Antibodies to MOG have ademyelination phenotype and affect oligodendrocyte cytoskeleton · cytoskeleton ABSTRACT Objective: To examine the clinical features of pediatric CNS demyelination
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FRACPDavid R Booth PhDLouise A Wienholt PhDKristina Prelog FRACPDamien R Clark FRACPGilles J Guillemin PhDChai K Lim PhDEmily K Mathey PhDFabienne Brilot PhD
Antibodies to MOG have a demyelinationphenotype and affect oligodendrocytecytoskeleton
ABSTRACT
Objective To examine the clinical features of pediatric CNS demyelination associated with pos-itive myelin oligodendrocyte glycoprotein (MOG) antibodies and to examine the functional effectsof MOG antibody on oligodendrocyte cytoskeleton
Methods WemeasuredMOGantibody using a fluorescence-activated cell sorting live cell-based assayin acute sera of 73 children with CNS demyelination (DEM) (median age 8 years range 13ndash153)followed for a median of 4 years We used MO313 cells to examine immunoglobulin (Ig) G effects onoligodendrocyte cytoskeleton using 3D deconvolution imaging
ResultsMOGantibodieswere found in 3173 patients with DEM (42) but in 024 controls At firstpresentation MOG antibodyndashpositive patients were more likely to have bilateral than unilateral opticneuritis (ON) (910 vs 15 respectively p 5 003) less likely to have brainstem findings (231 vs1642 p5 0005) more likely to have a raised erythrocyte sedimentation rate20 mmh (919 vs321 p 5 005) less likely to have intrathecal oligoclonal bands (016 vs 527 p 5 018) and lesslikely to be homozygous or heterozygous for human leukocyte antigen DRB11501 (318 vs 722p5 046) MOG antibody positivity varied according to clinical phenotype with ON and relapsing ONmost likely to be seropositive Two relapsingMOG antibodyndashpositive patients treated with mycophe-nolate mofetil remain in remission and have become MOG antibody seronegative Oligodendrocytesincubated with purified IgG from MOG antibodyndashpositive patients showed a striking loss of organi-zation of the thin filaments and the microtubule cytoskeleton as evidenced by F-actin and b-tubulinimmunolabelings
Conclusions MOG antibody may define a separate demyelination syndrome which has therapeu-tic implications MOG antibody has functional effects on oligodendrocyte cytoskeleton Neurol
Neuroimmunol Neuroinflammation 20141e12 doi 101212NXI0000000000000012
Recently autoantibodies that bind to cell surface antigens have been shown to be importantdiagnostic biomarkers in autoimmune brain disease including autoimmune encephalitis andautoimmune demyelination1ndash3 Myelin oligodendrocyte glycoprotein (MOG) is a minor com-ponent of myelin proteins but has been the focus of extensive research in demyelinating diseasesMOG is localized on the outermost surface of myelin and has a proposed role in the regulation
These authors contributed equally to the manuscript
From the Neuroimmunology Group (RCD EMT VM R-YAK NS K Pathmanandavel SR FB) Institute for Neuroscience andMuscle Research The Kids Research Institute at the Childrenrsquos Hospital at Westmead Sydney Medical School University of Sydney WestmeadAustralia Institute for Immunology and Allergy Research (DRB) Westmead Millenium Institute for Medical Research University of SydneyWestmead Australia Clinical Immunology (LAW) Royal Prince Alfred Hospital Sydney Medical School Immunology amp Infectious DiseasesUniversity of Sydney Camperdown Australia Department of Radiology (K Prelog) the Childrenrsquos Hospital at Westmead Australia Departmentof Paediatric Neurology (DRC) Womenrsquos and Childrenrsquos Hospital North Adelaide Australia Neuroinflammation Group (GJG CKL)MND and Neurodegenerative Diseases Research Centre Macquarie University Australian School of Advanced Medicine North Ryde Australiaand Neuroinflammation Group (EKM) Brain and Mind Research Institute University of Sydney Camperdown Australia
Go to Neurologyorgnn for full disclosures Funding information and disclosures deemed relevant by the authors if any are provided at the end ofthe article The Article Processing Charge was paid by the authors
This is an open access article distributed under the terms of the Creative Commons Attribution-Noncommercial No Derivative 30 License whichpermits downloading and sharing the work provided it is properly cited The work cannot be changed in any way or used commercially
Neurologyorgnn copy 2014 American Academy of Neurology 1
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
of microtubule stability4 Autoantibodiesagainst MOG (MOG antibodies) have beenshown to mediate demyelination in rodentsin ldquo2-hit modelsrdquo and also in primates5ndash8
The importance of MOG antibodies inhuman demyelinating disease has previouslybeen controversial predominantly due to theuse of antibody assays that denature proteinand alter conformation More recently usingcell-based assays high titer MOG antibody hasbeen unequivocally found in 20ndash40 of chil-dren with acute CNS demyelination9 In partic-ular MOG antibodies have been shown to beassociated with acute disseminated encephalomy-elitis (ADEM) and patients with neuromyelitisoptica (NMO)-like phenotypes who are negativefor NMO immunoglobulin (Ig) G10ndash16 How-ever detailed clinical and radiologic phenotypingassociated with MOG antibodies is still lackingand the role of MOG antibodies as a biomarkerin clinical practice is still not clear
Herein we further define the clinical signif-icance of MOG antibody as a biomarker andshow that MOG antibody can modify themicrotubule network and thin filaments ofoligodendrocytes
METHODS Patients and controls Patients The stored
acute serum (280degC) taken from 73 children during their first
episode of CNS demyelination (DEM) was used for this study
(median age 8 years range 13ndash153 37 females) All sera were
acute and before immune therapy The clinical and radiologic
features of 60 of the patients have been reported previously17
but the serologic investigation of this cohort has not been pre-
viously reported The patients were clinically phenotyped using
2013 consensus criteria18 The first episode of demyelination
was ADEM (n 5 28) transverse myelitis (TM n 5 15) optic
neuritis (ON n 5 15) and other clinically isolated syndrome
(CIS) excluding TM and ON (n 5 15) These other CIS
patients had polyfocal CIS cerebellar CIS brainstem CIS or
hemispheric CIS The acute MRI brain scans (n 5 70) and
MRI spine (n5 30) were reviewed and rated using MRI criteria
blinded to the laboratory findings as previously described using
McDonald KIDMUS Callen and Verhey criteria17 The
patients were followed for a median of 40 years (range 03ndash
137 years) At study census and classification 54 patients had a
monophasic disease (ADEM n 5 24 TM n 5 13 ON n 5 7
ldquoother CISrdquo n 5 10) Nineteen of 73 patients had a relapsing
Functional effects on human oligodendroglial cells MO313
cell line is an immortal human-human hybrid cell line that expresses
phenotypic characteristics of primary oligodendrocytes and was
created by fusing a 6-thioguanine-resistant mutant of the human
rhabdomyosarcoma RD (cancer of skeletal muscle) with adult
human oligodendrocytes by a lectin-enhanced polyethylene glycol
procedure24 Transduced human oligodendroglial MO313MOG1
and MO313Ctl cells were cultured as previously described24 and
were immunostained with described protocols (appendix e-1)2223
We purified human IgG or human MOG IgG from human sera
using protein G-agarose and Microcon (Millipore Billerica MA)
and human MOG bound to an activated N-hydroxysuccinimide
agarose column (GE Healthcare Little Chalfont United
Kingdom)2526 We also immunoabsorbed MOG antibodyndashpositive
sera by incubating 6 wells of live HEK293MOG1Ctl cells with MOG
antibodyndashpositive and ndashnegative sera22 In order to visualize effects of
protein Gndashpurified human IgGs on single cells MO313 cells were
seeded at low density and incubated with 6 mg of protein Gndashpurified
human IgGs from patient or control sera and HEK293MOG1-
immunoabsorbed MOG antibodyndashpositive serum for 45 minutes
at room temperature followed by goat anti-human IgG secondary
antibody for 15minutes at room temperature2728 After washing cells
were fixed and permeabilized and cytoskeleton filamentous actin
(F-actin marker of cytoskeleton thin filaments) and b-tubulin
2 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
(marker of cytoskeleton microtubule) were quantified using 3D
deconvolution microscopy (detailed methodology in appendix e-1)29
Human leukocyte antigen DRB11501 genotyping DNA
from patients with DEM (n 5 40) was extracted from saliva using
a saliva-specific DNA extraction kit according to the manufacturerrsquos
instructions (Oragene DNA Genotek Kanata Ontario Canada)
The rs9271366 single nucleotide polymorphism (SNP) that lies on
chromosome 6 at position 32694832 upstream of the allelic variation
(A G) was used to genotype the patients for human leukocyte
antigen (HLA)-DRB1150130 Patients with the allelic A are negative
for HLA-DRB11501 whereas patients with the allelic G are HLA-
DRB11501ndashpositive The SNP rs9271366 is always inherited with
the HLA-DRB11501 allele (complete linkage disequilibrium with
HLA-DRB11501)30 DNA was amplified using PCR (detailed
methodology in appendix e-1) and sequences were analyzed by the
software Sequencher (Gene Codes Corporation Ann Arbor MI)
Statistical analysis Chi-square with Yates correction test was usedto compare MOG antibody positivity between patient and control
groups and between patient subgroups and to compare binary clin-
ical and radiologic features between MOG antibodyndashpositive and ndash
negative groups Wilcoxon 2-sample test was used to compare ages
of patients in 2 groups No adjustment for multiple testing has been
performed The effects of IgG on F-actin and b-tubulin were
normally distributed thus means and SDs were analyzed with
the 2-tailed Student t test A p value less than 005 was
considered significant for the 2-tailed Student t test (MOG
antibody pathogenic effect)
RESULTS Surface MOG IgG antibody in children with
demyelinating diseases In order to align our detectionmethod to recent reports we first detected MOGantibody in serum using FACS assay andHEK cells ex-pressing human MOG1114163132 The mean fluores-cence intensity correlated with antibody concentration(figure 1A) Using the mean plus 3 SDs to establish thethreshold for positivity MOG antibodies were foundin 3173 (42) of the DEM group but in 024 con-trols (figure 1 BndashD p 5 0000) We have tested 57other medical and neurologic controls and all werenegative We also tested MOG antibody in CSF (fig-ure 1E) We had CSF available in 22 patients withDEM of which 5 were positive for MOG antibodyin serum Only 322 CSF samples were positive 2 ofwhich were serum positive and 1 of which was serumnegative (figure 1E) Serologic testing for AQP4 anti-bodies was performed in 64 patients with DEM andwas negative in all (data not shown)
Clinical and investigation findings according to serum
MOG antibody findings First episode We compared theclinical features according to MOG antibody statusduring the first episode of demyelination in detailand findings are presented in table e-1 Patients withpositive MOG antibody were younger (median age67 vs 104 years p5 006) and marginally more likelyto be female (1831 58 vs 1942 45 p 5 040)MOG antibody positivity was negatively associatedwith brainstem signs (231 vs 1642 42 p 5 0005)In patients with ON positive MOG antibody was
more common in patients with bilateral compared tounilateral ON (910 vs 15 respectively p 5 003 (fig-ure 1F) MOG antibodyndashpositive patients were morelikely to have a raised erythrocyte sedimentation rate(ESR) 20 mmh during the first episode than MOGantibodyndashnegative patients (919 vs 321 p 5 005)There was no correlation between age and elevatedESR in MOG antibodyndashpositive patients (figure 1G)Intrathecal oligoclonal bands were not present in theMOG antibodyndashpositive patients and were seen onlyin MOG antibodyndashnegative patients (016 vs 527p 5 018) MOG antibodyndashpositive patients were lesslikely to be homozygous or heterozygous for HLA-DRB11501 than patients with negative MOG anti-body (318 17 vs 722 32 p 5 046) Detailedblinded radiologic analysis of MRI brain (n 5 70) andMRI spine (n5 30) during the first episode showed nosignificant differences according to MOG antibody sta-tus (table e-2) There was no difference in MRI MScriteria usingMcDonald KIDMUS Callen and Verhey(table e-3)
Follow-up diagnosis To compare MOG antibodyfindings we compared the subgroups according to clas-sification at follow-up (figure 1 H and I) In the mon-ophasic subgroups MOG antibodies were positive in1124 (46) patients with ADEM 67 (86) patientswith ON 413 (31) patients with TM and 010(0) patients with ldquoother CISrdquo (defined in Methods)Patients with monophasic ON were more likely to beMOG antibodyndashpositive than the rest of the cohort(67 vs 2566 p 5 004) and patients with ldquootherCISrdquo were less likely to be MOG antibodyndashpositivethan the rest of the cohort (010 vs 3163 p 5
001) In the relapsing patients MOG antibody waspositive in 715 (47) of patients with MS and 34(75) patients with relapsing ON It is interesting thatthe 3 MOG antibodyndashpositive patients with relapsingON were negative for AQP4 antibody whereas theMOG antibodyndashnegative patient with relapsing ONwas not tested for AQP4 antibody due to insufficientserum In the relapsing patients (n 5 19) the MOGantibodyndashpositive patients were younger (median 56years range 26ndash142) than the ndashnegative patients(median 129 years range 25ndash153 p 5 003) TheMOG antibodyndashpositive patients had fewer relapses(22 relapses in 603 patient-years median annualizedrelapse rate 056) than the MOG antibodyndashnegativepatients (26 relapses in 39 patient-years median annu-alized relapse rate 065)
Longitudinal MOG antibody in 2 patients treated with
immune suppression Two recent patients (figure 2 andappendix e-2) with positive MOG antibodies who ful-filled 2013 criteria for MS18 were treated with myco-phenolate mofetil (MMF) which produced clinicalremission no new lesions on follow-up MRI scans
Neurology Neuroimmunology amp Neuroinflammation 3
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
Figure 1 Distribution of MOG IgG antibody in pediatric demyelinating diseases
(A) Antibody reactivity to myelin oligodendrocyte glycoprotein (MOG) was determined by flow cytometry live cell-based assay (B) Representative example offlow cytometry histograms for oneMOG antibodyndashpositive patient with a very high DMFI and (C) an intermediate DMFI MFI values are noted in the legend (D)Human surface MOG IgG antibody was detected in 3173 sera from patients with demyelinating diseases (DEM) and 024 controls (CTL) Magenta line ongraph represents the positivity threshold MOG antibody positivity is shown between brackets (E) Surface MOG antibody was detected in 322 CSF frompatients with DEM and 020CSF from patients with other neurologic diseases (CTL) Black circles represent patients with positiveMOG antibody in CSF andin serum (F) Distribution and number of MOG antibodyndashpositive patients in optic neuritis (ON) (G) Correlation between erythrocyte sedimentation rate andage in MOG antibodyndashpositive patients (H) Distribution and (I) number of MOG antibodyndashpositive patients in demyelinating diseases at follow-up Ab 5
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
and normalization of MOG antibodies (figure 2 andappendix e-2) In both patients upon treatment withMMF MOG antibody decreased below the thresholdof positivity (figure 2 A and B)
Loss of cytoskeleton organization by MOG antibody or
purified IgG from children with demyelinating diseases
Human oligodendrocyte MO313MOG1 cells expressedMOG at their surface whereas noMOG expression wasobserved on MO313Ctl cells (figure 3) MO313 cellsalso expressed oligodendrocyte markers such as 29 39-cyclic nucleotide 39-phosphodiesterase (CNPase) galac-tocerebroside (GalC) oligodendrocyte marker O4vimentin and c-series ganglioside-specific antigen(A2B5) (figure 3A) Myelin basic protein (MBP) aspecific marker of mature oligodendrocytes wasobserved only in PMA-differentiated MO313MOG1
cells (figure 3A) suggesting that undifferentiatedMO313 cells are immature oligodendrocytes Using
purified IgG and immunoaffinity-purified MOG IgGfromMOG antibodyndashpositive sera we immunolabeledHEK293MOG1 and HEK293Ctl cells on live cells byFACS (figure 3B) and on fixed MO313MOG1 cellsby immunocytochemistry (figure 3C) and showeda positive immunostaining compared to MOGantibodyndashnegative sera suggesting that proteinGndashpurified IgG includes MOG-specific IgG Due tosmall volumes of pediatric sera we used proteinGndashpurified IgG in pathogenic experiments Next wetreated fixed and live MO313MOG1Ctl cells with IgGfrom MOG antibodyndashpositive and ndashnegative patientswith DEM and healthy controls (HCs) Then all cellswere immunolabeled for b-tubulin (marker ofmicrotubule) or F-actin (marker of thin filaments) Wequantified results and expressed them by the F-actin andb-tubulin relative enrichments in the cytoplasm andperinuclear region over the entire cell All results were
Figure 2 Temporal distribution of MOG antibody in serum of 2 relapsing patients with demyelinating diseases
In both patient A (A) and patient B (B) upon treatment mycophenolate mofetil (MMF) myelin oligodendrocyte glycoprotein (MOG) antibody decreased towithin the healthy control range (below threshold of positivity) and these low titers were associated with remission Representative dot plot out of 3 experi-ments is shown Magenta lines on graphs represent the positivity threshold (obtained with 24 control samples) Black squares represent serum analysis dur-ing acute demyelination episodes and black circles represent sera during remission Type of demyelinating episode is shown on graph (C D) RepresentativeT2 axial MRI scans demonstrate demyelinating lesions during the first acute event and during convalescence Patient A (C) had globular deep white matterlesions on acute scan (left panel) which show residual gliosis on convalescent scan and no new lesions (right panel) Patient B (D) had inflammatory lesions inbasal ganglia and white matter on acute scan (left panel) with complete resolution on convalescent scan and no new lesions (right panel) Ab 5 antibodyADEM5 acute disseminated encephalomyelitis CEREB5 cerebellar episode Ig5 immunoglobulin MFI5mean fluorescence intensity ON5 optic neuritisTM 5 transverse myelitis
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
normalized using F-actin and b-tubulin in HCIgG-treated fixed cells Organization of themicrotubule and thin filament networks wassimilar in fixed cells incubated with HC (1006 101 and 100 6 226 respectively figure 4A and C) or DEM IgGs (1043 6 113 and858 6 318 respectively figure 4 A and C)
there was clear visualization of ldquostress-fibersrdquo andfilopedia after F-actin immunolabeling (figure 4Aupper panels) and bright mesh-like staining spreadout through the entire cell after b-tubulinimmunolabeling (figure 4A lower panels) Livecells treated by HC IgG displayed a smalldisorganization of F-actin thin filaments (916 144
Figure 3 Human oligodendroglial MO313MOG1 cells express markers of oligodendrocytes and are immunolabeled with protein G- and humanMOG-purified human IgG from MOG antibodyndashpositive DEM patients
