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DOI 10.1002/pmic.201300342
Proteomics 2014, 14, 784–794
RESEARCH ARTICLE
Proteomic analysis of human substantia nigra identifies novel candidates involved in Parkinson’s disease pathogenesis 3 ¨ ´ ˆ 1,2 , Virginie Licker1,2 , Natacha Turck2 , Eniko¨ Kovari , Karim Burkhardt4 , Melanie Cote 3 4 2 Maria Surini-Demiri , Johannes A. Lobrinus , Jean-Charles Sanchez and Pierre R. Burkhard1,2,5 1
Neuroproteomics Group, University Medical Center, Faculty of Medicine, Geneva University, Geneva, Switzerland 2 Translational Biomarker Group, University Medical Center, Faculty of Medicine, Geneva University, Geneva, Switzerland 3 Department of Psychiatry, Geneva University Hospitals, Geneva, Switzerland 4 Department of Pathology, Geneva University Hospitals, Geneva, Switzerland 5 Department of Neurology, Geneva University Hospitals, Geneva, Switzerland Parkinson’s disease (PD) pathology spreads throughout the brain following a region-specific process predominantly affecting the substantia nigra (SN) pars compacta. SN exhibits a progressive loss of dopaminergic neurons responsible for the major cardinal motor symptoms, along with the occurrence of Lewy bodies in the surviving neurons. To gain new insights into the underlying pathogenic mechanisms in PD, we studied postmortem nigral tissues dissected from pathologically confirmed PD cases (n = 5) and neurologically intact controls (n = 8). Using a high-throughput shotgun proteomic strategy, we simultaneously identified 1795 proteins with concomitant quantitative data. To date, this represents the most extensive catalog of nigral proteins. Of them, 204 proteins displayed significant expression level changes in PD patients versus controls. These were involved in novel or known pathogenic processes including mitochondrial dysfunction, oxidative stress, or cytoskeleton impairment. We further characterized four candidates that might be relevant to PD pathogenesis. We confirmed the differential expression of ferritin-L and seipin by Western blot and demonstrated the neuronal localization of gamma glutamyl hydrolase and nebulette by immunohistochemistry. Our preliminary findings suggest a role for nebulette overexpression in PD neurodegeneration, through mechanisms that may involve cytoskeleton dynamics disruption. All MS data have been deposited in the ProteomeXchange with identifier PXD000427 (http://proteomecentral.proteomexchange.org/dataset/PXD000427).
Received: August 6, 2013 Revised: December 13, 2013 Accepted: December 15, 2013
Keywords: Biomedicine / Nebulette / Parkinson’s disease / Postmortem tissue / Substantia nigra
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Correspondence: Dr. Pierre R. Burkhard, Department of Neurology, Geneva University Hospitals, 4, rue Gabrielle-Perret-Gentil, 1211 Geneva 14, Switzerland E-mail: [email protected] Fax: +41-22-372-83-32 Abbreviations: ␣-SYN, alpha-synuclein; BBB, blood brain barrier; BP, biological process; CC, cellular component; DA, dopamine;
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ECM, extracellular matrix; FE, fold enrichment; GGH, gamma glutamyl hydrolase; HCD, high-energy C-trap dissociation; LB, Lewy bodies; OT, orbitrap; PD, Parkinson’s disease; PMI, postmortem interval; SN, substantia nigra; TMT, tandem mass tag; WB, Western blot
Colour Online: See the article online to view Fig. 1 in colour.
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1
Introduction
Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the gradual dysfunction of the extrapyramidal motor system and clinically manifested by tremor at rest, rigidity, or bradykinesia. These cardinal motor symptoms stem from the striatal dopamine (DA) deficit proportional to the severe and progressive loss of midbrain melanized dopaminergic neurons projecting from the substantia nigra (SN) pars compacta. Susceptible nerve cells exhibit intracellular inclusions termed Lewy bodies (LB) formed by the abnormal aggregation of proteins including alphasynuclein (␣-SYN) [1]. Along with neuronal damage, a glial reaction is observed with the release of various inflammatory factors. At present, PD remains an incurable condition for which DA replacement therapy provides the most effective symptomatic relief during the first years of its administration. Importantly, the lack of sensitive and specific biomarkers available precludes an early and accurate PD diagnosis that is still based on patients’ clinical evaluation at a late pathological stage. Several lines of evidence indicate that oxidative stress, mitochondrial dysfunction, impaired protein degradation, excitotoxicity, apoptosis, or inflammation may participate to the successive events ultimately leading to neuronal death [2]. However, despite the multitude of scenarios elaborated, the precise sequence of biological events and molecular pathways underlying PD pathogenesis is still elusive. Reasons for this situation may involve a failure of traditional candidatebased studies to unravel the extremely complex nature of PD processes together with the inability to generate satisfactory animal models of PD, a human-specific disease. Recently, it was proposed to explore PD pathogenesis by global unbiased hypothesis-free “omic” approaches such as proteomics. Large-scale protein expression profiling analyses of human postmortem tissues were conducted that target brain components degenerating in PD including the SN, the frontal cortex, or the locus coeruleus [3–9]. These comparative studies of PD versus control proteomes confirmed several candidates but also revealed many novel ones triggering cell death in PD, which were involved in pathways such as oxidative stress or mitochondrial function. For example, using a 2-DE approach, our group recently identified cytosolic non specific dipeptidase 2 (CNDP2) as a novel key player in PD pathogenesis via a potential role in modulating the cellular response to oxidative stress [7]. Considerable efforts are still needed to improve PD pathogenesis understanding, a prerequisite for the establishment of disease-modifying therapies. We believe that the elucidation of the complex proteome alterations occurring in the SN of PD patients may provide a new source of PD-specific biological targets for diagnostic biomarkers, disease prevention, and therapeutic strategies. In the present study, we performed a high-throughput quantitative profiling of the SN proteome in PD patients compared to neurologically intact controls using a sixplex isobaric chemical labeling technique. We catalogued nearly 1800 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
proteins, which represents so far the largest nigral proteome map ever published. More than 200 proteins were found to be deregulated in PD cases versus controls, 21 of them with a significant fold change over 1.35. Some of them, including ferritin-light chain, seipin, nebulette, and gamma glutamyl hydrolase (GGH), may be interesting targets potentially involved early in PD pathogenesis.
2
Material and methods
2.1 Human brain tissues Thirteen human midbrain tissues (PD group: 5, and control group: 8) were obtained from the Division of Clinical Pathology and Psychiatry of the Geneva University Hospitals under a procedure approved by the local ethical committee (Table 1). Samples were either frozen at –80⬚C or fixed at 4⬚C in 15% formaldehyde for 4 weeks before being paraffin embedded. Written consents for brain autopsy and use for research purposes were signed by close relatives. PD cases were clinically diagnosed according to the United Kingdom PD Society Brain Bank criteria [10] and under symptomatic PD treatment before death, except patient P4 considered as an incidental case. PD diagnosis was confirmed neuropathologically by the observation of a severe nigral depigmentation due to neuronal loss and the presence of ␣-SYN immunoreactive inclusions. P4 may represent an early preclinical stage of PD [11], defined by the absence of symptomatic Parkinsonism but the presence of LB in the SN. Controls were cases without nigral abnormalities and with no previous history of neurological or psychiatric disorders. SN protein extracts were prepared as described in a previously published protocol [7]. Briefly, frozen tissues were cryosectioned in 18-m-thick slices and the SN—macroscopically recognizable by its dark pigmentation, was selectively dissected from slides with a scalpel. About 15 slices per sample were necessary to get 1 mg of nigral tissue, which was collected and kept at –80⬚C until analysis. 2.2 Quantitative sixplex TMT analysis 2.2.1 Sample preparation and TMT labeling Tissue samples from the PD (n = 3) and Control (n = 3) groups were matched for patients’ age and postmortem interval (PMI < 38 h) (Mann–whitney U test, p = 0.4 for age and p = 0.9 for PMI). One milligram of each sample was homogenized in 150 L of sample buffer (6 M Urea, 0.1 M TEAB pH 8.0, 0.1% CHAPS, 0.05% SDS) and protease inhibitors (Roche Applied Science, Basel, Switzerland), vortexed, sonicated, and centrifuged at 4⬚C to remove cellular debris. Protein concentrations were determined using the Bradford assay (Protein Assay, Bio-Rad, Hercules, CA). Fifty micrograms of proteins from each of the six samples were analyzed as described by Dayon et al. [12] with slight modifications. Equal amounts (1 g) of bovine www.proteomics-journal.com
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Table 1. Clinico-pathological data of human postmortem samples by experiment type
Experiment
Group
Sample
Sex
Age
Mean ± SD
TMT
Control
C1 C2 C3 P1* P2 P3*
M F F M M F
70 61 87 73 79 85
72.7 ± 13.2
C1 C3 C4 C5 P1* P3* P4* P5
M F M M M F M M
70 87 89 86 73 85 84 74
83 ± 8.8
C6 C7 C8 P1* P3* P4*
F M M M F M
92 88 82 73 85 84
Parkinson
WB
Control
Parkinson
IHC
Control
Parkinson
79 ± 6
79 ± 6.4
PMI
16 19 2
35 24 13 25 7 24
25.5 ± 9.7
16 2 0 8
35 13 23 31 25 24 38 25
29.7 ± 10.2
16 2 0
34 18 37 25 24 38
87.3 ± 5 80.7 ± 6.7
Mean ± SD
DD
24 ± 11 18.7 ± 10.1
28 ± 6.7
29 ± 7.8
DD, disease duration, PMI, postmortem interval. Asterisks indicate samples whose hemi-mesencephales were available both frozen and paraffin embedded.
-lactoglobulin (LACB) were spiked in each sample as an internal standard to control for an eventual experimental bias. Briefly, samples were reduced in 2.5 mM TCEP for 60 min at 37⬚C, alkylated in 10 mM iodoacetamide for 30 min in the dark at room temperature and digested overnight by trypsin (trypsin/protein ratio 1:25). The resulting peptide mixtures were tagged with the sixplex tandem mass tags (TMT) reagents (Thermo Scientific, Rockford, IL). Control samples C1, C2, C3 were labeled with TMTs 126.1, 128.1, and 130.1 and PD samples P1, P2, P3 with TMTs 127.1, 129.1, and 131.1, respectively. The six samples were pooled and dried for storage at –20⬚C.
(Thermo Electron, San Jose, CA) equipped with a NanoAcquity HPLC system (Waters Corporation, Milford, MA). Each sample was injected twice on two different days to obtain technical replicates and to increase protein coverage. Peptide separation and MS analysis were performed as described elsewhere [13]. To obtain precise peptide quantification without compromising peptide identification, ESI LTQ-OT MS analysis combined CID and high-energy C-trap dissociation (HCD) activation modes in the LTQ-OT. MS survey scans were acquired in the OT analyzer within an m/z window from 400 to 2000. A maximum of three precursor ions were selected for parallel fragmentation in CID or HCD and subsequent MS/MS detection in the LTQ or OT, respectively.
2.2.2 Off-gel peptide fractionation 2.2.4 Peptide and protein identification Peptides were then fractionated in-solution on the basis of their pI with an Agilent 3100 OFFGEL fractionator (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s protocol. Isoelectric focusing was done on a commercial 24 cm IPG pH 3−10 linear dry strip (GE Healthcare Biosciences AB, Uppsala, Sweden) with a 24 wells frame set until 50 kV/h was reached with a maximum current of 50 A and power of 200 mW. The 24 fractions were purified using C18 microspin columns (Harvard Apparatus, Holliston, MA), dried and stored at −20⬚C prior MS analysis. 2.2.3 LC-MS/MS analysis Each peptidic fraction was reconstituted and analyzed by LC−MS/MS on a LTQ Orbitrap (OT) XL mass spectrometer C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
MS data were processed using EasyProtConv [14]. Peak lists were generated from raw data, combining CID and HCD spectra for an optimized peptide identification and quantification [13]. The obtained peak list files were submitted to Easyprot, a platform that uses Phenyx (GeneBio, Geneva, Switzerland) for protein identification [14]. Searches were conducted against UniProt Swiss-Prot database (February 8, 2011, 525 207 entries) specifying Homo sapiens taxonomy. Trypsin was selected as the proteolytic enzyme, one missed cleavage was allowed, cysteine carbamidomethylations, TMT6 amino terminus, and TMT6 lysine were set as a fixed modification whereas oxidized methionine as variable. Peptide z-scores were then set to maintain a false positive peptide ratio below 1%. Proteins with at least two distinct peptide www.proteomics-journal.com
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sequences were selected. Proteins were clustered based on shared peptides indistinguishable by MS, with the protein entry containing the most peptides selected as the group reporter. 2.2.5 Protein relative quantification Publicly available Isobar R package was used to calculate protein ratios and select differentially regulated proteins between sample classes [15]. TMT6 reporter ion intensities were corrected for isotopic impurities as provided by the manufacturer and normalized imposing equal median intensity. Protein ratios were obtained by combining the reporter ion intensity ratios between all pairs of distinct classes (126, 128, 130 vs. 127, 129, 131) measured from MS/MS spectra. For each protein ratio, p-values were computed that model technical (ratio p-value) and biological variability (sample p-value) to estimate ratio accuracy and biological significance, respectively. The level of risk was set at 5%, requiring both p-values to be lower for differential expression selection. All details and mathematical demonstration are provided in Breitwieser et al. [15]. 2.3 Bioinformatics analysis The identified proteins were parsed by protein class through PANTHER online tool v7.2 [16], and GO cellular component (CC), and biological process (BP) annotations via DAVID webbased tool v6.7 [17, 18] or BINGO v2.0 plugin [19] from Cytoscape v2.6 [20]. For enrichment analyses, nigral datasets were compared with the default human reference dataset containing all GO annotations taxonomy. For each GO term, fold enrichment (FE) value was calculated as the ratio of its frequency in the SN proteome dataset compared to the default human proteome reference. Statistically overrepresented categories were determined using modified Fisher’s exact tests with p-value after Benjamini correction lower than 0.05. Categories with at least five associated proteins were selected. Of note, gene name correspondence for each identified protein group was used, as listed in Supporting Information File 1. 2.4 Western blotting verification The expression levels of ferritin light chain (ferritin-L) and seipin were assessed by Western blot (WB) using SN autopsy tissue from control (n = 4) and PD patients (n = 4) (see Table 1). Samples were matched for patients’ sex (3 M/1 F), age, and PMI (Mann–Whitney U test, p = 0.34 for age and p = 0.69 for PMI). Ten micrograms protein from each sample were separated on a 12.5% T SDS-PAGE and electrophoretically transferred on a nitrocellulose membrane using a Hoefer system. Blots were probed with rabbit polyclonal to ferritin-L (1:4000, ab69090, Abcam), rabbit polyclonal to seipin (1:2000, ab106793, Abcam) and mouse C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
monoclonal to actin (1:30 000, Sigma). Anti-rabbit and antimouse horseradish peroxidase conjugated secondary antibodies (Dako) were diluted at 1:2000. Detection of immunoreactive bands was performed using the chemiluminescent ECL Western Blotting System (GE Healthcare) and quantification of band volumes with TotalLab Quant (Nonlinear Dynamics). Actin was used as a reference protein for normalization and statistical analysis was performed using the two-tailed Mann–Whitney U t-test (p < 0.05).
2.5 Immunohistochemical analysis Immunohistochemical staining of ferritin-L, GGH, and nebulette was performed on paraffin-embedded SN tissue from control (n = 3) and PD patients (n = 3) (Table 1). Samples were matched for patients’ sex (2 M/1 F), age, and PMI (Mann– whitney U test, p = 0.4 for age and p = 1 for PMI). Sections were cut at 12 m before being deparaffinized in xylene and dehydrated in ethanol (60⬚). After microwave-induced antigen retrieval in citrate buffer, sections were incubated with 0.25% potassium permanganate in PBS followed by 1% potassium bisulfite/1% oxalic acid to bleach neuromelanin pigmentation. Then, sections were hybridized overnight at 4⬚C with rabbit polyclonal to ferritin-L (1:500, ab69090, Abcam), rabbit polyclonal to GGH (1:200, HPA025226, Sigma) or goat polyclonal to nebulette (1:200, NBP1-45223, Novus Biologicals) diluted in 0.3% Triton-X/1% BSA in PBS followed by 1 h at room temperature with anti-rabbit or anti-goat horseradish peroxidase conjugated secondary antibodies (Dako), diluted in 0.3% Triton-X/1% BSA in PBS. Staining was visualized using 3,3 -diaminobenzidine (DAB, Sigma) as a chromogen and sections were counterstained with Cresyl Violet. No staining was detected on negative controls, treated identically except for hybridization with primary antibody that was omitted.
3
Results
3.1 Quantitative MS analysis of protein profiles The shotgun proteomic experiment involving sixplex tandem mass tag (TMT6 ) labeling allowed the simultaneous identification and quantification of the SN proteome from three PD and three control patients. A total of 1795 proteins were identified with at least two distinct peptides. An overlap of 1183 protein identifications (67%) was observed between replicate injections. Proteins sharing the same set or subset of peptides were considered as a single entry (Supporting Information Appendix 1). Peptides assigned to multiple protein hits were not considered for quantification and 1794 of the 1795 identified protein groups could be quantified with at least one spectrum using Isobar package. Spiked-LACB quantification was checked for quality control and no particular experimental bias was observed. Coefficient of variations of LACB reporter relative intensities calculated for each www.proteomics-journal.com
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channel varied from 6 to 16%, in accordance with isobaric tagging performance [12]. Two hundred four proteins were found significantly differentially expressed (p < 0.05), with keratins being excluded from the analysis. Of them, 96 were overexpressed and 108 underexpressed in the SN of PD patients. Overall, quantified proteins exhibited a low intergroup variability, with about 95% of the ratios falling in a sharp range between 0.8 and 1.25. This indicates that the large majority of the detected proteins was expressed at equivalent levels in PD and control samples. All protein identification and quantification details including statistics are available in Supporting Information File 1 and Supporting Information Appendix 1. The MS data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000427 [21].
3.2 PANTHER class and GO term analyses 3.2.1 Nigral proteome To assess the nigral proteome composition, the 1795 identified proteins were parsed into PANTHER classes and GO CC. The major functional classes were hydrolase (representing 16% of annotated hits), enzyme modulator (13%), oxidoreductase (12%), transferase (12%), and cytoskeletal proteins (12%) (Supporting Information File 2). According to the GO CC analysis (Fig. 1A), the largest proportion (82%) of proteins was annotated as cytoplasmic with a preferential (>50%) distribution in “cytosol,” “mitochondrion,” or “cytoskeleton” structures. About 11% (193 over 1686) of the annotated proteins were parsed into GO terms specific to neurons including “synapse,” (6%) “axon,” (4%) or “neuronal cell body” (3%). To gain more insights in the SN-specific features, enrichment of significantly overrepresented GO BP in the SN proteome dataset was assessed by comparison with the complete human proteome annotations. The same analysis was performed using a dataset approximating the human “central” proteome that comprised 1124 proteins ubiquitously expressed and experimentally identified in seven human nonneuronal cells lines [22]. Overrepresented GO categories found in common between the two sets were discarded in order to highlight the processes occurring more specifically in the nigral tissue and to remove the influence of the common cellular proteome (Supporting Information File 2) [23]. Nine categories exhibited higher FE value in the SN than in the central proteome dataset when considering FE differences of at least one unit, all linked to mitochondrial function and oxidative phosphorylation. One hundred seventy-three GO BP terms were found to be specifically overrepresented (p < 0.001) in the SN dataset (Supporting Information File 2), some of which are shown in Fig. 1A. Thirty-one terms covering 20% of the annotated proteins were related to neuronal activities such as synaptic transmission, plasticity, organization, or development. Many enriched terms were linked to C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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the generation of ATP, through glycolysis, TCA cycle, and oxidative phosphorylation. Other main enriched categories included cytoskeleton organization, regulation, and transport, fatty acid and amino acid metabolisms, or response to oxidative stress. 3.2.2 Differentially expressed proteins To gain more information on the nature and function of the 204 differentially expressed nigral proteins and the specific processes significantly modulated in PD pathogenesis, the dataset was further characterized using PANTHER functional classes and GO terms. The major PANTHER protein classes were oxidoreductase (representing 15% of annotated hits), cytoskeletal protein (13%), hydrolase (13%), transferase (10%), nucleic acid-binding protein (9%) but also chaperone (5%) and many terms related to translation (Supporting Information File 3). Proteins related to oxidoreductase, cytoskeletal, and chaperone classes had a tendency to be overexpressed in PD, whereas proteins in the other cited classes seemed to be more prone to underexpression in PD. Sixty-five GO CC terms and 22 GO BP categories were found to be significantly overrepresented (p < 0.05) in the differential proteins dataset compared to the human proteome (Supporting Information File 3), with a selection of them shown in Fig. 1B. Overall, enrichment in GO terms related to cytoskeleton activities (cytoskeleton, regulation of cell component organization) was observed, with a predominant overexpression of proteins falling in these categories in PD. On the opposite, a prevailing underexpression of proteins associated to several other enriched GO categories was noticed in PD. These included energy metabolism and mitochondrial activities (i.e. generation of precursor metabolites, mitochondria), intracellular transport processes (i.e. vesiclemediated transport, transport regulation), synaptic activities (i.e. synapse, transmission of nerve impulse) or translation (i.e. ribosome, endoplasmic reticulum proteins, translational elongation). 3.3 Verification of protein expression levels by WB To reduce false positive rates in the selection of differentially expressed proteins for verification, we set a cutoff threshold at a fold change higher than 1.35 on all TMT ratios, determined according to Tan et al. method based on random ratios [24] (for calculation details see Supporting Information File 1). The resulting list of the 21 top-differentially expressed proteins is shown in Table 2. Among them, we verified (i) ferritinL, a previously known PD candidate whose expression level in PD brains was still controversial in the literature [25] and (ii) seipin, an original candidate whose biological importance was recently shown in various neurological syndromes but not in PD [26]. Semiquantitative WB analysis demonstrated a significant 1.7-fold increase of ferritin-L and a 2.5-fold decrease of seipin www.proteomics-journal.com
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Figure 1. GO analyses of nigral proteins. In A, GO analyses were performed on the total nigral dataset. A1 illustrates nigral protein distribution pattern in subcellular localization based on GO CC categories. Of note, the sum of percentage exceeds 100% because of multiple annotation terms associated to a single protein. A2 shows representative GO BP terms found significantly overrepresented in the nigral proteome (Benjamini correction, p < 0.001). Protein count refers to the number of annotated proteins in each category. In B, enrichment of the set of differentially expressed nigral proteins in significantly overrepresented GO CC (B1) or BP (B2) is shown (Benjamini correction, p < 0.05). For each GO term, fold enrichment is given, as well as the number of overexpressed and underexpressed proteins in the SN of PD patients.
expression levels (p < 0.05, Mann–Whitney t-test) in the SN of PD patients (n = 4) versus controls (n = 4), consistent with our proteomic analysis (Fig. 2). Results were confirmed using samples from the proteomic experiment (n = 4) as well as four independent samples including an incidental PD case (P4) potentially representing a preclinical stage of the disease (Table 1). Importantly, P4 exhibited protein expression levels similar to the other PD cases (n = 3). 3.4 Cellular localization of candidates by IHC Next, we determined the localization of ferritin-L and the two novel candidates GGH and nebulette by IHC, using available paraffin-embedded nigral tissues of PD (n = 3) and Control
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(n = 3) groups (Table 1). Representative IHC images of two patients (P1, C6) are shown in Fig. 2 (for other patients see Supporting Information Fig. 1). As expected, ferritin-L was predominantly expressed in oligodendrocytes and microglial cells. Differences between pathological and healthy staining patterns were difficult to assess given the large number of immunoreactive cells. Conversely, the expression of GGH and nebulette was observed in the remaining DA neurons, validating our whole tissue approach to detect changes in scarcer cell populations. GGH staining was strong and occurred predominantly in DA neuron cell bodies and processes in the SN of PD and control patients. A lower number of GGH immunoreactive neurons was observed in PD, most probably resulting from neuronal loss. This observation supports the decrease in GGH level observed by proteomics. Similarly,
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Table 2. Top-differentially expressed proteins in the SN of PD patients versus controls with a fold change over 1.35.
AC
Protein name
PD/C ratio
Main PD pathogenic hypothesis
Top-overexpressed proteins in the SN of PD patients O76041 Nebulette P21695 Glycerol-3-phosphate dehydrogenase [NAD(+)], cyt Q9UDY2 Tight junction protein ZO-2 P14136 Glial fibrillary acidic protein P04083 Annexin A1 P02792 Ferritin light chain P00966 Argininosuccinate synthase P42330 Aldo-keto reductase family 1 member C3 Q00796 Sorbitol dehydrogenase P52895 Aldo-keto reductase family 1 member C2 Q8N5M4 Tetratricopeptide repeat protein 9C
1.86 1.74 1.71 1.63 1.58 1.57 1.53 1.49 1.46 1.44 1.39
*Cytoskeleton structure/regulation Energy metabolism *Blood-brain barrier communication Glial activation and inflammation Glial activation and inflammation Oxidative stress Oxidative stress (NO), protein modification Oxidative stress Energy metabolism Oxidative stress Unknown
Top-underexpressed proteins in the SN of PD patients P01871 Ig mu chain C region Q92820 Gamma-glutamyl hydrolase O00241 Signal-regulatory protein beta-1 Q96G97 Seipin P69849 Nodal modulator 3 Q9NY65 Tubulin alpha-8 chain Q53FP2 Transmembrane protein 35 Q05639 Elongation factor 1-alpha 2 O14594 Neurocan core protein P30040 Endoplasmic reticulum resident protein 29
0.29 0.64 0.66 0.67 0.70 0.71 0.72 0.73 0.74 0.74
Glial activation and inflammation Unknown Glial activation and inflammation Apoptosis Unknown *Cytoskeleton structure/regulation Unknown Protein synthesis, apoptosis *ECM structure/regulation Protein folding
Associated main PD pathogenic hypotheses are listed, with asterisks representing less conventional or novel pathways. AC, Swissprot accession number.
Figure 2. Confirmation and localization of selected proteomic candidates in the SN of PD patients. Ferritin-L in panel A and seipin in panel B were verified by WB analysis and respectively found significantly increased (PD/C ratio of 1.7) and decreased (PD/C ratio of 0.4) in PD patients versus controls (p < 0.05, Mann–Whitney U test). Bar graphs represent average -actin normalized band volumes with SD, given in % of controls. IHC analysis for GGH, nebulette, and ferritin-L was performed in the SN of PD patients and controls, with panel C showing representative IHC of a control (patient C6: a, c, e) and PD case (patient P1: b, d, f). GGH and nebulette exhibited a predominant expression in DA neurons (i.e. arrows) whereas ferritin-L was predominantly expressed by glial cells.
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nebulette expression was mainly confined to DA neuron cytoplasm, with a staining appearing to be more pronounced in PD than control patients. This tends to confirm the increase in nebulette levels observed by proteomics in PD patients.
4
Discussion
4.1 Characterizing the SN proteome Taking advantage of a sensitive shotgun proteomic approach we obtained the most comprehensive picture of the human SN proteome so far, with 1795 proteins simultaneously identified and quantified. Comparison with previously published studies [3, 4, 8] revealed 1139 proteins newly associated with human SN (listed in Supporting Information File 1). A significant proportion of annotated proteins were parsed into GO categories related to neuronal components (11%) or activities (20%). GO overrepresentation analyses supported the notion that SN relies on high-energy supply and efficient anti-oxidant defenses, due to the particular morphological and physiological specificities of DA neurons [27, 28]. An important role for cytoskeletal organization, proper vesicular transport and trafficking, or amino acid cycle was also highlighted. Any perturbation in one or more of the above-cited processes might be particularly critical for SN correct function. SN proteome characterization represents a first step toward the understanding of the SN complexity and its specific features, which might hold the key to its vulnerability in PD.
4.2 Identifying protein alterations in the SN of PD patients Our proteomic experiment revealed a subset of 204 proteins modulated in PD. Twenty-four of them were found similarly differential by others [3, 4, 7, 8], with a majority of concordant ratios (i.e. peroxiredoxin 1, gluthatione S transferase P, neurocan, GFAP, CNDP2). Differences between proteomic studies may be explained by various means including sample heterogeneity (i.e. patient’s history, comorbidities), tissue quality and PMI as well as the lack of standardized protocols and reliable independent validation steps. Therefore, we evaluated protein expression of four selected candidates by WB (i.e. ferritin-L and seipin) and IHC (i.e. ferritin-L, nebulette, and GGH) experiments, which tend to confirm our proteomic results. Interestingly, IHC analyses indicated a preferential localization of nebulette and GGH inside neurons, confirming the ability of our approach to detect neuron-specific changes. Consistent with other proteomic findings [7,8], we demonstrated the overexpression of ferritin, in particular its light subunit, in the SN of PD patients. With a preferential localization in glial cell populations, ferritin-L levels may be augmented to bind iron released from dying DA neurons [29] or may reflect the mild gliosis occurring in PD. Importantly, we highlighted the potential involvement of three novel can C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
791 didates, GGH, seipin, and nebulette, whose link with PD had not been formally established. GGH is a key enzyme in folate metabolism whose decreased expression in PD SN could be due to neuronal loss, as assessed by IHC analysis. The ER-resident glycoprotein seipin, found underexpressed in PD, was already associated to neurological disorders (i.e. Silver syndrome, Charcot-Marie-Tooth syndrome variant) [26]. The protein was observed similarly diminished in the locus coeruleus of PD patients, another aminergic nucleus affected earlier than SN in the disease process [9]. Mutations in seipin were shown to result in protein misfolding, accumulation in the ER, unfolded protein response pathway activation, ERstress, protein translation diminution, and ultimately cell death. These observations suggest a role for seipin in PD pathogenesis through protein aggregation or ER-mediated stress. Nebulette, found overexpressed in PD, is a member of the nebulin actin-binding protein family [30] whose shorter splice variant Lasp-2 is strongly expressed in brain tissues [31]. Interestingly, Lasp-2 overexpression in pheocytochroma PC6 cell line was shown to inhibit neurite outgrowth in response to growth factors [32]. Together with its molecular scaffold role for actin, Lasp-2 may participate in the organization of focal adhesions, structures linking cellular cytoskeleton with extracellular matrix (ECM) [32]. Inappropriate interactions in these regions could lead to neurodegeneration through anoikis programmed cell death [33]. Altogether, proteome alterations observed in PD may reflect several known pathogenic processes involved in neurodegeneration as a cause or a consequence of it (Fig. 3). We observed an increase in proteins associated to oxidative stress as well as a decrease in proteins involved in mitochondrial function and energy metabolism failure, three interconnected processes able to disrupt protein homeostasis and cellular function. We demonstrated a preferential overexpression of cytoskeletal proteins in PD, important LB components whose defects were already mentioned in PD [34–36]. Protein aggregation is central to neurodegenerative diseases and our data point to disturbances in several mechanisms known to promote protein accumulation such as protein processing or transport. We found more than a hundred proteins overexpressed in the SN of PD, some of which may accumulate pathogenically including vimentin, known to be a LB component [37, 38]. Of note, we did not identify overexpression of the classical LB constituents ubiquitin or ␣-SYN, similarly to other proteomic studies [3, 4, 8, 9]. This may be explained by the fact that (i) ␣-SYN may not be as central in sporadic PD as it is in familial PD forms, (ii) ␣-SYN overexpression may be masked by DA neuronal cell loss in PD and (iii) our methodology may not allow a proper solubilization of LB or protein aggregates. We also noticed a general attenuation in protein translation in PD brains, which may result from protein misfolding, aggregation, or ER stress. Our data suggested some less conventional pathogenic pathways as well, such as blood brain barrier (BBB) or ECM impairments. Abnormalities in neurons’ surroundings, including glial cell populations, BBB or ECM, can critically influence in a positive or www.proteomics-journal.com
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Figure 3. Potential pathogenic pathways involved in PD according to our proteomic data. Proteins found overexpressed in PD are represented in red and those underexpressed in blue.
negative way the fate of resident neuronal cells. Alterations of the ECM matrix for example may affect synaptic morphology and function [39], whereas a defect in the BBB may constitute a causative factor of DA neuron degeneration [40]. Finally, we found a dysregulation in proteins for which very few or no functional information was available such as Tetratricopeptide repeat protein 9C or Nodal Modulator 3.
4.3 Assessing limitations Using a whole tissue approach, we analyzed signals coming from mixed cell populations, without being able to differentiate their individual contribution to the total signal. Changes in specific nigral subpopulations were thus probably attenuated or totally masked, translating into smaller fold change. For example, signals from DA neuronal cell population (representing up to 10% of total cells) might be diluted ten times and become hardly detectable. Moreover, with a tissue-based normalization (i.e. tissue weight and total protein concentration), the total number of DA neurons in PD samples was theoretically reduced of at least 70% compared to controls. Thus, the overexpression of proteins such as alpha-SYN in surviving PD DA neurons may have been counteracted by the higher number of these cells in control tissues. Of note, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
nebulette protein found to be predominantly expressed in neurons by IHC, might exhibit a real higher increase in PD than the 1.9-fold reported by proteomics. In addition, the observed underexpression in several neuronal proteins might reflect neuronal loss occurring in PD. GGH for instance, observed to be decreased in PD and shown to localize in DA neurons by IHC, did not seem to exhibit a particular decrease at the individual cell level, but rather in the total number of cells. As a major hurdle when dealing with human samples, the availability of postmortem tissue is limited in terms of sample number, quantity, or quality, precluding the design of statistically powerful case-control validation studies. In the context of a dramatic decline in autopsy rates worldwide [41], with Geneva University Hospitals being no exception, we obtained a total of 13 patient samples in this study, which is comparable to other studies published in the field [3, 4, 7–9]. Samples were carefully selected within several years when meeting strict clinical, neuropathological, and quality criteria including (i) patient’s clinical evaluation, (ii) macroand microscopical examination of the SN at autopsy for the presence of PD neuropathological hallmarks and (iii) PMI under 48 h to minimize protein degradation [42]. Collected mesencephales were either directly frozen (n = 7), paraffinembedded (n = 3), or both (hemi-mesencephales, n = 3). For www.proteomics-journal.com
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each experiment, patients were matched between groups for age, sex, and PMI as these factors could affect protein levels. Of note, the amount of SN tissue obtained per sample was restrained and sample P2 was consumed before the end of the verification step. For this reason, P2 was not used for WB and C2 was set aside to keep the number of samples even. In recent years, many proteomic studies were performed in animal models of PD, even overshadowing investigations in human tissue [43]. Because PD models developed until now cannot recapitulate all clinical and neuropathological features associated with sporadic PD, results from these studies may hardly been translated into the human condition [44,45]. Human postmortem tissues offer in our view a unique window into the specific abnormalities occurring in PD [46], although not necessarily reflecting the complex dynamics of the whole degenerative process. Importantly, changes occurring early in PD pathogenesis may be detectable in late PD stage brains as well. Indeed, we found similar levels of seipin and ferritinL in the classical PD cases (n = 3) than in the incidental case P4, which most likely represents a true preclinical form of PD. This is a particularly important finding in the context of early PD biomarker research. This also constitutes a valuable demonstration that some of the observed changes in the SN are unrelated to patient’s PD treatment, as the incidental case was not under levodopa or any other PD medications.
4.4
Conclusions
Overall, the complex proteome alterations emphasized in this study provide further insights into the underlying pathogenic processes at work in the SN of PD patients. Importantly, nebulette protein, found overexpressed in the remaining DA neurons of PD patients, may be a key pathogenic candidate whose modulation may impact neuronal survival through cytoskeleton dynamics alteration. The elucidation of the precise sequence of events triggering neurodegeneration in PD may ultimately provide new therapeutic targets and biomarkers for the treatment and prevention of PD. This work has been made possible through the generosity of the Memorial A. De Rotschild Foundation, the Edmond J. Safra Philantropic Foundation, the Gustaaf Hamburger Foundation and Swiss Parkinson. We acknowledge the PRIDE team for the deposition of our data to the ProteomeXchange Consortium. The MS data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000427 [21]. The authors have declared no conflict of interest.
5
References
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DOI 10.1002/pmic.201300342
Proteomics 2014, 14, 784–794
RESEARCH ARTICLE
Proteomic analysis of human substantia nigra identifies novel candidates involved in Parkinson’s disease pathogenesis 3 ¨ ´ ˆ 1,2 , Virginie Licker1,2 , Natacha Turck2 , Eniko¨ Kovari , Karim Burkhardt4 , Melanie Cote 3 4 2 Maria Surini-Demiri , Johannes A. Lobrinus , Jean-Charles Sanchez and Pierre R. Burkhard1,2,5 1
Neuroproteomics Group, University Medical Center, Faculty of Medicine, Geneva University, Geneva, Switzerland 2 Translational Biomarker Group, University Medical Center, Faculty of Medicine, Geneva University, Geneva, Switzerland 3 Department of Psychiatry, Geneva University Hospitals, Geneva, Switzerland 4 Department of Pathology, Geneva University Hospitals, Geneva, Switzerland 5 Department of Neurology, Geneva University Hospitals, Geneva, Switzerland Parkinson’s disease (PD) pathology spreads throughout the brain following a region-specific process predominantly affecting the substantia nigra (SN) pars compacta. SN exhibits a progressive loss of dopaminergic neurons responsible for the major cardinal motor symptoms, along with the occurrence of Lewy bodies in the surviving neurons. To gain new insights into the underlying pathogenic mechanisms in PD, we studied postmortem nigral tissues dissected from pathologically confirmed PD cases (n = 5) and neurologically intact controls (n = 8). Using a high-throughput shotgun proteomic strategy, we simultaneously identified 1795 proteins with concomitant quantitative data. To date, this represents the most extensive catalog of nigral proteins. Of them, 204 proteins displayed significant expression level changes in PD patients versus controls. These were involved in novel or known pathogenic processes including mitochondrial dysfunction, oxidative stress, or cytoskeleton impairment. We further characterized four candidates that might be relevant to PD pathogenesis. We confirmed the differential expression of ferritin-L and seipin by Western blot and demonstrated the neuronal localization of gamma glutamyl hydrolase and nebulette by immunohistochemistry. Our preliminary findings suggest a role for nebulette overexpression in PD neurodegeneration, through mechanisms that may involve cytoskeleton dynamics disruption. All MS data have been deposited in the ProteomeXchange with identifier PXD000427 (http://proteomecentral.proteomexchange.org/dataset/PXD000427).
Received: August 6, 2013 Revised: December 13, 2013 Accepted: December 15, 2013
Keywords: Biomedicine / Nebulette / Parkinson’s disease / Postmortem tissue / Substantia nigra
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Correspondence: Dr. Pierre R. Burkhard, Department of Neurology, Geneva University Hospitals, 4, rue Gabrielle-Perret-Gentil, 1211 Geneva 14, Switzerland E-mail: [email protected] Fax: +41-22-372-83-32 Abbreviations: ␣-SYN, alpha-synuclein; BBB, blood brain barrier; BP, biological process; CC, cellular component; DA, dopamine;
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ECM, extracellular matrix; FE, fold enrichment; GGH, gamma glutamyl hydrolase; HCD, high-energy C-trap dissociation; LB, Lewy bodies; OT, orbitrap; PD, Parkinson’s disease; PMI, postmortem interval; SN, substantia nigra; TMT, tandem mass tag; WB, Western blot
Colour Online: See the article online to view Fig. 1 in colour.
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1
Introduction
Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the gradual dysfunction of the extrapyramidal motor system and clinically manifested by tremor at rest, rigidity, or bradykinesia. These cardinal motor symptoms stem from the striatal dopamine (DA) deficit proportional to the severe and progressive loss of midbrain melanized dopaminergic neurons projecting from the substantia nigra (SN) pars compacta. Susceptible nerve cells exhibit intracellular inclusions termed Lewy bodies (LB) formed by the abnormal aggregation of proteins including alphasynuclein (␣-SYN) [1]. Along with neuronal damage, a glial reaction is observed with the release of various inflammatory factors. At present, PD remains an incurable condition for which DA replacement therapy provides the most effective symptomatic relief during the first years of its administration. Importantly, the lack of sensitive and specific biomarkers available precludes an early and accurate PD diagnosis that is still based on patients’ clinical evaluation at a late pathological stage. Several lines of evidence indicate that oxidative stress, mitochondrial dysfunction, impaired protein degradation, excitotoxicity, apoptosis, or inflammation may participate to the successive events ultimately leading to neuronal death [2]. However, despite the multitude of scenarios elaborated, the precise sequence of biological events and molecular pathways underlying PD pathogenesis is still elusive. Reasons for this situation may involve a failure of traditional candidatebased studies to unravel the extremely complex nature of PD processes together with the inability to generate satisfactory animal models of PD, a human-specific disease. Recently, it was proposed to explore PD pathogenesis by global unbiased hypothesis-free “omic” approaches such as proteomics. Large-scale protein expression profiling analyses of human postmortem tissues were conducted that target brain components degenerating in PD including the SN, the frontal cortex, or the locus coeruleus [3–9]. These comparative studies of PD versus control proteomes confirmed several candidates but also revealed many novel ones triggering cell death in PD, which were involved in pathways such as oxidative stress or mitochondrial function. For example, using a 2-DE approach, our group recently identified cytosolic non specific dipeptidase 2 (CNDP2) as a novel key player in PD pathogenesis via a potential role in modulating the cellular response to oxidative stress [7]. Considerable efforts are still needed to improve PD pathogenesis understanding, a prerequisite for the establishment of disease-modifying therapies. We believe that the elucidation of the complex proteome alterations occurring in the SN of PD patients may provide a new source of PD-specific biological targets for diagnostic biomarkers, disease prevention, and therapeutic strategies. In the present study, we performed a high-throughput quantitative profiling of the SN proteome in PD patients compared to neurologically intact controls using a sixplex isobaric chemical labeling technique. We catalogued nearly 1800 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
proteins, which represents so far the largest nigral proteome map ever published. More than 200 proteins were found to be deregulated in PD cases versus controls, 21 of them with a significant fold change over 1.35. Some of them, including ferritin-light chain, seipin, nebulette, and gamma glutamyl hydrolase (GGH), may be interesting targets potentially involved early in PD pathogenesis.
2
Material and methods
2.1 Human brain tissues Thirteen human midbrain tissues (PD group: 5, and control group: 8) were obtained from the Division of Clinical Pathology and Psychiatry of the Geneva University Hospitals under a procedure approved by the local ethical committee (Table 1). Samples were either frozen at –80⬚C or fixed at 4⬚C in 15% formaldehyde for 4 weeks before being paraffin embedded. Written consents for brain autopsy and use for research purposes were signed by close relatives. PD cases were clinically diagnosed according to the United Kingdom PD Society Brain Bank criteria [10] and under symptomatic PD treatment before death, except patient P4 considered as an incidental case. PD diagnosis was confirmed neuropathologically by the observation of a severe nigral depigmentation due to neuronal loss and the presence of ␣-SYN immunoreactive inclusions. P4 may represent an early preclinical stage of PD [11], defined by the absence of symptomatic Parkinsonism but the presence of LB in the SN. Controls were cases without nigral abnormalities and with no previous history of neurological or psychiatric disorders. SN protein extracts were prepared as described in a previously published protocol [7]. Briefly, frozen tissues were cryosectioned in 18-m-thick slices and the SN—macroscopically recognizable by its dark pigmentation, was selectively dissected from slides with a scalpel. About 15 slices per sample were necessary to get 1 mg of nigral tissue, which was collected and kept at –80⬚C until analysis. 2.2 Quantitative sixplex TMT analysis 2.2.1 Sample preparation and TMT labeling Tissue samples from the PD (n = 3) and Control (n = 3) groups were matched for patients’ age and postmortem interval (PMI < 38 h) (Mann–whitney U test, p = 0.4 for age and p = 0.9 for PMI). One milligram of each sample was homogenized in 150 L of sample buffer (6 M Urea, 0.1 M TEAB pH 8.0, 0.1% CHAPS, 0.05% SDS) and protease inhibitors (Roche Applied Science, Basel, Switzerland), vortexed, sonicated, and centrifuged at 4⬚C to remove cellular debris. Protein concentrations were determined using the Bradford assay (Protein Assay, Bio-Rad, Hercules, CA). Fifty micrograms of proteins from each of the six samples were analyzed as described by Dayon et al. [12] with slight modifications. Equal amounts (1 g) of bovine www.proteomics-journal.com
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Table 1. Clinico-pathological data of human postmortem samples by experiment type
Experiment
Group
Sample
Sex
Age
Mean ± SD
TMT
Control
C1 C2 C3 P1* P2 P3*
M F F M M F
70 61 87 73 79 85
72.7 ± 13.2
C1 C3 C4 C5 P1* P3* P4* P5
M F M M M F M M
70 87 89 86 73 85 84 74
83 ± 8.8
C6 C7 C8 P1* P3* P4*
F M M M F M
92 88 82 73 85 84
Parkinson
WB
Control
Parkinson
IHC
Control
Parkinson
79 ± 6
79 ± 6.4
PMI
16 19 2
35 24 13 25 7 24
25.5 ± 9.7
16 2 0 8
35 13 23 31 25 24 38 25
29.7 ± 10.2
16 2 0
34 18 37 25 24 38
87.3 ± 5 80.7 ± 6.7
Mean ± SD
DD
24 ± 11 18.7 ± 10.1
28 ± 6.7
29 ± 7.8
DD, disease duration, PMI, postmortem interval. Asterisks indicate samples whose hemi-mesencephales were available both frozen and paraffin embedded.
-lactoglobulin (LACB) were spiked in each sample as an internal standard to control for an eventual experimental bias. Briefly, samples were reduced in 2.5 mM TCEP for 60 min at 37⬚C, alkylated in 10 mM iodoacetamide for 30 min in the dark at room temperature and digested overnight by trypsin (trypsin/protein ratio 1:25). The resulting peptide mixtures were tagged with the sixplex tandem mass tags (TMT) reagents (Thermo Scientific, Rockford, IL). Control samples C1, C2, C3 were labeled with TMTs 126.1, 128.1, and 130.1 and PD samples P1, P2, P3 with TMTs 127.1, 129.1, and 131.1, respectively. The six samples were pooled and dried for storage at –20⬚C.
(Thermo Electron, San Jose, CA) equipped with a NanoAcquity HPLC system (Waters Corporation, Milford, MA). Each sample was injected twice on two different days to obtain technical replicates and to increase protein coverage. Peptide separation and MS analysis were performed as described elsewhere [13]. To obtain precise peptide quantification without compromising peptide identification, ESI LTQ-OT MS analysis combined CID and high-energy C-trap dissociation (HCD) activation modes in the LTQ-OT. MS survey scans were acquired in the OT analyzer within an m/z window from 400 to 2000. A maximum of three precursor ions were selected for parallel fragmentation in CID or HCD and subsequent MS/MS detection in the LTQ or OT, respectively.
2.2.2 Off-gel peptide fractionation 2.2.4 Peptide and protein identification Peptides were then fractionated in-solution on the basis of their pI with an Agilent 3100 OFFGEL fractionator (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s protocol. Isoelectric focusing was done on a commercial 24 cm IPG pH 3−10 linear dry strip (GE Healthcare Biosciences AB, Uppsala, Sweden) with a 24 wells frame set until 50 kV/h was reached with a maximum current of 50 A and power of 200 mW. The 24 fractions were purified using C18 microspin columns (Harvard Apparatus, Holliston, MA), dried and stored at −20⬚C prior MS analysis. 2.2.3 LC-MS/MS analysis Each peptidic fraction was reconstituted and analyzed by LC−MS/MS on a LTQ Orbitrap (OT) XL mass spectrometer C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
MS data were processed using EasyProtConv [14]. Peak lists were generated from raw data, combining CID and HCD spectra for an optimized peptide identification and quantification [13]. The obtained peak list files were submitted to Easyprot, a platform that uses Phenyx (GeneBio, Geneva, Switzerland) for protein identification [14]. Searches were conducted against UniProt Swiss-Prot database (February 8, 2011, 525 207 entries) specifying Homo sapiens taxonomy. Trypsin was selected as the proteolytic enzyme, one missed cleavage was allowed, cysteine carbamidomethylations, TMT6 amino terminus, and TMT6 lysine were set as a fixed modification whereas oxidized methionine as variable. Peptide z-scores were then set to maintain a false positive peptide ratio below 1%. Proteins with at least two distinct peptide www.proteomics-journal.com
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sequences were selected. Proteins were clustered based on shared peptides indistinguishable by MS, with the protein entry containing the most peptides selected as the group reporter. 2.2.5 Protein relative quantification Publicly available Isobar R package was used to calculate protein ratios and select differentially regulated proteins between sample classes [15]. TMT6 reporter ion intensities were corrected for isotopic impurities as provided by the manufacturer and normalized imposing equal median intensity. Protein ratios were obtained by combining the reporter ion intensity ratios between all pairs of distinct classes (126, 128, 130 vs. 127, 129, 131) measured from MS/MS spectra. For each protein ratio, p-values were computed that model technical (ratio p-value) and biological variability (sample p-value) to estimate ratio accuracy and biological significance, respectively. The level of risk was set at 5%, requiring both p-values to be lower for differential expression selection. All details and mathematical demonstration are provided in Breitwieser et al. [15]. 2.3 Bioinformatics analysis The identified proteins were parsed by protein class through PANTHER online tool v7.2 [16], and GO cellular component (CC), and biological process (BP) annotations via DAVID webbased tool v6.7 [17, 18] or BINGO v2.0 plugin [19] from Cytoscape v2.6 [20]. For enrichment analyses, nigral datasets were compared with the default human reference dataset containing all GO annotations taxonomy. For each GO term, fold enrichment (FE) value was calculated as the ratio of its frequency in the SN proteome dataset compared to the default human proteome reference. Statistically overrepresented categories were determined using modified Fisher’s exact tests with p-value after Benjamini correction lower than 0.05. Categories with at least five associated proteins were selected. Of note, gene name correspondence for each identified protein group was used, as listed in Supporting Information File 1. 2.4 Western blotting verification The expression levels of ferritin light chain (ferritin-L) and seipin were assessed by Western blot (WB) using SN autopsy tissue from control (n = 4) and PD patients (n = 4) (see Table 1). Samples were matched for patients’ sex (3 M/1 F), age, and PMI (Mann–Whitney U test, p = 0.34 for age and p = 0.69 for PMI). Ten micrograms protein from each sample were separated on a 12.5% T SDS-PAGE and electrophoretically transferred on a nitrocellulose membrane using a Hoefer system. Blots were probed with rabbit polyclonal to ferritin-L (1:4000, ab69090, Abcam), rabbit polyclonal to seipin (1:2000, ab106793, Abcam) and mouse C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
monoclonal to actin (1:30 000, Sigma). Anti-rabbit and antimouse horseradish peroxidase conjugated secondary antibodies (Dako) were diluted at 1:2000. Detection of immunoreactive bands was performed using the chemiluminescent ECL Western Blotting System (GE Healthcare) and quantification of band volumes with TotalLab Quant (Nonlinear Dynamics). Actin was used as a reference protein for normalization and statistical analysis was performed using the two-tailed Mann–Whitney U t-test (p < 0.05).
2.5 Immunohistochemical analysis Immunohistochemical staining of ferritin-L, GGH, and nebulette was performed on paraffin-embedded SN tissue from control (n = 3) and PD patients (n = 3) (Table 1). Samples were matched for patients’ sex (2 M/1 F), age, and PMI (Mann– whitney U test, p = 0.4 for age and p = 1 for PMI). Sections were cut at 12 m before being deparaffinized in xylene and dehydrated in ethanol (60⬚). After microwave-induced antigen retrieval in citrate buffer, sections were incubated with 0.25% potassium permanganate in PBS followed by 1% potassium bisulfite/1% oxalic acid to bleach neuromelanin pigmentation. Then, sections were hybridized overnight at 4⬚C with rabbit polyclonal to ferritin-L (1:500, ab69090, Abcam), rabbit polyclonal to GGH (1:200, HPA025226, Sigma) or goat polyclonal to nebulette (1:200, NBP1-45223, Novus Biologicals) diluted in 0.3% Triton-X/1% BSA in PBS followed by 1 h at room temperature with anti-rabbit or anti-goat horseradish peroxidase conjugated secondary antibodies (Dako), diluted in 0.3% Triton-X/1% BSA in PBS. Staining was visualized using 3,3 -diaminobenzidine (DAB, Sigma) as a chromogen and sections were counterstained with Cresyl Violet. No staining was detected on negative controls, treated identically except for hybridization with primary antibody that was omitted.
3
Results
3.1 Quantitative MS analysis of protein profiles The shotgun proteomic experiment involving sixplex tandem mass tag (TMT6 ) labeling allowed the simultaneous identification and quantification of the SN proteome from three PD and three control patients. A total of 1795 proteins were identified with at least two distinct peptides. An overlap of 1183 protein identifications (67%) was observed between replicate injections. Proteins sharing the same set or subset of peptides were considered as a single entry (Supporting Information Appendix 1). Peptides assigned to multiple protein hits were not considered for quantification and 1794 of the 1795 identified protein groups could be quantified with at least one spectrum using Isobar package. Spiked-LACB quantification was checked for quality control and no particular experimental bias was observed. Coefficient of variations of LACB reporter relative intensities calculated for each www.proteomics-journal.com
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channel varied from 6 to 16%, in accordance with isobaric tagging performance [12]. Two hundred four proteins were found significantly differentially expressed (p < 0.05), with keratins being excluded from the analysis. Of them, 96 were overexpressed and 108 underexpressed in the SN of PD patients. Overall, quantified proteins exhibited a low intergroup variability, with about 95% of the ratios falling in a sharp range between 0.8 and 1.25. This indicates that the large majority of the detected proteins was expressed at equivalent levels in PD and control samples. All protein identification and quantification details including statistics are available in Supporting Information File 1 and Supporting Information Appendix 1. The MS data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000427 [21].
3.2 PANTHER class and GO term analyses 3.2.1 Nigral proteome To assess the nigral proteome composition, the 1795 identified proteins were parsed into PANTHER classes and GO CC. The major functional classes were hydrolase (representing 16% of annotated hits), enzyme modulator (13%), oxidoreductase (12%), transferase (12%), and cytoskeletal proteins (12%) (Supporting Information File 2). According to the GO CC analysis (Fig. 1A), the largest proportion (82%) of proteins was annotated as cytoplasmic with a preferential (>50%) distribution in “cytosol,” “mitochondrion,” or “cytoskeleton” structures. About 11% (193 over 1686) of the annotated proteins were parsed into GO terms specific to neurons including “synapse,” (6%) “axon,” (4%) or “neuronal cell body” (3%). To gain more insights in the SN-specific features, enrichment of significantly overrepresented GO BP in the SN proteome dataset was assessed by comparison with the complete human proteome annotations. The same analysis was performed using a dataset approximating the human “central” proteome that comprised 1124 proteins ubiquitously expressed and experimentally identified in seven human nonneuronal cells lines [22]. Overrepresented GO categories found in common between the two sets were discarded in order to highlight the processes occurring more specifically in the nigral tissue and to remove the influence of the common cellular proteome (Supporting Information File 2) [23]. Nine categories exhibited higher FE value in the SN than in the central proteome dataset when considering FE differences of at least one unit, all linked to mitochondrial function and oxidative phosphorylation. One hundred seventy-three GO BP terms were found to be specifically overrepresented (p < 0.001) in the SN dataset (Supporting Information File 2), some of which are shown in Fig. 1A. Thirty-one terms covering 20% of the annotated proteins were related to neuronal activities such as synaptic transmission, plasticity, organization, or development. Many enriched terms were linked to C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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the generation of ATP, through glycolysis, TCA cycle, and oxidative phosphorylation. Other main enriched categories included cytoskeleton organization, regulation, and transport, fatty acid and amino acid metabolisms, or response to oxidative stress. 3.2.2 Differentially expressed proteins To gain more information on the nature and function of the 204 differentially expressed nigral proteins and the specific processes significantly modulated in PD pathogenesis, the dataset was further characterized using PANTHER functional classes and GO terms. The major PANTHER protein classes were oxidoreductase (representing 15% of annotated hits), cytoskeletal protein (13%), hydrolase (13%), transferase (10%), nucleic acid-binding protein (9%) but also chaperone (5%) and many terms related to translation (Supporting Information File 3). Proteins related to oxidoreductase, cytoskeletal, and chaperone classes had a tendency to be overexpressed in PD, whereas proteins in the other cited classes seemed to be more prone to underexpression in PD. Sixty-five GO CC terms and 22 GO BP categories were found to be significantly overrepresented (p < 0.05) in the differential proteins dataset compared to the human proteome (Supporting Information File 3), with a selection of them shown in Fig. 1B. Overall, enrichment in GO terms related to cytoskeleton activities (cytoskeleton, regulation of cell component organization) was observed, with a predominant overexpression of proteins falling in these categories in PD. On the opposite, a prevailing underexpression of proteins associated to several other enriched GO categories was noticed in PD. These included energy metabolism and mitochondrial activities (i.e. generation of precursor metabolites, mitochondria), intracellular transport processes (i.e. vesiclemediated transport, transport regulation), synaptic activities (i.e. synapse, transmission of nerve impulse) or translation (i.e. ribosome, endoplasmic reticulum proteins, translational elongation). 3.3 Verification of protein expression levels by WB To reduce false positive rates in the selection of differentially expressed proteins for verification, we set a cutoff threshold at a fold change higher than 1.35 on all TMT ratios, determined according to Tan et al. method based on random ratios [24] (for calculation details see Supporting Information File 1). The resulting list of the 21 top-differentially expressed proteins is shown in Table 2. Among them, we verified (i) ferritinL, a previously known PD candidate whose expression level in PD brains was still controversial in the literature [25] and (ii) seipin, an original candidate whose biological importance was recently shown in various neurological syndromes but not in PD [26]. Semiquantitative WB analysis demonstrated a significant 1.7-fold increase of ferritin-L and a 2.5-fold decrease of seipin www.proteomics-journal.com
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Figure 1. GO analyses of nigral proteins. In A, GO analyses were performed on the total nigral dataset. A1 illustrates nigral protein distribution pattern in subcellular localization based on GO CC categories. Of note, the sum of percentage exceeds 100% because of multiple annotation terms associated to a single protein. A2 shows representative GO BP terms found significantly overrepresented in the nigral proteome (Benjamini correction, p < 0.001). Protein count refers to the number of annotated proteins in each category. In B, enrichment of the set of differentially expressed nigral proteins in significantly overrepresented GO CC (B1) or BP (B2) is shown (Benjamini correction, p < 0.05). For each GO term, fold enrichment is given, as well as the number of overexpressed and underexpressed proteins in the SN of PD patients.
expression levels (p < 0.05, Mann–Whitney t-test) in the SN of PD patients (n = 4) versus controls (n = 4), consistent with our proteomic analysis (Fig. 2). Results were confirmed using samples from the proteomic experiment (n = 4) as well as four independent samples including an incidental PD case (P4) potentially representing a preclinical stage of the disease (Table 1). Importantly, P4 exhibited protein expression levels similar to the other PD cases (n = 3). 3.4 Cellular localization of candidates by IHC Next, we determined the localization of ferritin-L and the two novel candidates GGH and nebulette by IHC, using available paraffin-embedded nigral tissues of PD (n = 3) and Control
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(n = 3) groups (Table 1). Representative IHC images of two patients (P1, C6) are shown in Fig. 2 (for other patients see Supporting Information Fig. 1). As expected, ferritin-L was predominantly expressed in oligodendrocytes and microglial cells. Differences between pathological and healthy staining patterns were difficult to assess given the large number of immunoreactive cells. Conversely, the expression of GGH and nebulette was observed in the remaining DA neurons, validating our whole tissue approach to detect changes in scarcer cell populations. GGH staining was strong and occurred predominantly in DA neuron cell bodies and processes in the SN of PD and control patients. A lower number of GGH immunoreactive neurons was observed in PD, most probably resulting from neuronal loss. This observation supports the decrease in GGH level observed by proteomics. Similarly,
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Table 2. Top-differentially expressed proteins in the SN of PD patients versus controls with a fold change over 1.35.
AC
Protein name
PD/C ratio
Main PD pathogenic hypothesis
Top-overexpressed proteins in the SN of PD patients O76041 Nebulette P21695 Glycerol-3-phosphate dehydrogenase [NAD(+)], cyt Q9UDY2 Tight junction protein ZO-2 P14136 Glial fibrillary acidic protein P04083 Annexin A1 P02792 Ferritin light chain P00966 Argininosuccinate synthase P42330 Aldo-keto reductase family 1 member C3 Q00796 Sorbitol dehydrogenase P52895 Aldo-keto reductase family 1 member C2 Q8N5M4 Tetratricopeptide repeat protein 9C
1.86 1.74 1.71 1.63 1.58 1.57 1.53 1.49 1.46 1.44 1.39
*Cytoskeleton structure/regulation Energy metabolism *Blood-brain barrier communication Glial activation and inflammation Glial activation and inflammation Oxidative stress Oxidative stress (NO), protein modification Oxidative stress Energy metabolism Oxidative stress Unknown
Top-underexpressed proteins in the SN of PD patients P01871 Ig mu chain C region Q92820 Gamma-glutamyl hydrolase O00241 Signal-regulatory protein beta-1 Q96G97 Seipin P69849 Nodal modulator 3 Q9NY65 Tubulin alpha-8 chain Q53FP2 Transmembrane protein 35 Q05639 Elongation factor 1-alpha 2 O14594 Neurocan core protein P30040 Endoplasmic reticulum resident protein 29
0.29 0.64 0.66 0.67 0.70 0.71 0.72 0.73 0.74 0.74
Glial activation and inflammation Unknown Glial activation and inflammation Apoptosis Unknown *Cytoskeleton structure/regulation Unknown Protein synthesis, apoptosis *ECM structure/regulation Protein folding
Associated main PD pathogenic hypotheses are listed, with asterisks representing less conventional or novel pathways. AC, Swissprot accession number.
Figure 2. Confirmation and localization of selected proteomic candidates in the SN of PD patients. Ferritin-L in panel A and seipin in panel B were verified by WB analysis and respectively found significantly increased (PD/C ratio of 1.7) and decreased (PD/C ratio of 0.4) in PD patients versus controls (p < 0.05, Mann–Whitney U test). Bar graphs represent average -actin normalized band volumes with SD, given in % of controls. IHC analysis for GGH, nebulette, and ferritin-L was performed in the SN of PD patients and controls, with panel C showing representative IHC of a control (patient C6: a, c, e) and PD case (patient P1: b, d, f). GGH and nebulette exhibited a predominant expression in DA neurons (i.e. arrows) whereas ferritin-L was predominantly expressed by glial cells.
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nebulette expression was mainly confined to DA neuron cytoplasm, with a staining appearing to be more pronounced in PD than control patients. This tends to confirm the increase in nebulette levels observed by proteomics in PD patients.
