Proteomic Profiling of Plasma in Huntington's Disease Reveals

Proteomic Profiling of Plasma in Huntington’s Disease Reveals Neuroinflammatory Activation and Biomarker Candidates Annette Dalrymple,†,O Edward J. Wild,‡,O Richard Joubert,† Kirupa Sathasivam,§ Maria Bjo1 rkqvist,| Åsa Peterse´ n,| Graham S. Jackson,‡ Jeremy D. Isaacs,‡ Mark Kristiansen,‡ Gillian P. Bates,§ Blair R. Leavitt,⊥ Geoff Keir,# Malcolm Ward,† and Sarah J. Tabrizi*,‡ Proteome Sciences PLC, Cobham, Surrey, United Kingdom, Department of Neurodegenerative Disease, Institute of Neurology, London, United Kingdom, King’s College London School of Medicine, Guy’s Hospital, London, United Kingdom, Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund, Sweden, Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC, Canada, and Neuroimmunology Laboratory, National Hospital for Neurology and Neurosurgery, London, United Kingdom Received February 12, 2007

Huntington’s disease (HD) causes widespread CNS changes and systemic abnormalities including endocrine and immune dysfunction. HD biomarkers are needed to power clinical trials of potential treatments. We used multiplatform proteomic profiling to reveal plasma changes with HD progression. Proteins of interest were evaluated using immunoblotting and ELISA in plasma from 2 populations, CSF and R6/2 mice. The identified proteins demonstrate neuroinflammation in HD and warrant further investigation as possible biomarkers. Keywords: Huntington’s disease • plasma • neuroinflammation • proteomics • 2D electrophoresis • LC/MS/MS • MALDI-TOF • ELISA • biomarkers

Introduction Huntington’s disease (HD) is a devastating, progressive, neurodegenerative disease with autosomal dominant inheritance, characterized by cognitive decline, movement disorder, and behavioral abnormalities. It is caused by a CAG triplet repeat expansion in the gene encoding huntingtin, a ubiquitously expressed protein.1 The pathological hallmark of HD is striatal neuronal cell loss, but there are widespread changes in the CNS,1 and systemic abnormalities have been identified including endocrine dysfunction2 and immune activation.3 Peripheral abnormalities are of significance in their own right but may also reflect central pathology and, where implicated molecules cross the bloodbrain barrier, may exert central effects on brain pathogenesis. In addition, because of its slow clinical progression and the limitations of standard clinical rating scales,4 there is a need for biomarkers of onset and progression in HD to power clinical trials of potential disease-modifying treatments.5,6 Unlike in other degenerative diseases, diagnostic markers are not required because of the reliable genetic test for HD.6 Several candidate biomarkers in plasma have been proposed,7,8 but no * To whom correspondence should be addressed. Dr Sarah J Tabrizi, MRC Prion Unit, Institute of Neurology, National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK; Phone: 44-8451-555000; Fax: 44-207-676-2180; E-mail [email protected]. † Proteome Sciences PLC. ‡ Institute of Neurology. § King’s College London School of Medicine. | Wallenberg Neuroscience Center. ⊥ University of British Columbia. # National Hospital for Neurology and Neurosurgery. O These authors contributed equally as first author. 10.1021/pr0700753 CCC: $37.00

 2007 American Chemical Society

plasma proteins to date have been reported to be effective biomarkers for HD. Because of its ready accessibility, plasma is an appealing site for biomarker discovery and evaluation, while cerebrospinal fluid, although difficult to obtain, offers closer insights into the CNS milieu. Proteomic profiling has previously been applied to mouse models of HD and human brain tissue9 but has not been reported for human plasma or CSF. Using a multiplatform proteomic profiling approach with 2 different depletion techniques and protein identification methods, we identified 18 candidate proteins differentially expressed in human plasma at various stages of HD including pre-manifest gene carriers. Candidate proteins were assessed mechanistically in terms of known roles in the pathogenesis of HD and other neurodegenerative diseases. The most promising protein in this respect, clusterin, was evaluated further by immunoblotting and enzymelinked immunosorbent assay (ELISA) quantification in plasma samples from 2 independent populations, as well as in matched CSF and plasma samples. To investigate the neuroinflammatory processes highlighted by the proteomic discovery, we also investigated interleukin-6 (IL-6) levels in both human plasma and the R6/2 transgenic mouse model of HD using ELISA.

