Laurie Broadwater

Ashish Pandit

Sausan Azzam

Robert Clements

Jonathan Vadnal

Michael Sulak

V Wee

Ernest J Freeman

Roger B Gregory

Ph.D Jennifer Mcdonough
email: jmcdonou@kent.edu

Analysis of the mitochondrial proteome in multiple sclerosis cortex

Keywords: Mitochondrial proteomics, multiple sclerosis, experimental autoimmune encephalomyelitis Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS), principal component analysis (PCA), normal appearing gray matter (NAGM), Multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), myelin

Mitochondrial dysfunction has been proposed to play a role in the neuropathology of multiple sclerosis (MS). Previously, we reported significant alterations in the transcription of nuclear-encoded electron transport chain genes in MS and confirmed translational alterations for components of Complexes I and III that resulted in reductions in their activity. To more thoroughly and efficiently elucidate potential alterations in the expression of mitochondrial and related proteins, we have characterized the mitochondrial proteome in postmortem MS and control cortex using Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). Using principal component analysis (PCA) and hierarchical clustering techniques we were able to analyze the differential patterns of SELDI-TOF spectra to reveal clusters of peaks which distinguished MS from control samples. Four proteins in particular were responsible for distinguishing disease from control. Peptide fingerprint mapping unambiguously identified these differentially expressed proteins. Three proteins identified are involved in respiration including cytochrome c oxidase subunit 5b (COX5b), the brain specific isozyme of creatine kinase, and hemoglobin β-chain. The fourth protein identified was myelin basic protein (MBP). We then investigated whether these alterations were consistent in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We found that MBP was similarly altered in EAE but the respiratory proteins were not. These data indicate that while the EAE mouse model may mimic aspects of

Introduction

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system resulting in progressive physical and cognitive disability. In the MS brain, white matter lesions are a hallmark of the disease; however, cortical gray matter lesion load has been found to be extensive [START_REF] Bö | Grey Matter Pathology in Multiple Sclerosis[END_REF] and damage to neurons and axons correlates with progression and cognitive impairment [START_REF] Bjartmar | Neurological Disability Correlates with Spinal Cord Axonal Loss and Reduced N-Acetyl Aspartate in Chronic Multiple Sclerosis Patients[END_REF][START_REF] Mathiesen | Multi-slice Echo-planar Spectroscopic MR Imaging Provides Both Global and Local Metabolite Measures in Multiple Sclerosis[END_REF][START_REF] Rudick | Gray Matter Atrophy Correlates with MS Disability Progression Measured with MSFC but not EDSS[END_REF]. Several studies have implicated mitochondrial dysfunction as a possible mechanism in the development of MS related neuropathology. Nuclear magnetic resonance spectroscopy has identified decreases in the neuronal mitochondrial metabolite N-acetyl aspartate (NAA) in MS brain and decreases in NAA appears to precede neuronal atrophy, indicating that dysfunctional mitochondria may precede neurodegeneration [START_REF] Inglese | Indirect Evidence For Early Widespread Gray Matter Involvement in Relapsing-Remitting Multiple Sclerosis[END_REF][START_REF] Cader | Discordant White Matter N-Acetylaspartate and Diffusion MRI Measures Suggest that Chronic Dysfunction Contributes to Axonal Pathology in Multiple Sclerosis[END_REF]. Mitochondrial dysfunction has also been implicated in disease pathology in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS [7]. It has been reported that increased nitration of electron transport chain components leads to decreased mitochondrial activity in EAE [START_REF] Qi | Mitochondrial Protein Nitration Primes Neurodegeneration in Experimental Autoimmune Encephalomyelitis[END_REF] and that cyclophilin D, a modulator of the mitochondrial permeability transition pore, is protective [START_REF] Forte | Cyclophilin D inactivation protects axons in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis[END_REF].

Similarly, analyses of postmortem MS white matter have identified alterations to mitochondrial enzyme

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activity and damage to mitochondrial DNA in lesions [START_REF] Mahad | Mitochondrial Defects in Acute Multiple Sclerosis Lesions[END_REF][START_REF] Kalman | The Involvement of Mitochondria in the Pathogenesis of Multiple Sclerosis[END_REF][START_REF] Witte | Enhanced Number and Activity of Mitochondria in Multiple Sclerosis Lesions[END_REF]. Other studies have demonstrated defects in mitochondrial electron transport gene expression and function in normal appearing gray matter (NAGM) in postmortem MS cortex [START_REF] Pandit | Impaired Regulation of Electron Transport Chain Subunit Genes by Nuclear Respiratory Factor 2 in Multiple Sclerosis[END_REF][START_REF] Dutta | Mitochondrial Dysfunction as a Cause of Axonal Degeneration in Multiple Sclerosis Patients[END_REF]. These studies have identified transcriptional changes in important mitochondrial genes; however, translational or post-translational alterations in a multitude of other proteins may also impact mitochondrial function and energy production. Therefore, in the current report, we have applied Surface Enhanced Laser Desorption and Ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to mitochondrial fractions derived from MS cortex to obtain a more complete understanding of protein expression changes that could contribute to the mechanisms of mitochondrial dysfunction in MS. Studies of proteins originating from MS brain tissue may provide new insights into the pathogenesis and etiology of the disease. Previous proteomic analyses of MS tissue have focused on white matter, lesioned tissue, and cerebrospinal fluid (CSF) [START_REF] Avasarala | A Distinctive Molecular Signature of Multiple Sclerosis Derived from MALDI-TOF/MS and Serum Proteomic Pattern Analysis[END_REF][START_REF] Westman-Brinkmalm | Proteomics/peptodomics Tools to Find CSF Biomarkers for Neurodegenerative Diseases[END_REF][START_REF] Quintana | Systems Biology Approaches for the Study of Multiple Sclerosis[END_REF][START_REF] Zetterberg | Clinical Proteomics in Neurdegenerative Disorders[END_REF][START_REF] Han | Proteomic Analysis of Active Multiple Sclerosis Lesions Reveal Therapeutic Targets[END_REF][START_REF] Selmaj | Corpora Amylacea from Multiple Sclerosis Brain Tissue Consists of Aggregated Neuronal Cells[END_REF][START_REF] Liu | Quantative Proteomic Analysis of Cerebrospinal Fluid of Patients with Multiple Sclerosis[END_REF][START_REF] Chiasserini | CSF Proteome Analysis in Multiple Sclerosis Patients by Two-Dimensional Electrophoresis[END_REF][START_REF] D'aguanno | Differential Cerebro Spinal Fluid Proteome Investigtion of Leber Hereditry Optic Neuropathy (LHON) and Multiple Sclerosis[END_REF]. However, there is evidence from magnetic resonance imaging studies that the mechanisms involved in tissue damage are different in gray matter and white matter in MS [START_REF] Filippi | Magnetic resonance techniques to quantify tissue damage, tissue repair, and functional cortical reorganization in multiple sclerosis[END_REF][START_REF] Fisher | Gray matter atrophy in multiple sclerosis: a longitudinal study[END_REF]. Therefore studies detailing protein differential expression in normal appearing gray matter are necessary to provide additional insight into disease manifestation and progression as it relates to the neurodegenerative component of MS pathology.

Materials and Methods

Tissue Preparation and Protein Extraction:

Reagents used in the preparation of all buffers were obtained from Sigma-Aldrich (St. Louis, MO).

Brains were obtained from The Rocky Mountain MS Center Tissue Bank (Englewood, CO), the Brain and Spinal Fluid Resource Center (UCLA) and from The Kathleen Price Bryan Brain Bank (Durham, NC) under IRB protocol. Tissue was obtained by rapid autopsy and frozen at -70°C. This study includes two cohorts, each consisting of four control and four secondary progressive multiple sclerosis (SPMS) donors.

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Tissue from each cohort was homogenized on separate dates using identical protocols. MS and control brains have been matched primarily for brain region (parietal, Brodmann Area 1-3, and frontal cortex, Brodmann Area 9), and also for age, sex, and post mortem interval (PMI) as closely as possible. Frozen sections, 30 µm thick, from adjacent sides of the tissue blocks were immunostained for myelin proteolipid protein (PLP) to ensure the absence of demyelinated lesions as previously described [START_REF] Trapp | Axonal transection in the lesions of multiple sclerosis[END_REF]. Donor demographics are described in Table 1. Eight control and eight MS donors were used for the differential expression proteomic analyses while one additional control (C9) and MS donor (MS9) were used in western blotting for confirmation.

EAE was induced in C57Bl/6 mice by subcutaneous injection of MOG 35-55 as previously described [START_REF] Dasilva | Expression and regulation of matrix metalloproteinase-12 in experimental autoimmune encephalomyelitis and by bone marrow derived macrophages in vitro[END_REF]. EAE brains and brains from control littermates were prepared at The University of Calgary and sent frozen to Kent State University for analysis by Western blotting and SELDI-TOF-MS. Protein was isolated from whole brains and from cortex for Western analyses. EAE-afflicted mice were at peak clinical severity (Grade 4 of the 5 point scale) when sacrificed.

For postmortem human tissue, approximately 250mg tissue from frozen blocks was carefully excised excluding white matter. Tissue was homogenized using a Wheaton homogenizer with a Teflon ® pestle in whole cell homogenization buffer (20mM KCl, 3mM MgCl 2, 10mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) pH 7.9, 0.5% NP-40, 5% glycerol with protease inhibitors (P2714, Sigma-Aldrich, St. Louis, MO) in forty strokes. The homogenate was centrifuged for 10 minutes at 500xg at 4°C. The supernatant was removed and centrifuged at 10,000g for 30 minutes at 4°C. The pellet containing the mitochondrially enriched fractions was further purified by washing twice in 20mM phosphate buffered saline (PBS), pH 7.4. The mitochondrial pellet was lysed in mitochondrial lysis buffer (50mM Tris, 7M urea, 3% CHAPS with protease inhibitors) by vortexing for one minute and then incubating for 20 minutes at room temperature. The mitochondrial lysate was centrifuged for 10 minutes at 10,000g at 4°C. A modified Lowry assay was used to quantify the supernatant protein concentration. All samples were stored at -80°C until further analysis.

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Immunofluorescent Staining:

Frozen tissue blocks from postmortem MS and control brains were fixed in 4% paraformaldehyde for 24 hours and washed in PBS (2 x 24 hours). Fixed tissue blocks were sliced 75 µm thick perpendicular to the outer edge of the cortex (to include all cell layers) on a media cooled vibratome. Sections were stored in PBS in multiwell plates at 4°C in a humidified chamber until subjected to the staining procedure.

Thereafter, free-floating sections were incubated in goat anti-hemoglobin All images were acquired with isometric voxels at a resolution of 0.42 microns per pixel. The image stacks were z-projected using ImageJ (NIH, Bethesda, MD) and channels merged to show co-localized signal within pyramidal cells.

