Alterasi Pada Metabolisme, Peran Protein Dan Fungsi Sinapt

Proteomic analysis of parkin knockout mice: alterations inenergy metabolism, protein handling and synaptic function

Magali Periquet, Olga Corti, Sandrine Jacquier and Alexis Brice

INSERM U679, Hopital de la Salpetrie`re, AP-HP, 75013 Paris, France

Abstract

Parkin knockout (KO) mice show behavioural and biochemical

changes that reproduce some of the presymptomatic aspects of

Parkinsons disease, in the absence of neuronal degeneration.

To provide insight into the pathogenic mechanisms underlying

the preclinical stages of parkin-related parkinsonism, we sear-

ched for possible changes in the brain proteome of parkin KO

mice by means of fluorescence two-dimensional difference gel

electrophoresis and mass spectrometry. We identified 87 pro-

teins that differed in abundance between wild-type and parkin

KO mice by at least 45%. A high proportion of these proteins

were related to energy metabolism. The levels of several pro-

teins involved in detoxification, stress-related chaperones and

components of the ubiquitinproteasome pathway were also

altered. These differences might reflect adaptive mechanisms

aimed at compensating for the presence of reactive oxygen

species and the accumulation of damaged proteins inparkinKO

mice. Furthermore, the up-regulation of several members of the

membrane-associated guanylate kinase family of synaptic

scaffold proteins and several septins, including the Parkin

substrate cell division control related protein 1 (CDCRel-1), may

contribute to the abnormalities in neurotransmitter release

previously observed in parkin KO mice. This study provides

clues into possible compensatory mechanisms that protect

dopaminergic neurones from death in parkin KO mice and may

help us understand the preclinical deficits observed in parkin-

related parkinsonism.

Keywords: knockout mice, parkin, proteomic, two-dimen-

sional fluorescence difference gel electrophoresis.

J. Neurochem. (2005) 95, 12591276.

Parkinsons disease (PD) is a common neurodegenerativedisorder clinically characterized by resting tremor, rigidity andbradykinesia. These severe neurological symptoms are causedby the selective, progressive degeneration of dopaminergicneurones in the substantia nigra pars compacta. Lewy bodies ubiquitylated neuronal cytoplasmic inclusions are a patho-logical hallmark of PD (Forno 1987). Seven genes involved inrare monogenic forms of PD have been discovered in the past8 years (Dekker et al. 2003). Missense mutations in thea-synuclein gene, multiplications of a genomic region inclu-ding this gene (Singleton et al. 2003), as well as missensemutations in the recently discovered dardarin gene (Paisan-Ruiz et al. 2004; Zimprich et al. 2004) cause autosomaldominant forms of parkinsonism. The ubiquitin carboxyter-minal hydrolase L1 (UCH-L1) and Nurr-1 genes are alsopotentially involved in autosomal dominant parkinsoniansyndromes. However, their role in the pathogenesis of thedisease remains uncertain, because the corresponding muta-tions have been found in only a small number of families. Inaddition, the parkin, DJ-1 and PTEN-induced kinase 1(PINK1) genes are responsible for autosomal recessive formsof the disease (Dekker et al. 2003; Valente et al. 2004).

In 1998, the parkin gene was shown to be responsible for adistinct clinical and genetic entity in Japan, dened asautosomal recessive juvenile parkinsonism (Kitada et al.1998). A series of parkin exon rearrangements and pointmutations have since been identied in almost 50% of patientswith familial autosomal recessive early-onset parkinsonismfrom different populations, and in 15% of non-familial cases(Lucking et al. 2000; Lohmann et al. 2003; Periquet et al.

Received March 1, 2005; revised manuscript received May 10, 2005;accepted July 18, 2005.Address correspndence and reprint requests to Alexis Brice, INSERM

U679, Hopital de la Salpetrie`re, 47 Boulevard de lHopital, 75651 ParisCedex 13, France. E-mail: [email protected] used: AcCN, acetonitrile; CDCRel-1, cell division

control related protein 1; 2D DIGE, two-dimensional difference gelelectrophoresis; DTT, dithiothreitol; GTP2, glutathione S-transferase P2;IPG, immobilized pH gradient; KO, knockout; MAGUK, membrane-associated guanylate kinase; MALDITOF, matrix-assisted laserdesorption/ionizationtime of ight; MS, mass spectrometry; NSF,N-ethylmaleimide sensitive fusion protein; OTUB1, OTU-domain uba1-binding protein; PD, Parkinsons disease;PINK1, PTEN-induced kinase 1;UCH-L1, ubiquitin carboxyterminal hydrolase L1; WT, wild type.

Journal of Neurochemistry, 2005, 95, 12591276 doi:10.1111/j.1471-4159.2005.03442.x

2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276 1259

2003). These mutations are associated with a wide range ofages at onset and a broad phenotypic spectrum, includingcases clinically indistinguishable from idiopathic PD (Kleinet al. 2000; Lucking et al. 2000). Neuropathological descrip-tions of brains from patients with homozygous parkindeletions have reported specic dopaminergic neuronaldegeneration in the substantia nigra pars compacta in theabsence of Lewybodies (Mori et al. 1998; Hayashi et al.2000; van de Warrenburg et al. 2001; Gouider-Khouja et al.2003). However, Lewy body pathology and/or tau depositshave been described in a few individuals with compoundheterozygous mutations (Mori et al. 1998; Farrer et al. 2001).Parkin, a 465-amino acid protein with a ubiquitin-like

domain at its N-terminus and a C-terminal cysteine-richRING-IBR-RING motif, has E3 ubiquitinprotein ligaseactivity that promotes the ubiquitylation and proteasomaldegradation of specic protein substrates (Imai et al. 2000;Shimura et al. 2000; Zhang et al. 2000; Dev et al. 2003).Loss of Parkin function is thought to result in the progressiveaccumulation of non-ubiquitylated, potentially toxic sub-strates, leading to neurodegeneration. At least 10 Parkinsubstrates have been identied so far with roles in cellprocesses as diverse as cell signalling (Pael-R), cell cyclecontrol (cyclin E), protein biosynthesis (the p38 scaffoldsubunit of the multi-aminoacyl-tRNA synthetase compo-nent), cytoskeletal dynamics (a/b tubulin), and vesicular andsynaptic functions (CDCRel-1 and CDCRel-2, synaptotag-min IX, O-glycosylated a-synuclein, synphilin, the dopaminetransporter) (Hattori and Mizuno 2004; Jiang et al. 2004).However, the specic and potentially synergistic pathologicalroles of these substrates are unclear.We and others have recently generated a parkin knockout

(KO) mouse model to facilitate investigation of thepathogenic mechanisms underlying parkinsonism due toparkin gene mutations (Goldberg et al. 2003; Itier et al.2003; Von Coelln et al. 2004; Perez and Palmiter 2005).There was no evidence for a loss of nigrostriatal dopam-inergic neurones in these mice, but a number of behaviouraland biochemical changes were observed, including decitsin dopamine handling, reproducing some of the presymp-tomatic aspects of PD (Itier et al. 2003). This model istherefore of value for investigation of the functionalconsequences of parkin gene inactivation, including poten-tial compensatory mechanisms preventing the onset of aparkinsonian phenotype.To shed line on the molecular pathways affected in parkin

KO mice and identify potential Parkin substrates predicted toaccumulate in these mice, we performed a differential analysisof parkin KO and wild-type (WT) brain proteomes. Two-dimensional uorescence difference gel electrophoresis (2DDIGE)was chosen for this purpose, because it has proven to bevaluable for a number of biological applications, includingstudies related to neurodegenerative disorders such as schizo-phrenia and Huntingtons disease (Zabel et al. 2002; Swatton

et al. 2004). This technique involves the pre-electrophoreticlabelling of the protein samples to be compared withuorescent dyes, such as Cy2 and Cy5, which allowsstatistically valid quantication of a dynamic range of proteinconcentrations with high sensitivity (Patton 2000); thelabelled protein samples are pooled and mixed with aninternal standard prelabelled with Cy3 to normalize the resultsand reduce gel-to-gel variability. When used with the dedica-ted DeCyder analysis software (Amersham Bioscience Inc.,Amersham, UK), this technique permits the sensitive, massspectrometry (MS)-compatible and reproducible identicationof statistically signicant differences in the protein expressionproles of multiple samples examined simultaneously (Tongeet al. 2001; Gharbi et al. 2002; Yan et al. 2002).We combined this approach with sensitive matrix-assis-

ted laser desorption/ionizationtime of ight (MALDITOF) MS and tandem MS, to screen six WT and six parkinKO mice at 2 and 12 months, in two brain structures(cortex and striatum). Eighty-seven unique proteins wereidentied that differed in abundance between the brains ofparkin KO and WT mice. These differences provide newinformation concerning the molecular pathways that mightbe involved in the preclinical stages of parkin-relatedparkinsonism.

Experimental procedures

Animals and brain tissues

Studies were carried out on 2- and 12-month-old parkin KO (Itieret al. 2003) and WT mice with a pure 129SV background. Animalswere anaesthetized with sodium pentobarbital (60 mg/kg i.p.) and

perfused intercardially with saline. Brains were removed, and the

cortex and striatum tissues were dissected out and rapidly frozen.

Brain tissue was lyophilized for 48 h.

Preparation of protein samples

Lyophilized tissues (10 mg) were resuspended in 80 lL 0.032 MTris-HCl/Tris base containing 1.2% (v/v) Triton X-100. The

preparations were heated at 100C for 5 min, then homogenized,and 7.5 lL Dnase I and Rnase in 100 mM Pefabloc/100 mM EDTAwas gradually added. The mixture was incubated for 10 min, then

the proteins were solubilized by adding 105 mg urea, 38 mg

thiourea and 47 lL 21% CHAPS. Protein samples were kept atroom temperature (25C) for 10 min, gently vortexed, and thencentrifuged for 5 min at 14 000 g, followed by 45 min at100 000 g. Protein extracts were aliquoted and stored at )80C orimmediately run on rst-dimension gels.

