Proteomic Profiling of Cervical and Lumbar Spinal ... - ACS Publications

Sep 21, 2009 - ... Milano, Italy, and Department of Legal Medicine, Forensic and Pharmaco-Toxicological Sciences “A. .... observed in the wobbler pa...
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Proteomic Profiling of Cervical and Lumbar Spinal Cord Reveals Potential Protective Mechanisms in the Wobbler Mouse, a Model of Motor Neuron Degeneration Antonio Bastone,*,† Elena Fumagalli,† Paolo Bigini,† Pietro Perini,† Davide Bernardinello,† Alfredo Cagnotto,† Ilario Mereghetti,† Daniela Curti,‡ Mario Salmona,† and Tiziana Mennini† Department of Biochemistry and Molecular Pharmacology, “Mario Negri” Institute for Pharmacological Research, Milano, Italy, and Department of Legal Medicine, Forensic and Pharmaco-Toxicological Sciences “A. Fornari”, University of Pavia, Pavia, Italy Received June 30, 2009

The wobbler mouse is a model of selective motor neuron degeneration in the cervical spinal cord. Comparing cervical and lumbar tracts of control and diseased mice at the early stage of pathology by proteomic analysis, we identified 31 proteins by peptide mass fingerprint after tryptic digestion and MALDI-TOF analysis, that were differently represented among the four experimental groups. In healthy mice, patterns of protein expression differed between cervical and lumbar tract: proteins of cellular energetic metabolism pathway showed lower expression in the cervical tract, while cellular trafficking proteins were overrepresented. In wobbler mice, these differences disappeared and the expression pattern was similar between cervical and lumbar spinal cord. We found that most of the proteins differentially regulated in wobbler with respect to control cervical tract were related to astrogliosis or involved in glutamate-glutamine cycle, energy transduction and redox functions. Proteins overrepresented in the wobbler lumbar spinal cord were cytoskeleton proteins and cellular transport proteins, in particular the vesicle fusing ATPase and the isoform 2 of syntaxin-binding protein 1, involved in vesicle trafficking. We suggest that overexpression of proteins involved in vesicle trafficking, together with proteins counteracting mitochondrial dysfunction can have neuroprotective effects, preserving lumbar spinal cord motor neurons in wobbler mice. Keywords: MALDI-TOF analysis • motor neuron disease • syntaxin-binding protein 1 • vesicle fusing ATPase • wobbler mouse

Introduction Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by motor neuron death in brain and spinal cord, leading to progressive muscular atrophy and paralysis;1-3 the prognosis is negative, and the death of patients commonly occurs within 2-5 years after the diagnosis. Riluzole is the only pharmacological treatment approved, but its efficacy is very weak4 and the lack of knowledge of etiology hampers the accomplishment of an effective therapy. Human samples obtained from deceased ALS patients do not help in understanding the basic mechanisms involved in motor neuron death, not even in identifying early diagnostic markers. Widely used alternative models include cellular and animal models recapitulating the main pathological features of human ALS. The discovery that several mutations in the gene coding for the Cu-Zn superoxide dismutase type-1 (SOD1) were responsible for a familial form of ALS (fALS1)5 and the evidence that * To whom correspondence should be addressed. Dipartimento di Biochimica e Farmacologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano, Italy. Phone: +39 02 39014546. Fax: +39 02 39014744. E-mail: [email protected]. † “Mario Negri” Institute for Pharmacological Research. ‡ University of Pavia. 10.1021/pr900569d CCC: $40.75

 2009 American Chemical Society

mice expressing a high number of copies (>15) of hSOD1 develop a progressive form of motor neuron degenerative disorder similar to human ALS, mainly focused the attention of scientific community on this model.6,7 In the last years, new biochemical approaches like proteomic analysis and gene expression profiling have been developed to investigate possible etiopathological mechanisms. Several proteomic studies have been carried out on peripheral and CNS samples of SOD1 mice, allowing to clarify some aspects related to human disease.8-13 However, less than the 2% of total ALS cases are associated with SOD1mutation; thus, proteomic approach in different cellular or animal models of motor neuron degeneration, unrelated to SOD1 mutation, should be applied to obtain a wider overview of factors potentially relevant in the early pathological stage and to screen for disease biomarkers. The wobbler mouse14 is an alternative well-characterized animal model of motor neuron degeneration related to a point mutation in Vps54,15 a protein of the GARP complex involved in the retrograde transport of endosomes from the periphery to the Golgi apparatus. The wobbler disease is characterized by selective motor neuron death in the spinal cord; differently from ALS patients and SOD1 mice, in wobbler mice motor neuron loss is confined to the cervical spinal cord region, while Journal of Proteome Research 2009, 8, 5229–5240 5229 Published on Web 09/21/2009

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Bastone et al. 16,17

the lumbar spinal cord is undamaged. Symptoms appear around the 3rd-4th week of age, then the disease rapidly progresses until the 12th week of age. Although so far there is no evidence of a direct link between Vps54 mutation and ALS,18 the wobbler mice share many clinical, neuropathological and biochemical hallmarks either with ALS patients or SOD1 mice. Furthermore, the wobbler mouse enables to study in the same animal degenerating motor neurons in the cervical spinal cord and motor neurons that escape degeneration in the lumbar spinal cord. In the present study, we carried out a proteomic analysis of cervical and lumbar spinal cord in early symptomatic wobbler mice and age-matched control mice, to identify differently represented proteins possibly involved in mechanisms of motor neuron degeneration at the disease onset. The comparative analysis of these four groups enabled the characterization of possible markers of vulnerability in the cervical spinal cord of wobbler mice and the identification of possible protective factors in the lumbar spinal cord. For the most interesting identified proteins, results obtained by proteomic study were validated with experiments of cellular localization and enzymatic activity.

Materials and Methods Animals. Wobbler mice and healthy littermates (NFR strain, NIH, Animal Resources, Bethesda, MD) were bred at Charles River Italia (Calco, Lecco, Italy). Mice were housed at a temperature of 21 ( 1 °C with relative humidity of 55 ( 10% and a 12 h light/dark cycle. Procedures involving animals and their care were conducted according international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, December 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, US National Research Council 1996). The protocol for the use of laboratory animals was approved by the Italian Ministry of Health in 2007 (Approval number 12.1 BASE3). The wobbler (wr) mutation is recessive; thus, homozygous wobbler mice can be obtained only by mating heterozygous males and females mice. To select mice suitable for the study, genotypization of the progeny was carried out exploiting a restriction polymorphism in the Cct4 gene associated to the mutation at the wr locus as previously described.19 Homozygous wobbler (wr/wr) and control (+/+) mice were used, while heterozygous healthy mice (wr/+) were excluded. We used both males and females mice, since no sex-related differences were observed in the wobbler pathology. Mice were sacrificed at 4 weeks of age, corresponding to the early stage of wobbler disease. Two-Dimensional Gel Electrophoresis (2-DE). Three wobbler and three control mice were sacrificed and spinal cords rapidly dissected; the cervical and lumbar tracts were isolated and individually processed. Four experimental groups were obtained, each composed of three samples: wobbler cervical spinal cord (WrC), wobbler lumbar spinal cord (WrL), control cervical spinal cord (CtrC) and control lumbar spinal cord (CtrL). Tissue was homogenized in 2-D lysis buffer (10 mM TrisHCl, pH 7.4, 0.5% Zwittergen, 1 mM EDTA, protease inhibitors cocktail, CompleteC, Roche), sonicated, and centrifuged at 105 000g for 30 min, and supernatants were collected. Protein content was quantified using the Bradford method with BSA as standard. Proteins were precipitated with a chloroform/ methanol mixture and resuspended in a commercial solution (DeStreak solution; GE Healthcare) containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.5% carrier ampholytes and DeStreak 5230