Western Blotting:

Mitochondrially enriched or cytoplasmic samples were separated on NuPage ® 4-12%

Ion Exchange Fractionation:

The mitochondrial enriched protein extracts were further fractionated using ion exchange chromatography in a spin column format (column-UFC30HV00; centrifuge tube-UFC3000TB Millipore, Billerica, MA). Samples (100µg each) were equilibrated using buffer A (9M Urea, 2% CHAPS in 50mM

Tris HCl pH 9.0) and incubated for twenty minutes at room temperature. Buffer B (Buffer A diluted 1 part to 8 parts 50mM Tris HCl, pH 9.0) was used to dilute all samples to 200µL and then stored on ice while the remaining samples were prepared (30 minutes maximum). The quaternary ammonium anion exchange beads (Q ceramic HyperD F, Pall Biosepra, NewYork, NY) were equilibrated three times in buffer B. The samples were loaded onto the column/tube and mixed in an end to end fashion for 30 minutes at room temperature. After this initial incubation, the samples were centrifuged for 1 minute at 1000xg. This eluate was the flow through fraction. The column was removed from the centrifuge tube and placed in the next centrifuge tube labeled according to the wash buffer. The pH 9 elution buffer (50mM Tris HCl, pH 9.0, 0.1% octyl glucopyranoside (OGP), 200µL) was added to the column and mixed in an end to end fashion

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for 10 minutes, then centrifuged as before. This process was repeated for each buffer at pH 7.0, 5.0, 4.0, and 3.0, and a final organic solvent elution, yielding six fractions with pI ranges of greater than 9.0 in fraction 1, 7.0-9.0 in fraction 2, 5.0-7.0 in fraction 3, 4.0-5.0 in fraction 4, 3.0-4.0 in fraction 5, and less than 3.0 in fraction 6, respectively. Specific buffer compositions were as follows: pH 7.0 elution buffer; 50mM HEPES pH 7.0, 0.1% OGP, pH 5.0 elution buffer; 100mM sodium acetate pH 5.0, 0.1% OGP, pH 4.0 elution buffer; 100mM sodium acetate pH 4.0, 0.1% OGP, pH 3.0 elution buffer; 50mM sodium acetate pH 3.0, 0.1% OGP; organic elution buffer; 33.3% isopropyl alcohol (IPA), 16.7% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA). All protein solutions were stored on dry ice until return to the -80°C freezer.

SELDI-TOF-MS Acquisition:

All samples were mixed with a saturated α-cyano-4-hydroxycinnamic acid matrix solution consisting of 50% ACN and 0.5% TFA by volume with 0.6% OGP by weight in a 1:5 dilution. Each matrix diluted sample (1µL) was applied to a spot on an NP20 Proteinchip® (normal phase chromatographic surface, Ciphergen Biosystems, Fremont, CA) chip in a randomized manner. Mass spectra were acquired with a model PBSIIc SELDI-TOF mass spectrometer manufactured by Ciphergen Biosystems (Fremont, CA). The optimal sensitivity and laser intensity were established on a location not used in transient averaging. These values were then used to develop a spot protocol. This same protocol was applied to all chips. Data were acquired at a digitizer rate of 250.0MHz in positive ion mode with a chamber vacuum of less than 5x10 -07 torr. The source voltage was 20kV and the detector voltage was 2,700V. A total of 65 transients were averaged for each spectrum. All spectral processing (smoothing and baseline subtraction)

was performed with Proteinchip 3.1 Software (Ciphergen Biosystems, Fremont, CA). Spectra were calibrated externally over the appropriate mass range using at least three of the following standards:

recombinant Hirudin BHVK (6,964Da), bovine cytochrome c (12,230 Da) equine cardiac myoglobin (16,952 Da), bovine RBC carbonic anhydrase (29,024 Da) and S. cerevisae enolase [START_REF] Biagioli | Unexpected Expression of α-and β-globin in Mesencephalic Dopaminergic Neurons and Glial Cells[END_REF]670 Da). In order to make appropriate comparisons, all spectra were normalized against the total ion current [START_REF] Avasarala | A Distinctive Molecular Signature of Multiple Sclerosis Derived from MALDI-TOF/MS and Serum Proteomic Pattern Analysis[END_REF].

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Data Analysis:

Manual peak picking, using a signal to noise ratio of greater than 4.0, allowed for unbiased peak selection. Automatic peak detection in Ciphergen Express often neglects those features which appear as shoulders on larger peaks [START_REF] Carlson | Improving feature detection and analysis of surface-enhanced laser desorption/ionization-time of flight mass spectra[END_REF] and consequently manual peak selection across all spectra of the same fraction provided a more accurate assessment of the spectral features. Criterion for the selection of peaks across the entire data set required peak occurrence in 25% of the spectra with a signal to noise ratio greater than 4.0. Peak occurrence percentages were selected based upon the expected absence of some peaks from either the control or the MS group. Peaks selected for further evaluation and validation had a signal to noise ratio of greater than 3.0 and were present in the overexpressed sample group (control or MS). All statistical calculations were done with Ciphergen® Express software (Ciphergen Biosystems, Fremont, CA).

Univariate analysis included the non-parametric Mann-Whitney test. Applicable multivariate techniques included principal component analysis (PCA) and hierarchical clustering [START_REF] Eisen | Cluster Analysis and Display of Genomewide Expression Patterns[END_REF]. Peaks considered differentially expressed were those altered by at least 1.8 fold, p< 0.05.

Immunoprecipitation:

Anti-nitrotyrosine antibody (05-233 Millipore, Billerica, MA) was crosslinked to Protein G agarose beads using the Pierce Seize X kit according to manufacturers' instructions. Briefly, Protein A agarose beads were used to preclear mitochondrially enriched proteins. Protein G beads were covalently crosslinked to the anti-nitrotyrosine antibody using disuccinimidyl suberate and washed to remove any unreacted molecules. The beads were then incubated with mitochondrially enriched NAGM protein and any unbound proteins were removed by washing. The nitrated proteins were then eluted from the beads using a low pH wash. The eluant was then separated using pre-cast Bis-Tris gels (Invitrogen, Carlsbad, CA), stained with blue silver [START_REF] Candiano | Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis[END_REF] and destained in reverse osmosis water. Bands of interest (those increased in MS) were excised, dehydrated and sent to the University of Pittsburgh Genomics and Proteomics Core Facility for trypsin digestion and peptide fingerprint mapping.

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Antibody Pull Down Confirmation:

An antibody against COX5b (Proteintech Group, Chicago, IL, PN 11418-2-AP) were derivatized to 3µm diameter carboxylated polystyrene microspheres (Polysciences PN 09850) using a modified carbodiimide method [START_REF] Hermanson | Bioconjugate Techniques[END_REF]. Briefly, the beads were washed in 50mM MES These solutions were randomly spotted onto an NP-20 Proteinchip® at 0.5µL, air-dried and repeated.

Protein Purification and Peptide Fingerprint Mapping:

Individual ion exchange fractions were purified by gel electrophoresis on 16% acrylamide Tris glycine gels at 200V for 45 minutes [START_REF] Laemmli | Cleavage of Structural Proteins During Assembly of Head of Bacteriophage-T4[END_REF]. The gels were stained with coomassie blue [START_REF] Echan | Protein Detection in Gels Using Fixation[END_REF] and gel plugs of interest were removed. The gel plugs were destained [START_REF] Jiménez | In-Gel Digestion of Proteins for MALDI-MS Fingerprint Mapping[END_REF], dehydrated and stored in 1% acetic acid prior to trypsin digestion and peptide fingerprint mapping. Trypsin digestion followed by HPLC-ESI-MS/MS on an

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LCQ Deca XP mass spectrometer (ThermoFinnigan, San Jose, CA) was performed at the Genomics and Proteomics Core Laboratories at the University of Pittsburgh.

Results

Mitochondrial Protein Differential Expression in Postmortem MS Cortex:

Donor demographics are described in Table 1. Mitochondrial samples from either parietal or frontal cortex were obtained from NAGM as confirmed using PLP staining. Analysis of the mitochondrial fractions was divided into two separate groups or cohorts due to tissue sample availability at study onset. The first cohort of samples consisted of mitochondrial fractions obtained from four control and four MS brain slices (C1-4 and MS1-4). As additional brain tissue became available the analysis was extended to incorporate the second group, cohort 2, which also consisted of mitochondrial fractions derived from four control and four MS brain slices (C5-8 and MS5-8). The mitochondrial fractions were separated by ion-exchange fractionation and analyzed by SELDI-TOF-MS. Mass spectra were acquired from fractions with pI values between 9 and 3 and revealed multiple peaks. The Mann-Whitney test was used to identify peaks with significant differences in peak intensities. Filtering the data for proteins altered by at least 1.8 fold at the 0.05 significance level we identified nineteen peaks which were differentially expressed (see Supplemental Data, Table1). Cohort 1 contained twelve differentially expressed peaks, with five of these peaks from fraction 3 (pI 5.0-7.0) and five from fraction 6 (pI<3), and cohort 2 contained seven differentially expressed peaks, three of which originated in fraction 6 (pI <3).

These fractions were selected for peptide fingerprint mapping and further investigation. Fractions 4 (pI 4.0 -5.0) and 6 from cohort 2 were also selected for further purification and peptide fingerprint mapping.

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Analysis of Proteomic Data with Multivariate Statistics:

The application of hierarchical clustering to differential protein expression data identifies elements which may be linked in their occurrence. This unsupervised classification method can be used to identify and validate those biomarkers responsible for specific patterns and plays an essential role in current disease investigations. Since it is unlikely that one specific peak is responsible for the differential Principal component analysis (PCA) is a common technique used in the analysis of large data sets and uses computational methods for pattern recognition [35]. Mathematically, in this particular analysis, a PC is a linear combination of peaks from all samples included in the analysis. The contribution of a specific PC to a sample's variability is indicated by the sample score. Each sample has a single unique score from each PC. The first PC accounts for the greatest variability in the data set with each subsequent PC accounting for decreasing amounts of variability. Therefore, the first several PCs will reveal which variable contributes the most to the differences between the populations and the sample specific scores will relate that variability to each sample.

Applying PCA to this research enables the identification of key proteins which may be responsible for the differences between control and disease states. We used this particular analysis to generate two dimensional scatter plots of the principal component (PC) scores to aid in the visualization of the data reduction process. Variables with similar scores for a specific PC will segregate together in a scatter plot, allowing the identification of similar variables. Because PCs are uncorrelated by definition [START_REF] Jolliffe | Principal Component Analysis[END_REF], this process can be used to verify those factors which independently influence the data segregation.

