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A role for Apolipoprotein A-I in the pathogenesis of multiple sclerosis
A Role for Apolipoprotein A-I in the pathogenesis of Multiple Sclerosis Lindsay Meyers, Chassidy J. Groover, Joshua Douglas, Sangmin Lee, David Brand, Michael C. Levin, Lidia A. Gardner PII: DOI: Reference:
S0165-5728(14)00944-8 doi: 10.1016/j.jneuroim.2014.10.010 JNI 476019
To appear in:
Journal of Neuroimmunology
Received date: Revised date: Accepted date:
24 June 2014 20 October 2014 24 October 2014
Please cite this article as: Meyers, Lindsay, Groover, Chassidy J., Douglas, Joshua, Lee, Sangmin, Brand, David, Levin, Michael C., Gardner, Lidia A., A Role for Apolipoprotein A-I in the pathogenesis of Multiple Sclerosis, Journal of Neuroimmunology (2014), doi: 10.1016/j.jneuroim.2014.10.010
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ACCEPTED MANUSCRIPT A Role for Apolipoprotein A-I in the pathogenesis of Multiple Sclerosis
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Lindsay Meyers1, Chassidy J. Groover1, Joshua Douglas1, Sangmin Lee1,2, David Brand2, Michael C. Levin1,2, and Lidia A. Gardner1,2 Research Service VAMC, Memphis, TN, 38104, 2 Department of Neurology University of
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Tennessee Health Science Center, Memphis, TN, 38163.
Corresponding Author:
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Lidia A. Gardner, Ph.D. University of Tennessee Health Science Center
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Memphis, TN, 38163
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855 Monroe Avenue, Rm. 415
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Department of Neurology
Ph. +1(901) 5238990 Ext.6477 Email: [email protected]
1. Introduction
There are three general types of MS, relapsing remitting MS (RRMS), primary progressive MS (PPMS) and secondary progressive MS (SPMS). Clinically there are some clear differences between RRMS, SPMS and PPMS. For example, RRMS is two times more likely to occur in women than men, whereas the gender distribution in PPMS is about equal (Coyle 2005). Diagnosis of a specific MS type is largely dependent on presentation of neurological symptoms. The PPMS typically has a worse prognosis compared to RRMS (Pelfrey, Cotleur et al. 2002). It is not clear why some RRMS patients progress to the SPMS while others do not.
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ACCEPTED MANUSCRIPT We hypothesized that distinction between types of MS might be found in the differential expression of proteins in patient’s plasma. We analyzed protein expression in plasma of patients with
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three MS subtypes and age-matched controls. Proteins were separated by two-dimensional gel electrophoresis and identified with matrix-assisted laser desorption ionization (MALDI) time of flight
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(TOF) mass spectroscopy. The protein that was consistently different in MS samples compared to
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controls was identified as Apolipoprotein A-I (Apo A-I). Apo A-I is a constitutive anti-inflammatory
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molecule that recently has been shown to be a marker of clinical response to interferon-beta therapy in MS patients (Gandhi, McKay et al. 2010). Importantly, progressive MS patients had significantly
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lower plasma levels of Apo A-I expression compared to healthy controls, suggesting that high levels of Apo A-I are neuroprotective. According to our data, Apo A-I levels could not be used as a stand-
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alone diagnostic biomarker, however levels of this protein present in patient’s serum might be useful
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in assessing disease progression. We used experimental autoimmune encephalomyelitis (EAE) to
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validate our observation and test the hypothesis that mice deficient in Apo A-I would develop worse disease. EAE induction in mice deficient in murine Apo A-I resulted in worse disease compared to control animals. Taken together, these data indicates that Apo A-I might play an important role in the pathogenesis of MS.
2. Materials and Methods 2.1. Human samples Two hundred eight samples were utilized for Apo A-I studies from three different sources: 1) patients from the local clinic (53 RRMS, 15 SPMS, and 8 PPMS); 2) healthy adult volunteers from Memphis community (52 healthy control samples); 3) samples obtained from the “Accelerated Cure Project (www.acceleratedcure.org)” (35 SPMS; 45 PPMS). All samples were collected from fasting individuals (12h), according to the approved Institutional Review Board (IRB) protocol with patient
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ACCEPTED MANUSCRIPT consent. Controls were matched for age and gender to disease groups. Blood samples were drawn from the antecubital vein into heparin-coated (for plasma) and silicone coated (for serum) tubes. All
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samples were processed within 30 min from collection and stored at -80C for future use. 2.2. Enzyme Linked Immuno-Sorbent Assay (ELISA).
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Apo A-I levels were measured with a commercially available kit from Eagle Biosciences (Nashua,
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NH), and an ‘in house’ developed ELISA assays. Assays were done in duplicates. Briefly, diluted
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standards and samples (1:200 in PBS) were applied onto anti-Apo A-I pre-coated plate, incubated at 37C for 1 h. After each incubation step plates were washed four times in TBST (100mM Tris, pH
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7.4 + 0.02% tween). Secondary HRP conjugated antibodies were added to the plates for 1h at 37C. Plates were washed as described before and TMB/peroxide substrate was added for 30 min. Reaction
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was terminated with 0.5N sulfuric acid. The ‘in house’ direct ELISA had better sensitivity to Apo A-I
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protein. We utilized mouse monoclonal anti-Apo A-I antibodies (Abnova, Taiwan) to coat plates
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overnight. Plates were washed and blocked with 0.5% BSA prior to sample application and after each consecutive step. Samples were diluted as described above and incubated on the plate for 1h at 37C. HRP conjugated anti-Apo A-I antibodies were diluted in coating buffer (10mM PBS, pH 7.4) and applied to the plate. TMB substrate and stop solution were obtained from Thermo Fisher Scientific (Waltham, MA). Reaction optical densities were read at 450 nm in a microtiter plate reader (Molecular Devices, LLC). Apo A-I concentration was calculated based on the standard curve numbers with 4-parameter algorithm in Soft Max Pro software. 2.3. Antibodies Primary anti-human Apo A-I mouse monoclonal antibodies were obtained from Abnova (Taipei City, Taiwan). Anti-mouse Apo A-I goat polyclonal antibodies (Santa Cruz, CA) and secondary antimouse HRP antibodies were obtained from GE Healthcare (Pittsburg, PA). Apo A-I antibodies from
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ACCEPTED MANUSCRIPT Santa Cruz specifically recognize the mouse protein sequence; the epitope is located in the Cterminal domain of Apo A-I, where identity with human sequence is 50%.
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2.4. Protein electrophoresis For one-dimensional Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (1-D SDS-PAGE)
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plasma samples were diluted 8 times with 1xPBS and mixed with 2x-sample buffer. All samples
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were heated to 95C for 5 min to denature protein, chilled on ice for 2 min and loaded onto 12%
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SDS-PAGE gels (Bio-Rad, Hercules, CA). A total of 60µg of protein were loaded per well. Proteins were separated and transferred onto polyvinylidene difluoride (PVDF) membranes for Western
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blotting. Apo A-I was detected with primary anti-Apo A-I antibodies (1:2000), Abnova (Taipei City, Taiwan), and secondary anti-mouse HRP (1:10,000), GE Healthcare. Proteins were detected using
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either X-ray film, and/or digital imaging. Band intensity was quantified using Quantity One Software
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(Bio-Rad, Hercules, CA). Numbers were analyzed and graphed in Prism GraphPad software.
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2.4.1. Preparative IEF (Rotofor).
Plasma proteins were separated based on pI using Rotofor IEF (isoelectric focusing) with soluble ampholites in solution. Fractions were collected, washed and concentrated using Amicon filters with 10kDa membranes. Protein concentration of each fraction was measured on a Nanodrop spectrophotometer. Five micrograms of each fraction was then loaded onto 1-D SDS PAGE for protein expression analysis. Fractions that had differential expression were extracted and analyzed with MALDI-TOF. 2.4.2. Two-dimensional (2-D) SDS-PAGE. Plasma samples were albumin, IgG and salt depleted with a kit from Pierce (Rockford, IL). Protein concentration was measured on a Nanodrop spectrophotometer. Equal amounts of protein (150µg) for each sample were separated by 2D-PAGE using a pI range 4–7 strip and stained with SYPRO Ruby stain. The gels were scanned on Typhoon imager (GE Healthcare, Pittsburg, PA), and TIFF
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ACCEPTED MANUSCRIPT images were imported into PDQuest image analysis software (Bio-Rad, Hercules, CA). Protein spots were detected using the automated spot detection feature. The images were visually inspected at high
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resolution and corrected for undetected or incorrectly detected spots/artifacts. Upon completion of the image analysis, data were stored both as a 2D image and Gaussian image. The 2-D dataset
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consisted of 20 samples that were divided into four groups (controls, RRMS, SPMS and PPMS) of
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five samples in each. Within the “MatchSet” produced by the image analysis software, 5 Apo A-I
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spots were identified as being expressed in at least one of the 20 gels. We used the default normalization of data in the MatchSet that is based on total pixel quantity in valid spots. This
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normalization method assumes that few protein spots change within the experiment and that the changes averaged out across the whole gel. PDQuest software includes three statistical tests for data
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analysis, the Student’s t test (a parametric test) and the Mann-Whitney rank sum test and Wilcoxon
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signed rank test (both non-parametric tests). We used Mann-Whitney rank sum test. The intensity
2.5. Animals
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values that were generated in PDQuest were analyzed and graphed in Prism GraphPad software.
C57BL/6J and C57BL/6-Tg (APO A-I) 1Rub/J mice were purchased from Jackson Laboratories (Bar Harbor, ME). The Apo A-I deficient mice (C57BL/6-Tg (APO A-I) 1Rub/J) were originally developed in laboratory of Dr. Rubin (Laurence Berkley National Laboratories) and donated to Jackson Laboratories. These mice carry the human apolipoprotein A-I transgene and show a two-fold increase in total plasma cholesterol levels and greater than a four-fold decrease in the levels of mouse Apo A-I. Mice colonies were maintained by sister-brother mating’s under specific pathogen free (SPF) conditions at 24 ± 2°C with a light-controlled regimen (12h light/dark cycle). Tap water was provided ad libitum. All mice included in our EAE experiments were housed in the same facility and were on the same standard chow diet (Harlan, Laboratories, Indianapolis, IN) for at least 2 weeks prior experimental treatment.
