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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: brice@ccr.jussieu.frAbbreviations 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
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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
1262 M. Periquet et al.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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.
Proteomic analysis of parkin knockout mice 1263
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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+
1264 M. Periquet et al.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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)
SwissProt or gi accession Protein name
No. and status of spots
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.
Proteomic analysis of parkin knockout mice 1265
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
Table 3 Proteins differentially regulated in the cortex of WT and parkin KO mice
SwissProt or gi accession Protein name
No. and status of spots
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.
1266 M. Periquet et al.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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.
Proteomic analysis of parkin knockout mice 1267
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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.
1268 M. Periquet et al.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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.
Proteomic analysis of parkin knockout mice 1269
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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.
1270 M. Periquet et al.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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
Proteomic analysis of parkin knockout mice 1271
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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
iatu
mP
eriquetetal.
2005
A
DH
um
an
bra
inS
chonberg
eretal.
2001
+S
CZ
DH
um
an
bra
inP
rabakara
netal.
2004
+A
DG
SK
3b
transgenic
mic
eT
illem
anetal.
2002b
ST
B1_M
OU
SE
Synta
xin
-bin
din
gpro
tein
1+
/P
Dparkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
+P
DM
PT
Pm
ice
m
itochondria
SN
Jin
etal.
2005
SE
P7_M
OU
SE
Septin
7+
PD
parkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
S
CZ
DH
um
an
bra
inP
rabakara
netal.
2004
Pro
tein
fold
ing
HS
72_M
OU
SE
Heat-
shock-r
ela
ted
70-k
Da
pro
tein
2
PD
parkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
S
CZ
DH
um
an
bra
inP
rabakara
netal.
2004
na
AD
Tau
transgenic
mic
eT
illem
anetal.
2002a
Str
ess/d
eto
xifi
cation
AC
ON
_H
UM
AN
Aconitate
hydra
tase,
mitochondrial
+P
Dparkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
S
CZ
DH
um
an
bra
inP
rabakara
netal.
2004
DH
CA
_M
OU
SE
Carb
onylre
ducta
se
[NA
DP
H]
1+
PD
parkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
S
CZ
DH
um
an
bra
inP
rabakara
netal.
2004
+A
DD
SH
um
an
bra
inB
alc
zetal.
2001
LG
UL_M
OU
SE
Gly
oxala
se
I+
PD
parkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
+P
DM
PT
Pm
ice
m
itochondria
SN
Jin
etal.
2005
-
PD
parkin
KO
mic
e,
ventr
alm
idbra
inP
ala
cin
oetal.
2004
Cyto
skele
ton
DY
N1_M
OU
SE
Dynam
in-1
+/
PD
parkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
+/
SC
ZD
Hum
an
bra
inP
rabakara
netal.
2004
na
AD
Tau
transgenic
mic
eT
illem
anetal.
2002a
SP
CN
_M
OU
SE
Spectr
ina
chain
,bra
in+
PD
parkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
S
CZ
DH
um
an
bra
inP
rabakara
netal.
2004
TB
A1_M
OU
SE
Tubulin
achain
+P
Dparkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
A
DG
SK
3b
transgenic
mic
eT
illem
anetal.
2002b
S
CZ
DH
um
an
bra
inP
rabakara
netal.
2004
1272 M. Periquet et al.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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
7(C
ontinued)
Sw
issP
rot
or
giaccessio
nP
rote
innam
eS
tatu
sD
isease
Modeland/o
rtissue
Refe
rences
Pro
tein
degra
dation
UB
L1_M
OU
SE
Ubiq
uitin
carb
oxyte
rmin
al
hydro
lase
L1
+P
Dparkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
S
CZ
DH
um
an
bra
inS
chonberg
eretal.
2001
P
DA
DH
um
an
bra
inC
hoietal.
2004b
D
SM
ouse
ES
cells
Kadota
etal.
2004
+(O
xfo
rm)
AD
Hum
an
bra
inC
aste
gnaetal.
2002a
Pro
tein
bio
synth
esis
SY
Y_H
UM
AN
Tyro
syltR
NA
synth
eta
se
+P
Dparkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
S
CZ
DH
um
an
bra
inP
rabakara
netal.
2004
Am
ino
acid
synth
esis
GLN
A_M
OU
SE
Glu
tam
ine
synth
eta
se
P
Dparkin
KO
mic
e,
cort
exstr
iatu
mP
eriquetetal.
2005
+A
DG
SK
3b
transgenic
mic
eT
illem
anetal.
2002b
+(O
xfo
rm)
AD
Hum
an
bra
inC
aste
gnaetal.
2002a
Ox,
oxid
ized;
Gly
,gly
cosyla
ted;
PD
,P
ark
insons
dis
ease;
AD
,A
lzheim
ers
dis
ease;
DS
,D
ow
ns
syndro
me;
SC
ZD
,schiz
ophre
nia
;S
N,
substa
ntia
nig
ra;
+,
incre
ased
abundance,
,
decre
ased
abundance;
+/-
,altere
dele
ctr
ophore
tic
mobili
ty;
na,
data
not
availa
ble
.
Proteomic analysis of parkin knockout mice 1273
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 12591276
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).
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