Identification and Confirmation of Differentially Expressed Proteins in MS Cortex:

The unambiguous identification of specific differentially expressed peaks in cohort 1 by peptide fingerprint mapping was complicated by the low resolution of one dimensional gel electrophoresis and

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the complex mixture of analytes in the 10 kDa and 16 kDa regions. Each band contained at least two protein species as evidenced by the SELDI-TOF mass spectra. Regardless, the entire band of each sample was excised and submitted for peptide fingerprint mapping on a ThermoFinnigan LCQ Deca XP. Confirmation of the identification of the peak at 10.6 kDa as COX5b was obtained by employing COX5b antibody labeled beads. SELDI-TOF-MS spectra acquired both before and after protein solutions were exposed to this COX5b antibody confirmed that the differentially expressed peak at 10.6 kDa was

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COX5b as shown in Figure 3A. Relative peak intensity ratios were used to determine which peak resulted from COX5b. The 10.1 kDa peak was verified as unchanged before and after spectra by one way ANOVA and was therefore used as the normalization peak to determine which peak intensity changed as a result of exposure to the COX5b IgG beads. Representative spectra from one of three trials are shown in Figure 3A. Only the peak at 10.6kDa demonstrated a change in relative peak height. This molecular weight corresponds to the mass of COX5b after signal peptide cleavage.

We then performed western blotting experiments to confirm the protein expression differences identified by SELDI-MS as shown in Figure 3. In order to demonstrate the purity of our mitochondrial fractionation procedure, we performed western blots for expression of a neuronal marker, neurofilament, and the mitochondrial encoded protein cytochrome c oxidase subunit II (COX2), in MS and control cytoplasmic and mitochondrial fractions (Figure 3B). The altered expression of proteins identified in cohort 1 and in cohort 2, including MBP, COX5b, creatine kinase B (CKB) and the hemoglobin-β chain (Hbb), were confirmed by western blotting in mitochondrial fractions (Figure 3 C and D). While these proteins were not found to be significantly altered in both cohorts by SELDI-TOF-MS, quantitation of western blotting experiments confirmed a significant decrease in COX5b and significant increases in MBP, hemoglobin β, and creatine kinase B expression in mitochondrial fractions from samples across both cohort 1 and 2 and in additional samples (C9 and MS9) (Figure 3E). One control sample not included in the SELDI analysis but which was analyzed by western blot, C9, contained consistently increased mitochondrial content as indicated by the increase in COX2 expression (Figure 3C). MS samples still expressed increased levels of creatine kinase B relative to this control sample in spite of this increase in mitochondria. Further, COX5b expression was significantly decreased in MS samples even when C9 was not included in the analysis. COX5b and MBP were only expressed in mitochondrial fractions. Hemoglobin and creatine kinase B were detected in both mitochondrial and cytoplasmic fractions, but were not significantly increased in cytoplasmic fractions. Western blots were also performed for these proteins in cytoplasmic and mitochondrial fractions isolated from either whole brain

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or cortex from EAE and control mice. Representative western blots are shown in Figure 3C and D and quantitation in Figure 3E shows that only increased MBP expression is modeled in the EAE mouse. We did observe more than one isoform of MBP increased (two bands ~ 18-20 kDa) in the EAE mitochondrial fractions rather than the one isoform increased in human MS tissue at ~ 18 kDa (Figure 3C).

Further, we localized hemoglobin expression to neuronal cell bodies in MS postmortem tissue by immunofluorescent staining with antibodies to hemoglobin (red fluorescence) and neurofilament (green fluorescence) as shown in Figure 4. We observed intense hemoglobin staining in pyramidal neurons in MS cortex supporting our SELDI-TOF data indicating increased expression of hemoglobin in MS samples.

Increased Nitrotyrosine Modified Proteins in MS:

The detection of increased nitrated proteins in MS mitochondrial fractions was performed by immunoprecipitation with an antibody to nitrotyrosine. Eluates from the nitrotyrosine immunoprecipitation of control and MS samples were purified using one dimensional gel electrophoresis and differentially stained bands which were increased in intensity in the MS mitochondrial fraction between 20-25kDa and 40-45kDa were excised. Nitrated proteins identified using peptide fingerprint mapping are shown in Table 3. the protein sequence coverage was calculated at 39.1% and 45.0%, respectively (see Table 3).

Discussion

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In this study, we examined peaks in 96 SELDI-TOF mass spectra (six fractions from sixteen brain samples). Each mass spectrum was represented by over 65,000 data points. To decrease the complexity associated with such a large data set, we reduced the number of variables using principal component analysis and generated PC score scatter plots to more easily visualize the segregation of samples derived from MS cortex compared to those obtained from controls. In our first group (cohort 1), using PCA, we analyzed mitochondrial proteins eluted in fraction 3 and determined that the first two PCs accounted for 80% of the variability in the data set and allowed for segregation of samples obtained from MS or control cortex into two distinct groups with respect to the presence or absence of disease. The ability to separate disease from control suggests that the presence and/or absence of specific groups of peaks correlates with disease expression and that these peaks may be important in elucidating biological processes that are responsible for disease etiology or progression. In addition, we found that of the proteins separated out from fraction 3 of our first cohort, the peaks in the 10 kDa and 16 kDa range contributed the greatest amount to the separation of MS from control donors. The lack of 10 kDa peaks and excess of the 16kDa peak is most prominent in MS donors. Therefore the proteins within these peaks were selected for identification by peptide fingerprint mapping.

The peak at 16kDa was identified by peptide fingerprint mapping as MBP, a component of myelin. This was an unexpected result and one that was consistent in mitochondrial fractions from both controls and MS donors. MBP is a very basic protein which is normally associated with membranes and it is possible that MBP released from myelin during tissue homogenization was bound adventitiously to mitochondrial membranes and remained in the mitochondrial fraction even after the washing steps.

However, we consistently found increased levels of MBP associated with MS fractions and its presence is an important distinguishing factor separating controls from MS as determined in our principal component analysis. Other proteomic studies of mouse brain have also shown mitochondrial proteins, including several involved in the electron transport chain, as well as hemoglobin, and creatine kinase B in myelin preparations [START_REF] Taylor | Proteomic mapping provides powerful insights into functional myelin biology[END_REF][START_REF] Vanrobaeys | Profiling of Myelin Proteins by 2D Gel Electrophoresis and Multidimensional Liquid Chromatography Coupled to MALDI TOF-TOF Mass Spectrometry[END_REF][START_REF] Ishii | Human myelin proteome and comparative analysis with mouse myelin[END_REF][START_REF] Mugnaini | Distribution of Schwann cell cytoplasm and plasmalemmal vesicles (caveolae) in peripheral myelin sheaths. An electron microscopic study with thin sections and freeze-fracturing[END_REF][START_REF] Ravera | Evidence of aerobic ATP synthesis in isolated myelin vesicles[END_REF]. However, this is the first time that myelin basic protein has been

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observed associated with mitochondrial fractions. In addition, we see an increase in the amount of MBP associated with mitochondrial fractions in MS donors relative to controls by Western blot and have also shown that the expression of MBP isoforms is altered in the EAE mouse model of MS. These data suggest that MBP can be localized and associated with mitochondria both in normal and diseased brain, but the significance of this for MS disease pathology is not clear.

Peptide fingerprint mapping identified the protein isolated from fraction 3 of cohort 1 in the 10 kDa region as COX5b, a component of Complex IV of the electron transport chain. The expression of this subunit has also been shown to be decreased by mRNA expression microarray analysis [START_REF] Dutta | Mitochondrial Dysfunction as a Cause of Axonal Degeneration in Multiple Sclerosis Patients[END_REF]. Nuclear encoded mitochondrial proteins, including subunit 5b, are transcribed with a positively charged Nterminal signal sequence which is cleaved upon import into the appropriate mitochondrial location. The full length COX5b has a molecular weight of 13,696 Da and a pI of 9.07. However, upon removal of the 31 residue transit sequence, the molecular weight is reduced to 10,613 Da and the pI decreases to 6.33.

Both physical properties are in strong agreement with the differentially expressed species in fraction 3

(5.0-7.0). The loss of this subunit may play a role in mitochondrial dysfunction since the nuclear encoded subunits function in the assembly and maintenance of Complex IV [START_REF] Galati | Role of Nuclear-encoded Subunit Vb in the Assembly and Stability of Cytochrome c Oxidase Complex: Implications in Mitochondrial Dysfunction and ROS Production[END_REF]. Indeed, in null yeast mutants of subunit 4, 5a, 5b, 6c and 7a, Complex IV failed to assemble [START_REF] Capaldi | Structure and Assembly of Cytochrome c Oxidase[END_REF]. Additionally, Complex IV dysfunction may impact the production of free radicals, since it has been shown that various cytochrome c oxidase subunits provide protection from highly reactive peroxynitrite [START_REF] Fontanesi | Assembly of Mitochondrial Cytochrome c-Oxidase, a Complicated and Highly Regulated Cellular Process[END_REF].

Our differential protein expression analysis of mitochondrially enriched fractions from MS and control gray matter also demonstrated that the differentially expressed protein in fraction 4 from cohort 2, with a mass of 16 kDa, was consistent with hemoglobin-β. Notably, both hemoglobin-α2 and hemoglobin-β genes are upregulated in mononuclear blood cells from MS patients as reported recently in a study comparing monozygous twins discordant for multiple sclerosis [START_REF] Särkijärvi | Gene Expression Profiles in Finnish Twins with Multiple Sclerosis[END_REF]. This phenomenon appears to be extended to the brain, since our data show increased expression of hemoglobin-β in MS cortex.

Further, we localized hemoglobin expression to pyramidal neuronal cell bodies by immunofluorescent

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staining with antibodies to both hemoglobin and neurofilament. The discovery of hemoglobin expression in neurons, until recently considered specific to red blood cells, is novel. Other recent studies support our finding, including studies by Biagoli et al., (2009) [START_REF] Biagioli | Unexpected Expression of α-and β-globin in Mesencephalic Dopaminergic Neurons and Glial Cells[END_REF] and [START_REF] Richter | Neurons Express Hemoglobin α-and β-chains in Rat and Human Brains[END_REF] [START_REF] Richter | Neurons Express Hemoglobin α-and β-chains in Rat and Human Brains[END_REF] in which the expression of both α-and β-globin transcripts was shown in dopaminergic neurons from mouse, rat, and human brains. Functionally, alterations in hemoglobin expression may affect cellular energetics, since dopaminergic cell lines over expressing α-and β-globin exhibited changes in oxygen homeostasis and alterations in the expression of oxidative phosphorylation genes. These data support our finding of hemoglobin expression in the brain and support a potential role for hemoglobin in respiration.

Hemoglobin may also aid in protecting neurons from oxidative damage, since the ability of hemoglobin α 2 β 2 to scavenge peroxynitrite (ONOO -) in a variety of O 2 bound states has been suggested [START_REF] Herold | Reactions of Peroxynitrite with Globin Proteins and Their Possible Physiological Role[END_REF].