(A) Immunocytochemistry on fixed permeabilized cells showed that MO313MOG1 cells expressed oligodendrocyte markers (B) Protein G- and human MOG-immunopurified IgG from MOG antibodyndashpositive serum immunolabeled live HEK293MOG1 cells but did not immunolabel HEK293Ctl cells compared to MOGantibodyndashnegative serum Binding to cells was determined by flow cytometry Mean fluorescence intensity (MFI) values are shown in legends (C) ProteinG- and human MOG-purified IgG from MOG antibodyndashpositive serum also immunolabeled fixed unpermeabilized MO313MOG1 cells compared to MOGantibodyndashnegative serum Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy) Nuclei stainedwith 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm A2B55 c-series ganglioside-specific antigen A2B5 Ab5 antibody CNPase529 39-cyclic nucleotide39-phosphodiesterase DEM 5 demyelinating diseases GalC 5 galactocerebroside HEK 5 human embryonic kidney Ig 5 immunoglobulin MBP 5 myelinbasic protein MOG 5 myelin oligodendrocyte glycoprotein O4 5 oligodendrocyte marker O4
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
Figure 4 Purified IgG from DEM patients induces loss of cytoskeleton organization in live human oligodendroglial MO313MOG1 cells
F-actin (upper images) and b-tubulin (lower images) immunolabelings in human fixed (A) or live (B) oligodendroglial MO313MOG1 cells incubated with purifiedhealthy control (HC) IgG or MOG antibodyndashpositive DEM IgG Representative data are shown (volume projection of entire Z-stack acquired using deconvo-lution microscopy) Nuclei stained with 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm Dotted lines on right images represent contour of cells as deter-mined by differential interference contrast images (not shown) (C) Quantification of change in distribution of thin filament (F-actin upper scatter plot) andmicrotubule network (b-tubulin lower scatter plot) organization using 3D deconvolution microscopy F-actin and b-tubulin were majorly affected in live cellsincubated with DEM IgG Forty different cells (1 cell5 1 diamond) from 2 HC and 2 DEM patients out of 3 independent experiments are shown Results areexpressed as ratio of the relative enrichment (RE) between cytoplasm and nucleus and values shown are percentages relative to HC IgG FIX (100) Redbars represent mean Note that there also was a loss of organization of the thin filaments after HC IgG incubation on live cells compared to fixed conditionsbut to a lesser extent than after DEM IgG (D) Cell viability assay by flow cytometry on live human oligodendroglial MO313MOG1 cells incubated 45 minuteswith 6 mg of purified HC IgG or DEM IgG No difference in cell death was observed Dead cell percentages are noted in the legend 7AAD 5 7-amino-actinomycin D DEM 5 demyelinating diseases Ig 5 immunoglobulin MOG 5 myelin oligodendrocyte glycoprotein
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
p 00001 figure 4 B and C upper images)and undisturbed microtubular b-tubulin network(1074 6 321 figure 4 B and C) similar to thatobserved in fixed DEM and HC IgG-treated cells(figure 4A upper panels) Following treatment withDEM IgG on live MO313MOG1 cells we observed astriking loss of organization in both F-actin (7246 117 p 00001 figure 4 B and C) andb-tubulin networks (522 6 131 p 00001figure 4 B and C) in otherwise healthy-lookingundividing cells visualized by differential interferencecontrast imaging (figure 4B dotted line and data notshown) and analyzed by FACS by which there was nodifference in viability between live MO313MOG1 livecells treated with DEM IgG or HC IgG for45 minutes (figure 4D 8726 06 vs 8726 211) or10 hours (data not shown) No loss of cytoskeletonorganization was observed when live MO313Ctl cellswere incubated with HC IgG and MOG antibodyndashpositive DEM IgG nor when live MO313MOG1 cellswere incubated with MOG antibodyndashnegative DEMIgG (figure e-1 and appendix e-3) In additionsuccessful immunoabsorption of MOG antibodyndashpositive serum on HEK293MOG1 cells led to nochange in cytoskeleton organization whenimmunoabsorbed serum was incubated on liveMO313MOG1 cells (figure e-1 and appendix e-3)Overall after incubation with MOG antibodyndashpositive DEM IgG both F-actin and b-tubulinimmunolabelings appeared to be enriched withina perinuclear region in the center of the cellssurrounding the nucleus
DISCUSSION As in this study recent reports haveconsistently found that serum MOG antibody is pre-sent in a significant proportion of children with CNSdemyelinating disease whereas AQP4 antibodies arerare9 We believe that the clinical and investigationfindings in this report support the evolving conceptthat MOG antibodies may define a separate autoim-mune demyelinating syndrome that may be distinctfrom classic adult-onset MS MOG antibodies arepresent in up to 50 of children with ADEM as inthis report Proebstel et al33 found that ADEMMOGantibody seropositive children became seronegative inthe recovery period whereas children with relapsingMS typically remained seropositive More recentlyMOG antibodies have been found in children withON and in some patients with NMO phenotypenegative for NMO IgG1431 In this report we havestrengthened this association with ON and found thatpatients with bilateral rather than unilateral ON weremore likely to be MOG antibodyndashpositive BilateralON is a common ON phenotype in children but israre in adults in whom unilateral ON is the moretypical demyelinating event of MS In contrast
patients with brainstem signs and CIS other thanON and TM were rarely MOG antibodyndashpositiveAn elevated ESR was more common in MOGantibodyndashpositive patients An elevated ESR isatypical of MS and should raise suspicion of anautoimmune disorder or alternate inflammatorymimic of MS We showed that elevation of ESR inMOG antibodyndashpositive patients was independent ofage Intrathecal oligoclonal bands are one of the classicbiomarkers of MS and were absent in all MOGantibodyndashpositive patients and were present only inthe MOG antibodyndashnegative patients Only 17 ofthe MOG antibodyndashpositive patients had 1 or bothHLA-DRB11501 alleles the main MS susceptibilityallele which is the same as the previously reportedpercentage in the ldquonormalrdquo healthy population inAustralia34 whereas the MOG antibodyndashnegativepatients were more likely to have 1 or more HLA-DRB11501 allele This negative association wasnot statistically significant and larger cohorts arerequired to test whether this correlation is a realrather than a suspected phenomenon In contrastradiologic features did not appear to differentiateMOG antibodyndashpositive from ndashnegative patients inthis childhood cohort
The presence of MOG antibodies is unlikely tohave therapeutic implications in the first demyelinat-ing event but is more likely to be therapeuticallyimportant in the relapsing patient Although 2 recentpatients fulfilled criteria for MS the presence ofMOG antibodies and elevated ESR led us to treatthem more like NMO patients with the immune sup-pression agent MMF rather than conventional MSdisease-modifying therapies Both patients had noclinical or radiologic relapse for 12 months and 36months respectively and both became MOG anti-body seronegative Further studies are required tounderstand the longitudinal course of patients withMOG antibodyndashassociated relapsing disease and thetreatment approach required
Cell-based assays have been essential to detectMOG antibody with a high specificity9 especiallywhen the full-length MOG is expressed in cells ratherthan the C-terminal truncated MOG35 Our experi-ence with neuronal antigens informed us that FACSis sensitive and quantitative10132022333637 We detectedCSF MOG antibody in only 3 of 22 DEM samples ofwhich 5 were positive in serum This suggests CSF isless sensitive than serum for MOG antibody detectionwhich could be due to a lack of sensitivity or couldrepresent a genuine absence of intrathecal Ig synthesisin these patients Supporting this concept none of thepatients with positive MOG antibody had intrathecaloligoclonal bands
The pathogenic function of MOG antibody is stillunclear but over the recent years the detection of
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316
and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538
Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40
Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders
AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript
RCD and FB Acquisition analysis and interpretation of results RCD
EMT VM R-YAK NS SR KP LAW DRB K Prelog
DRC GJG CKL EKM FB Statistical analysis RCD EMT
FB Critical revisions of manuscript RCD EMT VM NS SR
KP DRB LAW K Prelog DRC GJG CKL EKM FB
ACKNOWLEDGMENTThe authors thank all the patients and family members who provided
samples for the study The authors thank Dr Maggie Wang for use of
the Flow Cytometry Core Facility of the Westmead Millennium Institute
(Australia) The authors thank Dr Hong Yu for use of the Imaging Core
Facility at the Westmead Research Hub
STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple
Sclerosis Research Foundation Multiple Sclerosis Research Australia
MS Angels Melbourne (Australia) and the Petre Foundation (Australia)
DISCLOSURERC Dale has received research funding from the Star Scientific Foundation
the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis
Research Australia NHMRC and the Petre Foundation has received hon-
oraria from Biogen Idec and is on advisory boards for Queensland Child-
renrsquos Medical Institute Research and MSARD EM Tantsis has received
a scholarship from the Australian Postgraduate Award program V Merheb
and R-YA Kumaran report no disclosures N Sinmaz has received a schol-
arship from the Australian Postgraduate Award program K Pathmanandavel
has received a scholarship from the Petre Foundation (Australia)
S Ramanathan has received a scholarship from the National Health and
Medical Research Council (Australia) DR Booth has received funding from
the National Health and Medical Research Council (Australia) the Australian
Research Council Multiple Sclerosis Research Australia and the Leukemia
Foundation has received honoraria and funding from Biogen Idec Merck
Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS
ONE LA Wienholt K Prelog and DR Clark report no disclosures
GJ Guillemin is the editor-in-chief for the International Journal for Trypto-
phan Research and has received research support from the Australian Research
Council CK Lim has received research funding from Multiple Sclerosis
Research Australia and Trish MS Research Foundation EK Mathey has
received funding from the National Health and Medical Research Council
(Australia) and the Rebecca L Cooper Medical Research Foundation
(Australia) F Brilot has received research funding from the Star Scientific
Foundation the Trish Multiple Sclerosis Research Foundation Multiple
Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre
Foundation and is an associate editor for the Journal of Visualized Experiments
Go to Neurologyorgnn for full disclosures
Received February 25 2014 Accepted in final form April 16 2014
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romyelitis optica Lancet Neurol 201211535ndash544
2 Vincent A Bien CG Irani SR Waters P Autoantibodies
associated with diseases of the CNS new developments
and future challenges Lancet Neurol 201110759ndash772
3 Wingerchuk DM Lennon VA Lucchinetti CF
Pittock SJ Weinshenker BG The spectrum of neuromy-
elitis optica Lancet Neurol 20076805ndash815
4 Johns TG Bernard CC The structure and function of
myelin oligodendrocyte glycoprotein J Neurochem
1999721ndash9
5 Linington C Bradl M Lassmann H Brunner C Vass K
Augmentation of demyelination in rat acute allergic
encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelinoligodendrocyte glycopro-
tein Am J Pathol 1988130443ndash454
6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous
relapsing-remitting EAE in the SJLJ mouse MOG-reactive
transgenic T cells recruit endogenous MOG-specific B cells
J Exp Med 20092061303ndash1316
7 Schluesener HJ Sobel RA Linington C Weiner HL A
monoclonal antibody against a myelin oligodendrocyte
glycoprotein induces relapses and demyelination in central
nervous system autoimmune disease J Immunol 1987
1394016ndash4021
8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB
Lee JI Genain CP Molecular characterization of antibody
specificities against myelinoligodendrocyte glycoprotein
in autoimmune demyelination Proc Natl Acad Sci U S A
2002998207ndash8212
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
of microtubule stability4 Autoantibodiesagainst MOG (MOG antibodies) have beenshown to mediate demyelination in rodentsin ldquo2-hit modelsrdquo and also in primates5ndash8
The importance of MOG antibodies inhuman demyelinating disease has previouslybeen controversial predominantly due to theuse of antibody assays that denature proteinand alter conformation More recently usingcell-based assays high titer MOG antibody hasbeen unequivocally found in 20ndash40 of chil-dren with acute CNS demyelination9 In partic-ular MOG antibodies have been shown to beassociated with acute disseminated encephalomy-elitis (ADEM) and patients with neuromyelitisoptica (NMO)-like phenotypes who are negativefor NMO immunoglobulin (Ig) G10ndash16 How-ever detailed clinical and radiologic phenotypingassociated with MOG antibodies is still lackingand the role of MOG antibodies as a biomarkerin clinical practice is still not clear
Herein we further define the clinical signif-icance of MOG antibody as a biomarker andshow that MOG antibody can modify themicrotubule network and thin filaments ofoligodendrocytes
METHODS Patients and controls Patients The stored
acute serum (280degC) taken from 73 children during their first
episode of CNS demyelination (DEM) was used for this study
(median age 8 years range 13ndash153 37 females) All sera were
acute and before immune therapy The clinical and radiologic
features of 60 of the patients have been reported previously17
but the serologic investigation of this cohort has not been pre-
viously reported The patients were clinically phenotyped using
2013 consensus criteria18 The first episode of demyelination
was ADEM (n 5 28) transverse myelitis (TM n 5 15) optic
neuritis (ON n 5 15) and other clinically isolated syndrome
(CIS) excluding TM and ON (n 5 15) These other CIS
patients had polyfocal CIS cerebellar CIS brainstem CIS or
hemispheric CIS The acute MRI brain scans (n 5 70) and
MRI spine (n5 30) were reviewed and rated using MRI criteria
blinded to the laboratory findings as previously described using
McDonald KIDMUS Callen and Verhey criteria17 The
patients were followed for a median of 40 years (range 03ndash
137 years) At study census and classification 54 patients had a
monophasic disease (ADEM n 5 24 TM n 5 13 ON n 5 7
ldquoother CISrdquo n 5 10) Nineteen of 73 patients had a relapsing
Functional effects on human oligodendroglial cells MO313
cell line is an immortal human-human hybrid cell line that expresses
phenotypic characteristics of primary oligodendrocytes and was
created by fusing a 6-thioguanine-resistant mutant of the human
rhabdomyosarcoma RD (cancer of skeletal muscle) with adult
human oligodendrocytes by a lectin-enhanced polyethylene glycol
procedure24 Transduced human oligodendroglial MO313MOG1
and MO313Ctl cells were cultured as previously described24 and
were immunostained with described protocols (appendix e-1)2223
We purified human IgG or human MOG IgG from human sera
using protein G-agarose and Microcon (Millipore Billerica MA)
and human MOG bound to an activated N-hydroxysuccinimide
agarose column (GE Healthcare Little Chalfont United
Kingdom)2526 We also immunoabsorbed MOG antibodyndashpositive
sera by incubating 6 wells of live HEK293MOG1Ctl cells with MOG
antibodyndashpositive and ndashnegative sera22 In order to visualize effects of
protein Gndashpurified human IgGs on single cells MO313 cells were
seeded at low density and incubated with 6 mg of protein Gndashpurified
human IgGs from patient or control sera and HEK293MOG1-
immunoabsorbed MOG antibodyndashpositive serum for 45 minutes
at room temperature followed by goat anti-human IgG secondary
antibody for 15minutes at room temperature2728 After washing cells
were fixed and permeabilized and cytoskeleton filamentous actin
(F-actin marker of cytoskeleton thin filaments) and b-tubulin
2 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
(marker of cytoskeleton microtubule) were quantified using 3D
deconvolution microscopy (detailed methodology in appendix e-1)29
Human leukocyte antigen DRB11501 genotyping DNA
from patients with DEM (n 5 40) was extracted from saliva using
a saliva-specific DNA extraction kit according to the manufacturerrsquos
instructions (Oragene DNA Genotek Kanata Ontario Canada)
The rs9271366 single nucleotide polymorphism (SNP) that lies on
chromosome 6 at position 32694832 upstream of the allelic variation
(A G) was used to genotype the patients for human leukocyte
antigen (HLA)-DRB1150130 Patients with the allelic A are negative
for HLA-DRB11501 whereas patients with the allelic G are HLA-
DRB11501ndashpositive The SNP rs9271366 is always inherited with
the HLA-DRB11501 allele (complete linkage disequilibrium with
HLA-DRB11501)30 DNA was amplified using PCR (detailed
methodology in appendix e-1) and sequences were analyzed by the
software Sequencher (Gene Codes Corporation Ann Arbor MI)
Statistical analysis Chi-square with Yates correction test was usedto compare MOG antibody positivity between patient and control
groups and between patient subgroups and to compare binary clin-
ical and radiologic features between MOG antibodyndashpositive and ndash
negative groups Wilcoxon 2-sample test was used to compare ages
of patients in 2 groups No adjustment for multiple testing has been
performed The effects of IgG on F-actin and b-tubulin were
normally distributed thus means and SDs were analyzed with
the 2-tailed Student t test A p value less than 005 was
considered significant for the 2-tailed Student t test (MOG
antibody pathogenic effect)
RESULTS Surface MOG IgG antibody in children with
demyelinating diseases In order to align our detectionmethod to recent reports we first detected MOGantibody in serum using FACS assay andHEK cells ex-pressing human MOG1114163132 The mean fluores-cence intensity correlated with antibody concentration(figure 1A) Using the mean plus 3 SDs to establish thethreshold for positivity MOG antibodies were foundin 3173 (42) of the DEM group but in 024 con-trols (figure 1 BndashD p 5 0000) We have tested 57other medical and neurologic controls and all werenegative We also tested MOG antibody in CSF (fig-ure 1E) We had CSF available in 22 patients withDEM of which 5 were positive for MOG antibodyin serum Only 322 CSF samples were positive 2 ofwhich were serum positive and 1 of which was serumnegative (figure 1E) Serologic testing for AQP4 anti-bodies was performed in 64 patients with DEM andwas negative in all (data not shown)
Clinical and investigation findings according to serum
MOG antibody findings First episode We compared theclinical features according to MOG antibody statusduring the first episode of demyelination in detailand findings are presented in table e-1 Patients withpositive MOG antibody were younger (median age67 vs 104 years p5 006) and marginally more likelyto be female (1831 58 vs 1942 45 p 5 040)MOG antibody positivity was negatively associatedwith brainstem signs (231 vs 1642 42 p 5 0005)In patients with ON positive MOG antibody was
