Oxidation and nitration of α-synuclein and their implications in neurodegenerative diseases

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Archives of Biochemistry and Biophysics xxx (2013) xxx–xxx

YABBI 6393 No. of Pages 8, Model 5G

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Archives of Biochemistry and Biophysics

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Oxidation and nitration of a-synuclein and their implicationsin neurodegenerative diseases

Cecilia Chavarría, José M. Souza ⇑Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, 11800Montevideo, Uruguay

a r t i c l e i n f o a b s t r a c t

2324252627282930313233

Article history:Received 11 October 2012and in revised form 7 February 2013Available online xxxx

Keywords:a-SynucleinMethionine sulfoxide3-NitrotyrosineLipid adductSynucleinopathies

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0003-9861/$ - see front matter � 2013 Published byhttp://dx.doi.org/10.1016/j.abb.2013.02.009

⇑ Corresponding author. Fax: +598 2924 9563.E-mail address: [email protected] (J.M. Souza).

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Synucleinopathies include Parkinson’s disease, dementia with Lewy bodies, Lewy body variant of Alzhei-mer’s disease and multiple system atrophy, among the most relevant diseases. All of these diseases arecharacterized by the presence of amyloid inclusions in neurons, which are rich in the aggregate a-synuc-lein protein. What is the biological mechanism concerned in the gain-of-function that implicates the par-ticipation of a-synuclein in these diseases? Post-translational modifications of a-synuclein induced bynitroxidative stress are a relevant hypothesis that may explain many of the experimental data. We willreview the biophysical and biochemical properties of a-synuclein, methionine residues oxidation, nitra-tion and oxidation of tyrosine residues in a-synuclein, and modifications of a-synuclein mediated by pro-teins and lipids under nitroxidative stress conditions. The biological consequences of these modificationsare analyzed in terms of the properties of a-synuclein oligomerization and fibrillation, degradation of a-synuclein and the implications in the immunological response.

� 2013 Published by Elsevier Inc.

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Introduction

The importance of the study of the biochemical and biophysicalproperties of a-synuclein (a-syn) derives from its connection witha number of diseases denominated synucleinopathies. The first linkappears when a 35-amino acid peptide was isolated from plaquesamples from patients with Alzheimer’s disease (AD). This peptidewas named the non-amyloid component (initially abbreviated asNAC), and was demonstrated that this peptide belongs to a largerprotein, which was later identified as a-syn [1]. The non-amyloidcomponent region corresponds to the hydrophobic core of thea-syn protein (Fig. 1). The second link was the discovery, in kin-dred with an autosomal dominant Parkinson’s disease (PD), of amissense mutation in the a-syn gene producing the amino acidsubstitution A53T in the protein [2]. Other a-syn gene alterationsare associated with diseases, autosomal dominant missense muta-tions A30T and E46K (Fig. 1) and trisomies of wild type a-syn gene,all causing PD or dementia with Lewy bodies (DLB) [3–5]. Synuc-leinopathies are characterized histopathologically by amyloidinclusions such as Lewy bodies and Lewy neurites in PD, dementiawith Lewy bodies and Lewy body variant of AD; and glial and neu-ronal cytoplasmatic inclusions in multiple system atrophy (MSA).Every one of these inclusions has a-syn as a major component,

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J.M. Souza, Arch. Biochem. Bio

independently of the familiar or sporadic clinical presentation[6–9].

a-Synuclein is a 140 amino acid protein with four tyrosine res-idues, four methionine residues and one residue of histidine, de-prived of cysteine as well as tryptophan residues (Fig. 1). Theprimary sequence divides the protein in three regions: the N-ter-minal region (amino acid 1–60), the central region (61–95) andthe C-terminal region (96–140) (Fig. 1). The N-terminal region isextremely conserved between the synuclein family proteins andshows a variable number of the KTKEGV repeats, also found inthe lipoproteins group. This region adopts an amphipathic a-helixstructure when the protein binds to phospholipids [10]. The centralregion is a hydrophobic zone that corresponds to the non-amyloidcomponent and holds the amyloidogenic properties of the proteinwhich allows a-syn to form fibrils, rich in b-sheets structures. TheC-terminal region is an acidic segment rich in glutamic, asparticand proline residues. This region determines the acidic isoelectricpoint of the a-syn (pI = 4.7) [11]. Observing the primary sequence,it becomes evident that all the missense mutations diseases-asso-ciated, reside in the N-terminal region. Others critical sites, such asmethionine and tyrosine residues are distributed in the regionsN- or C-terminal, not in the central region of a-syn (Fig. 1). Untilrecently, a-syn was valued as a heat-stable protein, with a mono-meric natively unfolded structure [12]. Although it is a random coilprotein, this monomer assumes different conformational struc-tures that hinder the amyloidogenic region. There are long-rangeinteractions between both the N- and C-terminal with the non-

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Fig. 1. Features of the primary sequence of a-synuclein. The amphiphatic N-terminal region is marked in blue (amino acids 1–60), the hydrophobic NAC (non-amyloidcomponent) region is indicated in green (amino acids 61–94) and the C-terminal region is in red (amino acids 96–140). The site of missense mutations associated with earlyonset of Parkinson’s disease is indicated with a blue arrow. Methionine, tyrosine and histidine, amino acids associated with nitroxidative modifications are highlighted inbold. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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amyloid component region (NAC). These interactions avoid NAC–NAC contact between different monomers, which is the basis forthe oligomerization and aggregation of a-syn. This is an intrinsicautoinhibitory mechanism that should be disrupted in order to fa-vor a-syn oligomers and fibrils formation [13]. The native-un-folded paradigm was disputed by the work of Selkoe’s laboratory,which showed that a-syn in its native state is a folded tetramer,rich in a-helix, with greater lipid-binding affinity than the mono-mer and resistant to fibril formation. In addition this tetramerica-syn is N-acetylated in vivo, at least in human erythrocytes wheremost of the characterization was done [14]. Soon, this work waschallenged and laboratories from different places concluded thata-syn is a native monomeric and unstructured protein in erythro-cytes and in the nervous system [15]. If a-syn is a native tetramerthen numerous questions arise: what is the structure of this pro-tein? Is the rate of dissociation of the tetramer too slow to reachequilibrium with the monomeric forms? And how could the mono-meric species form from the tetramer in the pathway tooligomerization?

a-Syn is a particularly abundant presynaptic protein, expressedin the neocortex, hippocampus, striatum, thalamus, and cerebel-lum [16]. The function of a-syn is not completely recognized. Thelocation and some experimental data suggest an association withpresinaptic vesicles [17]. The knockout mice for a-syn are quitenormal, but show an increase of dopamine release in response toelectrical stimuli [18]. a-Syn is upregulated in songbirds, duringthe periods of song-learning, this is a function related with synap-tic plasticity [19]. Furthermore, a chaperone-like activity and amodulation activity of phospholipases, have been also shown fora-syn [20–23]. The binding of a-syn at the Notch1 promoter, a fac-tor involved in adult neurogenesis, has been recently demon-strated. P53 and a-syn interact together to repress the Notch1transcription and consequently may produce abnormal neuronaldifferentiation [24].

This review will focus on nitroxidative modifications of a-syn aspost-translational modifications induced by an increase in oxygenand nitrogen reactive species. We analyze the conditions in orderto observe these modifications and their functional consequences,which may explain the development of synucleinopathies.