Lastly, in cohort 2 we identified a protein from fraction 6 as creatine kinase B (42 kDa). It has been observed previously that IgG from MS patients reacts with hemoblobin β, as well as with creatine kinase B and the expression of creatine kinase B has also been found in CSF of MS patients [START_REF] Nobem | Lumbar Cerebrospinal Fluid Proteome in Multiple Sclerosis: Characterization by Ultrafiltration, Liquid Chromatography, and Mass Spectrometry[END_REF][START_REF] Almeras | New antigenic candidates in Multiple Sclerosis: identification by serological proteome analysis[END_REF]. In myocardial infarction, serum levels of creatine kinase are used as an indicator of heart muscle damage. In MS, it may represent a similar marker for nervous tissue damage. However, we detected an almost 2 fold increase in expression in mitochondrial fractions obtained from nonlesion MS cortex. At this time, the significance and mechanistic importance of these findings are not clear. However, in neurons it has been suggested that creatine kinase B may serve to buffer fluctuations in ATP and levels of inorganic phosphate, as well as serve as a shuttle for high energy phosphate bonds between mitochondria and cytoplasm [START_REF] Andres | Functions and effects of creatine in the central nervous system[END_REF].

Mitochondrial dysfunction and neuroinflammation contribute to neuropathology not only in MS [START_REF] Van Horssen | Radical changes in Multiple Sclerosis pathogenesis[END_REF], but are also common to other complex neurodegenerative diseases such as Alzheimer's Disease (AD) and Parkinson's Disease (PD) [START_REF] Smith | Widespread peroxynitritemediated damage in Alzheimer's disease[END_REF][START_REF] Parker | Abnormalities of the electron transport chain in idiopathic Parkinson's disease[END_REF][START_REF] Lin | Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases[END_REF][START_REF] Zhu | Mitochondrial Dysfunction in Parkinson's Disease[END_REF][START_REF] Filippo | Mitochondria and the link between neuroinflammation and neurodegeneration[END_REF]. Additionally, several studies suggest that mitochondrial dysfunction is an early occurrence in neurodegeneration and molecular studies have found evidence of mutant proteins interacting with and preventing transport of nuclear encoded proteins to

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

mitochondria [START_REF] Reddy | Mitochondrial Medicine in Aging and Neurodegenerative Diseases[END_REF]. Consistent with our findings in this study and in previous studies [START_REF] Pandit | Impaired Regulation of Electron Transport Chain Subunit Genes by Nuclear Respiratory Factor 2 in Multiple Sclerosis[END_REF][START_REF] Dutta | Mitochondrial Dysfunction as a Cause of Axonal Degeneration in Multiple Sclerosis Patients[END_REF], the expression of mitochondrial electron transport chain proteins has also been shown to be decreased in PD in the brain with Complex I most consistently reported [START_REF] Mizuno | Deficiencies in Complex I subunits of the Respiratory Chain in Parkinson's Disease[END_REF][START_REF] Schapira | Mitochondrial complex I deficiency in Parkinson's disease[END_REF][START_REF] Hattori | Immunohistochemical Studies on Complex-I, Complex-II, Complex-III and Complex-IV of Mitochondria in Parkinson's Disease[END_REF]. In contrast, in AD, fewer mitochondria but increased cytochrome c oxidase (Complex IV) and mitochondrial DNA have been observed in neurons [START_REF] Hirai | Mitochondrial Abnormalities in Alzheimer's Disease[END_REF]. In AD, reactive oxygen species (ROS) may be a primary event mediating mitochondrial dysfunction [START_REF] Moreira | Alzheimer disease and the role of free radicals in the pathogenesis of the disease[END_REF] whereas in PD, a defect in mitochondria may mediate increased ROS as mitochondrial toxins can elicit neuronal pathology similar to PD [START_REF] Langston | Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis[END_REF][START_REF] Betarbet | Chronic systemic pesticide exposure reproduces features of Parkinson's disease[END_REF]. In MS, mitochondrial damage similar to what has been reported in both AD and PD has been observed. Damage to mitochondrial DNA and altered cytochrome c oxidase immunoreactivity similar to what has been observed in AD have been reported in axons in chronic active and inactive lesions [START_REF] Lu | Oxidative damage to mitochondrial DNA and activity of mitochondrial enzymes in chronic active lesions of multiple sclerosis[END_REF][START_REF] Mahad | Mitochondrial changes within axons in multiple sclerosis[END_REF] while gray matter pathology in MS is more consistent with PD, including reductions in electron transport chain subunits and mitochondrial activity [START_REF] Pandit | Impaired Regulation of Electron Transport Chain Subunit Genes by Nuclear Respiratory Factor 2 in Multiple Sclerosis[END_REF][START_REF] Dutta | Mitochondrial Dysfunction as a Cause of Axonal Degeneration in Multiple Sclerosis Patients[END_REF]. While the underlying mechanisms and events involved in mitochondrial dysfunction differ between these neurodegenerative diseases, the end result of these mitochondrial perturbations is similar resulting in increased ROS, loss of Ca 2+ homeostasis, and necrotic or apoptotic cell death [START_REF] Celsi | Mitochondria, calcium and cell death: A deadly triad in neurodegeneration[END_REF]. In MS these processes are exacerbated by demyelination which increases energy demand to maintain ion homeostasis through the energy dependent Na + /K + ATPase in neurons [START_REF] Waxman | Axonal conduction and injury in multiple sclerosis: the role of sodium channels[END_REF].

We detected increased nitration of electron transport chain subunits, creatine kinase B, and MBP in MS cortex. This is indicative of increased ROS and peroxynitrite mediated oxidative damage in MS and is consistent with previous studies [START_REF] Bizzozero | Evidence of nitrosative damage in the brain white matter of patients with multiple sclerosis[END_REF][START_REF] Pandit | Impaired Regulation of Electron Transport Chain Subunit Genes by Nuclear Respiratory Factor 2 in Multiple Sclerosis[END_REF] which reported increased levels of nitrated proteins in MS tissue when compared with controls. Nitration of mitochondrial electron transport chain subunits has also been identified in AD postmortem brain and in the spinal cord in EAE [START_REF] Sultana | Protein oxidation and lipid peroxidation in brain of subjects with Alzheimer's disease: insights into mechanism of neurodegeneration from redox proteomics[END_REF][START_REF] Qi | Mitochondrial Protein Nitration Primes Neurodegeneration in Experimental Autoimmune Encephalomyelitis[END_REF]. Increased protein nitration suggests an inflammatory mechanism is involved as inflammatory microglial cells express inducible nitric oxide synthase (iNOS) and release nitric oxide (NO) in MS lesions [START_REF] Colton | Microglia, an in vivo source of reactive oxygen species in the brain[END_REF][START_REF] Liu | Expression of inducible nitric oxide synthase and nitrotyrosine in multiple sclerosis lesions[END_REF]. This NO can diffuse and react with superoxide formed as a byproduct of mitochondrial respiration and create peroxynitrite which can also diffuse through tissue [START_REF] Beckman | The double-edged role of nitric oxide in brain function and superoxide-mediated injury[END_REF] and may account for our finding increased protein nitration

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

even in NAGM in MS [START_REF] Pandit | Impaired Regulation of Electron Transport Chain Subunit Genes by Nuclear Respiratory Factor 2 in Multiple Sclerosis[END_REF]. Alternatively, electron transport chain proteins may be oxidatively modified as a result of axonal transport through lesioned axonal segments. Either direct nitration of electron transport chain proteins by peroxynitrite or competitive inhibition of electron transport chain Complex IV by NO can lead to mitochondrial dysfunction [START_REF] Radi | Inhibition of mitochondrial electron transport by peroxynitrite[END_REF][START_REF] Sarti | Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell[END_REF].

Conclusions

Our data suggest that alterations in MBP expression and protein nitration in MS cortex involve an inflammatory mechanism shared with the EAE mouse model. The EAE mouse model is an autoimmune disease model, in which lesions similar to those observed in MS are observed upon immunization with myelin protein antigens. This model has been helpful in understanding inflammatory disease mechanisms in MS but does not reflect all aspects of MS neuropathology [77]. MS is both an autoimmune and a neurodegenerative disease, and while protein nitration and altered MBP expression are consistent in the EAE mouse model and in MS, we did not observe similar alterations in expression levels of COX5b, hemoglobin β chain, or creatine kinase B in EAE, indicating that a separate mechanism is also involved in MS pathology. This mechanism may involve cellular stress separate from that mediated by neuroinflammation such as metabolic stress which can lead to differential expression of mitochondrial enzymes and subunits of the electron transport chain [START_REF] Palmfeldt | Mitochondrial proteomics on human fibroblasts for identification of metabolic imbalance and cellular distress[END_REF]. The existence of separate mechanisms involved in mitochondrial dysfunction is not surprising given the heterogeneity and complexity of MS pathology [START_REF] Hu | The pathological spectrum of CNS inflammatory demyelinating diseases[END_REF] and is reflected in the disparate types of mitochondrial abnormalities reported in acute and chronic white matter lesions, in axons, and in gray matter in MS. Increased mitochondrial density and either increased Complex IV activity or protein has been reported in chronic active white matter lesions in axons and astrocytes [START_REF] Lu | Oxidative damage to mitochondrial DNA and activity of mitochondrial enzymes in chronic active lesions of multiple sclerosis[END_REF][START_REF] Witte | Enhanced Number and Activity of Mitochondria in Multiple Sclerosis Lesions[END_REF] and also in a subset of demyelinated axons in chronic inactive lesions [START_REF] Mahad | Mitochondrial changes within axons in multiple sclerosis[END_REF].

In contrast, decreased Complex IV activity and decreased immunoreactivity to Complex IV subunits have been reported in acute white matter lesions [START_REF] Mahad | Mitochondrial Defects in Acute Multiple Sclerosis Lesions[END_REF] and also in injured axons in chronic active lesions [START_REF] Mahad | Mitochondrial changes within axons in multiple sclerosis[END_REF].

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

These studies suggest an adaptive response by both neurons and glia to increase mitochondrial activity in response to demyelination, thereby increasing ROS, and also a defect in mitochondrial transport in damaged tissue. Our data however, examined NAGM rather than lesioned tissue and has confirmed our previous studies which found decreased expression of mitochondrial electron transport chain subunit genes in MS [START_REF] Pandit | Impaired Regulation of Electron Transport Chain Subunit Genes by Nuclear Respiratory Factor 2 in Multiple Sclerosis[END_REF][START_REF] Dutta | Mitochondrial Dysfunction as a Cause of Axonal Degeneration in Multiple Sclerosis Patients[END_REF] and has extended these findings and implicated an imbalance in the regulation of additional respiratory proteins in MS disease pathology. Our findings have important implications for the development of new neuroprotective therapies for MS.

