rna-binding protein altered expression and mislocalization in ms · in the present study,...

ARTICLE OPEN ACCESS

RNA-binding protein altered expression andmislocalization in MSKatsuhisa Masaki MD PhD Yoshifumi Sonobe PhD Ghanashyam Ghadge PhD Peter Pytel MD

Paula Lepine MSc Florian Pernin MSc Qiao-Ling Cui MD PhD Jack P Antel MD Stephanie Zandee PhD

Alexandre Prat MD PhD and Raymond P Roos MD

Neurol Neuroimmunol Neuroinflamm 20207e704 doi101212NXI0000000000000704

Correspondence

Dr Roos

rroosneurologybsduchicagoedu

AbstractObjectiveTo determine whether there are nuclear depletion and cellular mislocalization of RNA-bindingproteins (RBPs) transactivation response DNA-binding protein of 43 kDa (TDP-43) fused insarcoma (FUS) and polypyrimidine tractndashbinding protein (PTB) in MS as is the case inamyotrophic lateral sclerosis (ALS) and oligodendrocytes infected with Theiler murine en-cephalomyelitis virus (TMEV) we examined MS lesions and in vitro cultured primary humanbrainndashderived oligodendrocytes

MethodsNuclear depletion and mislocalization of TDP-43 FUS and PTB are thought to contribute tothe pathogenesis of ALS and TMEV demyelination The latter findings prompted us to in-vestigate these RBPs in the demyelinated lesions of MS and in in vitro cultured humanbrainndashderived oligodendrocytes under metabolic stress conditions

ResultsWe found (1) mislocalized TDP-43 in oligodendrocytes in active lesions in some patients withMS (2) decreased PTB1 expression in oligodendrocytes in mixed activeinactive de-myelinating lesions (3) decreased nuclear expression of PTB2 in neurons in cortical de-myelinating lesions and (4) nuclear depletion of TDP-43 in oligodendrocytes under metabolicstress induced by low glucoselow nutrient conditions compared with optimal cultureconditions

ConclusionTDP-43 has been found to have a key role in oligodendrocyte function and viability whereasPTB is important in neuronal differentiation suggesting that altered expression and mis-localization of these RBPs in MS lesions may contribute to the pathogenesis of demyelinationand neurodegeneration Our findings also identify nucleocytoplasmic transport as a target fortreatment

From the Department of Neurology (KM YS GG RPR) and Department of Pathology (PP) University of Chicago Medical Center IL Neuroimmunology Research Laboratory(PL SZ AP) Centre du Recherche du Centre Hospitalier de lrsquoUniversite deMontreal (CRCHUM) QC Canada andNeuroimmunology Unit (FP Q-LC JPA) Montreal NeurologicalInstitute McGill University QC Canada

Go to NeurologyorgNN for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

The pathologic mechanisms driving demyelination and neu-rodegeneration in MS remain poorly understood In thepresent study we investigated the expression and localizationof 3 RNA-binding proteins (RBPs)1 in MS and in culturedoligodendrocytes exposed to metabolic stress These RBPspredominantly reside in the nucleus but can shuttle into thecytoplasm

We previously found that RBP polypyrimidine tractndashbindingprotein (PTB) is mislocalized to the cytoplasm in Theilermurine encephalomyelitis virus (TMEV)-infected cellsmdashandhypothesized that this nuclear depletion plays a role inTMEV-induced disease pathogenesis23 Because amyo-trophic lateral sclerosis (ALS) and TMEV target similarneural cell types and because nuclear depletion and mis-localization of RBPs transactivation response DNA-bindingprotein of 43 kDa (TDP-43) and fused in sarcoma (FUS)have been implicated in ALS pathogenesis we next in-vestigated these RBPs in TMEV infections4 We subsequentlyfound that TDP-43 and FUS in addition to PTB were mis-localized in demyelinating lesions in TMEV-infected neuralcells including oligodendrocytes4

Because TMEV-induced demyelinating disease serves as anexperimental model of MS we next examined the expressionpattern and localization of these RBPs in MS We now reportnuclear depletion and mislocalization of TDP-43 and PTB inMS lesions and in vitro cultured oligodendrocytes Recentpublications stress the importance of TDP-43 for oligoden-drocyte survival and myelination5 and of PTB for neuronaldifferentiation67 suggesting a role for these RBPs in MSpathogenesis and the potential importance of nucleocyto-plasmic transport as a target for treatment

MethodsEthics statementThe study involved tissue from human subjects and was ap-proved by the University of Chicago Institutional Review Boardfor Clinical Research Informed written consent for an autopsyat the University of Chicago was obtained from an immediatemember of the deceasedrsquos family The autopsies on patients withMS8 from the Centre de Recherche du Centre Hospitalier delrsquoUniversite de Montreal had informed consent and were inaccordance with institutional guidelines and approval by thelocal Centre Hospitalier de lrsquoUniversite de Montreal ethicscommittee (HD04046 and BH07001)9 The use of tissue from

the Montreal Neurological Institute McGill University wasapproved by the McGill University Health Center ResearchEthics Board The human samples that were used are describedin the e-methods (linkslwwcomNXIA221)

Staging of demyelinating lesionsWe classified MS plaques into 3 stages10 based on the densityof macrophages (1) activemdashlesions densely and diffuselyinfiltrated with macrophages (2) mixed activeinactivemdashlesions with macrophages restricted to the periphery and (3)inactivemdashlesions with no increase in macrophage numberswithin the plaque We classified cortical plaques into 3 sub-types leukocortical (involving both white matter [WM] andcortex) intracortical and subpial (superficial cortical)

Semiquantitativeanalysis ofRBPmislocalizationTissue preparation and immunohistochemistryimmunofluorescence methods are described in the Supple-ment Sections from blocks of the cerebral cortex andWM in allMS cases were stained with 339-Diaminobenzidine or byfluorescence for RBPs that included TDP-43 PTB1 PTB2 andFUS A semiquantitative assessment of RBP nuclear depletionand mislocalization or decreased expression in demyelinatinglesions was performed by taking digital photographs witha complementary metal oxide semiconductor camera at a reso-lution of 1636 times 1088 pixels with a times20 (075NA) objective Atleast 3 different photographs of areas of 1 lesion that were morethan 1 mm apart from each other in x and y directions wererandomly taken for every demyelinating lesion At least 100neuronal or glial cells per each area were identified on the basisof cytologic features11 and scored based on the degree of mis-localization or decreased nuclear expression of RBPs comparedwith normal-appearing WM (NAWM) from the same casestained at the same timeminus no orminimal +mild (10ndash30 cells)++moderate (30ndash100 cells) +++ cases prominent (gt100 cells)(see table e-1 linkslwwcomNXIA220)