Experimental Section Subject Recruitment. All participants gave informed written consent, and the study protocol received the approval of local and national ethics review boards. Subjects with concomitant CNS disorders, significant medical comorbidity, known liver dysfunction, recent alcohol or substance abuse, and those taking medications or supplements suspected or known to interfere with the experimental methods used were excluded. Journal of Proteome Research 2007, 6, 2833-2840

2833

Published on Web 06/07/2007

research articles

Dalrymple et al.

Table 1. Clinical Characteristics of Subjects in Each Study for Discovery and Evaluation Experiments

experiment

Overall subject pool

disease stage

Control Premanifest Early Moderate Advanced 2D electrophoresis Control first plasma study HD patients 2D electrophoresis Control second plasma study Premanifest Early Moderate Advanced Immunoblotting Control Plasma Premanifest Early Moderate Clusterin ELISA Control Plasma Premanifest Early Moderate Clusterin ELISA Control Matched CSF and plasma Early Moderate IL-6 ELISA Control Plasma Premanifest Early Moderate a

number of female: subjects male

74 42 58 66 10 10 10a 55 14 15 15 10 15 15 15 15 18 18 19 18 9 9 11 34 16 23 23

(43:31) (24:18) (32:26) (38:28) (2:8) (8:2) (7:3) (28:27) (7:7) (9:6) (9:6) (2:8) (6:9) (7:8) (9:6) (9:6) (13:5) (12:6) (10:9) (13:5) (6:3) (4:5) (1:10) (19:15) (8:8) (14:9) (13:10)

mean age (range)

44 (21-74) 40 (27-61) 46 (29-67) 51 (23-77) 54 (40-68) 49 (29-67) 51 (31-70) 43 (22-68) 37 (30-46) 43 (29-51) 48 (24-62) 54 (40-68) 47 (24-68) 38 (30-46) 43 (29-51) 48 (24-62) 42 (21-74) 41 (27-61) 48 (29-63) 55 (35-77) 45 (25-66) 51 (38-64) 53 (38-72) 41 (21-67) 38 (30-61) 44 (29-67) 50 (23-69)

Four early, 4 moderate, 2 advanced HD patients.

In view of the likelihood of nutritional, infective, and inflammatory disorders in advanced HD, such patients were not included in validation experiments. Collection and Fractionation of Blood Samples. Subjects were recruited through the HD Multidisciplinary Clinic of the National Hospital for Neurology and Neurosurgery, London, U.K. Blood samples were collected from patients who had tested positive for the HD genetic mutation. Control subjects were partners and spouses of HD patients, at-risk individuals who tested negative for the HD mutation, and healthy individuals from the general population. Premanifest HD mutation carriers were identified according to the absence of diagnostic motor abnormalities on the Unified Huntington’s Disease Rating Scale.4 Patients with motor abnormalities were defined as having early, moderate, or advanced disease using the total functional capacity (TFC) scale (13-7, early; 6-3, moderate)10 assessed by experienced clinical raters. All samples were collected in EDTA tubes between 2 and 4 p.m. and fractionated within 2 h by density gradient centrifugation using a standard technique.11 Plasma aliquots were stored at -70 °C until use. Subjects’ clinical data are shown in Table 1. CAG repeat lengths were sized by PCR followed by capillary electrophoresis. Collection of Matched CSF and Blood Samples. CSF donors were recruited through the University of British Columbia HD Medical Clinic. Twenty subjects with HD and 10 control subjects, age-matched and lacking the HD mutation, were recruited (see Table 1). Gene-positive subjects were staged early or moderate according Independence Score as assessed by an experienced rater (100-80, early; 75-60, moderate).4 CSF was obtained by lumbar puncture, examined by microscopy, and centrifuged to remove cells, and the acellular portion was frozen at -80 °C. There was no significant contamination of CSF by blood cells (median erythrocyte count 0.5 × 106/L, range 0-171; median leukocyte count 1.0 × 106/L, range 0-17). Blood samples were obtained within an hour of lumbar puncture in EDTA tubes. Plasma was extracted by two-stage centrifugation and frozen at -80 °C. 2834