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reagent as reductant. Samples containing 0.5 mg of protein were run on IPG strips (13 cm long, pH 3-10 NL) using the IPG-phore system (GE Healthcare). A 70 000 Vh was reached at the end of isoelectric focusing. The IPG strips were equilibrated for 10 min in 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS and 65 mM DTT followed by 10 min equilibration in the same buffer containing 2.5% iodoacetamide instead of DTT. The IPG strips were embedded on 10% polyacrylamide gels and run for the second dimension using the SE660 system (GE Healthcare). The 2-D gels were stained with colloidal Coomassie (Gel Code Blue Stain, Pierce). Gel Image Analysis and Quantification. The 2-D gels were acquired with a scanner and computer gel image analysis was done using the Progenesis software (workstation version 2004.3; Nonlinear Dynamics). Each spot volume was intragel normalized to the total spots volume and an averaged value was calculated from the three samples of each group. The fold change was calculated as the ratio between the averaged volume of a spot and the averaged volume of the matched spot in the respective comparison-group. In-Gel Digestion, MALDI-TOF Mass Spectrometry (MS) Analysis, and Protein Identification. The selected spots were manually excised from 2-D gels and trypsin-digested as previously described.8 Briefly, spots were destained overnight with 40% ethanol in 25 mM ammonium bicarbonate, then washed with increasing concentrations of acetonitrile in distilled water. Tryptic digestion was carried out overnight with 12.5 ng/µL of sequencing modified porcine trypsin (Promega), at 33 °C in 10% acetonitrile in 25 mM ammonium bicarbonate. The reaction was stopped by adding trifluoroacetic acid (0.1% final). Spots excised from at least 2/3 different gels were processed separately. A total of 0.6 µL of tryptic digest was loaded on a SCOUT 384 multiprobe target and air-dried; before mass spectrometric analysis, 0.6 µL of the matrix R-cyano-4-hydroxycinnamic acid (Bruker Daltonics) was added and sample was air-dried. The remaining tryptic digest was desalted, concentrated with C18 ZipTip pipet tips (Millipore Corp.) and cocrystallized on the target with the matrix before mass spectrometric analysis. The stock solution of matrix was prepared as saturated solution in TA (50% acetonitrile containing 0.1% trifluoroacetic acid), and diluted 1:1 with TA before mixing with the sample. Mass mapping of tryptic peptides was done with a Reflex III MALDITOF mass spectrometer (Bruker Daltonics) equipped with a 337 nitrogen laser, operating in the positive ion, reflectron mode, with a delayed extraction of 200 ns. The reflector voltage was set to 23 kV and the detector voltage to 1.7 kV. External spectrometer calibration was performed using Bruker peptide standards (Bruker Daltonics). Spectra were acquired over the m/z range from 600 to 4000; 100-400 single laser shots were summed for each spectrum. All spectra were analyzed with flexAnalysis (flexAnalysis version 2.0, Bruker Daltonics) and internally calibrated using trypsin autolysis fragments, routinely obtaining accuracy better than 30 ppm. Monoisotopic peaks were annotated with flexAnalysis default parameters using a S/N threshold of 6, maximal number of peaks 100, quality factor threshold 30 and manually revised. Spectra originating from parallel protein digestions were compared pairwise to discard common peaks derived from trypsin autodigestion or from other contaminants. The peak lists for each protein were accomplished manually using both spectra from non-ZipTipped and ZipTipped samples. The peak lists generated were processed with the online databases search engine MASCOT

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Model of Motor Neuron Degeneration (version 2.2; www.matrixscience.com) or Aldente (version February 11, 2008; www.expasy.org), against the nonredundant protein public databases National Centre for Biotechnology Information (NCBI) (date February 18, 2009; 7 873 120 sequences; 2 713 143 868 residues) and UniProtKB/Swiss-Prot (release 56.8 of February 10, 2009; 410 518 entries; 148 080 998 residues). The following restrictions were applied: (a) Mus musculus (16 078 sequences), (b) (0.2 Da error, (c) trypsin digestion with up to one missing cleavage site, (d) cysteine carbamidomethylation as a fixed modification and methionine oxidation as a variable modification. Protein identification was validated when the MOWSE score was significant (p < 0.05) according to MASCOT.20 If peptides matched to multiple members of a protein family, we reported the accession number of the protein identified as the first hit (top rank) by MASCOT or Aldente. SDS-PAGE and Western Blot. Cervical and lumbar spinal cord tracts from wobbler and control mice were individually homogenized in 500 µL of homogenization buffer (50 mM TrisHCl, pH 7.4, 0.5% (v/v) TritonX-100, protease inhibitors cocktail, Complete C, Roche) and sonicated; protein content was quantified using the Bradford method with BSA as standard and adjusted to a final concentration of 2.5 µg protein/µL. Samples were diluted with Laemmli sample buffer and 20 µg protein/lane was loaded on a 10% polyacrylamide gel. Separated proteins were transferred by submersed electroblotting on PDVF membranes. Membranes were incubated in blocking buffer (TBS with 5% nonfat dry milk and 0.1% Tween20), washed and probed with goat anti-glutamine synthetase primary antibody (sc-6640, Santa Cruz Biotechnology; 1:200). Secondary horseradish peroxidase (HRP)-conjugated antibodies were added and reactive proteins were detected using the ECL Plus Western Blotting Detection kit (GE Healthcare) and visualized on photographic film. Membranes were reprobed with mouse anti-actin (MAB150 1R, Chemicon, 1:10000) to check protein loading. Immunofluorescence. Cervical and lumbar spinal cord tissues from 3 wobbler and 3 control mice were prepared as previously described.21 For isoform 2 of syntaxin-binding protein 1 (isoform 2 of Unc-18-1) and vesicle-fusing ATPase (NSF) experiments, 30 µm thick sections were incubated overnight at room temperature with 0.001% TritonX-100 in PBS and stained with goat anti-Unc-18-1 primary antibody (sc14557, Santa Cruz, 1:50) and goat anti-NSF primary antibody (sc-15917, Santa Cruz, 1:100). Sections were incubated with biotinylated anti-goat secondary antibody (Vectastain, 1:200) and immunofluorescence amplification was done with a tyramide amplification kit (TSA fluorescent system, Perkin-Elmer). The anti-Unc-18-1 polyclonal antibody used in the immunofluorescence experiments was tested in Western blot and correctly stained the spot identified as isoform 2 of syntaxinbinding protein 1 in 2-D gels, due to sequence identity of the target epitope in the two proteins (not shown). The selective expression of glutamine synthetase in neurons and astrocytes was investigated by colocalization experiments with goat antiglutamine synthetase primary antibody (sc-6640, Santa Cruz, 1:500) together with mouse anti-GFAP primary antibody (MAB12029, Immunological Sciences, 1:5000) or 530-615 NeuroTrace Fluorescent Nissl reagent (Molecular Probes, 1:1000). Appropriate fluorescent secondary antibodies (Alexa-488 and Alexa-Cy5, Molecular Probes, 1:1000) were incubated for 2 h at room temperature. To exclude cross-reactivity, each primary antibody was incubated with the secondary antibody associated

