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Neurobiology of Aging 29 (2008) 408–417

Early �-synuclein lipoxidation in neocortex in Lewy body diseasesEsther Dalfo, Isidre Ferrer ∗

Institut de Neuropatologia, Servei Anatomia Patologica, IDIBELL-Hospital Universitari de Bellvitge; Facultat de Medicina,Universitat de Barcelona; carrer Feixa Llarga sn, 08907, Hospitalet de Llobregat; Spain

Received 22 August 2006; received in revised form 30 September 2006; accepted 18 October 2006Available online 12 December 2006

bstract

Previous studies in Lewy body diseases (LBDs), including Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB), have shownxidative stress damage more extended than the expected for the distribution of Lewy pathology. Since malondialdehyde (MDA) can formdducts with lysine residues of proteins, MDA-Lys immunoprecipitation and �-synuclein immunoblotting has been carried out in frontalortex and substantia nigra homogenates from five patients with PD, five DLB, three iPD and seven aged-matched controls to decipher thextent of lipoxidized �-synuclein in LBDs. MDA-Lys-lipoxidation of �-synuclein in the substantia nigra and frontal cortex has been found inll DLB and PD cases examined, but also in the frontal cortex in 3/3 and in the substantia nigra in 2/3 cases with iPD. In addition, one controlase had MDA-Lys-modified �-synuclein in the frontal cortex, and another in the substantia nigra. This work provides evidence of extended

ipoxidative modification of �-synuclein in LBDs. Moreover, it demonstrates that �-synuclein lipoxidation is an early event in LBDs whichrecedes �-synuclein solubility modification and aggregation, and formation of Lewy bodies and neurites.

2006 Elsevier Inc. All rights reserved.

eywords: Parkinson’s disease; Cerebral cortex; Oxidative damage; �-Synuclein

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. Introduction

Parkinson’s disease (PD), characterized by resting tremor,lowness of initial movement, rigidity, and general posturalnstability, is one of the most prevalent neurodegenerativeisorders among the elderly population. The disease is patho-ogically defined by loss of neurons in the substantia nigraars compacta, locus ceruleus, other nuclei of the brain stem,asal nucleus of Meynert and amygdala, and by the presencef eosinophilic intraneuronal proteinaceous inclusions calledewy bodies and aberrant neurites (Forno, 1996; Jellingernd Mizuno, 2003). Parkinson-like pathology restricted tohe medulla oblongata and pons, associated or not with mild

idbrain involvement in the absence of motor symptoms,s known as (asymptomatic) incidental or pre-clinical PD

iPD) (Forno, 1996; Jellinger and Mizuno, 2003). Demen-ia with Lewy bodies (DLB) is manifested as progressiveognitive impairment, dementia and parkinsonism, and it is

∗ Corresponding author.E-mail address: 8082ifa@comb.es (I. Ferrer).

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197-4580/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.neurobiolaging.2006.10.022

haracterized by the additional widespread distribution ofBs and neurites in the cerebral cortex (Ince and McKeith,003; Ince et al., 1998). DLB is often accompanied bylzheimer’s disease (AD) and this is considered the com-on form; DLB with minimal A�-amyloid deposits and no

au pathology characterizes the pure form (Kosaka, 1990;osaka, 1993). PD and DLB are within the spectrum ofewy body diseases (LBDs). Staging of brain pathology

elated to sporadic PD has been proposed (Braak et al.,003). This instrumental classification is useful as it delin-ates the topography of lesions in the different stages andorrelates and matches with clinical symptoms in the majorityf cases. Thus stages 1 and 2 are coincidental with pre-linical PD, stages 3 and 4 may have PD, and stages 5nd 6 are manifested as PD with cognitive impairment andLB.�-Synuclein is the major component of protein aggre-

ates in Lewy bodies and aberrant neurites (Baba et al., 1998;ashimoto and Masliah, 1999; Iwatsubo, 2003; Spillantini et

l., 1998). Mutations in the �-synuclein gene (A53T, A30P,46K) are associated with familial PD and DLB (Kruger

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t al., 1998; Polymeropoulos et al., 1997; Zarranz et al.,004). Triplication or duplication of the �-synuclein locuss a cause of PD (Chartier-Harlin et al., 2004; Ibanez et al.,004; Nishioka et al., 2006; Singleton et al., 2003). Basedn these characteristics, LBDs have been categorized as �-ynucleinopathies.