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Nitroxidative modifications of a-synuclein

a-Synuclein methionine oxidation

Each and every amino acid in proteins are susceptible to oxida-tion, but their reactivity differs depending on the nature of the

Please cite this article in press as: C. Chavarría, J.M. Souza, Arch. Biochem. Bio

amino acid and the location in the protein structure [25]. Methio-nine (Met) and cysteine are the most readily oxidized amino acids,however in a-syn there are no cysteine residues, hence methio-nines and tyrosines are the main amino acids susceptible for oxida-tion (Figs. 1 and 2). Methionine is easily oxidized to methioninesulfoxide by different oxidizing agents which are produced in bio-logical systems, such as: hydrogen peroxide, hypochlorite, chlor-amines and peroxynitrite (Fig. 2) [26]. The particularcharacteristic of this oxidative modification is that it is reversiblein the sense that it can be reduced back to the native amino acidunder some specific circumstances. The oxidation of methioninecreates another asymmetric center, named Met-R(O) and Met-S(O), and these sulfoxides can be reduced back to methionine bymethionine sulfoxide reductases (Msr) [27,28]. The Msr are en-zymes that catalyze the reduction of methionine sulfoxide basedon the thioredoxin system. The MsrA enzyme is specific for theMet-S(O) enantiomer and the MsrB enzymes are specific for theMet-R(O) enantiomer [29]. The reversible oxidation of methionineto methionine sulfoxide occurs under physiological conditions, butalso another methionine modification can occur under some cir-cumstances. The irreversible oxidation to methionine sulfone israre and only takes place in the presence of strong oxidants(Fig. 2) [30]. This irreversible oxidation is often associated withpathophysiological conditions and results in functional impair-ment of the oxidized proteins [30,31]. In the brains of patients withidiopathic PD and AD, the methionine residues were irreversiblyoxidized to methionine sulfone in the DJ-1 protein [32].

Human a-syn has four methionine residues. Met 1 and 5, situ-ated in the N-terminal and Met 116 and 127 located in the C-termi-nal region of the protein (Fig 1). Incubating a-syn protein with avery high concentration of H2O2 (4% H2O2 for 20 min), leads tothe oxidation of the four methionine residues to methionine sulf-oxide and no other modification appears due to this treatment[33]. Using the thioflavin T fluorescent assay, it was demonstratedthat methionine-oxidized a-syn revealed no evidence of fibril for-mation when it was incubated [33]. A mixture between methio-nine-oxidized a-syn and non-oxidized a-syn in a 1:2 relationgenerates an increase in the lag time, while using a relation ratioof 1:4 completely inhibits fibrillization [33]. According to Fink’swork, it is believed that the inhibition of fibril formation by non-oxidized a-syn in the presence of the methionine-oxidized proteinoriginates the formation of soluble hetero-oligomers. These het-ero-oligomers are located off the fibrillization pathway [29]. Stud-ies with mutants containing a methionine to leucine substitutionwere created and the degree of inhibition of fibrillization by methi-onine-oxidized a-syn is proportional to the number of oxidized



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Fig. 2. Oxidative and nitrative modifications of tyrosine and methionine in a-syn.(a) 3-Nitrotyrosine is form due to the action of different nitrating systems. It isformed by peroxinitrite (ONOO�) in the presence of carbon dioxide (CO2) and alsoby the system formed by the myeloperoxidase (MPO) in the presence of nitrite(NO�2 ) and hydrogen peroxide (H2O2). 3-Hydroxytyrosine is a product of oxidationof tyrosine, and the formation of 3,3-dityrosine occurs due to the recombination oftwo tyrosil radicals to form this product of dimerization. (b) Methionine is easilyoxidized to methionine sulfoxide by different oxidizing agents, such as peroxinitrite(ONOO�), hydrogen peroxide (H2O2), hydroxyl radical (OH), carbonate radical (CO�3 )and hypochlorite (ClO�). The oxidation to methionine sulfoxide is reversible and itcan be reduced back to methionine by the action of methionine sulfoxidereductases (Msr). The reduction of methionine sulfoxide is based on the thioredoxinsystem. Trx: thioredoxin. Trx-(S)2: thioredoxin oxidized form. Trx-(SH)2: thiore-doxin reduced form. TR: thioredoxin reductase. In the presence of strong oxidants(e.g., chloramines) the irreversible oxidation of methionine to methionine sulfoneoccurs.

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methionines [34]. To understand the possible role of the oxidationof a-syn methionine and the pathogenesis of PD it can be assumedthat if methionine-oxidized a-syn stabilizes soluble oligomers off-pathway. This could lead to the accumulation of hetero-oligomersconstituted by a-syn and oxidized-a-syn. Furthermore, as it wasdemonstrated by numerous authors [35–41], the toxicity of a-syn oligomers could have a toxic impact on cellular environment[33].

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a-Synuclein tyrosine oxidation and nitration

It is well accepted that protein nitration is a post-translationalmodification in proteins and is a biomarker associated with oxida-


tive damage [42]. Replacement of a hydrogen atom in the 30 posi-tion of the tyrosine phenolic ring by a nitro group may happenunder multiple NO-dependent mechanisms (Fig. 2) [43]. A keyevent is the abstraction of one electron from the tyrosine residueto form a tyrosyl radical. This could be mediated by hydroxyl rad-ical, carbonate radical, oxo-metal center and NO2 radical, whichcome from peroxynitrite or heme peroxidase/H2O2/nitrite, andthen, the recombination of tyrosyl radical with NO2 radical to form3-nitrotyrosine. The presence of the intermediary tyrosyl radical,also explains the formation of 3,30-dityrosine due to recombinationof tyrosyl radicals promoted by nitrating agents such as peroxyni-trite and nitrogen dioxide (Fig. 2) [44–46].

a-Syn has four tyrosine residues in positions 39 (at the N-termi-nal region), 125, 133 and 136 (at the C-terminal region), all suscep-tible to be nitrated (Fig 1). a-Syn is extremely susceptible tonitrating agents, being nitrated even under substoichiometric con-centration of peroxynitrite. In addition to induce tyrosine nitration,peroxynitrite also induces 3,30-dityrosine formation via the oxida-tion of tyrosine residues, with the concomitant dimers and oligo-mer formation (Fig. 3) [47]. Pre-formed a-syn filaments treatedwith peroxynitrite resist denaturing condition such as urea orSDS [47], similar as in Lewy bodies purified from patients withPD and DLB [8]. In these diseases a-syn is found nitrated [48].The role of tyrosine nitration and oxidation in a-syn was disputedin favor of methionine oxidation, although there are no data yetshowing specific formation of a-syn methionine sulfoxide or sul-fone in synucleinopathies [29,49]. Nevertheless, in vivo it wasestablished that tyrosine residues are essential for a-syn aggrega-tion under oxidative stress conditions [50,51]. Nitration of Tyr-39leads to a high rate of oligomerization. Besides, mutation in thisresidue conduces to high levels of fibrilization [52]. Through stud-ies with mutants in Tyr-125 it was shown that the presence of Tyr-125 contributes to a-syn dimer formation after exposing the re-combinant protein to nitrating agents [53]. Nitrated a-syn mono-mers, dimers and oligomers were purified and assayed for fibrilformation. Monomeric or dimeric nitrated a-syn accelerates therate of fibril formation and also can seed and promote the fibrilla-tion of non-modified a-syn. On the other hand, nitrated a-syn olig-omers inhibit the fibril formation [54]. The yield of nitratedproteins in biological samples are usually low [55], hence the for-mation of low levels of monomeric nitrated a-syn and an excess ofunmodified a-syn is expected, at least at the beginning of a longasymptomatic period. Therefore, these monomeric or dimeric ni-trated a-syn forms may start the seeding for the formation of olig-omers and fibrils of unmodified a-syn that ultimately will developinto Lewy bodies or neurites in a long period of time. Immunohis-tochemical studies using monoclonal antibodies against nitrateda-syn revealed robust and abundant staining of numerous Lewybodies, Lewy neurites, glial cell inclusions, and neuroaxonal spher-oids in brains from diverse types of synucleinopathies [48].