Labelling of protein samples (DIGE)

Fluorescent dyes were conjugated to solubilized proteins via

N-hydroxysuccinimidyl linkages, such that 10% of each proteinwas labelled. Typically, 50 lg parkin KO or WT mouse proteinextract was labelled with 400 pmol cyanin dye Cy5 for parkin-KOand Cy2 for WT (Amersham Bioscience Inc.). As 2D-DIGE

analysis requires large amounts of protein, we used a pool of six

1260 M. Periquet et al.

2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276

mice to study the striatum proteome. We also pooled cortex samples,

to make it possible to compare the results obtained in the striatum

and cortex. A pool of all samples was also prepared and labelled

with Cy3, for use as an internal standard on all gels. Labeling

reactions were performed in the dark, at room temperature, for

30 min and were quenched by incubation with a 50-fold molar

excess of free lysine over dye for 10 min at room temperature.

Samples were diluted with an equal volume of solution containing

7 M urea, 2 M thiourea, 4% CHAPS, 67 mM dithiothreitol (DTT),

1% Pharmalyte 310 and 0.2% (v/v) Triton X-100.

2D gel electrophoresis

The rst dimension of electrophoresis was carried out with narrow

immobilized pH gradient gels (Immobiline Dry Strip, pH 4.56,

5.56.7, 69) on a horizontal electrophoresis apparatus (Multiphor

II; Amersham Pharmacia Biotechnology). Overlapping pH gradients

were used, to optimize protein resolution (Fig. 1). Immobilized pH

gradient (IPG) strips (0.5 3 80 mm), containing immobilinesNL 310, were rehydrated in a cassette containing 6 M urea, 2 M

thiourea, 1% (v/v) CHAPS, 0.5% Pharmalyte 310 and 0.4% DTT.

Isoelectric focusing was then performed by applying 50 lg labelled

proteins (for analytical gels) or 500 lg unlabelled proteins (forpreparative gels) to the anodic side. The samples were made to enter

the IPG strips by applying a low voltage gradient (50 V for 2 h,

100 V for 2 h, 300 V for 2 h). The gel was then run for 16 h at

2000 V and 18 h at 3500 V for the pH 4.56 and 5.56.7 gradients,

and for 1 h at 600 V and then 9 h at 3500 V for the pH 69

gradient. The strips were then equilibrated by incubating for 10 min

in 0.1 M Tris-HCl containing 0.5% (v/w) DTT, 36% urea and 30%

(v/w) glycerol, and then for a further 10 min in a solution of the

same composition except that DTT was replaced by 4.5%

iodoacetamide. In the second dimension, strips were subjected to

13% T, 2.54% C sodium dodecyl sulfatepolyacrylamide gel

elctrophoresis, using the Iso-Dalt apparatus (Hoeffer, San Francisco,

CA, USA; T corresponds to the total percentage concentration of

acrylamide and N,N-methylenebisacrylamide in the gel, and Ccorresponds to the concentration of N,N-methylenebisacrylamideas a percentage (by weight of acrylamide and N,N-methylenebis-acrylamide). Gels were run at 10C, 50 mA for 1.5 h, then at100 mA for 1.5 h and overnight at 185 mA. For each set of

conditions, we ran three analytical and two preparative gels in

parallel.

4.5(a)

(b)

200 kDa

15 kDa

MW

pl plpl6 6 96.75.5

Fig. 1 2D gels images showing the narrow range of overlapping pH

gradients. (a) Analytical gels. In order to optimize protein resolution,

the first dimension of electrophoresis was carried out with narrow

overlapping pH gradient gels (Immobiline Dry Strip, pH 4.56, 5.56.7,

69). Some 50 lg of each sample was labelled with cyanin dyes and

loaded on the analytic gels. (b) Preparative gels; 500 lg unlabelled

proteins were detected by staining with Sypro-Ruby dye after elec-

trophoresis. The patterns of staining for the cyanin dyes and Sypro-

Ruby dye were very similar, facilitating accurate matching and picking

on these preparative gels.

Proteomic analysis of parkin knockout mice 1261


Protein visualization

The Cy2, Cy3 and Cy5 components of each gel were imaged

individually using mutually exclusive excitation/emission wave-

lengths on a ProXpress (Perkin Elmer, Norwalk, CT, USA)

uorescent gel scanner. Gel images were normalized by adjusting

exposure times according to mean pixel values. Unlabelled proteins

(preparative gels) were detected by staining with Sypro-Ruby dye

(Molecular Probes, Eugene, OR, USA) after electrophoresis and

images were acquired as above.

Image analysis

For each condition, pools of six WT and six KO samples were

labelled with Cy2 and Cy5 respectively, and run on three replicate

gels together with a Cy3-labelled mixture of all 12 WT and KO

samples as the in-gel standard. The differential in-gel analysis

module of the DeCyder software was used to quantify protein spot

volumes for each in-gel image pair (Cy2Cy3, Cy5Cy3) and

express the values as a ratio (Cy2/Cy3, Cy5/Cy3). The DeCyder

biological variation analysis module was subsequently used to

match the protein spot maps of all replicate gels. This software

module calculates the average changes in the Cy2/Cy3 and Cy5/Cy3

ratios across gels and applies statistics (Students t-test) to associatea level of condence with each of those changes. Only the proteins

presenting variations in the Cy2/Cy3 and Cy5/Cy3 ratios exceeding

an arbitrary threshold of 1.45, corresponding to a change in

abundance of 45%, and with a p-value < 0.05, were considered to besignicantly different. Proteins were dened as up-regulated or

down-regulated if their abundance was higher or lower, respectively,

in parkin KO mice than in WT mice.

MS

As the molecular mass of the labelled protein is 0.5 kDa greaterthan that of the unmodied protein, we labelled the minimum

number of molecules for each protein and excised the unlabelled

protein spot rather than the labelled protein spot for mass

spectrometry. The differences observed in 2D DIGE analyses were

compared with Sypro-Ruby protein patterns, and spots were selected

for picking (Ettan spot picker; Amersham Biosciences, Little

Chalfont, UK) on the basis of this staining pattern. The patterns

of staining for the cyanin dyes and Sypro-Ruby dye were very

similar, facilitating accurate matching and picking (Fig. 1).

Up-regulated proteins were excised from a preparative gel contain-

ing a KO sample and down-regulated proteins were excised from a

preparative gel containing a WT sample.

The proteins were reduced with DTT (Sigma, Poole, UK),

alkylated with iodoacetamide and digested with trypsin (modied

trypsin, sequencing grade; Roche, Indianapolis, IN, USA) overnight

at 37C, using the automatic DIGESTPRO digester from ABIMED(Longenfeld, Germany). Tryptic digests were dried under vacuum in

a Speed-Vac. Samples were resuspended in 4 lL 0.1% formic acid.A 0.5-lL aliquot of each sample was used to measure automaticallythe mass ngerprint on a Bruker Reex III MALDITOF mass

spectrometer (Bruker-Daltonix GmbH, Bremen, Germany) in

positive ion reector mode using delayed extraction. The measured

tryptic peptide masses were transformed automatically, through the

MS BioTools program, into input used by Mascot software (Matrix

Science, London, UK) to search the National Center for Biotech-

nology Information (NCBI) database.

To conrm some of the ngerprints, tryptic digests were

separated by HPLC, using the LC-Packings system (San Francisco,

CA, USA), including an injector (Famos), a concentrator (Switchos)

and a pump (Ultimate). The ow rate was adjusted to 200 nL/min. A

gradient was used, starting at 2% Acetonitrile (AcCN) in 0.1%

formic acid for 1 min, increased to 50% AcCN over 40 min, and

nally to 90% AcCN over 10 min. A 1-lL aliquot was injected fromthe autosampler (in user dened program mode) into a

15 cm 75 lm fused silica column packed with PLRP-S 5 lm(Polymer Laboratories).

The LC system was connected to an ion trap mass spectrometer

(LCQ Deca; Finnigan Corp, San Jose, CA, USA), run by Xcalibur

software. The spray voltage was set at 2.1 kV, the temperature of the

ion transfer tube was set at 180C and the normalized collisionenergies were set at 35% for MS/MS. We used dynamic exclusion.

The sequences of the uninterpreted CID spectra were identied by

correlation with the peptide sequences present in the NCBI non-

redundant protein database, using the SpectrumMill program

(Millenium Pharmaceuticals, Cambridge, MA, USA).

Protein annotation and data handling

The MALDI-TOF MS and MSMS analyses that followed our 2D

DIGE approach were successful for about 74% (159 spots) of all

spots showing altered abundance in parkin KO mice. Owing to post-translational modications and the use of overlapping pH gradients,

the 159 protein spots identied in both the cortex and the striatum

from 2- and 12-month-old mice corresponded to 87 different

proteins. To ensure a high quality of annotation, the proteins

identied were compared with those in the Swissprot sequence

database, using the Blast2 algorithm. A relational database

containing protein annotations and experimental results was created

(Access; Microsoft Corporation, Redmond, WA, USA) to facilitate

analysis.