In vitro studiesIn vitro cultured primary oligodendrocytes using a previouslydescribed cell isolation procedure have been found to havea purity of gt90 and express mature oligodendrocyte genemarkers with few progenitor markers1213 The tissue is col-lected from surgical resections of nonmalignancy cases asso-ciated with epilepsy the tissue is derived from a site distantfrom visible pathology1213 Aliquots are routinely provided toa neuropathologist to exclude any distinct abnormalities Thetissue is mainly subcortical WM but does contain fragmentsof gray matter

GlossaryALS = amyotrophic lateral sclerosis FUS = fused in sarcoma LG = low glucose LPS = lipopolysaccharidemRNA =messengerRNA NAGM = normal-appearing gray matter NAWM = normal-appearing white matter PI = propidium iodide PPWM =periplaque white matter PTB = polypyrimidine tractndashbinding proteinRBP = RNA-binding protein TDP-43 = transactivationresponse DNA-binding protein of 43 kDa TMEV = Theiler murine encephalomyelitis virus TNFa = tumor necrosis factoralpha

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN

In the present study oligodendrocytes were isolated and cul-tured from samples of 4 surgical resections (3 adult cases 2males ages 57 and 38 years and 1 female age 51 years and 1pediatric case male age 7 years)1213 The isolation techniqueinvolved initial dissociation of tissue using trypsin digestionfollowed by Percoll gradient centrifugation to remove myelinThe total cell fraction was plated onto a noncoated flask thatwas kept overnight at 37degC to allow adhesion of the microgliafraction Floating cells were then recovered (gt90 were O4+)and plated into 12 well poly-L-lysine and extracellular matrixndashcoated chamber slides (30000 cells per well) in defined me-dium (referred to as N1) consisting of Dulbecco ModifiedEagle Medium-F12 (Sigma-Aldrich St Louis MO) supple-mented with N1 (Sigma-Aldrich) 001 bovine serum albu-min 1 penicillin-streptomycin B27 (Invitrogen BurlingtonON CA) platelet-derived growth factor with 2 A subunits (10ngmL) basic fibroblast growth factor (10 ngmL) and tri-iodothyronine (2 nM) (Sigma-Aldrich) After 4 days media inindividual chambers were replaced with fresh N1 or withDulbecco Modified Eagle Medium with 025 gL glucose (re-ferred to as low glucose [LG]) After an additional 2 or 6 daysof N1 or LG treatment cells were incubated with monoclonalO4 antibody and propidium iodide (PI) for 15 minutes at37degC Cells were fixed with 4 paraformaldehyde for 10minutes at room temperature and then stained with a second-ary antibody directed against O4 goat anti-mouse IgM con-jugated to AF-647 (SouthernBiotech Birmingham AL) Afterpermeabilization buffer with 01 Triton X-100 the cells werestained with polyclonal anti-TDP-43 antibody (ProteintechRosemont IL) for 1 hour at 37degC followed by goat anti-rabbitpolyclonal antibody conjugated to AF-488 andHoescht 33258(Invitrogen) for 30minutes Cells were then examined using anepifluorescent microscope (Zeiss Oberkochen Germany) todetermine the percent of O4 cells that were PI+ cells and ofcells that showed predominantly nuclear vs cytoplasmic dis-tribution of TDP-43 Data were derived by blinded observerscounting 75ndash100 cells per condition Data between LG andN1conditions were compared using a paired t test

Statistical analysisData were analyzed using GraphPad Prism version 70a andare expressed as mean plusmn standard error of the mean Signifi-cance was assessed using the Student t test and p values lessthan 005 were considered significant A 95 CI was calcu-lated for the difference in frequency of RBP mislocalization orlevel of RBP expression between a cell type in the de-myelinating lesion vs periplaque whitegray matter

Data availabilityAny data not published within the article will be shared byrequest from any qualified investigator in anonymized form

ResultsTDP-43 in ALSIn ALS TDP-43 is depleted from the nucleus in some motorneurons and localized in aggregates in the cytoplasm (figure 1A

arrows) whereas other neurons and glial cells have TDP-43 inits normal location in the nucleus (figure 1 A and B arrow-head) At times phosphorylated TDP-43 is present in the cy-toplasmic aggregates (figure 1C) In contrast FUS maintainedits normal nuclear localization in cells in the same region thathad cells with TDP-43 mislocalization (figure 1D) PTB1 wasnot detected in motor neurons (figure 1E) because it is knownto have a limited distribution in this cell type14 PTB2 waspresent in motor neurons but like FUS had a normal nuclearlocalization (figure 1F) In contrast to these findings in ALSa predominant nuclear localization of TDP-43 was present inneurons and oligodendrocytes in human control CNS tissuefrom a patient with myasthenia gravis (figure 1 G and H) Withimmunofluorescent staining we examined an additional case ofALS and another CNS control case from a patient with mus-cular dystrophy TDP-43 was normally expressed in the nucleusof some motor neurons in the ALS case whereas nuclear de-pletion of TDP-43 with skein-like inclusions was seen in thecytoplasm of other motor neurons (figure 1 IndashK) In thecontrol case TDP-43 was seen in nuclei of cortical neurons(figure 1L) and spinal cord motor neurons (figure 1M)

