat. 10:249–258; 1996.

[67] Sasaki, S.; Shibata, N.; Iwata, M. Neuronal nitric oxide synthaseimmunoreactivity in the spinal cord in amyotrophic lateral scle-rosis. Acta Neuropathol. (Berl.) 101:351–357; 2001.

[68] Anneser, J. M.; Cookson, M. R.; Ince, P. G.; Shaw, P. J.; Borasio,G. D. Glial cells of the spinal cord and subcortical white matterup-regulate neuronal nitric oxide synthase in sporadic amyotro-phic lateral sclerosis. Exp. Neurol. 171:418–421; 2001.

[69] Catania, M. V.; Aronica, E.; Yankaya, B.; Troost, D. Increasedexpression of neuronal nitric oxide synthase spliced variants inreactive astrocytes of amyotrophic lateral sclerosis human spinalcord. J. Neurosci. 21:RC148; 2001.

[70] Almer, G.; Guegan, C.; Teismann, P.; Naini, A.; Rosoklija, G.;Hays, A. P.; Chen, C.; Przedborski, S. Increased expression of thepro-inflammatory enzyme cyclooxygenase-2 in amyotrophic lat-eral sclerosis. Ann. Neurol. 49:176–185; 2001.

[71] Alam, Z. I.; Halliwell, B.; Jenner, P. No evidence for increasedoxidative damage to lipids, proteins, or DNA in Huntington’sdisease. J. Neurochem. 75:840–846; 2000.

[72] Browne, S. E.; Bowling, A. C.; MacGarvey, U.; Baik, M. J.;

802 M. F. BEAL

Berger, S. C.; Muqit, M. M. K.; Bird, E. D.; Beal, M. F. Oxidativedamage and metabolic dysfunction in Huntington’s disease: se-lective vulnerability of the basal ganglia. Ann. Neurol. 41:646–653; 1997.

[73] Bogdanov, M. B.; Andreassen, O. A.; Dedeoglu, A.; Ferrante,R. J.; Beal, M. F. Increased oxidative damage to DNA in a

transgenic mouse model of Huntington’s disease. J. Neurochem.79:1–5; 2001.

[74] Tabrizi, S. J.; Workman, J.; Hart, P. E.; Mangiarini, L.; Mahal, A.;Bates, G.; Cooper, J. M.; Schapira, A. H. Mitochondrial dysfunc-tion and free radical damage in the Huntington R6/2 transgenicmouse. Ann. Neurol. 47:80–86; 2000.

803Protein oxidation and neurodegeneration