Journal of Proteome Research • Vol. 6, No. 7, 2007

2D Gel Electrophoresis. Two independent 2D gel electrophoresis (2DE) studies were performed using different depletion and identification techniques to maximize sensitivity. For the first study (minimal depletion), plasma samples from 10 HD patients and 10 controls (Table 1) were depleted of albumin and immunoglobulins using an antibody-based spin column (GE healthcare, U.K.). For the second study (stringent depletion), plasma samples from 55 HD patients and 55 matched controls (Table 1) were depleted of the 6 most abundant proteins using Multiple Affinity Removal System (MARS) HPLC columns (Agilent Technologies, CA). Depleted protein pellets were re-suspended in 2D gel lysis buffer composed of 9.5 M urea, 2% CHAPS (USB Corporation, OH), 1% dithiothreitol, 0.8% Pharmalyte (pH 3-10, GE Healthcare), and Mini Complete Protease Inhibitor Cocktail (Roche, U.K.). The protein concentration of each plasma sample was determined using the Bradford assay (Bio-Rad, U.K.). Plasma samples (75 and 100 µg in first and second study, respectively) were loaded onto immobilized pH 3-10 nonlinear gradient strips, then separated in the second dimension using SDS polyacrylamide gels as described.12,13 Resultant 2DE gels were silver-stained with OWL silver stain (Insight Biotechnologies, U.K.), and image analysis was performed using ProgenesisTM Workstation, (version 2003.02, Nonlinear Dynamics, U.K.). Images were processed using the automatic wizard for spot detection, warping, and matching, followed by manual editing and optimal matching to the reference gel (>80% per gel). Following background subtraction and normalization to total spot volume, spot data from the first study were analyzed using Student’s t tests at the 95% confidence level. Spot data from the second study were imported into StatistiXL software (www.statistixl.com), and the mean of normalized volume, coefficient of variation, fold change, and Mann-Whitney test were determined (pairwise comparisons between groups). A program developed internally was used to select statistically significant spots automatically based on the following criteria: spots present within at least 60% of gels, g1.5-fold change, and p < 0.005. Significant spots from the first study were excised, prepared, and analyzed by LC/MS/MS as described.14 Proteins present in the spots were determined using Mascot search algorithm against the NCBI nonredundant (www.ncbi.nlm.nih.gov) and Swiss-Prot (expasy.org) databases. Significant spots from the second study were excised, prepared, and analyzed using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry as described.15 Mass spectra were matched with the Ms-Fit program (prospector.ucsf.edu), and the Swiss-Prot database was used to identify proteins. Semiquantitative Immunoblotting. On the basis of the results of the first 2DE experiment, the availability of quality commercial ELISA kits, and the identification in the literature of plausible mechanistic roles in HD pathogenesis for the proteins, clusterin and β-actin were selected for further analysis. The concentrations of R and β-clusterin and β-actin were determined by semiquantitative immunoblotting in 60 plasma samples (Table 1). Control experiments using a range of plasma dilutions and primary antibody dilutions were performed to ensure that quantification was within the linear range. For Rand β-clusterin, undepleted plasma samples (2 µg) were used; for β-actin, plasma samples were depleted using MARS HPLC columns, and 20 µg were used. Plasma was denatured in Laemmli sample buffer and size-separated using 12% trisglycine gels (National Diagnostics, U.K.). Following SDS-PAGE,

research articles

Proteomic Profiling of Plasma in Huntington’s Disease

Table 2. Proteins Identified by 2DE and MALDI-TOF To Be Regulated in HD Patients and Controls spot no.

151 357 376

578 660 661

725 789

259 283

Figure 1. Proteins identified by 2DE and LC/MS/MS. (A) Representative 2D gel region depicting spots 1713 and 1960 that were significantly upregulated in 10 HD patients compared with 10 controls. (B) Proteins identified by LC/MS/MS. Spot 1713 contained 2 proteins, β-actin and ApoA-IV; spot 1960 contained the R and β chains of clusterin.

proteins were transferred to polyvinylidene difluoride membrane (GE Healthcare). Transfer efficiency and equal loading of protein samples were assessed by incubating membranes with Ponceau red solution (Sigma) and gels post-transfer with EZblue solution (Sigma). Representative post-transfer gels and membranes are supplied as Supporting Information (Figures S1 and S2). Membranes were washed with PBS-T and incubated with blocking buffer, then with anti-R-clusterin (1:10 000, 05-354, Upstate, U.K.), anti-β-clusterin (1:40 000, sc-6419, Santa Cruz Biotechnology, CA), or anti-β-actin (1:250, A5316, Sigma) antibodies, followed by horseradish peroxidase-conjugated sheep anti-mouse (R-clusterin and β-actin, GE Healthcare) or rabbit anti-goat secondary antibodies (β-clusterin, Jackson laboratories, ME). For protein band visualization and quantification, membranes were incubated with ECL-plus, scanned, and analyzed using ImageQuant software (GE Healthcare). Linear regression analysis with coded variables for each subject group (control ) 1, premanifest )2, early ) 3, moderate ) 4) was used to identify significant trends with advancing disease. Data were reanalyzed using age as a covariate to account for any effect of age on clusterin concentration. Comparisons between individual subject groups were made using ANOVA with post-hoc Tukey HSD analysis. Clusterin ELISA. Clusterin concentrations in paired CSF and plasma samples (n ) 29, Table 1) and in a larger set of plasma samples (n ) 73, Table 1) were measured using a commercially available ELISA kit (RD194034200, BioVendor, Modrice, Czech Republic; manufacturer’s protocol). Absorbances were determined at 450 nm. Clusterin concentrations were determined from standard curves. Statistical analysis was performed as described for immunoblotting experiments. Human IL-6 ELISA. Plasma IL-6 concentrations in 96 samples (Table 1) were determined using IL-6 ELISA kits (EK-