Mutations in the �-synuclein gene, increased levelsf �-synuclein, and oxidative stress lead to �-synucleinggregation in vitro (Hashimoto et al., 1999; Narhi et al.,999; Paik et al., 2000). Yet oxidative stress is also aain contributory factor in the pathogenesis of PD and

elated �-synucleinopathies (Jenner, 2003; Markesberry etl., 2001). Studies in the substantia nigra and midbrainave shown decreased levels of reduced glutathione (Perryt al., 1982; Sian et al., 1994), increased Cu/ZN super-xide dismutase I and Mn superoxide dismutase (SOD2)rotein and mRNA levels (Ceballos et al., 1990; Marttilat al., 1988; Saggu et al., 1989), and increased protein car-onyls, lipid peroxides (Alam et al., 1997a,b; Floor andetzel, 1998), and 4-hydroxy-2-nonenal (Dexter et al., 1986;

oritaka et al., 1996), as well as changes in polyunsatu-ated fatty acids sustaining lipid peroxidation (Shelley, 1998),eading to increased generation of malondialdehyde andydroperoxides (Dexter et al., 1989). Advanced glycationnd products (AGE) have also been found in the substan-ia nigra and locus ceruleus in PD (Jenner, 1998). Finally,xidative RNA and DNA damage also occurs in the sub-tantia nigra in PD (Alam et al., 1997a,b; Castellani et al.,996). Oxidative stress has also been observed in other brainegions in LBDs. A generalized increase in protein car-onyls has been found in the telencephalon in PD (Alamt al., 1997a,b). Oxidative DNA damage, as revealed byncreased levels of 8-hydroxyguanine, occurs not only inhe substantia nigra but also in the basal ganglia and cere-ral cortex in PD (Sanchez-Ramos et al., 1994; Zhang etl., 1999). Indices of oxidative stress with altered mitochon-rial function have been observed in the cerebral cortex inPD (Dexter et al., 1994). Moreover, recent studies havehown mass spectrometric and immunochemical evidence ofbnormal lipid composition, increased lipoxidative damagey the markers malondialdehyde-lysine (MDA-Lys) and 4-ydroxynonenal-lysine, increased AGE modifications, andncreased RAGE cellular expression in the frontal cortex inBDs including iPD (Dalfo et al., 2005). Preliminary pro-

eomic studies have also revealed �-synuclein and SOD2s targets of lipoxidative damage in iPD (Dalfo et al.,005).

In line with our previous studies, the present work isocused on �-synuclein as a possible target of lipoxidativeamage in the substantia nigra and frontal cortex in LBDs.esults show not only MDA-Lys-lipoxidation of �-synuclein

n the substantia nigra and frontal cortex in all DLB and PD

ases examined, but also in the frontal cortex in 3/3 and in theubstantia nigra in 2/3 cases with iPD. This work provides forhe first time evidence of extended lipoxidative modificationf �-synuclein in LBDs.

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f Aging 29 (2008) 408–417 409

. Materials and methods

.1. Tissue samples

Clinically, all cases of PD had suffered from classicalD lasting from 8 to 15 years, and none of them had cog-itive impairment. Cases with DLB fulfilled the clinicalriteria proposed by the consortium on DLB internationalorkshop (McKeith et al., 2000, 1996). Brain samples werebtained from the Institute of Neuropathology and Universityf Barcelona Brain Banks.

The brains of five patients with PD, five DLB, three iPDnd seven aged-matched controls were obtained at autopsy,ollowing informed consent of the patients or their rela-ives and the approval of the local ethics committee. Bothenders were represented equally; age range was between0 and 88 years (mean age 73 years), and the averageime between death and tissue processing was 5.4 h. Onealf of the brain was immediately cut on coronal sec-ions, 1 cm thick, frozen on dry ice and stored at −80 ◦Cntil use. For morphological examinations, the brains werexed by immersion in 4% buffered formalin for 2 or 3eeks. The neuropathological study was carried out on de-axed 4-�m-thick paraffin sections of the frontal (area 8),rimary motor, primary sensory, parietal, temporal supe-ior, temporal inferior, anterior cingulated, anterior insular,nd primary and associative visual cortices; entorhinal cortexnd hippocampus; caudate, putamen and pallidum; medialnd posterior thalamus; subthalamus; Meynert nucleus;mygdala; midbrain (two levels), pons and medulla oblon-ata; and cerebellar cortex and dentate nucleus. The sectionsere stained with haematoxylin and eosin, luxol fast blue-luver Barrera, and, for immunohistochemistry to glialbrillary acidic protein, CD68 and Licopericum esculentum

ectin for microglia, A�-amyloid, pan-tau, phosphorylation-pecific tau Thr181, Ser202, Ser214, Ser262, Ser396nd Ser422, and �B-crystallin, �-synuclein and ubiqui-in.