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ACCEPTED MANUSCRIPT 2.6. Induction of EAE Only female mice (10-12 wks. old) were used in EAE experiments. All experimental procedures
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were carried out in accordance with approved animal protocol. EAE was induced by immunization with an emulsion of MOG35-55 (Myelin Oligodendrocyte Glycoprotein) in complete Freund's adjuvant
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(CFA), followed by administration of intraperitoneal pertussis toxin (PTX) in phosphate-buffered
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saline (PBS) (first on the day of immunization and then again the following day) according to the
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manufacture’s instructions (Hooke laboratories, St. Lawrence, MA). Four EAE experiments with 5 animals per group were conducted. EAE was scored on a 0-5 scale: 0 no disease, 1- limp tail and
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hind paw inhibition, 2- poor balance, limp tail, dragging hind paws, 3- limp tail with paralysis of one front and one hind paw, 4- mouse has minimal movement in the front paws, both hind paws are
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paralyzed, and 5-mouse is euthanized due to severe paralysis. At the onset of the disease solid food
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pellets were supplemented with raspberry gelatin cubes to help animals maintain body weight. The
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clinical score results were averaged from all experiments. 2.7. Flash –Visual Evoked Potentials (F-VEP) F-VEP was performed as previously described (Sullivan, Geisert et al. 2011). Briefly, after an overnight dark adaptation, mice were anesthetized with an intraperitoneal injection of ketamine/xylazine/urethane (25/10/800μg/g body weight). Body temperature was maintained at 37C with a heating pad. Pupils were dilated with 1% atropine. Platinum needle electrodes (Grass Technologies, West Warwick, RI) were placed approximately 3 mm lateral to lambda over the left and right cortex. Flashes of white light at an intensity of 1.0 cd·sec/m2 were presented in a Ganzfeld dome (Diagnosys, Lowell, MA). The flash frequency was 1 Hz with an inner sweep delay of 500msec. Each result was an average of 200 sweeps. The results were exported in to an Excel spreadsheet and averaged for quantification of amplitude and latency. Recordings were analyzed in GraphPad software as described below.
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ACCEPTED MANUSCRIPT 2.8. Statistical analysis One-way analysis of variance (ANOVA) followed by a pair-wise Bonferroni post hoc comparison
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test, with a p value ≤ 0.01 considered statistically significant, was used to compare relative band intensity counts, ELISA results, and F-VEPs. Statistical analysis was performed with Prism 4.0
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software (GraphPad, San Diego, CA).
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2.9. Immunohistochemistry
At the end of EAE experiments, mice were deeply anesthetized with ketamine/xylazine injection and
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perfused with 4%paraformaldehyde. Brain and spinal column were extracted and post-fixed in 4% paraformaldehyde over night at 4C. Spinal columns were decalcified in decalcifying solution
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(Thermo Fisher Scientific, Waltham, MA), washed in deionized water and processed for paraffin
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embedding. Paraffin slices were mounted onto glass slides for H&E (Hematoxylin/Eosin) and LFB
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(Luxol Fast Blue) staining. For Fluoro-Jade staining slides were de-waxed in xylene, immersed in 100% ethanol for 5 min, 70% alcohol for 2 min, and then rinsed with two 1-min changes if dd-H2O. Slides were then incubated with freshly made 0.06% potassium permanganate for 17 min and rinsed with two 1-min changes of ddH2O. The 0.001% Fluoro Jade staining solution was made in 0.1% acetic acid. Slides were stained for 30 min at room temperature in the dark, and then air-dried, dipped in xylene and covered with VectaShield mounting media (Vector Laboratories, Burlingame, CA). For Apo A- I staining, slides were deparaffinized in xylene (3X for 5 min), 100% Ethanol (3X for 5 min), 70% Ethanol (1X for 5 min), and H2O (2X for 5 min). Slides were blocked with donkey serum for 30 min at room temperature (RT) and incubated with primary goat polyclonal antibodies that recognize mouse Apo A-I protein (Santa Cruz, CA) over night at 4C. Slides were then washed with 1x TBST and secondary anti-goat TX Red (Vector Laboratories, Burlingame, CA) were applied for 2h at RT. After application of secondary antibodies, slides were washed 4X with TBST, cleared in
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ACCEPTED MANUSCRIPT xylene and mounted with DPX. We examined 20 fields per condition and used 10 fields for quantitative analysis. For quantitation purposes slides were examined under Axio Observer A1
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microscope (Carl Zeiss, Thornwood, NY). All sections were scored as follows: 0, no infiltration/demyelination (<50 cells); 1, mild infiltration/demyelination of nerve or nerve sheath (50–
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100 cells); 2, moderate infiltration/demyelination (100–150 cells); 3, severe
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infiltration/demyelination (150–200 cells); 4, massive infiltration/demyelination (>200 cells).
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2.10. Flow cytometry
Single cell suspensions of spleens were obtained 36 days post-immunization using standard
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technique (Zhou, Yuan et al. 2011). Spleen cell suspensions were obtained by hemolysis using sterile technique. Single-cell suspensions of spleenocytes (0.5x 106) were incubated with the following
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fluorescently labeled antibodies (CD3, CD4, CD19, CD25). Foxp3 staining was done on permeabilized cells with Foxp3 staining kit form eBioscience (San Diego, CA) according to the
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manufacturer’s protocol. All fluorescently labeled antibodies were purchased from BD Pharmingen (San Diego, CA). Labeled cells were washed, resuspended in 1% paraformaldehyde, and analyzed using a LSR-II flow cytometer.
2.11. Measurement of the cytokines
Cytokines were measured in the serum collected from experimental animals prior to sacrifice. We used mouse multi- analyte ELISArray kit from Qiagen (Valencia, CA). Briefly, 50µl of serum was mixed with equal amount of assay buffer, added to the plate and incubated for 2h at room temperature. Standard antigen cocktail containing cytokines of interest was used for quantification purposes. Plates were washed after each step. Detection antibodies were added and incubated for 1h then Avidin-HRP for 30 min. Plates were extensively washed, dried and TMB/peroxide substrate was added for 30 min. Reaction was stopped and read at 450 nm in a microtiter plate reader (Molecular
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ACCEPTED MANUSCRIPT Devices, LLC). Absorbance values outside of linear range of 0 - 2.5 were not included in the analysis.
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3. Results
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We used serum and plasma derived from blood collected from fasting MS patients (n=156: 53
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RRMS, 50 SPMS and 53 PPMS) and age-matched controls (n=57). Serum was primarily used for Western blot and ELISA measurements and plasma for 2-D analysis. We realized that high amounts
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of albumin and immunoglobulin (IgG) might mask other proteins present in human plasma. Therefore, the samples were albumin, salt and IgG depleted. Samples were then subjected to
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isoelectric focusing in Rotofor Cell (Bio-Rad, Hercules, CA) (Fig. 1A.) Fractionated samples were separated by molecular weight on SDS-PAGE gels (Fig. 1A, inset); bands were extracted and
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subjected to MALDI-TOF. The protein that was consistently different in MS samples compared to controls was identified as Apolipoprotein A-I (Apo A-I) with 97.8% positivity. To verify MALDI-
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TOF results, we performed 2-D electrophoresis on five samples from each MS type and controls. Apo A-I was identified on 2-D gels at molecular weight of 28kDa and pI range 5.3-5-6 on pH 4-7 strips (Fig. 1B, circle, Fig. 2B, shown control sample). The most striking difference in Apo A-I expression was identified between controls and PPMS (Fig. 1B, 2B). Samples were separated on 2-D gels, stained with SYPRO Ruby protein stain and scanned for analysis. Our sample set included 20 gels with 5 samples in each group. To verify Apo A-I expression, 2-D gels were transferred onto nitrocellulose membrane and probed with anti-Apo A-I antibodies (Fig. 1C). It is not clear why antibodies recognized only two out of five Apo A-I spots in controls and RRMS. There was limited reactivity in SPMS and none in PPMS samples. A possible explanation to this discrepancy might be in epitope specificity for Apo A-I expression. Not all of the spots on the 2-D gel might have had the perfect epitope match for this specific antibody. According to the reference database (SWISS2DPAGE) Apo A-I is represented in five major spots between 22873 and 23000 Da and pI range 9
ACCEPTED MANUSCRIPT 4.99-5.22 and four additional spots between 7490 and 9451 Da and pI range 7.27- 4.99, correspondingly. We analyzed five major protein spots as detected by SYPRO Ruby staining
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expressed on our gels at ~22,8 kDa, (Figs. 2A, B). Spots three and four were consistently different between controls and MS patients and correlated with disease type. Therefore, we focused our
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analysis on Apo A-I spots 3 and 4 (Figs. 2B, C). Protein intensity for each spot was averaged from
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five gels per group and groups were compared to each other and to controls using one-way ANOVA.
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The fold change of averaged normalized volumes was calculated between controls and PPMS patients (Fig. 2B, inset table). Patients with SPMS and PPMS had lower Apo A-I spot intensity
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compared to samples collected from RRMS. This analysis revealed that healthy age-matched controls had the highest Apo A-I spot intensity, followed by the RRMS, SPMS and PPMS groups. The PPMS
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group had lowest spot 3 and 4 intensity in comparison to any group. Finally, spots two and five had
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lower expression in comparison to controls but showed no correlation with disease type.
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Unlike 2-D analysis, 1-D SDS electrophoresis allows for simultaneous protein detection in multiple samples. Therefore, we subjected depleted serum samples to SDS-PAGE and subsequent Western blot analysis with anti-Apo A-I antibodies (Fig. 3A). Equal amounts of protein were loaded per well; separated proteins were transferred onto a nitrocellulose membrane and stained with Ponseau-S solution to label total protein load. Membranes were rinsed and probed with anti-Apo A-I antibodies. Bands were quantified with QuantityOne software (Bio-Rad, Hercules, CA). Quantitative Western blot analyses revealed differences between MS samples in comparison to controls. All MS patients had lower Apo A-I expression when compared to healthy controls (Fig. 3A). Among MS groups RRMS samples had highest ApoA-I expression and were statistically different (P<0.01) from progressive forms of disease. To answer the question if Apo A-I could serve as a biomarker of disease progression in MS, we measured Apo A-I expression in human serum and plasma by ELISA. Normal Apo A-I levels in 10
ACCEPTED MANUSCRIPT human plasma ranged between 0.9-1.3 g/L. We quantified Apo A-I expression in a set of 208 samples (Table 1). Patient samples were age-matched to controls. Commercially available ELISA (Eagle
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Biosciences) showed no statistically significant difference between the groups. In addition, Apo A-I values obtained by this method often exceeded normal Apo A-I range.
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To address this discrepancy we developed an “in house” direct sandwich ELISA. We utilized
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mouse monoclonal antibodies as the capture antibodies (Abnova, Taipei City, Taiwan), which were
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also used in the 1- and 2-D analyses (Figs. 1 and 2). These antibodies were conjugated to EZ-Link Plus activated peroxidase (Fisher Thermo Scientific, Waltham, MA). Results obtained with the ‘in
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house’ ELISA were more consistent with values seen in 1- and 2-D analyses and nearer to the reported range on Apo A-I plasma expression. According to our data, the average Apo A-I values in
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controls were 2.1g/L, with some samples actually exceeding the upper limit of detection (Fig. 3B).