291 297 608 614 699 703 783

a

protein or proteins in spot

observationa

A. Proteins Increased with Progressing HD Clinical Stage Complement component C7 precursor C < P Alpha-2-macroglobulin precursor Alpha-2-antiplasmin E control (p ) 0.0001); moderate > premanifest (p ) 0.0073). For β-clusterin: moderate > control (p ) 0.0082). β-Actin expression did not track with disease progression (data not shown). Clusterin ELISA. Reliability of the clusterin ELISA assay was demonstrated by its low mean coefficient of variation (7.1%). ELISA experiments confirmed increased clusterin expression with HD stage in samples from both patient populations studied and in both plasma and CSF (Figure 3). Statistically significant increases across subject groups from control through progressing disease were found in the plasma from U.K. subjects (p ) 0.0032) and plasma and matched CSF from Canadian subjects (p ) 0.0036 and p ) 0.00009, respectively). These trends all remained significant when allowing for any effect of age and sex on clusterin concentration in the regression analysis (p ) 0.0146, p ) 0.0352, and p ) 0.00003, respectively); clusterin concentration correlated only very weakly with age (R2 ) 0.0429). There were also significant differences between individual groups (ANOVA with post-hoc Tukey HSD analysis). For U.K. plasma: moderate > control (p


Figure 3. Quantitative analysis of clusterin concentrations in HD plasma and CSF using ELISA. (A) Box-and-whisker plot showing clusterin concentrations in plasma samples from U.K. subjects. Mean concentrations ( SD: control (C) 121 ( 25.6; premanifest HD (P) 120 ( 29.2; early HD (E) 129 ( 26.7; moderate HD (M) 149 ( 32.2. (B) Box-and-whisker plot showing clusterin concentrations in plasma samples from Canadian subjects. Mean concentrations ( SD: C 192 ( 30.8; E 223 ( 30.0; M 248 ( 52.4. (C) Box-and-whisker plot showing clusterin concentrations in matched CSF samples from Canadian subjects. Mean concentrations ( SD: C 1.50 ( 0.7; E 2.02 ( 0.5; M 2.64 ( 0.5. The overall trend for increasing clusterin across all groups, using linear regression analysis, was statistically significant in all three studies (##p ) 0.0032 for U.K. plasma, ##p ) 0.0036 for Canadian plasma, and ####p ) 0.00009 for Canadian CSF). Significant differences between individual groups are shown (ANOVA with post-hoc Tukey HSD test: *p < 0.05; ***p < 0.001).

) 0.0251); moderate > premanifest (p ) 0.0147). For Canadian plasma: moderate > control (p ) 0.0147). For CSF: moderate > control (p ) 0.0003). The apparent lower plasma clusterin concentration overall in U.K. plasma compared with Canadian plasma is likely due

research articles

Figure 4. IL-6 ELISA quantification in HD patients and R6/2 mice. (A) Plasma IL-6 concentration (ng/L) with standard error bars in controls and HD patients. Mean concentrations ( SD: control (C) 1.45 ( 0.733; premanifest HD (P) 1.93 ( 1.12; early HD (E) 1.71 ( 1.01; moderate HD (M) 2.89 ( 1.98. The overall trend for increasing IL-6 across all groups, using linear regression analysis, was statistically significant (###p ) 0.0004). Significant differences between individual groups are shown (ANOVA with posthoc Tukey HSD test: *p < 0.05; ***p < 0.001). (B) Serum IL-6 concentration (ng/L) with standard error bars from wild-type (WT) and R6/2 mice. n ) 15 for both groups at 4 weeks, 15 WT and 13 R6/2 at 8 weeks, and 15 WT and 17 R6/2 at 12 weeks. Serum IL-6 was significantly elevated in R6/2 compared with controls at 12 weeks (**p ) 0.0013, t test).