( 1 :

 1 100, Thermo Scientific, Rockford, IL) and mouse anti-neurofilament (1:500, SMI-32, Covance, Princeton, NJ) antibodies in PBS, 5% triton-X 100, 3% normal donkey serum for 24 hours. After 3 washes in PBS (10 minutes each) the tissue was incubated in donkey anti-mouse Alexa 488 (1:250, A-11055 Invitrogen, Carlsbad, CA) and donkey anti-goat Alexa 546 (1:250, A-11056 Invitrogen, Carlsbad, CA) in PBS for 12 hours. Following three ten minute washes in PBS, the sections were incubated in 50 mM ammonium acetate, 10mM cupric sulfate for 30 minutes to quench lipofuscin auto-fluorescence. Sections were next washed three times in PBS (10 minutes each), placed below coverslips under Vectashield ® mounting media (Invitrogen, Carlsbad, CA), sealed with clear nail polish, and refrigerated until microscopically imaged. Images were acquired using an Olympus FV500 confocal microscope equipped with four lasers (HeCd 442 nm, Ar 488 nm and 514 nm, HeNe 543 nm and HeNe 633 nm) and a 60X oil objective. Microscopic fields were acquired that clearly contained pyramidal cells as indicated by neurofilament staining. Green and red fluorescence image stacks were captured sequentially to prevent any channel bleed through, and spanned the physical section.

  (2-(N-morpholino)ethanesulfonic acid, Sigma PN M8250) twice. A 200mg/mL solution of EDC (N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride Fluka PN-03449) in 50mM MES was prepared. The washed beads (100µL) were incubated with the EDC solution such that the final concentration was 2mg/mL for approximately 2 minutes in a vortexer set on low speed. 200 µg of mitochondrially enriched NAGM protein was added to 500µl of the bead slurry. Protein and beads were incubated with constant mixing at room temperature for 3 hours. Beads were washed in 10mM Tris pH 8.0 0.5%BSA twice. Beads were stored in 150µL 10mM Tris pH 8.0 0.5%BSA at 4°C. Bright field microscopy was used to confirm low self-aggregation and antigenantibody activity of conjugated beads.Mitochondrial protein extracts from specific ion exchange fractions (2.5µL) were incubated with bead solution (2.0µL) in 10mM Tris pH 8.0 0.5%BSA to a final volume of 10µL for 1 hour with constant mixing at room temperature. Solutions from both before and after bead incubation were diluted 1/10 in a saturated α-cyano-4-hydroxycinnamic acid (CHCA; Sigma PN 145505-5g) aqueous solution consisting of 50% ACN (Fisher PN A996-4), 0.6% OGP (Sigma PN O8001-5g) and 0.5% TFA (JT Baker PN9470-00).

  expression between control and MS NAGM mitochondrially-enriched proteins, a technique which detects correlated sets of variables is needed to analyze this data. In this research, a modified Pearson productmoment correlation coefficient was employed as a distance measure and the average linkage method was used to perform a hierarchical cluster analysis of protein expression and compute a dendrogram. Nodes of the dendrogram join the most similar objects and the relative length of the branches is indirectly proportional to the similarity of those objects. The most significant feature in the hierarchical clustering analysis is the segregation of control and MS donors by disease state (fraction 3 and 6 from cohort 1 and fraction 6 from cohort 2 shown in Figure1 panels A, B, and C). Other fractions in this data set have only one or two differentially expressed peaks and therefore are not sufficiently complex to warrant the use of multivariate methods such as hierarchical clustering. Fraction 3 of cohort 1 contains two distinct nodes (Figure1, Panel A). The short length of the branches indicate that the peak intensities of the proteins in each node are highly correlated, suggesting a potential relationship between the peaks in each node. The highly correlated peaks of the upper node, containing peaks at 10.7, 10.6, 10.2, 10.1 and 9.9 kDa, have a correlation coefficient greater than 0.85. The lower node, containing peaks at 15.9, 15.8, 8.0, and 7.9 kDa, has a correlation coefficient greater than 0.94. Of the four peaks, three were differentially expressed at the 95% confidence level, with p values of 0.009, 0.076, 0.028 and 0.028, respectively. Fraction 6 of cohort 1 (Figure1, Panel B) also shows several highly correlated nodes. The upper middle node containing peaks at 22.9, 22.7, 11.5, 23.7, and 11.4 kDa have a correlation coefficient greater than 0.85, while the correlation coefficient of the lower middle node consisting of peaks at 9.8, 9.7, 12.6, 12.5 and 17.2 kDa is greater than 0.92. The linear correlation of the data combined with significant p values of A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 0.076, 0.028, 0.028, 0.016, and 0.076, respectively, indicate a group of possible biomarkers. Fraction 6 of Cohort 2 shows a distinct clustering by disease as well as several nodes (Figure 1, Panel C).

Figure 1 ,

 1 Figure 1, Panel A, B, and C display PC scatter plots of PC1 and PC2. Notice the segregation of the data into MS and control sample groups in each fraction. This technique also confirmed that other parameters such as brain region, gender, age or PMI were not responsible for data segregation. For all controlled variables, PCA verified that disease state was the only donor characteristic responsible for sample segregation.

  process and protease leading to this particular differentially expressed MBP fragment is currently under investigation.In cohort 2, proteins from fraction 4 and 6 were purified by electrophoresis and bands were removed at 16 kDa and 42 kDa, respectively. The protein at 42 kDa was identified as creatine kinase type B (UniProt accession number P12277) with a Mascot score of 70.26 from seven peptides with a sequence coverage of 27.11%. The peak at 16 kDa was identified as hemoglobin β-chain (UniProt accession number P68871) with a Mascot score of 60.25 from six peptides and a sequence coverage of 51.37%.The SELDI-TOF mass spectra for these four proteins are shown in Figure2, Panels A-D, and demonstrates the overexpression of hemoglobin β, MBP, and creatine kinase B, and decreased expression of COX5b in MS relative to controls.

  Identified in the bands exhibiting increased nitrotyrosine immunoreactivity in MS were the mitochondrial encoded catalytic subunit of Complex IV of the electron transport chain, cytochrome c oxidase subunit 2 (COX2), two subunits of Complex I (NDUA8 and NDUV2), a subunit of Complex III (UQCR2), 2', 3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), beta actin (ACTB), Creatine kinase B (CKB), and MBP. Several splice variants of MBP have isoforms in the 20kDa range. Ten peptides from MBP isoforms 3 and 5 ( MBP1 and MBP3), were detected and

Figure 1 .

 1 Figure 1. Multivariate analysis of cohort 1, fractions 3 and 6 and cohort 2 fraction 6 using principal component analysis and hierarchical clustering. Multivariate analyses including hierarchical clustering and principal component score scatter plots show the segregation of MS and control samples. Panel A: Cohort 1 fraction 3 analysis. Panel B: Cohort 1 Fraction 6 analysis. Panel C: Cohort 2 fraction 6 analysis. Peaks identified by peptide fingerprint mapping are indicated with asterisks.

Figure 2 .

 2 Figure 2. Proteomic differential expression of identified proteins. SELDI-TOF mass spectra showing the differential expression of a set of peaks. Panel A: The 16 kDa peak was identified as hemoglobin β chain, Panel B: The 42kDa peak was identified as Creatine Kinase type B, Panel C: The 10.6kDa peak was identified as COX5b, Panel D: The 16kDa peak was identified as MBP. Mass spectra of mitochondrial samples outlined in red are control samples and those outlined in blue are MS samples.

Figure 3 .Figure 4 .

 34 Figure 3. Confirmation of the identity of differentially expressed proteins. A. Mitochondrially enriched protein solutions were incubated with an antibody to COX5b covalently bound to beads. Aliquots analyzed by SELDI-TOF-MS before and after incubation confirm the identity of the mass spectral peak at 10.6 kDa as COX5b. The red box highlights the region of interest for these spectra. B. Representative western blot demonstrating the relative purity of the cellular fractionation. Western blots were performed on cytoplasmic (cyto) and mitochondrial (mito) fractions isolated from MS and control cortex, run side by side and blotted with an antibody to the neuron specific protein, neurofilament (NF), and an antibody to the mitochondrial encoded COX2 protein. C. Representative western blots show increased MBP, hemoglobin β (Hbb), and creatine kinase (CKB) and decreased COX5b in mitochondrial fractions isolated from MS cortex. Increased expression of MBP was also observed in the EAE mouse brain while CKB, Hbb, and COX5b were not similarly altered in EAE and MS. To control for protein loading, western blots were reprobed with antibodies to either Complex II (CII) or COX2. D. Representative western blots of hemoglobin β (Hbb) and creatine

Table 2

 2

	describes the identified differentially expressed peaks from both data sets listing fold change,
	Mann-Whitney p values, theoretical isoelectric point, theoretical molecular weight, cohort, mass to charge

ratio (m/z) and peptide fingerprint mapping results.

Table 2

 2

	lists the most probable identifications with Mascot scores, number of peptides, sequence
	coverage, probabilities and protein characteristics. The list of matches in the database for the 10 kDa
	protein fragment included cytochrome c oxidase subunit 5b (COX5b) (UniProt accession number
	P10606), an electron transport chain subunit of cytochrome c oxidase (Complex IV) with a Mascot score
	of 20.2 from two peptides detected and 12.24 % sequence coverage. The 15.9 kDa peak in fraction 3 was
	identified as myelin basic protein (MBP) (UniProt accession number P02686) with a Mascot score of
	80.21 from 10 peptides with a sequence coverage range of 25.7% for isoform 1 to over 49% for isoform 6
	(see

Table 2 )

 2 and almost certainly represents a proteolytic fragment of one of the MBP isoforms. The

Acknowledgements:

We would like to thank the Rocky Mountain MS Center, the Kathleen Price Bryan Brain Bank, and the Brain and Spinal Fluid Resource Center at UCLA for tissue. This research was funded by NIH Grant R21NS058921 (JM), a Grant from the Ohio Board of Regents, and by Acorn Ventures, Bellevue, WA (RBG).


Guido M Voets
email: g.voets@umcutrecht.nl

A C Fluit

Jelle Scharringa

James Cohen Stuart

Maurine A Leverstein-Van Hall

A set of multiplex PCRs for genotypic detection of extendedspectrum -lactamases, carbapenemases, plasmid-mediated AmpC -lactamases and OXA -lactamases

Keywords:

Introduction

The increasing prevalence of Gram-negative bacteria producing -lactamases, which hydrolyse third-generation cephalosporins or carbapenems, is a worldwide problem.

The most prevalent -lactamases are extended-spectrum -lactamases (ESBLs) of the CTX-M, TEM and SHV families [START_REF] Gniadkowski | Evolution of extended-spectrum -lactamases by mutation[END_REF]. However, the prevalence of Gram-negative bacteria that are resistant to third-generation cephalosporins owing to increased expression of chromosomal -lactamases, such as SME and IMI-1, or the acquisition of plasmid-mediated -lactamases, such as IMI-2, NDM-1, KPC, IMP, OXA, PER and VEB, as well as AmpC -lactamases is increasing [START_REF] Nordmann | Emerging carbapenemases in Gram-negative aerobes[END_REF][START_REF] Naas | Minor extended-spectrum -lactamases[END_REF][START_REF] Jacoby | AmpC -lactamases[END_REF][START_REF] Yong | Characterization of a new metallo--lactamase gene, bla NDM-1 , and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India[END_REF][START_REF] Walther-Rasmussen | Class A carbapenemases[END_REF].