more common in patients with bilateral compared tounilateral ON (910 vs 15 respectively p 5 003 (fig-ure 1F) MOG antibodyndashpositive patients were morelikely to have a raised erythrocyte sedimentation rate(ESR) 20 mmh during the first episode than MOGantibodyndashnegative patients (919 vs 321 p 5 005)There was no correlation between age and elevatedESR in MOG antibodyndashpositive patients (figure 1G)Intrathecal oligoclonal bands were not present in theMOG antibodyndashpositive patients and were seen onlyin MOG antibodyndashnegative patients (016 vs 527p 5 018) MOG antibodyndashpositive patients were lesslikely to be homozygous or heterozygous for HLA-DRB11501 than patients with negative MOG anti-body (318 17 vs 722 32 p 5 046) Detailedblinded radiologic analysis of MRI brain (n 5 70) andMRI spine (n5 30) during the first episode showed nosignificant differences according to MOG antibody sta-tus (table e-2) There was no difference in MRI MScriteria usingMcDonald KIDMUS Callen and Verhey(table e-3)
Follow-up diagnosis To compare MOG antibodyfindings we compared the subgroups according to clas-sification at follow-up (figure 1 H and I) In the mon-ophasic subgroups MOG antibodies were positive in1124 (46) patients with ADEM 67 (86) patientswith ON 413 (31) patients with TM and 010(0) patients with ldquoother CISrdquo (defined in Methods)Patients with monophasic ON were more likely to beMOG antibodyndashpositive than the rest of the cohort(67 vs 2566 p 5 004) and patients with ldquootherCISrdquo were less likely to be MOG antibodyndashpositivethan the rest of the cohort (010 vs 3163 p 5
001) In the relapsing patients MOG antibody waspositive in 715 (47) of patients with MS and 34(75) patients with relapsing ON It is interesting thatthe 3 MOG antibodyndashpositive patients with relapsingON were negative for AQP4 antibody whereas theMOG antibodyndashnegative patient with relapsing ONwas not tested for AQP4 antibody due to insufficientserum In the relapsing patients (n 5 19) the MOGantibodyndashpositive patients were younger (median 56years range 26ndash142) than the ndashnegative patients(median 129 years range 25ndash153 p 5 003) TheMOG antibodyndashpositive patients had fewer relapses(22 relapses in 603 patient-years median annualizedrelapse rate 056) than the MOG antibodyndashnegativepatients (26 relapses in 39 patient-years median annu-alized relapse rate 065)
Longitudinal MOG antibody in 2 patients treated with
immune suppression Two recent patients (figure 2 andappendix e-2) with positive MOG antibodies who ful-filled 2013 criteria for MS18 were treated with myco-phenolate mofetil (MMF) which produced clinicalremission no new lesions on follow-up MRI scans
Neurology Neuroimmunology amp Neuroinflammation 3
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
Figure 1 Distribution of MOG IgG antibody in pediatric demyelinating diseases
(A) Antibody reactivity to myelin oligodendrocyte glycoprotein (MOG) was determined by flow cytometry live cell-based assay (B) Representative example offlow cytometry histograms for oneMOG antibodyndashpositive patient with a very high DMFI and (C) an intermediate DMFI MFI values are noted in the legend (D)Human surface MOG IgG antibody was detected in 3173 sera from patients with demyelinating diseases (DEM) and 024 controls (CTL) Magenta line ongraph represents the positivity threshold MOG antibody positivity is shown between brackets (E) Surface MOG antibody was detected in 322 CSF frompatients with DEM and 020CSF from patients with other neurologic diseases (CTL) Black circles represent patients with positiveMOG antibody in CSF andin serum (F) Distribution and number of MOG antibodyndashpositive patients in optic neuritis (ON) (G) Correlation between erythrocyte sedimentation rate andage in MOG antibodyndashpositive patients (H) Distribution and (I) number of MOG antibodyndashpositive patients in demyelinating diseases at follow-up Ab 5
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
and normalization of MOG antibodies (figure 2 andappendix e-2) In both patients upon treatment withMMF MOG antibody decreased below the thresholdof positivity (figure 2 A and B)
Loss of cytoskeleton organization by MOG antibody or
purified IgG from children with demyelinating diseases
Human oligodendrocyte MO313MOG1 cells expressedMOG at their surface whereas noMOG expression wasobserved on MO313Ctl cells (figure 3) MO313 cellsalso expressed oligodendrocyte markers such as 29 39-cyclic nucleotide 39-phosphodiesterase (CNPase) galac-tocerebroside (GalC) oligodendrocyte marker O4vimentin and c-series ganglioside-specific antigen(A2B5) (figure 3A) Myelin basic protein (MBP) aspecific marker of mature oligodendrocytes wasobserved only in PMA-differentiated MO313MOG1
cells (figure 3A) suggesting that undifferentiatedMO313 cells are immature oligodendrocytes Using
purified IgG and immunoaffinity-purified MOG IgGfromMOG antibodyndashpositive sera we immunolabeledHEK293MOG1 and HEK293Ctl cells on live cells byFACS (figure 3B) and on fixed MO313MOG1 cellsby immunocytochemistry (figure 3C) and showeda positive immunostaining compared to MOGantibodyndashnegative sera suggesting that proteinGndashpurified IgG includes MOG-specific IgG Due tosmall volumes of pediatric sera we used proteinGndashpurified IgG in pathogenic experiments Next wetreated fixed and live MO313MOG1Ctl cells with IgGfrom MOG antibodyndashpositive and ndashnegative patientswith DEM and healthy controls (HCs) Then all cellswere immunolabeled for b-tubulin (marker ofmicrotubule) or F-actin (marker of thin filaments) Wequantified results and expressed them by the F-actin andb-tubulin relative enrichments in the cytoplasm andperinuclear region over the entire cell All results were
Figure 2 Temporal distribution of MOG antibody in serum of 2 relapsing patients with demyelinating diseases
In both patient A (A) and patient B (B) upon treatment mycophenolate mofetil (MMF) myelin oligodendrocyte glycoprotein (MOG) antibody decreased towithin the healthy control range (below threshold of positivity) and these low titers were associated with remission Representative dot plot out of 3 experi-ments is shown Magenta lines on graphs represent the positivity threshold (obtained with 24 control samples) Black squares represent serum analysis dur-ing acute demyelination episodes and black circles represent sera during remission Type of demyelinating episode is shown on graph (C D) RepresentativeT2 axial MRI scans demonstrate demyelinating lesions during the first acute event and during convalescence Patient A (C) had globular deep white matterlesions on acute scan (left panel) which show residual gliosis on convalescent scan and no new lesions (right panel) Patient B (D) had inflammatory lesions inbasal ganglia and white matter on acute scan (left panel) with complete resolution on convalescent scan and no new lesions (right panel) Ab 5 antibodyADEM5 acute disseminated encephalomyelitis CEREB5 cerebellar episode Ig5 immunoglobulin MFI5mean fluorescence intensity ON5 optic neuritisTM 5 transverse myelitis
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
normalized using F-actin and b-tubulin in HCIgG-treated fixed cells Organization of themicrotubule and thin filament networks wassimilar in fixed cells incubated with HC (1006 101 and 100 6 226 respectively figure 4A and C) or DEM IgGs (1043 6 113 and858 6 318 respectively figure 4 A and C)
there was clear visualization of ldquostress-fibersrdquo andfilopedia after F-actin immunolabeling (figure 4Aupper panels) and bright mesh-like staining spreadout through the entire cell after b-tubulinimmunolabeling (figure 4A lower panels) Livecells treated by HC IgG displayed a smalldisorganization of F-actin thin filaments (916 144
Figure 3 Human oligodendroglial MO313MOG1 cells express markers of oligodendrocytes and are immunolabeled with protein G- and humanMOG-purified human IgG from MOG antibodyndashpositive DEM patients
(A) Immunocytochemistry on fixed permeabilized cells showed that MO313MOG1 cells expressed oligodendrocyte markers (B) Protein G- and human MOG-immunopurified IgG from MOG antibodyndashpositive serum immunolabeled live HEK293MOG1 cells but did not immunolabel HEK293Ctl cells compared to MOGantibodyndashnegative serum Binding to cells was determined by flow cytometry Mean fluorescence intensity (MFI) values are shown in legends (C) ProteinG- and human MOG-purified IgG from MOG antibodyndashpositive serum also immunolabeled fixed unpermeabilized MO313MOG1 cells compared to MOGantibodyndashnegative serum Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy) Nuclei stainedwith 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm A2B55 c-series ganglioside-specific antigen A2B5 Ab5 antibody CNPase529 39-cyclic nucleotide39-phosphodiesterase DEM 5 demyelinating diseases GalC 5 galactocerebroside HEK 5 human embryonic kidney Ig 5 immunoglobulin MBP 5 myelinbasic protein MOG 5 myelin oligodendrocyte glycoprotein O4 5 oligodendrocyte marker O4
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
Figure 4 Purified IgG from DEM patients induces loss of cytoskeleton organization in live human oligodendroglial MO313MOG1 cells
F-actin (upper images) and b-tubulin (lower images) immunolabelings in human fixed (A) or live (B) oligodendroglial MO313MOG1 cells incubated with purifiedhealthy control (HC) IgG or MOG antibodyndashpositive DEM IgG Representative data are shown (volume projection of entire Z-stack acquired using deconvo-lution microscopy) Nuclei stained with 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm Dotted lines on right images represent contour of cells as deter-mined by differential interference contrast images (not shown) (C) Quantification of change in distribution of thin filament (F-actin upper scatter plot) andmicrotubule network (b-tubulin lower scatter plot) organization using 3D deconvolution microscopy F-actin and b-tubulin were majorly affected in live cellsincubated with DEM IgG Forty different cells (1 cell5 1 diamond) from 2 HC and 2 DEM patients out of 3 independent experiments are shown Results areexpressed as ratio of the relative enrichment (RE) between cytoplasm and nucleus and values shown are percentages relative to HC IgG FIX (100) Redbars represent mean Note that there also was a loss of organization of the thin filaments after HC IgG incubation on live cells compared to fixed conditionsbut to a lesser extent than after DEM IgG (D) Cell viability assay by flow cytometry on live human oligodendroglial MO313MOG1 cells incubated 45 minuteswith 6 mg of purified HC IgG or DEM IgG No difference in cell death was observed Dead cell percentages are noted in the legend 7AAD 5 7-amino-actinomycin D DEM 5 demyelinating diseases Ig 5 immunoglobulin MOG 5 myelin oligodendrocyte glycoprotein
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
p 00001 figure 4 B and C upper images)and undisturbed microtubular b-tubulin network(1074 6 321 figure 4 B and C) similar to thatobserved in fixed DEM and HC IgG-treated cells(figure 4A upper panels) Following treatment withDEM IgG on live MO313MOG1 cells we observed astriking loss of organization in both F-actin (7246 117 p 00001 figure 4 B and C) andb-tubulin networks (522 6 131 p 00001figure 4 B and C) in otherwise healthy-lookingundividing cells visualized by differential interferencecontrast imaging (figure 4B dotted line and data notshown) and analyzed by FACS by which there was nodifference in viability between live MO313MOG1 livecells treated with DEM IgG or HC IgG for45 minutes (figure 4D 8726 06 vs 8726 211) or10 hours (data not shown) No loss of cytoskeletonorganization was observed when live MO313Ctl cellswere incubated with HC IgG and MOG antibodyndashpositive DEM IgG nor when live MO313MOG1 cellswere incubated with MOG antibodyndashnegative DEMIgG (figure e-1 and appendix e-3) In additionsuccessful immunoabsorption of MOG antibodyndashpositive serum on HEK293MOG1 cells led to nochange in cytoskeleton organization whenimmunoabsorbed serum was incubated on liveMO313MOG1 cells (figure e-1 and appendix e-3)Overall after incubation with MOG antibodyndashpositive DEM IgG both F-actin and b-tubulinimmunolabelings appeared to be enriched withina perinuclear region in the center of the cellssurrounding the nucleus
DISCUSSION As in this study recent reports haveconsistently found that serum MOG antibody is pre-sent in a significant proportion of children with CNSdemyelinating disease whereas AQP4 antibodies arerare9 We believe that the clinical and investigationfindings in this report support the evolving conceptthat MOG antibodies may define a separate autoim-mune demyelinating syndrome that may be distinctfrom classic adult-onset MS MOG antibodies arepresent in up to 50 of children with ADEM as inthis report Proebstel et al33 found that ADEMMOGantibody seropositive children became seronegative inthe recovery period whereas children with relapsingMS typically remained seropositive More recentlyMOG antibodies have been found in children withON and in some patients with NMO phenotypenegative for NMO IgG1431 In this report we havestrengthened this association with ON and found thatpatients with bilateral rather than unilateral ON weremore likely to be MOG antibodyndashpositive BilateralON is a common ON phenotype in children but israre in adults in whom unilateral ON is the moretypical demyelinating event of MS In contrast
patients with brainstem signs and CIS other thanON and TM were rarely MOG antibodyndashpositiveAn elevated ESR was more common in MOGantibodyndashpositive patients An elevated ESR isatypical of MS and should raise suspicion of anautoimmune disorder or alternate inflammatorymimic of MS We showed that elevation of ESR inMOG antibodyndashpositive patients was independent ofage Intrathecal oligoclonal bands are one of the classicbiomarkers of MS and were absent in all MOGantibodyndashpositive patients and were present only inthe MOG antibodyndashnegative patients Only 17 ofthe MOG antibodyndashpositive patients had 1 or bothHLA-DRB11501 alleles the main MS susceptibilityallele which is the same as the previously reportedpercentage in the ldquonormalrdquo healthy population inAustralia34 whereas the MOG antibodyndashnegativepatients were more likely to have 1 or more HLA-DRB11501 allele This negative association wasnot statistically significant and larger cohorts arerequired to test whether this correlation is a realrather than a suspected phenomenon In contrastradiologic features did not appear to differentiateMOG antibodyndashpositive from ndashnegative patients inthis childhood cohort
The presence of MOG antibodies is unlikely tohave therapeutic implications in the first demyelinat-ing event but is more likely to be therapeuticallyimportant in the relapsing patient Although 2 recentpatients fulfilled criteria for MS the presence ofMOG antibodies and elevated ESR led us to treatthem more like NMO patients with the immune sup-pression agent MMF rather than conventional MSdisease-modifying therapies Both patients had noclinical or radiologic relapse for 12 months and 36months respectively and both became MOG anti-body seronegative Further studies are required tounderstand the longitudinal course of patients withMOG antibodyndashassociated relapsing disease and thetreatment approach required
Cell-based assays have been essential to detectMOG antibody with a high specificity9 especiallywhen the full-length MOG is expressed in cells ratherthan the C-terminal truncated MOG35 Our experi-ence with neuronal antigens informed us that FACSis sensitive and quantitative10132022333637 We detectedCSF MOG antibody in only 3 of 22 DEM samples ofwhich 5 were positive in serum This suggests CSF isless sensitive than serum for MOG antibody detectionwhich could be due to a lack of sensitivity or couldrepresent a genuine absence of intrathecal Ig synthesisin these patients Supporting this concept none of thepatients with positive MOG antibody had intrathecaloligoclonal bands
The pathogenic function of MOG antibody is stillunclear but over the recent years the detection of
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316
and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538
Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40
Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders
AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript
RCD and FB Acquisition analysis and interpretation of results RCD
EMT VM R-YAK NS SR KP LAW DRB K Prelog
DRC GJG CKL EKM FB Statistical analysis RCD EMT
FB Critical revisions of manuscript RCD EMT VM NS SR
KP DRB LAW K Prelog DRC GJG CKL EKM FB
ACKNOWLEDGMENTThe authors thank all the patients and family members who provided
samples for the study The authors thank Dr Maggie Wang for use of
the Flow Cytometry Core Facility of the Westmead Millennium Institute
(Australia) The authors thank Dr Hong Yu for use of the Imaging Core
Facility at the Westmead Research Hub
STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple
Sclerosis Research Foundation Multiple Sclerosis Research Australia
MS Angels Melbourne (Australia) and the Petre Foundation (Australia)
DISCLOSURERC Dale has received research funding from the Star Scientific Foundation
the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis
Research Australia NHMRC and the Petre Foundation has received hon-
oraria from Biogen Idec and is on advisory boards for Queensland Child-
renrsquos Medical Institute Research and MSARD EM Tantsis has received
a scholarship from the Australian Postgraduate Award program V Merheb
and R-YA Kumaran report no disclosures N Sinmaz has received a schol-
arship from the Australian Postgraduate Award program K Pathmanandavel
has received a scholarship from the Petre Foundation (Australia)
S Ramanathan has received a scholarship from the National Health and
Medical Research Council (Australia) DR Booth has received funding from
the National Health and Medical Research Council (Australia) the Australian
Research Council Multiple Sclerosis Research Australia and the Leukemia
Foundation has received honoraria and funding from Biogen Idec Merck
Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS
ONE LA Wienholt K Prelog and DR Clark report no disclosures
GJ Guillemin is the editor-in-chief for the International Journal for Trypto-
phan Research and has received research support from the Australian Research
Council CK Lim has received research funding from Multiple Sclerosis
Research Australia and Trish MS Research Foundation EK Mathey has
received funding from the National Health and Medical Research Council
(Australia) and the Rebecca L Cooper Medical Research Foundation
(Australia) F Brilot has received research funding from the Star Scientific
Foundation the Trish Multiple Sclerosis Research Foundation Multiple
Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre
Foundation and is an associate editor for the Journal of Visualized Experiments
Go to Neurologyorgnn for full disclosures
Received February 25 2014 Accepted in final form April 16 2014
REFERENCES1 Papadopoulos MC Verkman AS Aquaporin 4 and neu-
romyelitis optica Lancet Neurol 201211535ndash544
2 Vincent A Bien CG Irani SR Waters P Autoantibodies
associated with diseases of the CNS new developments
and future challenges Lancet Neurol 201110759ndash772
3 Wingerchuk DM Lennon VA Lucchinetti CF
Pittock SJ Weinshenker BG The spectrum of neuromy-
elitis optica Lancet Neurol 20076805ndash815
4 Johns TG Bernard CC The structure and function of
myelin oligodendrocyte glycoprotein J Neurochem
1999721ndash9
5 Linington C Bradl M Lassmann H Brunner C Vass K
Augmentation of demyelination in rat acute allergic
encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelinoligodendrocyte glycopro-
tein Am J Pathol 1988130443ndash454
6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous
relapsing-remitting EAE in the SJLJ mouse MOG-reactive
transgenic T cells recruit endogenous MOG-specific B cells