Metal-mediated a-synuclein oxidation

Iron deposits have been identified in Lewy bodies in the sub-stantia nigra [56], and elevated copper concentrations have beenreported in the cerebrospinal fluid of PD patients [57]. Several epi-demiologic studies connect the presence of high levels of metalswith the occurrence of PD [58,59].

Elevated Mn levels in the brain have also been associated withimpairment in iron homeostasis, excitotoxicity, mitochondrial dys-function, oxidative stress, induction of protein aggregation, as wellas alteration in the homeostatic condition of other divalent metalsthat share similar transporter systems with Mn [60]. For these rea-sons, abnormal metal-protein interactions have emerged as animportant potential mechanism in the pathophysiological eventsof PD.



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Fig. 3. Functional consequences of post-translational modifications in a-synuclein. The different a-syn conformations are described as: blue ball, random coil monomericform; blue square, a-helix rich forms (tetrameric and membrane-bound); b-sheet rich forms; NO�2 tyrosine nitrated a-syn; 3,30dityrosine crosslinked a-syn. Th17 isa subset of T helper lymphocytes that produce interleukin 17A and APC correspond to antigen-presenting cells. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)



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Metal-catalyzed oxidation may result in structural damage toproteins and has been implicated in aging and diseases, includingneurological disorders such as Alzheimer’s disease and amyotro-phic lateral sclerosis [61].

Particularly, a-syn is a protein susceptible to copper-catalyzedoxidation. Consequently, an extensive oligomerization and pre-cipitation of the protein has been observed in the presence ofcopper [61,62]. The reaction of copper-oxidation involves thereduction of Cu(II) by an electron donor and the conversion ofmolecular oxygen into reactive oxygen species which are respon-sible for inducing the oxidative modifications of the protein. Thismechanism is highly selective, it is a site-specific process, and in-volves the interaction of the protein with both oxidation states ofthe copper ion [63]. Cu(II) binds specifically to a-syn and is effec-tive in accelerating aggregation at physiologically relevant con-centrations. The a-syn Cu(I) complex can be reoxidized byoxygen, producing H2O2 as an agent that causes toxicity and neu-ronal cell death [63]. When recombinant a-syn was incubatedovernight at 37 �C with 10 lM Cu(II) or Fe(III) in the presenceof 300 lM H2O2, minimal oxidation of a-syn was observed. How-ever, in the presence of a reducing agent such as DTT, an increasein a-syn b-sheet rich oligomers was generated in the presence ofcopper and especially with iron. This correlates with an inhibitionin fibril formation [64].


The presence of two binding sites for Cu(II) at the N-terminus ofa-syn has been described. A high affinity binding site and a loweraffinity binding site for Cu(II) with a Kd of 0.20 and 50 lM, respec-tively [63,65–67]. In amyloid disorders, such as AD and prion dis-ease, copper binds to these proteins stoichiometrically and witha Kd in the submicromolar range, similar to a-syn [68]. The bindingof Cu(II) at the N-terminal region is recognized as a key event trig-gering the a-syn aggregation, although the impact of these struc-tural-affinity differences in this region, has not been elucidated[63]. The metals bind the C-terminal region with much lower affin-ities, Kd in the millimolar range, outside of the physiopathologicalrange expected [69]. Hence, a-syn should compete with other li-gands in order to be able to bind copper. This situation may takeplace and favor the a-syn-Cu(II) complexes formation, only whena perturbation of copper metabolism occurs, as may happen inWilson’s disease [70].

Manganese and other essential and toxic metals can increase di-rectly fibril formation by a-syn [71]. Mn also raises the expressionof a-syn in vitro [72,73] and chronic exposure leads to a-syn aggre-gation in vivo in neurons and glial cells of non-human primates[74]. One possible mechanism that could be supporting this factis that high concentration of manganese can induce oxidativestress [33]. The overexpression of a-syn can induce apoptosis sub-sequent to oxidative stress [30,73].



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As a general consequence of the oxidation process there is a fail-ure to degrade oxidized a-syn by the proteasome which underliesthe potential pathological consequences of the metal-catalyzedoxidation of a-syn [64], this issue is discussed later.

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Protein-mediated a-synuclein oxidation

Nitration and oxidation of a-syn can be mediated by proteins,as was shown by myeloperoxidase in the presence of H2O2 and ni-trite [47].1 Cytochrome c was immune-colocalized with a-syn inLewy bodies of patients with PD and others synucleinopathies[77]. In vitro experiments show that cytochrome c-H2O2 catalyzesa-syn crosslinking via 3,30-dityrosine formation, being tyrosines133 and 136 the more reactive tyrosine residues [78]. Similar resultswere obtained using the cytchrome c-cardiolipin-H2O2 system,where a crosslinking between cytchrome c and a-syn involving tyro-sine 74 from cytchrome c and 133 from a-syn was also found [79].Cytochrome c is not a true peroxidase but when Met80 is displacedits 6th coordination position with the iron heme by alkaline pH, car-diolipin binding, methionine oxidation or tyrosine nitration, in-creases its peroxidase activity [80–83]. Also cytochrome c has abasic pI (10.2) which may favor its interaction with the acidic a-syn. In what scenario may cytochrome c and a-syn interact? Cyto-chrome c is located in the intermembrane mitochondria space andcan be released to cytosol by proapoptotic signals. a-Syn is mainlya cytosolic protein but a fraction is associated with mitochondrialcomplex I due to a cryptic mitochondrial-targeted signal in its se-quence [84]. Using HeLa-containing a-syn cells and SH-SY5Y cellsstimulated with a proapoptotic stimulus (actinomycin D) or a pro-oxidant stimulus (tert-butyl hydroperoxide) it is possible to findcolocalization and aggregation of cytochrome c and a-syn. In addi-tion, under similar conditions there is a decrease in the apoptoticrate in the presence of a-syn, suggesting an anti-apoptotic role ofthe complex cytochrome c-cardiolipin-a-syn [79].

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Lipid-adduct formation in a-synuclein

Reactive oxygen and nitrogen species can initiate lipid peroxi-dation of membrane polyunsaturated fatty acids. Being a fractionof a-syn associated with membranes, is positioned to be a goodtarget for reactive lipid-peroxidation products. Association of a-syn with membranes protects the protein from tyrosine oxidationand nitration by peroxynitrite, although it can also promote dif-ferent a-syn-lipid adduct formation, inducing novels post-transla-tional a-syn modifications (Fig. 3) [85]. Incubation of a-syn with4-hydroxy-2-nonenal (HNE), an important aldehyde producedduring lipoperoxidation, induces covalent modification of the pro-tein, generating up to six HNE-adducted per a-syn [86]. However,at low and biological relevant HNE/protein ratio, only His-50 wasmodified through Michael addition product [85,86]. HNE-ad-ducted a-syn shows an increase in b-sheet structures and induceinhibition of fibrillation with the concomitant formation of solu-ble a-syn oligomers [86]. Through the use of tyrosine analog-membrane bound was possible to estimate the reaction rate be-tween lipid peroxyl radicals with the phenol moiety. Producinga phenoxyl radical which may form a Diels–Alder adducts[87,88]. The residue tyrosine-39 of a-syn is the most probable sitefor inducing this type of lipid-protein adduct, since the N-terminalregion form an amphipathic a-helix when the protein interactwith membranes.

