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Journal of Neurochemistry, 2005, 95, 12591276

doi:10.1111/j.1471-4159.2005.03442.x

Proteomic analysis of parkin knockout mice: alterations in


energy 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 searched 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 proteins 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 proteins 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 in parkin KO
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 parkinrelated parkinsonism.
Keywords: knockout mice, parkin, proteomic, two-dimensional fluorescence difference gel electrophoresis.
J. Neurochem. (2005) 95, 12591276.

Parkinsons disease (PD) is a common neurodegenerative


disorder clinically characterized by resting tremor, rigidity and
bradykinesia. These severe neurological symptoms are caused
by the selective, progressive degeneration of dopaminergic
neurones in the substantia nigra pars compacta. Lewy bodies
ubiquitylated neuronal cytoplasmic inclusions are a pathological hallmark of PD (Forno 1987). Seven genes involved in
rare monogenic forms of PD have been discovered in the past
8 years (Dekker et al. 2003). Missense mutations in the
a-synuclein gene, multiplications of a genomic region including this gene (Singleton et al. 2003), as well as missense
mutations in the recently discovered dardarin gene (PaisanRuiz et al. 2004; Zimprich et al. 2004) cause autosomal
dominant forms of parkinsonism. The ubiquitin carboxyterminal hydrolase L1 (UCH-L1) and Nurr-1 genes are also
potentially involved in autosomal dominant parkinsonian
syndromes. However, their role in the pathogenesis of the
disease remains uncertain, because the corresponding mutations have been found in only a small number of families. In
addition, the parkin, DJ-1 and PTEN-induced kinase 1
(PINK1) genes are responsible for autosomal recessive forms
of the disease (Dekker et al. 2003; Valente et al. 2004).

In 1998, the parkin gene was shown to be responsible for a


distinct clinical and genetic entity in Japan, dened as
autosomal recessive juvenile parkinsonism (Kitada et al.
1998). A series of parkin exon rearrangements and point
mutations have since been identied in almost 50% of patients
with familial autosomal recessive early-onset parkinsonism
from 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 Paris
Cedex 13, France. E-mail: brice@ccr.jussieu.fr
Abbreviations used: AcCN, acetonitrile; CDCRel-1, cell division
control related protein 1; 2D DIGE, two-dimensional difference gel
electrophoresis; DTT, dithiothreitol; GTP2, glutathione S-transferase P2;
IPG, immobilized pH gradient; KO, knockout; MAGUK, membraneassociated guanylate kinase; MALDITOF, matrix-assisted laser
desorption/ionizationtime of ight; MS, mass spectrometry; NSF,
N-ethylmaleimide sensitive fusion protein; OTUB1, OTU-domain uba1binding protein; PD, Parkinsons disease; PINK1, PTEN-induced kinase 1;
UCH-L1, ubiquitin carboxyterminal hydrolase L1; WT, wild type.

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1259

1260 M. Periquet et al.

2003). These mutations are associated with a wide range of


ages at onset and a broad phenotypic spectrum, including
cases clinically indistinguishable from idiopathic PD (Klein
et al. 2000; Lucking et al. 2000). Neuropathological descriptions of brains from patients with homozygous parkin
deletions have reported specic dopaminergic neuronal
degeneration in the substantia nigra pars compacta in the
absence 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 deposits
have been described in a few individuals with compound
heterozygous 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-rich
RING-IBR-RING motif, has E3 ubiquitinprotein ligase
activity that promotes the ubiquitylation and proteasomal
degradation 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 progressive
accumulation of non-ubiquitylated, potentially toxic substrates, leading to neurodegeneration. At least 10 Parkin
substrates have been identied so far with roles in cell
processes as diverse as cell signalling (Pael-R), cell cycle
control (cyclin E), protein biosynthesis (the p38 scaffold
subunit of the multi-aminoacyl-tRNA synthetase component), cytoskeletal dynamics (a/b tubulin), and vesicular and
synaptic functions (CDCRel-1 and CDCRel-2, synaptotagmin IX, O-glycosylated a-synuclein, synphilin, the dopamine
transporter) (Hattori and Mizuno 2004; Jiang et al. 2004).
However, the specic and potentially synergistic pathological
roles of these substrates are unclear.
We and others have recently generated a parkin knockout
(KO) mouse model to facilitate investigation of the
pathogenic mechanisms underlying parkinsonism due to
parkin 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 dopaminergic neurones in these mice, but a number of behavioural
and biochemical changes were observed, including decits
in dopamine handling, reproducing some of the presymptomatic aspects of PD (Itier et al. 2003). This model is
therefore of value for investigation of the functional
consequences of parkin gene inactivation, including potential compensatory mechanisms preventing the onset of a
parkinsonian phenotype.
To shed line on the molecular pathways affected in parkin
KO mice and identify potential Parkin substrates predicted to
accumulate in these mice, we performed a differential analysis
of parkin KO and wild-type (WT) brain proteomes. Twodimensional uorescence difference gel electrophoresis (2D
DIGE) was chosen for this purpose, because it has proven to be
valuable for a number of biological applications, including
studies related to neurodegenerative disorders such as schizophrenia and Huntingtons disease (Zabel et al. 2002; Swatton

et al. 2004). This technique involves the pre-electrophoretic


labelling of the protein samples to be compared with
uorescent dyes, such as Cy2 and Cy5, which allows
statistically valid quantication of a dynamic range of protein
concentrations with high sensitivity (Patton 2000); the
labelled protein samples are pooled and mixed with an
internal standard prelabelled with Cy3 to normalize the results
and reduce gel-to-gel variability. When used with the dedicated DeCyder analysis software (Amersham Bioscience Inc.,
Amersham, UK), this technique permits the sensitive, mass
spectrometry (MS)-compatible and reproducible identication
of statistically signicant differences in the protein expression
proles of multiple samples examined simultaneously (Tonge
et al. 2001; Gharbi et al. 2002; Yan et al. 2002).
We combined this approach with sensitive matrix-assisted laser desorption/ionizationtime of ight (MALDI
TOF) MS and tandem MS, to screen six WT and six parkin
KO mice at 2 and 12 months, in two brain structures
(cortex and striatum). Eighty-seven unique proteins were
identied that differed in abundance between the brains of
parkin KO and WT mice. These differences provide new
information concerning the molecular pathways that might
be involved in the preclinical stages of parkin-related
parkinsonism.