Quantitative western blot analyses

CDCRel-1 and calretinin protein levels were analysed in samples from

the cortex of 2- and 12-month-old mice respectively (n 5 forWTorparkinKOmice).We ran about 40 lg total protein extract in each laneof a 15-well, precast 412% gradient sodium dodecyl sulfate

polyacrylamide mini gel (Invitrogen, Carlsbad, CA, USA). After

electrophoresis, the proteins were transferred to nitrocellulose lters

(Protran, Schleicher & Schuell Bioscience, Dassel, Germany). The

lter was blocked by incubation in 0.2% Tween and 5% non-fat milk

powder in phosphate-buffered saline, followed by anti-calretinin

(1 : 10000; Swant, Bellinzona, Switzerland), anti-CDCRel1

(1 : 5000) or anti-actin (1 : 2000; gift from Sigma, St Louis, MO,

USA) antibodies. Secondary antibodies radiolabelled with 125I

(1 : 200; IM 131 and IM 134, Amersham Biosciences) were visual-

ized by phosphoimaging and quantied by Aida analysis software

(Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany).

Oxyblot analyses

Protein carbonyls were assayed by western blot analysis in brains of

2- and 12-month-old WT and KO mice, according to the

manufacturers instructions (Oxyblot; Chemicon, Temecula, CA,

USA). In brief, 15 lg protein from individual cortex and striatumextracts obtained in 50 mM Hepes containing 150 mM NaCl, 10%

glycerol, 1% Triton X-100, 100 mM NaF, 0.2 mM Na3VO4 and



complete protease inhibitors (Roche) was reacted with 2,4-di-

nitrophenylhydrazine and western blotted using a primary antibody

specic to dinitrophenylhydrazone-derivatized residues (Oxyblot;

Chemicon) and a 125I-labelled secondary antibody (Amersham

Biosciences) or a non-radioactive secondary antibody (Oxyblot;

Chemicon). Protein carbonyls were visualized by phosphoimaging

or revealed using enhanced chemioluminescence and quantied by

densitometry. Blots were subsequently reprobed for actin immuno-

reactvity (1 : 2000; Sigma) and revealed using enhanced chemio-

luminescence (Pierce, Rockford, IL, USA).

Results

Analysis of cortical and striatal mouse tissues at 2 and12 months of age using 2D DIGE technology led to theidentication of 159 differentially regulated protein spotsbetween WT and parkin KO mice. In 2D analysis, proteinsare frequently detected in more than one spot, indicating thepresence of either different isoforms and/or post-translationalmodications. In order to optimize protein resolution and tobetter detect these different isoforms, the rst dimension ofelectrophoresis was carried out with narrow overlapping pHgradient gels (Immobiline Dry Strip, pH 4.56, 5.56.7, 69;Fig. 1). The differentially regulated spots were rst analysedby MALDITOF MS, using peptide mass ngerprints anddatabase searches. Proteins not identied by this methodwere subjected to MS/MS, followed by a search of sequencedatabases. With these techniques, we identied 87 uniqueproteins that differed in abundance by at least 45% in WTand parkin KO mice (Tables 13). In 2-month-old mice, thenumber of proteins found to be differentially regulated in thecortex and the striatum and the proportions of up- and down-regulated proteins were similar (Table 1). In contrast, in12-month-old mice, the majority of the proteins with alteredabundance were found in the striatum and most of them wereup-regulated (p < 0.05) (Table 1). Approximately 20% (18of 87) of the identied proteins were differentially regulatedin both 2- and 12-month-old parkin KO mice and a similarpercentage (14/87) were dysregulated in both the cortex andthe striatum, at one or both of the ages examined (Tables 4and 5). Overall, 46 proteins that increased in abundance wereidentied, 31 that decreased in abundance, three that changedtheir pattern of regulation between 2 and 12 months, and

seven that had a change in their electrophoretic mobility. Themobility variants were represented by at least two independ-ent spots with different isoelectric points of a sameprotein subjected to opposite regulation in one and the sameexperiment (status +/ in Tables 25). They usually corres-pond to different isoforms of a protein that has undergonepost-translational modication, i.e. a shift in phosphorylationstatus, as illustrated in Fig. 2(c). Other examples of changesin staining intensity are illustrated in Fig. 2.Classication of the differentially regulated proteins

according to the specic keywords attributed to them in theSwissprot database (Table 6) showed that a certain propor-tion of the modulated proteins were linked to energymetabolism (glycolysis, ATP synthesis, avoprotein, hydro-gen ion transport, FAD, NAD/NADP, mitochondrion) andprotein processing pathways (heat shock, proteasome, pro-tein biosynthesis, ligase, chaperone, transit peptide, isom-erase). The frequent occurrence of the keywords kinase andGTP binding suggested that cell signalling pathways werelikely to be disrupted, together with vesicle trafcking andcytoskeletal dynamics processes in which proteins withkinase and GTPase activities play major roles.Extensive literature searches in Pubmed, to characterize

each protein, led to the identication of 12 distinctfunctional categories, each including at least two proteinsup- or down-regulated in the absence of the parkin gene(Fig. 3, Tables 2 and 3). Sixty-seven of the 87 identiedproteins fell into these categories; nine, represented byseveral spots with either increased or decreased abundance,a situation compatible with post-translational modicationsor a change in the pattern of regulation between 2- and12-month-old mice, were not included in Fig. 3. Elevenproteins could not be assigned to established functionalcategories based on their putative functions. Among theseproteins, the calcium-binding protein calretinin was found.Calretinin, a brain-specic, potentially neuroprotective pro-tein (Mura et al. 2000; Tsuboi et al. 2000), was down-regulated in the cortex of 12-month-old KO mice (Table 3).The down-regulation of this protein was conrmed byquantitative western blot following the one-dimensional gelelectrophoresis of protein samples from mouse cortex(Fig. 4).

Table 1 Number of proteins that differed

significantly in abundance in WT and parkin

KO mice Age (months)

Cortex Striatum

Increased Decreased Altered EM Increased Decreased Altered EM

2 15 12 5 19 18 3

12 4 4 0 23 5 0

The total number of matched protein spots that differed in abundance by at least 45% in WT and

KO samples is shown for 2- and 12-month-old mice. Note that the sum of these proteins differs

from the total number of 87 identified proteins, because some of them are regulated in two

structures or at two ages. Fold differences were calculated from the mean standardized abundance

of triplicate spots. Only means with p values < 0.05 were included. EM, electrophoretic mobility.



Table 2 Proteins differentially regulated in the striatum of WT and parkin KO mice

SwissProt or gi accession Protein name

No. and status of spots

Striatum

2 months

Striatum

12 months

Energy metabolism (n 17)ALFC_MOUSE Fructose-bisphosphate aldolase C [Fragment] 1

ATPA_MOUSE ATP synthase a chain, mitochondrial 3+ 2+

CISY_HUMAN Citrate synthase 1 1

DLDH_MOUSE Dihydrolipoamide dehydrogenase 1+

DHSA_HUMAN Succinate dehydrogenase flavoprotein subunit 1+

G3P_MOUSE Glyceraldehyde-3-phosphate dehydrogenase 2+

KAD3_MOUSE GTP:AMP phosphotransferase mitochondrial 1+ 1+

KPY2_MOUSE Pyruvate kinase, M2 isozyme 1/2 4

LDHA_MOUSE L-Lactate dehydrogenase A chain 1

MDHC_MOUSE Malate dehydrogenase, cytoplasmic 1

NUBM_HUMAN NADH-ubiquinone oxidoreductase 51-kDa subunit 1+

ODPB_RAT Pyruvate dehydrogenase E1 component b subunit 1

PGK1_MOUSE Phosphoglycerate kinase 1 *1

SCB2_MOUSE Succinyl-CoA ligase [GDP-forming] b-chain, mitochondrial 1+

THIL_RAT Acetyl-CoA acetyltransferase, mitochondrial 1+

UCR1_RAT Ubiquinol-cytochrome c reductase complex core protein I 1+

539926/13435978 Acetyl-CoA C-acetyltransferase 1+

Signal transduction (n 8)ARK1_RAT b-Adrenergic receptor kinase 1 *1

CRK_MOUSE Proto-oncogene C-crk 1

DPY2_MOUSE Dihydropyrimidinase-related protein-2 (CRMP-2) 5/2+ 5+

P2BA_MOUSE Ser/Thr protein phosphatase 2B catalytic subunit, a isoform 1+

PP1B_HUMAN Ser/Thr protein phosphatase PP1-b catalytic subunit 1+

PTNB_MOUSE Protein tyrosine phosphatase, non receptor type 1-1 1

143Z_MOUSE 14-3-3 prot f/d 1

MPP3_HUMAN MAGUK p55 subfamily member 3 1+

Vesicular trafficking (n 8)GDIR_HUMAN rho GDP-dissociation inhibitor 1 2+

NSF_MOUSE N-ethylmaleimide-sensitive fusion protein 1- 1+

ST1B_MOUSE Syntaxin 1B 1

STB1_MOUSE Syntaxin-binding protein 1 1 1+

Septin family

SEP5_MOUSE Septin 5 1+ 1+

SEP7_MOUSE Septin 7 1+

Y202_HUMAN Septin-like protein KIAA0202 1+

8922712 Hypothetical protein FLJ10849 (Septin 2 or 6 homologue) 1+ 1+

Protein folding (n 2)GR75_MOUSE Stress-70 protein 1+

HS7C_MOUSE Heat-shock cognate 71-kDa protein 1

Stress/detoxification (n 4)DHCA_MOUSE Carbonyl reductase [NADPH] 1 1+

GTP2_MOUSE Glutathione S-transferase P 2 *2

LGUL_MOUSE Glyoxalase I *1+

TRXB_MOUSE Thioredoxin reductase 1+

Cytoskeleton (n 4)AR20_HUMAN ARP2/3 complex 20-kDa subunit 1

CAP1_MOUSE Adenyl cyclase-associated protein 1 *1/1+

DYN1_MOUSE Dynamin-1 1 1+

TBA1_MOUSE Tubulin a1 chain 3+



This approach conrmed that a large proportion of thedifferentially regulated proteins were involved in energymetabolism (nup 9, ndown 10, nup/down 4), includingthe glycolytic pathway, the Krebs cycle and the mitochond-rial respiratory chain. The differentially regulated proteinsalso played roles in signal transduction pathways (nup 6, ndown 4, nup/down 1), vesicle trafcking (nup 6,ndown 1, nup/down 2), cytoskeletal dynamics (nup 2, ndown 2, nup/down 2), protein folding (nup 3,ndown 3) and degradation (nup 2, ndown 1), and theoxidative stress response and/or detoxication processes(nup 4, ndown 1). These latter changes occurred in theabsence of modications in protein oxidation, as revealed bythe western blot analysis of protein carbonyls in brain lysatesof 2-month-old (not shown) and 12-month-old WT and KO