Neuropathological characterization of PD was accordingo well-established neuropathological criteria (Jellinger and

izuno, 2003). Neuropathological characterization of DLBas according to consensus guidelines of the consortiumn DLB international workshop (Ince and McKeith, 2003;nce et al., 1998). To further refine �-synuclein pathology,taging of brain pathology related to sporadic PD proposedy Braak et al. was used in the present study. Basically,tages 1 and 2 affect the medulla oblongata plus the pontineegmentum; stage 3, the midbrain; stage 4, the basal prosen-ephalon and mesocortex; and stages 5 and 6, the neocortexBraak et al., 2003). Associated AD stages were furtherstablished depending on the amyloid deposition burdennd neurofibrillary pathology following the nomenclature

f Braak and Braak (Braak and Braak, 1999). Stages ofmyloid deposition refer to initial deposits in the basaleocortex (stage A), deposits extended to the associationreas of the neocortex (stage B), and heavy deposition

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hroughout the entire cortex (stage C). Stages of neurofib-illary pathology correspond to transentorhinal (I-II), limbicIII-IV) and neocortical (V and VI). Control cases wereonsidered in the absence of neurological symptoms andigns, and with no abnormalities in the neuropathologicaltudy.

No neurological symptoms were recorded in iPD cases.ll of them were admitted to the hospital for non-

elated conditions, mainly heart attack and infectiousespiratory diseases. Three patients with neuropatholog-cally verified PD-related changes (two men and oneoman) were here included. Neurons with Lewy bodies

nd �-synuclein-immunoreactive neurites were practicallyestricted to the dorsal motor nucleus and reticular formationf the medulla oblongata, raphe nuclei, gigantocellu-ar reticular nucleus and locus ceruleus in two cases.

few Lewy neurites and neurons with Lewy bodies,ogether with preservation in number of pigmented neu-ons and the absence of astrogliosis and microgliosis,ccurred in the third case. Lewy bodies and neurites inhe amygdala, nucleus basalis of Meynert, entorhinal cor-ex, cingulate cortex and neocortex were absent in thehree cases. Therefore, these cases corresponded to stages–3.

No neurological symptoms or metabolic disorders hadccurred in control cases. No abnormalities, including AD-ssociated changes or vascular disorders, were found in

ontrol cases.

The main neuropathological data in the present series areummarized in Table 1.

ETp

able 1ummary of cases

ase Disease Gender Age (y) Post-mortem (h)

1 Control M 63 72 Control M 69 83 Control M 79 74 Control F 65 45 Control F 82 116 Control F 80 117 Control 49 78 iPD M 76 29 iPD F 74 3

10 iPD M 72 211 PD M 66 512 PD M 81 513 PD M 88 214 PD M 70 915 PD F 60 416 DLB M 60 817 DLB M 68 1218 DLB M 71 619 DLB M 81 620 DLB M 85 7

tage related to Parkinson’s disease (PD) refers to the operational classification propangles (stages I-VI) and amyloid plaques (stages A–C) as proposed by Braak anarkinson disease; PD: Parkinson disease; DLB: dementia with Lewy bodies (note

hus considered as pure forms of DLB).