to a different primary antibody, without positive staining (not shown). Sections were observed with an Olympus Fluoview microscope BX61 with confocal system FV500. Enzymatic Activities and Oxygen Consumption Rate (QO2). Cervical and lumbar spinal cord tracts obtained from wobbler and control mice were individually homogenized with 0.32 M sucrose buffer containing 10 mM HEPES and 1 mM EGTA, pH 7.4. Succinate dehydrogenase, citrate synthase and mitochondrial creatine kinase activities in spinal cord homogenates were measured by spectrophotometric analysis, in a final volume of 1 mL, as previously described.22,23 Enzymatic activities were expressed as (nanomoles of product formed/ min)/mg of protein. Glutamine synthetase activity was evaluated as previously described24 and enzymatic activity was expressed as (micrograms of product formed/h)/mg of protein. Protein content in the homogenates was quantified using the commercial BCA kit (Pierce) or by the method of Lowry25 using BSA as standard. QO2 was evaluated in spinal cord homogenates as previously described23 in the presence of 5 mM succinate and 4 µM rotenone. Results were expressed as (nanomoles O2/min)/mg protein. Statistical Analysis. Statistical comparison of fold change in protein expression in 2D-gels was done using the Student’s t-test within the Progenesis software. Statistical comparison of results from functional assays was done using the two-way analysis of variance (ANOVA) with Bonferroni’s post-test or the paired Student’s t-test within the Prism software (GraphPad Software, Inc.; version 5.01 for Windows).

Results Using the warping and matching algorithm of Progenesis software, we compared protein expression in four experimental groups: cervical versus lumbar spinal cord in control mice (CtrC vs CtrL), cervical versus lumbar spinal cord in wobbler mice (WrC vs WrL), cervical spinal cord from wobbler mice versus cervical spinal cord from control mice (WrC vs CtrC), and lumbar spinal cord from wobbler mice versus lumbar spinal cord from control mice (WrL vs CtrL). Figure 1 shows the representative Coomassie stained 2-D gel for each experimental group. Differential analysis of the proteome recognized 33 spots, corresponding to 31 proteins, differently represented among the four experimental groups, that were identified by peptide mass fingerprint (PMF) after tryptic digestion and MALDI-TOF analysis. Proteins are listed in Table 1; protein name, accession number according to the Swiss-Prot and TrEMBL database, observed and calculated isoelectric point, observed and calculated molecular mass, percent of protein sequence coverage, score for search in a decoy database (MASCOT), experimental peptides matched and peptides searched are reported. Both dihydropyrimidinase-related protein 2 and pyruvate kinase isozyme M2 were identified in two different spots (spot nos. 8 and 9 and spots nos. 11 and 12, respectively; Table 1), that likely represent protein isoforms with different post-transductional modifications. In fact, both are cytoplasmic proteins and possess several phosphorylation sites, some experimentally tested and some inferred by similarity, as described by protein databases (Swiss-Prot), consistent with different migration properties in 2-DE gels. Identification of proteins by MALDI-TOF MS, which provides PMF but no sequence information, requires first high quality separation of proteins. In our study, separation of proteins by Journal of Proteome Research • Vol. 8, No. 11, 2009 5231

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Figure 1. Representative 2-D gels showing proteins distribution in cervical and lumbar spinal cord of wobbler and control mice. Proteins were separated by isoelectric focusing between pH 3-10 NL, followed by SDS-PAGE and stained with colloidal Commassie. Gels were digitally acquired and analyzed by computerassisted densitometry. Proteins differentially represented among the four experimental groups, identified by MALDI-TOF analysis, are highlighted and numbered from 1 to 33.

optimized 2-DE and the stringent parameters used to create the peak lists and to search database protein make our identification reliable. Comparison of cervical to lumbar spinal cord identified 8 proteins expressed in a significantly different amount in wobbler mice and a larger group of 23 proteins in control mice; 4 proteins were shared between these two groups (Table 2). Comparing cervical or lumbar spinal cord in wobbler and control mice, we identified 17 proteins differently represented in these two groups, with 6 shared proteins (Table 2). Because of the wide intrinsic variability of the proteomic assay, we focused on the proteins with significant fold change higher than 1.5. Individual proteins differently represented in the comparison groups can be divided into four major functional categories: cellular and energetic metabolism, cytoskeleton and structural proteins, neurotransmitter-related proteins, redox pathway (Table 2). In these categories, we further investigated some interesting/key proteins, to validate results obtained by proteomic analysis. Cellular and Energetic Metabolism. In general, enzymes involved in glycolysis, TCA cycle, and in mitochondrial activity were less represented in cervical than in lumbar spinal cord in control mice, but expressed at similar levels in the two regions of wobbler mice, mainly due to increased expression in wobbler cervical spinal cord. We further investigated if different protein levels were paralleled by changes in activity of enzymes 5232

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Bastone et al. involved in the mitochondrial respiratory chain. The oxygen consumption rate (QO2) measured in the presence of FADH2generating substrates succinate and rotenone was not significantly different in cervical and lumbar tracts comparing wobbler and control mice (Figure 2A). Succinate dehydrogenase flavoprotein subunit (complex II) was more represented in cervical than in lumbar tract of control mice (fold change +2), as well as in lumbar spinal cord of wobbler mice compared to controls (fold change +2.1); however, the higher protein levels did not result in significantly increased enzymatic activity that was similar in cervical and lumbar tracts comparing wobbler and control mice (Figure 2B). The succinate dehydrogenase activity appeared to be lower in the cervical tract of wobbler mice, although after succinate dehydrogenase activity was normalized to citrate synthase activity (Figure 2C), a marker of mitochondrial mass, no difference was evident. Creatine kinase expression was 1.9-fold higher in lumbar than in cervical tract in control mice. Increased levels were found comparing the cervical tract of wobbler and control mice (fold change +1.8), while levels in lumbar spinal cord were similar. Functional activity of creatine kinase in tissue homogenates was significantly lower in cervical than in lumbar spinal cord of control mice (p < 0.05, Figure 2D), consistently with the protein expression. Furthermore, in agreement with increased protein levels, we found that activity was significantly higher in the cervical tract of wobbler than control mice (p < 0.02; Figure 2D). Cytoskeleton and Structural Proteins. In general, we found that the most relevant changes of these proteins were represented by significant increase in the wobbler lumbar spinal cord. A representative protein was T-complex protein 1 subunit-β that showed a significant overexpression (fold change of +1.9) in lumbar spinal cord of wobbler compared to control mice. Of particular interest in this category, we found two proteins involved in cellular trafficking: the isoform 2 of syntaxin-binding protein 1, involved in synaptic vesicle docking and fusion, and the vesicle fusing ATPase, required for vesiclemediated transport. Both these proteins were significantly overrepresented in cervical than in lumbar tract of control mice (isoform 2 of syntaxin-binding protein 1, fold change +4.9; vesicle fusing ATPase, fold change +1.8). At variance, the expression levels were similar in cervical and lumbar spinal cord in wobbler mice, due to increase of isoform 2 of syntaxinbinding protein 1 (fold change +4.8) and vesicle-fusing ATPase (fold change +1.9) selectively in wobbler lumbar tract, while expression in cervical spinal cord was similar between wobbler and control mice (Table 2). Immunofluorescence experiments on cervical and lumbar spinal cord sections confirmed these results, showing higher immunofluorescence intensity in neurons of cervical than lumbar tract in control mice (Figure 3A,B; Figure 3E,F). In wobbler spinal cord sections, immunofluorescence for isoform 2 of syntaxin-binding protein 1 and vesiclefusing ATPase was increased both in cervical and lumbar sections (Figure 3C,D; Figure 3G,H). Either in wobbler or in controls, no appreciable difference was found in signal intensity between cervical and lumbar neurons. We also noted that immunoreactivity for both isoform 2 of syntaxin-binding protein 1 and vesicle-fusing ATPase was confined to neurons in spinal cord sections of both wobbler and control mice, identifiable from their typical cell morphology (Figure 3). Neurotransmitter-Related. The protein showing the major changes in this group was glutamine synthetase, involved in glutamate-glutamine cycle. Proteomic experiments showed that