mice (Fig. 5). Other categories included proteins involved inlipid metabolism (nup 3, ndown 2), amino acid andprotein biosynthesis pathways (nup 2, ndown 2), RNAprocessing (nup 1, ndown 1) and neurotransmitter hand-ling (nup 2).The proteins that increased in abundance in parkin KO

mice are potential substrates of Parkins E3 ubiquitinproteinligase activity. Three members of the membrane-associatedguanylate kinase (MAGUK) family of neuronal scaffoldingproteins (p55 subfamily members 2, 3 and 6) were foundamong the proteins found to be exclusively up-regulated inparkin KO mice. In addition, members of the septin family(CDCRel-1/septin5, septin 7, septin-like protein KIAA0202,hypothetical septin 2 or 6 homologue FLJ10849) (Tables 2and 3) were up-regulated both in the cortex and in the

Table 2 (Continued)



Striatum

2 months

Striatum

12 months

Protein degradation (n 3)PSB5_MOUSE Proteasome subunit beta type 5 1+

UBL1_MOUSE Ubiquitin carboxyterminal hydrolase L1 1+

18490720 Deubiquitinating enzyme OTUB1 1

Lipid metabolism (n 3)ACDV_MOUSE Acyl-CoA dehydrogenase, very-long-chain specific 1+

PCCA_RAT Propionyl CoA carboxylase a chain 1+

6678760 Lysophospholipase 1 1

Protein biosynthesis (n 2)SYY_HUMAN Tyrosyl tRNA synthetase 1+

SYTC_HUMAN Threonyl tRNA synthetase 1

Amino acid synthesis (n 2)GLNA_MOUSE Glutamine synthetase 1

SRR_MOUSE Serine racemase 1+

RNA processing (n 3)HE47_RAT Probable ATP-dependent RNA helicase p47 1

PCB2_MOUSE Poly(rC)binding protein 1+

Neurotransmitter metabolism (n 2)GABT_RAT 4-Aminobutyrate aminotransferase, mitochondrial 1+ 2+

TY3H_MOUSE Tyrosine 3-hydroxylase 1+

Others (n 7)ALBU_MOUSE Serum albumin 1

ARHY_MOUSE ADP-ribosylarginine hydrolase 1+

CAH2_MOUSE Carbonic anhydrase II 3

FCE2_MOUSE Low-affinity immunoglobulin epsilon FC receptor 1+

KPR1_HUMAN Ribose-phosphate pyrophosphokinase I 1

POR1_MOUSE Voltage-dependent anion-selective channel protein 1 2+

SPEE_MOUSE Spermidine synthase 1+

2D analysis allows the resolution of different isoforms and/or post-translational modifications of a same protein. Thus, several dysregulated spots

can correspond to a unique protein. These isoforms were in some cases all up-regulated (status +) or down-regulated (status ) in parkin KO mice,

or subjected to opposite regulation, owing to altered electrophoretic mobility (status +/). Owing to the use of overlapping pH gradients, the same

protein could also be detected twice in two consecutive pH gradients gels. In all the tables of the article, the number of spots corresponding to the

same protein is noted next to the status associated. *Proteins differing in abundance by a factor > 2.



Table 3 Proteins differentially regulated in the cortex of WT and parkin KO mice



Cortex 2 months Cortex 12 months

Energy metabolism (n 13)ATPA_MOUSE ATP synthase a chain, mitochondrial 1/1+ 2+

ATPB_MOUSE ATP synthase b chain, mitochondrial 1

DHSA_HUMAN Succinate dehydrogenase flavoprotein subunit 1+

ENOG_MOUSE c Enolase 1/1+

G3P_MOUSE Glyceraldehyde-3-phosphate dehydrogenase 3/1+

LDHA_MOUSE L-Lactate dehydrogenase A chain 1

MDHC_MOUSE Malate dehydrogenase, cytoplasmic 2

NUCM_HUMAN NADH-ubiquinone oxidoreductase 49-kDa subunit 1-

ODPA_MOUSE Pyruvate dehydrogenase E1 component a subunit 1+

PGK1_MOUSE Phosphoglycerate kinase 1 *1

UCR1_RAT Ubiquinol-cytochrome c reductase complex core protein I 1+

UCR2_MOUSE Ubiquinol-cytochrome c reductase complex core protein 2 1

128325765 NADH-ubiquinone oxidoreductase PDSW subunit HS homolog 1

Signal transduction (n 4)DPY2_MOUSE Dihydropyrimidinase-related protein-2 (CRMP-2) 2/3+

9910474 MAGUK p55 subfamily member 6 1+

7709986 Sumo-1 activating enzyme subunit 2 1+

MPP2_HUMAN MAGUK p55 subfamily member 2 1+

Vesicular trafficking (n 3)NSF_MOUSE N-ethylmaleimide sensitive fusion protein 1+

SEP7_MOUSE Septin 7 1+

SNX5_MOUSE Sorting nexin 5 *1+

Protein folding (n 6)GR75_MOUSE Stress-70 protein 2+

GR78_MOUSE 78-kDa glucose-regulated protein 1

HS72_MOUSE Heat-shock-related 70-kDa protein 2 1

HS7C_MOUSE Heat-shock cognate 71-kDa protein 2

OS94_MOUSE Heat-shock 70-related protein APG-1 1+

TCP2_MOUSE T-complex protein 1, a subunit B 1+

Stress/detoxification (n 2)ACON_HUMAN Aconitate hydratase, mitochondrial 2+

GTP2_MOUSE Glutathione S-transferase P 2 *1

Cytoskeleton (n 2)ACTG_HUMAN c-Actin 1

SPCN_MOUSE Spectrin a chain, brain 2+

Protein degradation (n 1)UBA1_MOUSE Ubiquitin-activating enzyme E1 type 1 1+ 1+

Lipid metabolism (n 2)CAO1_MOUSE Acyl-coenzyme A oxidase 1, peroxisomal 1+

MTE1_MOUSE Acyl CoA thioester hydrolase 1

Others (n 5)A1A3_RAT Sodium/potassium-transporting ATPase a-3 chain 1

CLB2_MOUSE Calretinin 1

DD19_MOUSE ATP-dependent RNA helicase DDX19, dead box protein 1+

MO25_MOUSE No known function 1

POR1_MOUSE Voltage-dependent anion-selective channel protein 1 1/1+

2D analysis allows the resolution of different isoforms and/or post-translational modifications of a same protein. Thus, several dysregulated spots

can correspond to a unique protein. These isoforms were in some cases all up-regulated (status +) or down-regulated (status ) in parkin KO mice,

or subjected to opposite regulation, owing to altered electrophoretic mobility (status +/). Owing to the use of overlapping pH gradients, the same

protein could also be detected twice in two consecutive pH gradients gels. In all the tables of the article, the number of spots corresponding to the

same protein is noted next to the status associated. *Proteins differing in abundance by a factor > 2.



striatum, at both ages (Tables 4 and 5). In particular, vespots, migrating with the same molecular weight but withdifferent isoelectric points, were identied as the Parkinsubstrate CDCRel-1/septin5 (M. Duchesne, personal com-munication). The abundance of one of these spots increasedby at least 45% in the striatum of 2- and 12-month-old parkinKO mice compared with WT mice (Tables 2 and 4). Inaddition, the levels of a second spot were increased by 33%in the striatum of 12-month-old mice (p < 0.021). As wedecided arbitrarily to take into consideration only the mostsignicant protein abundance changes (> 45%), this proteinis not listed in the tables presented. A reproducible increasein total CDCRel-1 levels was conrmed in individual cortexsamples (Fig. 4) and pooled striata (data not shown) of

parkin KO mice, resolved by conventional one-dimensionalgel electrophoresis and analysed by quantitative westernblotting. However, this increase did not reach statisticalsignicance. This is probably due to the fact that the vedifferent CDCRel-1 isoforms with identical molecular weightmigrate as a single band on one-dimensional gels.Additional Parkin substrates showed altered abundance in

our parkin KO model. Huynh et al. (2003) reported thatsynaptotagmin XI and synaptotagmin I are ubiquitylated byParkin. In our study, synaptotagmin I was found to beup-regulated by 40% (p < 0.001) in the cortex of 2-month-old mice (data not shown). In addition, several spotscorresponding to the tubulin a-1 chain (Ren et al. 2003)were increased in abundance in the striatum of 12-month-oldparkin KO mice (Table 2). Finally, we also analysed thelevels of our previously identied Parkin substrate p38 (Cortiet al. 2003) by one-dimensional western blotting, although itwas not identied as being altered in abundance in our 2D

Table 5 Proteins differentially regulated in cortex and striatum at 2

and/or 12 months

SwissProt or

gi accession

Age

(months)

No. and status

of spots Fold

Energy metabolism

ATPA_MOUSE 2/12 1/8+ > 1.45

DHSA_HUMAN 2 2+ > 1.45

G3P_MOUSE 2 3/3+ >1.45

LDHA_MOUSE 2/12 2 > 1.45

MDHC_MOUSE 2/12 3 > 1.45

PGK1_MOUSE 2/12 2 > 1.45

UCR1_RAT 2/12 2+ > 1.45

Signal transduction

DPY2_MOUSE 2/12 7/10+ > 1.45

Vesicular trafficking

NSF_MOUSE 2/12 12+ > 1.45

SEP7_MOUSE 2/12 2+ > 1.45

Protein folding

GR75_MOUSE 12 3+ > 1.45

HS7C_MOUSE 2 3 > 1.45

Stress/detoxification

GTP2_MOUSE 2 3 > 2.00

Others

POR1_MOUSE 2/12 1/3+ > 1.45

2D analysis allows the resolution of different isoforms and/or post-

translational modifications of a same protein. Thus, several dysregu-

lated spots can correspond to a unique protein. These isoforms were

in some cases all up- (status +) or down-regulated (status -) in parkin

KO mice, or subjected to opposite regulation, owing to altered elec-

trophoretic mobility (status +/-). Owing to the use of overlapping pH

gradients, the same protein could also be detected twice in two con-

secutive pH gradient gels. In all the tables of the article, the number of

spots corresponding to the same protein is noted next to the status

associated.