J Exp Med 20092061303ndash1316
7 Schluesener HJ Sobel RA Linington C Weiner HL A
monoclonal antibody against a myelin oligodendrocyte
glycoprotein induces relapses and demyelination in central
nervous system autoimmune disease J Immunol 1987
1394016ndash4021
8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB
Lee JI Genain CP Molecular characterization of antibody
specificities against myelinoligodendrocyte glycoprotein
in autoimmune demyelination Proc Natl Acad Sci U S A
2002998207ndash8212
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
(marker of cytoskeleton microtubule) were quantified using 3D
deconvolution microscopy (detailed methodology in appendix e-1)29
Human leukocyte antigen DRB11501 genotyping DNA
from patients with DEM (n 5 40) was extracted from saliva using
a saliva-specific DNA extraction kit according to the manufacturerrsquos
instructions (Oragene DNA Genotek Kanata Ontario Canada)
The rs9271366 single nucleotide polymorphism (SNP) that lies on
chromosome 6 at position 32694832 upstream of the allelic variation
(A G) was used to genotype the patients for human leukocyte
antigen (HLA)-DRB1150130 Patients with the allelic A are negative
for HLA-DRB11501 whereas patients with the allelic G are HLA-
DRB11501ndashpositive The SNP rs9271366 is always inherited with
the HLA-DRB11501 allele (complete linkage disequilibrium with
HLA-DRB11501)30 DNA was amplified using PCR (detailed
methodology in appendix e-1) and sequences were analyzed by the
software Sequencher (Gene Codes Corporation Ann Arbor MI)
Statistical analysis Chi-square with Yates correction test was usedto compare MOG antibody positivity between patient and control
groups and between patient subgroups and to compare binary clin-
ical and radiologic features between MOG antibodyndashpositive and ndash
negative groups Wilcoxon 2-sample test was used to compare ages
of patients in 2 groups No adjustment for multiple testing has been
performed The effects of IgG on F-actin and b-tubulin were
normally distributed thus means and SDs were analyzed with
the 2-tailed Student t test A p value less than 005 was
considered significant for the 2-tailed Student t test (MOG
antibody pathogenic effect)
RESULTS Surface MOG IgG antibody in children with
demyelinating diseases In order to align our detectionmethod to recent reports we first detected MOGantibody in serum using FACS assay andHEK cells ex-pressing human MOG1114163132 The mean fluores-cence intensity correlated with antibody concentration(figure 1A) Using the mean plus 3 SDs to establish thethreshold for positivity MOG antibodies were foundin 3173 (42) of the DEM group but in 024 con-trols (figure 1 BndashD p 5 0000) We have tested 57other medical and neurologic controls and all werenegative We also tested MOG antibody in CSF (fig-ure 1E) We had CSF available in 22 patients withDEM of which 5 were positive for MOG antibodyin serum Only 322 CSF samples were positive 2 ofwhich were serum positive and 1 of which was serumnegative (figure 1E) Serologic testing for AQP4 anti-bodies was performed in 64 patients with DEM andwas negative in all (data not shown)
Clinical and investigation findings according to serum
MOG antibody findings First episode We compared theclinical features according to MOG antibody statusduring the first episode of demyelination in detailand findings are presented in table e-1 Patients withpositive MOG antibody were younger (median age67 vs 104 years p5 006) and marginally more likelyto be female (1831 58 vs 1942 45 p 5 040)MOG antibody positivity was negatively associatedwith brainstem signs (231 vs 1642 42 p 5 0005)In patients with ON positive MOG antibody was
more common in patients with bilateral compared tounilateral ON (910 vs 15 respectively p 5 003 (fig-ure 1F) MOG antibodyndashpositive patients were morelikely to have a raised erythrocyte sedimentation rate(ESR) 20 mmh during the first episode than MOGantibodyndashnegative patients (919 vs 321 p 5 005)There was no correlation between age and elevatedESR in MOG antibodyndashpositive patients (figure 1G)Intrathecal oligoclonal bands were not present in theMOG antibodyndashpositive patients and were seen onlyin MOG antibodyndashnegative patients (016 vs 527p 5 018) MOG antibodyndashpositive patients were lesslikely to be homozygous or heterozygous for HLA-DRB11501 than patients with negative MOG anti-body (318 17 vs 722 32 p 5 046) Detailedblinded radiologic analysis of MRI brain (n 5 70) andMRI spine (n5 30) during the first episode showed nosignificant differences according to MOG antibody sta-tus (table e-2) There was no difference in MRI MScriteria usingMcDonald KIDMUS Callen and Verhey(table e-3)
Follow-up diagnosis To compare MOG antibodyfindings we compared the subgroups according to clas-sification at follow-up (figure 1 H and I) In the mon-ophasic subgroups MOG antibodies were positive in1124 (46) patients with ADEM 67 (86) patientswith ON 413 (31) patients with TM and 010(0) patients with ldquoother CISrdquo (defined in Methods)Patients with monophasic ON were more likely to beMOG antibodyndashpositive than the rest of the cohort(67 vs 2566 p 5 004) and patients with ldquootherCISrdquo were less likely to be MOG antibodyndashpositivethan the rest of the cohort (010 vs 3163 p 5
001) In the relapsing patients MOG antibody waspositive in 715 (47) of patients with MS and 34(75) patients with relapsing ON It is interesting thatthe 3 MOG antibodyndashpositive patients with relapsingON were negative for AQP4 antibody whereas theMOG antibodyndashnegative patient with relapsing ONwas not tested for AQP4 antibody due to insufficientserum In the relapsing patients (n 5 19) the MOGantibodyndashpositive patients were younger (median 56years range 26ndash142) than the ndashnegative patients(median 129 years range 25ndash153 p 5 003) TheMOG antibodyndashpositive patients had fewer relapses(22 relapses in 603 patient-years median annualizedrelapse rate 056) than the MOG antibodyndashnegativepatients (26 relapses in 39 patient-years median annu-alized relapse rate 065)
Longitudinal MOG antibody in 2 patients treated with
immune suppression Two recent patients (figure 2 andappendix e-2) with positive MOG antibodies who ful-filled 2013 criteria for MS18 were treated with myco-phenolate mofetil (MMF) which produced clinicalremission no new lesions on follow-up MRI scans
Neurology Neuroimmunology amp Neuroinflammation 3
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
Figure 1 Distribution of MOG IgG antibody in pediatric demyelinating diseases
(A) Antibody reactivity to myelin oligodendrocyte glycoprotein (MOG) was determined by flow cytometry live cell-based assay (B) Representative example offlow cytometry histograms for oneMOG antibodyndashpositive patient with a very high DMFI and (C) an intermediate DMFI MFI values are noted in the legend (D)Human surface MOG IgG antibody was detected in 3173 sera from patients with demyelinating diseases (DEM) and 024 controls (CTL) Magenta line ongraph represents the positivity threshold MOG antibody positivity is shown between brackets (E) Surface MOG antibody was detected in 322 CSF frompatients with DEM and 020CSF from patients with other neurologic diseases (CTL) Black circles represent patients with positiveMOG antibody in CSF andin serum (F) Distribution and number of MOG antibodyndashpositive patients in optic neuritis (ON) (G) Correlation between erythrocyte sedimentation rate andage in MOG antibodyndashpositive patients (H) Distribution and (I) number of MOG antibodyndashpositive patients in demyelinating diseases at follow-up Ab 5
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
and normalization of MOG antibodies (figure 2 andappendix e-2) In both patients upon treatment withMMF MOG antibody decreased below the thresholdof positivity (figure 2 A and B)
Loss of cytoskeleton organization by MOG antibody or
purified IgG from children with demyelinating diseases
Human oligodendrocyte MO313MOG1 cells expressedMOG at their surface whereas noMOG expression wasobserved on MO313Ctl cells (figure 3) MO313 cellsalso expressed oligodendrocyte markers such as 29 39-cyclic nucleotide 39-phosphodiesterase (CNPase) galac-tocerebroside (GalC) oligodendrocyte marker O4vimentin and c-series ganglioside-specific antigen(A2B5) (figure 3A) Myelin basic protein (MBP) aspecific marker of mature oligodendrocytes wasobserved only in PMA-differentiated MO313MOG1
cells (figure 3A) suggesting that undifferentiatedMO313 cells are immature oligodendrocytes Using
purified IgG and immunoaffinity-purified MOG IgGfromMOG antibodyndashpositive sera we immunolabeledHEK293MOG1 and HEK293Ctl cells on live cells byFACS (figure 3B) and on fixed MO313MOG1 cellsby immunocytochemistry (figure 3C) and showeda positive immunostaining compared to MOGantibodyndashnegative sera suggesting that proteinGndashpurified IgG includes MOG-specific IgG Due tosmall volumes of pediatric sera we used proteinGndashpurified IgG in pathogenic experiments Next wetreated fixed and live MO313MOG1Ctl cells with IgGfrom MOG antibodyndashpositive and ndashnegative patientswith DEM and healthy controls (HCs) Then all cellswere immunolabeled for b-tubulin (marker ofmicrotubule) or F-actin (marker of thin filaments) Wequantified results and expressed them by the F-actin andb-tubulin relative enrichments in the cytoplasm andperinuclear region over the entire cell All results were
Figure 2 Temporal distribution of MOG antibody in serum of 2 relapsing patients with demyelinating diseases
In both patient A (A) and patient B (B) upon treatment mycophenolate mofetil (MMF) myelin oligodendrocyte glycoprotein (MOG) antibody decreased towithin the healthy control range (below threshold of positivity) and these low titers were associated with remission Representative dot plot out of 3 experi-ments is shown Magenta lines on graphs represent the positivity threshold (obtained with 24 control samples) Black squares represent serum analysis dur-ing acute demyelination episodes and black circles represent sera during remission Type of demyelinating episode is shown on graph (C D) RepresentativeT2 axial MRI scans demonstrate demyelinating lesions during the first acute event and during convalescence Patient A (C) had globular deep white matterlesions on acute scan (left panel) which show residual gliosis on convalescent scan and no new lesions (right panel) Patient B (D) had inflammatory lesions inbasal ganglia and white matter on acute scan (left panel) with complete resolution on convalescent scan and no new lesions (right panel) Ab 5 antibodyADEM5 acute disseminated encephalomyelitis CEREB5 cerebellar episode Ig5 immunoglobulin MFI5mean fluorescence intensity ON5 optic neuritisTM 5 transverse myelitis
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
normalized using F-actin and b-tubulin in HCIgG-treated fixed cells Organization of themicrotubule and thin filament networks wassimilar in fixed cells incubated with HC (1006 101 and 100 6 226 respectively figure 4A and C) or DEM IgGs (1043 6 113 and858 6 318 respectively figure 4 A and C)
there was clear visualization of ldquostress-fibersrdquo andfilopedia after F-actin immunolabeling (figure 4Aupper panels) and bright mesh-like staining spreadout through the entire cell after b-tubulinimmunolabeling (figure 4A lower panels) Livecells treated by HC IgG displayed a smalldisorganization of F-actin thin filaments (916 144
Figure 3 Human oligodendroglial MO313MOG1 cells express markers of oligodendrocytes and are immunolabeled with protein G- and humanMOG-purified human IgG from MOG antibodyndashpositive DEM patients
(A) Immunocytochemistry on fixed permeabilized cells showed that MO313MOG1 cells expressed oligodendrocyte markers (B) Protein G- and human MOG-immunopurified IgG from MOG antibodyndashpositive serum immunolabeled live HEK293MOG1 cells but did not immunolabel HEK293Ctl cells compared to MOGantibodyndashnegative serum Binding to cells was determined by flow cytometry Mean fluorescence intensity (MFI) values are shown in legends (C) ProteinG- and human MOG-purified IgG from MOG antibodyndashpositive serum also immunolabeled fixed unpermeabilized MO313MOG1 cells compared to MOGantibodyndashnegative serum Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy) Nuclei stainedwith 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm A2B55 c-series ganglioside-specific antigen A2B5 Ab5 antibody CNPase529 39-cyclic nucleotide39-phosphodiesterase DEM 5 demyelinating diseases GalC 5 galactocerebroside HEK 5 human embryonic kidney Ig 5 immunoglobulin MBP 5 myelinbasic protein MOG 5 myelin oligodendrocyte glycoprotein O4 5 oligodendrocyte marker O4
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
Figure 4 Purified IgG from DEM patients induces loss of cytoskeleton organization in live human oligodendroglial MO313MOG1 cells
F-actin (upper images) and b-tubulin (lower images) immunolabelings in human fixed (A) or live (B) oligodendroglial MO313MOG1 cells incubated with purifiedhealthy control (HC) IgG or MOG antibodyndashpositive DEM IgG Representative data are shown (volume projection of entire Z-stack acquired using deconvo-lution microscopy) Nuclei stained with 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm Dotted lines on right images represent contour of cells as deter-mined by differential interference contrast images (not shown) (C) Quantification of change in distribution of thin filament (F-actin upper scatter plot) andmicrotubule network (b-tubulin lower scatter plot) organization using 3D deconvolution microscopy F-actin and b-tubulin were majorly affected in live cellsincubated with DEM IgG Forty different cells (1 cell5 1 diamond) from 2 HC and 2 DEM patients out of 3 independent experiments are shown Results areexpressed as ratio of the relative enrichment (RE) between cytoplasm and nucleus and values shown are percentages relative to HC IgG FIX (100) Redbars represent mean Note that there also was a loss of organization of the thin filaments after HC IgG incubation on live cells compared to fixed conditionsbut to a lesser extent than after DEM IgG (D) Cell viability assay by flow cytometry on live human oligodendroglial MO313MOG1 cells incubated 45 minuteswith 6 mg of purified HC IgG or DEM IgG No difference in cell death was observed Dead cell percentages are noted in the legend 7AAD 5 7-amino-actinomycin D DEM 5 demyelinating diseases Ig 5 immunoglobulin MOG 5 myelin oligodendrocyte glycoprotein
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
p 00001 figure 4 B and C upper images)and undisturbed microtubular b-tubulin network(1074 6 321 figure 4 B and C) similar to thatobserved in fixed DEM and HC IgG-treated cells(figure 4A upper panels) Following treatment withDEM IgG on live MO313MOG1 cells we observed astriking loss of organization in both F-actin (7246 117 p 00001 figure 4 B and C) andb-tubulin networks (522 6 131 p 00001figure 4 B and C) in otherwise healthy-lookingundividing cells visualized by differential interferencecontrast imaging (figure 4B dotted line and data notshown) and analyzed by FACS by which there was nodifference in viability between live MO313MOG1 livecells treated with DEM IgG or HC IgG for45 minutes (figure 4D 8726 06 vs 8726 211) or10 hours (data not shown) No loss of cytoskeletonorganization was observed when live MO313Ctl cellswere incubated with HC IgG and MOG antibodyndashpositive DEM IgG nor when live MO313MOG1 cellswere incubated with MOG antibodyndashnegative DEMIgG (figure e-1 and appendix e-3) In additionsuccessful immunoabsorption of MOG antibodyndashpositive serum on HEK293MOG1 cells led to nochange in cytoskeleton organization whenimmunoabsorbed serum was incubated on liveMO313MOG1 cells (figure e-1 and appendix e-3)Overall after incubation with MOG antibodyndashpositive DEM IgG both F-actin and b-tubulinimmunolabelings appeared to be enriched withina perinuclear region in the center of the cellssurrounding the nucleus
DISCUSSION As in this study recent reports haveconsistently found that serum MOG antibody is pre-sent in a significant proportion of children with CNSdemyelinating disease whereas AQP4 antibodies arerare9 We believe that the clinical and investigationfindings in this report support the evolving conceptthat MOG antibodies may define a separate autoim-mune demyelinating syndrome that may be distinctfrom classic adult-onset MS MOG antibodies arepresent in up to 50 of children with ADEM as inthis report Proebstel et al33 found that ADEMMOGantibody seropositive children became seronegative inthe recovery period whereas children with relapsingMS typically remained seropositive More recentlyMOG antibodies have been found in children withON and in some patients with NMO phenotypenegative for NMO IgG1431 In this report we havestrengthened this association with ON and found thatpatients with bilateral rather than unilateral ON weremore likely to be MOG antibodyndashpositive BilateralON is a common ON phenotype in children but israre in adults in whom unilateral ON is the moretypical demyelinating event of MS In contrast
patients with brainstem signs and CIS other thanON and TM were rarely MOG antibodyndashpositiveAn elevated ESR was more common in MOGantibodyndashpositive patients An elevated ESR isatypical of MS and should raise suspicion of anautoimmune disorder or alternate inflammatorymimic of MS We showed that elevation of ESR inMOG antibodyndashpositive patients was independent ofage Intrathecal oligoclonal bands are one of the classicbiomarkers of MS and were absent in all MOGantibodyndashpositive patients and were present only inthe MOG antibodyndashnegative patients Only 17 ofthe MOG antibodyndashpositive patients had 1 or bothHLA-DRB11501 alleles the main MS susceptibilityallele which is the same as the previously reportedpercentage in the ldquonormalrdquo healthy population inAustralia34 whereas the MOG antibodyndashnegativepatients were more likely to have 1 or more HLA-DRB11501 allele This negative association wasnot statistically significant and larger cohorts arerequired to test whether this correlation is a realrather than a suspected phenomenon In contrastradiologic features did not appear to differentiateMOG antibodyndashpositive from ndashnegative patients inthis childhood cohort
The presence of MOG antibodies is unlikely tohave therapeutic implications in the first demyelinat-ing event but is more likely to be therapeuticallyimportant in the relapsing patient Although 2 recentpatients fulfilled criteria for MS the presence ofMOG antibodies and elevated ESR led us to treatthem more like NMO patients with the immune sup-pression agent MMF rather than conventional MSdisease-modifying therapies Both patients had noclinical or radiologic relapse for 12 months and 36months respectively and both became MOG anti-body seronegative Further studies are required tounderstand the longitudinal course of patients withMOG antibodyndashassociated relapsing disease and thetreatment approach required
Cell-based assays have been essential to detectMOG antibody with a high specificity9 especiallywhen the full-length MOG is expressed in cells ratherthan the C-terminal truncated MOG35 Our experi-ence with neuronal antigens informed us that FACSis sensitive and quantitative10132022333637 We detectedCSF MOG antibody in only 3 of 22 DEM samples ofwhich 5 were positive in serum This suggests CSF isless sensitive than serum for MOG antibody detectionwhich could be due to a lack of sensitivity or couldrepresent a genuine absence of intrathecal Ig synthesisin these patients Supporting this concept none of thepatients with positive MOG antibody had intrathecaloligoclonal bands