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Model of Motor Neuron Degeneration

Table 1. Proteins Differently Represented in the Cervical and Lumbar Spinal Cord of Wobbler and Control Mice, Identified by Proteomic Analysisa spot

protein name

AC

pI obs

pI calc

Mw obs

Mw calc

cov

score

pm/ps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28b 29 30 31 32 33

Aconitate hydratase Mitochondrial inner membrane protein Vesicle-fusing ATPase Glycerol-3-phosphate dehydrogenase Succinate dehydrogenase flavoprotein subunit Isoform 2 of syntaxin-binding protein 1 Transketolase Dihydropyriminidase related protein 2 Dihydropyriminidase related protein 2 Dihydropyriminidase related protein 3 Pyruvate kinase isozyme M2 Pyruvate kinase isozyme M2 Protein disulfide isomerase A3 Vacuolar ATP synthase sub B, brain isoform D-3-phosphoglycerate dehydrogenase T-complex protein 1 subunit β Glutamate dehydrogenase 1 ATP synthase R chain Synaptic vesicle membrane protein VAT-1 homologue 4-Aminobutyrate aminotransferase NADH-ubiquinone oxidoreductase 49 kDa subunit Glutamine synthetase Creatine kinase, ubiquitous mitochondrial Phosphoglycerate kinase 1 Fructose-biphosphate aldolase C Fructose-biphosphate aldolase A Isoform 2 of NAD-dependent deacetylase sirtuitin 2 NAD-dependent deacetylase sirtuitin 2 Glyceraldehyde-3-phosphate dehydrogenase Phosphoglycerate mutase 1 Glutathione S-tranferase Mu 1 Peroxyredoxin-6 ATP synthase D chain

Q99KI0 Q8CAQ8 P46460 Q64521 Q8K2B3 O08599-2 P40142 O08553 O08553 Q62188 P52480 P52480 P27773 P62814 Q61753 P80314 P26443 Q03265 Q62465 P61922 Q91WD5 P15105 P30275 P09411 P05063 P05064 Q8 VDQ8-2 Q8 VDQ8 P16858 Q9DBJ1 P10649 O08709 Q9DCX2

7.5 6.1 6.7 6.2 6.6 6.7 7.5 6.2 6.4 6.3 7.6 7.2 5.9 5.5 6.4 6.3 7.0 8.1 6.1 7.5 6.1 6.8 7.6 8.1 7.2 8.5 6.4 6.9 8.5 7.0 8.2 6.5 5.4

7.4 6.2 6.5 5.8 6.3 6.3 7.2 6.0 6.0 6.0 7.4 7.4 5.8 5.6 6.1 6.0 6.7 8.3 6.0 7.2 5.9 6.5 7.3 7.5 6.8 8.4 6.6 5.2 8.5 6.8 8.1 6.2 5.5

90 90 80 75 70 70 70 65 65 63 63 60 58 56 56 55 54 54 53 52 46 43 43 42 40 40 38 37 36 30 29 29 26

82 84 83 76 68 69 68 62 62 62 58 58 54 56 56 57 56 55 43 53 49 42 43 44 40 39 40 43 36 29 26 26 19

48 45 43 40 49 46 38 67 69 48 53 45 49 51 34 38 55 64 42 54 45 52 60 50 68 66 54 57 48 80 65 69 81

279 316 219 206 347 327 230 300 341 199 219 192 211 207 168 105 229 247 120 192 171 166 177 193 207 218 132 207 136 184 177 170 131

35/68 34/53 36/74 29/59 31/40 27/32 22/38 30/53 40/66 20/72 34/73 25/49 26/57 23/49 19/49 18/58 32/62 41/85 14/46 26/57 20/42 26/87 26/76 18/33 22/76 24/73 17/72 20/52 18/50 24/69 15/40 14/39 13/47

a Spot number as shown in Figure 1. Protein name and accession number (AC) are reported according to the Swiss-Prot and TrEMBL data base. pI obs, observed isoelectric point (pI); pI calc, calculated pI; Mw obs, observed molecular mass (Mw); Mw calc, calculated Mw; cov, percentage of sequence coverage by experimental PMF; score, probability score in Mascot program; pm/ps, experimental peptides matched and experimental peptides searched. b Spot no. 28, identified as NAD-dependent deacetylase sirtuitin 2, shows experimental pI different from the calculated pI (6.8 vs 5.2). This spot might represent an isoform or a proteolytic product of NAD-dependent deacetylase sirtuitin 2, not yet published.

protein expression was significantly higher in cervical than in lumbar spinal cord of wobbler mice (fold change +1.8); the pattern was inverted in control mice, and glutamine synthetase was lower in the cervical than in lumbar tract (fold change -2.4). Moreover, expression was markedly increased in wobbler than in control cervical spinal cord (fold change +5.2), while levels in lumbar spinal cord were similar in wobbler and control mice. We further investigated the protein levels and activity by Western blot and enzymatic assay, and the cellular localization by immunofluorescence. The different distribution of glutamine synthetase in the four experimental groups was confirmed by Western blot experiments (Figure 4B). Glutamine synthetase activity was significantly increased by about 2-fold in homogenate of cervical spinal cord in wobbler with respect to control mice (5.3 ( 0.2 and 3.1 ( 0.2 (µg of product/h)/mg protein, respectively; p < 0.001, n ) 3, Figure 4A). Activity was also significantly higher in wobbler cervical tract than in lumbar tract of both wobbler and control mice, that showed similar values (3.8 ( 0.3 and 3.7 ( 0.6 (µg of product/h)/mg protein, respectively; p < 0.01, n ) 3, Figure 4A). In control mice, glutamine synthetase activity was not significantly higher in the lumbar tract; thus, activity followed a different pattern in the cervical and lumbar tracts comparing wobbler and control mice (F for interaction p > 0.0017). With immunofluorescence

experiments on cervical spinal cord sections, we investigated glutamine synthetase expression in astrocytes (identified by GFAP-immunostaining) and neurons (identified by Neuro Trace staining). In control mice, a majority of GFAP-positive cells were localized in the white matter (fibrillary astrocytes) (Figure 4C); few astrocytes showed glutamine synthetase positivity (Figure 4D) mostly in the gray matter, suggesting that in physiological conditions glutamine synthetase is mainly expressed in protoplasmic astrocytes, as shown by colocalization of GFAP and glutamine synthetase (Figure 4E). GFAP expression was markedly increased in astrocytes of wobbler mice (Figure 4F); GFAP-positive astrocytes also showed a strong increase in glutamine synthetase expression (Figure 4G), both in the white and in the gray matter as demonstrated by staining colocalization (Figure 4H). We also observed that glutamine synthetase expression was not homogeneous in all astrocytes (Figure 4I, arrowheads). Colocalization experiments between Nissl and glutamine synthetase staining showed no expression of the enzyme in neuronal cells, both in wobbler and controls (not shown). In this group of differentially regulated proteins, also the distribution of 4-aminobutyrate aminotransferase, involved in GABA degradation, was significantly changed: the protein was selectively overrepresented in both cervical and lumbar spinal Journal of Proteome Research • Vol. 8, No. 11, 2009 5233