to the different blood fractionation techniques employed in the two populations. IL-6 ELISA. ELISA quantification of IL-6 in human plasma demonstrated a statistically significant trend to increase across advancing disease groups (p ) 0.0004) as well as significantly increased IL-6 in moderate HD compared with both controls and premanifest HD (Figure 4A). This trend remained significant when allowing for any effect of age and sex on IL-6 concentration in the regression analysis (p ) 0.0023); IL-6 concentration correlated only very weakly with age (R2 ) 0.0700). There were also significant differences between individual groups (ANOVA with post-hoc Tukey HSD analysis): moderate > control (p ) 0.0004); moderate > premanifest (p ) 0.0227). ELISA data from R6/2 HD transgenic mice confirmed these findings, with mean serum IL-6 concentration significantly higher in R6/2 than wild-type at 12 weeks (p ) 0.0013) (Figure 4B). Journal of Proteome Research • Vol. 6, No. 7, 2007 2837

research articles Discussion Immune Activation in HD Identified by Proteomic Discovery. Several of the 18 proteins identified by our 2 proteomic discovery techniques in human plasma that track with disease progression are involved in regulation of the innate immune system, which is known to be activated in HD.3 Alpha-2-macroglobulin (A2M) is an acute-phase protein, whose release is stimulated by IL-6.18 C7 and C9 are components of membrane attack complex (MAC), whose formation is modulated by clusterin.19 Clusterin, which was also shown to track with disease progression by semiquantitative immunoblotting and ELISA in plasma and CSF, is found in almost all mammalian tissues and biofluids but is differentially expressed by certain cell types, and tissue-specific isoforms exist. Its expression is upregulated in a variety of physiological and pathological states including apoptosis and response to injury. It is implicated in diverse mechanisms of cytoprotection, membrane recycling, and regulation of membrane attack complex formation. A unified role has been proposed as a heatshock or chaperone protein with cytoprotective properties.20 Our IL-6 ELISA findings in human plasma and the R6/2 mouse confirm, and may demonstrate, a unifying trigger for, the presence of innate immune activation in HD. IL-6 is a proinflammatory cytokine that induces the release of acute-phase proteins including alpha-2-macroglobulin.18 The acute-phase response leads to the activation of the complement cascade via C3 and hence the production of downstream factors (such as C7 and C9) and modulating factors such as clusterin.18 This peripheral immune activation may have important consequences. Inflammatory factors have profound physiological effects that may explain features of the HD phenotype. Il6, for instance, is involved in the regulation of energy balance by decreasing food intake and increasing energy expenditure, possibly through its action on the hypothalamus.21 Il-6 also triggers corticosteroid release by acting on the hypothalamic/ pituitary/adrenal axis,22 which may explain the derangement of the hypothalamic-pituitary-adrenal axis observed in R6/2 mice and humans.2 Together, these effects may contribute to such phenotypic phenomena as the unexplained weight loss observed in HD.23 Neuroinflammation and HD Pathogenesis. Molecules produced by peripheral tissues as a result of disease processes may cross the blood-brain barrier and exert effects on the CNS. Neuroinflammation likely occurs as a result of locally and systemically produced factors. Complement activation and production of C7 have been demonstrated by microglia in the striatum of HD patients.24 The classical complement pathway is activated by amyloid-β (Aβ) components; amyloid and complement proteins (including the MAC) colocalize in Alzheimer’s disease (AD) brain,24 and MAC components introduced into the brain of live rats induce neurodegeneration.25 Apoptotic cells bind complement, activating the classical pathway, suggesting a possible mechanism of complement activation and bystander damage in HD.24 The importance of neuroinflammation in HD is underscored by human positron-emission tomography studies showing microglial activation in premanifest and early HD patients.26,27 Clusterin and A2M are intimately involved in complement regulation, and both have links to pathogenesis in neurodegeneration. Complement components are synthesized locally in AD brain, and brain-derived clusterin is an inhibitor of MAC formation.19 Clusterin and complement component mRNA are 2838


Dalrymple et al.