Table 4 Proteins differentially regulated at both 2 and 12 months

SwissProt/gi accession

No. and status

of spots Fold

Energy metabolism

ATPA_MOUSE 1/8+ > 1.45

CISY_HUMAN 2 > 1.45

KAD3_MOUSE 2+ > 1.45

KPY2_MOUSE 5/2+ > 1.45

LDHA_MOUSE 2 > 1.45

MDHC_MOUSE 3 > 1.45

PGK1_MOUSE 2 > 2.00

UCR1_RAT 2+ > 1.45

Signal transduction

DPY2_MOUSE 7-/10+ > 1.45

Vesicular trafficking

NSF_MOUSE 1/2+ >1.45

STB1_MOUSE 1/1+ > 1.45

Septin family

SEP5_MOUSE 2+ > 1.45

SEP7_MOUSE 2+ > 1.45

8922712 2+ > 1.45

Cytoskeleton

DYN1_MOUSE 1/1+ > 1.45

Protein degradation

UBA1_MOUSE 2+ > 1.45

Neurotransmitter metabolism

GABT_RAT 3+ > 1.45

Others

POR1_MOUSE 1/3+ > 1.45

2D analysis allows the resolution of different isoforms and/or post-

translational modifications of a same protein. Thus, several dysregu-

lated spots can correspond to a unique protein . These isoforms were

in some cases all up-regulated (status +) or down-regulated (status )

in parkin KO mice, or subjected to opposite regulation, owing to altered

electrophoretic mobility (status +/). Owing to the use of overlapping

pH gradients, the same protein could also be detected twice in two

consecutive pH gradients gels. In all the tables of the article, the

number of spots corresponding to the same protein is noted next to the

fold difference associated.



DIGE analysis. The quantitative analysis of protein samplesfrom individual striata from 12-month-old KO and WT micedid not reveal any signicant change in abundance of p38levels (Fig. 4).

Discussion

Nowadays, 2D DIGE is the most powerful 2D polyacryla-mide gel electrophoresis-based approach for widespreadprotein proling. We used this recent technology to compareprotein expression proles in parkin KO and WT mice, usinga pool of samples as an internal standard and the dedicatedDeCyder analysis software developed by Amersham Bio-sciences (Tonge et al. 2001; Gharbi et al. 2002; Yan et al.2002). Owing to the large amount of material required for 2DDIGE, our analyses were performed on pools of brainextracts obtained from six animals for each condition. Thisexperimental paradigm gives an appropriate indication of the

average biological differences between groups of samples,although it does not provide information on inter-animalvariation. The validity of this approach was demonstrated ina previous study showing that similar results are obtainedwhen individual animals or pooled samples are analysed by2D DIGE (Tonge et al. 2001). We found that 87 proteinsdiffered in abundance by at least 45% in parkin KO and WTmice.Classication of these proteins led to the identication of

12 major functional categories affected by inactivation of theparkin gene. The most frequently represented categoryincluded proteins related to energy metabolism, particularlyto the glycolytic pathway, the Krebs cycle and the mitoch-ondrial respiratory chain. This functional class has also beenshown to be affected at the mRNA and/or protein levels inother models of cell degeneration and in neurodegenerativediseases (Loring et al. 2001; Gozal et al. 2002; Napolitanoet al. 2002; Seong et al. 2002; Tilleman et al. 2002a, 2002b;Xie et al. 2002; Kuhn et al. 2003). These proteins wereeither up- or down-regulated, or subject to post-translationalmodication, such that the overall consequences of theirdifferential regulation are difcult to predict. In the absenceof a neurodegenerative phenotype, these changes probably

Fig. 2 2D gel images showing selected differentially regulated pro-

teins. (a) An up-regulated spot (boxed protein) identified as aconitase

hydratase, which was 1.97 times more abundant in the cortex of

2-month-old KO than in WT mice. (b). A down-regulated spot (boxed

protein) identified as phosphoglycerate kinase 1, which was 2.09 times

less abundant in the cortex of 2-month-old KO than in WT mice. (c)

Two isoforms of a chain ATP synthase protein, which were regulated

differently in the cortex at the age of 2 months. The first isoform was

down-regulated by at least 60% ( 1.66), whereas the second morebasic variant was up-regulated to the same extent ( 1.63), suggestinga shift due to phosphorylation or other post-translational modifications.

Squares and circles indicate downward and upward changes in parkin

KO mice respectively.

Table 6 Functions of differentially regulated proteins assessed by

keyword analysis

Occurrences SwissProt keyword Confidence index

16 Glycolysis 20.66

6 Heat_shock 13.34

5 ATP_synthesis 13.34

7 Flavoprotein 7.37

5 Hydrogen_ion_transport 6.46

5 Proteasome 6.25

8 Ligase 6.16

5 FAD 5.56

8 Lyase 5.52

7 NADP 5.29

5 Protein_biosynthesis 5.13

8 Chaperone 4.85

8 NAD 4.71

18 Transit_peptide 4.68

8 Kinase 4.64

11 Acetylation 4.04

25 Oxidoreductase 3.86

7 Magnesium 3.79

6 Isomerase 3.58

9 GTP binding 3.37

22 Mitochondrion 3.21

Only keywords occurring more than four times were taken into account

and their relative significance (confidence index 3) was determinedby normalizing the observed frequency of each keyword to the relative

frequency in the Swissprot database as a whole. Only entries for

mouse proteins were considered.



reect an adaptive regulation of cellular energy in parkin KOmice, as suggested by the altered abundance of enzymesinvolved in glycolysis (glyceraldehyde-3-dehydrogenase,c-enolases, pyruvate kinase) and energy regeneration(subunits of the mitochondrial ATP synthase).Several lines of evidence suggest that dysfunction of the

ubiquitin-dependent proteasomal degradation pathway plays

a major role in the pathophysiology of both familial andsporadic PD. Changes in proteins related to this pathwayhave previously been reported at the transcriptional level incell and mouse models of PD (Cadet et al. 2001; Ryu et al.2002; Kuhn et al. 2003). In parkin KO mice, the abundanceof several stress-induced chaperones was altered, includingheat-shock protein 70-related proteins, the osmotic stressprotein Osp94 and the T complex protein 1, which plays arole in the folding of actin and tubulin, and probably also ofother cytoskeletal proteins (Dunn et al. 2001). Of note,several cytoskeletal proteins were altered in abundance inparkin KO mice, including the Parkin substrate tubulin a-1chain (Ren et al. 2003). In addition, the PD-associatedprotein UCH-L1, and the proteasome subunit b type 5 weremore abundant in parkin KO mice, whereas the deubiquityenzyme OTU-domain Uba1-binding protein (OTUB1) wasless abundant. These modications were paralleled bychanges in the levels of several enzymes linked to cellularstress and detoxication processes. In particular, the level ofthe antioxidant protein Glutathione S-transferase P2 (GTP2)was decreased in both striatum and cortex from 2-month-oldparkin KO mice, whereas other proteins (carbonyl reductase,glyoxalase I, thioredoxin reductase) known to be protectiveagainst oxidative stress-induced neurodegeneration (Chenet al. 2004) increased in abundance in these mice. Thesechanges might reect an adaptive response to high concen-trations of free radicals, as suggested by our previousobservation that reduced glutathione concentrations are highin both the striatum and in fetal mesencephalic neuronalcultures from parkin KO mice (Itier et al. 2003). In youngand aged parkin KO mice, however, the levels of protein

Fig. 3 Functional distribution of proteins

differentially regulated in parkin KO and WT

mice. The major functional categories of all

the proteins identified in cortex and striatum

are plotted in a lateral bar graph as a per-

centage of the total that increased in

abundance (right) and the total that

decreased in abundance (left). Only func-

tional categories with two or more members

are shown for proteins differing in abun-

dance by at least 45% in the two types of

mice. Proteins with several isoforms, some

of which were up-regulated and some of

which were down-regulated, were exclu-

ded.

Fig. 4 Quantitative western blot analyses of CDCRel-1, calretinin and

p38 in the cortex of WT and parkin KO mice. A slight but reproducible

increase in CDCRel-1 protein levels was observed in KO mice,

whereas calretinin protein levels were significantly reduced. In con-

trast, p38 levels were similar in WT and KO mice. Data were obtained

by normalizing the relative intensities of CDCRel-1, calretinin and p38

signals to the intensity of the respective actin signal in each sample.