f Aging 29 (2008) 408–417

.2. MDA–Lys immunoprecipitation and immunoblot

Samples (0.1 g) from control and diseased cases wereomogenized in a glass homogenizer in 10 volumesf ice-cold immunoprecipitation (IP) buffer (PBS, 1 mMDTA, 50 mM sodium orthovanadate and a tablet of pro-

ease inhibitors (Roche, Madrid, Spain) and centrifuged at000 × g for 10 min at 4 ◦C. The S1 fraction was pre-clearedith protein G-sepharose (Amersham Biosciences, Madrid,pain) for 1 h at 4 ◦C while shaking. Protein concentra-

ions were determined using the BCA method with bovineerum albumin (BSA) as a standard. Equal aliquots of pre-leared S1 (2 mg) were incubated with mouse monoclonalnti-MDA-Lys antibody (JaiCA, Deltaclon, Madrid, Spain) at◦C overnight. 40 �l of 1:1 (v/v) protein G-sepharose beadsere as added for 2 h. The immune complexes were collectedy centrifugation and washed three times with IP buffer. Theellet was re-suspended in 20 �L of 4× sample buffer andhe immunocomplexes were processed for 10% SDS-PAGElectrophoresis and Western blotting to nitrocellulose mem-ranes (BioRad, Barcelona, Spain). Two membranes werelectrophoresed in parallel. While one was incubated withabbit polyclonal anti-MDA-Lys (Deltaclon, Madrid, Spain)t a dilution of 1:1000 as a positive control for the immuno-recipitation process, the other was incubated with rabbitolyclonal anti-�-synuclein (Chemicon, Bionova, Madrid,pain) at a dilution of 1:4000. Proteins were detected by the

CL chemiluminescence method (Amersham Biosciences).otal homogenates and immunoprecipitated samples wererocessed in parallel with a lane containing the antibody

Braak stages �A4 amyloid AD NFT Braak stages PD

0 0 00 0 00 II 00 I 0A III 00 I 0A 0 0A 0 2A 0 30 0 20 I 3A II 40 II 40 0 4A 0 4A I 6B 0 6B 0 5A I 6B II 6

osed by Braak et al. (2003). AD changes include presence of neurofibrillaryd Braak (1999). P-m: post-mortem; M: male; F: female; iPD: incidental

that all cases with DLB have no or restricted AD-related pathology and are

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sed for immunoprecipitation and protein G-sepharose witho sample, and another lane containing brain sample plusrotein G-sepharose.

.3. α-Synuclein solubility and aggregation

Brain samples (0.2 g) of the frontal cortex of patients withPD, PD, DLB and controls were homogenized in a glassomogenizer in 1.5 mL of ice-cold PBS (sodium phosphateuffer, pH 7.0, plus protease inhibitors), sonicated, and cen-rifuged at 2650 × 3g at 4 ◦C for 10 min. The pellet wasiscarded and the resulting supernatant was ultracentrifugedt 100,000 × 3g at 4 ◦C for 1 h. The supernatant (S2) was kepts the PBS-soluble fraction (cytosolic fraction). The resultingellet was re-suspended in a solution of PBS, pH 7.0, con-aining 0.5% sodium deoxycholate, 1% Triton and 0.1% SDS,nd it was ultracentrifuged at 100,000 × 3g at 4 ◦C for 1 h.he resulting supernatant (S3) was kept as the deoxycholate-oluble fraction. The corresponding pellet was re-suspendedn a solution of 2% SDS in PBS and maintained at room tem-erature for 2 h. Immediately afterward, the samples wereentrifuged at 100,000 × g at 25 ◦C for 1 h and the result-ng supernatant (S4) was the SDS-soluble fraction. Equalmounts of each fraction were mixed with reducing sampleuffer and processed for 10% SDS-PAGE electrophoresis andestern blot analysis. The membranes were incubated with

nti-�-synuclein (Chemicon) at a dilution of 1:4000. Pro-ein bands were visualized with the ECL method (Amershamioscience).

.4. α-Synuclein immunoprecipitation andmmunoblotting

This was carried out in samples (0.3 g) of the frontal cortexarea 8) of controls, iPD, PD and DLB cases. The samplesere homogenized in a glass homogenizer in 1.5 ml of ice-

old immunoprecipitation buffer (Hepes 20 mM pH 7.2, 1%riton X-100, 1% sodium deoxycholate, 0.2% SDS, 150 mMaCl, 1 mM sodium orthovanadate, 1 mM sodium fluoride,0% glycerol, 10 �g/ml aprotinin, 1 mM phenylmethylsul-onyl fluoride), sonicated and centrifuged at 5000 rpm for0 min at 4 ◦C. The S1 fraction was pre-cleared with pro-ein G-sepharose (Amersham) for 1 h Protein concentrationas determined using the BCA method (Pierce) with bovine