upregulated in HD brain and localize to sites of severe pathology including the caudate.28,29 In AD, clusterin colocalizes with Aβ in neuritic plaques, is found reversibly complexed with Aβ in CSF, and inhibits the aggregation of Aβ in vitro.19 Clusterin levels are elevated in CSF in AD.30 Clusterin has been shown to cross the blood-brain barrier,29 and our data confirm that its concentration in CSF rises in parallel with that in plasma in HD. A2M is upregulated in reactive astrocytes during brain injury.31 It binds strongly to Aβ, localizes to senile plaques in AD, is neuroprotective to cells exposed to Aβ toxicity, and mediates Aβ degradation and clearance by endocytosis.31,32 A link with HD pathogenesis is plausible: both huntingtin fragments in HD and β-amyloid aggregates in AD are ubiquitinated,33 and A2M-bound proteins are lysed intracellularly.34 A2M, along with complement proteins, was recently shown by proteomic analysis to be significantly elevated in plasma in Alzheimers disease.35 Among the non-inflammatory proteins identified, alpha-2antiplasmin is neuroprotective against excitotoxic neuronal death and has been suggested to have therapeutic potential in stroke.36 Afamin, which showed decreased production with advancing HD stage, has binding affinity for vitamin E and has also been shown to be neuroprotective against oxidative stress.37 Identification of Novel Biomarker Candidates. The proteins identified by our discovery experiments may have the ability to function individually or together as biomarkers of HD. An ideal HD biomarker would directly measure a disease process occurring in the brain, but there is a valuable role for peripheral biomarkers of neurodegenerative diseases, over and above the ready accessibility of plasma compared with brain tissue or CSF. First, some of the changes we see in plasma may be due to CNS processes causing direct leakage into plasma. We are investigating the possibility of identifying brain-specific isoforms in plasma to study this. Second, a single mechanism arising from the HD triplet repeat expansion may produce parallel changes in both central and peripheral tissues, causing plasma levels of a protein to mirror those in the CNS. Third, as discussed above, peripherally produced molecules may exert central effects on the brain. To function as desired, to reduce duration, cost, and sample size requirements for clinical trials of potential disease-modifying treatments, a candidate biomarker must be obtainable from easily accessible biofluid, must track linearly with disease progression, and should have mechanistic links to disease pathogenesis in order to respond predictably to disease modification.6 The proteins highlighted by our study appear to meet these basic requirements. Clusterin, in particular, which we have evaluated by two techniques in two independent populations in both plasma and CSF, has promise as a biomarker for HD. Our mouse IL-6 data also suggest a possible translational biomarker for testing therapeutic candidates across species, though the nonlinear pattern of change in both human and mouse likely reflects the multifactorial regulation of neuroinflammatory pathways. For large-scale validation and later clinical use, it is likely that a combination of several clinical, neuroimaging, and biochemical biomarkers will be necessary to track disease progression in HD.6 The potential markers we have identified warrant further investigation in large longitudinal cohort studies, as well as in concert with potential disease-modifying interventions.


Acknowledgment. This study was financially supported by the High Q Foundation, the Wellcome Trust (66270), the Segerfalk Foundation, and the U.K. Department of Health. B.R.L. is a Canadian Institutes of Health Research ClinicianScientist. S.J.T. is a U.K. Department of Health National Clinician Scientist. We thank members of the Euro-HD Biomarkers working group and the High Q Biomarkers Consortium for useful discussions; the staff of the HD Multidisciplinary clinics in London and Vancouver, and the patients and controls who donated samples; Chris Frost for statistical advice and input; all members of the Proteome Sciences research team; and Ray Young who prepared the figures. Supporting Information Available: Representative post-transfer membranes and post-transfer SDS-PAGE gels for R- and β-clusterin; and representative control 2D gel region depicting spots 1713 and 1960 that were significantly upregulated in 10 HD patients compared with 10 controls. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Huntington’s Disease, 3rd ed.; Bates, G., Harper P. S., Jones L., Eds.; Oxford University Press: Oxford, 2002. (2) Bjorkqvist, M.; Petersen, A.; Bacos, K.; Isaacs, J.; Norlen, P.; Gil, J.; Popovic, N.; Sundler, F.; Bates, G. P.; Tabrizi, S. J., et al. Progressive alterations in the hypothalamic-pituitary-adrenal axis in the R6/2 transgenic mouse model of Huntington’s disease. Hum. Mol. Genet. 2006, 15, 1713-1721. (3) Leblhuber, F.; Walli, J.; Jellinger, K.; Tilz, G. P.; Widner, B.; Laccone, F.; Fuchs, D. Activated immune system in patients with Huntington’s disease. Clin. Chem. Lab. Med. 1998, 36, 747-750. (4) The Huntington Study Group. Unified Huntington’s Disease Rating Scale: reliability and consistency. Mov. Disord. 1996, 11, 136-142. (5) Handley, O. J.; Naji, J. J.; Dunnett, S. B.; Rosser, A. E. Pharmaceutical, cellular and genetic therapies for Huntington’s disease. Clin. Sci. 2006, 110, 73-88. (6) Henley, S. M.; Bates, G. P.; Tabrizi, S. J. Biomarkers for neurodegenerative diseases. Curr. Opin. Neurol. 2005, 18, 698-705. (7) Hersch, S. M.; Gevorkian, S.; Marder, K.; Moskowitz, C.; Feigin, A.; Cox, M.; Como, P.; Zimmerman, C.; Lin, M.; Zhang, L., et al. Creatine in Huntington disease is safe, tolerable, bioavailable in brain and reduces serum 8OH2′dG. Neurology 2006, 66, 250252. (8) Borovecki, F.; Lovrecic, L.; Zhou, J.; Jeong, H.; Then, F.; Rosas, H. D.; Hersch, S. M.; Hogarth, P.; Bouzou, B.; Jensen, R. V., et al. Genome-wide expression profiling of human blood reveals biomarkers for Huntington’s disease. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11023-11028. (9) Zabel, C.; Chamrad, D. C.; Priller, J.; Woodman, B.; Meyer, H. E.; Bates, G. P.; Klose, J. Alterations in the Mouse and Human Proteome Caused by Huntington’s Disease. Mol. Cell. Proteomics 2002, 1, 366-375. (10) Shoulson, I. Huntington disease: functional capacities in patients treated with neuroleptic and antidepressant drugs. Neurology 1981, 31, 1333-1335. (11) Boyum, A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. Suppl. 1968, 97, 77-89. (12) Weekes, J.; Wheeler, C. H.; Yan, J. X.; Weil, J.; Eschenhagen, T.; Scholtysik, G.; Dunn, M. J. Bovine dilated cardiomyopathy: proteomic analysis of an animal model of human dilated cardiomyopathy. Electrophoresis 1999, 20, 898-906. (13) Heinke, M. Y.; Wheeler, C. H.; Yan, J. X.; Amin, V.; Chang, D.; Einstein, R.; Dunn, M. J.; dos Remedios, C. G. Changes in myocardial protein expression in pacing-induced canine heart failure. Electrophoresis 1999, 20, 2086-2093. (14) Standen, C. L.; Perkinton, M. S.; Byers, H. L.; Kesavapany, S.; Lau, K. F.; Ward, M.; McLoughlin, D.; Miller, C. C. The neuronal adaptor protein Fe65 is phosphorylated by mitogen-activated protein kinase (ERK1/2). Mol. Cell. Neurosci. 2003, 24, 851-857.