The mean CDCRel-1/actin, calretinin/actin and p38/actin ratios were

set arbitrarily at 1. Values are expressed as mean SEM (n 5animals per group). *p < 0.05 versus WT (Students t-test). Results

representative of at least three independent experiments are shown.



carbonyls were comparable to those of WT mice, indicatingthat antioxidant defences and/or detoxication processes areefcient in these animals.Several proteins involved in vesicle trafcking or function

were also affected in parkin KO mice, including componentsof the SNARE (soluble N-ethylmaleimide sensitive fusionprotein (NSF) attachment protein receptors) complex: NSF,syntaxin 1B and syntaxin-binding protein 1. In particular,four members of the septin protein family (which is involved

in vesicle transport and exocytosis, but also in cytokinesis,protein scaffolding and several other cellular processes),including the Parkin substrate septin5/CDCRel-1, were moreabundant in parkin KO mice than in WT mice. Members ofthis protein family accumulate in neurobrillary tangles andglial brils in Alzheimers disease, and in lewy bodies in PD(Kitada et al. 1998; Ihara et al. 2003). CDCRel-1 and therelated protein CDCRel-2 also accumulate in the brains ofPD sufferers with parkin gene mutations (Zhang et al. 2000;Choi et al. 2003). Regulation of the degradation of synapticvesicle-associated proteins by Parkin, which is present onsynaptic vesicles (Kubo et al. 2001), may modulate neuro-transmitter release. Loss of this function might be partiallyresponsible for the observed abnormalities in dopaminergicand glutamatergic neurotransmission in parkin KO mice(Itier et al. 2003). The up-regulation of three members of theMAGUK p55 subfamily of synaptic scaffolding proteins mayalso be involved in these changes in synaptic function.Interestingly, a previous study demonstrated a direct inter-action between Parkin and the MAGUK family membercalcium/calmodulin-dependent serine protein kinase (CASK)and suggested that CASK might target Parkin to specializedfunctional membrane domains where it may modulate theactivity of NMDA receptors (Fallon et al. 2002).While this work was in progress, Palacino et al. (2004)

reported a differential proteomic analysis of another parkinKO mouse model, using conventional 2D gel technologyand silver-stained gels. Surprisingly, they found consistentdecreases in the abundance of only 13 proteins and alteredelectrophoretic mobility of an additional one. Decreasedabundance of proteins involved in mitochondrial function,i.e subunits of complexes I and IV, was associated with areduction in the respiratory capacity of striatal mitochon-dria from parkin KO mice. This mouse model alsoexhibited decreased levels of proteins involved in protec-tion against oxidative stress, decreased serum antioxidantcapacity and, in contrast to our model, increased proteinand lipid peroxidation. Only two (pyruvate dehydrogenaseE1a1 and glyoxalase I) of the 13 proteins identied inPalacinos study were also found to be changed inabundance in our parkin KO mice. These proteins showeda decrease in abundance in Palacinos study, whereas theywere increased in abundance in our parkin KO mice,raising the possibility that these proteins represent falsepositives in either or both studies. However, this surprisingdiscordance might also result from the identication ofdifferent protein isoforms or post-translational variants,which would be dysregulated differently in the two studies,as was the case in a previous report (Choi et al. 2004b).Indeed, our proteomic analysis was performed using abroader range of pH gradients, which might have led to theidentication of protein isoforms that were not resolved onthe 310 pH gradient gel used by Palacino. The consid-erably greater number of proteins identied in our study

(a)

(b)

Fig. 5 Levels of protein carbonyls are similar in the cortex and stria-

tum of WT and parkin KO mice. Analysis of individual cortex (a) and

striatum (b) samples from 12-month-old animals revealed comparable

levels of oxidatively damaged proteins in WT and parkin KO mice.

Equivalent protein loading was confirmed by western blotting using an

anti-actin antibody. C, protein samples in which 2,4-din-

itrophenylhydrazine was omitted; M, molecular weight protein stand-

ard; BSA, oxidatively modified bovine seum albumin.



Table

7P

rote

ins

diffe

rentially

regula

ted

betw

eenparkin

KO

and

WT

mic

eand

identified

inoth

er

pro

teom

icstu

die

sanaly

zin

gneuro

degenera

tive

models

or

dis

ord

ers

Sw

issP

rot

or

giaccessio

nP

rote

innam

eS

tatu

sD

isease

Modeland/o

rtissue

Refe

rences

Energ

ym

eta

bolis

m

ALF

C_M

OU

SE

Fru

cto

se-b

isphosphate

ald

ola

se

C

PD

parkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

[Fra

gm

ent]

S

CZ

DH

um

an

bra

inP

rabakara

netal.

2004

AT

PA

_M

OU

SE

AT

Psynth

asea

chain

,m

itochondrial

+P

Dparkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

na

AD

Tau

transgenic

mic

eT

illem

anetal.

2002a

AT

PB

_M

OU

SE

AT

Psynth

aseb

chain

,m

itochondrial

P

Dparkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

+A

DH

um

an

bra

inT

sujietal.

2002

D

SH

um

an

bra

inK

imetal.

2000

DLD

H_M

OU

SE

Dih

ydro

lipoam

ide

dehydro

genase

P

Dparkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

na

AD

Tau

transgenic

mic

eT

illem

anetal.

2002a

DH

SA

_H

UM

AN

Succin

ate

dehydro

genase

flavopro

tein

+A

Dparkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

subunit

+A

DG

SK

3b

transgenic

mic

eT

illem

anetal.

2002b

EN

OG

_M

OU

SE

cE

nola

se

/+

PD

parkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

+A

DH

um

an

bra

inS

chonberg

eretal.

2001

S

CZ

DH

um

an

bra

inP

rabakara

netal.

2004

na

AD

Tau

transgenic

mic

eT

illem

anetal.

2002a

G3P

_M

OU

SE

Gly

cera

ldehyde

3-p

hosphate

dehydro

genase

/+

PD

parkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

+A

DH

um

an

bra

inS

chonberg

eretal.

2001

P

DM

PT

Pm

ice

m

itochondria

SN

Jin

etal.

2005

S

CZ

DH

um

an

bra

inP

rabakara

netal.

2004

na

AD

Tau

transgenic

mic

eT

illem

anetal.

2002a

NU

CM

_H

UM

AN

NA

DH

-ubiq

uin

one

oxid

ore

ducta

se

A

Dparkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

49-k

Da

subunit

A

DG

SK

3b

transgenic

mic

eT

illem

anetal.

2002b

OD

PA

_M

OU

SE

Pyru

vate

dehydro

genase

E1

com

ponent

+P

Dparkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

asubunit

S

CZ

DH

um

an

bra

inP

rabakara

netal.

2004

P

DM

PT

Pm

ice

m

itochondria

SN

Jin

etal.

2005

P

Dparkin

KO

mic

e,

ventr

alm

idbra

inP

ala

cin

oetal.

2004

UC

R1_R

AT

Ubiq

uin

ol-cyto

chro

mec

reducta

se

com

ple

x+

AD

parkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

core

pro

tein

I

AD

Hum

an

bra

inK

imetal.

2000

S

CZ

DH

um

an

bra

inP

rabakara

netal.

2004

Sig

naltr

ansduction

DP

Y2_M

OU

SE

Dih

ydro

pyrim

idin

ase-r

ela

ted

pro

tein

-2+

/P

Dparkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

(CR

MP

-2)

A

DH

um

an

bra

inT

sujietal.

2002

A

DH

um

an

bra

inS

chonberg

eretal.

2001

(G

lyfo

rm)

AD

Hum

an

bra

inK

annin

enetal.

2004

S

CZ

DH

um

an

bra

inP

rabakara

netal.

2004

+(O

xfo

rm)

AD

P

DH

um

an

bra

inC

hoietal.

2004a

+(O

xfo

rm)

AD

Hum

an

bra

inC

aste

gnaetal.

2002b

+A

DG

SK

3b

transgenic

mic

eT

illem

anetal.

2002b



Table

7(C

ontinued)

Sw

issP

rot

or

giaccessio

nP

rote

innam

eS

tatu

sD

isease

Modeland/o

rtissue

Refe

rences

P2B

A_M

OU

SE

Ser/

Thr

pro

tein

phosphata

se

2B

cata

lytic

+P

Dparkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

subunit,a

isofo

rm+

AD

GS

K3b

transgenic

mic

eT

illem

anetal.

2002b

143Z

_M

OU

SE

14-3

-3pro

tf/

d

PD

parkin

KO

mic

e,

cort

exstr

iatu

mP

eriquetetal.

2005

P

DM

PT

Pm

ice

m

itochondria

SN

Jin

etal.

2005

na

AD

Tau

transgenic

mic

eT

illem

anetal.