1 There is evidence that myeloperoxidase is expressed in microglia in neurode-generative diseases and also that myeloperoxidase is involved in MPTP toxicity inmice [75,76].


Functional consequences of a-synuclein nitroxidativemodifications

Amyloidogenic properties of a-synuclein

The transformation of amyloidogenic proteins from the mono-meric state into fibrillar aggregates seems to progress via differentintermediates called protofibrils, protofilaments or oligomers, allthese structures are rich in b-sheets (Fig. 3) [89–91]. Fibrillationof a-syn follows a nucleation-dependent process involving majorstructural rearrangement which leads to the formation of a-syn fi-brils [92]. Prior to the formation of the fibril, several non-fibrillaroligomeric aggregates, or protofibrils, were identified [37]. Thespherical protofibrils are thought to undergo head-to-tail associa-tions to form elongated chain-like [93], and ring-like protofibrillarspecies (Fig. 3) [94].

a-Syn fibril formation occurs through discrete oligomeric inter-mediates that disappear upon fibril formation. Evidence has shownthat these oligomeric intermediates are the pathogenic speciesresponsible for a-syn toxicity [35–41]. The presence of a-syndeposits in the brains of ‘‘symptomatic’’ transgenic mice are non-fibrillar [40] and it was shown that neurons containing Lewybodies appear to be better than the neighboring neurons. Thesedata, support the hypothesis that a-syn oligomers are the toxicsspecies underlying the molecular basis of synucleinopathies [95–97]. Therefore, cellular processes that lead to either increased for-mation of dimers and/or oligomers or, alternatively, decreasedclearance of these species, may be associated with a-syn mediatedtoxicity [97].

The shape and molecular weight of a-syn intermediates thatparticipate in the fibrillization process was characterized usingelectron and atomic force microscopy [98]. These protofibrillarintermediates are structurally heterogeneous and it was describedthat protofibrillar intermediates exist with annular, tubular andrectangular structures [99]. These protofibrills are consumed dur-ing the fibril formation process because they act as seeds on thea-syn fibril formation pathway [98]. Several factors could modu-late the formation and stability of the a-syn oligomeric intermedi-ates, including mutations, post-translational modifications andinteraction with related proteins. The interaction with phospholip-ids and polyunsaturated fatty acids in the membrane also plays animportant role in the process of a-syn oligomerization [50,100–103]. As mentioned above, nitroxidative modifications of a-synmay have a role in induction of oligomer and/or fibril formationof a-syn (Fig. 3).

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Membrane pore formations

A toxic role for the oligomeric a-syn intermediates was pro-posed, though the exact mechanism by which these intermediateslead to cell toxicity is still unclear. It is worth to mention that olig-omers formed from several proteins associated with neurodegen-erative disorders (such as Ab-peptide, prion protein, a-syn) sharesimilar features and all form pore-like structures with membranepermeabilization under certain circumstances. This could be a gen-eral mechanism of generating toxicity for amyloidogenic proteins.

a-Syn oligomers can bind tightly to phosphatidylglycerol vesi-cles via a b-sheet like structure, consequently this binding resultsin membrane permeation [98]. Permeabilization may occur as a re-sult of membrane perturbation via the formation of a pore-likestructure of about 2.5 nm in diameter. Annular and tubular proto-fibrillar a-syn intermediates are consistent with pore-like struc-tures of the same size [104]. Furthermore, membrane poreformation capacity is specific for oligomers and was practicallynot observed when monomeric and fibrillar a-syn were analyzed.



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To demonstrate that oligomers are the key intermediates responsi-ble for pore formation, an antibody that specifically reacts witholigomers was used. In the presence of this antibody, the processof pore formation was inhibited [105]. Immersion in the mem-brane of the a-syn mutant A53T occurs more rapidly in compari-son with the wild type a-syn [106].

Using a model of a planar lipid bilayer and measuring the distri-bution of conductance states obtained in different fusion events, itwas shown that these pores were permanently open (Fig. 3) [105].The formation of pore-like structures might result in abnormal cal-cium influx contributing to neurodegeneration [107–110]. An in-crease of cytosolic calcium may specifically activate constitutiveforms of nitric oxide synthase and hence the increased productionof nitric oxide. Nitric oxide may also increase superoxide produc-tion due to mitochondrial respiratory chain inhibition. This mech-anism could promote nitration and oxidation reactions [111]. Inregards with the stoichiometries required for a-syn forming mem-brane pores, it was demonstrated that complete coating of themembrane vesicle is not required for permeabilization [104]. Lipidadduct formation between a-syn and lipid peroxidation productsmay help to stabilize membrane pore structures produced by a-syn.

Proteasome and lysosome degradation of nitroxidated a-synuclein

Lewy bodies in PD and synucleinopathies are recognized byanti-ubiquitin antibodies, establishing a relationship betweenintracellular proteins degradation and synucleinopathies[112,113]. An E3 ubiquitin ligase, parkin, shows mutations associ-ated with some juvenile PD, demonstrating that the proteasomepathway could be implicated in the development of PD [114]. Ni-trated monomers of a-syn are more slowly degraded by the 20Sproteasome than the unmodified protein. Degradation by calpainI, a calcium-dependent-non-lysosomal protease, of nitrated mono-mers of a-syn also occurs at a slower rate compared with control[54]. Aggregated proteins also are eliminated by autophagy, form-ing an autophagolysosome where their content is degraded[115,116]. Oxidation and nitration of a-syn inhibit the degradationby the autophagy pathway [117]. It was demonstrated, that oxi-dized and nitrated proteins were degraded faster by the protea-some than the unmodified proteins [118–120]. In the case ofnitroxidative-modified a-syn, the data support the opposite behav-ior. Modified a-syn is resistant to the degradation and so, thesespecies may have an intracellular increase in half-life and concen-tration, which may promote oligomerization and toxicity by a-syn(Fig. 3).

Immunological response induced by nitrated a-synuclein

Protein post-translational modifications can induce an immu-nological response, it is a common method to detect nitratedprotein on western blots or in tissue by using antibodies anti-3-nitrotyrosine [121]. In addition, nitrated-peptides can induceCD4+T response by the antigen-presenting cells during the antigenprocessing [122]. In an experimental model of PD using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin,it is possible to detect nitrated a-syn in mice treated with MPTP[123]. Gendelman’s group have shown elegantly that nitrated a-syn elicit an immunological response in mice that exacerbate MPTPtoxicity. The presence of nitrated a-syn in the cervical lymph nodesof MPTP-treated mice was demonstrated [124]. Immunization ofmice with nitrated a-syn activates a subset of CD4+T cells, Th17that produces IL-17A and attenuates CD4+CD25+ regulatory T cells[125,126]. As a consequence, there is an increase microglia activa-tion with the concomitant production of pro-inflammatory cyto-kines and an increase production of reactive oxygen and nitrogen


species (Fig. 3)[125]. Production of endogenous anti-3-nitrotyro-sine antibodies, were detected in plasma of patients with acutelung injury. By this way, exhibiting an autoimmunological re-sponse against endogenous nitrated proteins during an acute dis-ease [127]. In the case of a-syn, small amount of nitrated a-synmay start an autoimmunological response, mediated by Th17 cells,which exacerbates microglia activation with induction of addi-tional tyrosine nitration of a-syn, creating a positive feedback loop(Fig. 3). This hypothesis accords with the attenuation of the toxic-ity induced by MPTP in knockout mice for the inducible nitric oxidesynthase (iNOS) [128] and the increases in a-syn nitration andaggregation associate with the overexpression of iNOS [129].