Several multiplex polymerase chain reactions (PCRs) for the detection of lactamase families have been published [START_REF] Dallenne | Development of a set of multiplex PCR assays for the detection of genes encoding important lactamases in Enterobacteriaceae[END_REF][START_REF] Woodford | Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp[END_REF][START_REF] Mendes | Rapid detection and identification of metallo--lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis[END_REF]. In particular, the set of multiplex PCRs by Dallenne et al. [START_REF] Dallenne | Development of a set of multiplex PCR assays for the detection of genes encoding important lactamases in Enterobacteriaceae[END_REF] detects a broad range of epidemiologically important lactamase families. However, this set does not detect the SME, IMI, NDM, NMC-A, GIM, SIM and SPM -lactamase families as well as several OXA groups within the OXA family.

The aim of this study was to develop a set of multiplex PCRs for the detection of the majority of genes encoding clinically important -lactamases causing thirdgeneration cephalosporin or carbapenem resistance using a single amplification protocol.

To achieve this aim, seven multiplex PCRs were developed for the detection of plasmid-mediated AmpC -lactamases (ACC, ACT, DHA, CMY, FOX, LAT, MIR and 24, 48, 1, 2, 51, 4 and 58) using Super Taq polymerase (HT Biotechnology Ltd., Cambridge, UK) (Table 1). For detection of genes encoding CTX-M, TEM, SHV, GES, VEB, PER, KPC, VIM and IMP -lactamase families, four multiplex PCRs of Dallenne et al. (multiplex I, II, IV and VI) [START_REF] Dallenne | Development of a set of multiplex PCR assays for the detection of genes encoding important lactamases in Enterobacteriaceae[END_REF] were evaluated using the same amplification protocol as used for our PCRs, including Super Taq polymerase instead of the AmpliTaq polymerase (Sigma-Aldrich, St-Quentin-Fallavier, France) originally used by the authors.

Material and methods

Primers were designed using the Lasergene 8 program (DNASTAR, Madison, WI).

Optimisation of the amplification conditions of each PCR, both the newly presented PCRs in this paper as well as the complementary PCRs of Dallenne et al. [START_REF] Dallenne | Development of a set of multiplex PCR assays for the detection of genes encoding important lactamases in Enterobacteriaceae[END_REF], resulted in the following PCR protocol. DNA was isolated using a NucleoSpin ® Tissue Kit (Macherey-Nagel, Dϋren, Germany) according to the manufacturer's instructions. DNA concentrations of the samples were equalised to ca. 50 ng/L and 1 L was added to each multiplex PCR in a 25 L reaction mixture containing 1 PCR buffer, 50 M of each deoxynucleotide triphosphate, 1 U Super Taq and a variable concentration of primers and MgCl 2 (Table 1). Amplification was carried out as follows: initial denaturation for 1 min at 94 C, 30 cycles of 30 s at 94 C, 40 s at 60 C and 1 min at 72 C, and a final elongation step for 1 min at 72 C. Amplification products were visualised after running at 100 V for 1 h on a 1% agarose gel containing GelRed TM (Biotium Inc., Hayward, CA). A 1-kb DNA ladder

Results and discussion

Amplification products of the expected sizes were obtained with all control strains, confirming the group specificity of the primers (Fig. 1). In addition, the expected amplification products were also obtained with the adapted complementary PCRs described by Dallenne et al. [START_REF] Dallenne | Development of a set of multiplex PCR assays for the detection of genes encoding important lactamases in Enterobacteriaceae[END_REF] (data not shown). To determine the specificity of the primers for allotypes for which no positive control strain was available, in silico PCR simulation was performed. All allotypes to which the primers anneal, allowing for one mismatch except for the 3'-nucleotide, and allotypes with more mismatches but in the same target gene group were identified (Table 1). In silico PCR simulation was also performed to check for any unwanted cross-reaction with members of the Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa. The It should be noted that the MGC2 multiplex PCR yielded a 160-bp non-specific amplification product (Fig. 1B, lanes 1 and 2). DNA sequencing showed that this product was identical to part of a CMY-2 group gene in the control strain (lanes 1 and 2). In six isolates that were expected to remain negative, another 160-bp nonspecific amplification product was noted. DNA sequencing showed that this product was part of an ACT-like AmpC (GenBank accession no. EF078894.1) (data not shown). The CMY-2 group gene amplification resulted from annealing of the ACT-GCM2-R primer and CY2-GC2M-F primer to genes of the CMY-2 group despite several mismatches. The ACT-like AmpC gene amplification resulted from annealing of the ACT-GCM2 primers to the ACT-like AmpC gene. However, these amplification products were not considered to be a problem for interpretation of the multiplex PCR because they are smaller than the expected specific products. Furthermore, the amplification products were derived from a CMY-2 group or ACT-like gene, which are both targets of the MGC2 multiplex PCR. In conclusion, we describe a set of multiplex PCRs that, when used in combination with the multiplexes I, II, IV and VI of Dallenne et al. [START_REF] Dallenne | Development of a set of multiplex PCR assays for the detection of genes encoding important lactamases in Enterobacteriaceae[END_REF], can detect a wide range of -lactamases using the same amplification conditions. This enables detection of the majority of clinically important -lactamases causing third-generation cephalosporin or carbapenem resistance. a All allotypes without brackets have no or one mismatch with the primers, except for the 3'-nucleotide, designed for this target group.

b Primer sequence from David Livermore, Health Protection Agency, UK (pers. corr.).

c CMY-2, -4 to -7, -12 to -16, -18, -21, -23, -24, -27 to -36, -38 to -41, -43 to -45, -47 to -49, -53 to -55, and -59.

d Also known as the EBC group [START_REF] Perez-Perez | Detection of plasmid-mediated AmpC lactamase genes in clinical isolates by using multiplex PCR[END_REF].

e Also known as the CIT group [START_REF] Perez-Perez | Detection of plasmid-mediated AmpC lactamase genes in clinical isolates by using multiplex PCR[END_REF] A c c e p t e d g OXA-4, -7, -10, -11, -13, -14, -16, -17, -19, -28, -56, -74, -101, -129, -142, -145 and -147.

  of Gram-negative micro-organisms to third-generation cephalosporins and carbapenems owing to -lactamases is an increasing problem. Although the CTX-M, TEM and SHV extended-spectrum -lactamases (ESBLs) are most widely disseminated, other -lactamase families have also recently emerged, such as plasmid-mediated AmpC -lactamases and carbapenemases. Here we describe a new set of multiplex polymerase chain reactions (PCRs) with one amplification protocol enabling detection of 25 prevalent -lactamase families, including ESBLs, carbapenemases, plasmid-mediated AmpC -lactamases and OXA -lactamases.

  metallo-carbapenemases (GIM, NDM, SIM and SPM), serine carbapenemases (IMI, SME and NMC-A) and OXA -lactamases (OXA groups 23,

6 (

 6 Invitrogen, Breda, The Netherlands) was used as a size marker. DNA sequencing was performed by BaseClear (Leiden, The Netherlands). In silico PCR simulations were performed using nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and MegAlign (DNASTAR Lasergene 8).

  3'-end of the SPM-GBM-R primer anneals with 21 of 27 bp to the ACC deaminase gene of a Pseudomonas spp. (GenBank accession no. EU520401) and the 3'-end of O58-GD3M-R anneals with 17 of 22 bp to the biotin sulfoxide reductase gene of Escherichia coli (GenBank accession no. CP001846) and also with 17 of 22 bp to the molybdopterin guanine dinucleotide-containing S/N-oxide reductase gene of E. coli (GenBank accession no. CP001637). Cross-reaction of the SPM-GBM-R primer was not a problem with the P. aeruginosa positive control. Cross-reactions of O58-GD3M-R may result in poorer resolution of the MGD3 multiplex PCR when used in combination with E. coli.

  A c c e p t e d M a n u s c r i p t Page 7 of 20 A c c e p t e d M a n u s c r i p t 7

Fig. 1 .

 1 Fig. 1. Multiplex polymerase chain reactions (PCRs). All multiplex PCR products

1 OXA- 2 ,

 12 -3, -15, -21, -32, -34, -36, -46, -53, -141, -144, -161 and [OXA-118, chain reaction; NCTC, National Collection of Type Cultures.

M a n u s c r i p t 8 f

 8 OXA-51, -64 to -71, -75 to -80, -82 to -84, -86 to -95, -98 to -100, -106 to -113, -115 to -117, -128, -130 to -132, -138, -144, -148 to -150, and -172 to -180.

Acknowledgments

The authors wish to thank Patrice Nordmann for kindly providing isolates Nord-03 and Nord-04; Kyungwon Lee for isolate PSA097; and Ana Gales for isolate PSA098.

Funding


Jaana Maarit

PhD, LicSci (Pharm Korhonen

PhD Arja Helin-Salmivaara

PhD Risto Huupponen

Keywords: adherence, drug use pattern, claims data, misclassification

Purpose Knowledge of different usage patterns emerging during a long-term statin therapy is limited.

We aimed at characterizing statin use, including rates of reinitiation after extended periods of non-use, transitions between good and poor adherence, and the effect of the length of a drug-free period on identification of new use.

Methods Persons aged 45-84 purchasing statins in 1997 and 2007 were identified through the Finnish prescription register. New users in 1997, including those with no statin purchases during the preceding 3 years, were followed-up until institutionalization, death or Dec 31, 2007. Reinitiation rates after ≥180 days with no refill for statins were analyzed using survival analysis. Annual adherence levels and the prevalences of good (proportion of days covered ≥0.80) and poor (<0.80) adherence were calculated for 10 one-year periods since initiation. For statin users in 2007, purchases were captured over the previous 10 years.

Results 32,760 persons initiated statin treatment in 1997, 48.1% of whom discontinued it for at least 180 days by the end of follow-up. Of the discontinuers, 46.7% restarted the treatment within 1 year and 88.7% by the end of follow-up. Of those followed-up for 10 years or longer, 51.8% accumulated 6 or more years of treatment with good adherence. In 2007, 27.7% of the initiators having no statin purchases within the previous year had refills during the preceding 10 years.

Conclusion

Statin use is dynamic. This should be taken into consideration in clinical practice and when studying the incidence, patterns and health outcomes of statin use.