The pathogenic function of MOG antibody is stillunclear but over the recent years the detection of
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316
and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538
Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40
Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders
AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript
RCD and FB Acquisition analysis and interpretation of results RCD
EMT VM R-YAK NS SR KP LAW DRB K Prelog
DRC GJG CKL EKM FB Statistical analysis RCD EMT
FB Critical revisions of manuscript RCD EMT VM NS SR
KP DRB LAW K Prelog DRC GJG CKL EKM FB
ACKNOWLEDGMENTThe authors thank all the patients and family members who provided
samples for the study The authors thank Dr Maggie Wang for use of
the Flow Cytometry Core Facility of the Westmead Millennium Institute
(Australia) The authors thank Dr Hong Yu for use of the Imaging Core
Facility at the Westmead Research Hub
STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple
Sclerosis Research Foundation Multiple Sclerosis Research Australia
MS Angels Melbourne (Australia) and the Petre Foundation (Australia)
DISCLOSURERC Dale has received research funding from the Star Scientific Foundation
the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis
Research Australia NHMRC and the Petre Foundation has received hon-
oraria from Biogen Idec and is on advisory boards for Queensland Child-
renrsquos Medical Institute Research and MSARD EM Tantsis has received
a scholarship from the Australian Postgraduate Award program V Merheb
and R-YA Kumaran report no disclosures N Sinmaz has received a schol-
arship from the Australian Postgraduate Award program K Pathmanandavel
has received a scholarship from the Petre Foundation (Australia)
S Ramanathan has received a scholarship from the National Health and
Medical Research Council (Australia) DR Booth has received funding from
the National Health and Medical Research Council (Australia) the Australian
Research Council Multiple Sclerosis Research Australia and the Leukemia
Foundation has received honoraria and funding from Biogen Idec Merck
Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS
ONE LA Wienholt K Prelog and DR Clark report no disclosures
GJ Guillemin is the editor-in-chief for the International Journal for Trypto-
phan Research and has received research support from the Australian Research
Council CK Lim has received research funding from Multiple Sclerosis
Research Australia and Trish MS Research Foundation EK Mathey has
received funding from the National Health and Medical Research Council
(Australia) and the Rebecca L Cooper Medical Research Foundation
(Australia) F Brilot has received research funding from the Star Scientific
Foundation the Trish Multiple Sclerosis Research Foundation Multiple
Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre
Foundation and is an associate editor for the Journal of Visualized Experiments
Go to Neurologyorgnn for full disclosures
Received February 25 2014 Accepted in final form April 16 2014
REFERENCES1 Papadopoulos MC Verkman AS Aquaporin 4 and neu-
romyelitis optica Lancet Neurol 201211535ndash544
2 Vincent A Bien CG Irani SR Waters P Autoantibodies
associated with diseases of the CNS new developments
and future challenges Lancet Neurol 201110759ndash772
3 Wingerchuk DM Lennon VA Lucchinetti CF
Pittock SJ Weinshenker BG The spectrum of neuromy-
elitis optica Lancet Neurol 20076805ndash815
4 Johns TG Bernard CC The structure and function of
myelin oligodendrocyte glycoprotein J Neurochem
1999721ndash9
5 Linington C Bradl M Lassmann H Brunner C Vass K
Augmentation of demyelination in rat acute allergic
encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelinoligodendrocyte glycopro-
tein Am J Pathol 1988130443ndash454
6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous
relapsing-remitting EAE in the SJLJ mouse MOG-reactive
transgenic T cells recruit endogenous MOG-specific B cells
J Exp Med 20092061303ndash1316
7 Schluesener HJ Sobel RA Linington C Weiner HL A
monoclonal antibody against a myelin oligodendrocyte
glycoprotein induces relapses and demyelination in central
nervous system autoimmune disease J Immunol 1987
1394016ndash4021
8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB
Lee JI Genain CP Molecular characterization of antibody
specificities against myelinoligodendrocyte glycoprotein
in autoimmune demyelination Proc Natl Acad Sci U S A
2002998207ndash8212
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
Figure 1 Distribution of MOG IgG antibody in pediatric demyelinating diseases
(A) Antibody reactivity to myelin oligodendrocyte glycoprotein (MOG) was determined by flow cytometry live cell-based assay (B) Representative example offlow cytometry histograms for oneMOG antibodyndashpositive patient with a very high DMFI and (C) an intermediate DMFI MFI values are noted in the legend (D)Human surface MOG IgG antibody was detected in 3173 sera from patients with demyelinating diseases (DEM) and 024 controls (CTL) Magenta line ongraph represents the positivity threshold MOG antibody positivity is shown between brackets (E) Surface MOG antibody was detected in 322 CSF frompatients with DEM and 020CSF from patients with other neurologic diseases (CTL) Black circles represent patients with positiveMOG antibody in CSF andin serum (F) Distribution and number of MOG antibodyndashpositive patients in optic neuritis (ON) (G) Correlation between erythrocyte sedimentation rate andage in MOG antibodyndashpositive patients (H) Distribution and (I) number of MOG antibodyndashpositive patients in demyelinating diseases at follow-up Ab 5
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
and normalization of MOG antibodies (figure 2 andappendix e-2) In both patients upon treatment withMMF MOG antibody decreased below the thresholdof positivity (figure 2 A and B)
Loss of cytoskeleton organization by MOG antibody or
purified IgG from children with demyelinating diseases
Human oligodendrocyte MO313MOG1 cells expressedMOG at their surface whereas noMOG expression wasobserved on MO313Ctl cells (figure 3) MO313 cellsalso expressed oligodendrocyte markers such as 29 39-cyclic nucleotide 39-phosphodiesterase (CNPase) galac-tocerebroside (GalC) oligodendrocyte marker O4vimentin and c-series ganglioside-specific antigen(A2B5) (figure 3A) Myelin basic protein (MBP) aspecific marker of mature oligodendrocytes wasobserved only in PMA-differentiated MO313MOG1
cells (figure 3A) suggesting that undifferentiatedMO313 cells are immature oligodendrocytes Using
purified IgG and immunoaffinity-purified MOG IgGfromMOG antibodyndashpositive sera we immunolabeledHEK293MOG1 and HEK293Ctl cells on live cells byFACS (figure 3B) and on fixed MO313MOG1 cellsby immunocytochemistry (figure 3C) and showeda positive immunostaining compared to MOGantibodyndashnegative sera suggesting that proteinGndashpurified IgG includes MOG-specific IgG Due tosmall volumes of pediatric sera we used proteinGndashpurified IgG in pathogenic experiments Next wetreated fixed and live MO313MOG1Ctl cells with IgGfrom MOG antibodyndashpositive and ndashnegative patientswith DEM and healthy controls (HCs) Then all cellswere immunolabeled for b-tubulin (marker ofmicrotubule) or F-actin (marker of thin filaments) Wequantified results and expressed them by the F-actin andb-tubulin relative enrichments in the cytoplasm andperinuclear region over the entire cell All results were
Figure 2 Temporal distribution of MOG antibody in serum of 2 relapsing patients with demyelinating diseases
In both patient A (A) and patient B (B) upon treatment mycophenolate mofetil (MMF) myelin oligodendrocyte glycoprotein (MOG) antibody decreased towithin the healthy control range (below threshold of positivity) and these low titers were associated with remission Representative dot plot out of 3 experi-ments is shown Magenta lines on graphs represent the positivity threshold (obtained with 24 control samples) Black squares represent serum analysis dur-ing acute demyelination episodes and black circles represent sera during remission Type of demyelinating episode is shown on graph (C D) RepresentativeT2 axial MRI scans demonstrate demyelinating lesions during the first acute event and during convalescence Patient A (C) had globular deep white matterlesions on acute scan (left panel) which show residual gliosis on convalescent scan and no new lesions (right panel) Patient B (D) had inflammatory lesions inbasal ganglia and white matter on acute scan (left panel) with complete resolution on convalescent scan and no new lesions (right panel) Ab 5 antibodyADEM5 acute disseminated encephalomyelitis CEREB5 cerebellar episode Ig5 immunoglobulin MFI5mean fluorescence intensity ON5 optic neuritisTM 5 transverse myelitis
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
normalized using F-actin and b-tubulin in HCIgG-treated fixed cells Organization of themicrotubule and thin filament networks wassimilar in fixed cells incubated with HC (1006 101 and 100 6 226 respectively figure 4A and C) or DEM IgGs (1043 6 113 and858 6 318 respectively figure 4 A and C)
there was clear visualization of ldquostress-fibersrdquo andfilopedia after F-actin immunolabeling (figure 4Aupper panels) and bright mesh-like staining spreadout through the entire cell after b-tubulinimmunolabeling (figure 4A lower panels) Livecells treated by HC IgG displayed a smalldisorganization of F-actin thin filaments (916 144
Figure 3 Human oligodendroglial MO313MOG1 cells express markers of oligodendrocytes and are immunolabeled with protein G- and humanMOG-purified human IgG from MOG antibodyndashpositive DEM patients
(A) Immunocytochemistry on fixed permeabilized cells showed that MO313MOG1 cells expressed oligodendrocyte markers (B) Protein G- and human MOG-immunopurified IgG from MOG antibodyndashpositive serum immunolabeled live HEK293MOG1 cells but did not immunolabel HEK293Ctl cells compared to MOGantibodyndashnegative serum Binding to cells was determined by flow cytometry Mean fluorescence intensity (MFI) values are shown in legends (C) ProteinG- and human MOG-purified IgG from MOG antibodyndashpositive serum also immunolabeled fixed unpermeabilized MO313MOG1 cells compared to MOGantibodyndashnegative serum Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy) Nuclei stainedwith 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm A2B55 c-series ganglioside-specific antigen A2B5 Ab5 antibody CNPase529 39-cyclic nucleotide39-phosphodiesterase DEM 5 demyelinating diseases GalC 5 galactocerebroside HEK 5 human embryonic kidney Ig 5 immunoglobulin MBP 5 myelinbasic protein MOG 5 myelin oligodendrocyte glycoprotein O4 5 oligodendrocyte marker O4
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
Figure 4 Purified IgG from DEM patients induces loss of cytoskeleton organization in live human oligodendroglial MO313MOG1 cells
F-actin (upper images) and b-tubulin (lower images) immunolabelings in human fixed (A) or live (B) oligodendroglial MO313MOG1 cells incubated with purifiedhealthy control (HC) IgG or MOG antibodyndashpositive DEM IgG Representative data are shown (volume projection of entire Z-stack acquired using deconvo-lution microscopy) Nuclei stained with 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm Dotted lines on right images represent contour of cells as deter-mined by differential interference contrast images (not shown) (C) Quantification of change in distribution of thin filament (F-actin upper scatter plot) andmicrotubule network (b-tubulin lower scatter plot) organization using 3D deconvolution microscopy F-actin and b-tubulin were majorly affected in live cellsincubated with DEM IgG Forty different cells (1 cell5 1 diamond) from 2 HC and 2 DEM patients out of 3 independent experiments are shown Results areexpressed as ratio of the relative enrichment (RE) between cytoplasm and nucleus and values shown are percentages relative to HC IgG FIX (100) Redbars represent mean Note that there also was a loss of organization of the thin filaments after HC IgG incubation on live cells compared to fixed conditionsbut to a lesser extent than after DEM IgG (D) Cell viability assay by flow cytometry on live human oligodendroglial MO313MOG1 cells incubated 45 minuteswith 6 mg of purified HC IgG or DEM IgG No difference in cell death was observed Dead cell percentages are noted in the legend 7AAD 5 7-amino-actinomycin D DEM 5 demyelinating diseases Ig 5 immunoglobulin MOG 5 myelin oligodendrocyte glycoprotein
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
p 00001 figure 4 B and C upper images)and undisturbed microtubular b-tubulin network(1074 6 321 figure 4 B and C) similar to thatobserved in fixed DEM and HC IgG-treated cells(figure 4A upper panels) Following treatment withDEM IgG on live MO313MOG1 cells we observed astriking loss of organization in both F-actin (7246 117 p 00001 figure 4 B and C) andb-tubulin networks (522 6 131 p 00001figure 4 B and C) in otherwise healthy-lookingundividing cells visualized by differential interferencecontrast imaging (figure 4B dotted line and data notshown) and analyzed by FACS by which there was nodifference in viability between live MO313MOG1 livecells treated with DEM IgG or HC IgG for45 minutes (figure 4D 8726 06 vs 8726 211) or10 hours (data not shown) No loss of cytoskeletonorganization was observed when live MO313Ctl cellswere incubated with HC IgG and MOG antibodyndashpositive DEM IgG nor when live MO313MOG1 cellswere incubated with MOG antibodyndashnegative DEMIgG (figure e-1 and appendix e-3) In additionsuccessful immunoabsorption of MOG antibodyndashpositive serum on HEK293MOG1 cells led to nochange in cytoskeleton organization whenimmunoabsorbed serum was incubated on liveMO313MOG1 cells (figure e-1 and appendix e-3)Overall after incubation with MOG antibodyndashpositive DEM IgG both F-actin and b-tubulinimmunolabelings appeared to be enriched withina perinuclear region in the center of the cellssurrounding the nucleus
DISCUSSION As in this study recent reports haveconsistently found that serum MOG antibody is pre-sent in a significant proportion of children with CNSdemyelinating disease whereas AQP4 antibodies arerare9 We believe that the clinical and investigationfindings in this report support the evolving conceptthat MOG antibodies may define a separate autoim-mune demyelinating syndrome that may be distinctfrom classic adult-onset MS MOG antibodies arepresent in up to 50 of children with ADEM as inthis report Proebstel et al33 found that ADEMMOGantibody seropositive children became seronegative inthe recovery period whereas children with relapsingMS typically remained seropositive More recentlyMOG antibodies have been found in children withON and in some patients with NMO phenotypenegative for NMO IgG1431 In this report we havestrengthened this association with ON and found thatpatients with bilateral rather than unilateral ON weremore likely to be MOG antibodyndashpositive BilateralON is a common ON phenotype in children but israre in adults in whom unilateral ON is the moretypical demyelinating event of MS In contrast
patients with brainstem signs and CIS other thanON and TM were rarely MOG antibodyndashpositiveAn elevated ESR was more common in MOGantibodyndashpositive patients An elevated ESR isatypical of MS and should raise suspicion of anautoimmune disorder or alternate inflammatorymimic of MS We showed that elevation of ESR inMOG antibodyndashpositive patients was independent ofage Intrathecal oligoclonal bands are one of the classicbiomarkers of MS and were absent in all MOGantibodyndashpositive patients and were present only inthe MOG antibodyndashnegative patients Only 17 ofthe MOG antibodyndashpositive patients had 1 or bothHLA-DRB11501 alleles the main MS susceptibilityallele which is the same as the previously reportedpercentage in the ldquonormalrdquo healthy population inAustralia34 whereas the MOG antibodyndashnegativepatients were more likely to have 1 or more HLA-DRB11501 allele This negative association wasnot statistically significant and larger cohorts arerequired to test whether this correlation is a realrather than a suspected phenomenon In contrastradiologic features did not appear to differentiateMOG antibodyndashpositive from ndashnegative patients inthis childhood cohort
The presence of MOG antibodies is unlikely tohave therapeutic implications in the first demyelinat-ing event but is more likely to be therapeuticallyimportant in the relapsing patient Although 2 recentpatients fulfilled criteria for MS the presence ofMOG antibodies and elevated ESR led us to treatthem more like NMO patients with the immune sup-pression agent MMF rather than conventional MSdisease-modifying therapies Both patients had noclinical or radiologic relapse for 12 months and 36months respectively and both became MOG anti-body seronegative Further studies are required tounderstand the longitudinal course of patients withMOG antibodyndashassociated relapsing disease and thetreatment approach required
Cell-based assays have been essential to detectMOG antibody with a high specificity9 especiallywhen the full-length MOG is expressed in cells ratherthan the C-terminal truncated MOG35 Our experi-ence with neuronal antigens informed us that FACSis sensitive and quantitative10132022333637 We detectedCSF MOG antibody in only 3 of 22 DEM samples ofwhich 5 were positive in serum This suggests CSF isless sensitive than serum for MOG antibody detectionwhich could be due to a lack of sensitivity or couldrepresent a genuine absence of intrathecal Ig synthesisin these patients Supporting this concept none of thepatients with positive MOG antibody had intrathecaloligoclonal bands
The pathogenic function of MOG antibody is stillunclear but over the recent years the detection of
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316
and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538
Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40
Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders
AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript
RCD and FB Acquisition analysis and interpretation of results RCD
EMT VM R-YAK NS SR KP LAW DRB K Prelog
DRC GJG CKL EKM FB Statistical analysis RCD EMT
FB Critical revisions of manuscript RCD EMT VM NS SR
KP DRB LAW K Prelog DRC GJG CKL EKM FB
ACKNOWLEDGMENTThe authors thank all the patients and family members who provided
samples for the study The authors thank Dr Maggie Wang for use of
the Flow Cytometry Core Facility of the Westmead Millennium Institute
(Australia) The authors thank Dr Hong Yu for use of the Imaging Core
Facility at the Westmead Research Hub
STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple
Sclerosis Research Foundation Multiple Sclerosis Research Australia
MS Angels Melbourne (Australia) and the Petre Foundation (Australia)
DISCLOSURERC Dale has received research funding from the Star Scientific Foundation
the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis
Research Australia NHMRC and the Petre Foundation has received hon-
oraria from Biogen Idec and is on advisory boards for Queensland Child-
renrsquos Medical Institute Research and MSARD EM Tantsis has received
a scholarship from the Australian Postgraduate Award program V Merheb
and R-YA Kumaran report no disclosures N Sinmaz has received a schol-
arship from the Australian Postgraduate Award program K Pathmanandavel
has received a scholarship from the Petre Foundation (Australia)
S Ramanathan has received a scholarship from the National Health and
Medical Research Council (Australia) DR Booth has received funding from