Final remarks

During the aging process there is a decrease in the numbers ofneurons in certain brain regions; strikingly this process happens inthe substantia nigra of midbrain. However, this natural processdoes not induce symptoms. In a group of patients, there is a steeperdecrease of dopaminergic neurons, and when a threshold isreached the typical extrapyramidal symptoms appears, such as inPD [130]. Most patients with PD are described as idiopathic formsof the disease, meaning no specific cause. There are some factorsassociated with PD, including genetic causes, toxins and head trau-ma [131] (http://en.wikipedia.org/wiki/Parkinson%27s_disease).The list of toxins include: paraquat, rotenone, MPTP, manganese,mercury, copper, carbon monoxide, cyanide, carbon sulfide, tolu-ene and N-hexane. Many of these toxins are related with mito-chondrial damage and production of oxygen- and nitrogen-reactive species. There is an overlapping between PD, synucleinop-athies such as DLB and Alzheimer’s disease. Alzheimer’s diseasepatients may develop extrapyramidal signs during their sicknessand patients with PD may develop a dementia in the later stagesof the disease. Dementia with Lewy bodies patients show progres-sive dementia, extrapyramidal signs and visual hallucinationsamong its main symptoms [132]. Beneath this overlapping be-tween different diseases, a common pathophysiological mecha-nism may underlay.

The inheritable synucleinopathies associated with missensemutations in a-syn gene and the abundant presence of a-syn inthe Lewy bodies in sporadic- or genetic-associated synucleinopa-thies, strongly supports a gain-of-function of a-syn and a key rolein these diseases. Nitrated monomeric and/or dimeric a-syn aregood candidates to begin the seeding of the aggregate forms ofa-syn. However, the ill-defined oligomeric forms are probablythe proximal toxic species. Oxidative modified-a-syn, such asmethionine sulfoxide or sulfone or lipid-adducted species, maystabilize the oligomeric and toxic forms of a-syn, although exper-imental data are now scarce. Nitroxidative modified-a-syn, mainlynitrated a-syn, may be responsible for the initial and continuousproduction of nitric oxide and superoxide, and so peroxynitritegeneration, by microglia activation in the midbrain. Nitrated a-syn processed by the antigen-presenting cells may, in certain pa-tients, select a group of T lymphocytes (Th17) which suppressthe activity of regulatory T cells and exacerbate microglia activa-tion. PD and other synucleinopathies behave as chronic inflamma-tory diseases, nourishing by a positive loop of nitrated a-syn, Th17activation, microglia activation and more nitrated a-syn.

Acknowledgements

We thank Dr. Rafael Radi for a critical reading and comments.We acknowledge Mrs. Mariana Souza Sapiro for her careful reviewof the manuscript.


http://en.wikipedia.org/wiki/Parkinson%27s_disease


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This work was supported by grants Agencia Nacional de Inves-tigación e Innovación - Fondo Clemente Estable to JMS and a schol-arship to CC by Agencia Nacional de Investigación e Innovación,and also by Programa de Desarrollo de las Ciencias Básicas(PEDECIBA).

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References

[1] K. Ueda, H. Fukushima, E. Masliah, Y. Xia, A. Iwai, M. Yoshimoto, D.A. Otero, J.Kondo, Y. Ihara, T. Saitoh, Proc. Natl. Acad. Sci. USA 90 (1993) 11282–11286.

[2] M.H. Polymeropoulos, C. Lavedan, E. Leroy, S.E. Ide, A. Dehejia, A. Dutra, B.Pike, H. Root, J. Rubenstein, R. Boyer, E.S. Stenroos, S. Chandrasekharappa, A.Athanassiadou, T. Papapetropoulos, W.G. Johnson, A.M. Lazzarini, R.C.Duvoisin, G. Di Iorio, L.I. Golbe, R.L. Nussbaum, Science 276 (1997) 2045–2047.

[3] R. Kruger, W. Kuhn, T. Muller, D. Woitalla, M. Graeber, S. Kosel, H. Przuntek,J.T. Epplen, L. Schols, O. Riess, Nat. Genet. 18 (1998) 106–108.

[4] J.J. Zarranz, J. Alegre, J.C. Gomez-Esteban, E. Lezcano, R. Ros, I. Ampuero, L.Vidal, J. Hoenicka, O. Rodriguez, B. Atares, V. Llorens, E. Gomez Tortosa, T. delSer, D.G. Munoz, J.G. de Yebenes, Ann. Neurol. 55 (2004) 164–173.

[5] A.B. Singleton, M. Farrer, J. Johnson, A. Singleton, S. Hague, J. Kachergus, M.Hulihan, T. Peuralinna, A. Dutra, R. Nussbaum, S. Lincoln, A. Crawley, M.Hanson, D. Maraganore, C. Adler, M.R. Cookson, M. Muenter, M. Baptista, D.Miller, J. Blancato, J. Hardy, K. Gwinn-Hardy, Science 302 (2003) 841.

[6] M.G. Spillantini, R.A. Crowther, R. Jakes, M. Hasegawa, M. Goedert, Proc. Natl.Acad. Sci. USA 95 (1998) 6469–6473.

[7] M.G. Spillantini, M.L. Schmidt, V.M. Lee, J.Q. Trojanowski, R. Jakes, M. Goedert,Nature 388 (1997) 839–840.

[8] M. Baba, S. Nakajo, P.H. Tu, T. Tomita, K. Nakaya, V.M. Lee, J.Q. Trojanowski, T.Iwatsubo, Am. J. Pathol. 152 (1998) 879–884.

[9] M.G. Spillantini, R.A. Crowther, R. Jakes, N.J. Cairns, P.L. Lantos, M. Goedert,Neurosci. Lett. 251 (1998) 205–208.

[10] W.S. Davidson, A. Jonas, D.F. Clayton, J.M. George, J. Biol. Chem. 273 (1998)9443–9449.

[11] W. Hoyer, T. Antony, D. Cherny, G. Heim, T.M. Jovin, V. Subramaniam, J. Mol.Biol. 322 (2002) 383–393.

[12] P.H. Weinreb, W. Zhen, A.W. Poon, K.A. Conway, P.T. Lansbury Jr.,Biochemistry 35 (1996) 13709–13715.

[13] C.W. Bertoncini, Y.S. Jung, C.O. Fernandez, W. Hoyer, C. Griesinger, T.M. Jovin,M. Zweckstetter, Proc. Natl. Acad. Sci. USA 102 (2005) 1430–1435.

[14] T. Bartels, J.G. Choi, D.J. Selkoe, Nature 477 (2011) 107–110.[15] B. Fauvet, M.K. Mbefo, M.B. Fares, C. Desobry, S. Michael, M.T. Ardah, E. Tsika,

P. Coune, M. Prudent, N. Lion, D. Eliezer, D.J. Moore, B. Schneider, P. Aebischer,O.M. El-Agnaf, E. Masliah, H.A. Lashuel, J. Biol. Chem. 287 (2012) 15345–15364.

[16] A. Iwai, E. Masliah, M. Yoshimoto, N. Ge, L. Flanagan, H.A. de Silva, A. Kittel, T.Saitoh, Neuron 14 (1995) 467–475.