erum albumin as a standard. Equal aliquots of pre-clearedample were incubated with anti-�-synuclein antibody (Neo-arkers) and 35 �l of 1:1 (v/v) protein G-sepharose for 4 h at◦C. The immune complexes were collected by centrifuga-

ion and washed five times with a buffer containing 20 mMris–HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl,mM sodium orthovanadate, 10% glycerol and 1% Non-

det P-40 (Sigma). The pellet was re-suspended with 20 �l

f reducing sample buffer, and the immunocomplexes wererocessed for 10% SDS-PAGE electrophoresis and Westernlot analysis. The membranes were incubated with anti-�-ynuclein (Chemicon) used at a dilution of 1:2000, or with

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f Aging 29 (2008) 408–417 411

nti-ubiquitin (Dako, Barcelona, Spain) used at a dilutionf 1:100. The protein bands were visualized using the ECLethod (Amersham). Total homogenates and immunopre-

ipitated samples were processed in parallel. An additionalane contained the antibody used for immunoprecipitationound to protein G-sepharose with no sample, and anotherane with sample plus protein G-sepharose without the anti-ody used for immunoprecipitation were used as controls ofhe immunoprecipitation.

. Results

.1. General comments

Representative images of �-synuclein pathology in therontal cortex and substantia nigra in the present series arehown in Fig. 1. Lewy bodies and aberrant neurites are absentn control cases and in cases 8 and 10 with iPD. A few-synuclein-immunoractive neurites and cytoplasmic inclu-ions in substantia nigra neurons are seen in case 9. Largeumbers of Lewy bodies and neurites occur in the sub-tantia nigra in PD, whereas the frontal cortex is devoid of-synuclein inclusions. Finally, Lewy bodies and aberranteurites are abundant in the substantia nigra and frontal cortexn DLB.

.2. MDA-Lys-modified α-synuclein in control and LBD

Western blots of total homogenates of the frontal cor-ex and substantia nigra from control and LBD cases weremmunostained for �-synuclein; bands of higher moleculareight were also seen in DLB cases as detailed elsewhere

Dexter et al., 1986). MDA-Lys-immunoprecipitated samplesmmunostained with anti-MDA-Lys showed dense bands ofariable molecular weight due to non-specific binding andeak bands at about 17 kDa after long exposure in diseased

ases (data not shown).MDA-Lys-immunoprecipitates of the same samples

mmunostained with anti-�-synuclein antibody revealed apecific band at the appropriate molecular weight of 17 kDa,hereas no specific immunoreaction was seen in the lanes

orresponding to anti-�-synuclein antibody and protein G-epharose with no sample and in the lane corresponding to theample with protein G-sepharose without anti-�-synuclein.

Immunoprecipitated samples of the frontal cortex andubstantia nigra from cases with iPD run in parallel withmmunoprecipitated samples from controls revealed a weakand corresponding to �-synuclein in both the frontal cortexnd substantia nigra (Fig. 2).

Similarly, MDA-Lys-modified �-synuclein was alsobserved in the frontal cortex and substantia nigra in PD cases

Fig. 3). In the same line, MDA-Lys-immunoprecipitatedamples of the frontal cortex and substantia nigra in DLBases, but not in controls run in parallel, showed a band of-synuclein (Fig. 4, upper panel). Interestingly, the density

412 E. Dalfo, I. Ferrer / Neurobiology of Aging 29 (2008) 408–417

Fig. 1. �-Synuclein pathology in the frontal cortex (area 8) (A, C, E, G) and substantia nigra pars compacta (B, D, F, H) in control (A and B), incidentalParkinson’s disease (iPD) (C and D), Parkinson’s disease (E and F) and Dementia with Lewy bodies (G and H). Note the presence of isolated aberrant neurites,a lein-imP gra ands cortexw .

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nd granular deposits in a single neuron in iPD (case 9), numerous �-synucD (case 10), and numerous Lewy bodies and neurites in the substantia niubstantia nigra and frontal cortex in control (case 4) and in the frontal cortexith haematoxylin. A, C, E, G, H bar in H = 25 �m; B, D, F bar in F = 50 �m

f the band was variable from one case to another and thisas not related with the degree of synuclein pathology in the

rontal cortex as revealed by the Braak stage of the diseaseFig. 4, lower panel).

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ig. 2. Western blots of total homogenates from control (HC) and incidental Paramples (IPHC and IPiPD, respectively) immunostained with anti-�-synuclein antiPiPDs but no IPCs show weak band corresponding to �-synuclein in both the frono specific immunoreaction is seen in the lanes corresponding to anti-�-synuclein

nd in the lane corresponding to the sample with protein G-sepharose without anti-�ot specific. Control, case 2 and iPD, case 8 in Table 1.

munoreactive cytoplasmic and neuritic inclusions in the substantia nigra infrontal cortex in DLB (case 19). No synuclein aggregates are seen in thein iPD and PD. �-synuclein immunohistochemistry slightly counterstained

MDA-Lys-modified �-synuclein was seen in the frontalortex and substantia nigra in all cases of DLB and in allases of PD. Lipoxidized synuclein was also observed in therontal cortex in the three cases with iPD, but only in two

kinson’s disease (HiPD), and MDA-Lys-immunoprecipitates of the samebody reveal a specific band at the appropriate molecular weight of 17 kDa.tal cortex and substantia nigra in MDA-Lys-immunoprecipitated samples.antibody and protein G-sepharose with no sample (Anti-MDA + Prot G),-synuclein (Sample + Prot G). Upper bands of higher molecular weight are

E. Dalfo, I. Ferrer / Neurobiology of Aging 29 (2008) 408–417 413

Fig. 3. Western blots of total homogenates from control (HC) and Parkinson’s disease (HPD), and MDA-Lys-immunoprecipitates of the same samples (IPHCand IPPD, respectively) immunostained with anti-�-synuclein antibody, reveal specific bands at the appropriate molecular weight of 17 kDa. IPPDs but no IPCsshow a band corresponding to �-synuclein in both the frontal cortex and substantia nigra in MDA-Lys-immunoprecipitated-samples. Control, case 3 and PD,case 12 in Table 1.

Fig. 4. Western blots of total homogenates from control (HC) and Dementia with Lewy bodies (DLB), and MDA-Lys-immunoprecipitates of the same samples( ody, reb rtex anc in Tabler tified by

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IPHC and IPDLB, respectively) immunostained with anti-�-synuclein antibut no IPCs show a band corresponding to �-synuclein in both the frontal coontrol, case 1 and DLB, case 18. Lower panel, DLB cases 16, 17 and 19elated with the amount of synuclein pathology in the frontal cortex as iden

ases in the substantia nigra. Case 10 in Table 1 exhibited

DA-Lys-modified �-synuclein in the frontal cortex but not

n the substantia nigra.Intriguingly, two control cases showed curious patterns.

ase 5 showed MDA-Lys-modified �-synuclein in the frontal

ig. 5. Western blots of total homogenates of the frontal cortex fromontrol (HC) and incidental Parkinson’s cases (iPD), and MDA-Lys-mmunoprecipitates of the same samples (IPHC and IPiPD, respectively)mmunostained with anti-�-synuclein antibody, reveal specific bands at theppropriate molecular weight of 17 kDa. Both iPD cases and one controlright) show MDA-Lys-modified �-synuclein in the frontal cortex. Controlase on the left: 4, control case on the right, 5; iPD, cases 9 and 10 in Table 1.

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veal specific bands at the appropriate molecular weight of 17 kDa. IPDLBsd substantia nigra in MDA-Lys-immunoprecipitated-samples. Upper panel:

1. Note that the lower density of the lane corresponding to case 19 is notthe Braak stage (see Table 1).

ortex (but not in the substantia nigra) (Fig. 5). Case 7 bore

ipoxidized �-synuclein in the substantia nigra, but not in therontal cortex (Fig. 6). In both cases, the pattern expectedor iPD and PD cases run in parallel did not differ from the

ig. 6. Western blots of total homogenates of the substantia nigra from con-rol (HC) and Parkinson’s cases (PD), and MDA-Lys-immunoprecipitatesf the same samples (IPHC and IPPD, respectively) immunostained withnti-�-synuclein antibody, reveal specific bands at the appropriate moleculareight of 17 kDa. Both PD cases and one control (right) show MDA-Lys-odified �-synuclein in the substantia nigra. Control case on the left: 6,

ontrol case on the right, 7; PD, cases 13 and 14 in Table 1.