the National Health and Medical Research Council (Australia) the Australian
Research Council Multiple Sclerosis Research Australia and the Leukemia
Foundation has received honoraria and funding from Biogen Idec Merck
Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS
ONE LA Wienholt K Prelog and DR Clark report no disclosures
GJ Guillemin is the editor-in-chief for the International Journal for Trypto-
phan Research and has received research support from the Australian Research
Council CK Lim has received research funding from Multiple Sclerosis
Research Australia and Trish MS Research Foundation EK Mathey has
received funding from the National Health and Medical Research Council
(Australia) and the Rebecca L Cooper Medical Research Foundation
(Australia) F Brilot has received research funding from the Star Scientific
Foundation the Trish Multiple Sclerosis Research Foundation Multiple
Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre
Foundation and is an associate editor for the Journal of Visualized Experiments
Go to Neurologyorgnn for full disclosures
Received February 25 2014 Accepted in final form April 16 2014
REFERENCES1 Papadopoulos MC Verkman AS Aquaporin 4 and neu-
romyelitis optica Lancet Neurol 201211535ndash544
2 Vincent A Bien CG Irani SR Waters P Autoantibodies
associated with diseases of the CNS new developments
and future challenges Lancet Neurol 201110759ndash772
3 Wingerchuk DM Lennon VA Lucchinetti CF
Pittock SJ Weinshenker BG The spectrum of neuromy-
elitis optica Lancet Neurol 20076805ndash815
4 Johns TG Bernard CC The structure and function of
myelin oligodendrocyte glycoprotein J Neurochem
1999721ndash9
5 Linington C Bradl M Lassmann H Brunner C Vass K
Augmentation of demyelination in rat acute allergic
encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelinoligodendrocyte glycopro-
tein Am J Pathol 1988130443ndash454
6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous
relapsing-remitting EAE in the SJLJ mouse MOG-reactive
transgenic T cells recruit endogenous MOG-specific B cells
J Exp Med 20092061303ndash1316
7 Schluesener HJ Sobel RA Linington C Weiner HL A
monoclonal antibody against a myelin oligodendrocyte
glycoprotein induces relapses and demyelination in central
nervous system autoimmune disease J Immunol 1987
1394016ndash4021
8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB
Lee JI Genain CP Molecular characterization of antibody
specificities against myelinoligodendrocyte glycoprotein
in autoimmune demyelination Proc Natl Acad Sci U S A
2002998207ndash8212
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
and normalization of MOG antibodies (figure 2 andappendix e-2) In both patients upon treatment withMMF MOG antibody decreased below the thresholdof positivity (figure 2 A and B)
Loss of cytoskeleton organization by MOG antibody or
purified IgG from children with demyelinating diseases
Human oligodendrocyte MO313MOG1 cells expressedMOG at their surface whereas noMOG expression wasobserved on MO313Ctl cells (figure 3) MO313 cellsalso expressed oligodendrocyte markers such as 29 39-cyclic nucleotide 39-phosphodiesterase (CNPase) galac-tocerebroside (GalC) oligodendrocyte marker O4vimentin and c-series ganglioside-specific antigen(A2B5) (figure 3A) Myelin basic protein (MBP) aspecific marker of mature oligodendrocytes wasobserved only in PMA-differentiated MO313MOG1
cells (figure 3A) suggesting that undifferentiatedMO313 cells are immature oligodendrocytes Using
purified IgG and immunoaffinity-purified MOG IgGfromMOG antibodyndashpositive sera we immunolabeledHEK293MOG1 and HEK293Ctl cells on live cells byFACS (figure 3B) and on fixed MO313MOG1 cellsby immunocytochemistry (figure 3C) and showeda positive immunostaining compared to MOGantibodyndashnegative sera suggesting that proteinGndashpurified IgG includes MOG-specific IgG Due tosmall volumes of pediatric sera we used proteinGndashpurified IgG in pathogenic experiments Next wetreated fixed and live MO313MOG1Ctl cells with IgGfrom MOG antibodyndashpositive and ndashnegative patientswith DEM and healthy controls (HCs) Then all cellswere immunolabeled for b-tubulin (marker ofmicrotubule) or F-actin (marker of thin filaments) Wequantified results and expressed them by the F-actin andb-tubulin relative enrichments in the cytoplasm andperinuclear region over the entire cell All results were
Figure 2 Temporal distribution of MOG antibody in serum of 2 relapsing patients with demyelinating diseases
In both patient A (A) and patient B (B) upon treatment mycophenolate mofetil (MMF) myelin oligodendrocyte glycoprotein (MOG) antibody decreased towithin the healthy control range (below threshold of positivity) and these low titers were associated with remission Representative dot plot out of 3 experi-ments is shown Magenta lines on graphs represent the positivity threshold (obtained with 24 control samples) Black squares represent serum analysis dur-ing acute demyelination episodes and black circles represent sera during remission Type of demyelinating episode is shown on graph (C D) RepresentativeT2 axial MRI scans demonstrate demyelinating lesions during the first acute event and during convalescence Patient A (C) had globular deep white matterlesions on acute scan (left panel) which show residual gliosis on convalescent scan and no new lesions (right panel) Patient B (D) had inflammatory lesions inbasal ganglia and white matter on acute scan (left panel) with complete resolution on convalescent scan and no new lesions (right panel) Ab 5 antibodyADEM5 acute disseminated encephalomyelitis CEREB5 cerebellar episode Ig5 immunoglobulin MFI5mean fluorescence intensity ON5 optic neuritisTM 5 transverse myelitis
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
normalized using F-actin and b-tubulin in HCIgG-treated fixed cells Organization of themicrotubule and thin filament networks wassimilar in fixed cells incubated with HC (1006 101 and 100 6 226 respectively figure 4A and C) or DEM IgGs (1043 6 113 and858 6 318 respectively figure 4 A and C)
there was clear visualization of ldquostress-fibersrdquo andfilopedia after F-actin immunolabeling (figure 4Aupper panels) and bright mesh-like staining spreadout through the entire cell after b-tubulinimmunolabeling (figure 4A lower panels) Livecells treated by HC IgG displayed a smalldisorganization of F-actin thin filaments (916 144
Figure 3 Human oligodendroglial MO313MOG1 cells express markers of oligodendrocytes and are immunolabeled with protein G- and humanMOG-purified human IgG from MOG antibodyndashpositive DEM patients
(A) Immunocytochemistry on fixed permeabilized cells showed that MO313MOG1 cells expressed oligodendrocyte markers (B) Protein G- and human MOG-immunopurified IgG from MOG antibodyndashpositive serum immunolabeled live HEK293MOG1 cells but did not immunolabel HEK293Ctl cells compared to MOGantibodyndashnegative serum Binding to cells was determined by flow cytometry Mean fluorescence intensity (MFI) values are shown in legends (C) ProteinG- and human MOG-purified IgG from MOG antibodyndashpositive serum also immunolabeled fixed unpermeabilized MO313MOG1 cells compared to MOGantibodyndashnegative serum Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy) Nuclei stainedwith 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm A2B55 c-series ganglioside-specific antigen A2B5 Ab5 antibody CNPase529 39-cyclic nucleotide39-phosphodiesterase DEM 5 demyelinating diseases GalC 5 galactocerebroside HEK 5 human embryonic kidney Ig 5 immunoglobulin MBP 5 myelinbasic protein MOG 5 myelin oligodendrocyte glycoprotein O4 5 oligodendrocyte marker O4
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
Figure 4 Purified IgG from DEM patients induces loss of cytoskeleton organization in live human oligodendroglial MO313MOG1 cells
F-actin (upper images) and b-tubulin (lower images) immunolabelings in human fixed (A) or live (B) oligodendroglial MO313MOG1 cells incubated with purifiedhealthy control (HC) IgG or MOG antibodyndashpositive DEM IgG Representative data are shown (volume projection of entire Z-stack acquired using deconvo-lution microscopy) Nuclei stained with 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm Dotted lines on right images represent contour of cells as deter-mined by differential interference contrast images (not shown) (C) Quantification of change in distribution of thin filament (F-actin upper scatter plot) andmicrotubule network (b-tubulin lower scatter plot) organization using 3D deconvolution microscopy F-actin and b-tubulin were majorly affected in live cellsincubated with DEM IgG Forty different cells (1 cell5 1 diamond) from 2 HC and 2 DEM patients out of 3 independent experiments are shown Results areexpressed as ratio of the relative enrichment (RE) between cytoplasm and nucleus and values shown are percentages relative to HC IgG FIX (100) Redbars represent mean Note that there also was a loss of organization of the thin filaments after HC IgG incubation on live cells compared to fixed conditionsbut to a lesser extent than after DEM IgG (D) Cell viability assay by flow cytometry on live human oligodendroglial MO313MOG1 cells incubated 45 minuteswith 6 mg of purified HC IgG or DEM IgG No difference in cell death was observed Dead cell percentages are noted in the legend 7AAD 5 7-amino-actinomycin D DEM 5 demyelinating diseases Ig 5 immunoglobulin MOG 5 myelin oligodendrocyte glycoprotein
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
p 00001 figure 4 B and C upper images)and undisturbed microtubular b-tubulin network(1074 6 321 figure 4 B and C) similar to thatobserved in fixed DEM and HC IgG-treated cells(figure 4A upper panels) Following treatment withDEM IgG on live MO313MOG1 cells we observed astriking loss of organization in both F-actin (7246 117 p 00001 figure 4 B and C) andb-tubulin networks (522 6 131 p 00001figure 4 B and C) in otherwise healthy-lookingundividing cells visualized by differential interferencecontrast imaging (figure 4B dotted line and data notshown) and analyzed by FACS by which there was nodifference in viability between live MO313MOG1 livecells treated with DEM IgG or HC IgG for45 minutes (figure 4D 8726 06 vs 8726 211) or10 hours (data not shown) No loss of cytoskeletonorganization was observed when live MO313Ctl cellswere incubated with HC IgG and MOG antibodyndashpositive DEM IgG nor when live MO313MOG1 cellswere incubated with MOG antibodyndashnegative DEMIgG (figure e-1 and appendix e-3) In additionsuccessful immunoabsorption of MOG antibodyndashpositive serum on HEK293MOG1 cells led to nochange in cytoskeleton organization whenimmunoabsorbed serum was incubated on liveMO313MOG1 cells (figure e-1 and appendix e-3)Overall after incubation with MOG antibodyndashpositive DEM IgG both F-actin and b-tubulinimmunolabelings appeared to be enriched withina perinuclear region in the center of the cellssurrounding the nucleus
DISCUSSION As in this study recent reports haveconsistently found that serum MOG antibody is pre-sent in a significant proportion of children with CNSdemyelinating disease whereas AQP4 antibodies arerare9 We believe that the clinical and investigationfindings in this report support the evolving conceptthat MOG antibodies may define a separate autoim-mune demyelinating syndrome that may be distinctfrom classic adult-onset MS MOG antibodies arepresent in up to 50 of children with ADEM as inthis report Proebstel et al33 found that ADEMMOGantibody seropositive children became seronegative inthe recovery period whereas children with relapsingMS typically remained seropositive More recentlyMOG antibodies have been found in children withON and in some patients with NMO phenotypenegative for NMO IgG1431 In this report we havestrengthened this association with ON and found thatpatients with bilateral rather than unilateral ON weremore likely to be MOG antibodyndashpositive BilateralON is a common ON phenotype in children but israre in adults in whom unilateral ON is the moretypical demyelinating event of MS In contrast
patients with brainstem signs and CIS other thanON and TM were rarely MOG antibodyndashpositiveAn elevated ESR was more common in MOGantibodyndashpositive patients An elevated ESR isatypical of MS and should raise suspicion of anautoimmune disorder or alternate inflammatorymimic of MS We showed that elevation of ESR inMOG antibodyndashpositive patients was independent ofage Intrathecal oligoclonal bands are one of the classicbiomarkers of MS and were absent in all MOGantibodyndashpositive patients and were present only inthe MOG antibodyndashnegative patients Only 17 ofthe MOG antibodyndashpositive patients had 1 or bothHLA-DRB11501 alleles the main MS susceptibilityallele which is the same as the previously reportedpercentage in the ldquonormalrdquo healthy population inAustralia34 whereas the MOG antibodyndashnegativepatients were more likely to have 1 or more HLA-DRB11501 allele This negative association wasnot statistically significant and larger cohorts arerequired to test whether this correlation is a realrather than a suspected phenomenon In contrastradiologic features did not appear to differentiateMOG antibodyndashpositive from ndashnegative patients inthis childhood cohort
The presence of MOG antibodies is unlikely tohave therapeutic implications in the first demyelinat-ing event but is more likely to be therapeuticallyimportant in the relapsing patient Although 2 recentpatients fulfilled criteria for MS the presence ofMOG antibodies and elevated ESR led us to treatthem more like NMO patients with the immune sup-pression agent MMF rather than conventional MSdisease-modifying therapies Both patients had noclinical or radiologic relapse for 12 months and 36months respectively and both became MOG anti-body seronegative Further studies are required tounderstand the longitudinal course of patients withMOG antibodyndashassociated relapsing disease and thetreatment approach required
Cell-based assays have been essential to detectMOG antibody with a high specificity9 especiallywhen the full-length MOG is expressed in cells ratherthan the C-terminal truncated MOG35 Our experi-ence with neuronal antigens informed us that FACSis sensitive and quantitative10132022333637 We detectedCSF MOG antibody in only 3 of 22 DEM samples ofwhich 5 were positive in serum This suggests CSF isless sensitive than serum for MOG antibody detectionwhich could be due to a lack of sensitivity or couldrepresent a genuine absence of intrathecal Ig synthesisin these patients Supporting this concept none of thepatients with positive MOG antibody had intrathecaloligoclonal bands
The pathogenic function of MOG antibody is stillunclear but over the recent years the detection of
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316
and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538
Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40
Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders
AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript
RCD and FB Acquisition analysis and interpretation of results RCD
EMT VM R-YAK NS SR KP LAW DRB K Prelog
DRC GJG CKL EKM FB Statistical analysis RCD EMT
FB Critical revisions of manuscript RCD EMT VM NS SR
KP DRB LAW K Prelog DRC GJG CKL EKM FB
ACKNOWLEDGMENTThe authors thank all the patients and family members who provided
samples for the study The authors thank Dr Maggie Wang for use of
the Flow Cytometry Core Facility of the Westmead Millennium Institute
(Australia) The authors thank Dr Hong Yu for use of the Imaging Core
Facility at the Westmead Research Hub
STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple
Sclerosis Research Foundation Multiple Sclerosis Research Australia
MS Angels Melbourne (Australia) and the Petre Foundation (Australia)
DISCLOSURERC Dale has received research funding from the Star Scientific Foundation
the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis
Research Australia NHMRC and the Petre Foundation has received hon-
oraria from Biogen Idec and is on advisory boards for Queensland Child-
renrsquos Medical Institute Research and MSARD EM Tantsis has received
a scholarship from the Australian Postgraduate Award program V Merheb
and R-YA Kumaran report no disclosures N Sinmaz has received a schol-
arship from the Australian Postgraduate Award program K Pathmanandavel
has received a scholarship from the Petre Foundation (Australia)
S Ramanathan has received a scholarship from the National Health and
Medical Research Council (Australia) DR Booth has received funding from
the National Health and Medical Research Council (Australia) the Australian
Research Council Multiple Sclerosis Research Australia and the Leukemia
Foundation has received honoraria and funding from Biogen Idec Merck
Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS
ONE LA Wienholt K Prelog and DR Clark report no disclosures
GJ Guillemin is the editor-in-chief for the International Journal for Trypto-
phan Research and has received research support from the Australian Research
Council CK Lim has received research funding from Multiple Sclerosis
Research Australia and Trish MS Research Foundation EK Mathey has
received funding from the National Health and Medical Research Council
(Australia) and the Rebecca L Cooper Medical Research Foundation
(Australia) F Brilot has received research funding from the Star Scientific
Foundation the Trish Multiple Sclerosis Research Foundation Multiple
Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre
Foundation and is an associate editor for the Journal of Visualized Experiments
Go to Neurologyorgnn for full disclosures
Received February 25 2014 Accepted in final form April 16 2014
REFERENCES1 Papadopoulos MC Verkman AS Aquaporin 4 and neu-
romyelitis optica Lancet Neurol 201211535ndash544
2 Vincent A Bien CG Irani SR Waters P Autoantibodies
associated with diseases of the CNS new developments
and future challenges Lancet Neurol 201110759ndash772
3 Wingerchuk DM Lennon VA Lucchinetti CF
Pittock SJ Weinshenker BG The spectrum of neuromy-
elitis optica Lancet Neurol 20076805ndash815
4 Johns TG Bernard CC The structure and function of
myelin oligodendrocyte glycoprotein J Neurochem
1999721ndash9
5 Linington C Bradl M Lassmann H Brunner C Vass K
Augmentation of demyelination in rat acute allergic
encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelinoligodendrocyte glycopro-
tein Am J Pathol 1988130443ndash454
6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous
relapsing-remitting EAE in the SJLJ mouse MOG-reactive
transgenic T cells recruit endogenous MOG-specific B cells
J Exp Med 20092061303ndash1316
7 Schluesener HJ Sobel RA Linington C Weiner HL A
monoclonal antibody against a myelin oligodendrocyte
glycoprotein induces relapses and demyelination in central
nervous system autoimmune disease J Immunol 1987
1394016ndash4021
8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB
Lee JI Genain CP Molecular characterization of antibody
specificities against myelinoligodendrocyte glycoprotein
in autoimmune demyelination Proc Natl Acad Sci U S A
2002998207ndash8212
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
normalized using F-actin and b-tubulin in HCIgG-treated fixed cells Organization of themicrotubule and thin filament networks wassimilar in fixed cells incubated with HC (1006 101 and 100 6 226 respectively figure 4A and C) or DEM IgGs (1043 6 113 and858 6 318 respectively figure 4 A and C)
there was clear visualization of ldquostress-fibersrdquo andfilopedia after F-actin immunolabeling (figure 4Aupper panels) and bright mesh-like staining spreadout through the entire cell after b-tubulinimmunolabeling (figure 4A lower panels) Livecells treated by HC IgG displayed a smalldisorganization of F-actin thin filaments (916 144