Altered localization and expression of RBPs inoligodendrocytes in WM plaquesTDP-43 was mislocalized to the cytoplasm in glial cells inactive demyelinating lesions from patients MS3 and 13 toa moderate degree (table e-1 linkslwwcomNXIA220)(MS3mdashfigure 2 AndashH) Double immunofluorescence dem-onstrated that this mislocalization was present in CNPase-positive oligodendrocytes to a significant extent (figure 2 Iand J) the nuclear depletion and cytoplasmic mislocalizationwere statistically significantly greater when compared witholigodendrocytes in the periplaque WM (PPWM) (95 CI3189ndash6161 p = 00003) (figure 2 I and J) Similar findingswere also present in all 3 active demyelinating plaques in thecase of MS13 (figure e-1 linkslwwcomNXIA219) Cellswith TDP-43 mislocalization had normal morphology and noevidence of cleaved caspase-3 staining suggesting that theseoligodendrocytes were not dying Although the oligoden-drocytes in active plaques in the CNS tissue from MS3 and13 exhibited TDP-43 mislocalization this was not the casewith the active plaques from a biopsy of a tumefactive MSlesion in MS4 and from another MS case with 3 active pla-ques No abnormalities were found with respect to the normalnuclear localization and expression of FUS in active plaques

In addition to our finding of mislocalization of TDP-43 insome active plaques there was decreased expression ofPTB1 in oligodendrocytes in mixed activeinactive de-myelinating lesions (MS 2 6 10ndash13 table e-1 linkslwwcomNXIA220) however cytoplasmic mislocalization ofPTB1 was not seen in these lesions In addition TDP-43 andFUS were present in the nucleus in mixed activeinactivedemyelinating lesions The decreased expression of PTB1ranged from mild to prominent (table e-1) Although PTB1had its expected nuclear staining in NAWM in the case ofMS10 (figure 3 B and E) there was markedly decreased

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 3

expression in the nuclei and cytoplasm of CNPase-positiveoligodendroglia in demyelinated and partly remyelinatedlesions (figure 3 C D F and G) this lesion had macro-phages present in the periphery as typical of active-inactiveplaques The decrease in expression was statistically signifi-cant compared with oligodendrocytes in the PPWM (95CI 2865ndash6485 p = 00007) (figure 3H) Cells with de-creased PTB1 expression had normal morphology and therewas no evidence of caspase-3 staining In the case of MS6PTB1 expression was also diminished in mixed activeinactive demyelinating lesion (figure 3 IndashK) again macro-phages were present in the periphery of this plaque Thedecrease in expression was statistically significant comparedwith oligodendrocytes in the PPWM (95 CI 2860ndash5540p = 00003) (figure 3K)

Alteration of RBPs in cortical plaquesLeukocortical mixed activeinactive demyelinating lesions frompatients MS2 8 10ndash12 had mild to moderate diminution ofPTB2 expression in neurons within the demyelinated areacompared with neurons in adjacent normal-appearing graymatter (NAGM) (MS2mdashfigure 4 AndashH MS10mdashfigure 4IndashL) (table e-1 linkslwwcomNXIA220) Although the ex-pression of PTB2 was decreased in the nucleus in these cellsthere was no evidence of cytoplasmic mislocalization or aggre-gate formation of PTB2 (figure 4 EndashG) The decrease in PTB2expression in cortical neurons in leukocortical plaques in thecase ofMS2 and 10 was statistically significant compared withcortical neurons in the periplaque gray matter (MS2 95 CI4222ndash7228 p lt 00001 MS10 95 CI 4070ndash6780 p lt00001) (figure 4 H and L) In contrast to these findings theexpression of TDP-43 and FUS in neurons in leukocortical

Figure 1 Expression pattern of RNA-binding proteins in ALS and a control patient and two patients with normal CNS

(AndashF) A case of sporadic ALS (A) TDP-43 is expressed normally in the nucleus of some spinal cord motor neurons (arrowhead) but depleted from the nucleus ofaffected motor neurons forming aggregates (large arrows) (B) TDP-43 is in its normal nuclear location in glial cells in spinal cord white matter (C) A cytoplasmicinclusion in spinal cordmotor neurons contains pTDP-43 As expected in normalmotor neurons (D) FUS is present in thenucleus (E) PTB1 is not present and (F) PTB2is present in the nucleus (G and H) A CNS control patient withmyasthenia gravis Expression of TDP-43 is mainly seen in the nuclei of cortical neurons (G) and whitematter oligodendrocytes (H) (IndashK) An additional case of sporadic ALS (I) Immunofluorescent staining for TDP-43 shows normal expression in the nucleus of 1 spinalcordmotor neuron (arrow) but mislocalization to the cytoplasm (arrowhead) in another neuron (J and K) Highmagnification view of the 2motor neurons shown inpanel I with normal nuclear expressionof TDP-43 in 1neuron (J) but nuclear depletionof TDP-43 alongwith a skein-like cytoplasmic inclusion in another neuron (K) (LandM) ACNS control patientwithmuscular dystrophy TDP-43 expression is seen in the nucleus of cortical neurons (L) and spinal cordmotor neurons (M) Scale bars50μm(AandB) 20μm(CndashG I) and10μm(H JndashM)ALS= amyotrophic lateral sclerosis FUS= fused in sarcomaPTB= polypyrimidine tractndashbindingprotein pTDP-43=phosphorylated TDP-43 TDP-43 = transactivation response DNA-binding protein of 43 kDa


plaques and of TDP-43 FUS and PTB2 in neurons of intra-cortical and subpial plaques had a normal expression in thenuclei and did not differ from that in NAGM (table e-1)

TDP-43 proteinopathy in in vitro primaryhuman oligodendrocytesWe cultured primary human oligodendrocytes in a LG me-dium to model metabolic stress conditions thought to occurin MS lesions1213 As shown in figure 5A there is only a low

level of cell death under N1 conditions at day 2 in cultureLevels modestly increase under LG conditions as previouslydescribed1213 By day 6 however there was a significantincrease in cell death under LG conditions The percent ofO4 cells showing nuclear depletion of TDP-43 was signifi-cantly increased in the LG-treated cultures compared withthe N1 counterparts (mean 46 for LG vs 17 for N1 p =0032 n = 4 figure 5B and illustrated in figure 5C) Nucleardepletion was observed in virtually all PI+ cells as shown