Experimental procedures
Animals and brain tissues
Studies were carried out on 2- and 12-month-old parkin KO (Itier
et al. 2003) and WT mice with a pure 129SV background. Animals
were 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 M
Tris-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 EDTA
was 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 at
room temperature (25C) for 10 min, gently vortexed, and then
centrifuged for 5 min at 14 000 g, followed by 45 min at
100 000 g. Protein extracts were aliquoted and stored at )80C or
immediately 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 protein
was labelled. Typically, 50 lg parkin KO or WT mouse protein
extract was labelled with 400 pmol cyanin dye Cy5 for parkin-KO
and Cy2 for WT (Amersham Bioscience Inc.). As 2D-DIGE
analysis requires large amounts of protein, we used a pool of six

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

Proteomic analysis of parkin knockout mice 1261

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 immobilines
NL 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 (for


preparative 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 C
corresponds to the concentration of N,N-methylenebisacrylamide
as a percentage (by weight of acrylamide and N,N-methylenebisacrylamide). Gels were run at 10C, 50 mA for 1.5 h, then at
100 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.

(a)
4.5

pl

5.5

pl

6.7

pl

200 kDa

MW

15 kDa
(b)

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 electrophoresis. The patterns of staining for the cyanin dyes and SyproRuby dye were very similar, facilitating accurate matching and picking
on these preparative gels.

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

1262 M. Periquet et al.

Protein visualization
The Cy2, Cy3 and Cy5 components of each gel were imaged
individually using mutually exclusive excitation/emission wavelengths 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 associate
a 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 be
signicantly 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 greater
than 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 containing 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 automatically
the 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 Biotechnology 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 from
the 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 collision
energies 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 nonredundant 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 posttranslational 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 for WT or
parkin KO mice). We ran about 40 lg total protein extract in each lane
of 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 visualized 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 striatum
extracts obtained in 50 mM Hepes containing 150 mM NaCl, 10%
glycerol, 1% Triton X-100, 100 mM NaF, 0.2 mM Na3VO4 and

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Proteomic analysis of parkin knockout mice 1263

complete protease inhibitors (Roche) was reacted with 2,4-dinitrophenylhydrazine 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 immunoreactvity (1 : 2000; Sigma) and revealed using enhanced chemioluminescence (Pierce, Rockford, IL, USA).

Results

Analysis of cortical and striatal mouse tissues at 2 and


12 months of age using 2D DIGE technology led to the
identication of 159 differentially regulated protein spots
between WT and parkin KO mice. In 2D analysis, proteins
are frequently detected in more than one spot, indicating the
presence of either different isoforms and/or post-translational
modications. In order to optimize protein resolution and to
better detect these different isoforms, the rst dimension of
electrophoresis was carried out with narrow overlapping pH
gradient gels (Immobiline Dry Strip, pH 4.56, 5.56.7, 69;
Fig. 1). The differentially regulated spots were rst analysed
by MALDITOF MS, using peptide mass ngerprints and
database searches. Proteins not identied by this method
were subjected to MS/MS, followed by a search of sequence
databases. With these techniques, we identied 87 unique
proteins that differed in abundance by at least 45% in WT
and parkin KO mice (Tables 13). In 2-month-old mice, the
number of proteins found to be differentially regulated in the
cortex and the striatum and the proportions of up- and downregulated proteins were similar (Table 1). In contrast, in
12-month-old mice, the majority of the proteins with altered
abundance were found in the striatum and most of them were
up-regulated (p < 0.05) (Table 1). Approximately 20% (18
of 87) of the identied proteins were differentially regulated
in both 2- and 12-month-old parkin KO mice and a similar
percentage (14/87) were dysregulated in both the cortex and
the striatum, at one or both of the ages examined (Tables 4
and 5). Overall, 46 proteins that increased in abundance were
identied, 31 that decreased in abundance, three that changed
their pattern of regulation between 2 and 12 months, and
Table 1 Number of proteins that differed
significantly in abundance in WT and parkin
KO mice

seven that had a change in their electrophoretic mobility. The


mobility variants were represented by at least two independent spots with different isoelectric points of a same
protein subjected to opposite regulation in one and the same
experiment (status +/ in Tables 25). They usually correspond to different isoforms of a protein that has undergone
post-translational modication, i.e. a shift in phosphorylation
status, as illustrated in Fig. 2(c). Other examples of changes
in staining intensity are illustrated in Fig. 2.
Classication of the differentially regulated proteins
according to the specic keywords attributed to them in the
Swissprot database (Table 6) showed that a certain proportion of the modulated proteins were linked to energy
metabolism (glycolysis, ATP synthesis, avoprotein, hydrogen ion transport, FAD, NAD/NADP, mitochondrion) and
protein processing pathways (heat shock, proteasome, protein biosynthesis, ligase, chaperone, transit peptide, isomerase). The frequent occurrence of the keywords kinase and
GTP binding suggested that cell signalling pathways were
likely to be disrupted, together with vesicle trafcking and
cytoskeletal dynamics processes in which proteins with
kinase and GTPase activities play major roles.
Extensive literature searches in Pubmed, to characterize
each protein, led to the identication of 12 distinct
functional categories, each including at least two proteins
up- or down-regulated in the absence of the parkin gene
(Fig. 3, Tables 2 and 3). Sixty-seven of the 87 identied
proteins fell into these categories; nine, represented by
several spots with either increased or decreased abundance,
a situation compatible with post-translational modications
or a change in the pattern of regulation between 2- and
12-month-old mice, were not included in Fig. 3. Eleven
proteins could not be assigned to established functional
categories based on their putative functions. Among these
proteins, the calcium-binding protein calretinin was found.
Calretinin, a brain-specic, potentially neuroprotective protein (Mura et al. 2000; Tsuboi et al. 2000), was downregulated in the cortex of 12-month-old KO mice (Table 3).
The down-regulation of this protein was conrmed by
quantitative western blot following the one-dimensional gel
electrophoresis of protein samples from mouse cortex
(Fig. 4).