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The RRMS samples had an average of 1.6g/L, which is also within the normal range. Average Apo
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A-I levels in SPMS samples were 0.9g/L and PPMS were between 0.5-0.9g/L (Fig. 3B). Each type of MS showed statistical significance compared to healthy controls (Fig 3B). Subjects that were on medication affecting Apo A-I levels, such as statins and NSAIDs (non-steroidal anti-inflammatory drugs) were excluded from the analysis. We hypothesized that Apo A-I might have a protective role in MS due to its anti-inflammatory activity. To test our hypothesis, we utilized wild type (WT) and Apo A-I transgenic (Tg) mice (C57BL/6-Tg (Apo A-I) 1Rub/J). These animals have four times less mouse Apo A-I in the circulation compared to the wild type mice (C57BL/6J) due to the presence of human Apo A-I transgene regulated by a human promoter (Rubin, Ishida et al. 1991). These mice have increased amount of total ApoA-I in their serum, however the expression of mouse ApoA-I is four fold reduced (Fig. 4 A, B. The protein sequence of mouse (264 AA) and human (267AA) Apo A-I is highly homologous, except at its C-terminal domain, where the two sequences share only 50-80% homology. 11
ACCEPTED MANUSCRIPT We utilized antibodies produced to the epitope located between amino acids 250 and 264 of the mouse Apo A-I protein (Santa Cruz, CA). These antibodies recognized endogenous mouse Apo A-I
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protein in the serum, liver and the spinal cord of the WT mice and serum of the Apo A-I deficient animals (Fig. 4A, B). In contrast, there was no mouse Apo-A-I protein detected in the liver or spinal
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cord of Tg mice. We utilized the same antibodies to confirm Apo A-I expression on histological
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spinal cord sections (Fig. 4C), which showed expression of Apo-A-I protein in WT but not in Tg
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mice. Phenotypically, Tg mice do not differ from the WT animals (C57BL/6J). Therefore these mice present a suitable model to investigate how low levels of Apo A-I affect EAE progression. We
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initiated EAE in Apo A-I deficient and WT animals and recorded clinical scores (Fig. 5A). Mice (1012 weeks old) were injected with MOG35-55 according to the manufacture’s instructions (Hooke
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laboratories, St. Lawrence, MA). Disease onset was between days 12 and 15 after injections. Both
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groups developed EAE, however mice deficient in murine Apo A-I exhibited higher EAE scores (Fig.
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5A, P<0.001), more vacuolization in the spinal cord and damage to optic nerves in comparison to controls (Fig. 5C). Larger vacuolization was seen on both H&E and LFB stained spinal cord tissue (Fig. 5C, Fig. 6). Vacuolization could be a sign of neuronal degeneration or changes in myelin. Therefore we calculated demyelination scores by scoring each quadrant of a spinal cord for the presence and absence of demyelination. For each group 10 lumbar spinal sections were scored 36 days after EAE induction. Results are expressed as the mean pathological score SEM. Naïve mice no demyelination, C57BL/6J - 2.5 0.2 and Apo A-I deficient - 4 0.2. Vacuolization was scored on high magnification images of the spinal cord. The number of vacuoles were calculated per filed view quadrant as follows: 1 - 5 vacuoles = score of 1, 5 - 6 vacuoles = score of 2, 10 - 30 vacuoles = score of 3. ApoA-I deficient mice had higher demyelination score (3. 4 0.2) in comparison to wild type mice (2.9 0.2), Fig. 6.
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ACCEPTED MANUSCRIPT Degenerating neurons were visualized with Fluoro-Jade B, a known marker of neurodegeneration, Fig. 5C (Schmued and Hopkins 2000; Bian, Wei et al. 2007; Ehara and Ueda
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2009; Ravikumar, Jain et al. 2012). Tissue collected from naïve mice did not uptake Fluoro-Jade B staining. Fluoro-Jade staining was more prominent in brain (not shown) and spinal cord sections of
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Apo A-I deficient in contrast to wild type animals (Fig. 5C). These data are in agreement with the
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demyelination scores. Thus, the Tg mice showed evidence of tissue damage (vacuolization) that
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appears to be the result of both damaged myelin and neurodegeneration. We repeated these experiments six times and obtained similar results.
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Optic neuritis is a common MS symptom. Therefore we recorded optic nerve function with FVEP tests (Fig. 5B). F-VEPs were measured in the visual cortex of naïve, Apo A-I deficient and wild
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type mice. The classical N1 and P1 peaks in the F-VEP averaged waveforms were seen in naïve and
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wild type (C57BL/6J) mice. For a signal to reach the visual cortex both the retinal ganglion cells
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soma and axon must be intact. EAE animals had severely diminished P2 and N2 signals. Apo A-I deficient animals had delayed latency (Fig. 5B) and a number of these mice had no detectable signal at all. The average amplitude and the latency of the N1 and P1 peaks were quantified. Wild type mice had P1 (25.74.2μV) and N1 (58.3 6.1μV) peak amplitudes. Naïve Tg mice had P1 (15 4.8μV) and N1 (45 5.2μV) peak amplitudes. Apo A-I deficient EAE mice had no detectable peaks between 0-500ms. Both groups of mice with EAE had significantly different F-VEP recordings from naïve mice, P<0.001. In addition, Apo A-I deficient mice developed necrotizing tail lesions that were not seen in the control animals (data not shown). Similar lesions were observed in exclusively neurologically affected gelatinase B deficient mice with EAE (Dubois, Masure et al. 1999). These data suggests that Apo A-I deficient transgenic animals had worse EAE compared to wild-type control mice.
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ACCEPTED MANUSCRIPT Knowing that T - cell differentiation plays an important role in MS and EAE, we used flow cytometry to characterize populations of T – cells isolated from the spleens of these animals with
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flow cytometry (Fig. 7A). According to our data, Apo A-I deficient mice showed an insignificant trend for increased number of regulatory T cells (CD25+/Foxp3+) compared to WT and naive mice.
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We also used ELISA to measure the expression of different cytokines in these animals and found that
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Apo A-I deficient mice had statistically significant differences in the expression of IL -2, IL-23, INF.
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TNF- and TGF-ß levels showed a similar trend, consistent with inflammatory response to EAE (Fig. 7B). IL-17A levels were not statistically different between the wild type and Apo A-I deficient mice.
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Taken together, these observations support our hypothesis that Apo A-I might play a role in EAE, an
4. Discussion
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animal model of MS pathogenesis.
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Human brain has high lipid content; therefore lipids and their turnover are good candidates as contributors to diseases of the CNS. However, the importance of lipids and their metabolism in MS has been understudied. Myelin associated glycoproteins have been studied in connection with autoantibodies to MOG (myelin oligodendrocyte glycoprotein) and MBP (myelin basic protein) (Bernard, Johns et al. 1997; Genain, Cannella et al. 1999; Haas, Hug et al. 2005) for several decades now. One recent study proposed that sulfatides (major component of myelin lipids) suppress T cells activation and prevent potential induction of autoimmunity within the CNS (Mycko, Sliwinska et al. 2014). Disturbances in lipid metabolism can lead to myelin loss, neuronal degeneration and metabolic distress (Levin 2014). Weinstock–Guttman showed an association between serum lipid profiles and disability and MRI outcomes in MS (Weinstock-Guttman, Zivadinov et al. 2011). It is well known that Apo A-I plays an important protective role in atherosclerosis, cardiovascular and cognitive diseases, however its function in MS has not been fully investigated. In humans elevated 14
ACCEPTED MANUSCRIPT Apo A-I levels have been associated with APOA1 A allele. Specifically, carriers of this allele performed significantly better on semantic verbal fluency and the Stroop interference tests (Koutsis,
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Panas et al. 2009). The authors of this study evaluated 138 patients with MS and 43 controls and concluded that they found an association of the ApoA1 -75G/A promoter polymorphism with
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cognitive performance in MS (Koutsis, Panas et al. 2009). In addition, Apo A-I may play a role in
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neuronal regeneration (Vollbach, Heun et al. 2005) by acting as a constitutive anti-inflammatory
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factor (Burger and Dayer 2002). Apo A-I has also been described as a biomarker of clinical response to interferon-beta (INF-ß) treatment in a small cohort of MS patients (Gandhi, McKay et al.). A
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different study measured Apo A-I levels before and after INF-ß treatment in MS patients and found that patients, who suffered relapses or progressed in their EDSS had lower plasma Apo A-I levels,
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compared to their basal values (Sena, Pedrosa et al. 2000). These studies point to an important role
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that Apo A-I might play in MS pathogenesis, however none measured Apo A-I levels in different MS
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subtypes or examined correlation with disease progression. We identified that Apo A-I is differentially expressed in serum and plasma of MS patients and age-matched controls. According to our data the average Apo A-I levels detected in all MS types were lower than in controls. Expression of Apo A-I was remarkably different in progressive MS patients, particularly by Western blot. Perhaps a longitudinal study could answer a question if low Apo A-I levels could predict or influence the development of SPMS in RRMS patients. The experiments that we undertook in a mouse model of MS (EAE) supported our hypothesis that Apo A-I plays a beneficial role in MS. We chose Apo A-I deficient animals instead of knockouts because they have reduced Apo A-I levels of endogenous murine Apo A-I, much like MS patients. To the best of our knowledge we are the first to initiate EAE in these animals and show that reduction in Apo A-I expression results in greater neurological deficits on clinical, electrophysiological and histological levels. The limited amount of endogenous murine Apo A-I in
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ACCEPTED MANUSCRIPT the spinal cord of Tg mice correlated with higher demyelination and degeneration scores, indicating a possible protective role of this protein in the CNS. MS is the most common immune-mediated
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disease of the CNS (Levin, Lee et al. 2013). It is possible that inflammation in the periphery exacerbated by the low Apo A-I levels could trigger faster Th2 cell penetration of into the CNS,
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soliciting neuronal demyelination and degeneration.
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The F-VEP is an appropriate electrophysiological test to study functional loss in optic nerve.
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(Diem, Sattler et al. 2005; Sullivan, Geisert et al. 2011; You, Klistorner et al. 2011). If either the RGC soma or axon is compromised the signal cannot reach the visual cortex and therefore is not
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detected by F-VEP (Sabel and Aschoff 1993). At the time of sacrifice the majority of Apo A-I deficient mice had minimal cortical response as measured by F-VEP. The EAE wild type mice also
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had impaired F-VEP recordings in comparison to control group. However these mice had detectable
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N1 and P1 amplitudes and almost normal latencies, indicating extensive cell death, but normal
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synaptic connectivity in the remaining cells. These data suggests that Apo A-I deficient mice had more neuroaxonal loss in comparison to the wild-type animals. It is now well established that the IL-23/IL17 pathway is critical for the development of autoimmune diseases (Hunter 2005; Iwakura and Ishigame 2006; McKenzie, Kastelein et al. 2006; van der Fits, Mourits et al. 2009). IL-23 can induce chronic inflammation through activation of IL17/Th-IL17 cells, or via myeloid cell activation and production of cytokines IL-1 and IL-6. (Iwakura and Ishigame 2006). More studies are needed to clearly define the role Apo A-I plays in autoimmunity. Our results show that expression of IL-23 was statistically higher in transgenic compared to control mice. IL-17A levels were up regulated in both EAE C57BL/6J and Apo A-I mice in comparison to naïve animals (data not shown). However they were not statistically different between the two EAE groups. Cytokine secretion has specific pattern along the different phases of
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ACCEPTED MANUSCRIPT EAE (Almolda, Costa et al. 2011). Measurement of specific cytokines at different time points could provide a better insight into which particular pathway is activated in Apo A-I deficient mice.