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ost common profiles observed in diseased brains. As seen inable 1, cases 5 and 7 were aged 82 and 49 years, respectively,

hus ruling out old age as the causative factor of �-synucleinipoxidation in these cases.

.3. α-Synuclein solubility and aggregation in control,PD, PD and DLB

�-Synuclein was recovered in the cytosolic fraction, ando a lesser extent in the deoxycholate fraction, in control, iPDnd PD samples. In addition, �-synuclein was recovered inhe SDS fraction only in DLB (Fig. 7).

No �-synuclein aggregates were seen in the frontal cor-ex in control and iPD cases. Discrete �-synuclein bands of

olecular weight between 50 and 70 kDa were found in theytosolic fraction in PD. �-synuclein aggregates were presentn the deoxycholate, and particularly in the SDS fraction onlyn DLB cases (Fig. 7).

.4. α-Synuclein immunoprecipitation andmmunoblotting

Total homogenates and �-synuclein-immunoprecipitatedamples of the frontal cortex were immunoblotted with-synuclein to validate the immunoprecipitation. Similarensity bands of �-synuclein 17 kDa were recovered inotal homogenates of control and diseased brains. Totalomogenates blotted with anti-ubiquitin showed strong andomogeneous immunoreactivity at 66 kDa in control, iPDnd PD samples, whereas several bands of variable molecular

eight were found in DLB. �-synuclein-immunoprecipitated

amples blotted for anti-ubiquitin revealed no specific stain-ng thus suggesting no ubiquitilation of non-aggegatedynuclein of 17 kDa (Fig. 8).

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ig. 7. Solubility and aggregation of �-synuclein in frontal cortex homogenates ofnd Dementia with Lewy Bodies (DLB). A specific band of 17 kDa is recovered in tDxc) fractions in CTL, iPD, PD and DLB. �-Synuclein is also recovered in the SDetected in the Cytosolic fraction in PD, and to a large extent in the Dxc and SDS f

f Aging 29 (2008) 408–417

. Discussion

MDA is one of the most abundant lipoperoxidation prod-cts in cells, and it can also be produced endogenously viarostaglandin biosynthesis (Esterbauer et al., 1991). MDA islso reactive with amino acids and proteins under certainhysiological conditions (Esterbauer et al., 1991; Uchida,000). MDA can form adducts primarily with lysine residuesf proteins and also, to some extent, with histidine, tyrosine,rginine and methionine residues (Esterbauer et al., 1991;chida, 2000). It has been suggested that at least 80% ofDA in tissues is reversibly bound to proteins (Slatter et al.,

004; Slatter et al., 2000).The present results have shown MDA-Lys modifications

f �-synuclein in the frontal cortex in one case and in theubstantia nigra in another with no clinical symptoms ando neuropathological evidence of LBD pathology, thus indi-ating that �-synuclein may be lipoxidized in an unknownercentage of individuals who might otherwise be considereds controls on the basis of clinical data and neuropathologi-al findings. That the presence of MDA-Lys-modified proteinay be age-related is not sustained in this short series, as one

f the cases was 49 years old. Whether some individualsearing MDA-Lys-modified �-synuclein might be prone touffering later DLB is a matter of further speculation.

Lipoxidized �-synuclein was found in the frontal cortexn the three cases with iPD and in the substantia nigra in/3 cases. Yet MDA-Lys-modified �-synuclein occurs in therontal cortex and in the substantia nigra in all cases withD, and in the frontal cortex and substantia nigra in all cases

ith DLB. Since no AD-related pathology occurred in the

elected group of iPD, PD and DLB cases (pure form ofLB), oxidative modifications in DLB can not be attributed

o other concomitant degenerative diseases.

control (CTL), incidental Parkinson disease (iPD), Parkinson disease (PD)he phosphate-buffered saline soluble (Cyt) and in the deoxycholate-solubleS fraction only in DLB. In addition, bands of higher molecular weights areractions only in DLB.