Figure 2 Altered expression of TDP-43 in oligodendrocytes of MS plaques

(AndashG) An active and demyelinating lesion fromMS3 (A) Hematoxylin and eosin staining shows perivascular inflammation withmononuclear cell infiltration (B and C)Immunostaining for proteolipid protein andCD68 identifies amarked loss ofmyelinwith dense infiltration ofmacrophages (D)Numerous CD68-positivemacrophagesareseen in theperivascularareaandparenchymaof the lesioncenter (E) Foamymacrophagesphagocytosingproteolipidprotein-positivemyelindebris (FndashH)Boundaryof active demyelinating lesionofMS3 (F) Thedashed line shows the approximateboundary between thedemyelinating lesion andPPWM (G)Highmagnification viewof the square in the demyelinating lesion shown in panel F TDP-43 is nuclear depleted andmislocalized to the cytoplasm in some of the glial cells in the demyelinatinglesion (H) Highmagnification viewof the square in PPWM TDP-43 is predominantly localized to the nuclei of glial cells in PPWM (I) Double immunofluorescence showsmislocalization of TDP-43 in CNPase-positive oligodendrocytes at the edge of a demyelinating plaque (arrowheads) (J) TDP-43 nuclear depletion and cytoplasmicmislocalization inCNPase-positiveoligodendrocytes TDP-43mislocalization inoligodendrocytes is significantly greater indemyelinating lesions thanPPWMEachdot forthisquantitationandsubsequentonesrepresentsaphotographfromaseparate regionof theplaqueScalebars1mm(AndashC) 50μm(DndashF I) and20μm(GandH)DAPI=496-diamidino-2-phenylindole PPWM = periplaque white matter TDP-43 = transactivation response DNA-binding protein of 43 kDa


with the PI+ cells in figure 5C Of note the percent of PIminus

cells with nuclear depletion was greater in LG cultures vs N1cultures (mean 44 for LG vs 14 for N1 p = 0014 n = 3data not shown)

DiscussionAbnormalities of expression and localization of RBPs havebeen described in a number of diseases including ALSHuntington disease and viral infections1 In the present study

we focused on TDP-43 FUS and PTB because these RBPshave an important impact on RNA biology and also becausetheir mislocalization is thought to influence the pathogenesisof ALS and TMEV infections

TDP-43 is a ubiquitously expressed RBP that predominantlyresides in the nucleus but shuttles across the nuclear mem-brane in association with messenger RNAs (mRNAs)15 Ahallmark of almost all cases of ALS is disruption of nucleo-cytoplasmic trafficking with resultant nuclear depletion

Figure 3 Reduced expression of PTB1 in oligodendrocytes of demyelinating lesions

(AndashH) Mixed activeinactive and demyelinating subcortical white matter lesion from MS10 (AndashD) Double immunostaining for PTB1 (brown) and MBP (pink)(A) Macroscopic view of demyelinating subcortical lesion Immunoreactivity for MBP is sharply demarcated in a subcortical area that has no detectable PTB1(B) Immunoreactivity for PTB1 is present in the nucleus of glial cells including oligodendrocytes in NAWM (C) Nuclear expression of PTB1 is diminished in anincompletely remyelinated area (D) Expression of PTB1 is markedly decreased in glial cell nuclei in the demyelinated lesion (EndashG) Double immunofluorescence forPTB1 and CNPase Although immunoreactivity for PTB1 is preserved in the nuclei of CNPase-positive oligodendrocytes inNAWM (E) nuclear PTB1 is decreased in theremyelinated area (F) and markedly depleted in oligodendrocytes in the lesion center (G) (H) Frequency of decreased PTB1 expression in CNPase-positive oligo-dendrocytes Decreased PTB1 in oligodendrocytes is significantly more frequent in demyelinating lesions than PPWM (IndashK) Mixed activeinactive and demyelinatingwhitematter lesion fromMS6 (I) Double immunofluorescence shows that nuclear expression of PTB1 ismarkedly diminished in the demyelinating lesion (J) Highermagnification of PTB1 expression in the demyelinating lesion andNAWM Although PTB1 is localized in nuclei of CNPase-positive oligodendrocytes of NAWM PTB1 isdecreased in CNPase-positive oligodendrocytes of the demyelinating lesion (K) Decreased PTB1 expression in oligodendrocytes in active-inactive demyelinatinglesions is significantlymore frequent than inPPWMScalebars 1mm(A) 100μm(I) 50μm(BndashD) 20μm(J) and10μm(EndashG)NAWM=normal-appearingwhitematterDAPI = 496-diamidino-2-phenylindole MBP = myelin basic protein PPWM = periplaque white matter PTB = polypyrimidine tractndashbinding protein


Figure 4 Reduced expression of PTB2 in neurons of cortical demyelinating lesions

(A and B) Serial sections of a mixed activeinactive demyelinating lesion (A) Hematoxylin and eosin stain shows hypocellularity and (B) immunos-taining for proteolipid protein shows sharply demarcated periventricular demyelination (C) High magnification view of graywhite matter (GMWM)interface shown in panel B Cortical demyelination is seen in the GM The dashed line shows the approximate boundary between normal-appearingGM and demyelinated GM (D) CD68-positive macrophages are restricted to the periphery of the lesion (arrowheads) (E) High magnification withimmunofluorescent staining of the region of the dashed line (at edge of cortical demyelination) shown in panel C Above the dashed line in the normal-appearing GM PTB2 has a normal nuclear expression in neurons In contrast the expression of PTB2 is markedly decreased in neurons in thedemyelinated GM below the dashed line (F and G) Higher-magnification view of the region shown in panel E (F) Normal-appearing (myelinated) GMwhich is above the dashed line shows normal expression of PTB2 in the nuclei of neurons In contrast (G) which is the demyelinated GM below thedashed line shows decreased PTB2 expression in nuclei of neurons (H) Frequency of decreased PTB2 expression in cortical neurons Decreased PTB2in cortical neurons is significantly more frequent in cortical demyelinating lesions than the PPGM (IndashL) Leukocortical mixed activeinactive de-myelinating lesion MS10 (I) Hematoxylin and eosin stain shows subcortical WM lesion (J) A region of the cortex above the dashed line and within thelesion is demyelinated (K) A higher magnification of the region within the rectangle in panel J includes the boundary of cortical demyelination PTB2expression is diminished in the demyelinated region of the cortex whereas PTB2 is preserved in the cortical neurons in the same layer of the PPGM (L)Frequency of decreased PTB2 expression in cortical neurons Decreased PTB2 in cortical neurons is significantly more frequent in cortical de-myelinating lesions than the PPGM Scale bars 5 mm (A and B) 1 mm (C D I and J) 100 μm (K) 50 μm (E) and 10 μm (F and G) DAPI = 496-diamidino-2-phenylindole GM = gray matter PLP = proteolipid protein PPGM = periplaque gray matter PTB = polypyrimidine tractndashbinding protein WM = whitematter


cytoplasmic mislocalization aggregation cleavage and phos-phorylation of TDP-43 in neural cells16ndash18 The decreasedexpression and mislocalization or TDP-43 are thought tocause abnormalities of splicing and RNA metabolism and addto nucleocytoplasmic transport disruption thereby contrib-uting to ALS pathogenesis19ndash22 It is likely that cytoplasmicmislocalization of other RBPs in addition to TDP-43 adds tothe cellular dysfunction in ALS23