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CD4+ T cells specific for myelin peptides mediate EAE. T-cells or the cytokines they secrete could alter neuronal function or damage neuronal and axonal membranes, which can be reflected in
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increased clinical scores (Haines, Inglese et al. 2011). Our data suggests that in the setting of Th1
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polarized cells (which secrete IFN-) Apo A-I deficient mice experienced exacerbated EAE. These
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animals also had increased levels of other inflammatory cytokines - TNF and IL-23 (Th17 inducer). These data suggest that low Apo A-I levels have profound effect on cytokine expression and
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subsequently on the nervous system. IFN-γ is thought to play an important role in the activation of encephalitogenic T-cells and CNS inflammation (Berthou, Duverger et al. 1996). IFN-γ and TGF-ß
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induce regulatory T-cells (CD4+CD25+Tregs) in EAE. We expected to see lower numbers of Tregs in
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Tg mice, however found no difference in frequency between the wild type and Tg mice. The slight
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increase in Tregs in Tg mice that we saw in our study could represent a compensatory (homeostatic) mechanism. Haas et al., showed equal distribution of Tregs in blood and cerebrospinal fluid of MS patients and controls. However suppressive potency of patient derived CD4+CD25+ Tregs was impaired compared to controls (Haas, Hug et al. 2005). Regulatory T-cells (Tregs) are characteristically associated with activation and expression of transcription factor Foxp3 (Rubin, Ishida et al. 1991; Hori, Nomura et al. 2003; Mukherjee, Locke et al. 2008). In general, increase in CD4+CD25+ Tregs indicates Th2 differentiation and has been observed in glatiramer acetate (Copaxone) treated mice and humans (Hong, Li et al. 2005; Weber, Prod'homme et al. 2007). IFN-γ plays a crucial role in the self-regulatory mechanisms of the immune system in response to acute inflammation through the induction of transcription factor Foxp3 (Hong, Li et al. 2005). However the actual mechanism of the added severity in low Apo A-I condition remains to be elucidated.
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ACCEPTED MANUSCRIPT Maintaining high Apo A-I levels might be beneficial for MS patients. Since Apo A-I is a major HDL component, use of statin medication could be beneficial. However clinical trials in MS patients
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delivered controversial results, especially in the area of new relapses and brain plaque formation (Maier, De Jonge et al. 2009; Markovic-Plese, Jewells et al. 2009; Chataway 2012; Chataway,
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Schuerer et al. 2014). Statins are known to lower LDL and improve triglyceride profiles. A meta-
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analysis of 32,258 patients from 37 randomized trials found that in addition to lowering LDL levels
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statin drugs (rosuvastatin, atorvastatin and simvastatin) raised HDL-C and Apo A-I levels (Barter, Brandrup-Wognsen et al. 2010). High dose (80mg) of oral atorvastatin decreased LDL in MS patients
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but had no effect on HDL (Paul, Waiczies et al. 2008). A recent MS-STAT clinical trial has shown that high dose simvastatin attenuates brain atrophy (Chataway, Schuerer et al. 2014). The main
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reason behind brain atrophy is believed to be a neuroaxonal loss. The reduction in brain atrophy in
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this trial was associated with reduction in total cholesterol levels. The authors did not directly
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measure Apo A-I levels in MS patients, however because of the known effects of simvastatin on cholesterol metabolism (Matthan, Giovanni et al. 2003), the data strongly suggest that HDL and Apo A-I levels were higher in simvastatin group, where cholesterol was reduced from 5.5 mmol/L to 4.1mmol/L and brain atrophy was reduced by 43%. This study however didn’t show any effect on immune markers such as Th 17, INF, IL-4, IL-10, and CD4+FoxP3. Therefore these data suggest that statins (even at a high dose) have a very limited positive effect on MS. This could be due to the fact that statins have been shown to increase reactive oxygen species, elevate lipid peroxidation and induce oxidative DNA damage in human peripheral blood lymphocytes (Gajski, Garaj-Vrhovac et al. 2008; Qi, Zheng et al. 2013). Increased lipid peroxidation is associated with disease exacerbation periods and lesion pathogenesis in MS patients (Toshniwal and Zarling 1992). Therefore a different type of drug, acting upon a different mechanism of Apo A-I activation might provide more benefit for progressive MS patients.
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5. Conclusion Our data supports the hypothesis that Apo A-I plays an important role in MS pathogenesis. Through
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a series of clinical, electrophysiological and histological experiments we have shown that increased
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Apo A-I levels are beneficial for neuroaxonal protection and are associated with less neurological
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deficit in EAE, an MS animal model. The exact mechanism of Apo A-I involvement in CNS has to be studied further in order to identify positive regulators of Apo A-I biogenesis and its involvement
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in MS.
Acknowledgments
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The authors thank Drs. Tonia Rex and Natalya Lenchik for the assistance and guidance in performing
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and analyzing the F-VEPs, 2-D PAGE, and Flow cytometry respectfully. This work was funded by
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the pilot grant from the National Multiple Sclerosis Society (to LAG) and the VA Merit Review Award (to MCL).
Figure captions
Color preference on the Web only. Figure 1. A. Plasma protein separation in Rotofor cell and protein identification (inset). B. Twodimensional analysis of Apo A-I protein expression in human plasma (control (left) and PPMS (right)). C. Apo A-I protein detection (SYPRO ruby protein stain) and Western blot analysis with anti-ApoA1 antibodies. Antibodies had high affinity to Apo A-I in Control and RRMS samples, but not in SPMS or PPMS. Figure 2. A. Apo A-I expression on 2-D gel (zoomed area with five distinct spots from control sample). B. Five samples per group were analyzed and quantified for ApoA-I. The most significant
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ACCEPTED MANUSCRIPT differences were found in the expression of spots 3 and 4. Inset table shows fold change between control and disease groups. C. Graphical representation of Apo A-I protein expression in analyzed
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samples. Statistically significant differences were observed between controls and all disease groups for spot 3 and between RRMS and progressive MS samples for spot 4. ApoA-I expression in spot 3
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was different between controls and patients with progressive disease (SPMS and PPMS).
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Figure 3. A. Western Blot quantification of Apo A-I expression. 60µg of pre-cleared plasma was
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loaded onto SDS-PAGE and probed with anti-ApoA1 antibodies. Band intensity was quantified in QuantityOne software (Bio-Rad, Hercules, CA). Twenty five samples per groups were analyzed.
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Representative images (above the graph) are shown for each group. B. Apo A-I levels measured in serum of MS patients and controls with ELISA.
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Figure 4. Endogenous ApoA-I expression in wild type and transgenic mice. A. Coomassie blue
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stained SDS-PAGE. Lane 1-Molecular weight marker; lanes 2-5 samples obtained from wild type
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animals (2-serum, 3-brain, 4-liver, 5- spinal cord); lanes 6-9 samples from ApoA-I deficient mouse (6-serum, 7-brain, 8 - liver, 9 - spinal cord). ApoA-I is expressed at 28kDA. B. Western blot detection of mouse ApoA-I protein in different tissue. Apo A-I is not expressed in brain, but is present in the spinal cord of the wild-type animal in contrast to Apo A-I deficient mouse. C. Histology of the lumbar spinal cord sections in naive C57BL/6J and ApoA-I deficient animals. Primary goat polyclonal antibodies that recognize mouse Apo A-I protein (Santa Cruz, CA) were used for detection. Secondary antibodies were anti-goat TxRed (Vector laboratories, Burlingame, CA). Wild type mice have prominent Apo A-I expression in comparison to transgenic mice (arrows). Scale bar = 50µM. Figure 5. Apo A-I deficient mice exhibit more EAE signs in comparison to control animals A. EAE induced mice show first disease signs on day 14. Scale 0-4. B. Flash-VEP recording in naïve and EAE induced wild type and Apo A-I deficient mice. C. Histopathology of the spinal cord. Scale
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ACCEPTED MANUSCRIPT bar=50µm. Luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining of the paraffin embedded spinal cord tissue. LFB identifies demyelinated regions (stained purple) and increased vacuolization
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(red arrows) in Apo A-I deficient animals. Fluoro-Jade B staining identifies neuronal degeneration seen in C57BL/6J and Apo A-I deficient mice but not in Naïve animals.
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Figure 6. Lumbar section of the spinal cord of ApoA-I deficient mice displays increased
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vacuolization. Lower panel represents higher magnification (20x) of the regions of interest (red
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squares). Arrows indicated vacuoles containing cellular debris. Vacuolization was scored on high magnification images. Number of vacuoles were calculated per filed view quadrant as follows: 1 - 5
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vacuoles = score of 1, 5 - 6 vacuoles = score of 2, 10 - 30 vacuoles = score of 3. ApoA-I deficient mice had higher demyelination score (3. 4 0.2) in comparison to wild type mice (2.9 0.2), Scale
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bar=20µm.
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Figure 7. A. Flow cytometry analysis of the T cells expressed in experimental animals. No
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significant increase in regulatory T cells was detected. B. Cytokine expression in wild type and ApoA-I deficient mice. Serum was collected from experimental animals prior to sacrifice on day 36. Multianalyte ELISArray (Qiagen) was used to detect Th1 vs. Th2 cytokines. Results were analyzed in GraphPad prism software utilizing student t-test. Star indicates significant difference between the groups. *=P<0.05, **=P<0.01.
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Markovic-Plese, S., V. Jewells, et al. (2009). "Combining beta interferon and atorvastatin may increase disease activity in multiple sclerosis." Neurology 72(22): 1965; author reply 1965-1966. Matthan, N. R., A. Giovanni, et al. (2003). "Impact of simvastatin, niacin, and/or antioxidants on cholesterol metabolism in CAD patients with low HDL." J Lipid Res 44(4): 800-806. McKenzie, B. S., R. A. Kastelein, et al. (2006). "Understanding the IL-23-IL-17 immune pathway." Trends Immunol 27(1): 17-23. Mukherjee, R., K. T. Locke, et al. (2008). "Novel peroxisome proliferator-activated receptor alpha agonists lower lowdensity lipoprotein and triglycerides, raise high-density lipoprotein, and synergistically increase cholesterol excretion with a liver X receptor agonist." J Pharmacol Exp Ther 327(3): 716-726. Mycko, M. P., B. Sliwinska, et al. (2014). "Brain glycolipids suppress T helper cells and inhibit autoimmune demyelination." J Neurosci 34(25): 8646-8658. Paul, F., S. Waiczies, et al. (2008). "Oral high-dose atorvastatin treatment in relapsing-remitting multiple sclerosis." PLoS One 3(4): e1928. Pelfrey, C. M., A. C. Cotleur, et al. (2002). "Sex differences in cytokine responses to myelin peptides in multiple sclerosis." J Neuroimmunol 130(1-2): 211-223. Qi, X. F., L. Zheng, et al. (2013). "HMG-CoA reductase inhibitors induce apoptosis of lymphoma cells by promoting ROS generation and regulating Akt, Erk and p38 signals via suppression of mevalonate pathway." Cell Death Dis 4: e518. Ravikumar, M., S. Jain, et al. (2012). "An organotypic spinal cord slice culture model to quantify neurodegeneration." J Neurosci Methods 211(2): 280-288. Rubin, E. M., B. Y. Ishida, et al. (1991). "Expression of human apolipoprotein A-I in transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high density lipoprotein size subclasses." Proc Natl Acad Sci U S A 88(2): 434-438. Sabel, B. A. and A. Aschoff (1993). "Functional recovery and morphological changes after injury to the optic nerve." Neuropsychobiology 28(1-2): 62-65. Schmued, L. C. and K. J. Hopkins (2000). "Fluoro-Jade: novel fluorochromes for detecting toxicant-induced neuronal degeneration." Toxicol Pathol 28(1): 91-99. Sena, A., R. Pedrosa, et al. (2000). "Interferon beta1a therapy changes lipoprotein metabolism in patients with multiple sclerosis." Clin Chem Lab Med 38(3): 209-213. Sullivan, T. A., E. E. Geisert, et al. (2011). "Systemic adeno-associated virus-mediated gene therapy preserves retinal ganglion cells and visual function in DBA/2J glaucomatous mice." Hum Gene Ther 22(10): 1191-1200. Toshniwal, P. K. and E. J. Zarling (1992). "Evidence for increased lipid peroxidation in multiple sclerosis." Neurochem Res 17(2): 205-207. van der Fits, L., S. Mourits, et al. (2009). "Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis." J Immunol 182(9): 5836-5845. Vollbach, H., R. Heun, et al. (2005). "APOA1 polymorphism influences risk for early-onset nonfamiliar AD." Ann Neurol 58(3): 436-441. Weber, M. S., T. Prod'homme, et al. (2007). "Type II monocytes modulate T cell-mediated central nervous system autoimmune disease." Nat Med 13(8): 935-943. Weinstock-Guttman, B., R. Zivadinov, et al. (2011). "Serum lipid profiles are associated with disability and MRI outcomes in multiple sclerosis." J Neuroinflammation 8: 127. You, Y., A. Klistorner, et al. (2011). "Latency delay of visual evoked potential is a real measurement of demyelination in a rat model of optic neuritis." Invest Ophthalmol Vis Sci 52(9): 6911-6918. Zhou, B., J. Yuan, et al. (2011). "Administering human adipose-derived mesenchymal stem cells to prevent and treat experimental arthritis." Clin Immunol 141(3): 328-337.