Figure 3 Human oligodendroglial MO313MOG1 cells express markers of oligodendrocytes and are immunolabeled with protein G- and humanMOG-purified human IgG from MOG antibodyndashpositive DEM patients
(A) Immunocytochemistry on fixed permeabilized cells showed that MO313MOG1 cells expressed oligodendrocyte markers (B) Protein G- and human MOG-immunopurified IgG from MOG antibodyndashpositive serum immunolabeled live HEK293MOG1 cells but did not immunolabel HEK293Ctl cells compared to MOGantibodyndashnegative serum Binding to cells was determined by flow cytometry Mean fluorescence intensity (MFI) values are shown in legends (C) ProteinG- and human MOG-purified IgG from MOG antibodyndashpositive serum also immunolabeled fixed unpermeabilized MO313MOG1 cells compared to MOGantibodyndashnegative serum Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy) Nuclei stainedwith 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm A2B55 c-series ganglioside-specific antigen A2B5 Ab5 antibody CNPase529 39-cyclic nucleotide39-phosphodiesterase DEM 5 demyelinating diseases GalC 5 galactocerebroside HEK 5 human embryonic kidney Ig 5 immunoglobulin MBP 5 myelinbasic protein MOG 5 myelin oligodendrocyte glycoprotein O4 5 oligodendrocyte marker O4
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
Figure 4 Purified IgG from DEM patients induces loss of cytoskeleton organization in live human oligodendroglial MO313MOG1 cells
F-actin (upper images) and b-tubulin (lower images) immunolabelings in human fixed (A) or live (B) oligodendroglial MO313MOG1 cells incubated with purifiedhealthy control (HC) IgG or MOG antibodyndashpositive DEM IgG Representative data are shown (volume projection of entire Z-stack acquired using deconvo-lution microscopy) Nuclei stained with 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm Dotted lines on right images represent contour of cells as deter-mined by differential interference contrast images (not shown) (C) Quantification of change in distribution of thin filament (F-actin upper scatter plot) andmicrotubule network (b-tubulin lower scatter plot) organization using 3D deconvolution microscopy F-actin and b-tubulin were majorly affected in live cellsincubated with DEM IgG Forty different cells (1 cell5 1 diamond) from 2 HC and 2 DEM patients out of 3 independent experiments are shown Results areexpressed as ratio of the relative enrichment (RE) between cytoplasm and nucleus and values shown are percentages relative to HC IgG FIX (100) Redbars represent mean Note that there also was a loss of organization of the thin filaments after HC IgG incubation on live cells compared to fixed conditionsbut to a lesser extent than after DEM IgG (D) Cell viability assay by flow cytometry on live human oligodendroglial MO313MOG1 cells incubated 45 minuteswith 6 mg of purified HC IgG or DEM IgG No difference in cell death was observed Dead cell percentages are noted in the legend 7AAD 5 7-amino-actinomycin D DEM 5 demyelinating diseases Ig 5 immunoglobulin MOG 5 myelin oligodendrocyte glycoprotein
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
p 00001 figure 4 B and C upper images)and undisturbed microtubular b-tubulin network(1074 6 321 figure 4 B and C) similar to thatobserved in fixed DEM and HC IgG-treated cells(figure 4A upper panels) Following treatment withDEM IgG on live MO313MOG1 cells we observed astriking loss of organization in both F-actin (7246 117 p 00001 figure 4 B and C) andb-tubulin networks (522 6 131 p 00001figure 4 B and C) in otherwise healthy-lookingundividing cells visualized by differential interferencecontrast imaging (figure 4B dotted line and data notshown) and analyzed by FACS by which there was nodifference in viability between live MO313MOG1 livecells treated with DEM IgG or HC IgG for45 minutes (figure 4D 8726 06 vs 8726 211) or10 hours (data not shown) No loss of cytoskeletonorganization was observed when live MO313Ctl cellswere incubated with HC IgG and MOG antibodyndashpositive DEM IgG nor when live MO313MOG1 cellswere incubated with MOG antibodyndashnegative DEMIgG (figure e-1 and appendix e-3) In additionsuccessful immunoabsorption of MOG antibodyndashpositive serum on HEK293MOG1 cells led to nochange in cytoskeleton organization whenimmunoabsorbed serum was incubated on liveMO313MOG1 cells (figure e-1 and appendix e-3)Overall after incubation with MOG antibodyndashpositive DEM IgG both F-actin and b-tubulinimmunolabelings appeared to be enriched withina perinuclear region in the center of the cellssurrounding the nucleus
DISCUSSION As in this study recent reports haveconsistently found that serum MOG antibody is pre-sent in a significant proportion of children with CNSdemyelinating disease whereas AQP4 antibodies arerare9 We believe that the clinical and investigationfindings in this report support the evolving conceptthat MOG antibodies may define a separate autoim-mune demyelinating syndrome that may be distinctfrom classic adult-onset MS MOG antibodies arepresent in up to 50 of children with ADEM as inthis report Proebstel et al33 found that ADEMMOGantibody seropositive children became seronegative inthe recovery period whereas children with relapsingMS typically remained seropositive More recentlyMOG antibodies have been found in children withON and in some patients with NMO phenotypenegative for NMO IgG1431 In this report we havestrengthened this association with ON and found thatpatients with bilateral rather than unilateral ON weremore likely to be MOG antibodyndashpositive BilateralON is a common ON phenotype in children but israre in adults in whom unilateral ON is the moretypical demyelinating event of MS In contrast
patients with brainstem signs and CIS other thanON and TM were rarely MOG antibodyndashpositiveAn elevated ESR was more common in MOGantibodyndashpositive patients An elevated ESR isatypical of MS and should raise suspicion of anautoimmune disorder or alternate inflammatorymimic of MS We showed that elevation of ESR inMOG antibodyndashpositive patients was independent ofage Intrathecal oligoclonal bands are one of the classicbiomarkers of MS and were absent in all MOGantibodyndashpositive patients and were present only inthe MOG antibodyndashnegative patients Only 17 ofthe MOG antibodyndashpositive patients had 1 or bothHLA-DRB11501 alleles the main MS susceptibilityallele which is the same as the previously reportedpercentage in the ldquonormalrdquo healthy population inAustralia34 whereas the MOG antibodyndashnegativepatients were more likely to have 1 or more HLA-DRB11501 allele This negative association wasnot statistically significant and larger cohorts arerequired to test whether this correlation is a realrather than a suspected phenomenon In contrastradiologic features did not appear to differentiateMOG antibodyndashpositive from ndashnegative patients inthis childhood cohort
The presence of MOG antibodies is unlikely tohave therapeutic implications in the first demyelinat-ing event but is more likely to be therapeuticallyimportant in the relapsing patient Although 2 recentpatients fulfilled criteria for MS the presence ofMOG antibodies and elevated ESR led us to treatthem more like NMO patients with the immune sup-pression agent MMF rather than conventional MSdisease-modifying therapies Both patients had noclinical or radiologic relapse for 12 months and 36months respectively and both became MOG anti-body seronegative Further studies are required tounderstand the longitudinal course of patients withMOG antibodyndashassociated relapsing disease and thetreatment approach required
Cell-based assays have been essential to detectMOG antibody with a high specificity9 especiallywhen the full-length MOG is expressed in cells ratherthan the C-terminal truncated MOG35 Our experi-ence with neuronal antigens informed us that FACSis sensitive and quantitative10132022333637 We detectedCSF MOG antibody in only 3 of 22 DEM samples ofwhich 5 were positive in serum This suggests CSF isless sensitive than serum for MOG antibody detectionwhich could be due to a lack of sensitivity or couldrepresent a genuine absence of intrathecal Ig synthesisin these patients Supporting this concept none of thepatients with positive MOG antibody had intrathecaloligoclonal bands
The pathogenic function of MOG antibody is stillunclear but over the recent years the detection of
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316
and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538
Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40
Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders
AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript
RCD and FB Acquisition analysis and interpretation of results RCD
EMT VM R-YAK NS SR KP LAW DRB K Prelog
DRC GJG CKL EKM FB Statistical analysis RCD EMT
FB Critical revisions of manuscript RCD EMT VM NS SR
KP DRB LAW K Prelog DRC GJG CKL EKM FB
ACKNOWLEDGMENTThe authors thank all the patients and family members who provided
samples for the study The authors thank Dr Maggie Wang for use of
the Flow Cytometry Core Facility of the Westmead Millennium Institute
(Australia) The authors thank Dr Hong Yu for use of the Imaging Core
Facility at the Westmead Research Hub
STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple
Sclerosis Research Foundation Multiple Sclerosis Research Australia
MS Angels Melbourne (Australia) and the Petre Foundation (Australia)
DISCLOSURERC Dale has received research funding from the Star Scientific Foundation
the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis
Research Australia NHMRC and the Petre Foundation has received hon-
oraria from Biogen Idec and is on advisory boards for Queensland Child-
renrsquos Medical Institute Research and MSARD EM Tantsis has received
a scholarship from the Australian Postgraduate Award program V Merheb
and R-YA Kumaran report no disclosures N Sinmaz has received a schol-
arship from the Australian Postgraduate Award program K Pathmanandavel
has received a scholarship from the Petre Foundation (Australia)
S Ramanathan has received a scholarship from the National Health and
Medical Research Council (Australia) DR Booth has received funding from
the National Health and Medical Research Council (Australia) the Australian
Research Council Multiple Sclerosis Research Australia and the Leukemia
Foundation has received honoraria and funding from Biogen Idec Merck
Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS
ONE LA Wienholt K Prelog and DR Clark report no disclosures
GJ Guillemin is the editor-in-chief for the International Journal for Trypto-
phan Research and has received research support from the Australian Research
Council CK Lim has received research funding from Multiple Sclerosis
Research Australia and Trish MS Research Foundation EK Mathey has
received funding from the National Health and Medical Research Council
(Australia) and the Rebecca L Cooper Medical Research Foundation
(Australia) F Brilot has received research funding from the Star Scientific
Foundation the Trish Multiple Sclerosis Research Foundation Multiple
Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre
Foundation and is an associate editor for the Journal of Visualized Experiments
Go to Neurologyorgnn for full disclosures
Received February 25 2014 Accepted in final form April 16 2014
REFERENCES1 Papadopoulos MC Verkman AS Aquaporin 4 and neu-
romyelitis optica Lancet Neurol 201211535ndash544
2 Vincent A Bien CG Irani SR Waters P Autoantibodies
associated with diseases of the CNS new developments
and future challenges Lancet Neurol 201110759ndash772
3 Wingerchuk DM Lennon VA Lucchinetti CF
Pittock SJ Weinshenker BG The spectrum of neuromy-
elitis optica Lancet Neurol 20076805ndash815
4 Johns TG Bernard CC The structure and function of
myelin oligodendrocyte glycoprotein J Neurochem
1999721ndash9
5 Linington C Bradl M Lassmann H Brunner C Vass K
Augmentation of demyelination in rat acute allergic
encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelinoligodendrocyte glycopro-
tein Am J Pathol 1988130443ndash454
6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous
relapsing-remitting EAE in the SJLJ mouse MOG-reactive
transgenic T cells recruit endogenous MOG-specific B cells
J Exp Med 20092061303ndash1316
7 Schluesener HJ Sobel RA Linington C Weiner HL A
monoclonal antibody against a myelin oligodendrocyte
glycoprotein induces relapses and demyelination in central
nervous system autoimmune disease J Immunol 1987
1394016ndash4021
8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB
Lee JI Genain CP Molecular characterization of antibody
specificities against myelinoligodendrocyte glycoprotein
in autoimmune demyelination Proc Natl Acad Sci U S A
2002998207ndash8212
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
Figure 4 Purified IgG from DEM patients induces loss of cytoskeleton organization in live human oligodendroglial MO313MOG1 cells
F-actin (upper images) and b-tubulin (lower images) immunolabelings in human fixed (A) or live (B) oligodendroglial MO313MOG1 cells incubated with purifiedhealthy control (HC) IgG or MOG antibodyndashpositive DEM IgG Representative data are shown (volume projection of entire Z-stack acquired using deconvo-lution microscopy) Nuclei stained with 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm Dotted lines on right images represent contour of cells as deter-mined by differential interference contrast images (not shown) (C) Quantification of change in distribution of thin filament (F-actin upper scatter plot) andmicrotubule network (b-tubulin lower scatter plot) organization using 3D deconvolution microscopy F-actin and b-tubulin were majorly affected in live cellsincubated with DEM IgG Forty different cells (1 cell5 1 diamond) from 2 HC and 2 DEM patients out of 3 independent experiments are shown Results areexpressed as ratio of the relative enrichment (RE) between cytoplasm and nucleus and values shown are percentages relative to HC IgG FIX (100) Redbars represent mean Note that there also was a loss of organization of the thin filaments after HC IgG incubation on live cells compared to fixed conditionsbut to a lesser extent than after DEM IgG (D) Cell viability assay by flow cytometry on live human oligodendroglial MO313MOG1 cells incubated 45 minuteswith 6 mg of purified HC IgG or DEM IgG No difference in cell death was observed Dead cell percentages are noted in the legend 7AAD 5 7-amino-actinomycin D DEM 5 demyelinating diseases Ig 5 immunoglobulin MOG 5 myelin oligodendrocyte glycoprotein
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
p 00001 figure 4 B and C upper images)and undisturbed microtubular b-tubulin network(1074 6 321 figure 4 B and C) similar to thatobserved in fixed DEM and HC IgG-treated cells(figure 4A upper panels) Following treatment withDEM IgG on live MO313MOG1 cells we observed astriking loss of organization in both F-actin (7246 117 p 00001 figure 4 B and C) andb-tubulin networks (522 6 131 p 00001figure 4 B and C) in otherwise healthy-lookingundividing cells visualized by differential interferencecontrast imaging (figure 4B dotted line and data notshown) and analyzed by FACS by which there was nodifference in viability between live MO313MOG1 livecells treated with DEM IgG or HC IgG for45 minutes (figure 4D 8726 06 vs 8726 211) or10 hours (data not shown) No loss of cytoskeletonorganization was observed when live MO313Ctl cellswere incubated with HC IgG and MOG antibodyndashpositive DEM IgG nor when live MO313MOG1 cellswere incubated with MOG antibodyndashnegative DEMIgG (figure e-1 and appendix e-3) In additionsuccessful immunoabsorption of MOG antibodyndashpositive serum on HEK293MOG1 cells led to nochange in cytoskeleton organization whenimmunoabsorbed serum was incubated on liveMO313MOG1 cells (figure e-1 and appendix e-3)Overall after incubation with MOG antibodyndashpositive DEM IgG both F-actin and b-tubulinimmunolabelings appeared to be enriched withina perinuclear region in the center of the cellssurrounding the nucleus
DISCUSSION As in this study recent reports haveconsistently found that serum MOG antibody is pre-sent in a significant proportion of children with CNSdemyelinating disease whereas AQP4 antibodies arerare9 We believe that the clinical and investigationfindings in this report support the evolving conceptthat MOG antibodies may define a separate autoim-mune demyelinating syndrome that may be distinctfrom classic adult-onset MS MOG antibodies arepresent in up to 50 of children with ADEM as inthis report Proebstel et al33 found that ADEMMOGantibody seropositive children became seronegative inthe recovery period whereas children with relapsingMS typically remained seropositive More recentlyMOG antibodies have been found in children withON and in some patients with NMO phenotypenegative for NMO IgG1431 In this report we havestrengthened this association with ON and found thatpatients with bilateral rather than unilateral ON weremore likely to be MOG antibodyndashpositive BilateralON is a common ON phenotype in children but israre in adults in whom unilateral ON is the moretypical demyelinating event of MS In contrast
patients with brainstem signs and CIS other thanON and TM were rarely MOG antibodyndashpositiveAn elevated ESR was more common in MOGantibodyndashpositive patients An elevated ESR isatypical of MS and should raise suspicion of anautoimmune disorder or alternate inflammatorymimic of MS We showed that elevation of ESR inMOG antibodyndashpositive patients was independent ofage Intrathecal oligoclonal bands are one of the classicbiomarkers of MS and were absent in all MOGantibodyndashpositive patients and were present only inthe MOG antibodyndashnegative patients Only 17 ofthe MOG antibodyndashpositive patients had 1 or bothHLA-DRB11501 alleles the main MS susceptibilityallele which is the same as the previously reportedpercentage in the ldquonormalrdquo healthy population inAustralia34 whereas the MOG antibodyndashnegativepatients were more likely to have 1 or more HLA-DRB11501 allele This negative association wasnot statistically significant and larger cohorts arerequired to test whether this correlation is a realrather than a suspected phenomenon In contrastradiologic features did not appear to differentiateMOG antibodyndashpositive from ndashnegative patients inthis childhood cohort
The presence of MOG antibodies is unlikely tohave therapeutic implications in the first demyelinat-ing event but is more likely to be therapeuticallyimportant in the relapsing patient Although 2 recentpatients fulfilled criteria for MS the presence ofMOG antibodies and elevated ESR led us to treatthem more like NMO patients with the immune sup-pression agent MMF rather than conventional MSdisease-modifying therapies Both patients had noclinical or radiologic relapse for 12 months and 36months respectively and both became MOG anti-body seronegative Further studies are required tounderstand the longitudinal course of patients withMOG antibodyndashassociated relapsing disease and thetreatment approach required
Cell-based assays have been essential to detectMOG antibody with a high specificity9 especiallywhen the full-length MOG is expressed in cells ratherthan the C-terminal truncated MOG35 Our experi-ence with neuronal antigens informed us that FACSis sensitive and quantitative10132022333637 We detectedCSF MOG antibody in only 3 of 22 DEM samples ofwhich 5 were positive in serum This suggests CSF isless sensitive than serum for MOG antibody detectionwhich could be due to a lack of sensitivity or couldrepresent a genuine absence of intrathecal Ig synthesisin these patients Supporting this concept none of thepatients with positive MOG antibody had intrathecaloligoclonal bands