In the present study we demonstrate a number of abnormal-ities in expression and localization of RBPs inMS lesions and inin vitro cultured oligodendrocytes We found that TDP-43 wasmislocalized in oligodendrocytes in demyelinated lesions inMS as was the case in TMEV infections Of note TDP-43 isknown to bind to 100s of mRNAs including mRNAs encodingproteolipid protein myelin basic protein myelin oligoden-drocyte glycoprotein and myelin-associated glycoprotein andto play a key role in RNA metabolism and splicing21 Impor-tantly and relevant to our findings is a recent report that anexperimental decrease in expression of TDP-43 in mature oli-godendrocytes in mice leads to demyelination and RIPK1-mediated necroptosis of oligodendrocytes5 of note nec-roptosis has been reported to occur in MS and experimentalmodels of MS24 In the case of ALS TDP-43 nuclear depletionand mislocalization are associated with posttranslational mod-ifications of this protein and rarely are a result of mutation ofthis gene however the study of Wang et al5 indicates thatTDP-43 knockdown alone can lead to a reduction in myelin

gene expression and is indispensable for oligodendrocyte sur-vival and myelination These findings of Wang et al5 suggestthat nuclear depletion and mislocalization of TDP-43 in MSlesions would similarly lead or contribute to demyelination andin some cases death of oligodendrocytes

We found a decrease in PTB1 in oligodendrocytes in mixedactiveinactive demyelinating lesions and a decrease in PTB2 inneurons in cortical plaques PTB1 and PTB2 are paralogousRBPs that are encoded by related genes6 PTB1 is not expressedin mature neurons and muscle whereas PTB2 is expressed inthese cells and others These RBPs function in regulating al-ternative splicing and also play a role in translation mRNAstability and polyadenylation The control of splicing is espe-cially important in the CNS because of the myriad of mRNAisoforms that have key roles in development and functionSplicing in oligodendrocytes and neurons in MS demyelinatedregions is likely affected by the nuclear depletion and cyto-plasmicmislocalization of PTB Importantly PTB is involved inthe differentiation of neural precursor cells67 In this way PTB2nuclear depletion and cytoplasmicmislocalization in neurons ofcortical plaques may contribute to neurodegeneration and thecognitive decline associated with MS

In summary we found that there is disruption of TDP-43 andPTB expression and localization that varies in different neuralcell types in MS plaques It is not unlikely that other RBPs aredepleted in the nucleus and mislocalized to the cytoplasm in

Figure 5 Nuclear depletion of TDP-43 in human primary oligodendrocytes cultured in vitro under LG conditions

Data were derived from 3 adult cases and 1 pe-diatric casewith no history ofMS (A) Cell death ofhuman oligodendrocytes was assessed using PIstaining at 2 and 6 days under optimal (N1) andmetabolic stress (LG) culture conditions (B) De-pletion of nuclear expression of TDP-43 in cul-tures of oligodendrocytes after 2 days oftreatment with LG compared with N1 conditionSolid lines (adult cases) and dashed line (pediat-ric case) connect cultures of oligodendrocytesobtained from the same biopsy tissue (C) Rep-resentative images showing immunostained oli-godendrocytes 2 days under N1 and LGconditions From left side DAPI (blue) O4 (gray)TDP-43 (green) and TDP-43 merged with PI(green and red respectively) Arrows show cellswith nuclear expression of TDP-43 arrowheadsshow TDP-43 nuclear-depleted PIminus cells starsshow TDP-43 nuclear-depleted PI+ cells Scalebar = 10 μm p lt 005 p lt 001 DAPI = 496-diamidino-2-phenylindole LG = low glucose PI =propidium iodide TDP-43 = transactivation re-sponse DNA-binding protein of 43 kDa


MS however the important known activities of TDP-43 andPTB suggest that the abnormalities we identified in these 2RBPs will have significant effects This variation may haveresulted from differences in the protein composition of thenuclear pore complex in different cell types25 Furthermoredifferent subtypes of MS lesions may manifest continuingchanges of RBP abnormalities over time because of the dynamicnature of demyelinating lesions and the varying inflammatorymilieu It may be that this changeable and very dynamic natureof MS lesions may have been the reason that active plaquesfrom MS14 had a normal localization of TDP-43 Also ofimportance is the fact that MS is a heterogeneous diseasemdashandtherefore it is not surprising that forms of MS that are differentfrom classical and typical cases may not share the same RBPabnormalities seen inmore prototypic cases ofMS Perhaps thiswas the reason that an active plaque from a biopsy froma tumefactive lesion of MS4 (in a patient who had only 1additional clinical problem over decades of observation) hada normal localization of TDP-43 (table e-1 linkslwwcomNXIA220)

The nucleocytoplasmic transport abnormalities in MS that weidentifiedmay have resulted from a number of possible causesProbably most relevant are reports that inflammation can leadto mislocalization of proteins in neural cells Correia et al26

found that mislocalization of TDP-43 occurred in culturedmicroglia and astrocytes following exposure to lipopolysac-charide (LPS) motor neuronndashlike NSC-34 cells after treat-ment with tumor necrosis factor alpha (TNFa) and motorneurons of mutant TDP-43 transgenic mice following LPSintraperitoneal injections Kim et al27 reported that treatingneuronal cultures with glutamate and TNFa led to mis-localization of HDAC1 with resultant axonal damage Thelatter investigators also detected abnormal cytoplasmic lo-calization of HDAC1 in damaged axons in patients with MSand in mice with cuprizone-induced demyelination Salapaet al28 found that interferon-γ led to cytoplasmic mis-localization of heterogeneous nuclear ribonucleoprotein(hnRNP) A1 an RBP These investigators also reported thatneurons in a region of an MS brain (in which no pathologywas described) had nuclear depletion and cytoplasmicmislocalization of hnRNP A1 which was aggregated instress granules A more recent publication by Salapa et al29

found mislocalization of hnRNP A1 and TDP-43 in spinalcord neurons in experimental allergic encephalomyelitis thehnRNP A1 mislocalization correlated with the clinical scoreand presence of infiltrates of CD3+ cells secreting interferon-γ