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Table1. Demographic data for study subjects. Gender F
Subjects on Medications
Average Age
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RRMS
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ACCEPTED MANUSCRIPT Highlights
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We identified differential expression of Apo A-I protein in human plasma MS patients on average had less Apo A-I compared to healthy controls The lowest expression of Apo A-I was found in progressive MS patients Mouse deficient in murine Apo A-I had worse EAE disease, an animal model for MS
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S0165-5728(14)00944-8 doi: 10.1016/j.jneuroim.2014.10.010 JNI 476019
To appear in:
Journal of Neuroimmunology
Received date: Revised date: Accepted date:
24 June 2014 20 October 2014 24 October 2014
Please cite this article as: Meyers, Lindsay, Groover, Chassidy J., Douglas, Joshua, Lee, Sangmin, Brand, David, Levin, Michael C., Gardner, Lidia A., A Role for Apolipoprotein A-I in the pathogenesis of Multiple Sclerosis, Journal of Neuroimmunology (2014), doi: 10.1016/j.jneuroim.2014.10.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A Role for Apolipoprotein A-I in the pathogenesis of Multiple Sclerosis
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Lindsay Meyers1, Chassidy J. Groover1, Joshua Douglas1, Sangmin Lee1,2, David Brand2, Michael C. Levin1,2, and Lidia A. Gardner1,2 Research Service VAMC, Memphis, TN, 38104, 2 Department of Neurology University of
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Tennessee Health Science Center, Memphis, TN, 38163.
Corresponding Author:
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Lidia A. Gardner, Ph.D. University of Tennessee Health Science Center
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Memphis, TN, 38163
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855 Monroe Avenue, Rm. 415
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Department of Neurology
Ph. +1(901) 5238990 Ext.6477 Email: [email protected]
1. Introduction
There are three general types of MS, relapsing remitting MS (RRMS), primary progressive MS (PPMS) and secondary progressive MS (SPMS). Clinically there are some clear differences between RRMS, SPMS and PPMS. For example, RRMS is two times more likely to occur in women than men, whereas the gender distribution in PPMS is about equal (Coyle 2005). Diagnosis of a specific MS type is largely dependent on presentation of neurological symptoms. The PPMS typically has a worse prognosis compared to RRMS (Pelfrey, Cotleur et al. 2002). It is not clear why some RRMS patients progress to the SPMS while others do not.
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ACCEPTED MANUSCRIPT We hypothesized that distinction between types of MS might be found in the differential expression of proteins in patient’s plasma. We analyzed protein expression in plasma of patients with
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three MS subtypes and age-matched controls. Proteins were separated by two-dimensional gel electrophoresis and identified with matrix-assisted laser desorption ionization (MALDI) time of flight
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(TOF) mass spectroscopy. The protein that was consistently different in MS samples compared to
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controls was identified as Apolipoprotein A-I (Apo A-I). Apo A-I is a constitutive anti-inflammatory
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molecule that recently has been shown to be a marker of clinical response to interferon-beta therapy in MS patients (Gandhi, McKay et al. 2010). Importantly, progressive MS patients had significantly
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lower plasma levels of Apo A-I expression compared to healthy controls, suggesting that high levels of Apo A-I are neuroprotective. According to our data, Apo A-I levels could not be used as a stand-
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alone diagnostic biomarker, however levels of this protein present in patient’s serum might be useful
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in assessing disease progression. We used experimental autoimmune encephalomyelitis (EAE) to
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validate our observation and test the hypothesis that mice deficient in Apo A-I would develop worse disease. EAE induction in mice deficient in murine Apo A-I resulted in worse disease compared to control animals. Taken together, these data indicates that Apo A-I might play an important role in the pathogenesis of MS.
2. Materials and Methods 2.1. Human samples Two hundred eight samples were utilized for Apo A-I studies from three different sources: 1) patients from the local clinic (53 RRMS, 15 SPMS, and 8 PPMS); 2) healthy adult volunteers from Memphis community (52 healthy control samples); 3) samples obtained from the “Accelerated Cure Project (www.acceleratedcure.org)” (35 SPMS; 45 PPMS). All samples were collected from fasting individuals (12h), according to the approved Institutional Review Board (IRB) protocol with patient
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ACCEPTED MANUSCRIPT consent. Controls were matched for age and gender to disease groups. Blood samples were drawn from the antecubital vein into heparin-coated (for plasma) and silicone coated (for serum) tubes. All
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samples were processed within 30 min from collection and stored at -80C for future use. 2.2. Enzyme Linked Immuno-Sorbent Assay (ELISA).
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Apo A-I levels were measured with a commercially available kit from Eagle Biosciences (Nashua,
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NH), and an ‘in house’ developed ELISA assays. Assays were done in duplicates. Briefly, diluted
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standards and samples (1:200 in PBS) were applied onto anti-Apo A-I pre-coated plate, incubated at 37C for 1 h. After each incubation step plates were washed four times in TBST (100mM Tris, pH
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7.4 + 0.02% tween). Secondary HRP conjugated antibodies were added to the plates for 1h at 37C. Plates were washed as described before and TMB/peroxide substrate was added for 30 min. Reaction
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was terminated with 0.5N sulfuric acid. The ‘in house’ direct ELISA had better sensitivity to Apo A-I
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protein. We utilized mouse monoclonal anti-Apo A-I antibodies (Abnova, Taiwan) to coat plates
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overnight. Plates were washed and blocked with 0.5% BSA prior to sample application and after each consecutive step. Samples were diluted as described above and incubated on the plate for 1h at 37C. HRP conjugated anti-Apo A-I antibodies were diluted in coating buffer (10mM PBS, pH 7.4) and applied to the plate. TMB substrate and stop solution were obtained from Thermo Fisher Scientific (Waltham, MA). Reaction optical densities were read at 450 nm in a microtiter plate reader (Molecular Devices, LLC). Apo A-I concentration was calculated based on the standard curve numbers with 4-parameter algorithm in Soft Max Pro software. 2.3. Antibodies Primary anti-human Apo A-I mouse monoclonal antibodies were obtained from Abnova (Taipei City, Taiwan). Anti-mouse Apo A-I goat polyclonal antibodies (Santa Cruz, CA) and secondary antimouse HRP antibodies were obtained from GE Healthcare (Pittsburg, PA). Apo A-I antibodies from
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ACCEPTED MANUSCRIPT Santa Cruz specifically recognize the mouse protein sequence; the epitope is located in the Cterminal domain of Apo A-I, where identity with human sequence is 50%.
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2.4. Protein electrophoresis For one-dimensional Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (1-D SDS-PAGE)
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plasma samples were diluted 8 times with 1xPBS and mixed with 2x-sample buffer. All samples
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were heated to 95C for 5 min to denature protein, chilled on ice for 2 min and loaded onto 12%
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SDS-PAGE gels (Bio-Rad, Hercules, CA). A total of 60µg of protein were loaded per well. Proteins were separated and transferred onto polyvinylidene difluoride (PVDF) membranes for Western
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blotting. Apo A-I was detected with primary anti-Apo A-I antibodies (1:2000), Abnova (Taipei City, Taiwan), and secondary anti-mouse HRP (1:10,000), GE Healthcare. Proteins were detected using
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either X-ray film, and/or digital imaging. Band intensity was quantified using Quantity One Software
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(Bio-Rad, Hercules, CA). Numbers were analyzed and graphed in Prism GraphPad software.
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2.4.1. Preparative IEF (Rotofor).
Plasma proteins were separated based on pI using Rotofor IEF (isoelectric focusing) with soluble ampholites in solution. Fractions were collected, washed and concentrated using Amicon filters with 10kDa membranes. Protein concentration of each fraction was measured on a Nanodrop spectrophotometer. Five micrograms of each fraction was then loaded onto 1-D SDS PAGE for protein expression analysis. Fractions that had differential expression were extracted and analyzed with MALDI-TOF. 2.4.2. Two-dimensional (2-D) SDS-PAGE. Plasma samples were albumin, IgG and salt depleted with a kit from Pierce (Rockford, IL). Protein concentration was measured on a Nanodrop spectrophotometer. Equal amounts of protein (150µg) for each sample were separated by 2D-PAGE using a pI range 4–7 strip and stained with SYPRO Ruby stain. The gels were scanned on Typhoon imager (GE Healthcare, Pittsburg, PA), and TIFF
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ACCEPTED MANUSCRIPT images were imported into PDQuest image analysis software (Bio-Rad, Hercules, CA). Protein spots were detected using the automated spot detection feature. The images were visually inspected at high
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resolution and corrected for undetected or incorrectly detected spots/artifacts. Upon completion of the image analysis, data were stored both as a 2D image and Gaussian image. The 2-D dataset
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consisted of 20 samples that were divided into four groups (controls, RRMS, SPMS and PPMS) of
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five samples in each. Within the “MatchSet” produced by the image analysis software, 5 Apo A-I
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spots were identified as being expressed in at least one of the 20 gels. We used the default normalization of data in the MatchSet that is based on total pixel quantity in valid spots. This
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normalization method assumes that few protein spots change within the experiment and that the changes averaged out across the whole gel. PDQuest software includes three statistical tests for data
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analysis, the Student’s t test (a parametric test) and the Mann-Whitney rank sum test and Wilcoxon
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signed rank test (both non-parametric tests). We used Mann-Whitney rank sum test. The intensity
2.5. Animals
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values that were generated in PDQuest were analyzed and graphed in Prism GraphPad software.