Cortex

Striatum

Age (months)

Increased

Decreased

Altered EM

Increased

Decreased

Altered EM

2
12

15
4

12
4

5
0

19
23

18
5

3
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.

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

1264 M. Periquet et al.

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


No. and status of spots

SwissProt or gi accession
Energy metabolism (n 17)
ALFC_MOUSE
ATPA_MOUSE
CISY_HUMAN
DLDH_MOUSE
DHSA_HUMAN
G3P_MOUSE
KAD3_MOUSE
KPY2_MOUSE
LDHA_MOUSE
MDHC_MOUSE
NUBM_HUMAN
ODPB_RAT
PGK1_MOUSE
SCB2_MOUSE
THIL_RAT
UCR1_RAT
539926/13435978
Signal transduction (n 8)
ARK1_RAT
CRK_MOUSE
DPY2_MOUSE
P2BA_MOUSE
PP1B_HUMAN
PTNB_MOUSE
143Z_MOUSE
MPP3_HUMAN
Vesicular trafficking (n 8)
GDIR_HUMAN
NSF_MOUSE
ST1B_MOUSE
STB1_MOUSE
Septin family
SEP5_MOUSE
SEP7_MOUSE
Y202_HUMAN
8922712
Protein folding (n 2)
GR75_MOUSE
HS7C_MOUSE
Stress/detoxification (n 4)
DHCA_MOUSE
GTP2_MOUSE
LGUL_MOUSE
TRXB_MOUSE
Cytoskeleton (n 4)
AR20_HUMAN
CAP1_MOUSE
DYN1_MOUSE
TBA1_MOUSE

Striatum
2 months

Protein name

Fructose-bisphosphate aldolase C [Fragment]


ATP synthase a chain, mitochondrial
Citrate synthase
Dihydrolipoamide dehydrogenase
Succinate dehydrogenase flavoprotein subunit
Glyceraldehyde-3-phosphate dehydrogenase
GTP:AMP phosphotransferase mitochondrial
Pyruvate kinase, M2 isozyme
L-Lactate dehydrogenase A chain
Malate dehydrogenase, cytoplasmic
NADH-ubiquinone oxidoreductase 51-kDa subunit
Pyruvate dehydrogenase E1 component b subunit
Phosphoglycerate kinase 1
Succinyl-CoA ligase [GDP-forming] b-chain, mitochondrial
Acetyl-CoA acetyltransferase, mitochondrial
Ubiquinol-cytochrome c reductase complex core protein I
Acetyl-CoA C-acetyltransferase

1
3+
1
1+
1+
2+
1+
1/2

b-Adrenergic receptor kinase 1


Proto-oncogene C-crk
Dihydropyrimidinase-related protein-2 (CRMP-2)
Ser/Thr protein phosphatase 2B catalytic subunit, a isoform
Ser/Thr protein phosphatase PP1-b catalytic subunit
Protein tyrosine phosphatase, non receptor type 1-1
14-3-3 prot f/d
MAGUK p55 subfamily member 3

*1
1
5/2+

rho GDP-dissociation inhibitor 1


N-ethylmaleimide-sensitive fusion protein
Syntaxin 1B
Syntaxin-binding protein 1

Striatum
12 months

2+
1

1+
4
1
1

1+
1
*1
1+
1+
1+
1+

5+
1+
1+

1
1
1+

11
1

Septin 5
Septin 7
Septin-like protein KIAA0202
Hypothetical protein FLJ10849 (Septin 2 or 6 homologue)

1+

1+

Stress-70 protein
Heat-shock cognate 71-kDa protein

Carbonyl reductase [NADPH] 1


Glutathione S-transferase P 2
Glyoxalase I
Thioredoxin reductase

1+
*2
*1+
1+

ARP2/3 complex 20-kDa subunit


Adenyl cyclase-associated protein 1
Dynamin-1
Tubulin a1 chain

1
*1/1+
1

2+
1+
1+
1+
1+
1+
1+
1+

1+
3+

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

Proteomic analysis of parkin knockout mice 1265

Table 2 (Continued)
No. and status of spots

SwissProt or gi accession
Protein degradation (n 3)
PSB5_MOUSE
UBL1_MOUSE
18490720
Lipid metabolism (n 3)
ACDV_MOUSE
PCCA_RAT
6678760
Protein biosynthesis (n 2)
SYY_HUMAN
SYTC_HUMAN
Amino acid synthesis (n 2)
GLNA_MOUSE
SRR_MOUSE
RNA processing (n 3)
HE47_RAT
PCB2_MOUSE
Neurotransmitter metabolism (n 2)
GABT_RAT
TY3H_MOUSE
Others (n 7)
ALBU_MOUSE
ARHY_MOUSE
CAH2_MOUSE
FCE2_MOUSE
KPR1_HUMAN
POR1_MOUSE
SPEE_MOUSE

Striatum
2 months

Protein name

Proteasome subunit beta type 5


Ubiquitin carboxyterminal hydrolase L1
Deubiquitinating enzyme OTUB1
Acyl-CoA dehydrogenase, very-long-chain specific
Propionyl CoA carboxylase a chain
Lysophospholipase 1
Tyrosyl tRNA synthetase
Threonyl tRNA synthetase

Striatum
12 months

1+
1+
1
1+
1+
1
1+
1

Glutamine synthetase
Serine racemase

Probable ATP-dependent RNA helicase p47


Poly(rC)binding protein

1
1+

4-Aminobutyrate aminotransferase, mitochondrial


Tyrosine 3-hydroxylase

1+

Serum albumin
ADP-ribosylarginine hydrolase
Carbonic anhydrase II
Low-affinity immunoglobulin epsilon FC receptor
Ribose-phosphate pyrophosphokinase I
Voltage-dependent anion-selective channel protein 1
Spermidine synthase

1+

2+
1+

1+
3
1+
1
2+
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.