[17] D.D. Murphy, S.M. Rueter, J.Q. Trojanowski, V.M. Lee, J. Neurosci. 20 (2000)3214–3220.

[18] A. Abeliovich, Y. Schmitz, I. Farinas, D. Choi-Lundberg, W.H. Ho, P.E. Castillo,N. Shinsky, J.M. Verdugo, M. Armanini, A. Ryan, M. Hynes, H. Phillips, D.Sulzer, A. Rosenthal, Neuron 25 (2000) 239–252.

[19] J.M. George, H. Jin, W.S. Woods, D.F. Clayton, Neuron 15 (1995) 361–372.[20] J.M. Souza, B.I. Giasson, V.M. Lee, H. Ischiropoulos, FEBS Lett. 474 (2000) 116–

119.[21] M. Ahn, S. Kim, M. Kang, Y. Ryu, T.D. Kim, Biochem. Biophys. Res. Commun.

346 (2006) 1142–1149.[22] J.E. Payton, R.J. Perrin, W.S. Woods, J.M. George, J. Mol. Biol. 337 (2004) 1001–

1009.[23] V. Narayanan, Y. Guo, S. Scarlata, Biochemistry 44 (2005) 462–470.[24] P. Desplats, B. Spencer, L. Crews, P. Patel, D. Morvinski-Friedmann, K. Kosberg,

S. Roberts, C. Patrick, B. Winner, J. Winkler, E. Masliah, J. Biol. Chem. (2012).[25] E.R. Stadtman, Annu. Rev. Biochem. 62 (1993) 797–821.[26] W. Vogt, Free Radic. Biol. Med. 18 (1995) 93–105.[27] J. Moskovitz, H. Weissbach, N. Brot, Proc. Natl. Acad. Sci. USA 93 (1996) 2095–

2099.[28] H. Sun, J. Gao, D.A. Ferrington, H. Biesiada, T.D. Williams, T.C. Squier,

Biochemistry 38 (1999) 105–112.[29] C.B. Glaser, G. Yamin, V.N. Uversky, A.L. Fink, Biochim. Biophys. Acta 1703

(2005) 157–169.[30] T. Hoshi, S. Heinemann, J. Physiol. 531 (2001) 1–11.[31] E.R. Stadtman, H. Van Remmen, A. Richardson, N.B. Wehr, R.L. Levine,

Biochim. Biophys. Acta 1703 (2005) 135–140.[32] J. Choi, M.C. Sullards, J.A. Olzmann, H.D. Rees, S.T. Weintraub, D.E. Bostwick,

M. Gearing, A.I. Levey, L.S. Chin, L. Li, J. Biol. Chem. 281 (2006) 10816–10824.[33] V.N. Uversky, G. Yamin, P.O. Souillac, J. Goers, C.B. Glaser, A.L. Fink, FEBS Lett.

517 (2002) 239–244.[34] M.J. Hokenson, V.N. Uversky, J. Goers, G. Yamin, L.A. Munishkina, A.L. Fink,

Biochemistry 43 (2004) 4621–4633.[35] F. Chiti, C.M. Dobson, Annu. Rev. Biochem. 75 (2006) 333–366.[36] K.A. Conway, S.J. Lee, J.C. Rochet, T.T. Ding, R.E. Williamson, P.T. Lansbury Jr.,

Proc. Natl. Acad. Sci. USA 97 (2000) 571–576.[37] M.S. Goldberg, P.T. Lansbury Jr., Nat. Cell Biol. 2 (2000) E115–119.


[38] D.P. Karpinar, M.B. Balija, S. Kugler, F. Opazo, N. Rezaei-Ghaleh, N. Wender,H.Y. Kim, G. Taschenberger, B.H. Falkenburger, H. Heise, A. Kumar, D. Riedel, L.Fichtner, A. Voigt, G.H. Braus, K. Giller, S. Becker, A. Herzig, M. Baldus, H.Jackle, S. Eimer, J.B. Schulz, C. Griesinger, M. Zweckstetter, EMBO J. 28 (2009)3256–3268.

[39] H.A. Lashuel, P.T. Lansbury Jr., Q. Rev. Biophys. 39 (2006) 167–201.[40] E. Masliah, E. Rockenstein, I. Veinbergs, M. Mallory, M. Hashimoto, A. Takeda,

Y. Sagara, A. Sisk, L. Mucke, Science 287 (2000) 1265–1269.[41] B. Winner, R. Jappelli, S.K. Maji, P.A. Desplats, L. Boyer, S. Aigner, C. Hetzer, T.

Loher, M. Vilar, S. Campioni, C. Tzitzilonis, A. Soragni, S. Jessberger, H. Mira, A.Consiglio, E. Pham, E. Masliah, F.H. Gage, R. Riek, Proc. Natl. Acad. Sci. USA 1084194-4199.

[42] J.M. Souza, G. Peluffo, R. Radi, Free Radic. Biol. Med. (2008).[43] R. Radi, Proc. Natl. Acad. Sci. USA 101 (2004) 4003–4008.[44] B. Alvarez, G. Ferrer-Sueta, B.A. Freeman, R. Radi, J. Biol. Chem. 274 (1999)

842–848.[45] H. Ischiropoulos, Arch. Biochem. Biophys. 356 (1998) 1–11.[46] S. Pennathur, V. Jackson-Lewis, S. Przedborski, J.W. Heinecke, J. Biol. Chem.

274 (1999) 34621–34628.[47] J.M. Souza, B.I. Giasson, Q. Chen, V.M. Lee, H. Ischiropoulos, J. Biol. Chem. 275

(2000) 18344–18349.[48] B.I. Giasson, J.E. Duda, I.V. Murray, Q. Chen, J.M. Souza, H.I. Hurtig, H.

Ischiropoulos, J.Q. Trojanowski, V.M. Lee, Science 290 (2000) 985–989.[49] G. Yamin, V.N. Uversky, A.L. Fink, FEBS Lett. 542 (2003) 147–152.[50] E.H. Norris, B.I. Giasson, H. Ischiropoulos, V.M. Lee, J. Biol. Chem. 6 (2003) 6.[51] W. Zhou, C.R. Freed, J. Biol. Chem. 279 (2004) 10128–10135.[52] S.R. Danielson, J.M. Held, B. Schilling, M. Oo, B.W. Gibson, J.K. Andersen, Anal.

Chem. 81 (2009) 7823–7828.[53] T. Takahashi, H. Yamashita, T. Nakamura, Y. Nagano, S. Nakamura, Brain Res.

938 (2002) 73–80.[54] R. Hodara, E.H. Norris, B.I. Giasson, A.J. Mishizen-Eberz, D.R. Lynch, V.M. Lee,

H. Ischiropoulos, J. Biol. Chem. 279 (2004) 47746–47753.[55] J.M. Souza, G. Peluffo, R. Radi, Free Radic. Biol. Med. 45 (2008) 357–366.[56] R.J. Castellani, S.L. Siedlak, G. Perry, M.A. Smith, Acta Neuropathol. 100 (2000)

111–114.[57] H.S. Pall, A.C. Williams, D.R. Blake, J. Lunec, J.M. Gutteridge, M. Hall, A. Taylor,

Lancet 2 (1987) 238–241.[58] J.M. Gorell, C.C. Johnson, B.A. Rybicki, E.L. Peterson, G.X. Kortsha, G.G. Brown,

R.J. Richardson, Neurology 48 (1997) 650–658.[59] J. Zayed, S. Ducic, G. Campanella, J.C. Panisset, P. Andre, H. Masson, M. Roy,

Can. J. Neurol. Sci. 17 (1990) 286–291.[60] A.B. Bowman, G.F. Kwakye, E.H. Hernandez, M. Aschner, J. Trace Elem. Med.