C57BL/6J and C57BL/6-Tg (APO A-I) 1Rub/J mice were purchased from Jackson Laboratories (Bar Harbor, ME). The Apo A-I deficient mice (C57BL/6-Tg (APO A-I) 1Rub/J) were originally developed in laboratory of Dr. Rubin (Laurence Berkley National Laboratories) and donated to Jackson Laboratories. These mice carry the human apolipoprotein A-I transgene and show a two-fold increase in total plasma cholesterol levels and greater than a four-fold decrease in the levels of mouse Apo A-I. Mice colonies were maintained by sister-brother mating’s under specific pathogen free (SPF) conditions at 24 ± 2°C with a light-controlled regimen (12h light/dark cycle). Tap water was provided ad libitum. All mice included in our EAE experiments were housed in the same facility and were on the same standard chow diet (Harlan, Laboratories, Indianapolis, IN) for at least 2 weeks prior experimental treatment.
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ACCEPTED MANUSCRIPT 2.6. Induction of EAE Only female mice (10-12 wks. old) were used in EAE experiments. All experimental procedures
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were carried out in accordance with approved animal protocol. EAE was induced by immunization with an emulsion of MOG35-55 (Myelin Oligodendrocyte Glycoprotein) in complete Freund's adjuvant
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(CFA), followed by administration of intraperitoneal pertussis toxin (PTX) in phosphate-buffered
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saline (PBS) (first on the day of immunization and then again the following day) according to the
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manufacture’s instructions (Hooke laboratories, St. Lawrence, MA). Four EAE experiments with 5 animals per group were conducted. EAE was scored on a 0-5 scale: 0 no disease, 1- limp tail and
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hind paw inhibition, 2- poor balance, limp tail, dragging hind paws, 3- limp tail with paralysis of one front and one hind paw, 4- mouse has minimal movement in the front paws, both hind paws are
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paralyzed, and 5-mouse is euthanized due to severe paralysis. At the onset of the disease solid food
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pellets were supplemented with raspberry gelatin cubes to help animals maintain body weight. The
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clinical score results were averaged from all experiments. 2.7. Flash –Visual Evoked Potentials (F-VEP) F-VEP was performed as previously described (Sullivan, Geisert et al. 2011). Briefly, after an overnight dark adaptation, mice were anesthetized with an intraperitoneal injection of ketamine/xylazine/urethane (25/10/800μg/g body weight). Body temperature was maintained at 37C with a heating pad. Pupils were dilated with 1% atropine. Platinum needle electrodes (Grass Technologies, West Warwick, RI) were placed approximately 3 mm lateral to lambda over the left and right cortex. Flashes of white light at an intensity of 1.0 cd·sec/m2 were presented in a Ganzfeld dome (Diagnosys, Lowell, MA). The flash frequency was 1 Hz with an inner sweep delay of 500msec. Each result was an average of 200 sweeps. The results were exported in to an Excel spreadsheet and averaged for quantification of amplitude and latency. Recordings were analyzed in GraphPad software as described below.
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ACCEPTED MANUSCRIPT 2.8. Statistical analysis One-way analysis of variance (ANOVA) followed by a pair-wise Bonferroni post hoc comparison
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test, with a p value ≤ 0.01 considered statistically significant, was used to compare relative band intensity counts, ELISA results, and F-VEPs. Statistical analysis was performed with Prism 4.0
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software (GraphPad, San Diego, CA).
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2.9. Immunohistochemistry
At the end of EAE experiments, mice were deeply anesthetized with ketamine/xylazine injection and
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perfused with 4%paraformaldehyde. Brain and spinal column were extracted and post-fixed in 4% paraformaldehyde over night at 4C. Spinal columns were decalcified in decalcifying solution
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(Thermo Fisher Scientific, Waltham, MA), washed in deionized water and processed for paraffin
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embedding. Paraffin slices were mounted onto glass slides for H&E (Hematoxylin/Eosin) and LFB
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(Luxol Fast Blue) staining. For Fluoro-Jade staining slides were de-waxed in xylene, immersed in 100% ethanol for 5 min, 70% alcohol for 2 min, and then rinsed with two 1-min changes if dd-H2O. Slides were then incubated with freshly made 0.06% potassium permanganate for 17 min and rinsed with two 1-min changes of ddH2O. The 0.001% Fluoro Jade staining solution was made in 0.1% acetic acid. Slides were stained for 30 min at room temperature in the dark, and then air-dried, dipped in xylene and covered with VectaShield mounting media (Vector Laboratories, Burlingame, CA). For Apo A- I staining, slides were deparaffinized in xylene (3X for 5 min), 100% Ethanol (3X for 5 min), 70% Ethanol (1X for 5 min), and H2O (2X for 5 min). Slides were blocked with donkey serum for 30 min at room temperature (RT) and incubated with primary goat polyclonal antibodies that recognize mouse Apo A-I protein (Santa Cruz, CA) over night at 4C. Slides were then washed with 1x TBST and secondary anti-goat TX Red (Vector Laboratories, Burlingame, CA) were applied for 2h at RT. After application of secondary antibodies, slides were washed 4X with TBST, cleared in
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ACCEPTED MANUSCRIPT xylene and mounted with DPX. We examined 20 fields per condition and used 10 fields for quantitative analysis. For quantitation purposes slides were examined under Axio Observer A1
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microscope (Carl Zeiss, Thornwood, NY). All sections were scored as follows: 0, no infiltration/demyelination (<50 cells); 1, mild infiltration/demyelination of nerve or nerve sheath (50–
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100 cells); 2, moderate infiltration/demyelination (100–150 cells); 3, severe
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infiltration/demyelination (150–200 cells); 4, massive infiltration/demyelination (>200 cells).
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2.10. Flow cytometry
Single cell suspensions of spleens were obtained 36 days post-immunization using standard
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technique (Zhou, Yuan et al. 2011). Spleen cell suspensions were obtained by hemolysis using sterile technique. Single-cell suspensions of spleenocytes (0.5x 106) were incubated with the following
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fluorescently labeled antibodies (CD3, CD4, CD19, CD25). Foxp3 staining was done on permeabilized cells with Foxp3 staining kit form eBioscience (San Diego, CA) according to the
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manufacturer’s protocol. All fluorescently labeled antibodies were purchased from BD Pharmingen (San Diego, CA). Labeled cells were washed, resuspended in 1% paraformaldehyde, and analyzed using a LSR-II flow cytometer.
2.11. Measurement of the cytokines
Cytokines were measured in the serum collected from experimental animals prior to sacrifice. We used mouse multi- analyte ELISArray kit from Qiagen (Valencia, CA). Briefly, 50µl of serum was mixed with equal amount of assay buffer, added to the plate and incubated for 2h at room temperature. Standard antigen cocktail containing cytokines of interest was used for quantification purposes. Plates were washed after each step. Detection antibodies were added and incubated for 1h then Avidin-HRP for 30 min. Plates were extensively washed, dried and TMB/peroxide substrate was added for 30 min. Reaction was stopped and read at 450 nm in a microtiter plate reader (Molecular
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ACCEPTED MANUSCRIPT Devices, LLC). Absorbance values outside of linear range of 0 - 2.5 were not included in the analysis.
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3. Results
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We used serum and plasma derived from blood collected from fasting MS patients (n=156: 53
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RRMS, 50 SPMS and 53 PPMS) and age-matched controls (n=57). Serum was primarily used for Western blot and ELISA measurements and plasma for 2-D analysis. We realized that high amounts
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of albumin and immunoglobulin (IgG) might mask other proteins present in human plasma. Therefore, the samples were albumin, salt and IgG depleted. Samples were then subjected to
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isoelectric focusing in Rotofor Cell (Bio-Rad, Hercules, CA) (Fig. 1A.) Fractionated samples were separated by molecular weight on SDS-PAGE gels (Fig. 1A, inset); bands were extracted and
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subjected to MALDI-TOF. The protein that was consistently different in MS samples compared to controls was identified as Apolipoprotein A-I (Apo A-I) with 97.8% positivity. To verify MALDI-
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TOF results, we performed 2-D electrophoresis on five samples from each MS type and controls. Apo A-I was identified on 2-D gels at molecular weight of 28kDa and pI range 5.3-5-6 on pH 4-7 strips (Fig. 1B, circle, Fig. 2B, shown control sample). The most striking difference in Apo A-I expression was identified between controls and PPMS (Fig. 1B, 2B). Samples were separated on 2-D gels, stained with SYPRO Ruby protein stain and scanned for analysis. Our sample set included 20 gels with 5 samples in each group. To verify Apo A-I expression, 2-D gels were transferred onto nitrocellulose membrane and probed with anti-Apo A-I antibodies (Fig. 1C). It is not clear why antibodies recognized only two out of five Apo A-I spots in controls and RRMS. There was limited reactivity in SPMS and none in PPMS samples. A possible explanation to this discrepancy might be in epitope specificity for Apo A-I expression. Not all of the spots on the 2-D gel might have had the perfect epitope match for this specific antibody. According to the reference database (SWISS2DPAGE) Apo A-I is represented in five major spots between 22873 and 23000 Da and pI range 9
ACCEPTED MANUSCRIPT 4.99-5.22 and four additional spots between 7490 and 9451 Da and pI range 7.27- 4.99, correspondingly. We analyzed five major protein spots as detected by SYPRO Ruby staining
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expressed on our gels at ~22,8 kDa, (Figs. 2A, B). Spots three and four were consistently different between controls and MS patients and correlated with disease type. Therefore, we focused our
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analysis on Apo A-I spots 3 and 4 (Figs. 2B, C). Protein intensity for each spot was averaged from
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five gels per group and groups were compared to each other and to controls using one-way ANOVA.
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The fold change of averaged normalized volumes was calculated between controls and PPMS patients (Fig. 2B, inset table). Patients with SPMS and PPMS had lower Apo A-I spot intensity
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compared to samples collected from RRMS. This analysis revealed that healthy age-matched controls had the highest Apo A-I spot intensity, followed by the RRMS, SPMS and PPMS groups. The PPMS
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group had lowest spot 3 and 4 intensity in comparison to any group. Finally, spots two and five had
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lower expression in comparison to controls but showed no correlation with disease type.
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Unlike 2-D analysis, 1-D SDS electrophoresis allows for simultaneous protein detection in multiple samples. Therefore, we subjected depleted serum samples to SDS-PAGE and subsequent Western blot analysis with anti-Apo A-I antibodies (Fig. 3A). Equal amounts of protein were loaded per well; separated proteins were transferred onto a nitrocellulose membrane and stained with Ponseau-S solution to label total protein load. Membranes were rinsed and probed with anti-Apo A-I antibodies. Bands were quantified with QuantityOne software (Bio-Rad, Hercules, CA). Quantitative Western blot analyses revealed differences between MS samples in comparison to controls. All MS patients had lower Apo A-I expression when compared to healthy controls (Fig. 3A). Among MS groups RRMS samples had highest ApoA-I expression and were statistically different (P<0.01) from progressive forms of disease. To answer the question if Apo A-I could serve as a biomarker of disease progression in MS, we measured Apo A-I expression in human serum and plasma by ELISA. Normal Apo A-I levels in 10
ACCEPTED MANUSCRIPT human plasma ranged between 0.9-1.3 g/L. We quantified Apo A-I expression in a set of 208 samples (Table 1). Patient samples were age-matched to controls. Commercially available ELISA (Eagle
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Biosciences) showed no statistically significant difference between the groups. In addition, Apo A-I values obtained by this method often exceeded normal Apo A-I range.