This approach conrmed that a large proportion of the


differentially regulated proteins were involved in energy
metabolism (nup 9, ndown 10, nup/down 4), including
the glycolytic pathway, the Krebs cycle and the mitochondrial respiratory chain. The differentially regulated proteins
also 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 the
oxidative stress response and/or detoxication processes
(nup 4, ndown 1). These latter changes occurred in the
absence of modications in protein oxidation, as revealed by
the western blot analysis of protein carbonyls in brain lysates
of 2-month-old (not shown) and 12-month-old WT and KO

mice (Fig. 5). Other categories included proteins involved in


lipid metabolism (nup 3, ndown 2), amino acid and
protein biosynthesis pathways (nup 2, ndown 2), RNA
processing (nup 1, ndown 1) and neurotransmitter handling (nup 2).
The proteins that increased in abundance in parkin KO
mice are potential substrates of Parkins E3 ubiquitinprotein
ligase activity. Three members of the membrane-associated
guanylate kinase (MAGUK) family of neuronal scaffolding
proteins (p55 subfamily members 2, 3 and 6) were found
among the proteins found to be exclusively up-regulated in
parkin 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 2
and 3) were up-regulated both in the cortex and in the

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

1266 M. Periquet et al.

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


No. and status of spots
SwissProt or gi accession
Energy metabolism (n 13)
ATPA_MOUSE
ATPB_MOUSE
DHSA_HUMAN
ENOG_MOUSE
G3P_MOUSE
LDHA_MOUSE
MDHC_MOUSE
NUCM_HUMAN
ODPA_MOUSE
PGK1_MOUSE
UCR1_RAT
UCR2_MOUSE
128325765
Signal transduction (n 4)
DPY2_MOUSE
9910474
7709986
MPP2_HUMAN
Vesicular trafficking (n 3)
NSF_MOUSE
SEP7_MOUSE
SNX5_MOUSE
Protein folding (n 6)
GR75_MOUSE
GR78_MOUSE
HS72_MOUSE
HS7C_MOUSE
OS94_MOUSE
TCP2_MOUSE
Stress/detoxification (n 2)
ACON_HUMAN
GTP2_MOUSE
Cytoskeleton (n 2)
ACTG_HUMAN
SPCN_MOUSE
Protein degradation (n 1)
UBA1_MOUSE
Lipid metabolism (n 2)
CAO1_MOUSE
MTE1_MOUSE
Others (n 5)
A1A3_RAT
CLB2_MOUSE
DD19_MOUSE
MO25_MOUSE
POR1_MOUSE

Protein name

Cortex 2 months

Cortex 12 months

ATP synthase a chain, mitochondrial


ATP synthase b chain, mitochondrial
Succinate dehydrogenase flavoprotein subunit
c Enolase
Glyceraldehyde-3-phosphate dehydrogenase
L-Lactate dehydrogenase A chain
Malate dehydrogenase, cytoplasmic
NADH-ubiquinone oxidoreductase 49-kDa subunit
Pyruvate dehydrogenase E1 component a subunit
Phosphoglycerate kinase 1
Ubiquinol-cytochrome c reductase complex core protein I
Ubiquinol-cytochrome c reductase complex core protein 2
NADH-ubiquinone oxidoreductase PDSW subunit HS homolog

1/1+
1
1+
1/1+
3/1+
1
2
11+
*1
1+
1

2+

Dihydropyrimidinase-related protein-2 (CRMP-2)


MAGUK p55 subfamily member 6
Sumo-1 activating enzyme subunit 2
MAGUK p55 subfamily member 2

2/3+
1+
1+
1+

N-ethylmaleimide sensitive fusion protein


Septin 7
Sorting nexin 5

1+
1+
*1+

Stress-70 protein
78-kDa glucose-regulated protein
Heat-shock-related 70-kDa protein 2
Heat-shock cognate 71-kDa protein
Heat-shock 70-related protein APG-1
T-complex protein 1, a subunit B

1
1
2
1+

Aconitate hydratase, mitochondrial


Glutathione S-transferase P 2

2+
*1

c-Actin
Spectrin a chain, brain

1
2+

Ubiquitin-activating enzyme E1 type 1

1+

Acyl-coenzyme A oxidase 1, peroxisomal


Acyl CoA thioester hydrolase

1+

Sodium/potassium-transporting ATPase a-3 chain


Calretinin
ATP-dependent RNA helicase DDX19, dead box protein
No known function
Voltage-dependent anion-selective channel protein 1

2+

1+

1+

1
1+
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.