Our in vitro culture conditions were selected to model met-abolic stress conditions that are thought to occur in MSlesions1213 Cui et al previously showed that these conditionswere associated with an initial withdrawal of cell processesmodeling the dying-back of oligodendrocyte processes ob-served inMS lesions (and cuprizone-induced demyelination)30

and TMEV-induced demyelination31 These changes werereversible if culture conditions were restored within a sub-sequent 48 hours If continued past this time however

significant cell death occurs by 6 days as shown in figure 5Awith activation of an autophagy response In the current studywe found TDP-43 nuclear depletion was increased under LGconditions after 2 days in culture a time when cell death levelsas detected by PI staining were low Importantly we specifi-cally observed nuclear depletion in cells that were still PInegative in addition to PI+ cells that also showed nucleardepletion of TDP-43 One of the oligodendrocyte cultureswas obtained from a child Although oligodendrocyte me-tabolism varies depending on the age of the individual theresults from the pediatric case importantly parallel those ofthe 3 adults

The in vitro oligodendrocyte results are consistent with in situdata showing nuclear depletion of TDP-43 in oligoden-drocytes with intact oligodendrocyte cell bodies Further-more TNFa has been found to lead to dying-back of culturedoligodendrocytes although in this case it was observed innewborn ratndashderived oligodendrocytes12 The combined invitro and in situ results suggest that the TDP-43 nuclear de-pletion reflects a cellular stress response that could be medi-ated both by metabolic conditions and inflammatorymediators of MS lesions Of note no difference in TDP-43transcripts was found in a microarray data set derived fromoligodendrocytes under N1 vs LG conditions for 2 days12

suggesting that any change in TDP-43 protein levels is a resultof translational regulation perhaps from stress such as fromLG32 or inflammatory factors triggering the integrated stressresponse Activation of the integrated stress response hasbeen previously implicated in the pathogenesis of MS33

Our results suggest that correcting the expression and local-ization of RBPs in MS may ameliorate disease In additionthis direction may lead to normal localization of key tran-scription factors and proteins that are required for efficientmyelination and remyelination in oligodendrocytes and oli-godendrocyte precursor cells34ndash37 Importantly nuclear ex-port inhibitors have been found to attenuate myelinoligodendrocyte glycoproteinndashinduced experimental auto-immune encephalomyelitis (and kainic acidndashinduced axonaldamage) by limiting areas of myelin damage preserving my-elinated and unmyelinated axon integrity and decreasing in-flammation38 Nuclear export inhibitors have also been foundto attenuate disease and to be neuroprotective in experimentalmodels of ALS39 including a mutant TDP-43mouse model19

and Huntington disease40 (which like ALS has abnormalitiesof nucleocytoplasmic transport) Furthermore nucleocyto-plasmic transport is being targeted in patients with cancer inaddition to neurologic diseasesmdashand a clinical trial witha nuclear export inhibitor is in progress in ALS

Altered nucleocytoplasmic transport leading to abnormalexpression and mislocalization of RBPs and other macro-molecules may not only contribute to the demyelination andneurodegeneration inMS but also underlie a number of otherdisease states both infectious and noninfectious The avail-ability of drugs that target nucleocytoplasmic transport may


provide new and novel treatment possibilities for thesedisorders

AcknowledgmentThe authors thank the UCLA Human Brain and Spinal FluidResource Center and the Rocky Mountain MS Center TissueBank (supported in part by a grant from the National MultipleSclerosis Society) for specimens from autopsied MS cases

Study fundingNIH National Institute of NINDS (R21NS096569) (RPR)Amyotrophic Lateral Sclerosis Association (RPR) Pro-gressive MS Alliance (BRAVEin MS) (JPA) LohengrinFoundation Steps4 Doug John and Patricia McDonald andBarbara and Marc Posner

DisclosureKMasaki Y Sonobe G Ghadge P Pytel P Lepine F PerninQ-L Cui JP Antel S Zandee A Prat and RP Roos reportno disclosures Go to NeurologyorgNN for full disclosures

Publication historyThis manuscript was previously posted on bioRxiv doi101101829457 Received October 28 2019 Accepted in finalform February 7 2020

References

1 Nussbacher JK Batra R Lagier-Tourenne C Yeo GW RNA-binding proteins inneurodegeneration seq and you shall receive Trends Neurosci 201538226ndash236doi 2101016jtins201510021003

2 Michiels T Roos RP Theilerrsquos virus central nervous system infection In Ehrenfeld EDomingo E Roos RP editors The Picornaviruses Washington DC ASM Press2010411ndash430

3 Pilipenko EV Viktorova EG Guest ST et al Cell-specific proteins regulate viral RNAtranslation and virus-induced disease EMBO J 2001206899ndash6908 doi 68101093emboj682068236899

4 Masaki K Sonobe Y Ghadge G et al TDP-43 proteinopathy in Theilerrsquos murineencephalomyelitis virus infection PLoS Pathog 201915e1007574

5 Wang J Ho WY Lim K et al Cell-autonomous requirement of TDP-43 an ALSFTD signature protein for oligodendrocyte survival and myelination Proc Natl AcadSci USA 2018115E10941ndashE10950 doi 1091011073pnas1809821115

6 Hu J Qian H Xue Y Fu XD PTBnPTB master regulators of neuronal fate inmammals Biophys Rep 20184204ndash214

7 Linares AJ Lin CH Damianov A et al The splicing regulator PTBP1 controls theactivity of the transcription factor Pbx1 during neuronal differentiation Elife 20154e09268

8 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 revisions to the McDonald criteria Ann Neurol 201169292ndash302

9 Dhaeze T Tremblay L Lachance C et al CD70 defines a subset of proinflammatoryand CNS-pathogenic TH1TH17 lymphocytes and is overexpressed in multiplesclerosis Cell Mol Immunol 201916652ndash665