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To address this discrepancy we developed an “in house” direct sandwich ELISA. We utilized
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mouse monoclonal antibodies as the capture antibodies (Abnova, Taipei City, Taiwan), which were
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also used in the 1- and 2-D analyses (Figs. 1 and 2). These antibodies were conjugated to EZ-Link Plus activated peroxidase (Fisher Thermo Scientific, Waltham, MA). Results obtained with the ‘in
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house’ ELISA were more consistent with values seen in 1- and 2-D analyses and nearer to the reported range on Apo A-I plasma expression. According to our data, the average Apo A-I values in
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controls were 2.1g/L, with some samples actually exceeding the upper limit of detection (Fig. 3B).
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The RRMS samples had an average of 1.6g/L, which is also within the normal range. Average Apo
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A-I levels in SPMS samples were 0.9g/L and PPMS were between 0.5-0.9g/L (Fig. 3B). Each type of MS showed statistical significance compared to healthy controls (Fig 3B). Subjects that were on medication affecting Apo A-I levels, such as statins and NSAIDs (non-steroidal anti-inflammatory drugs) were excluded from the analysis. We hypothesized that Apo A-I might have a protective role in MS due to its anti-inflammatory activity. To test our hypothesis, we utilized wild type (WT) and Apo A-I transgenic (Tg) mice (C57BL/6-Tg (Apo A-I) 1Rub/J). These animals have four times less mouse Apo A-I in the circulation compared to the wild type mice (C57BL/6J) due to the presence of human Apo A-I transgene regulated by a human promoter (Rubin, Ishida et al. 1991). These mice have increased amount of total ApoA-I in their serum, however the expression of mouse ApoA-I is four fold reduced (Fig. 4 A, B. The protein sequence of mouse (264 AA) and human (267AA) Apo A-I is highly homologous, except at its C-terminal domain, where the two sequences share only 50-80% homology. 11
ACCEPTED MANUSCRIPT We utilized antibodies produced to the epitope located between amino acids 250 and 264 of the mouse Apo A-I protein (Santa Cruz, CA). These antibodies recognized endogenous mouse Apo A-I
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protein in the serum, liver and the spinal cord of the WT mice and serum of the Apo A-I deficient animals (Fig. 4A, B). In contrast, there was no mouse Apo-A-I protein detected in the liver or spinal
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cord of Tg mice. We utilized the same antibodies to confirm Apo A-I expression on histological
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spinal cord sections (Fig. 4C), which showed expression of Apo-A-I protein in WT but not in Tg
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mice. Phenotypically, Tg mice do not differ from the WT animals (C57BL/6J). Therefore these mice present a suitable model to investigate how low levels of Apo A-I affect EAE progression. We
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initiated EAE in Apo A-I deficient and WT animals and recorded clinical scores (Fig. 5A). Mice (1012 weeks old) were injected with MOG35-55 according to the manufacture’s instructions (Hooke
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laboratories, St. Lawrence, MA). Disease onset was between days 12 and 15 after injections. Both
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groups developed EAE, however mice deficient in murine Apo A-I exhibited higher EAE scores (Fig.
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5A, P<0.001), more vacuolization in the spinal cord and damage to optic nerves in comparison to controls (Fig. 5C). Larger vacuolization was seen on both H&E and LFB stained spinal cord tissue (Fig. 5C, Fig. 6). Vacuolization could be a sign of neuronal degeneration or changes in myelin. Therefore we calculated demyelination scores by scoring each quadrant of a spinal cord for the presence and absence of demyelination. For each group 10 lumbar spinal sections were scored 36 days after EAE induction. Results are expressed as the mean pathological score SEM. Naïve mice no demyelination, C57BL/6J - 2.5 0.2 and Apo A-I deficient - 4 0.2. Vacuolization was scored on high magnification images of the spinal cord. The number of vacuoles were calculated per filed view quadrant as follows: 1 - 5 vacuoles = score of 1, 5 - 6 vacuoles = score of 2, 10 - 30 vacuoles = score of 3. ApoA-I deficient mice had higher demyelination score (3. 4 0.2) in comparison to wild type mice (2.9 0.2), Fig. 6.
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ACCEPTED MANUSCRIPT Degenerating neurons were visualized with Fluoro-Jade B, a known marker of neurodegeneration, Fig. 5C (Schmued and Hopkins 2000; Bian, Wei et al. 2007; Ehara and Ueda
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2009; Ravikumar, Jain et al. 2012). Tissue collected from naïve mice did not uptake Fluoro-Jade B staining. Fluoro-Jade staining was more prominent in brain (not shown) and spinal cord sections of
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Apo A-I deficient in contrast to wild type animals (Fig. 5C). These data are in agreement with the
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demyelination scores. Thus, the Tg mice showed evidence of tissue damage (vacuolization) that
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appears to be the result of both damaged myelin and neurodegeneration. We repeated these experiments six times and obtained similar results.
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Optic neuritis is a common MS symptom. Therefore we recorded optic nerve function with FVEP tests (Fig. 5B). F-VEPs were measured in the visual cortex of naïve, Apo A-I deficient and wild
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type mice. The classical N1 and P1 peaks in the F-VEP averaged waveforms were seen in naïve and
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wild type (C57BL/6J) mice. For a signal to reach the visual cortex both the retinal ganglion cells
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soma and axon must be intact. EAE animals had severely diminished P2 and N2 signals. Apo A-I deficient animals had delayed latency (Fig. 5B) and a number of these mice had no detectable signal at all. The average amplitude and the latency of the N1 and P1 peaks were quantified. Wild type mice had P1 (25.74.2μV) and N1 (58.3 6.1μV) peak amplitudes. Naïve Tg mice had P1 (15 4.8μV) and N1 (45 5.2μV) peak amplitudes. Apo A-I deficient EAE mice had no detectable peaks between 0-500ms. Both groups of mice with EAE had significantly different F-VEP recordings from naïve mice, P<0.001. In addition, Apo A-I deficient mice developed necrotizing tail lesions that were not seen in the control animals (data not shown). Similar lesions were observed in exclusively neurologically affected gelatinase B deficient mice with EAE (Dubois, Masure et al. 1999). These data suggests that Apo A-I deficient transgenic animals had worse EAE compared to wild-type control mice.
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ACCEPTED MANUSCRIPT Knowing that T - cell differentiation plays an important role in MS and EAE, we used flow cytometry to characterize populations of T – cells isolated from the spleens of these animals with
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flow cytometry (Fig. 7A). According to our data, Apo A-I deficient mice showed an insignificant trend for increased number of regulatory T cells (CD25+/Foxp3+) compared to WT and naive mice.
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We also used ELISA to measure the expression of different cytokines in these animals and found that
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Apo A-I deficient mice had statistically significant differences in the expression of IL -2, IL-23, INF.
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TNF- and TGF-ß levels showed a similar trend, consistent with inflammatory response to EAE (Fig. 7B). IL-17A levels were not statistically different between the wild type and Apo A-I deficient mice.
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Taken together, these observations support our hypothesis that Apo A-I might play a role in EAE, an
4. Discussion
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animal model of MS pathogenesis.
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Human brain has high lipid content; therefore lipids and their turnover are good candidates as contributors to diseases of the CNS. However, the importance of lipids and their metabolism in MS has been understudied. Myelin associated glycoproteins have been studied in connection with autoantibodies to MOG (myelin oligodendrocyte glycoprotein) and MBP (myelin basic protein) (Bernard, Johns et al. 1997; Genain, Cannella et al. 1999; Haas, Hug et al. 2005) for several decades now. One recent study proposed that sulfatides (major component of myelin lipids) suppress T cells activation and prevent potential induction of autoimmunity within the CNS (Mycko, Sliwinska et al. 2014). Disturbances in lipid metabolism can lead to myelin loss, neuronal degeneration and metabolic distress (Levin 2014). Weinstock–Guttman showed an association between serum lipid profiles and disability and MRI outcomes in MS (Weinstock-Guttman, Zivadinov et al. 2011). It is well known that Apo A-I plays an important protective role in atherosclerosis, cardiovascular and cognitive diseases, however its function in MS has not been fully investigated. In humans elevated 14
ACCEPTED MANUSCRIPT Apo A-I levels have been associated with APOA1 A allele. Specifically, carriers of this allele performed significantly better on semantic verbal fluency and the Stroop interference tests (Koutsis,
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Panas et al. 2009). The authors of this study evaluated 138 patients with MS and 43 controls and concluded that they found an association of the ApoA1 -75G/A promoter polymorphism with
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cognitive performance in MS (Koutsis, Panas et al. 2009). In addition, Apo A-I may play a role in
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neuronal regeneration (Vollbach, Heun et al. 2005) by acting as a constitutive anti-inflammatory
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factor (Burger and Dayer 2002). Apo A-I has also been described as a biomarker of clinical response to interferon-beta (INF-ß) treatment in a small cohort of MS patients (Gandhi, McKay et al.). A
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different study measured Apo A-I levels before and after INF-ß treatment in MS patients and found that patients, who suffered relapses or progressed in their EDSS had lower plasma Apo A-I levels,
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compared to their basal values (Sena, Pedrosa et al. 2000). These studies point to an important role
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that Apo A-I might play in MS pathogenesis, however none measured Apo A-I levels in different MS
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subtypes or examined correlation with disease progression. We identified that Apo A-I is differentially expressed in serum and plasma of MS patients and age-matched controls. According to our data the average Apo A-I levels detected in all MS types were lower than in controls. Expression of Apo A-I was remarkably different in progressive MS patients, particularly by Western blot. Perhaps a longitudinal study could answer a question if low Apo A-I levels could predict or influence the development of SPMS in RRMS patients. The experiments that we undertook in a mouse model of MS (EAE) supported our hypothesis that Apo A-I plays a beneficial role in MS. We chose Apo A-I deficient animals instead of knockouts because they have reduced Apo A-I levels of endogenous murine Apo A-I, much like MS patients. To the best of our knowledge we are the first to initiate EAE in these animals and show that reduction in Apo A-I expression results in greater neurological deficits on clinical, electrophysiological and histological levels. The limited amount of endogenous murine Apo A-I in
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ACCEPTED MANUSCRIPT the spinal cord of Tg mice correlated with higher demyelination and degeneration scores, indicating a possible protective role of this protein in the CNS. MS is the most common immune-mediated
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disease of the CNS (Levin, Lee et al. 2013). It is possible that inflammation in the periphery exacerbated by the low Apo A-I levels could trigger faster Th2 cell penetration of into the CNS,
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soliciting neuronal demyelination and degeneration.
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The F-VEP is an appropriate electrophysiological test to study functional loss in optic nerve.