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

Proteomic analysis of parkin knockout mice 1267

Table 4 Proteins differentially regulated at both 2 and 12 months

SwissProt/gi accession
Energy metabolism
ATPA_MOUSE
CISY_HUMAN
KAD3_MOUSE
KPY2_MOUSE
LDHA_MOUSE
MDHC_MOUSE
PGK1_MOUSE
UCR1_RAT
Signal transduction
DPY2_MOUSE
Vesicular trafficking
NSF_MOUSE
STB1_MOUSE
Septin family
SEP5_MOUSE
SEP7_MOUSE
8922712
Cytoskeleton
DYN1_MOUSE
Protein degradation
UBA1_MOUSE
Neurotransmitter metabolism
GABT_RAT
Others
POR1_MOUSE

No. and status


of spots

Fold

1/8+
2
2+
5/2+
2
3
2
2+

>
>
>
>
>
>
>
>

1.45
1.45
1.45
1.45
1.45
1.45
2.00
1.45

7-/10+

> 1.45

1/2+
1/1+

>1.45
> 1.45

2+
2+
2+

> 1.45
> 1.45
> 1.45

1/1+

> 1.45

2+

> 1.45

3+

> 1.45

1/3+

> 1.45

2D analysis allows the resolution of different isoforms and/or posttranslational 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
fold difference associated.

striatum, at both ages (Tables 4 and 5). In particular, ve


spots, migrating with the same molecular weight but with
different isoelectric points, were identied as the Parkin
substrate CDCRel-1/septin5 (M. Duchesne, personal communication). The abundance of one of these spots increased
by at least 45% in the striatum of 2- and 12-month-old parkin
KO mice compared with WT mice (Tables 2 and 4). In
addition, the levels of a second spot were increased by 33%
in the striatum of 12-month-old mice (p < 0.021). As we
decided arbitrarily to take into consideration only the most
signicant protein abundance changes (> 45%), this protein
is not listed in the tables presented. A reproducible increase
in total CDCRel-1 levels was conrmed in individual cortex
samples (Fig. 4) and pooled striata (data not shown) of

Table 5 Proteins differentially regulated in cortex and striatum at 2


and/or 12 months
SwissProt or
gi accession
Energy metabolism
ATPA_MOUSE
DHSA_HUMAN
G3P_MOUSE
LDHA_MOUSE
MDHC_MOUSE
PGK1_MOUSE
UCR1_RAT
Signal transduction
DPY2_MOUSE
Vesicular trafficking
NSF_MOUSE
SEP7_MOUSE
Protein folding
GR75_MOUSE
HS7C_MOUSE
Stress/detoxification
GTP2_MOUSE
Others
POR1_MOUSE

Age
(months)

No. and status


of spots

Fold

2/12
2
2
2/12
2/12
2/12
2/12

1/8+
2+
3/3+
2
3
2
2+

> 1.45
> 1.45
>1.45
> 1.45
> 1.45
> 1.45
> 1.45

2/12

7/10+

> 1.45

2/12
2/12

12+
2+

> 1.45
> 1.45

12
2

3+
3

> 1.45
> 1.45

> 2.00

2/12

1/3+

> 1.45

2D analysis allows the resolution of different isoforms and/or posttranslational modifications of a same protein. Thus, several dysregulated 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 electrophoretic mobility (status +/-). Owing to the use of overlapping pH
gradients, the same protein could also be detected twice in two consecutive 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.

parkin KO mice, resolved by conventional one-dimensional


gel electrophoresis and analysed by quantitative western
blotting. However, this increase did not reach statistical
signicance. This is probably due to the fact that the ve
different CDCRel-1 isoforms with identical molecular weight
migrate as a single band on one-dimensional gels.
Additional Parkin substrates showed altered abundance in
our parkin KO model. Huynh et al. (2003) reported that
synaptotagmin XI and synaptotagmin I are ubiquitylated by
Parkin. In our study, synaptotagmin I was found to be
up-regulated by 40% (p < 0.001) in the cortex of 2-monthold mice (data not shown). In addition, several spots
corresponding to the tubulin a-1 chain (Ren et al. 2003)
were increased in abundance in the striatum of 12-month-old
parkin KO mice (Table 2). Finally, we also analysed the
levels of our previously identied Parkin substrate p38 (Corti
et al. 2003) by one-dimensional western blotting, although it
was not identied as being altered in abundance in our 2D

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

1268 M. Periquet et al.

Table 6 Functions of differentially regulated proteins assessed by


keyword analysis

Fig. 2 2D gel images showing selected differentially regulated proteins. (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 more
basic variant was up-regulated to the same extent ( 1.63), suggesting
a shift due to phosphorylation or other post-translational modifications.
Squares and circles indicate downward and upward changes in parkin
KO mice respectively.

DIGE analysis. The quantitative analysis of protein samples


from individual striata from 12-month-old KO and WT mice
did not reveal any signicant change in abundance of p38
levels (Fig. 4).
Discussion

Nowadays, 2D DIGE is the most powerful 2D polyacrylamide gel electrophoresis-based approach for widespread
protein proling. We used this recent technology to compare
protein expression proles in parkin KO and WT mice, using
a pool of samples as an internal standard and the dedicated
DeCyder analysis software developed by Amersham Biosciences (Tonge et al. 2001; Gharbi et al. 2002; Yan et al.
2002). Owing to the large amount of material required for 2D
DIGE, our analyses were performed on pools of brain
extracts obtained from six animals for each condition. This
experimental paradigm gives an appropriate indication of the

Occurrences

SwissProt keyword

Confidence index

16
6
5
7
5
5
8
5
8
7
5
8
8
18
8
11
25
7
6
9
22

Glycolysis
Heat_shock
ATP_synthesis
Flavoprotein
Hydrogen_ion_transport
Proteasome
Ligase
FAD
Lyase
NADP
Protein_biosynthesis
Chaperone
NAD
Transit_peptide
Kinase
Acetylation
Oxidoreductase
Magnesium
Isomerase
GTP binding
Mitochondrion

20.66
13.34
13.34
7.37
6.46
6.25
6.16
5.56
5.52
5.29
5.13
4.85
4.71
4.68
4.64
4.04
3.86
3.79
3.58
3.37
3.21

Only keywords occurring more than four times were taken into account
and their relative significance (confidence index 3) was determined
by 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.

average biological differences between groups of samples,


although it does not provide information on inter-animal
variation. The validity of this approach was demonstrated in
a previous study showing that similar results are obtained
when individual animals or pooled samples are analysed by
2D DIGE (Tonge et al. 2001). We found that 87 proteins
differed in abundance by at least 45% in parkin KO and WT
mice.
Classication of these proteins led to the identication of
12 major functional categories affected by inactivation of the
parkin gene. The most frequently represented category
included proteins related to energy metabolism, particularly
to the glycolytic pathway, the Krebs cycle and the mitochondrial respiratory chain. This functional class has also been
shown to be affected at the mRNA and/or protein levels in
other models of cell degeneration and in neurodegenerative
diseases (Loring et al. 2001; Gozal et al. 2002; Napolitano
et al. 2002; Seong et al. 2002; Tilleman et al. 2002a, 2002b;
Xie et al. 2002; Kuhn et al. 2003). These proteins were
either up- or down-regulated, or subject to post-translational
modication, such that the overall consequences of their
differential regulation are difcult to predict. In the absence
of a neurodegenerative phenotype, these changes probably

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

Proteomic analysis of parkin knockout mice 1269

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 percentage of the total that increased in
abundance (right) and the total that
decreased in abundance (left). Only functional categories with two or more members
are shown for proteins differing in abundance 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 excluded.

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 contrast, 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 5
animals per group). *p < 0.05 versus WT (Students t-test). Results
representative of at least three independent experiments are shown.

reect an adaptive regulation of cellular energy in parkin KO


mice, as suggested by the altered abundance of enzymes
involved 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 and


sporadic PD. Changes in proteins related to this pathway
have previously been reported at the transcriptional level in
cell and mouse models of PD (Cadet et al. 2001; Ryu et al.
2002; Kuhn et al. 2003). In parkin KO mice, the abundance
of several stress-induced chaperones was altered, including
heat-shock protein 70-related proteins, the osmotic stress
protein Osp94 and the T complex protein 1, which plays a
role in the folding of actin and tubulin, and probably also of
other cytoskeletal proteins (Dunn et al. 2001). Of note,
several cytoskeletal proteins were altered in abundance in
parkin KO mice, including the Parkin substrate tubulin a-1
chain (Ren et al. 2003). In addition, the PD-associated
protein UCH-L1, and the proteasome subunit b type 5 were
more abundant in parkin KO mice, whereas the deubiquity
enzyme OTU-domain Uba1-binding protein (OTUB1) was
less abundant. These modications were paralleled by
changes in the levels of several enzymes linked to cellular
stress and detoxication processes. In particular, the level of
the antioxidant protein Glutathione S-transferase P2 (GTP2)
was decreased in both striatum and cortex from 2-month-old
parkin KO mice, whereas other proteins (carbonyl reductase,
glyoxalase I, thioredoxin reductase) known to be protective
against oxidative stress-induced neurodegeneration (Chen
et al. 2004) increased in abundance in these mice. These
changes might reect an adaptive response to high concentrations of free radicals, as suggested by our previous
observation that reduced glutathione concentrations are high
in both the striatum and in fetal mesencephalic neuronal
cultures from parkin KO mice (Itier et al. 2003). In young
and aged parkin KO mice, however, the levels of protein

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

1270 M. Periquet et al.

(a)

(b)

Fig. 5 Levels of protein carbonyls are similar in the cortex and striatum 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-dinitrophenylhydrazine was omitted; M, molecular weight protein standard; BSA, oxidatively modified bovine seum albumin.

carbonyls were comparable to those of WT mice, indicating


that antioxidant defences and/or detoxication processes are
efcient in these animals.
Several proteins involved in vesicle trafcking or function
were also affected in parkin KO mice, including components
of the SNARE (soluble N-ethylmaleimide sensitive fusion
protein (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 more
abundant in parkin KO mice than in WT mice. Members of
this protein family accumulate in neurobrillary tangles and
glial brils in Alzheimers disease, and in lewy bodies in PD
(Kitada et al. 1998; Ihara et al. 2003). CDCRel-1 and the
related protein CDCRel-2 also accumulate in the brains of
PD sufferers with parkin gene mutations (Zhang et al. 2000;
Choi et al. 2003). Regulation of the degradation of synaptic
vesicle-associated proteins by Parkin, which is present on
synaptic vesicles (Kubo et al. 2001), may modulate neurotransmitter release. Loss of this function might be partially
responsible for the observed abnormalities in dopaminergic
and glutamatergic neurotransmission in parkin KO mice
(Itier et al. 2003). The up-regulation of three members of the
MAGUK p55 subfamily of synaptic scaffolding proteins may
also be involved in these changes in synaptic function.
Interestingly, a previous study demonstrated a direct interaction between Parkin and the MAGUK family member
calcium/calmodulin-dependent serine protein kinase (CASK)
and suggested that CASK might target Parkin to specialized
functional membrane domains where it may modulate the
activity of NMDA receptors (Fallon et al. 2002).
While this work was in progress, Palacino et al. (2004)
reported a differential proteomic analysis of another parkin
KO mouse model, using conventional 2D gel technology
and silver-stained gels. Surprisingly, they found consistent
decreases in the abundance of only 13 proteins and altered
electrophoretic mobility of an additional one. Decreased
abundance of proteins involved in mitochondrial function,
i.e subunits of complexes I and IV, was associated with a
reduction in the respiratory capacity of striatal mitochondria from parkin KO mice. This mouse model also
exhibited decreased levels of proteins involved in protection against oxidative stress, decreased serum antioxidant
capacity and, in contrast to our model, increased protein
and lipid peroxidation. Only two (pyruvate dehydrogenase
E1a1 and glyoxalase I) of the 13 proteins identied in
Palacinos study were also found to be changed in
abundance in our parkin KO mice. These proteins showed
a decrease in abundance in Palacinos study, whereas they
were increased in abundance in our parkin KO mice,
raising the possibility that these proteins represent false
positives in either or both studies. However, this surprising
discordance might also result from the identication of
different 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 a
broader range of pH gradients, which might have led to the
identication of protein isoforms that were not resolved on
the 310 pH gradient gel used by Palacino. The considerably greater number of proteins identied in our study

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

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

Succinate dehydrogenase flavoprotein


subunit
c Enolase

Glyceraldehyde 3-phosphate dehydrogenase

NADH-ubiquinone oxidoreductase
49-kDa subunit
Pyruvate dehydrogenase E1 component
a subunit

Ubiquinol-cytochrome c reductase complex


core protein I

DHSA_HUMAN

ENOG_MOUSE

G3P_MOUSE

NUCM_HUMAN

UCR1_RAT

Signal transduction
DPY2_MOUSE

Dihydropyrimidinase-related protein-2
(CRMP-2)

Dihydrolipoamide dehydrogenase

DLDH_MOUSE

ODPA_MOUSE

ATP synthase b chain, mitochondrial

Fructose-bisphosphate aldolase C
[Fragment]
ATP synthase a chain, mitochondrial

Protein name

ATPB_MOUSE

ATPA_MOUSE

Energy metabolism
ALFC_MOUSE

SwissProt or
gi accession

+/

(Gly form)

+ (Ox form)
+ (Ox form)
+

+
na

na
+
+
/+
+

na
/+
+

na

Status

PD
AD
AD
AD
SCZD
ADPD
AD
AD

PD
SCZD
PD
AD
PD
AD
DS
PD
AD
AD
AD
PD
AD
SCZD
AD
PD
AD
PD
SCZD
AD
AD
AD
PD
SCZD
PD
PD
AD
AD
SCZD

Disease

parkin KO mice, cortexstriatum


Human brain
Human brain
Human brain
Human brain
Human brain
Human brain
GSK3b transgenic mice

parkin KO mice, cortexstriatum


Human brain
parkin KO mice, cortexstriatum
Tau transgenic mice
parkin KO mice, cortexstriatum
Human brain
Human brain
parkin KO mice, cortexstriatum
Tau transgenic mice
parkin KO mice, cortexstriatum
GSK3b transgenic mice
parkin KO mice, cortexstriatum
Human brain
Human brain
Tau transgenic mice
parkin KO mice, cortexstriatum
Human brain
MPTP mice mitochondria SN
Human brain
Tau transgenic mice
parkin KO mice, cortexstriatum
GSK3b transgenic mice
parkin KO mice, cortexstriatum
Human brain
MPTP mice mitochondria SN
parkin KO mice, ventral midbrain
parkin KO mice, cortexstriatum
Human brain
Human brain

Model and/or tissue

Table 7 Proteins differentially regulated between parkin KO and WT mice and identified in other proteomic studies analyzing neurodegenerative models or disorders

Periquet et al. 2005


Tsuji et al. 2002
Schonberger et al. 2001
Kanninen et al. 2004
Prabakaran et al. 2004
Choi et al. 2004a
Castegna et al. 2002b
Tilleman et al. 2002b

Periquet et al. 2005


Prabakaran et al. 2004
Periquet et al. 2005
Tilleman et al. 2002a
Periquet et al. 2005
Tsuji et al. 2002
Kim et al. 2000
Periquet et al. 2005
Tilleman et al. 2002a
Periquet et al. 2005
Tilleman et al. 2002b
Periquet et al. 2005
Schonberger et al. 2001
Prabakaran et al. 2004
Tilleman et al. 2002a
Periquet et al. 2005
Schonberger et al. 2001
Jin et al. 2005
Prabakaran et al. 2004
Tilleman et al. 2002a
Periquet et al. 2005
Tilleman et al. 2002b
Periquet et al. 2005
Prabakaran et al. 2004
Jin et al. 2005
Palacino et al. 2004
Periquet et al. 2005
Kim et al. 2000
Prabakaran et al. 2004

References

Proteomic analysis of parkin knockout mice 1271

Tubulin a chain

TBA1_MOUSE

Dynamin-1

Cytoskeleton
DYN1_MOUSE

Spectrin a chain, brain

Glyoxalase I

LGUL_MOUSE

SPCN_MOUSE

Carbonyl reductase [NADPH] 1

Aconitate hydratase, mitochondrial

Stress/detoxification
ACON_HUMAN

DHCA_MOUSE

Heat-shock-related 70-kDa protein 2

Septin 7

Syntaxin-binding protein 1

N-ethylmaleimide sensitive fusion


protein

Ser/Thr protein phosphatase


2B catalytic
subunit, a isoform
14-3-3 prot f/d

Protein name

Protein folding
HS72_MOUSE

SEP7_MOUSE

STB1_MOUSE

Vesicular trafficking
NSF_MOUSE

143Z_MOUSE

P2BA_MOUSE

SwissProt or
gi accession

Table 7 (Continued)

+/
+/
na
+

+
+
+
-

na

PD
SCZD
AD
PD
SCZD
PD
AD
SCZD

PD
SCZD
PD
SCZD
ADDS
PD
PD
PD

PD
SCZD
AD

PD
AD
SCZD
AD
PD
PD
PD
SCZD

AD
PD
PD
AD

na
+/

+
+
+/
+
+

PD

Disease

Status

parkin KO mice, cortexstriatum


Human brain
Tau transgenic mice
parkin KO mice, cortexstriatum
Human brain
parkin KO mice, cortexstriatum
GSK3b transgenic mice
Human brain

parkin KO mice, cortexstriatum


Human brain
parkin KO mice, cortexstriatum
Human brain
Human brain
parkin KO mice, cortexstriatum
MPTP mice mitochondria SN
parkin KO mice, ventral midbrain

parkin KO mice, cortexstriatum


Human brain
Tau transgenic mice

parkin KO mice, cortexstriatum


Human brain
Human brain
GSK3b transgenic mice
parkin KO mice, cortexstriatum
MPTP mice mitochondria SN
parkin KO mice, cortexstriatum
Human brain