Biol. 25 (2011) 191–203.[61] J.R. Requena, D. Groth, G. Legname, E.R. Stadtman, S.B. Prusiner, R.L. Levine,

Proc. Natl. Acad. Sci. USA 98 (2001) 7170–7175.[62] S.R. Paik, H.J. Shin, J.H. Lee, Arch. Biochem. Biophys. 378 (2000) 269–277.[63] A. Binolfi, E.E. Rodriguez, D. Valensin, N. D’Amelio, E. Ippoliti, G. Obal, R.

Duran, A. Magistrato, O. Pritsch, M. Zweckstetter, G. Valensin, P. Carloni, L.Quintanar, C. Griesinger, C.O. Fernandez, Inorg. Chem. 49 (2010) 10668–10679.

[64] N.B. Cole, D.D. Murphy, J. Lebowitz, L. Di Noto, R.L. Levine, R.L. Nussbaum, J.Biol. Chem. 280 (2005) 9678–9690.

[65] A. Binolfi, G.R. Lamberto, R. Duran, L. Quintanar, C.W. Bertoncini, J.M. Souza, C.Cervenansky, M. Zweckstetter, C. Griesinger, C.O. Fernandez, J. Am. Chem. Soc.130 (2008) 11801–11812.

[66] M.S. Jackson, J.C. Lee, Inorg. Chem. 48 (2009) 9303–9307.[67] J.C. Lee, H.B. Gray, J.R. Winkler, J. Am. Chem. Soc. 130 (2008) 6898–6899.[68] R.M. Rasia, C.W. Bertoncini, D. Marsh, W. Hoyer, D. Cherny, M. Zweckstetter,

C. Griesinger, T.M. Jovin, C.O. Fernandez, Proc. Natl. Acad. Sci. USA 102 (2005)4294–4299.

[69] A. Binolfi, R.M. Rasia, C.W. Bertoncini, M. Ceolin, M. Zweckstetter, C.Griesinger, T.M. Jovin, C.O. Fernandez, J. Am. Chem. Soc. 128 (2006) 9893–9901.

[70] A. Ala, A.P. Walker, K. Ashkan, J.S. Dooley, M.L. Schilsky, Lancet 369 (2007)397–408.

[71] V.N. Uversky, J. Li, A.L. Fink, J. Biol. Chem. 276 (2001) 44284–44296.[72] T. Cai, H. Che, T. Yao, Y. Chen, C. Huang, W. Zhang, K. Du, J. Zhang, Y. Cao, J.

Chen, W. Luo, Toxicol. Sci. 119 (2010) 169–177.[73] Y. Li, L. Sun, T. Cai, Y. Zhang, S. Lv, Y. Wang, L. Ye, Brain Res. Bull. 81 (2010)

428–433.[74] T.R. Guilarte, Neurotoxicology 31 572–574.[75] D.L. Lefkowitz, S.S. Lefkowitz, Free Radic. Biol. Med. 45 (2008) 726–731.[76] D.K. Choi, S. Pennathur, C. Perier, K. Tieu, P. Teismann, D.C. Wu, V. Jackson-

Lewis, M. Vila, J.P. Vonsattel, J.W. Heinecke, S. Przedborski, J. Neurosci. 25(2005) 6594–6600.

[77] M. Hashimoto, A. Takeda, L.J. Hsu, T. Takenouchi, E. Masliah, J. Biol. Chem. 274(1999) 28849–28852.

[78] R.A. Ruf, E.A. Lutz, I.G. Zigoneanu, G.J. Pielak, Biochemistry 47 (2008) 13604–13609.

[79] H. Bayir, A.A. Kapralov, J. Jiang, Z. Huang, Y.Y. Tyurina, V.A. Tyurin, Q. Zhao,N.A. Belikova, J. Biol. Chem. 284 (2009) 15951–15969.

[80] Y.R. Chen, L.J. Deterding, B.E. Sturgeon, K.B. Tomer, R.P. Mason, J. Biol. Chem.277 (2002) 29781–29791.

[81] L.V. Basova, I.V. Kurnikov, L. Wang, V.B. Ritov, N.A. Belikova, Vlasova, II, A.A.Pacheco, D.E. Winnica, J. Peterson, H. Bayir, D.H. Waldeck, V.E. Kagan,Biochemistry 46 (2007) 3423–3434.



704705706707708709710711712713714715716717718719720721722723724725726727728729730731732733734735736737738739740741742743744745746747748749750751752753754755

756757758759760761762763764765766767768769770771772773774775776777778779780781782783784785786787788789790791792793794795796797798799800801802803804

805



5 March 2013

[82] N.A. Belikova, Y.A. Vladimirov, A.N. Osipov, A.A. Kapralov, V.A. Tyurin, M.V.Potapovich, L.V. Basova, J. Peterson, I.V. Kurnikov, V.E. Kagan, Biochemistry 45(2006) 4998–5009.

[83] J.M. Souza, L. Castro, A.M. Cassina, C. Batthyany, R. Radi, Methods Enzymol.441 (2008) 197–215.

[84] L. Devi, V. Raghavendran, B.M. Prabhu, N.G. Avadhani, H.K.Anandatheerthavarada, J. Biol. Chem. 283 (2008) 9089–9100.

[85] A. Trostchansky, S. Lind, R. Hodara, T. Oe, I.A. Blair, H. Ischiropoulos, H. Rubbo,J.M. Souza, Biochem. J. 393 (2006) 343–349.

[86] Z. Qin, D. Hu, S. Han, S.H. Reaney, D.A. Di Monte, A.L. Fink, J. Biol. Chem. 282(2007) 5862–5870.

[87] S. Bartesaghi, J. Wenzel, M. Trujillo, M. Lopez, J. Joseph, B. Kalyanaraman, R.Radi, Chem. Res. Toxicol. 23 (2010) 821–835.

[88] R. Shchepin, M.N. Moller, H.Y. Kim, D.M. Hatch, S. Bartesaghi, B.Kalyanaraman, R. Radi, N.A. Porter, J. Am. Chem. Soc. 132 (2010) 17490–17500.

[89] A.L. Fink, Acc. Chem. Res. 39 (2006) 628–634.[90] R. Kayed, E. Head, J.L. Thompson, T.M. McIntire, S.C. Milton, C.W. Cotman, C.G.

Glabe, Science 300 (2003) 486–489.[91] M.J. Volles, S.J. Lee, J.C. Rochet, M.D. Shtilerman, T.T. Ding, J.C. Kessler, P.T.

Lansbury Jr., Biochemistry 40 (2001) 7812–7819.[92] S.J. Wood, J. Wypych, S. Steavenson, J.C. Louis, M. Citron, A.L. Biere, J. Biol.

Chem. 274 (1999) 19509–19512.[93] K.A. Conway, J.D. Harper, P.T. Lansbury Jr., Biochemistry 39 (2000) 2552–

2563.[94] T.T. Ding, S.J. Lee, J.C. Rochet, P.T. Lansbury Jr., Biochemistry 41 (2002) 10209–

10217.[95] L.S. Forno, J. Neuropathol. Exp. Neurol. 55 (1996) 259–272.[96] M.M. Tompkins, E.J. Basgall, E. Zamrini, W.D. Hill, Am. J. Pathol. 150 (1997)

119–131.[97] T.F. Outeiro, P. Putcha, J.E. Tetzlaff, R. Spoelgen, M. Koker, F. Carvalho, B.T.