10 Kuhlmann T Ludwin S Prat A Antel J Bruck W Lassmann H An updated histologicalclassification system for multiple sclerosis lesions Acta Neuropathol 201713313ndash24

11 Garcia-Cabezas MA John YJ Barbas H Zikopoulos B Distinction of neurons gliaand endothelial cells in the cerebral cortex an algorithm based on cytological featuresFront Neuroanat 201610107

12 Cui QL Khan D Rone M et al Sublethal oligodendrocyte injury a reversible con-dition in multiple sclerosis Ann Neurol 201781811ndash824

13 Rone MB Cui QL Fang J et al Oligodendrogliopathy in multiple sclerosis lowglycolytic metabolic rate promotes oligodendrocyte survival J Neurosci 2016364698ndash4707

14 Boutz PL Stoilov P Li Q et al A post-transcriptional regulatory switch in poly-pyrimidine tract-binding proteins reprograms alternative splicing in developingneurons Genes Dev 2007211636ndash1652 doi 16101101gad1558107

15 Buratti E Brindisi A Giombi M et al TDP-43 binds heterogeneous nuclear ribo-nucleoprotein AB through its C-terminal tail an important region for the inhibitionof cystic fibrosis transmembrane conductance regulator exon 9 splicing J Biol Chem200528037572ndash37584

16 NeumannM SampathuDMKwong LK et al Ubiquitinated TDP-43 in frontotemporallobar degeneration and amyotrophic lateral sclerosis Science 2006314130ndash133

17 Arai T Hasegawa M Akiyama H et al TDP-43 is a component of ubiquitin-positivetau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateralsclerosis Biochem Biophys Res Commun 2006351602ndash611

18 Igaz LM Kwong LK Xu Y et al Enrichment of C-terminal fragments in TAR DNA-binding protein-43 cytoplasmic inclusions in brain but not in spinal cord of fronto-temporal lobar degeneration and amyotrophic lateral sclerosis Am J Pathol 2008173182ndash194

19 Chou CC Zhang Y Umoh ME et al TDP-43 pathology disrupts nuclear porecomplexes and nucleocytoplasmic transport in ALSFTD Nat Neurosci 201821228ndash239

20 Lagier-Tourenne C Polymenidou M Cleveland DW TDP-43 and FUSTLSemerging roles in RNA processing and neurodegeneration HumMol Genet 201019R46ndashR64

21 Polymenidou M Lagier-Tourenne C Hutt KR et al Long pre-mRNA depletion andRNA missplicing contribute to neuronal vulnerability from loss of TDP-43 NatNeurosci 201114459ndash468

22 Purice MD Taylor JP Linking hnRNP function to ALS and FTD pathology FrontNeurosci 201812326

23 Conlon EG Fagegaltier D Agius P et al Unexpected similarities between C9ORF72and sporadic forms of ALSFTD suggest a common disease mechanism Elife 2018737754 doi 3771037554eLife37754

24 Ofengeim D Ito Y Najafov A et al Activation of necroptosis in multiple sclerosisCell Rep 2015101836ndash1849 doi 18101016jcelrep201518021051

25 Raices M DrsquoAngelo MA Nuclear pore complex composition a new regulator oftissue-specific and developmental functions Nat RevMol Cell Biol 201213687ndash699doi 6101038nrm3461

26 Correia AS Patel P Dutta K Julien JP Inflammation induces TDP-43 mislocalizationand aggregation PLoS One 201510e0140248 doi 01402100141371journalpone0140248

27 Kim JY Shen S Dietz K et al HDAC1 nuclear export induced by pathologicalconditions is essential for the onset of axonal damage Nat Neurosci 201013180ndash189

28 Salapa HE Johnson C Hutchinson C et al Dysfunctional RNA binding proteins andstress granules in multiple sclerosis J Neuroimmunol 2018324149ndash156 doi 101016jjneuroim201810081015

29 Salapa HE Libner CD Levin MC Dysfunctional RNA-binding protein biology andneurodegeneration in experimental allergic encephalomyelitis J Neurosci Res 201998704ndash717 doi 101002jnr24554

Appendix Authors

Name Location Contribution

KatsuhisaMasaki MDPhD

University of ChicagoMedical Center

Conception and design ofthe study and drafting ofthe manuscript

YoshifumiSonobe PhD


Conception and design ofthe study

GhanashyamGhadge PhD



Peter PytelMD


Acquisition of samplesand analysis of the data

Paula LepineMSc

Centre du Recherche duCentre Hospitalier delrsquoUniversite de Montreal


FlorianPernin MSc

McGill University Acquisition of samplesand analysis of the dataand drafting of themanuscript

Qiao-Ling CuiMD PhD

McGill University Acquisition of samplesand analysis of the data

Jack P AntelMD


StephanieZandee PhD



AlexandrePrat MD PhD



Raymond PRoos MD


Conception and design ofthe study and drafting themanuscript


30 Ludwin SK Johnson ES Evidence for a ldquodying-backrdquo gliopathy in demyelinatingdisease Ann Neurol 19819301ndash305

31 Rodriguez M Virus-induced demyelination in mice ldquodying backrdquo of oligoden-drocytes Mayo Clin Proc 198560433ndash438

32 Yang RWek SAWek RC Glucose limitation induces GCN4 translation by activationof Gcn2 protein kinase Mol Cell Biol 2000202706ndash2717

33 Way SW Popko B Harnessing the integrated stress response for the treatment ofmultiple sclerosis Lancet Neurol 201615434ndash443

34 Dai J Bercury KK Jin W Macklin WB Olig1 acetylation and nuclear export mediateoligodendrocyte development J Neurosci 20153515875ndash15893

35 Gottle P Kury P Intracellular protein shuttling a mechanism relevant for myelinrepair in multiple sclerosis Int J Mol Sci 20151615057ndash15085

36 Gottle P Sabo JK Heinen A et al Oligodendroglial maturation is dependent onintracellular protein shuttling J Neurosci 201535906ndash919

37 Nakahara J Kanekura K Nawa M et al Abnormal expression of TIP30 and arrestednucleocytoplasmic transport within oligodendrocyte precursor cells in multiplesclerosis J Clin Invest 2009119169ndash181

38 Haines JD Herbin O de la Hera B et al Nuclear export inhibitors avert pro-gression in preclinical models of inflammatory demyelination Nat Neurosci 201518511ndash520