Introduction

Statins have been shown to reduce the risk of cardiovascular events in a wide variety of high-risk patients [START_REF] Baigent | Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins[END_REF][START_REF] Brugts | The benefits of statins in people without established cardiovascular disease but with cardiovascular risk factors: meta-analysis of randomised controlled trials[END_REF]; however, benefits of statin therapy may be compromised because of suboptimal persistence with and adherence to it [START_REF] Shalev | Continuation of statin treatment and all-cause mortality[END_REF][START_REF] Perreault | Impact of better adherence to statin agents in the primary prevention of coronary artery disease[END_REF][START_REF] Corrao | Results of retrospective database analysis of adherence to statin therapy and risk of nonfatal ischemic heart disease in daily practice in Italy[END_REF]. One quarter to 50% of new statin users discontinue the therapy during the first year [START_REF] Benner | Long-term persistence in use of statin therapy in elderly patients[END_REF][START_REF] Mantel-Teeuwisse | Long term persistence with statin treatment in daily medical practice[END_REF][START_REF] Perreault | Persistence and determinants of statin therapy among middle-aged patients for primary and secondary prevention[END_REF][START_REF] Helin-Salmivaara | Long-term persistence with statin therapy: a nation-wide register study[END_REF]. In Finland, 44% of persons initiating statin use in 1995 continued it for 10 years or longer [START_REF] Helin-Salmivaara | Long-term persistence with statin therapy: a nation-wide register study[END_REF].

The pattern of statin use remained stable in 1995-2004, with a slight improvement in persistence rates for the initiators in 1998-2004 [START_REF] Helin-Salmivaara | Pattern of statin use among 10 cohorts of new users from 1995 to 2004: A register-based nationwide study[END_REF]. Information on discontinuation rates is important also because the interruption of statin use may exert an excess risk of cardiovascular events and death in some patients groups [START_REF] Risselada | Withdrawal of statins and risk of subarachnoid hemorrhage[END_REF][START_REF] Daskapoulou | Discontinuation of statin therapy following an acute myocardial infarction: a population-based study[END_REF], potentially attributable to a biologic rebound phenomenon [START_REF] Pineda | Statin rebound or withdrawal syndrome: Does it exist[END_REF]. However, studies ending the follow-up at the first extended break in refills are likely to underestimate the patient's cumulative statin exposure. Actually, the studies reporting on reinitation rates indicate that the majority of the stoppers resume statin use later on in their life [START_REF] Hudson | Comparison of measures of medication persistency using a prescription drug database[END_REF][START_REF] Brookhart | Physician follow-up and provider continuity are associated with long-term medication adherence[END_REF].

Observational studies on statin effectiveness typically employ new-user designs, restricting a study cohort to initiators. The new-user design allows for estimation of the effects associated with drug initiation and adjustment for confounding due to predictors of persistence with drug therapy, and also avoids overadjustment for potential consequences of drug treatment [START_REF] Ray | Evaluating medication effects outside clinical trials: New-user designs[END_REF]. Nevertheless, no consensus exists on the required time period without statin use before the first observed use. In administrative database studies, periods without any statin (or any lipid-lowering drug [START_REF] Perreault | Impact of better adherence to statin agents in the primary prevention of coronary artery disease[END_REF]) dispensations prior to the cohort entry have ranged from 3-6 months [START_REF] Scranton | Statin use and fracture risk[END_REF][START_REF] Willke | Differences in cardiovascular event rates between atorvastatin and simvastatin among new users: managed-care experience[END_REF] to 1-3 years [START_REF] Shalev | Continuation of statin treatment and all-cause mortality[END_REF][START_REF] Perreault | Impact of better adherence to statin agents in the primary prevention of coronary artery disease[END_REF][START_REF] Corrao | Results of retrospective database analysis of adherence to statin therapy and risk of nonfatal ischemic heart disease in daily practice in Italy[END_REF][START_REF] Smeeth | Effect of statins on a wide range of health outcomes: a cohort study validated by comparison with randomized trials[END_REF][START_REF] Chodick | Persistence with statins and incident cataract: a population-based historical cohort study[END_REF]. The choice of this so called washout period is likely to be motivated by the reimbursement, prescribing and dispensing rules of the healthcare system through which the data for a specific study are generated. Especially when studying refill patterns, it may be reasonable to select the washout period according to the characteristics of the data source; however, differences in washout periods across studies may hamper the comparison of the risk estimates of health outcomes obtained in them.

The aim of this study was two-fold: to characterize the patterns of long-term statin use, including the rates of reinitiation after extended periods of non-use and transitions between good and poor adherence, and to describe the effect of the length of drug-free period on identification of new use, in samples of statin initiators in Finland.

Methods

Reimbursement for and pricing of statins in Finland

In Finland, statins are available on prescription only. The National Health Insurance Scheme, run by the Social Insurance Institution of Finland (SII), provides prescription drug coverage for all about 5 million community-dwelling residents of the country [START_REF] Furu | The Nordic countries as a cohort for pharmacoepidemiological research[END_REF]. The drug reimbursement system consists of three refund categories (basic, lower special and higher special refund) graded according to medical criteria based on the severity of the illness and the necessity of the drug therapy [START_REF] Peura | Pharmaceutical Pricing and Reimbursement Information Project[END_REF]. Statins are reimbursed under the basic refund category except for the patients entitled to lower special refund due to dyslipidemia associated with coronary artery disease (CAD) (since 2000) or familial hypercholesterolemia. The copayment for the basic refund category was 10 euros per purchase and 50% of the balance of the remaining price until the end of 2005. For the lower special refund category, the respective figures were 5 euros and 25%. Thereafter, 42% and 72% of the price of each drug has been reimbursed. Generic simvastatin has been available since 2003 after which the prices of simvastatin products decreased dramatically. Beginning October 2006, the prescribing of less expensive statins was promoted by a restriction of the reimbursement of the most expensive ones, atorvastatin and rosuvastatin. These two statins remained reimbursable only for treatment of severe disorders of lipid metabolism in high risk patients who were not able to use less expensive statins due to intolerance or ineffectiveness [START_REF] Martikainen | Impact of restricted reimbursement on the use of statins in Finland. A register-based study[END_REF]. In practice, users of atorvastatin and rosuvastatin were entitled to reimbursement only after they had tried less expensive statins first.

Data sources

Since 1994, the SII has maintained an electronic pharmacy claims database called the Prescription Register. The Register contains records of all drug purchases reimbursed to residents in non-institutional settings [START_REF] Furu | The Nordic countries as a cohort for pharmacoepidemiological research[END_REF]. For each drug, dispensing date, the WHO Anatomical Therapeutic Chemical (ATC) classification code [START_REF] Who | About the ATC/DDD system[END_REF], strength, and quantity dispensed are recorded. For each person, information on birth and death dates and gender is available. Persons institutionalized permanently, including those staying in public nursing homes and hospitals for >90 days without interruption, are not eligible for reimbursements, and their purchases are not registered. These persons can be identified from a separate SII register.

Because of the reliance on the above administrative data, the expression statin use in this report refers to purchasing statins or being dispensed statin prescriptions in a pharmacy. The actual use of statins could not be verified.

Study samples

There were two separate samples. The first study sample consisted of persons initiating statin therapy (ATC code C10AA) during the year 1997. We defined an initiator (new statin user) as a person being dispensed a statin prescription without having been dispensed one in the preceding 3 years (1095 days).

As a sensitivity analysis, the above definition was relaxed so that all persons with no dispensations for statins during the preceding year (365 days) were considered as initiators. We followed the initiators up until they died, were institutionalized permanently according to the SII register, or reached the end of follow-up or Dec 31, 2007, whichever came first.

For identifying discontinuation dates, we created a drug coverage data file for each initiator. We linked consecutive prescriptions together based on dispending dates and the number of days' supply of each prescription, assuming a dosage of one tablet per day. If a prescription was filled when there should have been unused tablets from the previous dispensation, we assumed use of the new supply to begin after the end of the previous supply. We defined discontinuation as failing to fill a prescription within 180 days of finishing the previous one. In sensitivity analyses, this gap was defined as 90 and 270 days. According to the Finnish drug reimbursement scheme, a maximum of 3 months' supply of medication can be reimbursed at one transaction; therefore, gaps applicable in other reimbursement systems may be too short in the Finnish context. We analyzed statins as a group, considering switching between various statins as continuation of the therapy.

We operationalized adherence as the proportion of days covered (PDC) [START_REF] Andrade | Methods for evaluation of medication adherence and persistence using automated databases[END_REF]. Based on reimbursed statin purchases and assuming a dosage of one tablet per day, we obtained the PDC with a statin for each year (365-day period) of follow-up by dividing the number of days on which the person had a statin on-hand by 365. Good adherence refers to PDC >0.80 [START_REF] Benner | Long-term persistence in use of statin therapy in elderly patients[END_REF][START_REF] Simpson | A meta-analysis of the association between adherence to drug therapy and mortality[END_REF].

The second sample included all persons who had purchased statins in 2007. For this sample, we defined new statin users by varying the requirement for the length of the period without statin dispensations prior to the first prescription in 2007 from 3 months [START_REF] Scranton | Statin use and fracture risk[END_REF] up to 9 years. We calculated percentages of persons misclassified in each case, defining the persons with no statin dispensations during the previous 10 or more years as true new users.

Statistical analysis

For comparing characteristics of those qualifying and not qualifying as statin initiators when employing different purchase-free gaps, we used the χ 2 square test and the t-test. For the discontinuers in the 1997 sample, we estimated the distribution of the time until a person refilled a statin prescription with the Kaplan-Meier method, using SAS software (version 9.1, SAS Institute Inc., Cary, NC). There was no legal requirement for approval by an ethics committee since we used de-identified data and did not contact the persons.

Results

Characteristics of the initiators in 1997

A cohort of 32,760 statin initiators, i.e. those with no statin purchases during the preceding 3 years, aged 45-84 in 1997 was identified. The initiators had a mean age of 63 years; slightly less than half were male (Table 1). As identified in the SII Special Reimbursement Register, 10,682 (32.6%) had an established CAD and 88 (0.3%) had familial hypercholesterolemia. The initial statin was most commonly simvastatin (Table 1). The mean follow-up time was 8.2 years (SD, 3.4), 10,357 (31.6%) being censored because of institutionalization and 2825 (8.6%) because of death.

In sensitivity analyses, we relaxed the definition for new users to include all those with no statin dispensations during the preceding 1-year period: the number of 45-84 year-old initiators identified this way was 36,581 (Table 1). Compared with those qualifying as initiators with the 3-year gap (Table 1), the 3821 persons qualifying with the 1-year but not with the 3-year gap were more commonly male (55.5%) and somewhat younger (mean age, 62.3 years; SD, 8.5 years). In addition, they initiated their statin therapy more commonly with lovastatin (23.7%) and less commonly with simvastatin (40.8%) or fluvastatin (29.0%, p-values for all comparisons <0.0001).