2002a

Vesic

ula

rtr

affi

ckin

g

NS

F_M

OU

SE

N-e

thylm

ale

imid

esensitiv

efu

sio

n

pro

tein

+/

PD

parkin

KO

mic

e,

cort

exstr

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might be explained by technological differences. First,cyanin dye staining is more sensitive than silver stainingand has a greater quantication range, extending linearlyover four orders of magnitude (Patton 2000). Second, thepooled internal standard used with the 2D DIGE technol-ogy allows detection of changes in protein abundance thatcannot be seen in pairwise comparisons of individualbiological samples (Friedman et al. 2004). Third, the useof overlapping pH gradients in our study increased theresolution of 2D gels. Finally, differences in the number,nature and status of the differentially regulated proteinsmight also result from differences in the tissues and agesanalysed. Palacinos study was conducted on ventralmidbrain of 8-month-old mice, whereas our proteomicanalysis was performed on cortex and striatum from 2- and12-month-old mice.In conclusion, we have identied changes in the abun-

dance of a large number of proteins belonging to variousfunctional categories in our parkin KO model. Several ofthese functional categories have already been linked to cell/animal models of PD as well as to other neurodegenerativeconditions in previous differential gene expression orproteomic analyses, raising the question of their specicity.The categories most reproducibly affected at the mRNAand/or protein levels in these studies include proteinsinvolved in energy metabolism (Loring et al. 2001; Gozalet al. 2002; Napolitano et al. 2002; Seong et al. 2002;Tilleman et al. 2002a, 2002b; Xie et al. 2002; Kuhn et al.2003), protein folding and degradation (Cadet et al. 2001;Ryu et al. 2002; Kuhn et al. 2003) and detoxicationprocesses (Balcz et al. 2001; Prabakaran et al. 2004). Otherfunctional classes, such as proteins implicated in vesicletrafcking, cytoskeletal dynamics and protein folding anddegradation, were also frequently identied in severalproteomic analyses on brains from patients affected byschizophrenia, Downs syndrome or Alzheimers disease(Schonberger et al. 2001; Castegna et al. 2002a, 2002b;Choi et al. 2004a; Tilleman et al. 2002a, 2002b; Kadotaet al. 2004; Prabakaran et al. 2004; Jin et al. 2005).Detailed analysis of the protein abundance changes reportedin the various proteomic studies available so far in the eldof neurodegeneration and psychiatric conditions (Table 7)revealed that 26 of the 87 proteins identied in our parkinKO model (30%) were also affected in other studies. Theseproteins cover the major functional classes shown in Fig. 3.Therefore, in general, common pathways appear to bealtered in these models, although the nature of the affectedproteins is often different. However, our study also hints atthe possible specic involvement of members of the septinand MAGUK protein families in parkin-related PD. Indeed,only one previous study reported the down-regulation of aseptin (septin7) in human brain lysates from patients withschizophrenia (Prabakaran et al. 2004) (Table 7) and, to ourknowledge, alterations in MAGUK protein abundance wereTa

ble

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not observed in any of the previously reported differentialproteomic analyses.Overall, although further functional studies are required to

validate the proteins identied in our study and link themmechanistically to the molecular events underlying parkin-related Parkinsons disease, these data already constitute avaluable reference bank for future investigations into thepathological mechanisms involved in the early stages of thisdisease.

Acknowledgements

We thank Lydia Guennec for technical assistance, Frederic Darios

and Francisco Araujo for helpful discussions, and Merle Ruberg for

critical reading of the manuscript. This work was supported by the

Fondation pour la Recherche Medicale, the VERUM foundation,

Fondation de France and APOPIS (Abnormal proteins in the

pathogenesis of neurodegenerative disorders an integrated project

funded by the EU under the Sixth Framework Programme; Priority:

Life Science for Health, contract no. LSHM-CT-2003-503330).

References

Balcz B., Kirchner L., Cairns N., Fountoulakis M. and Lubec G. (2001)Increased brain protein levels of carbonyl reductase and alcoholdehydrogenase in Down syndrome and Alzheimers disease.J Neural Transm Suppl. 2001; (61), 193201.

Cadet J. L., Jayanthi S., McCoy M. T., Vawter M. and Ladenheim B.(2001) Temporal proling of methamphetamine-induced changesin gene expression in the mouse brain: evidence from cDNA array.Synapse 41, 4048.

Castegna A., Aksenov M., Aksenova M., Thongboonkerd V., Klein J. B.,Pierce W. M., Booze R., Markesbery W. R. and Buttereld D. A.(2002a) Proteomic identication of oxidatively modied proteinsin Alzheimers disease brain. Part I: creatine kinase BB, glutaminesynthase, and ubiquitin carboxy-terminal hydrolase L-1. FreeRadic. Biol. Med. 33, 562571.

Castegna A., Aksenov M., Thongboonkerd V., Klein J. B., Pierce W. M.,Booze R., Markesbery W. R. and Buttereld D. A. (2002b) Proteo-mic identication of oxidatively modied proteins in Alzheimersdisease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71. J. Neurochem. 82, 15241532.

Chen F., Wollmer M. A., Hoerndli F., Munch G., Kuhla B., Rogaev E. I.,Tsolaki M., Papassotiropoulos A. and Gotz J. (2004) Role forglyoxalase I in Alzheimers disease. Proc. Natl Acad. Sci. USA101, 76877692.

Choi J., Forster M. J., McDonald S. R., Weintraub S. T., Carroll C. A.and Gracy R. W. (2004a) Proteomic identication of specicoxidized proteins in ApoE-knockout mice: relevance to Alzhei-mers disease. Free Radic. Biol. Med. 36, 11551162.

Choi J., Levey A. I., Weintraub S. T., Rees H. D., Gearing M., Chin L. S.and Li L. (2004b) Oxidative modications and down-regulation ofubiquitin carboxyl-terminal hydrolase L1 associated with idio-pathic Parkinsons and Alzheimers diseases. J. Biol. Chem. 279,13 25613 264.

Choi P., Snyder H., Petrucelli L. et al. (2003) SEPT5_v2 is a parkin-binding protein. Brain Res. Mol. Brain Res. 117, 179189.

Corti O., Hampe C., Koutnikova H. et al. (2003) The p38 subunit of theaminoacyltRNA synthetase complex is a Parkin substrate: linking

protein biosynthesis and neurodegeneration. Hum. Mol. Genet. 12,14271437.

DekkerM. C., Bonifati V. and vanDuijn C.M. (2003) Parkinsons disease:piecing together a genetic jigsaw. Brain 126, 17221733.

Dev K. K., van der Putten H., Sommer B. and Rovelli G. (2003) Part I:parkin-associated proteins and Parkinsons disease. Neurophar-macology 45, 113.

Dunn A. Y., Melville M. W. and Frydman J. (2001) Review: cellularsubstrates of the eukaryotic chaperonin TRiC/CCT. J. Struct. Biol.135, 176184.

Fallon L., Moreau F., Croft B. G., Labib N., Gu W. J. and Fon E. A.(2002) Parkin and CASK/LIN-2 associate via a PDZ-mediatedinteraction and are co-localized in lipid rafts and postsynapticdensities in brain. J. Biol. Chem. 277, 486491.

Farrer M., Chan P., Chen R. et al. (2001) Lewy bodies and parkin-sonism in families with parkin mutations. Ann. Neurol. 50,293300.

Forno L. S. (1987) The Lewy body in Parkinsons disease. Adv. Neurol.45, 3543.

Friedman D. B., Hill S., Keller J. W., Merchant N. B., Levy S. E., CoffeyR. J. and Caprioli R. M. (2004) Proteome analysis of human coloncancer by two-dimensional difference gel electrophoresis and massspectrometry. Proteomics 4, 793811.

Gharbi S., Gaffney P., Yang A., Zvelebil M. J., Cramer R., WatereldM. D. and Timms J. F. (2002) Evaluation of two-dimensionaldifferential gel electrophoresis for proteomic expression analysisof a model breast cancer cell system. Mol. Cell. Proteomics 1,9198.

Goldberg M. S., Fleming S. M., Palacino J. J. et al. (2003) Parkin-decient mice exhibit nigrostriatal decits but not loss of dopam-inergic neurons. J. Biol. Chem. 278, 43 62843 635.

Gouider-Khouja N., Larnaout A., Amouri R., Sfar S., Belal S.,Ben Hamida C., Ben Hamida M., Hattori N., Mizuno Y. andHentati F. (2003) Autosomal recessive parkinsonism linked toparkin gene in a Tunisian family. Clinical, genetic and pathologicalstudy. Parkinsonism Relat. Disord. 9, 247251.

Gozal E., Gozal D., Pierce W. M., Thongboonkerd V., Scherzer J. A.,Sachleben L. R. Jr, Brittian K. R., Guo S. Z., Cai J. and Klein J. B.(2002) Proteomic analysis of CA1 and CA3 regions of rat hippo-campus and differential susceptibility to intermittent hypoxia.J. Neurochem. 83, 331345.

Hattori N. and Mizuno Y. (2004) Pathogenetic mechanisms of parkin inParkinsons disease. Lancet 364, 722724.

Hayashi S., Wakabayashi K., Ishikawa A., Nagai H., Saito M.,Maruyama M., Takahashi T., Ozawa T., Tsuji S. and Takahashi H.(2000) An autopsy case of autosomal-recessive juvenile parkin-sonism with a homozygous exon 4 deletion in the parkin gene.Mov. Disord. 15, 884888.

Huynh D. P., Scoles D. R., Nguyen D. and Pulst S. M. (2003) Theautosomal recessive juvenile Parkinson disease gene product,parkin, interacts with and ubiquitinates synaptotagmin XI. Hum.Mol. Genet. 12, 25872597.

Ihara M., Tomimoto H., Kitayama H., Morioka Y., Akiguchi I.,Shibasaki H., Noda M. and Kinoshita M. (2003) Association of thecytoskeletal GTP-binding protein Sept4/H5 with cytoplasmicinclusions found in Parkinsons disease and other synucleinopa-thies. J. Biol. Chem. 278, 24 09524 102.

Imai Y., Soda M. and Takahashi R. (2000) Parkin suppresses unfoldedprotein stress-induced cell death through its E3 ubiquitinproteinligase activity. J. Biol. Chem. 275, 35 66135 664.

Itier J. M., Ibanez P., Mena M. A. et al. (2003) Parkin gene inactivationalters behaviour and dopamine neurotransmission in the mouse.Hum. Mol. Genet. 12, 22772291.



Jiang H., Jiang Q. and Feng J. (2004) Parkin increases dopamine uptakeby enhancing the cell surface expression of dopamine transporter.J. Biol. Chem. 279, 54 38054 386.