5234 0.593 ( 0.090 0.286 ( 0.040 1.595 ( 0.143 0.608 ( 0.084 0.047 ( 0.006 1.123 ( 0.091 0.573 ( 0.085 1.354 ( 0.161 0.036 ( 0.001 0.055 ( 0.009 0.621 ( 0.066 0.387 ( 0.057 0.096 ( 0.007 0.320 ( 0.021 0.079 ( 0.007 0.133 ( 0.018 0.106 ( 0.003

0.125 ( 0.007 0.549 ( 0.054 0.034 ( 0.003 0.038 ( 0.013 0.106 ( 0.007 0.046 ( 0.010 0.125 ( 0.042 0.108 ( 0.011 0.139 ( 0.020 0.093 ( 0.010 0.403 ( 0.065 0.483 ( 0.061 0.146 ( 0.019 0.177 ( 0.025 0.168 ( 0.009 0.033 ( 0.007

0.287 ( 0.029 0.262 ( 0.033 0.853 ( 0.061 0.323 ( 0.031 0.057 ( 0.003 0.386 ( 0.039 0.235 ( 0.047 0.940 ( 0.072 0.031 ( 0.004 0.093 ( 0.009 0.257 ( 0.031 0.304 ( 0.037 0.051 ( 0.006 0.174 ( 0.025 0.158 ( 0.020 0.041 ( 0.004 0.163 ( 0.014

0.212 ( 0.006 0.989 ( 0.074 0.034 ( 0.004 0.189 ( 0.028 0.082 ( 0.004 0.045 ( 0.007 0.185 ( 0.025 0.117 ( 0.011 0.246 ( 0.017 0.062 ( 0.006 0.222 ( 0.025 0.202 ( 0.032 0.050 ( 0.012 0.166 ( 0.009 0.132 ( 0.008 0.063 ( 0.001

Dihydropyrimidinase related protein 2 Dihydropyrimidinase related protein 2 Dihydropyrimidinase related protein 3 Isoform 2 of syntaxin-binding protein1 Mitochondrial inner membrane protein Isoform 2 of NAD-dependent deacetylase sirtuitin-2 NAD-dependent deacetylase sirtuitin-2 T-complex protein 1 subunit-β Vesicle-fusing ATPase

4-Aminobutyrate aminotransferase Glutamate dehydrogenase 1 Glutamine synthetase

Glutathione S-transferase Mu 1 Piroxiredoxin-6 Protein disulfide-isomerase A3 Synaptic vesicle membrane protein VAT-1 homologue

Journal of Proteome Research • Vol. 8, No. 11, 2009

a

difference

0.097 ( 0.009 0.148 ( 0.012

0.408 ( 0.045 0.474 ( 0.050 0.089 ( 0.004 0.310 ( 0.023 0.170 ( 0.024

0.058 ( 0.004 0.106 ( 0.013

0.090 ( 0.011 1.146 ( 0.140 0.561 ( 0.037 1.527 ( 0.147

-3.3 1.5

-2.4 -1.4 -1.9 -1.8 2.0

-1.1 1.7

1.2 -2.9 -2.4 -1.4

Energetic Metabolism 0.703 ( 0.054 -2.1 0.341 ( 0.016 -1.1 1.255 ( 0.185 -1.9 0.740 ( 0.071 -1.9

WrL

0.04b 0.04b 0.01b 0.005b 0.71 0.02b 0.02b

Neurotransmitter-Related 0.141 ( 0.014 0.192 ( 0.015 -1.5 0.446 ( 0.053 0.481 ( 0.033 -1.8 1.052 ( 0.082 0.585 ( 0.076 -2.4 Redox Pathway 0.116 ( 0.015 0.145 ( 0.021 0.239 ( 0.029 0.259 ( 0.019 0.137 ( 0.017 0.165 ( 0.012 0.054 ( 0.011 0.077 ( 0.010

-2.9 -1.1 -1.3 1.9

0.26 0.56 0.01b

0.248 ( 0.026 0.208 ( 0.023 0.269 ( 0.039

b

-1.2 -1.1 -1.2 -1.4

-1.4 -1.1 1.8

1.1 -1.7 1.2

1.4 1.0 -1.5 1.0 -1.4 -1.2

-1.1 -1.3

-1.1 1.0 -1.1 -1.1 1.0

-1.3 1.1

-1.0 -1.0 -1.1 -1.1

-1.4 -1.3 -1.3 -1.3

difference

0.31 0.58 0.23 0.17

0.05b 0.60 0.01b

0.25 0.01b 0.33

0.14 0.89 0.005b 0.98 0.03b 0.41

0.35 0.18

0.53 0.95 0.45 0.58 0.94

0.04b 0.33

0.91 0.96 0.76 0.56

0.02b 0.01b 0.25 0.13

2.3 1.4 1.0 -1.2

2.3 2.0 5.2

1.5 1.0 1.3

1.3 1.0 1.7 -1.0 1.0 1.4

2.1 -1.4

1.4 1.8 1.6 1.6 1.1

1.4 1.3

1.6 2.9 2.2 1.5

1.8 1.0 1.2 1.8

difference

0.01b 0.05 0.80 0.44

0.002b 0.01b 7 × 10-5b

0.01b 0.87 0.08

0.20 0.94 0.02b 0.85 0.79 0.31

0.001b 0.08

0.03b 0.001b 0.02b 0.03b 0.57

0.08 0.05b

0.005b 7 × 10-5b 0.14 0.002b

0.003b 0.85 0.21 0.01b

p

WrC vs CtrC

fold change

p

WrC vs WrL

Significant p-values (p < 0.05); Student’s t-test.

9 × 10-5b 0.003b 0.97 0.003b 0.02b 0.97

0.01b 0.03b

0.002b 0.16 0.002b 0.004b 0.01b

0.34 0.02b

0.21 3 × 10-4b 0.01b 0.06

0.02b 0.66 0.003b 0.02b

p

1.5 1.1 1.8

0.284 ( 0.011 0.120 ( 0.007 0.323 ( 0.032

Cytoskeleton and Structural Proteins 0.285 ( 0.044 0.204 ( 0.017 1.7 0.995 ( 0.050 0.977 ( 0.110 1.8 0.057 ( 0.006 0.088 ( 0.005 -1.0 0.183 ( 0.004 0.182 ( 0.018 4.9 0.086 ( 0.011 0.122 ( 0.006 -1.3 0.062 ( 0.013 0.075 ( 0.006 -1.0

0.086 ( 0.007 0.115 ( 0.018

0.373 ( 0.027 0.535 ( 0.012 0.081 ( 0.008 0.286 ( 0.033 0.172 ( 0.011

0.044 ( 0.004 0.122 ( 0.002

0.089 ( 0.007 1.138 ( 0.068 0.509 ( 0.154 1.429 ( 0.062

Cellular and 0.505 ( 0.036 0.269 ( 0.010 0.997 ( 0.084 0.575 ( 0.063

WrC

CtrC vs CtrL

WrC, wobbler cervical spinal cord; CtrC, control cervical spinal cord; WrL, wobbler lumbar spinal cord; CtrL, control lumbar spinal cord;