Patterns of statin use in the 1997 cohort

Of the original cohort (n=32,760), 15,759 or almost every second (48.1%) had at least one statin-free period lasting for 180 days or longer during the follow-up. The number of initiators with at least one 90day statin-free gap was 20,840 (63.6%) and the number of those with at least a 270-day gap was 13,165 (40.2%), respectively. Of those with 90-, 180-and 270-day gaps, estimated 62.4%, 46.7% and 36.7% reinitiated statin therapy within 1 year after the end of the first gap (Fig 1). By the end of follow-up, the reinitiation rates were 92.3%, 88.7% and 86.2%, respectively.

Of those surviving at least 365 days since statin initiation (n=30,893), 14,886 (48.2%) had good first-year adherence (PDC >0.80, Table 2) while 2874 (9.3%) purchased only one statin prescription by the end of the first year. The proportion of those with good adherence increased up to 58.8% by the tenth year of follow-up. The proportion of those with continuous good adherence throughout the 10 years (17.1%) was the same as the proportion of those never achieving good adherence in any of the 10 years (17.3%) (Fig 2). Altogether 51.8% of those followed up for 10 years used statins with good adherence for a total of 6 years or longer, and 69.2% for 3 years or longer (total number of 1-year gaps in good adherence at most 4 and 7, respectively).

Sensitivity analyses revealed similar patterns of discontinuation, reinitiation and adherence in the cohort identified using the relaxed definition of new use (n=36,518). The proportions of initiators with at least one statin-free period lasting 90, 180, and 270 days during the follow-up were 64.7%, 49.2% and 41.1%. Estimated 62.8%, 47.7% and 38.2% of those with 90-, 180-and 270-day gaps restarted the therapy within 1 year after the end of the first gap. By the end of follow-up, the estimated probabilities of reinitiation were 92.5%, 89.1% and 86.8%, respectively. Of those surviving at least one year since initiation (n=34,517), 47.2% had good adherence and 9.7% purchased only one prescription during that year. One half (51.1%) of those followed up 10 or more years after initiation (n=22,486) accumulated at least 6 oneyear periods of good adherence.

Characteristics and previous statin purchases of the initiators in 2007

In total, 520,894 Finns aged 45-84 purchased statins in 2007. Of the 78,512 new statin users defined as those who had not purchased statins during the preceding 3 years, 89.3% initiated with simvastatin (Table 1). Every fifth (21.0%) of the 99,348 initiators defined as those with a 1-year purchase-free period did not qualify as such when using a 3-year gap. These 20,836 initiators differed from those with no dispensations during the previous 3 years (Table 1) being older (mean age, 64.4; SD, 9.7 years) and less commonly male (49.2%). Among them the initial statin was less commonly simvastatin (70.4%) and more commonly atorvastatin (11.0%) rosuvastatin (5.6%), fluvastatin (5.7%), pravastatin (4.1%), lovastatin (2.0%) or a combination of simvastain and ezetimibe (1.2%) (p-values for all comparisons <0.0001).

Overall, the number of new users decreased rapidly with the increasing length of statin-free period required before the first statin purchase in 2007: 27.7% of the new users defined as those with a 1-year purchase-free period did not qualify as such when using a 10-year gap (Fig 3). When using a 1-year gap, women aged 65-84 were somewhat more commonly (34.1%) misclassified as new users than were men of the same age (30.1%) or women and men aged 45-64 (24.1% and 24.8%).

Discussion

Our observations based on unselected populations of statin initiators from a country with universal prescription drug coverage support the view that long-term statin use is dynamic [START_REF] Brookhart | Physician follow-up and provider continuity are associated with long-term medication adherence[END_REF]. More specifically, we observed that most statin users had at least 1 extended period of non-use during an average of 8.2 years of follow-up. The majority (86-92%) of those who stopped statin use, however, reinitiated it.

Accordingly, almost 30% of persons with no statin purchases within 1 year preceding the initation (a conventional definition for new use) were not statin-naíve when the required period of no statin purchases was extended to the previous 10 years. Finally, while only every sixth statin initiator maintained good adherence and was therefore supposedly exposed to an optimal statin therapy throughout the whole 10year period, every second accumulated altogether 6 or more years of such exposure.

Incidence of 90-and 180-day treatment gaps and also the rates of reinitiation after the first gap were slightly higher in our study than in a Canadian study observing persons who initiated statin therapy between Jan 1997 and June 2004 [START_REF] Brookhart | Physician follow-up and provider continuity are associated with long-term medication adherence[END_REF]. This may partly be explained by the differences in reimbursement rules affecting the refill patterns, the typical duration of a statin prescription being 1 month in Canada and 3 months in Finland. Indeed, our sensitivity analyses showed that the estimated rates of discontinuation and reinitiation depend considerably on the gap length chosen, supporting our view that applying shorter gap lengths may overestimate the rate of interruptions in statin use in the Finnish context. Interestingly, our estimates on discontinuation and restarting of and good adherence to statin therapy were only marginally affected by the definition of new use in 1997. However, while about 10% of the 1997 sample defined as new statin users with a conventional definition of no statin purchases during the preceding year had purchased statins within the previous 3 years, the respective proportion in the 2007 sample was already about 20%. In addition, the differences between those qualifying as new users with the 1-year gap but not with the 3-year gap seemed to be larger in 2007 than in 1997. One reason for the differences in the distribution of the initiating statin in 2007 may be the reimbursement restriction of atorvastatin and rosuvastatin [START_REF] Martikainen | Impact of restricted reimbursement on the use of statins in Finland. A register-based study[END_REF]. In practice, the restriction meant that the initiators of atorvastatin and rosuvastatin with no trial with less expensive statins did not receive reimbursement for their statin and their purchases did not appear in the Prescription Register either. About half of those using atorvastatin and 40% of those using rosuvastatin in 2006 switched to simvastatin or another less expensive statin by the end of 2007 [START_REF] Martikainen | Impact of restricted reimbursement on the use of statins in Finland. A register-based study[END_REF]. It is possible that not all statin purchases by the switchers were captured in the Prescription Register and some switchers may have appeared as new users in 2007. Therefore, the rates of misclassification with the shortest washout periods might be exceptionally high in 2007. Overall, our findings indicate that comparisons of incidence rates of statin use across time periods, population subgroups and between studies using different statin-free periods should be done cautiously.

Our observations on the differences in cohort characteristics according to the definition of new use may have implications especially for studies assessing acute health outcomes of statin use. For example, when an inception cohort of statin initiators in 2007 is defined based on 1 year versus 3 or more years of no statin purchases, the cohort is more likely to include older women due to their greater chance of being misclassified. Consequently, for example, the estimates of relative risks of potential adverse events such as rise in liver enzymes and myopathies with the peak in incidence during the first year of treatment [START_REF] Hippisley-Cox | Unintended effects of statins in men and women in England and Wales: population based cohort study using the QResearch database[END_REF] may be more biased in this than other population subgroups.

Our study has limitations. Despite their obvious generalizability to all ambulatory statin users in Finland and to those in other countries with similar health care and drug coverage systems, the results could be different, for example, in heterogenous North American populations with various prescription drug coverage plans [START_REF] Schneeweiss | Adherence to statin therapy under drug cost sharing in patients with and without acute myocardial infarction: a population-based natural experiment[END_REF]. Furthermore, we may have misclassified statin use, persistence and adherence for many reasons. First, we did not have data on hospitalizations nor is the drug use in hospital or other institutional care captured in the Register, potentially leading to overestimation of gaps in statin use and underestimation of adherence. Second, we assumed that the dosage for all users was 1 tablet per day.

Because of splitting of tablets, we may have underestimated the adherence; however, splitting of tablets seems rare [START_REF] Dormuth | Factors related to splitting tablets of HMG-CoA reductase inhibitors: A population based analysis in British Columbia[END_REF]. Conversely, prescribing of dosages of more than 1 tablet per day would have lead to overestimation of adherence and persistence. Finally, we approximated the patients' actual drug use based on dispensations, and therefore we could not be sure if and how the dispensed statins were used. Neither 2 for the numbers of patients in follow-up).

Figure legends Fig 1 Fig 2

 legends12 Figure legends

Fig 3

 3 Fig 3 Proportions (%) of persons misclassified as new users in 2007 when using statin-free periods of different lengths, by age and sex. Reference group: those with no statin dispensation during the preceding ≥10 years.

Table 1

 1 Characteristics of statin initiators, defined using 1-and 3-year gaps of no statin purchases before the first purchase in 1997 and 2007 Statin initiator defined as a person with no statin purchases within the preceding 1095 days and b as a person with no statin purchases within the preceding 365 days.

		1997		2007
	Characteristic	3-year gap a	1-year gap b	3-year gap a	1-year gap b
	No. of persons	32 760	36 581	78 512	99 348
	Male, n (%)	15 534 (47.4)	17 653 (48.3)	41 891 (53.4)	52 142 (52.5)
	Age, mean (SD), y	63.0 (8.9)	63.0 (8.9)	62.5 (9.6)	62.9 (9.7)
	Age, n (%)
	45-54 y	6685 (20.4)	7440 (20.3)	17 635 (22.5)	21 156 (21.3)
	55-64 y	10 937 (33.4)	12 310 (33.7)	30 368 (38.7)	37 876 (38.1)
	65-74 y	11 983 (36.6)	13 317 (36.4)	19 499 (24.8)	25 420 (25.6)
	75-84 y	3155 (9.6)	3514 (9.6)	11 010 (14.0)	14 896 (15.0)
	Initial statin, n (%)
	Simvastatin	14 952 (45.6)	16 513 (45.1)	70 123 (89.3)	84 787 (85.3)
	Fluvastatin	10 362 (31.6)	11 268 (30.8)	2170 (2.8)	3364 (3.4)
	Lovastatin	5372 (16.4)	6478 (17.7)	299 (0.4)	724 (0.7)
	Pravastatin	2074 (6.3)	2322 (6.4)	987 (1.3)	1832 (1.8)
	Atorvastatin	NA	NA	2750 (3.5)	5032 (5.1)
	Rosuvastatin	NA	NA	1661 (2.1)	2833 (2.9)
	Simvastatin+Ezetimibe	NA	NA	522 (0.7)	776 (0.8)

a

were we able to address the reasons why patients stopped or restarted their statin use [START_REF] Brookhart | Physician follow-up and provider continuity are associated with long-term medication adherence[END_REF][START_REF] Steiner | Can we identify clinical predictors of medication adherence . . . and should we?[END_REF].

In conclusion, statin use is dynamic and reinitiations after treatment gaps are common. This should be taken into consideration in clinical practice and especially when studying the incidence, patterns and health outcomes of statin use.

Aknowledgements

This study was funded by a grant (October 26, 2007) from the Social Insurance Institution (SII) of Finland (Maarit Jaana Korhonen and Arja Helin-Samivaara).

We thank Kristiina Tyrkkö and Hilkka Ruuska of the SII for their expertise in extracting and managing the administrative data.