research articles (15) Joubert, R.; Strub, J. M.; Zugmeyer, S.; Kobi, D.; Carte, N.; Van Dorsselaer, A.; Boucherie, H.; Jaquet-Guffreund, L. Identification by mass spectrometry of two-dimensional gel electrophoresisseparated proteins extracted from lager brewing yeast. Electrophoresis 2001, 22, 2969-2982. (16) Woodman, B.; Butler, R.; Landles, C.; Lupton, M.; Tse, J.; Hockly, E.; Moffitt, H.; Sathasivam K.; Bates G. The HdhQ150/Q150 knockin mouse model of HD and the R6/2 exon 1 model develop comparable and widespread molecular phenotypes. Brain Res. Bull. 2007, 72, 83-97. (17) Hockly, E.; Woodman, B.; Mahal, A.; Lewis, C. M.; Bates, G. Standardization and statistical approaches to therapeutic trials in the R6/2 mouse. Brain Res. Bull. 2003, 61, 469-479. (18) Han, M. Cytokines and the hepatic acute phase response. J. Pathol. 1997, 181, 257-266. (19) Choi-Miura, N.-H.; Oda, T. Relationship between multifunctional protein clusterin and Alzheimer disease. Neurobiol. Aging 1996, 17, 717-722. (20) Jones, S. E.; Jomary, C. Clusterin. Int. J. Biochem. Cell Biol. 2002, 34, 427-431. (21) Cancello, R.; Tounian, A.; Poitou, C.; Clement, K. Adiposity signals, genetic and body weight regulation in humans. Diabetes Metab. 2004, 30, 215-227. (22) Perlstein, R. S.; Mougey, E. H.; Jackson, W. E.; Neta, R. Interleukin-1 and interleukin-6 act synergistically to stimulate the release of adrenocorticotropic hormone in vivo. Lymphokine Cytokine Res. 1991, 10, 141-146. (23) Sanberg, P. R.; Fibiger, H. C.; Mark, R. F. Body weight and dietary factors in Huntington’s disease patients compared with matched controls. Med. J. Aust. 1981, 1, 407-409. (24) Gasque, P.; Dean, Y. D.; McGreal, E. P.; VanBeek, J.; Morgan, B. P. Complement components of the innate immune system in health and disease in the CNS. Immunopharmacology 2000, 49, 171-186. (25) Xiong, Z.-Q.; Qian, W.; Suzuki, K.; McNamara, J. O. Formation of complement membrane attack complex in mammalian cerebral cortex evokes seizures and neurodegeneration. J. Neurosci. 2003, 23, 955-960. (26) Pavese, N.; Gerhard, A.; Tai, Y. F.; Ho, A. K.; Turkheimer, F.; Barker, R. A.; Brooks, D. J.; Piccini, P. Microglial activation correlates with severity in Huntington disease: a clinical and PET study. Neurology 2006, 66, 1638-1643. (27) Tai, Y.; Pavese, N.; Gerhard, A.; Tabrizi, S.; Barker, R.; Brooks, D.; Piccini, P. Microglial activation in presymptomatic Huntington’s disease gene carriers: a combined C-11-PK11195 and C-11raclopride PET study. Neurology 2006, 66, A134. (28) Hodges, A.; Strand, A. D.; Aragaki, A. K.; Kuhn, A.; Sengstag, T.; Hughes, G.; Elliston, L. A.; Hartog, C.; Goldstein, D. R.; Thu, D., et al. Regional and cellular gene expression changes in human Huntington’s disease brain. Hum. Mol. Genet. 2006, 15, 965977. (29) Duguid, J. R.; Bohmont, C. W.; Liu, N.; Tourtellotte, W. W. Changes in Brain Gene Expression Shared by Scrapie and Alzheimer Disease. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 72607264. (30) Nilselid, A. M.; Davidsson, P.; Nagga, K.; Andreasen, N.; Fredman, P.; Blennow, K. Clusterin in cerebrospinal fluid: Analysis of carbohydrates and quantification of native and glycosylated forms. Neurochem. Int. 2006, 48, 718-728. (31) Du, Y.; Bales, K. R.; Dodel, R. C.; Liu, X.; Glinn, M. A.; Horn, J. W.; Little, S. P.; Paul, S. M. Alpha 2-macroglobulin attenuates beta-amyloid peptide 1-40 fibril formation and associated neurotoxicity of cultured fetal rat cortical neurons. J. Neurochem. 1998, 70, 1182-1188. (32) Narita, M.; Holtzman, D. M.; Schwartz, A. L.; Bu, G. Alpha 2-macroglobulin complexes with and mediates the endocytosis of beta-amyloid peptide via cell surface low-density lipoprotein receptor-related protein. J. Neurochem. 1997, 69, 1904-1911. (33) Gutekunst, C.-A.; Li, S.-H.; Yi, H.; Mulroy, J. S.; Kuemmerle, S.; Jones, R.; Rye, D.; Ferrante, R. J.; Hersch, S. M.; Li, X.-J. Nuclear and Neuropil aggregates in Huntington’s disease: relationship to neuropathology. J. Neurosci. 1999, 19, 2522-2534. (34) Borth, W. Alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics. FASEB J. 1992, 6, 33453353. (35) Hye, A.; Lynham, S.; Thambisetty, M.; Causevic, M.; Campbell, J.; Byers, H. L.; Hooper, C.; Rijsdijk, F.; Tabrizi, S. J.; Banner, S.,

Journal of Proteome Research • Vol. 6, No. 7, 2007 2839

research articles et al. Proteome-based plasma biomarkers for Alzheimer’s disease. Brain 2006, 129, 3042-3050. (36) Campbell, S. J.; Finlay, M.; Clements, J. M.; Wells, G.; Miller, K. M.; Perry, V. H.; Anthony, D. C. Reduction of excitotoxicity and associated leukocyte recruitment by a broad-spectrum matrix metalloproteinase inhibitor. J. Neurochem. 2004, 89, 1378-1386.

2840


Dalrymple et al. (37) Heiser, M.; Hutter-Paier, B.; Jerkovic, L.; Pfragner, R.; Windisch, M.; Becker-Andre, M.; Dieplinger, H. Vitamin E binding protein afamin protects neuronal cells in vitro. J. Neural Transm. Suppl. 2002, 337-345.

PR0700753

Proteomic Profiling of Plasma in Huntington's Disease Reveals

Recommend Documents