Jin J., Meredith G. E., Chen L., Zhou Y., Xu J., Shie F. S., Lockhart P.and Zhang J. (2005) Quantitative proteomic analysis of mitoch-ondrial proteins: relevance to Lewy body formation and Parkin-sons disease. Brain Res. Mol. Brain Res. 134, 119138.

Kadota M., Nishigaki R., Wang C. C., Toda T., Shirayoshi Y., Inoue T.,Gojobori T., Ikeo K., Rogers M. S. and Oshimura M. (2004)Proteomic signatures and aberrations of mouse embryonic stemcells containing a single human chromosome 21 in neuronal dif-ferentiation: an in vitro model of Down syndrome. Neuroscience129, 325335.

Kanninen K., Goldsteins G., Aurrola S., Alafuzoff I. and Koistinaho J.(2004) Glycosylation changes in Alzheimers disease as revealedby a promeotic approach. Neurosci. Lett. 367, 235240.

Kim S. H., Vikolinsky R., Coiins N. and Cubec G. (2000) Decreasedlevels of complex III core protein I and complex V beta chain inbrains from patients with Alzheimers disease and Down Syn-drome. Cell. Mol. Life. Sci. 57, 18101816.

Kitada T., Asakawa S., Hattori N., Matsumine H., Yamamura Y.,Minoshima S., Yokochi M., Mizuno Y. and Shimizu N. (1998)Mutations in the parkin gene cause autosomal recessive juvenileparkinsonism. Nature 392, 605608.

Klein C., Pramstaller P. P., Kis B. et al. (2000) Parkin deletions in afamily with adult-onset, tremor-dominant parkinsonism: expandingthe phenotype. Ann. Neurol. 48, 6571.

Kubo S. I., Kitami T., Noda S., Shimura H., Uchiyama Y., Asakawa S.,Minoshima S., Shimizu N., Mizuno Y. and Hattori N. (2001) Parkinis associated with cellular vesicles. J. Neurochem. 78, 4254.

Kuhn K., Wellen J., Link N., Maskri L., Lubbert H. and Stichel C. C.(2003) The mouse MPTP model: gene expression changes indopaminergic neurons. Eur. J. Neurosci. 17, 112.

Lohmann E., Periquet M., Bonifati V. et al. (2003) How much pheno-typic variation can be attributed to parkin genotype? Ann. Neurol.54, 176185.

Loring J. F., Wen X., Lee J. M., Seilhamer J. and Somogyi R. (2001) Agene expression prole of Alzheimers disease. DNA Cell Biol. 20,683695.

Lucking C. B., Durr A., Bonifati V. et al. (2000) Association betweenearly-onset Parkinsons disease and mutations in the parkin gene.French Parkinsons Disease Genetics Study Group. N. Engl.J. Med. 342, 15601567.

Mori H., Kondo T., Yokochi M., Matsumine H., Nakagawa-Hattori Y.,Miyake T., Suda K. and Mizuno Y. (1998) Pathologic and bio-chemical studies of juvenile parkinsonism linked to chromosome6q. Neurology 51, 890892.

Mura A., Feldon J. and Mintz M. (2000) The expression of the calciumbinding protein calretinin in the rat striatum: effects of dopaminedepletion and L-DOPA treatment. Exp. Neurol. 164, 322332.

Napolitano M., Centonze D., Calce A., Picconi B., Spiezia S., Gulino A.,Bernardi G. and Calabresi P. (2002) Experimental parkinsonismmodulates multiple genes involved in the transduction of dopam-inergic signals in the striatum. Neurobiol. Dis. 10, 387395.

Paisan-Ruiz C., Jain S., Evans E. W. et al. (2004) Cloning of the genecontaining mutations that cause PARK8-linked Parkinsons dis-ease. Neuron 44, 595600.

Palacino J. J., Sagi D., Goldberg M. S., Krauss S., MotZ. C., Wacker M.,Klose J. and Shen J. (2004) Mitochondrial dysfunction and oxi-dative damage in parkin-decient mice. J. Biol. Chem. 279, 1861418 622.

Patton W. F. (2000) A thousand points of light: the application ofuorescence detection technologies to two-dimensional gel elec-trophoresis and proteomics. Electrophoresis 21, 11231144.

Perez F. A. and Palmiter R. D. (2005) Parkin-decient mice are not arobust model of parkinsonism. Proc. Natl Acad. Sci. USA 102,21742179.

Periquet M., Latouche M., Lohmann E. et al. (2003) Parkin mutationsare frequent in patients with isolated early-onset parkinsonism.Brain 126, 12711278.

Prabakaran S., Swatton J. E., Ryan M. M. et al. (2004) Mitochondrialdysfunction in schizophrenia: evidence for compromised brainmetabolism and oxidative stress. Mol. Psychiatry 9, 643.

Ren Y., Zhao J. and Feng J. (2003) Parkin binds to alpha/beta tubulin andincreases their ubiquitination and degradation. J. Neurosci. 23,33163324.

Ryu E. J., Harding H. P., Angelastro J. M., Vitolo O. V., Ron D. andGreene L. A. (2002) Endoplasmic reticulum stress and the unfol-ded protein response in cellular models of Parkinsons disease.J. Neurosci. 22, 10 69010 698.

Schonberger S. J., Edgar P. F., Kydd R., Faull R. L. and Cooper G. J.(2001) Proteomic analysis of the brain in Alzheimers disease:molecular phenotype of a complex disease process. Proteomics 1,15191528.

Seong J. K., Kim do K., Choi K. H., Oh S. H., Kim K. S., Lee S. S. andUm H. D. (2002) Proteomic analysis of the cellular proteinsinduced by adaptive concentrations of hydrogen peroxide in humanU937 cells. Exp. Mol. Med. 34, 374378.

Shimura H., Hattori N., Kubo S. et al. (2000) Familial Parkinson diseasegene product, parkin, is a ubiquitinprotein ligase. Nat. Genet. 25,302305.

Singleton A. B., Farrer M., Johnson J. et al. (2003) Alpha-synucleinlocus triplication causes Parkinsons disease. Science 302, 841.

Swatton J. E., Prabakaran S., Karp N. A., Lilley K. S. and Bahn S.(2004) Protein proling of human postmortem brain using2-dimensional uorescence difference gel electrophoresis (2-DDIGE). Mol. Psychiatry 9, 128143.

Tilleman K., Stevens I., Spittaels K., Haute C. V., Clerens S., Van DenBergh G., Geerts H., Van Leuven F., Vandesande F. and Moens L.(2002a) Differential expression of brain proteins in glycogensynthase kinase-3 transgenic mice: a proteomics point of view.Proteomics 2, 94104.

Tilleman K., Van den Haute C., Geerts H., van Leuven F., Esmans E.L. and Moens L. (2002b) Proteomics analysis of the neuro-degeneration in the brain of tau transgenic mice. Proteomics 2,656665.

Tonge R., Shaw J., Middleton B., Rowlinson R., Rayner S., Young J.,Pognan F., Hawkins E., Currie I. and Davison M. (2001) Validationand development of uorescence two-dimensional differential gelelectrophoresis proteomics technology. Proteomics 1, 377396.

Tsuboi K., Kimber T. A. and Shults C. W. (2000) Calretinin-containingaxons and neurons are resistant to an intrastriatal 6-hydroxydop-amine lesion. Brain Res. 866, 5564.

Tsuji T., Shiozaki A., Kohno R., Yoshizato K. and Shimohama S. (2002)Proteonic proling and neurodegeneration in Alzheimers disease.Neurochem. Res. 27, 12451253.

Valente E. M., Abou-Sleiman P. M., Caputo V. et al. (2004) Hereditaryearly-onset Parkinsons disease caused by mutations in PINK1.Science 304, 11581160.

Von Coelln R., Thomas B., Savitt J. M., Lim K. L., Sasaki M., Hess E. J.,Dawson V. L. and Dawson T. M. (2004) Loss of locus coeruleusneurons and reduced startle in parkin null mice. Proc. Natl Acad.Sci. USA 101, 10 74410 749.

van de Warrenburg B. P., Lammens M., Lucking C. B., Denee P.,Wesseling P., Booij J., Praamstra P., Quinn N., Brice A. andHorstink M. W. (2001) Clinical and pathologic abnormalities in afamily with parkinsonism and parkin gene mutations. Neurology56, 555557.



Xie T., Tong L., Barrett T., Yuan J., Hatzidimitriou G., McCann U. D.,Becker K. G., Donovan D. M. and Ricaurte G. A. (2002) Changesin gene expression linked to methamphetamine-induced dopam-inergic neurotoxicity. J. Neurosci. 22, 274283.

Yan J. X., Devenish A. T., Wait R., Stone T., Lewis S. and Fowler S.(2002) Fluorescence two-dimensional difference gel electrophor-esis and mass spectrometry based proteomic analysis of Escheri-chia coli. Proteomics 2, 16821698.

Zabel C., Chamrad D. C., Priller J., Woodman B., Meyer H. E., Bates G.P. and Klose J. (2002) Alterations in the mouse and human prot-

eome caused by Huntingtons disease. Mol. Cell. Proteomics 1,366375.

Zhang Y., Gao J., Chung K. K., Huang H., Dawson V. L. and Dawson T.M. (2000) Parkin functions as an E2-dependent ubiquitinproteinligase and promotes the degradation of the synaptic vesicle-asso-ciated protein, CDCrel-1. Proc. Natl Acad. Sci. USA 97, 13 35413 359.

Zimprich A., Biskup S., Leitner P. et al. (2004) Mutations in LRRK2cause autosomal-dominant parkinsonism with pleomorphicpathology. Neuron 44, 601607.



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