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(Diem, Sattler et al. 2005; Sullivan, Geisert et al. 2011; You, Klistorner et al. 2011). If either the RGC soma or axon is compromised the signal cannot reach the visual cortex and therefore is not
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detected by F-VEP (Sabel and Aschoff 1993). At the time of sacrifice the majority of Apo A-I deficient mice had minimal cortical response as measured by F-VEP. The EAE wild type mice also
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had impaired F-VEP recordings in comparison to control group. However these mice had detectable
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N1 and P1 amplitudes and almost normal latencies, indicating extensive cell death, but normal
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synaptic connectivity in the remaining cells. These data suggests that Apo A-I deficient mice had more neuroaxonal loss in comparison to the wild-type animals. It is now well established that the IL-23/IL17 pathway is critical for the development of autoimmune diseases (Hunter 2005; Iwakura and Ishigame 2006; McKenzie, Kastelein et al. 2006; van der Fits, Mourits et al. 2009). IL-23 can induce chronic inflammation through activation of IL17/Th-IL17 cells, or via myeloid cell activation and production of cytokines IL-1 and IL-6. (Iwakura and Ishigame 2006). More studies are needed to clearly define the role Apo A-I plays in autoimmunity. Our results show that expression of IL-23 was statistically higher in transgenic compared to control mice. IL-17A levels were up regulated in both EAE C57BL/6J and Apo A-I mice in comparison to naïve animals (data not shown). However they were not statistically different between the two EAE groups. Cytokine secretion has specific pattern along the different phases of
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ACCEPTED MANUSCRIPT EAE (Almolda, Costa et al. 2011). Measurement of specific cytokines at different time points could provide a better insight into which particular pathway is activated in Apo A-I deficient mice.
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CD4+ T cells specific for myelin peptides mediate EAE. T-cells or the cytokines they secrete could alter neuronal function or damage neuronal and axonal membranes, which can be reflected in
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increased clinical scores (Haines, Inglese et al. 2011). Our data suggests that in the setting of Th1
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polarized cells (which secrete IFN-) Apo A-I deficient mice experienced exacerbated EAE. These
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animals also had increased levels of other inflammatory cytokines - TNF and IL-23 (Th17 inducer). These data suggest that low Apo A-I levels have profound effect on cytokine expression and
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subsequently on the nervous system. IFN-γ is thought to play an important role in the activation of encephalitogenic T-cells and CNS inflammation (Berthou, Duverger et al. 1996). IFN-γ and TGF-ß
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induce regulatory T-cells (CD4+CD25+Tregs) in EAE. We expected to see lower numbers of Tregs in
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Tg mice, however found no difference in frequency between the wild type and Tg mice. The slight
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increase in Tregs in Tg mice that we saw in our study could represent a compensatory (homeostatic) mechanism. Haas et al., showed equal distribution of Tregs in blood and cerebrospinal fluid of MS patients and controls. However suppressive potency of patient derived CD4+CD25+ Tregs was impaired compared to controls (Haas, Hug et al. 2005). Regulatory T-cells (Tregs) are characteristically associated with activation and expression of transcription factor Foxp3 (Rubin, Ishida et al. 1991; Hori, Nomura et al. 2003; Mukherjee, Locke et al. 2008). In general, increase in CD4+CD25+ Tregs indicates Th2 differentiation and has been observed in glatiramer acetate (Copaxone) treated mice and humans (Hong, Li et al. 2005; Weber, Prod'homme et al. 2007). IFN-γ plays a crucial role in the self-regulatory mechanisms of the immune system in response to acute inflammation through the induction of transcription factor Foxp3 (Hong, Li et al. 2005). However the actual mechanism of the added severity in low Apo A-I condition remains to be elucidated.
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ACCEPTED MANUSCRIPT Maintaining high Apo A-I levels might be beneficial for MS patients. Since Apo A-I is a major HDL component, use of statin medication could be beneficial. However clinical trials in MS patients
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delivered controversial results, especially in the area of new relapses and brain plaque formation (Maier, De Jonge et al. 2009; Markovic-Plese, Jewells et al. 2009; Chataway 2012; Chataway,
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Schuerer et al. 2014). Statins are known to lower LDL and improve triglyceride profiles. A meta-
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analysis of 32,258 patients from 37 randomized trials found that in addition to lowering LDL levels
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statin drugs (rosuvastatin, atorvastatin and simvastatin) raised HDL-C and Apo A-I levels (Barter, Brandrup-Wognsen et al. 2010). High dose (80mg) of oral atorvastatin decreased LDL in MS patients
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but had no effect on HDL (Paul, Waiczies et al. 2008). A recent MS-STAT clinical trial has shown that high dose simvastatin attenuates brain atrophy (Chataway, Schuerer et al. 2014). The main
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reason behind brain atrophy is believed to be a neuroaxonal loss. The reduction in brain atrophy in
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this trial was associated with reduction in total cholesterol levels. The authors did not directly
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measure Apo A-I levels in MS patients, however because of the known effects of simvastatin on cholesterol metabolism (Matthan, Giovanni et al. 2003), the data strongly suggest that HDL and Apo A-I levels were higher in simvastatin group, where cholesterol was reduced from 5.5 mmol/L to 4.1mmol/L and brain atrophy was reduced by 43%. This study however didn’t show any effect on immune markers such as Th 17, INF, IL-4, IL-10, and CD4+FoxP3. Therefore these data suggest that statins (even at a high dose) have a very limited positive effect on MS. This could be due to the fact that statins have been shown to increase reactive oxygen species, elevate lipid peroxidation and induce oxidative DNA damage in human peripheral blood lymphocytes (Gajski, Garaj-Vrhovac et al. 2008; Qi, Zheng et al. 2013). Increased lipid peroxidation is associated with disease exacerbation periods and lesion pathogenesis in MS patients (Toshniwal and Zarling 1992). Therefore a different type of drug, acting upon a different mechanism of Apo A-I activation might provide more benefit for progressive MS patients.
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5. Conclusion Our data supports the hypothesis that Apo A-I plays an important role in MS pathogenesis. Through
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a series of clinical, electrophysiological and histological experiments we have shown that increased
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Apo A-I levels are beneficial for neuroaxonal protection and are associated with less neurological
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deficit in EAE, an MS animal model. The exact mechanism of Apo A-I involvement in CNS has to be studied further in order to identify positive regulators of Apo A-I biogenesis and its involvement
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in MS.
Acknowledgments
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The authors thank Drs. Tonia Rex and Natalya Lenchik for the assistance and guidance in performing
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and analyzing the F-VEPs, 2-D PAGE, and Flow cytometry respectfully. This work was funded by
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the pilot grant from the National Multiple Sclerosis Society (to LAG) and the VA Merit Review Award (to MCL).
Figure captions
Color preference on the Web only. Figure 1. A. Plasma protein separation in Rotofor cell and protein identification (inset). B. Twodimensional analysis of Apo A-I protein expression in human plasma (control (left) and PPMS (right)). C. Apo A-I protein detection (SYPRO ruby protein stain) and Western blot analysis with anti-ApoA1 antibodies. Antibodies had high affinity to Apo A-I in Control and RRMS samples, but not in SPMS or PPMS. Figure 2. A. Apo A-I expression on 2-D gel (zoomed area with five distinct spots from control sample). B. Five samples per group were analyzed and quantified for ApoA-I. The most significant
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ACCEPTED MANUSCRIPT differences were found in the expression of spots 3 and 4. Inset table shows fold change between control and disease groups. C. Graphical representation of Apo A-I protein expression in analyzed
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samples. Statistically significant differences were observed between controls and all disease groups for spot 3 and between RRMS and progressive MS samples for spot 4. ApoA-I expression in spot 3
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was different between controls and patients with progressive disease (SPMS and PPMS).
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Figure 3. A. Western Blot quantification of Apo A-I expression. 60µg of pre-cleared plasma was
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loaded onto SDS-PAGE and probed with anti-ApoA1 antibodies. Band intensity was quantified in QuantityOne software (Bio-Rad, Hercules, CA). Twenty five samples per groups were analyzed.
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Representative images (above the graph) are shown for each group. B. Apo A-I levels measured in serum of MS patients and controls with ELISA.
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Figure 4. Endogenous ApoA-I expression in wild type and transgenic mice. A. Coomassie blue
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stained SDS-PAGE. Lane 1-Molecular weight marker; lanes 2-5 samples obtained from wild type
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animals (2-serum, 3-brain, 4-liver, 5- spinal cord); lanes 6-9 samples from ApoA-I deficient mouse (6-serum, 7-brain, 8 - liver, 9 - spinal cord). ApoA-I is expressed at 28kDA. B. Western blot detection of mouse ApoA-I protein in different tissue. Apo A-I is not expressed in brain, but is present in the spinal cord of the wild-type animal in contrast to Apo A-I deficient mouse. C. Histology of the lumbar spinal cord sections in naive C57BL/6J and ApoA-I deficient animals. Primary goat polyclonal antibodies that recognize mouse Apo A-I protein (Santa Cruz, CA) were used for detection. Secondary antibodies were anti-goat TxRed (Vector laboratories, Burlingame, CA). Wild type mice have prominent Apo A-I expression in comparison to transgenic mice (arrows). Scale bar = 50µM. Figure 5. Apo A-I deficient mice exhibit more EAE signs in comparison to control animals A. EAE induced mice show first disease signs on day 14. Scale 0-4. B. Flash-VEP recording in naïve and EAE induced wild type and Apo A-I deficient mice. C. Histopathology of the spinal cord. Scale
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ACCEPTED MANUSCRIPT bar=50µm. Luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining of the paraffin embedded spinal cord tissue. LFB identifies demyelinated regions (stained purple) and increased vacuolization
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(red arrows) in Apo A-I deficient animals. Fluoro-Jade B staining identifies neuronal degeneration seen in C57BL/6J and Apo A-I deficient mice but not in Naïve animals.
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Figure 6. Lumbar section of the spinal cord of ApoA-I deficient mice displays increased
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vacuolization. Lower panel represents higher magnification (20x) of the regions of interest (red
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squares). Arrows indicated vacuoles containing cellular debris. Vacuolization was scored on high magnification images. Number of vacuoles were calculated per filed view quadrant as follows: 1 - 5
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vacuoles = score of 1, 5 - 6 vacuoles = score of 2, 10 - 30 vacuoles = score of 3. ApoA-I deficient mice had higher demyelination score (3. 4 0.2) in comparison to wild type mice (2.9 0.2), Scale
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bar=20µm.
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Figure 7. A. Flow cytometry analysis of the T cells expressed in experimental animals. No
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significant increase in regulatory T cells was detected. B. Cytokine expression in wild type and ApoA-I deficient mice. Serum was collected from experimental animals prior to sacrifice on day 36. Multianalyte ELISArray (Qiagen) was used to detect Th1 vs. Th2 cytokines. Results were analyzed in GraphPad prism software utilizing student t-test. Star indicates significant difference between the groups. *=P<0.05, **=P<0.01.
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Table1. Demographic data for study subjects. Gender F
Subjects on Medications
Average Age
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50.1
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28
RRMS
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22
31
SPMS
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PPMS
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We identified differential expression of Apo A-I protein in human plasma MS patients on average had less Apo A-I compared to healthy controls The lowest expression of Apo A-I was found in progressive MS patients Mouse deficient in murine Apo A-I had worse EAE disease, an animal model for MS
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