39 Zhang K Donnelly CJ Haeusler AR et al The C9orf72 repeat expansion disruptsnucleocytoplasmic transport Nature 201552556ndash61

40 Grima JC Daigle JG Arbez N et al Mutant huntingtin disrupts the nuclear porecomplex Neuron 20179493ndash107e6


DOI 101212NXI000000000000070420207 Neurol Neuroimmunol Neuroinflamm

Katsuhisa Masaki Yoshifumi Sonobe Ghanashyam Ghadge et al RNA-binding protein altered expression and mislocalization in MS

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

The pathologic mechanisms driving demyelination and neu-rodegeneration in MS remain poorly understood In thepresent study we investigated the expression and localizationof 3 RNA-binding proteins (RBPs)1 in MS and in culturedoligodendrocytes exposed to metabolic stress These RBPspredominantly reside in the nucleus but can shuttle into thecytoplasm

We previously found that RBP polypyrimidine tractndashbindingprotein (PTB) is mislocalized to the cytoplasm in Theilermurine encephalomyelitis virus (TMEV)-infected cellsmdashandhypothesized that this nuclear depletion plays a role inTMEV-induced disease pathogenesis23 Because amyo-trophic lateral sclerosis (ALS) and TMEV target similarneural cell types and because nuclear depletion and mis-localization of RBPs transactivation response DNA-bindingprotein of 43 kDa (TDP-43) and fused in sarcoma (FUS)have been implicated in ALS pathogenesis we next in-vestigated these RBPs in TMEV infections4 We subsequentlyfound that TDP-43 and FUS in addition to PTB were mis-localized in demyelinating lesions in TMEV-infected neuralcells including oligodendrocytes4

Because TMEV-induced demyelinating disease serves as anexperimental model of MS we next examined the expressionpattern and localization of these RBPs in MS We now reportnuclear depletion and mislocalization of TDP-43 and PTB inMS lesions and in vitro cultured oligodendrocytes Recentpublications stress the importance of TDP-43 for oligoden-drocyte survival and myelination5 and of PTB for neuronaldifferentiation67 suggesting a role for these RBPs in MSpathogenesis and the potential importance of nucleocyto-plasmic transport as a target for treatment

MethodsEthics statementThe study involved tissue from human subjects and was ap-proved by the University of Chicago Institutional Review Boardfor Clinical Research Informed written consent for an autopsyat the University of Chicago was obtained from an immediatemember of the deceasedrsquos family The autopsies on patients withMS8 from the Centre de Recherche du Centre Hospitalier delrsquoUniversite de Montreal had informed consent and were inaccordance with institutional guidelines and approval by thelocal Centre Hospitalier de lrsquoUniversite de Montreal ethicscommittee (HD04046 and BH07001)9 The use of tissue from

the Montreal Neurological Institute McGill University wasapproved by the McGill University Health Center ResearchEthics Board The human samples that were used are describedin the e-methods (linkslwwcomNXIA221)

Staging of demyelinating lesionsWe classified MS plaques into 3 stages10 based on the densityof macrophages (1) activemdashlesions densely and diffuselyinfiltrated with macrophages (2) mixed activeinactivemdashlesions with macrophages restricted to the periphery and (3)inactivemdashlesions with no increase in macrophage numberswithin the plaque We classified cortical plaques into 3 sub-types leukocortical (involving both white matter [WM] andcortex) intracortical and subpial (superficial cortical)

Semiquantitativeanalysis ofRBPmislocalizationTissue preparation and immunohistochemistryimmunofluorescence methods are described in the Supple-ment Sections from blocks of the cerebral cortex andWM in allMS cases were stained with 339-Diaminobenzidine or byfluorescence for RBPs that included TDP-43 PTB1 PTB2 andFUS A semiquantitative assessment of RBP nuclear depletionand mislocalization or decreased expression in demyelinatinglesions was performed by taking digital photographs witha complementary metal oxide semiconductor camera at a reso-lution of 1636 times 1088 pixels with a times20 (075NA) objective Atleast 3 different photographs of areas of 1 lesion that were morethan 1 mm apart from each other in x and y directions wererandomly taken for every demyelinating lesion At least 100neuronal or glial cells per each area were identified on the basisof cytologic features11 and scored based on the degree of mis-localization or decreased nuclear expression of RBPs comparedwith normal-appearing WM (NAWM) from the same casestained at the same timeminus no orminimal +mild (10ndash30 cells)++moderate (30ndash100 cells) +++ cases prominent (gt100 cells)(see table e-1 linkslwwcomNXIA220)

In vitro studiesIn vitro cultured primary oligodendrocytes using a previouslydescribed cell isolation procedure have been found to havea purity of gt90 and express mature oligodendrocyte genemarkers with few progenitor markers1213 The tissue is col-lected from surgical resections of nonmalignancy cases asso-ciated with epilepsy the tissue is derived from a site distantfrom visible pathology1213 Aliquots are routinely provided toa neuropathologist to exclude any distinct abnormalities Thetissue is mainly subcortical WM but does contain fragmentsof gray matter

GlossaryALS = amyotrophic lateral sclerosis FUS = fused in sarcoma LG = low glucose LPS = lipopolysaccharidemRNA =messengerRNA NAGM = normal-appearing gray matter NAWM = normal-appearing white matter PI = propidium iodide PPWM =periplaque white matter PTB = polypyrimidine tractndashbinding proteinRBP = RNA-binding protein TDP-43 = transactivationresponse DNA-binding protein of 43 kDa TMEV = Theiler murine encephalomyelitis virus TNFa = tumor necrosis factoralpha


























































References






























Appendix Authors





YoshifumiSonobe PhD






Peter PytelMD



Paula LepineMSc



FlorianPernin MSc


Qiao-Ling CuiMD PhD


Jack P AntelMD


StephanieZandee PhD






Raymond PRoos MD





























Reprints




























































References






























Appendix Authors





YoshifumiSonobe PhD






Peter PytelMD



Paula LepineMSc



FlorianPernin MSc


Qiao-Ling CuiMD PhD


Jack P AntelMD


StephanieZandee PhD






Raymond PRoos MD





























Reprints





























Reprints




rna-binding protein altered expression and mislocalization in ms · in the present study,...

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