4
Discussion
4.1 Characterizing the SN proteome Taking advantage of a sensitive shotgun proteomic approach we obtained the most comprehensive picture of the human SN proteome so far, with 1795 proteins simultaneously identified and quantified. Comparison with previously published studies [3, 4, 8] revealed 1139 proteins newly associated with human SN (listed in Supporting Information File 1). A significant proportion of annotated proteins were parsed into GO categories related to neuronal components (11%) or activities (20%). GO overrepresentation analyses supported the notion that SN relies on high-energy supply and efficient anti-oxidant defenses, due to the particular morphological and physiological specificities of DA neurons [27, 28]. An important role for cytoskeletal organization, proper vesicular transport and trafficking, or amino acid cycle was also highlighted. Any perturbation in one or more of the above-cited processes might be particularly critical for SN correct function. SN proteome characterization represents a first step toward the understanding of the SN complexity and its specific features, which might hold the key to its vulnerability in PD.
4.2 Identifying protein alterations in the SN of PD patients Our proteomic experiment revealed a subset of 204 proteins modulated in PD. Twenty-four of them were found similarly differential by others [3, 4, 7, 8], with a majority of concordant ratios (i.e. peroxiredoxin 1, gluthatione S transferase P, neurocan, GFAP, CNDP2). Differences between proteomic studies may be explained by various means including sample heterogeneity (i.e. patient’s history, comorbidities), tissue quality and PMI as well as the lack of standardized protocols and reliable independent validation steps. Therefore, we evaluated protein expression of four selected candidates by WB (i.e. ferritin-L and seipin) and IHC (i.e. ferritin-L, nebulette, and GGH) experiments, which tend to confirm our proteomic results. Interestingly, IHC analyses indicated a preferential localization of nebulette and GGH inside neurons, confirming the ability of our approach to detect neuron-specific changes. Consistent with other proteomic findings [7,8], we demonstrated the overexpression of ferritin, in particular its light subunit, in the SN of PD patients. With a preferential localization in glial cell populations, ferritin-L levels may be augmented to bind iron released from dying DA neurons [29] or may reflect the mild gliosis occurring in PD. Importantly, we highlighted the potential involvement of three novel can C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
791 didates, GGH, seipin, and nebulette, whose link with PD had not been formally established. GGH is a key enzyme in folate metabolism whose decreased expression in PD SN could be due to neuronal loss, as assessed by IHC analysis. The ER-resident glycoprotein seipin, found underexpressed in PD, was already associated to neurological disorders (i.e. Silver syndrome, Charcot-Marie-Tooth syndrome variant) [26]. The protein was observed similarly diminished in the locus coeruleus of PD patients, another aminergic nucleus affected earlier than SN in the disease process [9]. Mutations in seipin were shown to result in protein misfolding, accumulation in the ER, unfolded protein response pathway activation, ERstress, protein translation diminution, and ultimately cell death. These observations suggest a role for seipin in PD pathogenesis through protein aggregation or ER-mediated stress. Nebulette, found overexpressed in PD, is a member of the nebulin actin-binding protein family [30] whose shorter splice variant Lasp-2 is strongly expressed in brain tissues [31]. Interestingly, Lasp-2 overexpression in pheocytochroma PC6 cell line was shown to inhibit neurite outgrowth in response to growth factors [32]. Together with its molecular scaffold role for actin, Lasp-2 may participate in the organization of focal adhesions, structures linking cellular cytoskeleton with extracellular matrix (ECM) [32]. Inappropriate interactions in these regions could lead to neurodegeneration through anoikis programmed cell death [33]. Altogether, proteome alterations observed in PD may reflect several known pathogenic processes involved in neurodegeneration as a cause or a consequence of it (Fig. 3). We observed an increase in proteins associated to oxidative stress as well as a decrease in proteins involved in mitochondrial function and energy metabolism failure, three interconnected processes able to disrupt protein homeostasis and cellular function. We demonstrated a preferential overexpression of cytoskeletal proteins in PD, important LB components whose defects were already mentioned in PD [34–36]. Protein aggregation is central to neurodegenerative diseases and our data point to disturbances in several mechanisms known to promote protein accumulation such as protein processing or transport. We found more than a hundred proteins overexpressed in the SN of PD, some of which may accumulate pathogenically including vimentin, known to be a LB component [37, 38]. Of note, we did not identify overexpression of the classical LB constituents ubiquitin or ␣-SYN, similarly to other proteomic studies [3, 4, 8, 9]. This may be explained by the fact that (i) ␣-SYN may not be as central in sporadic PD as it is in familial PD forms, (ii) ␣-SYN overexpression may be masked by DA neuronal cell loss in PD and (iii) our methodology may not allow a proper solubilization of LB or protein aggregates. We also noticed a general attenuation in protein translation in PD brains, which may result from protein misfolding, aggregation, or ER stress. Our data suggested some less conventional pathogenic pathways as well, such as blood brain barrier (BBB) or ECM impairments. Abnormalities in neurons’ surroundings, including glial cell populations, BBB or ECM, can critically influence in a positive or www.proteomics-journal.com
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Figure 3. Potential pathogenic pathways involved in PD according to our proteomic data. Proteins found overexpressed in PD are represented in red and those underexpressed in blue.
negative way the fate of resident neuronal cells. Alterations of the ECM matrix for example may affect synaptic morphology and function [39], whereas a defect in the BBB may constitute a causative factor of DA neuron degeneration [40]. Finally, we found a dysregulation in proteins for which very few or no functional information was available such as Tetratricopeptide repeat protein 9C or Nodal Modulator 3.
4.3 Assessing limitations Using a whole tissue approach, we analyzed signals coming from mixed cell populations, without being able to differentiate their individual contribution to the total signal. Changes in specific nigral subpopulations were thus probably attenuated or totally masked, translating into smaller fold change. For example, signals from DA neuronal cell population (representing up to 10% of total cells) might be diluted ten times and become hardly detectable. Moreover, with a tissue-based normalization (i.e. tissue weight and total protein concentration), the total number of DA neurons in PD samples was theoretically reduced of at least 70% compared to controls. Thus, the overexpression of proteins such as alpha-SYN in surviving PD DA neurons may have been counteracted by the higher number of these cells in control tissues. Of note, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
nebulette protein found to be predominantly expressed in neurons by IHC, might exhibit a real higher increase in PD than the 1.9-fold reported by proteomics. In addition, the observed underexpression in several neuronal proteins might reflect neuronal loss occurring in PD. GGH for instance, observed to be decreased in PD and shown to localize in DA neurons by IHC, did not seem to exhibit a particular decrease at the individual cell level, but rather in the total number of cells. As a major hurdle when dealing with human samples, the availability of postmortem tissue is limited in terms of sample number, quantity, or quality, precluding the design of statistically powerful case-control validation studies. In the context of a dramatic decline in autopsy rates worldwide [41], with Geneva University Hospitals being no exception, we obtained a total of 13 patient samples in this study, which is comparable to other studies published in the field [3, 4, 7–9]. Samples were carefully selected within several years when meeting strict clinical, neuropathological, and quality criteria including (i) patient’s clinical evaluation, (ii) macroand microscopical examination of the SN at autopsy for the presence of PD neuropathological hallmarks and (iii) PMI under 48 h to minimize protein degradation [42]. Collected mesencephales were either directly frozen (n = 7), paraffinembedded (n = 3), or both (hemi-mesencephales, n = 3). For www.proteomics-journal.com
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each experiment, patients were matched between groups for age, sex, and PMI as these factors could affect protein levels. Of note, the amount of SN tissue obtained per sample was restrained and sample P2 was consumed before the end of the verification step. For this reason, P2 was not used for WB and C2 was set aside to keep the number of samples even. In recent years, many proteomic studies were performed in animal models of PD, even overshadowing investigations in human tissue [43]. Because PD models developed until now cannot recapitulate all clinical and neuropathological features associated with sporadic PD, results from these studies may hardly been translated into the human condition [44,45]. Human postmortem tissues offer in our view a unique window into the specific abnormalities occurring in PD [46], although not necessarily reflecting the complex dynamics of the whole degenerative process. Importantly, changes occurring early in PD pathogenesis may be detectable in late PD stage brains as well. Indeed, we found similar levels of seipin and ferritinL in the classical PD cases (n = 3) than in the incidental case P4, which most likely represents a true preclinical form of PD. This is a particularly important finding in the context of early PD biomarker research. This also constitutes a valuable demonstration that some of the observed changes in the SN are unrelated to patient’s PD treatment, as the incidental case was not under levodopa or any other PD medications.
4.4
Conclusions
Overall, the complex proteome alterations emphasized in this study provide further insights into the underlying pathogenic processes at work in the SN of PD patients. Importantly, nebulette protein, found overexpressed in the remaining DA neurons of PD patients, may be a key pathogenic candidate whose modulation may impact neuronal survival through cytoskeleton dynamics alteration. The elucidation of the precise sequence of events triggering neurodegeneration in PD may ultimately provide new therapeutic targets and biomarkers for the treatment and prevention of PD. This work has been made possible through the generosity of the Memorial A. De Rotschild Foundation, the Edmond J. Safra Philantropic Foundation, the Gustaaf Hamburger Foundation and Swiss Parkinson. We acknowledge the PRIDE team for the deposition of our data to the ProteomeXchange Consortium. The MS data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000427 [21]. The authors have declared no conflict of interest.
5
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