E. Dalfo, I. Ferrer / Neurobiology of Aging 29 (2008) 408–417 415

Fig. 8. �-Synuclein immunoprecipitation, and �-synuclein (A) and ubiquitin immunoblotting (B) in the frontal cortex in control, iPD, PD and DLB. Totalhomogenates are indicated as HC, HiPD, HPD and HDLB, whereas immunoprecipitates are labeled as IPC, IPiPD, IPPD and IPDLB, respectively, for control,i indicatw recipita� in the tw ted �-sy

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mdd

r�pfraamet�ta

PD, PD and DLB cases. Lanes marked as ProtG + S and ProtG + anti-�-synithout sample, and they are used as internal controls. �-Synuclein immunop-synuclein. The band at 36 kDa is not specific as it is found in all IPs andith anti-ubiquitin show no ubiquitin-immunoreactivity of immunoprecipia

Together, these findings suggest that lipoxidation acts atarly stages in both Lewy body-affected and Lewy body-non-ffected regions in LBDs, and that �-synuclein lipoxidations a constant event with disease progression. Regarding possi-le glycoxidation of �-synuclein, preliminary studies in ouraboratory have shown no evidence of AGE an, CML andEL modifications of �-synuclein in Lewy body diseases.

Oxidation and nitration of �-synuclein have been pro-osed as one of the mechanisms responsible for the formationf cross-linked �-synuclein oligomers (Dickson, 1999;iasson et al., 1999; Souza et al., 2000). The result of �-

ynuclein cross-linking by oxidation or nitration would behe formation of SDS-stable dimers and oligomers, whereas-synuclein self-oligomerization can be induced by oxida-

ive agents (Paik et al., 2000). Interestingly, antioxidantompounds show potent anti-�-synuclein-fibrillogenic and-synuclein-fibril-destabilizing effects, which is in accord

ith the enhancement of �-synuclein fibril formation byxidation (Ono and Yamada, 2006). Molecules such as tan-ic, rosmarinic and ferulic acids, curcumin, and tetracyclinenhibit �-synuclein fibril formation in a dose-dependent

eMws

e protein G with sample without the antibody and protein G with antibodytion reveals a specific band at about 17 kDa corresponding to non-aggregatedwo internal control lanes. Similar immunoprecipitated membranes blottednuclein in control and in diseased cases.

anner. Moreover, these molecules have been shown toestabilize preformed �-synuclein fibrils, also in a dose-ependent manner (Ono and Yamada, 2006).

In the present context, lipid peroxidation acquires majorelevance, in the sense that �-synuclein, the hallmark of-synucleinopathies, is being modified by MDA-Lys, inde-endently of the presence of �-synuclein aggregates in theorm of Lewy bodies and �-synuclein-immunoreactive neu-ites. Furthermore, MDA-Lys modifications in �-synucleinre not accompanied by changes in the solubility andggregation of �-synuclein, as revealed by biochemicalethods, corroborating and extending previous results

mphasizing the independence of lipoxidative damage andhe presence of �-synuclein aggregates in PD and related-synucleinopathies (Dalfo et al., 2005). Differences in

he level, type and temporal sequence of the oxidativelterations appear to result in both inhibitory and stimulatory

ffects on protein fibrillogenesis (Norris and Giasson, 2005).DA-Lys-modified �-synuclein is always present in areasith Lewy bodies and aberrant neurites, but lipoxidized �-

ynuclein is also present in the frontal cortex in PD and in the

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16 E. Dalfo, I. Ferrer / Neurobi

ajority of cases with pre-clinical PD. Therefore, it may benferred that lipoxidized �-synuclein is necessary but not suf-cient to the development of Lewy aggregates. Moreover, theresent findings suggest that ubiquitilation of abnormal non-ggregated �-synuclein is a late event in �-synucleinopathiess shown by using immunoprecipitation and immunoblottingethods.These observations give support not only to the con-

ept that lipoxidation precedes �-synuclein aggregationn LBDs (Dalfo et al., 2005), but also to the idea thatxidatively-altered proteins are present in the cerebral cortexn pre-clinical PD.

cknowledgements

This work was funded by grants from the Spanish Min-stry of Health, Instituto de Salud Carlos III PI05/1570 andI05/2214, and supported by the European Commissionnder the Sixth Framework Programme (BrainNet EuropeI, LSHM-CT-2004-503039). We thank T. Yohannan for edi-orial help.

There is no conflict of interest including any financial, per-onal or other relationships with other people or organizationsithin the three years of beginning the work.Brain samples were obtained from the Institute of Neu-

opathology and University of Barcelona Brain Banksollowing the guidelines and approval of the local Ethic Com-ittees.

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