GSK3b transgenic mice


parkin KO mice, cortexstriatum
MPTP mice mitochondria SN
Tau transgenic mice

parkin KO mice, cortexstriatum

Model and/or tissue

Periquet et al. 2005


Prabakaran et al. 2004
Tilleman et al. 2002a
Periquet et al. 2005
Prabakaran et al. 2004
Periquet et al. 2005
Tilleman et al. 2002b
Prabakaran et al. 2004

Periquet et al. 2005


Prabakaran et al. 2004
Periquet et al. 2005
Prabakaran et al. 2004
Balcz et al. 2001
Periquet et al. 2005
Jin et al. 2005
Palacino et al. 2004

Periquet et al. 2005


Prabakaran et al. 2004
Tilleman et al. 2002a

Periquet et al. 2005


Schonberger et al. 2001
Prabakaran et al. 2004
Tilleman et al. 2002b
Periquet et al. 2005
Jin et al. 2005
Periquet et al. 2005
Prabakaran et al. 2004

Tilleman et al. 2002b


Periquet et al. 2005
Jin et al. 2005
Tilleman et al. 2002a

Periquet et al. 2005

References

1272 M. Periquet et al.

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

Ox, oxidized; Gly,glycosylated; PD, Parkinsons disease; AD, Alzheimers disease; DS, Downs syndrome; SCZD, schizophrenia; SN, substantia nigra; +, increased abundance, , decreased
abundance; +/-, altered electrophoretic mobility; na, data not available.

Periquet et al. 2005


Tilleman et al. 2002b
Castegna et al. 2002a
parkin KO mice, cortexstriatum
GSK3b transgenic mice
Human brain
PD
AD
AD
Glutamine synthetase
Amino acid synthesis
GLNA_MOUSE

+
+ (Ox form)

Periquet et al. 2005


Prabakaran et al. 2004
parkin KO mice, cortexstriatum
Human brain
PD
SCZD
+

Tyrosyl tRNA synthetase


Protein biosynthesis
SYY_HUMAN

Periquet et al. 2005


Schonberger et al. 2001
Choi et al. 2004b
Kadota et al. 2004
Castegna et al. 2002a
parkin KO mice, cortexstriatum
Human brain
Human brain
Mouse ES cells
Human brain
PD
SCZD
PDAD
DS
AD
+

+ (Ox form)
Ubiquitin carboxyterminal
hydrolase L1
Protein degradation
UBL1_MOUSE

SwissProt or
gi accession

Table 7 (Continued)

Protein name

Status

Disease

Model and/or tissue

References

Proteomic analysis of parkin knockout mice 1273

might be explained by technological differences. First,


cyanin dye staining is more sensitive than silver staining
and has a greater quantication range, extending linearly
over four orders of magnitude (Patton 2000). Second, the
pooled internal standard used with the 2D DIGE technology allows detection of changes in protein abundance that
cannot be seen in pairwise comparisons of individual
biological samples (Friedman et al. 2004). Third, the use
of overlapping pH gradients in our study increased the
resolution of 2D gels. Finally, differences in the number,
nature and status of the differentially regulated proteins
might also result from differences in the tissues and ages
analysed. Palacinos study was conducted on ventral
midbrain of 8-month-old mice, whereas our proteomic
analysis was performed on cortex and striatum from 2- and
12-month-old mice.
In conclusion, we have identied changes in the abundance of a large number of proteins belonging to various
functional categories in our parkin KO model. Several of
these functional categories have already been linked to cell/
animal models of PD as well as to other neurodegenerative
conditions in previous differential gene expression or
proteomic analyses, raising the question of their specicity.
The categories most reproducibly affected at the mRNA
and/or protein levels in these studies include proteins
involved in energy metabolism (Loring et al. 2001; Gozal
et 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 detoxication
processes (Balcz et al. 2001; Prabakaran et al. 2004). Other
functional classes, such as proteins implicated in vesicle
trafcking, cytoskeletal dynamics and protein folding and
degradation, were also frequently identied in several
proteomic analyses on brains from patients affected by
schizophrenia, Downs syndrome or Alzheimers disease
(Schonberger et al. 2001; Castegna et al. 2002a, 2002b;
Choi et al. 2004a; Tilleman et al. 2002a, 2002b; Kadota
et al. 2004; Prabakaran et al. 2004; Jin et al. 2005).
Detailed analysis of the protein abundance changes reported
in the various proteomic studies available so far in the eld
of neurodegeneration and psychiatric conditions (Table 7)
revealed that 26 of the 87 proteins identied in our parkin
KO model (30%) were also affected in other studies. These
proteins cover the major functional classes shown in Fig. 3.
Therefore, in general, common pathways appear to be
altered in these models, although the nature of the affected
proteins is often different. However, our study also hints at
the possible specic involvement of members of the septin
and MAGUK protein families in parkin-related PD. Indeed,
only one previous study reported the down-regulation of a
septin (septin7) in human brain lysates from patients with
schizophrenia (Prabakaran et al. 2004) (Table 7) and, to our
knowledge, alterations in MAGUK protein abundance were

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

1274 M. Periquet et al.

not observed in any of the previously reported differential


proteomic analyses.
Overall, although further functional studies are required to
validate the proteins identied in our study and link them
mechanistically to the molecular events underlying parkinrelated Parkinsons disease, these data already constitute a
valuable reference bank for future investigations into the
pathological mechanisms involved in the early stages of this
disease.
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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