Aconitate hydratase ATP synthase D chain ATP synthase R chain Creatine kinase, ubiquitous mitochondrial D-3-phosphoglycerate dehydrogenase Fructose-bisphosphate aldolase A Fructose-bisphosphate aldolase C Glyceraldehyde-3-phosphate dehydrogenase Glycerol-3-phosphate dehydrogenase NADH-ubiquinone oxidoreductase 49 kDa subunit Phosphoglycerate kinase 1 Phosphoglycerate mutase 1 Pyruvate kinase isozyme M2 Pyruvate kinase isozyme M2 Succinate dehydrogenase flavoprotein subunit Transketolase Vacuolar ATP synthase subunit B brain isoform

CtrL

CtrC

protein name

normalized spot volume ( SEM

Table 2. Quantification of Differences in Protein Levels in the Cervical and Lumbar Tracts of Spinal Cord of Wobbler and Control Micea

-1.0 1.5 -1.0 2.3

2.1 1.2 1.2

2.0 1.9 1.9

1.6 1.8 2.6 4.8 1.1 1.6

-1.4 1.4

-1.5 1.3 -1.1 -1.0 2.1

1.6 1.9

1.9 1.0 -1.0 1.1

1.2 1.2 -1.3 1.2

0.96 0.04b 0.85 0.01b

0.002b 0.33 0.33

0.05b 0.01b 0.03b

0.005b 0.01b 9 × 10-5b 7 × 10-4b 0.14 0.05b

0.12 0.04b

0.04b 0.16 0.37 0.74 0.01b

0.002b 0.02b

0.01b 0.90 0.91 0.46

0.34 0.25 0.19 0.27

p

WrL vs CtrL difference

research articles Bastone et al.

research articles

Model of Motor Neuron Degeneration

Figure 2. Oxygen consumption rate (QO2) and mitochondrial enzymatic activities in spinal cord of control and wobbler mice. QO2 (A), succinate dehydrogenase (B), citrate synthase (C) and creatine kinase (D) activities were measured in spinal cord homogenate from cervical or lumbar spinal cord of control and wobbler mice at 4 weeks of age. Histograms represent the mean ( SE of 5-7 independent experiments performed in duplicate. Results are expressed in (nmol/min)/mg protein. Creatine kinase activity was significantly increased in wobbler than in control mice cervical spinal cord (**p < 0.02, paired Student’s t-test) and in lumbar as compared to cervical tract only in control mice (§p < 0.05, paired Student’s t-test).

Figure 3. Isoform 2 of syntaxin-binding protein 1 and vesicle-fusing ATPase staining in cervical and lumbar spinal cord of control and wobbler mice. (A) Representative pictures showing the isoform 2 of syntaxin-binding protein 1 staining in sections of the cervical spinal cord of control mice revealed selective localization in neuronal cells with brighter peripheral spots. (B) The staining intensity was lower in neurons of the lumbar spinal cord in healthy mice. In wobbler mice, the intensity for isoform 2 of syntaxinbinding protein 1 was higher than in control mice; staining intensity was similar in cervical (C) and lumbar neurons (D). Vesiclefusing ATPase was mainly localized in cytoplasm of large neurons in the cervical spinal cord of control mice (E), with lower immunoreactivity in lumbar spinal cord neurons (F). No immunoreactivity was found in the white matter. In spinal cord, sections of wobbler mice both cervical (G) and lumbar (H) neurons showed strong neuronal immunoreactivity for vesicle-fusing ATPase. Scale bar: 50 µm.

cord in wobbler with respect to control mice (fold change +2.3 and +2.1, respectively), while the distribution between cervical and lumbar spinal cord was unchanged comparing wobbler and control mice, being overrepresented in lumbar spinal cord. Redox Pathway. Among proteins involved in redox reactions, we found a significant increased expression of glutathione S-transferase Mu 1 selectively in the cervical tract in wobbler compared to control mice (fold change +2.4), while levels in the lumbar spinal cord were similar in the two groups.

Discussion The wobbler mouse, carrying a mutation in the vps54 gene,15 is a useful model of motor neuron degenerative diseases such as ALS and spinal muscular atrophy.17,26 To date, only one disease-specific nonsynonymous mutation was identified in the human vps54 gene among 197 ALS patients.18 In wobbler mice, motor neuron loss occurs selectively in the cervical spinal cord, while motor neurons in the lumbar tract are spared.17,27 Indeed, Journal of Proteome Research • Vol. 8, No. 11, 2009 5235

research articles

Bastone et al.

Figure 4. Glutamine synthetase activity and expression in spinal cord of control and wobbler mice. (A) Activity of glutamine synthetase was measured in tissue homogenates of cervical and lumbar spinal cord from wobbler and control mice. Values represent mean ( SE of 3 independent experiments performed in triplicate. Activity was significantly higher in wobbler than in control cervical spinal cord (***p < 0.001; two-way ANOVA) and in cervical than in lumbar wobbler mice spinal cord ($$p < 0.01; two-way ANOVA). (B) Western blot immunoreactivity for glutamine synthetase was higher in the cervical tract of wobbler mice (lanes 3 and 4) than in control mice (lanes 1 and 2). Actin was used as control for equal protein loading. Glutamine synthetase immunofluorescent staining was evaluated in cervical spinal cord sections of 4 week old control (C-E) and wobbler mice (F-H). In control mice, GFAP-positive cells were mainly located in the white matter (W.M.) (C), with less positive cells in the gray matter (G.M.). (D) Only few cells were immunoreactive for glutamine synthetase in control mice spinal cord. (E) Colocalization of GFAP and glutamine synthetase showed selective expression of this enzyme in astrocytes, although immunoreactivity was restricted to few astrocytes. (F) Reactive gliosis in wobbler mice greatly extended the area and intensity of GFAP immunoreactivity in the whole cervical spinal cord. (G) Glutamine synthetase immunoreactivity was also markedly increased in the gray and white matter. (H) Colocalization of the two staining showed that a vast majority of GFAPpositive cells were also glutamine synthetase-positive in both gray and white matter. The heterogeneity of glutamine synthetase expression in astrocytes (I, arrowheads) was shown at higher magnification. Scale bar: C-H, 80 µm; I, 30 µm.

this region can be considered an “inner control” and used to investigate protective or detrimental factors for motor neurons, also in comparison with matching tissues from healthy littermates. A 2-DE-based proteomic approach enabled us to find different patterns of protein distribution in the two tracts of spinal cord, both in control and in wobbler mice, at early symptomatic stage of the disease (see Table 3). We identified some proteins that can be relevant in maintaining tissue integrity and that might explain, at least in part, the selective vulnerability of cervical motor neurons in wobbler mice. The first interesting evidence we found is that protein distribution was different in the two tracts of spinal cord in control mice (Table 3 class I), probably due to different metabolic demands in these different regions in physiological 5236

Journal of Proteome Research • Vol. 8, No. 11, 2009

conditions. Proteins involved in glutamate degradation/ recycling, enzymes involved in cell energy metabolism and redox pathway were prevalently overrepresented in lumbar than in cervical spinal cord, while in the cervical tract, we found that overrepresented proteins were involved in cellular trafficking and mitochondrial respiratory chain complexes (Table 3 class I). Interestingly, these regional differences in protein levels were generally lost in wobbler mice, characterized by similar levels of expression in the two regions of the spinal cord (Tables 2 and 3 class II). This unbalanced protein distribution in wobbler mice spinal cord was due to increased expression of enzymes involved in the cell energy metabolism, antioxidant pathway and cytoskeletal proteins (Table 3 class IV and V). According to our findings, some of the proteins we identified

research articles

Model of Motor Neuron Degeneration

Table 3. Differently Represented Proteins with Significant Fold Change Higher than 1.5 Are Divided in Five Classes According to the Different Comparison Groupsa protein name

localization

function

fold change

Class I: Proteins Differently Represented in the Cervical with Respect to Lumbar Spinal Cord in Control Mice Transketolase C, ER(1.5%) pentose phosphate pathway (nonoxidative) Glutathione-S-transferase Mu1 C antioxidant Fructose-biphosphate aldolase A C glycolysis Fructose-biphosphate aldolase C C glycolysis Phosphoglycerate kinase 1 C glycolysis Glutamine synthetase C glutamate recycling Aconitate hydratase M tricarboxylic acid cycle ATP synthase R chain M energy transduction Creatine kinase M energy transduction Pyruvate kinase isozyme M2 C glycolysis Glutamate dehydrogenase 1 M glutamate degradation Isoform 2 of syntaxin-binding protein1 C synaptic vesicle docking and fusion Succinate dehydrogenase flavoprotein subunit M tricarboxylic acid cycle Synaptic vesicle membrane protein VAT-1 homologue V oxidoreductase Dihydropyrimidinase related protein 2 C CNS development Vesicle fusing ATPase C vesicle-mediated transport to Golgi NADH-ubiquinone oxidoreductase 49 kDa subunit M energy transduction

-3.3 -2.9 -2.9 -2.4 -2.4 -2.4 -2.1 -1.9 -1.9 -1.8 -1.8 +4.9 +2.0 +1.9 +1.8 +1.8 +1.7

Class II: Proteins Differently Represented in the Cervical with Respect to Lumbar Spinal Cord in Wobbler Mice T-complex protein 1 subunit β C chaperone Glutamine synthetase C glutamate recycling

-1.7 +1.8

Class III: Proteins Overrepresented in Both Cervical and Lumbar Spinal Cord of Wobbler with Respect to Control Mice cervical 4-Aminobutyrate aminotransferase M GABA degradation +2.3 Dihydropyrimidinase related protein 3 C CNS development +1.7 D-3-phosphoglycerate dehydrogenase C? amino-acid biosynthesis +1.6 NAD-dependent deacetylase sirtuitin-2 C cell cycle +1.5

lumbar +2.1 +2.6 +1.9 +2.0

Class IV: Proteins Overrepresented in the Cervical Spinal Cord of Wobbler Compared to Control Mice Glutamine synthetase C glutamate recycling Fructose-biphosphate aldolase A C glycolysis Glutathione-S-transferase Mu1 C antioxidant Transketolase C, ER(1.5%) pentose phosphate pathway (nonoxidative) Glutamate dehydrogenase 1 M glutamate degradation Creatine kinase M energy transduction Phosphoglycerate mutase 1 ? glycolysis Aconitate hydratase M tricarboxylic acid cycle Pyruvate kinase isozyme M2 C? glycolysis

+5.2 +2.9 +2.3 +2.1 +2.0 +1.8 +1.8 +1.8 +1.6

Class V: Proteins Overrepresented in the Lumbar Spinal Cord of Wobbler Compared to Control Mice Isoform 2 of syntaxin-binding protein 1 C synaptic vesicle docking and fusion Synaptic vesicle membrane protein VAT-1 homologue Mem ATPase; dehydrogenases/reductase Succinate dehydrogenase flavoprotein subunit M tricarboxylic acid cycle NADH-ubiquinone oxidoreductase M energy transduction Vesicle fusing ATPase C vesicle-mediated transport to Golgi T-complex protein 1 subunit β C chaperone Dihydropyrimidinase related protein 2 C CNS development Glycerol-3-phosphate dehydrogenase M lipid metabolism NAD-dependent deacetylase sirtuitin-2 C cell cycle

+4.8 +2.3 +2.1 +1.9 +1.9 +1.9 +1.8 +1.6 +1.6

a

Protein cellular localization and function are reported. N, nucleus; ER, endoplasmic reticulum; mem, membrane; M, mitochondria; C, cytoplasm.

in the energy metabolic pathway, redox function and chaperonine families were also reported to be differently represented in other proteomic profiling studies on the mutant SOD1 transgenic mouse, on cellular models of ALS8,10-12,28,29 and in otherneurodegenerativediseasesnotinvolvingmotorneurons,30-32 suggesting the participation of common biochemical pathways in wobbler disease and other models of neurodegeneration. A mechanism potentially relevant in neurodegenerative processes can involve proteins related to the antioxidant pathways; for example, the glutathione-S-transferase M1 protein exerts an antioxidant activity and also inhibits the apoptotic pathway mediated by ASK1, c-JNK and p38.33 We recently reported an increased phosphorylation of JNK and p38 in the

spinal cord of early symptomatic wobbler mice;34 so the upregulation of glutathione-S-transferase in cervical spinal cord of wobbler compared to control mice that was reported in this study (Table 3 class IV) may represent an attempt to counterbalance the high-oxidant environment and stress-activated protein kinase death pathway. Notably, up-regulation of this antioxidant protein is unique to wobbler mice since this protein is reduced in cells expressing the mutant hSOD1.28 Levels of proteins involved in anaerobic glycolytic metabolism, pentose cycle and creatine metabolism were also higher in the cervical tract of wobbler than control mice (Table 3 class IV), a result likely related to the motor neuron degeneration and reactive gliosis in the cervical spinal cord of wobbler mice Journal of Proteome Research • Vol. 8, No. 11, 2009 5237

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Bastone et al. 34-36

already at this early stage of disease. In particular, we observed a marked increase in glutamine synthetase, an astrocyte-specific enzyme playing a key role in glutamateglutamine recycling between neurons and astrocytes. Our results indicate that modifications involved both protein levels and activity. The higher expression of glutamine synthetase and GFAP in wobbler mice cervical spinal cord (Figure 4) confirmed the presence of activated astrocytes, a typical feature of wobbler pathology. Higher levels of glutamine synthetase expression and activity may suggest the presence of alterations in both glutamate and glutamine homeostasis, participating in mechanisms of motor neuron degeneration related to excitotoxic insults, as suggested by other reports37 (Bonanno et al., in press). Interestingly, glutamine synthetase expression was not homogeneous in all astrocytes (Figure 4I, arrowheads); however, our results did not confirm the previously reported lack of glutamine synthetase positivity in strongly GFAP-positive astrocytes in the spinal cord gray matter.38 In general, we can reason that proteins differently represented in cervical spinal cord of wobbler with respect to control mice are related to the effects of Vps54 mutation in a more vulnerable tissue. The basis of this vulnerability might reside in the different protein distribution observed between cervical and lumbar tracts in control mice. A further consideration arising from our results is that proteins overexpressed in both cervical and lumbar spinal cord of wobbler compared to control mice are likely related to the presence of the Vps54 mutation, but not relevant for the degeneration of motor neurons, occurring selectively in the cervical tract (Table 3 class III). Among these proteins, dihydropyrimidinase related protein-3, that interacts with synaptic vesicle protein 2 and regulates vesicle function in the growth cone, and NAD-dependent deacetylase sirtuitin-2, an enzyme involved in regulation of the cytoskeleton, seem to suggests a counteracting mechanism to defective vesicle trafficking in the wobbler disease. This is the first report showing that proteins that selectively differed between the lumbar tract of wobbler and control mice were at variance with that found comparing the cervical tracts (Table 3 class V). Since the lumbar spinal cord in wobbler mice is undamaged in spite of the presence of the Vps54 mutation, these proteins might help protect motor neurons from degeneration. Cellular mechanisms involved in energy production are known to be commonly altered in neurodegenerative diseases; in particular, mitochondrial complex I dysfunctions impair the respiratory chain, leading to ROS overproduction, DNA damage, lipid peroxidation and protein dysfunction. In line with these findings, we previously reported decreased oxygen consumption and decreased complex I activity in the cervical spinal cord of 4- and 12-week-old wobbler mice.23 In this study, we found that expression of NADH-ubiquinone oxidoreductase 49-kDa subunit and succinate dehydrogenase flavoprotein subunit, two proteins involved in the complex I and complex II of the mitochondrial respiratory chain, was significantly increased in wobbler than in control lumbar spinal cord (Table 3 class V). Furthermore, levels of these two enzymes were slightly higher in lumbar than in cervical spinal cord of wobbler mice, compared to respective regions in control mice, and this difference may sustain the mitochondrial function and contribute to motor neuron protection. Thus, the higher levels of proteins of complex I and, to a lesser extent, of complex II might represent an adaptive mechanism in the lumbar tract of wobbler mice aimed at maintaining functional respiratory 5238

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activity. In spite of increased protein levels, we found lower or unchanged mitochondrial activity in wobbler than in control mice, a discrepancy that could be ascribed to post-transductional changes modifying the catalytic activity of complex I in wobbler spinal cord. Other pathways are described to be altered in neurodegenerative processes, one of them linked to cellular transport and trafficking. Here, we showed that some protein associated to the cytoskeleton and cellular trafficking were selectively overrepresented in the wobbler lumbar spinal cord. In particular, the level of T-complex protein 1 subunit-β, a molecular chaperone involved in the folding of actin and in tubulin biogenesis,39 was significantly increased in wobbler lumbar spinal cord (Table 3 class V). In axons of lumbar motor neurons, the subunits of the T-complex protein 1 belong to the “slow component b” of axonal transport, together with the molecular chaperone Hsc73 and actin.39 It has been reported that all components of axonal transport were slowed in the affected forelimb motor neurons of wobbler mice, while only the fast anterograde axonal transport was specifically reduced in hindlimb motor neurons, with no changes in the slow and in retrograde component.40 Thus, the up-regulation of T-complex protein 1 we identified in the lumbar tract of wobbler mice spinal cord can be a protective mechanism that may explain the integrity of the slow component of anterograde and retrograde transport in lumbar motor neurons and the selective vulnerability of cervical motor neurons. An important and original finding of this study was the overexpression of two syntaxin-binding proteins involved in cellular trafficking, vesicle fusing ATPase and isoform 2 of syntaxin-binding protein1 (Table 3 class V), especially in light of the finding that the vsp54 mutation in wobbler disease affects a GARP protein involved in vesicles trafficking from the late endosomes to the trans-Golgi network.41 The vesicle-fusing ATPase is required for vesicle-mediated transport, catalyzing the fusion of transported vesicles within the Golgi cisternae, and is required for transport from the endoplasmic reticulum to the Golgi stacks. Both proteomic and immunofluorescence experiments showed a marked overexpression in the lumbar tract of wobbler spinal cord. In mutant SOD1 transgenic mice, vesicle fusing ATPase was significantly lower in the hippocampus, a region where neuronal loss is documented.42 The isoform 2 of syntaxin-binding protein 1 participates in neurotransmission and in cytoskeletal dynamics.43,44 Also in this case, we evidenced a significant overexpression of isoform 2 of syntaxin-binding protein 1 in the lumbar spinal cord of wobbler mice, while no changes were observed comparing the cervical tract in wobbler and control mice. The significant overexpression of both these proteins was selective in wobbler lumbar spinal cord, suggesting a protective mechanisms active only in the lumbar spinal cord tract, possibly leading to motor neurons preservation. Different results were found in mutant SOD1 transgenic mice, where syntaxin-binding protein 1 was significantly lower in the hippocampus, suggesting synaptic and neuronal loss.42 Reduced expression of syntaxin-binding protein also correlated with impaired motor function in 129X1/ SvJ mice.45 The increased levels of vesicle fusing ATPase and isoform 2 of syntaxin-binding protein1 in wobbler mice spinal cord can also be relevant in counteracting modifications in glutamatemediated neurotransmission. We recently reported an increased glutamate release in the cervical spinal cord of wobbler mice, without changes in the lumbar tract (Bonanno, G., et al.,

Model of Motor Neuron Degeneration in press). Since exocytotic release is dependent on the vesicle turnover in the nerve terminal, the Vps54 mutation in wobbler mice might lead to alteration in the mechanisms regulating vesicle availability and, subsequently, the exocytotic release machinery. These syntaxin binding proteins can inhibit the exocytotic neurotransmitter release by binding syntaxin, and their selective increase in the nonaffected spinal cord region in wobbler mice could represent a compensatory mechanism helping in maintenance of normal neurotransmission. Thus, in wobbler mice, the overexpression of protein involved in cellular trafficking in lumbar spinal cord might represent a protective factor that counteracts the changes linked to the Vps54 mutation. In conclusion, in wobbler mice, we identified different pattern of proteins expression between the affected cervical spinal cord tract and the lumbar tract, where no signs of motor neuron degeneration were evident. These changes were already present at the early symptomatic stage of the disease. The proteins overrepresented in the cervical tract mainly reflect the astrogliosis, and are involved in glutamate metabolism, energy transduction and redox function; proteins overrepresented in the wobbler lumbar spinal cord were cytoskeleton proteins involved in vesicular and axonal transport and proteins involved in mitochondrial function. We can conclude that the selective overexpression of proteins involved in these two important pathways, the cellular transport and the energetic metabolism, may have neuroprotective effects and significantly contribute in maintaining integrity and functionality of motor neurons in the lumbar region of the wobbler spinal cord. Abbreviations: 2-D, two-dimensional; 2-DE, two-dimensional gel electrophoresis; ALS, amyotrophic lateral sclerosis; ASK1, apoptosis-signaling kinase 1; CNS, central nervous system; ctr, control; GARP, Golgi-associated retrograde protein; GFAP, glial fibrillary acidic protein; Hsc73, heat shock protein 73; IPG, immobilized pH gradient; JNK, Jun N-terminal kinase; MOWSE, molecular weight search; MS, mass spectrometry; p38, protein 38; PMF, peptide mass fingerprint; QO2, oxygen consumption rate; ROS, reactive oxygen species; SOD1, superoxide dismutase 1; TCA, tricarboxylic acid; Vps54, vesicular protein sorting 54; wr, wobbler.

Acknowledgment. This study was partly supported by Cariplo Foundation (Grant GUARD) and Ministero della Salute (BIOMAR project). Authors thank Dr. Sara Barbera for technical support. Supporting Information Available: Parameters for peak list and protein search condition. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Beghi, E.; Mennini, T.; Bendotti, C.; Bigini, P.; Logroscino, G.; Chio, A.; Hardiman, O.; Mitchell, D.; Swingler, R.; Traynor, B. J.; Al-Chalabi, A. The heterogeneity of amyotrophic lateral sclerosis: A possible explanation of treatment failure. Curr. Med. Chem. 2007, 14 (30), 3185–3200. (2) Boillee, S.; Vande Velde, C.; Cleveland, D. W. ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron 2006, 52 (1), 39–59. (3) Goodall, E. F.; Morrison, K. E. Amyotrophic lateral sclerosis (motor neuron disease): proposed mechanisms and pathways to treatment. Expert Rev. Mol. Med. 2006, 8 (11), 1–22. (4) Miller, R. G.; Mitchell, J. D.; Lyon, M.; Moore, D. H. Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Amyotrophic Lateral Scler. Other Motor Neuron Disord. 2003, 4 (3), 191–206.

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