Hyman, P.J. McLean, PLoS One 3 (2008) e1867.[98] H.A. Lashuel, B.M. Petre, J. Wall, M. Simon, R.J. Nowak, T. Walz, P.T. Lansbury

Jr., J. Mol. Biol. 322 (2002) 1089–1102.[99] R. Kayed, Y. Sokolov, B. Edmonds, T.M. McIntire, S.C. Milton, J.E. Hall, C.G.

Glabe, J. Biol. Chem. 279 (2004) 46363–46366.[100] K. Assayag, E. Yakunin, V. Loeb, D.J. Selkoe, R. Sharon, Am. J. Pathol. 171

(2007) 2000–2011.[101] K. Beyer, Acta Neuropathol. 112 (2006) 237–251.[102] K. Beyer, Cell Biochem. Biophys. 47 (2007) 285–299.[103] R.J. Perrin, W.S. Woods, D.F. Clayton, J.M. George, J. Biol. Chem. 11 (2001) 11.[104] M.J. Volles, P.T. Lansbury Jr., Biochemistry 41 (2002) 4595–4602.[105] M. Kostka, T. Hogen, K.M. Danzer, J. Levin, M. Habeck, A. Wirth, R. Wagner,

C.G. Glabe, S. Finger, U. Heinzelmann, P. Garidel, W. Duan, C.A. Ross, H.Kretzschmar, A. Giese, J. Biol. Chem. 283 (2008) 10992-11003.

[106] I.F. Tsigelny, Y. Sharikov, W. Wrasidlo, T. Gonzalez, P.A. Desplats, L. Crews, B.Spencer, E. Masliah, FEBS J. (2012).

[107] K.M. Danzer, D. Haasen, A.R. Karow, S. Moussaud, M. Habeck, A. Giese, H.Kretzschmar, B. Hengerer, M. Kostka, J. Neurosci. 27 (2007) 9220–9232.

[108] H.Y. Kim, M.K. Cho, A. Kumar, E. Maier, C. Siebenhaar, S. Becker, C.O.Fernandez, H.A. Lashuel, R. Benz, A. Lange, M. Zweckstetter, J. Am. Chem. Soc.131 (2009) 17482–17489.


[109] A. Quist, I. Doudevski, H. Lin, R. Azimova, D. Ng, B. Frangione, B. Kagan, J.Ghiso, R. Lal, Proc. Natl. Acad. Sci. USA 102 (2005) 10427–10432.

[110] I.F. Tsigelny, P. Bar-On, Y. Sharikov, L. Crews, M. Hashimoto, M.A. Miller, S.H.Keller, O. Platoshyn, J.X. Yuan, E. Masliah, FEBS J. 274 (2007) 1862–1877.

[111] R. Radi, A. Cassina, R. Hodara, C. Quijano, L. Castro, Free Radic. Biol. Med. 33(2002) 1451–1464.

[112] M.L. Schmidt, J. Murray, V.M. Lee, W.D. Hill, A. Wertkin, J.Q. Trojanowski, Am.J. Pathol. 139 (1991) 53–65.

[113] W.P. Gai, H.X. Yuan, X.Q. Li, J.T. Power, P.C. Blumbergs, P.H. Jensen, Exp.Neurol. 166 (2000) 324–333.

[114] T. Kitada, S. Asakawa, N. Hattori, H. Matsumine, Y. Yamamura, S. Minoshima,M. Yokochi, Y. Mizuno, N. Shimizu, Nature 392 (1998) 605–608.

[115] J.L. Webb, B. Ravikumar, J. Atkins, J.N. Skepper, D.C. Rubinsztein, J. Biol. Chem.278 (2003) 25009–25013.

[116] K.A. Malkus, H. Ischiropoulos, Neurobiol. Dis. 46 (2012) 732–744.[117] M. Martinez-Vicente, Z. Talloczy, S. Kaushik, A.C. Massey, J. Mazzulli, E.V.

Mosharov, R. Hodara, R. Fredenburg, D.C. Wu, A. Follenzi, W. Dauer, S.Przedborski, H. Ischiropoulos, P.T. Lansbury, D. Sulzer, A.M. Cuervo, J. Clin.Invest. 118 (2008) 777–788.

[118] A.J. Gow, D. Duran, S. Malcolm, H. Ischiropoulos, FEBS Lett. 385 (1996) 63–66.[119] J.M. Souza, I. Choi, Q. Chen, M. Weisse, E. Daikhin, M. Yudkoff, M. Obin, J. Ara,

J. Horwitz, H. Ischiropoulos, Arch. Biochem. Biophys. 380 (2000) 360–366.[120] T. Grune, I.E. Blasig, N. Sitte, B. Roloff, R. Haseloff, K.J. Davies, J. Biol. Chem.

273 (1998) 10857–10862.[121] Y.Z. Ye, M. Strong, Z.Q. Huang, J.S. Beckman, Methods Enzymol. 269 (1996)

201–209.[122] J. Herzog, Y. Maekawa, T.P. Cirrito, B.S. Illian, E.R. Unanue, Proc. Natl. Acad.

Sci. USA 102 (2005) 7928–7933.[123] S. Przedborski, Q. Chen, M. Vila, B.I. Giasson, R. Djaldatti, S. Vukosavic, J.M.

Souza, V. Jackson-Lewis, V.M. Lee, H. Ischiropoulos, J. Neurochem. 76 (2001)637–640.

[124] E.J. Benner, R. Banerjee, A.D. Reynolds, S. Sherman, V.M. Pisarev, V. Tsiperson,C. Nemachek, P. Ciborowski, S. Przedborski, R.L. Mosley, H.E. Gendelman,PLoS One 3 (2008) e1376.

[125] A.D. Reynolds, D.K. Stone, J.A. Hutter, E.J. Benner, R.L. Mosley, H.E.Gendelman, J. Immunol. 184 (2010) 2261–2271.

[126] A.D. Reynolds, D.K. Stone, R.L. Mosley, H.E. Gendelman, J. Immunol. 182(2009) 4137–4149.

[127] L. Thomson, J. Christie, C. Vadseth, P.N. Lanken, X. Fu, S.L. Hazen, H.Ischiropoulos, Am. J. Respir. Cell Mol. Biol. 36 (2007) 152–157.

[128] G.T. Liberatore, V. Jackson-Lewis, S. Vukosavic, A.S. Mandir, M. Vila, W.G.McAuliffe, V.L. Dawson, T.M. Dawson, S. Przedborski, Nat. Med. 5 (1999)1403–1409.

[129] D.K. Stone, T. Kiyota, R.L. Mosley, H.E. Gendelman, Neurosci. Lett. 523 (2012)167–173.

[130] J.M. Fearnley, A.J. Lees, Brain 114 (Pt 5) (1991) 2283–2301.[131] V. Kumar, A.K. Abbas, N. Fausto, J. Aster, Robbins and Cotran Pathologic Basis

of Disease, Elsevier Health Sciences, 2009.[132] J.E. Galvin, V.M. Lee, J.Q. Trojanowski, Arch. Neurol. 58 (2001) 186–190.



Oxidation and nitration of α-synuclein and their implications in neurodegenerative diseases

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Transcript of Oxidation and nitration of α-synuclein and their implications in neurodegenerative diseases