The pathogenic function of MOG antibody is stillunclear but over the recent years the detection of
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316
and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538
Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40
Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders
AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript
RCD and FB Acquisition analysis and interpretation of results RCD
EMT VM R-YAK NS SR KP LAW DRB K Prelog
DRC GJG CKL EKM FB Statistical analysis RCD EMT
FB Critical revisions of manuscript RCD EMT VM NS SR
KP DRB LAW K Prelog DRC GJG CKL EKM FB
ACKNOWLEDGMENTThe authors thank all the patients and family members who provided
samples for the study The authors thank Dr Maggie Wang for use of
the Flow Cytometry Core Facility of the Westmead Millennium Institute
(Australia) The authors thank Dr Hong Yu for use of the Imaging Core
Facility at the Westmead Research Hub
STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple
Sclerosis Research Foundation Multiple Sclerosis Research Australia
MS Angels Melbourne (Australia) and the Petre Foundation (Australia)
DISCLOSURERC Dale has received research funding from the Star Scientific Foundation
the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis
Research Australia NHMRC and the Petre Foundation has received hon-
oraria from Biogen Idec and is on advisory boards for Queensland Child-
renrsquos Medical Institute Research and MSARD EM Tantsis has received
a scholarship from the Australian Postgraduate Award program V Merheb
and R-YA Kumaran report no disclosures N Sinmaz has received a schol-
arship from the Australian Postgraduate Award program K Pathmanandavel
has received a scholarship from the Petre Foundation (Australia)
S Ramanathan has received a scholarship from the National Health and
Medical Research Council (Australia) DR Booth has received funding from
the National Health and Medical Research Council (Australia) the Australian
Research Council Multiple Sclerosis Research Australia and the Leukemia
Foundation has received honoraria and funding from Biogen Idec Merck
Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS
ONE LA Wienholt K Prelog and DR Clark report no disclosures
GJ Guillemin is the editor-in-chief for the International Journal for Trypto-
phan Research and has received research support from the Australian Research
Council CK Lim has received research funding from Multiple Sclerosis
Research Australia and Trish MS Research Foundation EK Mathey has
received funding from the National Health and Medical Research Council
(Australia) and the Rebecca L Cooper Medical Research Foundation
(Australia) F Brilot has received research funding from the Star Scientific
Foundation the Trish Multiple Sclerosis Research Foundation Multiple
Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre
Foundation and is an associate editor for the Journal of Visualized Experiments
Go to Neurologyorgnn for full disclosures
Received February 25 2014 Accepted in final form April 16 2014
REFERENCES1 Papadopoulos MC Verkman AS Aquaporin 4 and neu-
romyelitis optica Lancet Neurol 201211535ndash544
2 Vincent A Bien CG Irani SR Waters P Autoantibodies
associated with diseases of the CNS new developments
and future challenges Lancet Neurol 201110759ndash772
3 Wingerchuk DM Lennon VA Lucchinetti CF
Pittock SJ Weinshenker BG The spectrum of neuromy-
elitis optica Lancet Neurol 20076805ndash815
4 Johns TG Bernard CC The structure and function of
myelin oligodendrocyte glycoprotein J Neurochem
1999721ndash9
5 Linington C Bradl M Lassmann H Brunner C Vass K
Augmentation of demyelination in rat acute allergic
encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelinoligodendrocyte glycopro-
tein Am J Pathol 1988130443ndash454
6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous
relapsing-remitting EAE in the SJLJ mouse MOG-reactive
transgenic T cells recruit endogenous MOG-specific B cells
J Exp Med 20092061303ndash1316
7 Schluesener HJ Sobel RA Linington C Weiner HL A
monoclonal antibody against a myelin oligodendrocyte
glycoprotein induces relapses and demyelination in central
nervous system autoimmune disease J Immunol 1987
1394016ndash4021
8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB
Lee JI Genain CP Molecular characterization of antibody
specificities against myelinoligodendrocyte glycoprotein
in autoimmune demyelination Proc Natl Acad Sci U S A
2002998207ndash8212
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
p 00001 figure 4 B and C upper images)and undisturbed microtubular b-tubulin network(1074 6 321 figure 4 B and C) similar to thatobserved in fixed DEM and HC IgG-treated cells(figure 4A upper panels) Following treatment withDEM IgG on live MO313MOG1 cells we observed astriking loss of organization in both F-actin (7246 117 p 00001 figure 4 B and C) andb-tubulin networks (522 6 131 p 00001figure 4 B and C) in otherwise healthy-lookingundividing cells visualized by differential interferencecontrast imaging (figure 4B dotted line and data notshown) and analyzed by FACS by which there was nodifference in viability between live MO313MOG1 livecells treated with DEM IgG or HC IgG for45 minutes (figure 4D 8726 06 vs 8726 211) or10 hours (data not shown) No loss of cytoskeletonorganization was observed when live MO313Ctl cellswere incubated with HC IgG and MOG antibodyndashpositive DEM IgG nor when live MO313MOG1 cellswere incubated with MOG antibodyndashnegative DEMIgG (figure e-1 and appendix e-3) In additionsuccessful immunoabsorption of MOG antibodyndashpositive serum on HEK293MOG1 cells led to nochange in cytoskeleton organization whenimmunoabsorbed serum was incubated on liveMO313MOG1 cells (figure e-1 and appendix e-3)Overall after incubation with MOG antibodyndashpositive DEM IgG both F-actin and b-tubulinimmunolabelings appeared to be enriched withina perinuclear region in the center of the cellssurrounding the nucleus
DISCUSSION As in this study recent reports haveconsistently found that serum MOG antibody is pre-sent in a significant proportion of children with CNSdemyelinating disease whereas AQP4 antibodies arerare9 We believe that the clinical and investigationfindings in this report support the evolving conceptthat MOG antibodies may define a separate autoim-mune demyelinating syndrome that may be distinctfrom classic adult-onset MS MOG antibodies arepresent in up to 50 of children with ADEM as inthis report Proebstel et al33 found that ADEMMOGantibody seropositive children became seronegative inthe recovery period whereas children with relapsingMS typically remained seropositive More recentlyMOG antibodies have been found in children withON and in some patients with NMO phenotypenegative for NMO IgG1431 In this report we havestrengthened this association with ON and found thatpatients with bilateral rather than unilateral ON weremore likely to be MOG antibodyndashpositive BilateralON is a common ON phenotype in children but israre in adults in whom unilateral ON is the moretypical demyelinating event of MS In contrast
patients with brainstem signs and CIS other thanON and TM were rarely MOG antibodyndashpositiveAn elevated ESR was more common in MOGantibodyndashpositive patients An elevated ESR isatypical of MS and should raise suspicion of anautoimmune disorder or alternate inflammatorymimic of MS We showed that elevation of ESR inMOG antibodyndashpositive patients was independent ofage Intrathecal oligoclonal bands are one of the classicbiomarkers of MS and were absent in all MOGantibodyndashpositive patients and were present only inthe MOG antibodyndashnegative patients Only 17 ofthe MOG antibodyndashpositive patients had 1 or bothHLA-DRB11501 alleles the main MS susceptibilityallele which is the same as the previously reportedpercentage in the ldquonormalrdquo healthy population inAustralia34 whereas the MOG antibodyndashnegativepatients were more likely to have 1 or more HLA-DRB11501 allele This negative association wasnot statistically significant and larger cohorts arerequired to test whether this correlation is a realrather than a suspected phenomenon In contrastradiologic features did not appear to differentiateMOG antibodyndashpositive from ndashnegative patients inthis childhood cohort
The presence of MOG antibodies is unlikely tohave therapeutic implications in the first demyelinat-ing event but is more likely to be therapeuticallyimportant in the relapsing patient Although 2 recentpatients fulfilled criteria for MS the presence ofMOG antibodies and elevated ESR led us to treatthem more like NMO patients with the immune sup-pression agent MMF rather than conventional MSdisease-modifying therapies Both patients had noclinical or radiologic relapse for 12 months and 36months respectively and both became MOG anti-body seronegative Further studies are required tounderstand the longitudinal course of patients withMOG antibodyndashassociated relapsing disease and thetreatment approach required
Cell-based assays have been essential to detectMOG antibody with a high specificity9 especiallywhen the full-length MOG is expressed in cells ratherthan the C-terminal truncated MOG35 Our experi-ence with neuronal antigens informed us that FACSis sensitive and quantitative10132022333637 We detectedCSF MOG antibody in only 3 of 22 DEM samples ofwhich 5 were positive in serum This suggests CSF isless sensitive than serum for MOG antibody detectionwhich could be due to a lack of sensitivity or couldrepresent a genuine absence of intrathecal Ig synthesisin these patients Supporting this concept none of thepatients with positive MOG antibody had intrathecaloligoclonal bands
The pathogenic function of MOG antibody is stillunclear but over the recent years the detection of
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316
and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538
Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40
Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders
AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript
RCD and FB Acquisition analysis and interpretation of results RCD
EMT VM R-YAK NS SR KP LAW DRB K Prelog
DRC GJG CKL EKM FB Statistical analysis RCD EMT
FB Critical revisions of manuscript RCD EMT VM NS SR
KP DRB LAW K Prelog DRC GJG CKL EKM FB
ACKNOWLEDGMENTThe authors thank all the patients and family members who provided
samples for the study The authors thank Dr Maggie Wang for use of
the Flow Cytometry Core Facility of the Westmead Millennium Institute
(Australia) The authors thank Dr Hong Yu for use of the Imaging Core
Facility at the Westmead Research Hub
STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple
Sclerosis Research Foundation Multiple Sclerosis Research Australia
MS Angels Melbourne (Australia) and the Petre Foundation (Australia)
DISCLOSURERC Dale has received research funding from the Star Scientific Foundation
the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis
Research Australia NHMRC and the Petre Foundation has received hon-
oraria from Biogen Idec and is on advisory boards for Queensland Child-
renrsquos Medical Institute Research and MSARD EM Tantsis has received
a scholarship from the Australian Postgraduate Award program V Merheb
and R-YA Kumaran report no disclosures N Sinmaz has received a schol-
arship from the Australian Postgraduate Award program K Pathmanandavel
has received a scholarship from the Petre Foundation (Australia)
S Ramanathan has received a scholarship from the National Health and
Medical Research Council (Australia) DR Booth has received funding from
the National Health and Medical Research Council (Australia) the Australian
Research Council Multiple Sclerosis Research Australia and the Leukemia
Foundation has received honoraria and funding from Biogen Idec Merck
Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS
ONE LA Wienholt K Prelog and DR Clark report no disclosures
GJ Guillemin is the editor-in-chief for the International Journal for Trypto-
phan Research and has received research support from the Australian Research
Council CK Lim has received research funding from Multiple Sclerosis
Research Australia and Trish MS Research Foundation EK Mathey has
received funding from the National Health and Medical Research Council
(Australia) and the Rebecca L Cooper Medical Research Foundation
(Australia) F Brilot has received research funding from the Star Scientific
Foundation the Trish Multiple Sclerosis Research Foundation Multiple
Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre
Foundation and is an associate editor for the Journal of Visualized Experiments
Go to Neurologyorgnn for full disclosures
Received February 25 2014 Accepted in final form April 16 2014
REFERENCES1 Papadopoulos MC Verkman AS Aquaporin 4 and neu-
romyelitis optica Lancet Neurol 201211535ndash544
2 Vincent A Bien CG Irani SR Waters P Autoantibodies
associated with diseases of the CNS new developments
and future challenges Lancet Neurol 201110759ndash772
3 Wingerchuk DM Lennon VA Lucchinetti CF
Pittock SJ Weinshenker BG The spectrum of neuromy-
elitis optica Lancet Neurol 20076805ndash815
4 Johns TG Bernard CC The structure and function of
myelin oligodendrocyte glycoprotein J Neurochem
1999721ndash9
5 Linington C Bradl M Lassmann H Brunner C Vass K
Augmentation of demyelination in rat acute allergic
encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelinoligodendrocyte glycopro-
tein Am J Pathol 1988130443ndash454
6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous
relapsing-remitting EAE in the SJLJ mouse MOG-reactive
transgenic T cells recruit endogenous MOG-specific B cells
J Exp Med 20092061303ndash1316
7 Schluesener HJ Sobel RA Linington C Weiner HL A
monoclonal antibody against a myelin oligodendrocyte
glycoprotein induces relapses and demyelination in central
nervous system autoimmune disease J Immunol 1987
1394016ndash4021
8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB
Lee JI Genain CP Molecular characterization of antibody
specificities against myelinoligodendrocyte glycoprotein
in autoimmune demyelination Proc Natl Acad Sci U S A
2002998207ndash8212
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316
and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538
Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40
Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders
AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript
RCD and FB Acquisition analysis and interpretation of results RCD
EMT VM R-YAK NS SR KP LAW DRB K Prelog
DRC GJG CKL EKM FB Statistical analysis RCD EMT
FB Critical revisions of manuscript RCD EMT VM NS SR
KP DRB LAW K Prelog DRC GJG CKL EKM FB
ACKNOWLEDGMENTThe authors thank all the patients and family members who provided
samples for the study The authors thank Dr Maggie Wang for use of
the Flow Cytometry Core Facility of the Westmead Millennium Institute
(Australia) The authors thank Dr Hong Yu for use of the Imaging Core
Facility at the Westmead Research Hub
STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple
Sclerosis Research Foundation Multiple Sclerosis Research Australia
MS Angels Melbourne (Australia) and the Petre Foundation (Australia)
DISCLOSURERC Dale has received research funding from the Star Scientific Foundation
the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis
Research Australia NHMRC and the Petre Foundation has received hon-
oraria from Biogen Idec and is on advisory boards for Queensland Child-
renrsquos Medical Institute Research and MSARD EM Tantsis has received
a scholarship from the Australian Postgraduate Award program V Merheb
and R-YA Kumaran report no disclosures N Sinmaz has received a schol-
arship from the Australian Postgraduate Award program K Pathmanandavel
has received a scholarship from the Petre Foundation (Australia)
S Ramanathan has received a scholarship from the National Health and
Medical Research Council (Australia) DR Booth has received funding from
the National Health and Medical Research Council (Australia) the Australian
Research Council Multiple Sclerosis Research Australia and the Leukemia
Foundation has received honoraria and funding from Biogen Idec Merck
Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS
ONE LA Wienholt K Prelog and DR Clark report no disclosures
GJ Guillemin is the editor-in-chief for the International Journal for Trypto-
phan Research and has received research support from the Australian Research
Council CK Lim has received research funding from Multiple Sclerosis
Research Australia and Trish MS Research Foundation EK Mathey has
received funding from the National Health and Medical Research Council
(Australia) and the Rebecca L Cooper Medical Research Foundation
(Australia) F Brilot has received research funding from the Star Scientific
Foundation the Trish Multiple Sclerosis Research Foundation Multiple
Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre
Foundation and is an associate editor for the Journal of Visualized Experiments
Go to Neurologyorgnn for full disclosures
Received February 25 2014 Accepted in final form April 16 2014
REFERENCES1 Papadopoulos MC Verkman AS Aquaporin 4 and neu-
romyelitis optica Lancet Neurol 201211535ndash544
2 Vincent A Bien CG Irani SR Waters P Autoantibodies
associated with diseases of the CNS new developments
and future challenges Lancet Neurol 201110759ndash772
3 Wingerchuk DM Lennon VA Lucchinetti CF
Pittock SJ Weinshenker BG The spectrum of neuromy-
elitis optica Lancet Neurol 20076805ndash815
4 Johns TG Bernard CC The structure and function of
myelin oligodendrocyte glycoprotein J Neurochem
1999721ndash9
5 Linington C Bradl M Lassmann H Brunner C Vass K
Augmentation of demyelination in rat acute allergic
encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelinoligodendrocyte glycopro-
tein Am J Pathol 1988130443ndash454
6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous
relapsing-remitting EAE in the SJLJ mouse MOG-reactive
transgenic T cells recruit endogenous MOG-specific B cells
J Exp Med 20092061303ndash1316
7 Schluesener HJ Sobel RA Linington C Weiner HL A
monoclonal antibody against a myelin oligodendrocyte
glycoprotein induces relapses and demyelination in central
nervous system autoimmune disease J Immunol 1987
1394016ndash4021
8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB
Lee JI Genain CP Molecular characterization of antibody
specificities against myelinoligodendrocyte glycoprotein
in autoimmune demyelination Proc Natl Acad Sci U S A
2002998207ndash8212
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
9 Reindl M Di Pauli F Rostasy K Berger T The spectrum
of MOG autoantibody-associated demyelinating diseases
Nat Rev Neurol 20139455ndash461
10 Brilot F Dale RC Selter RC et al Antibodies to native
myelin oligodendrocyte glycoprotein in children with
inflammatory demyelinating central nervous system dis-
ease Ann Neurol 200966833ndash842
11 Di Pauli F Mader S Rostasy K et al Temporal dynamics
of anti-MOG antibodies in CNS demyelinating diseases
Clin Immunol 2011138247ndash254
12 Lalive PH Hausler MG Maurey H et al Highly reactive
ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm
Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton
Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte
This information is current as of May 22 2014
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1
httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm