placental transfer of nmdar antibodies causes reversible

ARTICLE OPEN ACCESS

Placental transfer of NMDAR antibodies causesreversible alterations in miceAnna Garcıa-Serra MSc Marija Radosevic PhD Anika Pupak MSc Veronica Brito PhD Jose Rıos MSc

Esther Aguilar BS Estibaliz Maudes MSc Helena Arintildeo MD PhD Marianna Spatola MD PhD

Francesco Mannara PhD Marta Pedrentildeo PhD Bastien Joubert MD PhD Silvia Gines PhD

Jesus Planaguma PhD and Josep Dalmau MD PhD

Neurol Neuroimmunol Neuroinflamm 20218e915 doi101212NXI0000000000000915

Correspondence

Dr Dalmau

jdalmaucliniccat

AbstractObjectiveTo determine whether maternofetal transfer of NMDA receptor (NMDAR) antibodies haspathogenic effects on the fetus and offspring we developed a model of placental transfer ofantibodies

MethodsPregnant C57BL6J mice were administered via tail vein patientsrsquo or controlsrsquo immunoglobulinG (IgG) on days 14ndash16 of gestation when the placenta is able to transport IgG and theimmature fetal blood-brain barrier is less restrictive to IgG crossing Immunohistochemical andDiOlistic (gene gun delivery of fluorescent dye) staining confocal microscopy standardizeddevelopmental and behavioral tasks and hippocampal long-term potentiation were used todetermine the antibody effects

ResultsIn brains of fetuses patientsrsquo IgG but not controlsrsquo IgG bound to NMDAR causing a decreasein NMDAR clusters and cortical plate thickness No increase in neonatal mortality was ob-served but offspring exposed in utero to patientsrsquo IgG had reduced levels of cell-surface andsynaptic NMDAR increased dendritic arborization decreased density of mature (mushroom-shaped) spines microglial activation and thinning of brain cortical layers IIndashIV with cellularcompaction These animals also had a delay in innate reflexes and eye opening and duringfollow-up showed depressive-like behavior deficits in nest building poor motor coordinationand impaired social-spatial memory and hippocampal plasticity Remarkably all these para-digms progressively improved (becoming similar to those of controls) during follow-up untiladulthood

ConclusionsIn this model placental transfer of patientsrsquo NMDAR antibodies caused severe but reversiblesynaptic and neurodevelopmental alterations Reversible antibody effects may contribute to theinfrequent and limited number of complications described in children of patients who developanti-NMDAR encephalitis during pregnancy

These authors are joint senior authors

From the Institut drsquoInvestigacions Biomediques August Pi i Sunyer (IDIBAPS) (AG-S MR AP VB EA EM HA MS FM MP BJ SG JP JD) Hospital Clınic Universitat deBarcelona Departament de Biomedicina (AP VB SG) Facultat de Medicina Institut de Neurociencies Universitat de Barcelona Centro de Investigacion Biomedica en Red sobreEnfermedades Neurodegenerativas (CIBERNED) (AP VB SG) Madrid Medical Statistics Core Facility (JR) IDIBAPS and Hospital Clınic Barcelona Biostatistics Unit (JR) School ofMedicine Universitat Autonoma de Barcelona Centro de Investigaciones Biomedicas en Red de Enfermedades Raras (CIBERER) (JD) Madrid Spain Department of Neurology (JD)University of Pennsylvania Philadelphia and Institucio Catalana de Recerca i Estudis Avanccedilats (ICREA) (JD) Barcelona Spain

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

The Article Processing Charge was funded by the ISCIII

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

Anti-NMDA receptor (NMDAR) encephalitis is a neuronalantibody-mediated disease that associates with prominentneurologic and psychiatric symptoms1 Most clinical seriesshow that 60ndash80 of patients are women of childbearingage2ndash4 who sometimes develop the encephalitis duringpregnancy56 In addition to the obstetric complications thatcan arise from the severity of the disease5ndash7 there is theconcern that patientrsquos antibodies can reach the fetal brain andcause synaptic and neurodevelopmental alterations Thisconcern is supported by the demonstration of the pathoge-nicity of the antibodies in cultured neurons8ndash10 and in amouse model of cerebroventricular transfer of patientsrsquo anti-bodies11 Moreover peritoneal injection of a human NMDARmonoclonal antibody to pregnant mice resulted in highneonatal mortality and long-lasting irreversible neuro-developmental deficits in the offspring12

Therefore considering the severity of patientsrsquo symptomspathogenicity of the antibodies and the fact that NMDARsignaling regulates neuronal maturation13 migration14 andsynaptogenesis15 one would expect severe abnormalities inthe offspring of patients with anti-NMDAR encephalitisHowever the clinical experience is markedly different otherthan a few exceptional cases16 most reports indicate that theoffspring of these patients appear to have good outcomes6

Indeed among the limited number of problems reported inthe babies most are described as transient17 or attributed tomedications6 These discrepancies led us to investigate in amodel of maternofetal transfer of patientsrsquo immunoglobulin G(IgG) antibodies the effects on synaptic NMDAR dendriticcomplexity cortical development and microglial activation infetal and postnatal brains We also determined whether miceexposed in utero to patientsrsquo IgG had impairment of memorybehavior and hippocampal long-term plasticity and whetherthe antibody effects persisted or resolved from newborn toadulthood stages

MethodsMost of the methods and techniques used here have beenpreviously reported1118 and are described in appendix e-1(linkslwwcomNXIA341)

Human serum samples IgG purificationand immunoabsorptionIgG was isolated by ammonium sulfate precipitation fromserum of 7 patients with anti-NMDAR encephalitis and 7healthy blood donors All patients with anti-NMDAR

encephalitis were women (median age 20 years range 16ndash26years) with anti-NMDAR encephalitis The presence ofNMDAR antibodies (NMDAR-abs) was determined with ratbrain immunohistochemistry and a cell-based assay (CBA)1

To rule out the presence of other antibodies pooled IgG frompatients was immunoabsorbed with HEK293T cells express-ing GluN12B and the reactivity determined with brain im-munohistochemistry CBA of GluN12B and live neuronsshowing with all 3 techniques abrogation of reactivity1018 Weconfirmed that immunoabsorbed patientsrsquo IgG no longerdecreased the levels of NMDARs after 12-hour incubation asreported19

Animals infusion of IgG tissue processing anddetermination of antibodies in blood frompregnant mice and fetusesAnimal care and processing of brain of fetuses and offspringwere performed as reported11 Overall 54 pregnant C57BL6J mice 165 fetuses and 187 pups were used for behavioralelectrophysiologic morphologic and synaptic brain studies

Pooled IgG (800 μg) from patients or controls was injectedvia tail vein to pregnant mice on days 14 15 and 16 (E14E15 and E16) of gestation (figure 1A) These experimentswere planned according to the window of time in which theneonatal receptor (FcRn) which allows IgG transplacentaltransfer is expressed in placental tissue and the immaturefetal blood-brain barrier (BBB) does not restrict the crossingof IgG (by day E16 the BBB becomes more restrictive)20

The presence of human NMDAR-abs in blood from pregnantmice and fetuses was demonstrated with immunolabeling oflive rat hippocampal neurons in culture and conventionalCBA1 Antibody titers were obtained by serial sample dilutionusing CBA

Determination of human IgG NMDAR clustersand cortical plate thickness in fetal brain tissueTo determine whether human NMDAR-abs injected topregnant mice reached the brain of fetuses 5-μm-thick fetalbrain sections were immunostained for human IgG usingAlexa Fluor 488 goat anti-human IgG as above Quantifica-tion of fluorescence intensity was performed using the publicdomain Fiji ImageJ software (fijiscFiji)8 Confirmation thatpatientsrsquo IgG was bound to NMDAR was done byimmunoprecipitation18

The effects of patientsrsquo antibodies on total cell-surface andsynaptic NMDAR clusters and postsynaptic density protein95 (PSD95) were examined using immunohistochemistry

GlossaryBBB = blood-brain barrier CBA = cell-based assay fEPSP = field excitatory postsynaptic potential GEE = generalizedestimated equation GLM = generalized linear model IgG = immunoglobulin G LTP = long-term potentiation NMDAR =NMDA receptor NMDAR-ab = NMDAR antibody NOL = novel object location PD = postnatal day PPI = prepulseinhibition PSD95 = postsynaptic density protein 95

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 1 | January 2021 NeurologyorgNN

and confocal microscopy11 The thickness of the cortical platewas measured using 4 6-diamidino-2-phenylindole (DAPI)-

stained fetal brain sections and quantified using Fiji ImageJsoftware

Figure 1 Patientsrsquo antibodies intravenously administered to pregnant mice reach fetal brain and bind to NMDAR

(A) Diagram of the experimental design IV injection of pooled IgG was performed on gestational days E14 E15 and E16 Brain and blood samples from thefetus or offspring were collected on E16 E17 and E18 and PD 21 PD 70 and PD 155 blood samples of pregnantmice were collected on E16ndashE18 Brain tissuestudies were performed on the same time points and electrophysiology on PD 21 and PD 70 Newborns were assessed daily for neurodevelopmentalmilestones from birth until PD 21 After breastfeeding withdrawal mice underwent a battery of behavioral tests at age 1 2 and 4months Gestational periodismarked in pink and postnatal period in green (B) Representative images of HEK293T cells expressing GluN12B immunolabeledwith human IgG containedin serum of fetuses (E17) exposed to controlsrsquo or patientsrsquo IgG Scale bar = 10 μm (C and D) Representative human IgG immunolabeling in E17 fetal brainsections of animals exposed in utero to controlsrsquo (C) or patientsrsquo (D) IgG the 2 insets (small squares in C and D developing hippocampus) are shown enlargedon the right of each panel Scale bar whole brain = 100 μm insets = 10 μm (E) Quantification of human IgG immunofluorescence intensity in the developinghippocampus of fetal brains obtained on gestational days E16-E18 exposed to controlsrsquo IgG or patientsrsquo IgG Controlsrsquo IgG E16 n = 5 E17 n = 13 E18 n = 8patientsrsquo IgG E16 n = 9 E17 n = 16 E18 n = 8 Median intensity of IgG immunofluorescence in brain of mice exposed to controlsrsquo IgG was defined as 100Data presented in box plots show the median 25th and 75th percentiles whiskers indicate minimum and maximum Significance of treatment effect wasassessed by the Mann-Whitney U test p lt 00001 (F) Immunoprecipitated NMDAR-bound IgG from E17 and E18 brains of fetuses exposed in utero tocontrolsrsquoor patientsrsquo IgG Lanebrain tissue corresponds to homogenate ofmouse brain usedhere as control The band at100 KDa corresponds to theGluN1subunit of the NMDAR Each lane corresponds to the immunoprecipitation of the brains from 4 fetuses per each indicated condition IgG = immunoglobulinG NMDAR = NMDA receptor PD = postnatal day

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 1 | January 2021 3

Determination of human IgG NMDAR clusterssynaptic density cortical layer thickness andmicroglial activation in offspringThe presence of human IgG in the brain of offspring andthe density of NMDAR were examined as indicated forfetal brains (time points shown in figure 1A) NMDARclusters were quantified from a total of 15 images acquiredfrom CA1 CA3 and dentate gyrus of the hippocampus (5different areas per region)

The density of glutamatergic synapses was assessed with im-munohistochemistry using presynaptic (Bassoon) and post-synaptic (Homer1)markers and the corresponding secondaryantibodies (appendix e-1 linkslwwcomNXIA341)

Cortical layer thickness was measured in sections of brainsimmunostained with specific cortical layer markers including amonoclonal mouse anti-CUTL1 for layers IIndashIV and apolyclonal sheep anti-FoxP2 for layer VI followed by theappropriate secondary antibodies The layersrsquo thickness wasquantified using Fiji ImageJ software Cell density in CUTL1-labeled layers was measured on brain sections using DAPIstaining with spots algorithm in Imaris software

Microglial activation was assessed in brain sections using amonoclonal rat antibody against CD68 (pan-macrophage)and a polyclonal rabbit antibody against Iba-1 (activatedmicroglia) The CD68- or Iba-1ndashstained surface density wasquantified using Imaris software

Dendritic complexity and spinemorphology analysisThese studies were performed using DiOlistic (gene gundelivery of fluorescent dye) staining and confocal microscopy(appendix e-1 linkslwwcomNXIA341) Dendritic com-plexity was examined with Sholl analysis in cortical and CA1hippocampal pyramidal neurons21 Spine morphology wasexamined in DiI-labeled segments of apical secondary den-drites of cortical and CA1 pyramidal neurons Quantificationof mushroom-shaped dendritic spines was performed with thespine classification algorithm (Imaris software)

Field potential recordings hippocampal long-term potentiation and paired-pulse facilitationAcute sections of the hippocampus of the offspring were usedto determine long-term plasticity by the classical paradigm ofstimulation at the Schaffer collateral pathway and recordingthe field potentials at CA1 synapses19

Neurobehavioral assessment in postnatal andadult stagesFrom birth to weaning (defined as breastfeeding with-drawal on postnatal day [PD] 21) mice were assesseddaily for achievement of developmental milestones using amodified Fox battery similar to that previously repor-ted22 From weaning to adulthood (155 days) subsets of

mice underwent a comprehensive battery of memory andbehavioral tests (appendix e-1 linkslwwcomNXIA341and figure e-1 linkslwwcomNXIA342)

Statistical analysisComparison of human IgG intensity confocal densities ofNMDAR PSD95 Homer1 Bassoon Iba-1 CD68 andmushroom-shaped spines between mice exposed to pa-tientsrsquo IgG vs controlsrsquo IgG was performed with the Mann-Whitney U test as non-normally distributed parametersComparison of cortical plate thickness cortical layers IIndashIV and VI cortical cell density and electrophysiologic fieldexcitatory postsynaptic potential (fEPSP) slope changesbetween the 2 experimental groups was performed usingindependent sample t tests Sholl analysis data were ana-lyzed with a generalized linear model (GLM) for globaland time point effects For behavioral paradigms longi-tudinal analyses were performed by generalized estimatedequations (GEEs) using an AR(1) matrix to account forintraindividual correlations All models include litter sizegroup and group by time as fixed factors Time to rootingear detachment auditory startle and eye opening wereanalyzed with the Mann-Whitney U test All experimentswere assessed for outliers with the ROUT method apply-ing Q = 1 In all analyses we used a 2-sided type I error of5 All tests were performed using GraphPad Prism(version 8 GraphPad Inc San Diego CA) or SPSS(version 25 IBM Corp Armonk NY) for GLM and GEEmodels Mean values or estimated mean values (bodyweight righting reflex and negative geotaxis for the GEEmodel) were presented with plusmnSEM

Ethical approvalWritten informed consent was obtained from all patientsthe study was approved by the local institutional reviewboard at Hospital Clınic Barcelona (HCB20180192)Animal studies were approved by the Ethical Committee ofthe University of Barcelona (201063UE) and Spanish(RD 532013) regulations

Data availabilityData supporting these findings are available on reasonablerequest

ResultsPresence of human NMDAR-abs in serum ofpregnant mice and fetusesSerum samples collected on E16 E17 and E18 from pregnantmice and fetuses exposed to patientsrsquo IgG but not those ex-posed to controlsrsquo IgG showed human IgG binding to cellsurface of live hippocampal neuronal cultures andHEK293T cells expressing GluN1GluN2B (figure 1B) Thehuman NMDAR antibody titer (obtained by serial dilution ofsamples in CBA) was 1160ndash1320 in pregnant mice and 180ndash1160 in fetuses (data not shown)


Patientsrsquo IgG reach fetal brain causing areduction of synaptic NMDAR and thinning ofthe cortical plateOn days E16ndash18 fetuses exposed to patientsrsquo IgG showedincreased human IgG fluorescence in the developing hippo-campus compared with those exposed to controlsrsquo IgG (figure1 CndashE) Immunoprecipitation experiments confirmed thepresence of NMDAR bound to IgG in the brain precipitate(figure 1F) Analysis of the density of NMDAR clustersshowed that fetuses harboring patientsrsquo IgG had a significantdecrease in cluster density of total cell-surface and synapticNMDARs (figure 2 A and D) The cortical plate of thesefetuses was also thinner on E17 and E18 compared with thatof controls (figure 2 G and J)

Offspring exposed in utero to patientsrsquo IgGhave decreased NMDAR and synaptic densitythinning of cortical layers andmicroglial activationOn PD 21 and 70 mice exposed in utero to patientsrsquo IgG nolonger had significant brain deposits of IgG (not shown)Despite this the reduction of total cell-surface NMDARclusters persisted until day 70 (figure 2 B and C) with sub-sequent normalization so that by day 155 the levels weresimilar to those of controls (not shown) In contrast the levelsof synaptic NMDAR normalized faster they were decreasedon day 21 (figure 2E) but were normal on days 70 and 155(figure 2F not shown) PSD95 cluster density was not af-fected at any time point (data not shown)

Mice exposed to patientsrsquo IgG but not controlsrsquo IgG showedthinning of cortical layers IIndashIV (assessed with CUTL1FoxP2staining) on PD 21 (figure 2 H and K) that was no longerpresent on PD 70 (figure 2 I and L) This transient thinning ofcortical layers IIndashIV was associated with an increase in celldensity in these layers on PD 21 (p = 00065 data not shown)

On PD 21 the density of glutamatergic synapses (assessed bycolocalization of Homer1Bassoon clusters) was decreased inmice exposed in utero to patientsrsquo IgG (figure 3 A and B)These effects were no longer seen on day 70 (not shown)

On PD 21 and 70 there was an increase of 57 and 53 inactivated microglia (assessed by expression of Iba-1 in so-matosensory cortex) in mice exposed in utero to patientsrsquo IgG(figure 3 C and D) This difference in activated microglia wasno longer present on PD 155 (not shown) No significantdifference in the total number of microglial cells (assessed byexpression of CD68) was noted between groups of mice atany time point examined (figure 3E)

Exposure to patientsrsquo IgG causes alterations indendritic complexity spine density and spinemorphology in cortical and hippocampalpyramidal neuronsDendritic arborization was assessed by Sholl analysis (figure 4A and B) On PD 21 the Sholl analysis in cortical and

hippocampal areas showed a higher dendritic arborization inmice exposed in utero to patientsrsquo IgG (figure 4 C and D left)No differences were noted on PD 70 (figure 4 C and D right)

The density of mushroom-shaped (mature) dendritic spineswas assessed in the cerebral cortex and hippocampus using theImaris standard parameters for dendritic spine classification(example shown in figure 4 EndashG) On PD 21 the density ofthese spines was significantly decreased in mice exposed inutero to patientsrsquo IgG (cortex figure 4H left hippocampusfigure 4I left) The changes were no longer seen on PD 70(cortex figure 4H right and hippocampus figure 4I right)

Intrauterine exposure to patientsrsquo IgG causes areversible impairment in hippocampal long-term potentiation formationAssessment of fEPSPs showed that on PD 21 mice that hadbeen exposed in utero to patientsrsquo IgG had impairment inlong-term potentiation (LTP) formation compared with miceexposed in utero to controlsrsquo IgG (figure e-2 AndashC linkslwwcomNXIA343) This change in LTP formation was nolonger detected on PD 70 (figure e-2 DndashF) Paired-pulsefacilitation studies at 50 and 100 milliseconds of interstimuliinterval (which assess presynaptic release mechanisms) werenot different between groups of animals (data not shown)

Neurodevelopmental delay due to in uteroexposure to NMDAR-absNewborn pups exposed in utero to patientsrsquo IgG showedmultiple alterations including transient decrease of weight(figure 5A) longer times for proper body righting (figure 5B)and negative geotaxis (figure 5C) delayed eye opening(figure 5D) and longer time in open field locomotion (notshown p lt 001) Neonatal mortality occurred in litters ofmice exposed in utero to controlsrsquo IgG (26) or patientsrsquo IgG(25) which is in line with general litter loss in laboratorymice23 No significant differences were observed in ear de-tachment tactile rooting response and auditory startle (datanot shown)

Spontaneous reversal of behavioral alterationsfrom early to mature adulthoodAt age 1 month mice exposed in utero to patientsrsquo IgGshowed a significant decrease in the novel object location(NOL) index indicating impairment in visuospatial memory(figure 5E) a higher amount of unused material for nestbuilding (figure 5F) and a longer time of exploration of afamiliar intruder mouse indicating impairment in socialmemory (figure 5G trial 4) Assessment of motor co-ordination and balance showed that mice exposed in utero topatientsrsquo IgG had shorter times on the accelerating rotarod(figure 5H) No significant differences were noted in prepulseinhibition (PPI) of acoustic startle reaction locomotor ac-tivity or adult body weight at age 1 month (data not shown)

At age 2 months the alterations of NOL nest building andsocial interaction in mice that had been exposed to patientsrsquo


IgG were no longer present (figure 5 I and J social in-teraction not shown) PPI and locomotor activity remainedunaffected at age 2 months (not shown) However mice ex-posed in utero to patientsrsquo IgG showed a shorter time on theaccelerating rotarod (data not shown p = 00292) and longer

time of immobility in the tail suspension test (which assessescore helplessness) (figure 5K)

At age 4 months (adulthood) no significant difference wasnoted between mice exposed in utero to patientsrsquo IgG and

Figure 2 Intrauterine exposure to patientsrsquo IgG causes a transient reduction of cell-surface NMDARs and thinning of thecortical plate and cortical layers IIndashIV

(AndashF) Quantification of total cell-surface (AndashC) and synaptic (DndashF) NMDAR cluster density in brains obtained on gestational days E16 E17 and E18 (A andD) PD21 (B and E) and PD 70 (C and F) from animals exposed in utero to controlsrsquo IgG or patientsrsquo IgG Median of NMDAR clusters in mice exposed to controlsrsquo IgGwas defined as 100 Controlsrsquo IgG E16 n = 6 E17 n = 15 E18 n = 7 PD 21 n = 11 PD 70 n = 9 patientsrsquo IgG E16 n = 13 E17 n = 21 E18 n = 5 PD 21 n = 14 PD70 n = 10 (G) Representative cortical plate thickness (assessed with DAPI) in E17 and E18 brains of fetuses exposed in utero to controlsrsquo or patientsrsquo IgG Scalebars = 100 μm (HndashI) Representative sections of the somatosensory cortex (H PD 21 I PD 70) of animals exposed in utero to controlsrsquo or patientsrsquo IgG showingcortical layers IIndashIV (CUTL1 positive green fluorescence) and cortical layer VI (FoxP2 positive red fluorescence) arrows indicate the thickness of the indicatedlayers Scale bars = 100 μm (J) Quantification of cortical plate thickness in E16 E17 and E18 brains of fetuses exposed in utero to controlsrsquo IgG or patientsrsquo IgGControlsrsquo IgG E16 n = 4 E17 n = 11 E18 n = 10 patientsrsquo IgG E16 n = 8 E17 n = 14 E18 n = 8 (K and L) Quantification of the indicated cortical layersrsquo thickness(K PD 21 L PD 70) in animals exposed to controlsrsquo IgG or patientrsquos IgG Controlsrsquo IgG PD 21 n = 7 PD 70 n = 8 patientsrsquo IgG PD 21 n = 15 PD 70 n = 10 For JndashLthe mean thickness of the cortical plate or the indicated cortical layers in mice exposed in utero to controlsrsquo IgG was defined as 100 Data presented in boxplots show the median 25th and 75th percentiles whiskers indicate minimum and maximum Significance of treatment effect was assessed by the Mann-Whitney U test in AndashF and independent sample t test in JndashL p lt 005 p lt 001 p lt 00001 IgG = immunoglobulin G NMDAR = NMDA receptor PD =postnatal day


those exposed to controlsrsquo IgG in any of the following teststail suspension (figure 5L) rotarod locomotor activity NOLPPI nest building and social interaction (not shown)

DiscussionWe show that antibodies from patients with anti-NMDAR en-cephalitis infused to pregnant mice reached the fetal braincaused multiple alterations ranging from NMDAR synapticchanges to delayed neurodevelopment and behavioral deficits inthe offspring and that all these alterations progressively resolvedafter birth (summary in table e-1 linkslwwcomNXIA341) As

expected from previous experience with adult mice in whichpatientsrsquo IgG was infused in the cerebroventricular system1119

we observed a robust antibody-mediated decrease in NMDARclusters in the brain of the fetuses These effects occurred quickly(noted 2 days after injecting the antibodies to the mothers) andthe observed reduction of NMDAR clusters and impairment ofmemory and hippocampal long-term plasticity were detectableduring the first postnatal month

In addition hippocampal and cortical neurons from miceexposed in utero to patientsrsquo IgG showed an increase indendritic arborization accompanied by a reduction of mature

Figure 3 Intrauterine exposure to patientsrsquo IgG causes a reversible decrease in synaptic density and increase in activatedmicroglia

(A) Representative z-stacks of brain sections obtained on PD 21 from mice exposed in utero to controlsrsquo or patientsrsquo IgG immunostained for Homer1 andBassoon expression Colocalization of both markers is defined as a glutamatergic synapse Scale bar = 2 μm (B) Quantification of synaptic density (Homer1Bassoon colocalization) in brains obtained on PD 21 from animals exposed in utero to controlsrsquo IgG or patientsrsquo IgG Median density of Homer1Bassoonsynapses in mice exposed in utero to controlsrsquo IgG was defined as 100 Controlsrsquo IgG n = 11 patientsrsquo IgG n = 14 (C) Representative CD68 (macrophagespecific) and Iba-1 (activated microglia specific) immunostainings in brains obtained on PD 21 frommice exposed in utero to controlsrsquo or patientsrsquo IgG Scalebar = 20 μm (D and E) Quantification of Iba-1 (D) and CD68 (E) immunostaining in brains obtained at PD 21 and PD 70 frommice exposed to controlsrsquo IgG orpatientsrsquo IgG Median surface density of immunostaining inmice exposed in utero to controlsrsquo IgGwas defined as 100 Controlsrsquo IgG PD 21 n = 11 PD 70 n =9 patientsrsquo IgG PD 21 n = 14 PD 70 n = 10 Data presented in box plots show the median 25th and 75th percentiles whiskers indicate the minimum andmaximum Significance of treatment effect was assessed by theMann-WhitneyU test p lt 001 p lt 00001 IgG = immunoglobulin G PD =postnatal day


Figure 4 Intrauterine exposure to patientsrsquo IgG causes a transient increase in dendritic complexity and decrease in spinedensity

(A and B) Representative confocal image (A) and Sholl analysis (B) of a DiI-stained CA1 hippocampal pyramidal neuron showing in B the 10-μm spacedconcentric rings used for quantification of dendritic complexity Scale bar in A = 50 μm (C and D) Number of dendrite intersections according to the indicateddistance to the neuronal soma in cortical and hippocampal neurons examined at PD 21 and PD 70 of mice exposed in utero to controlsrsquo (Black) or patientsrsquo(blue) IgG Number ofmice exposed in utero to controlsrsquo IgG PD 21 n = 6 PD 70 n = 4 number ofmice exposed in utero to patientsrsquo IgG PD 21 n = 6 PD 70 n =4 Per each animal condition and brain region 4 neuronswere examined Data presented asmean plusmn SEM Significance of treatment effect was assessed by ageneralized lineal model p lt 005 p lt 001 p lt 0001 p lt 00001 (EndashG) Representative images by confocal microscopy of a DiI-stained CA1hippocampal pyramidal neuron (E) and a secondary apical dendrite (F) with morphologically classified dendritic spines (G mushroom-shaped spines ingreen) Scale bar in E = 50 μm scale bar in F = 5 μm (H and I) Quantification of mushroom dendritic spine density in cortical and hippocampal neuronsexamined at PD 21 and PD 70 in mice exposed in utero to controlsrsquo or patientsrsquo IgG The median density of mushroom spines (number of spinesμm) in miceexposed to controlsrsquo IgG was defined as 100 Total number of dendrites examinedtotal number of mice exposed to controlsrsquo IgG PD 21 cortex n = 1226hippocampus n = 1206 PD 70 cortex n = 814 and hippocampus n = 814 Total dendritesmice exposed to patientsrsquo IgG PD 21 cortex n = 1416hippocampus n = 1486 PD 70 cortex n = 704 and hippocampus n = 724 Data presented in box plots show themedian 25th and 75th percentiles whiskersindicateminimumandmaximum Significance of treatment effect was assessed by theMann-WhitneyU test in H and I p lt 005 IgG = immunoglobulin G PD= postnatal day


(mushroom-shaped) dendritic spines and synaptic densityThese alterations have not been reported in previous modelsof immunodepletion of NMDAR and likely contributed to theimpairment of hippocampal LTP The effect of NMDARhypofunction on dendritic arborization and the morphologyof the spines are not well understood In a cortex-specificGluN1 knockout model the cortical neurons in layer IVelaborated exuberant dendritic specializations24 however in astudy using MK801 (an NMDAR antagonist) during earlypostnatal development there was a transient decrease indendritic arborization in hippocampal pyramidal neurons thatreversed when MK801 was discontinued25 Our findings

resemble those of the cortex-specific GluN1 knockout al-though in our immune model the alterations were reversible

We believe that the increased dendritic arborization observedin animals exposed in utero to patientsrsquo IgG was likely causedby the reduction of NMDAR levels along with microglialactivation Any perinatal immune challenge such as thebinding of antibodies to NMDAR in the fetal brain can resultin microglial activation26 and consequently affect microglia-mediated synaptic pruning This normal microglial function isalso triggered by neural activity in the developing brain2728

For example impaired microglia-neuron interactions in

Figure 5 Intrauterine exposure to patientsrsquo IgG causes transient developmental delay and behavioral alterations inoffspring

(AndashD) Developmental milestone assessment (from birth to PD 21) in mice that were exposed in utero to controlsrsquo (Black) or patientsrsquo (blue) IgG including (A)body weight (B) time needed for body righting using the surface righting reflex test (PD 1 3 5 7 9 and 11) (C) time needed for 180deg turning to head upposition using the negative geotaxis reflex (PD 2 4 and 6) and (D) scored eye opening (PD 12ndash16) Number of mice exposed in utero to controlsrsquo IgG = 36number ofmice exposed to patientsrsquo IgG = 40 Data fromAndashCare represented as estimatedmean plusmn SEM inferred by theGEEmodels described inMethods andin D are represented as median Significance of treatment effect was assessed by a GEE adjusted model (AndashC) and Mann-Whitney U test (D) p lt 005 p lt0001 (EndashL) Battery of behavioral tests performed at the indicated time points including novel object location index (E 1month I 2months) nest building test(F 1 month J 2 months) social interaction test (G 1 month) rotarod test (H 1 month) and tail suspension test (K 2 months L 4 months) For EndashI and Knumber of mice exposed to controlsrsquo IgG = 18 and mice exposed to patientsrsquo IgG = 18 For J and L number of mice exposed to controlsrsquo IgG = 8 and miceexposed to patientsrsquo IgG = 9 Data from panels E F and IndashL are represented as box plots with median 25th and 75th percentiles whiskers indicate minimumandmaximum values data from G and H are represented as mean plusmn SEM For all behavioral tests significance of treatment effect was assessed by GEE p lt005 p lt 001 p lt 0001 GEE = generalized estimated equation IgG = immunoglobulin G PD = postnatal day


CX3CR1-deficient mice showed delayed synaptic functionalmaturation29 and immature and redundant connectivity thelatter attributed to reduced synaptic pruning30 These alter-ations spontaneously reverted by PD 30ndash402930 similar toour findings

In our model the brain of fetuses and offspring exposed inutero to patientsrsquo IgG had a reduction in the thickness of thecortical plate and cortical layers IIndashIV In mice glutamateserves as a chemoattractant for neurons in the developingcortex signaling cells to migrate into the cortical plate throughreceptor activation14 Studies with NMDAR antagonists(MK801 or APV) have shown that they block neuronal cellmigration from the ventricular zone into the cortical platedelaying neuronal migration to the upper cortical layers(IIndashIII)1431 In our immune-mediated model the transientdecrease in thickness of cortical layers IIndashIV was associatedwith an increase in cell density in the upper cortical layers thatlikely contributed to the restoration of the cortical layerthickness after PD 21 These findings are somewhat similar tothose obtained in models of genetic disruption of other pro-teins and receptors where the decrease in cortical thickness isreversible and associates with an increase in cell density in theupper cortical layers3233 For example loss of MeCP2 in theRett syndrome model results in a reduction in thickness ofsomatosensory cortical layers IIndashIII and VI with a total rescueof thickness after treatment with mirtazapine during PD28ndash4233 In another model based on a constitutive reductionin central serotonin (VMAT2sert-cre mice) the total corticalthickness reduction at PD 7 spontaneously recovered by PD1432 Like this model our anti-NMDAR model associateswith a temporary delay in the development of the uppercortical layers along with increased compaction of theselayers

Preceding the indicated memory deficit the newborn pupsexposed in utero to patientsrsquo IgG showed impaired innatereflexes and developmental delay Similar impairment of na-tive reflexes and developmental delay have been reported inGluN1 gene-manipulated mice34 and newborn rats treatedwith a competitive NMDAR antagonist35

A previous model of transplacental transfer of a human-derived NMDARmonoclonal antibody showed high neonatalmortality (27) impairment of native reflexes and long-lasting neuropathologic alterations without evidence of re-versibility12 In contrast we did not observe increased neo-natal mortality and the symptoms and developmentalalterations spontaneously improved Moreover although themonoclonal antibody model did not assess dendritic arbori-zation spine density cortical development and number ofcell surface and synaptic NMDAR clusters we found thatpatientsrsquo IgG antibodies robustly altered all these paradigmsalong with a dramatic decrease in NMDAR clusters andhippocampal LTP Thus the reversibility of these alterationsand associated symptoms cannot be attributed to a milderphenotype The reasons for the discrepancies between our

model in which serum NMDAR-abs representative of severalpatients were used and the model in which a human mono-clonal NMDAR-ab was used are unclear It is likely that themonoclonal antibody model lacking the repertoire NMDAR-abs of patients is less representative of the disease Never-theless the outcome of newborns from pregnant patients withanti-NMDAR encephalitis6 is remarkably different from thatobtained with the mouse monoclonal antibody model12

Our study does not allow generalization of the notion thatanti-NMDAR encephalitis during pregnancy does not repre-sent a risk for serious effects in the offspring For example ourmodel does not associate with encephalitis of the motherswhich in our experience6 is the most important risk factor forobstetric complications and potential brain damage of thenewborns However the low frequency of antibody-mediatedcomplications in newborns of patients with anti-NMDARencephalitis is puzzling and may also be related to the timingsof IgG placental transfer and BBB restriction properties forIgG crossing into the brain In humans placental transfer ofmaternal antibodies starts at 12ndash13 weeks of pregnancy36ndash38

that is approximately the same time the BBB of the fetus startsto become more restrictive to the passage of albumin (andlikely Igs) to the brain39 It is unknown whether this timewindow and the efficiency of the fetal BBB are affected by thepresence of maternal encephalitis or systemic inflammationFuture studies with pregnant patients who develop anti-NMDAR encephalitis and close follow-up of the babies areneeded to determine the frequency of deficits (if presentmostly overlooked until now) and the timing of improve-ment Keeping these limitations in mind the current modelsuggests that reversibility of some of the antibody-mediatedalterations can potentially occur Moreover the model can bemanipulated to assess the effects of systemic inflammationand provides also the possibility to determine whether in-hibitors of FcRn have potential utility in preventing the po-tential neonatal abnormalities reported here

AcknowledgmentThe authors thank Dr Myrna R Rosenfeld (IDIBAPSUniversity of Barcelona) for critical review of the manuscriptand useful suggestions andMercedes Alba Eva Caballero andAraceli Mellado (IDIBAPS Hospital Clınic University ofBarcelona) for their technical support

Study fundingThis study was funded by Plan Nacional de I+D+I and cofi-nanced by the Instituto de Salud Carlos III (ISCIII)mdashSubdireccion General de Evaluacion y Fomento de la Inves-tigacion Sanitaria and the Fondo Europeo de Desarrollo Re-gional (ISCIII-FEDER 1700234) Project Integrative ofExcellence (PIE 1600014) Centro de Investigacion Bio-medica en Red de Enfermedades Raras (CIBERER CB1500010) ldquoLa Caixardquo Foundation (ID 100010434 under theagreement LCFPRHR1752150001) The Safra Founda-tion (JD) and Fundacio CELLEX (JD) Centres deRecerca de Catalunya (CERCA) program by La Generalitat


de Catalunya (JR) Pla Estrategic de Recerca i Innovacio enSalut (PERIS SLT0021600346 JP) Ministerio deEconomia y Competitividad (RTI2018-094374-B-I00 SG)Basque Government Doctoral Fellowship Program (PRE_2019_1_0255 EM) and Agencia de Gestio drsquoAjuts Uni-versitaris i de Recerca (FI-AGAUR) grant program by LaGeneralitat de Catalunya (2018FI_B_00487 AP 2019FI_B1 00212 AG-S)

DisclosureA Garcıa-Serra M Radosevic A Pupak V Brito J Rıos EAguilar E Maudes H Arintildeo M Spatola F Mannara MPedrentildeo B Joubert S Gines and J Planaguma report nodisclosures J Dalmau receives royalties from Athena Diag-nostics for the use of Ma2 as an autoantibody test and fromEuroimmun for the use of NMDA GABAB receptor GABAAreceptor DPPX and IgLON5 as autoantibody tests Go toNeurologyorgNN for full disclosures

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 13 2020 Accepted in final form August 26 2020

References1 Dalmau J Gleichman AJ Hughes EG et al Anti-NMDA-receptor encephalitis case

series and analysis of the effects of antibodies Lancet Neurol 200871091ndash10982 Titulaer MJ McCracken L Gabilondo I et al Treatment and prognostic factors for

long-term outcome in patients with anti-NMDA receptor encephalitis an observa-tional cohort study Lancet Neurol 201312157ndash165

3 Irani SR Bera K Waters P et al N-methyl-D-aspartate antibody encephalitis tem-poral progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes Brain 20101331655ndash1667

4 Viaccoz A Desestret V Ducray F et al Clinical specificities of adult male patients withNMDA receptor antibodies encephalitis Neurology 201482556ndash563

5 Kumar MA Jain A Dechant VE et al Anti-N-methyl-D-aspartate receptor enceph-alitis during pregnancy Arch Neurol 201067884ndash887

6 Joubert B Garcia-Serra A Planaguma J et al Pregnancy outcomes in anti-NMDAreceptor encephalitis case series Neurol Neuroimmunol Neuroinflamm 20207e668

7 Xiao X Gui S Bai P et al Anti-NMDA-receptor encephalitis during pregnancy a casereport and literature review J Obstet Gynaecol Res 201743768ndash774

8 Hughes EG Peng X Gleichman AJ et al Cellular and synaptic mechanisms of anti-NMDA receptor encephalitis J Neurosci 2010305866ndash5875

9 Mikasova L De Rossi P Bouchet D et al Disrupted surface cross-talk betweenNMDA and Ephrin-B2 receptors in anti-NMDA encephalitis Brain 20121351606ndash1621

10 Moscato EH Peng X Jain A Parsons TD Dalmau J Balice-Gordon RJ Acutemechanisms underlying antibody effects in anti-N-methyl-D-aspartate receptor en-cephalitis Ann Neurol 201476108ndash119

11 Planaguma J Leypoldt F Mannara F et al Human N-methyl D-aspartate receptorantibodies alter memory and behaviour in mice Brain 201513894ndash109

12 Jurek B Chayka M Kreye J et al Human gestational N-methyl-d-aspartate receptorautoantibodies impair neonatal murine brain function Ann Neurol 201986656ndash670

13 Hirasawa T Wada H Kohsaka S Uchino S Inhibition of NMDA receptors inducesdelayed neuronal maturation and sustained proliferation of progenitor cells duringneocortical development J Neurosci Res 200374676ndash687

Appendix Authors

Name Location Contribution

AnnaGarcıa-Serra MSc

IDIBAPS Hospital ClınicUniversitat de BarcelonaSpain

Major role in theacquisition of dataanalyzed the data anddrafted the manuscript forintellectual content

MarijaRadosevicPhD


Major role in theacquisition of dataanalyzed the data andrevised the manuscript forintellectual content

AnikaPupak MSc

IDIBAPS Hospital Clınicand Institut deNeurociencies Universitatde Barcelona andCIBERNED Madrid Spain

Major role in theacquisition of data andrevised the manuscript forintellectual content

VeronicaBrito PhD



Jose RıosMSc

Medical Statistics CoreFacility IDIBAPS andHospital Clınic Barcelonaand Biostatistics UnitFaculty of MedicineUniversitat Autonoma deBarcelona Spain

Analyzed the data andrevised the manuscript forintellectual content

EstherAguilar BS



EstibalizMaudesMSc



Appendix (continued)


HelenaArintildeo MDPhD


Performed IVadministrations andrevised the manuscript forintellectual content

MariannaSpatolaMDPhD


Performedimmunoprecipitationtechnique and revised themanuscript for intellectualcontent

FrancescoMannaraPhD


Revised the manuscript forintellectual content

MartaPedrentildeoPhD



BastienJoubert MDPhD



Silvia GinesPhD



JesusPlanagumaPhD


Major role in theacquisition of datadesigned andconceptualized the studyand revised themanuscriptfor intellectual content

JosepDalmauMD PhD

IDIBAPS Hospital ClınicUniversitat de BarcelonaICREA CIBERER MadridSpain and University ofPennsylvania Philadelphia

Designed andconceptualized the studyand drafted themanuscript for intellectualcontent


14 Behar TN Scott CA Greene CL et al Glutamate acting at NMDA receptors stim-ulates embryonic cortical neuronal migration J Neurosci 1999194449ndash4461

15 Tolias KF Bikoff JB Burette A et al The Rac1-GEF Tiam1 couples the NMDAreceptor to the activity-dependent development of dendritic arbors and spinesNeuron 200545525ndash538

16 Jagota P Vincent A Bhidayasiri R Transplacental transfer of NMDA receptor anti-bodies in an infant with cortical dysplasia Neurology 2014821662ndash1663

17 Hilderink M Titulaer MJ Schreurs MW Keizer K Bunt JE Transient anti-NMDARencephalitis in a newborn infant due to transplacental transmission Neurol Neuro-immunol Neuroinflamm 20152e126

18 Planaguma J Haselmann H Mannara F et al Ephrin-B2 prevents N-methyl-D-aspartate receptor antibody effects on memory and neuroplasticity Ann Neurol 201680388ndash400

19 Mannara F Radosevic M Planaguma J et al Allosteric modulation of NMDA re-ceptors prevents the antibody effects of patients with anti-NMDAR encephalitis Brain20201432709ndash2720

20 Braniste V Al-Asmakh M Kowal C et al The gut microbiota influences blood-brainbarrier permeability in mice Sci Transl Med 20146263ra158

21 Ferreira TA Blackman AV Oyrer J et al Neuronal morphometry directly frombitmap images Nat Methods 201411982ndash984

22 Hill JM Lim MA Stone MM Developmental milestones in the newborn mouse InGozes I ed Neuromethods Totowa Humana Press 2008131ndash149

23 Weber EM Algers B Wurbel H Hultgren J Olsson IA Influence of strain and parityon the risk of litter loss in laboratory mice Reprod Domest Anim 201348292ndash296

24 Datwani A Iwasato T Itohara S Erzurumlu RS NMDA receptor-dependent patterntransfer from afferents to postsynaptic cells and dendritic differentiation in the barrelcortex Mol Cell Neurosci 200221477ndash492

25 Elhardt M Martinez L Tejada-Simon MV Neurochemical behavioral and archi-tectural changes after chronic inactivation of NMDA receptors in mice Neurosci Lett2010468166ndash171

26 Paolicelli RC Ferretti MT Function and dysfunction of microglia during brain de-velopment consequences for synapses and neural circuits Front Synaptic Neurosci201799

27 Schafer DP Lehrman EK Kautzman AG et al Microglia sculpt postnatal neuralcircuits in an activity and complement-dependent manner Neuron 201274691ndash705

28 Zuo Y Yang G Kwon E Gan WB Long-term sensory deprivation prevents dendriticspine loss in primary somatosensory cortex Nature 2005436261ndash265

29 Hoshiko M Arnoux I Avignone E Yamamoto N Audinat E Deficiency of themicroglial receptor CX3CR1 impairs postnatal functional development of thalamo-cortical synapses in the barrel cortex J Neurosci 20123215106ndash15111

30 Paolicelli RC Bolasco G Pagani F et al Synaptic pruning by microglia is necessary fornormal brain development Science 20113331456ndash1458

31 Reiprich P Kilb W Luhmann HJ Neonatal NMDA receptor blockade disturbsneuronal migration in rat somatosensory cortex in vivo Cereb Cortex 200515349ndash358

32 Narboux-Neme N Angenard G Mosienko V et al Postnatal growth defects in micewith constitutive depletion of central serotonin ACS Chem Neurosci 20134171ndash181

33 Bittolo T Raminelli CA Deiana C et al Pharmacological treatment with mirtazapinerescues cortical atrophy and respiratory deficits in MeCP2 null mice Sci Rep 2016619796

34 Single FN Rozov A Burnashev N et al Dysfunctions in mice by NMDA receptorpoint mutations NR1(N598Q) and NR1(N598R) J Neurosci 2000202558ndash2566

35 Mikulecka A Mares P NMDA receptor antagonists impair motor performance inimmature rats Psychopharmacology (Berl) 2002162364ndash372

36 Malek A Sager R Kuhn P Nicolaides KH Schneider H Evolution of maternofetaltransport of immunoglobulins during human pregnancy Am J Reprod Immunol199636248ndash255

37 Simister NE Story CM Chen HL Hunt JS An IgG-transporting Fc receptorexpressed in the syncytiotrophoblast of human placenta Eur J Immunol 1996261527ndash1531

38 Dancis J Lind J Oratz M Smolens J Vara P Placental transfer of proteins in humangestation Am J Obstet Gynecol 196182167ndash171

39 Virgintino D Robertson D Benagiano V et al Immunogold cytochemistry of theblood-brain barrier glucose transporter GLUT1 and endogenous albumin in thedeveloping human brain Brain Res Dev Brain Res 200012395ndash101


DOI 101212NXI000000000000091520218 Neurol Neuroimmunol Neuroinflamm

Anna Garciacutea-Serra Marija Radosevic Anika Pupak et al Placental transfer of NMDAR antibodies causes reversible alterations in mice

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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

Anti-NMDA receptor (NMDAR) encephalitis is a neuronalantibody-mediated disease that associates with prominentneurologic and psychiatric symptoms1 Most clinical seriesshow that 60ndash80 of patients are women of childbearingage2ndash4 who sometimes develop the encephalitis duringpregnancy56 In addition to the obstetric complications thatcan arise from the severity of the disease5ndash7 there is theconcern that patientrsquos antibodies can reach the fetal brain andcause synaptic and neurodevelopmental alterations Thisconcern is supported by the demonstration of the pathoge-nicity of the antibodies in cultured neurons8ndash10 and in amouse model of cerebroventricular transfer of patientsrsquo anti-bodies11 Moreover peritoneal injection of a human NMDARmonoclonal antibody to pregnant mice resulted in highneonatal mortality and long-lasting irreversible neuro-developmental deficits in the offspring12

Therefore considering the severity of patientsrsquo symptomspathogenicity of the antibodies and the fact that NMDARsignaling regulates neuronal maturation13 migration14 andsynaptogenesis15 one would expect severe abnormalities inthe offspring of patients with anti-NMDAR encephalitisHowever the clinical experience is markedly different otherthan a few exceptional cases16 most reports indicate that theoffspring of these patients appear to have good outcomes6

Indeed among the limited number of problems reported inthe babies most are described as transient17 or attributed tomedications6 These discrepancies led us to investigate in amodel of maternofetal transfer of patientsrsquo immunoglobulin G(IgG) antibodies the effects on synaptic NMDAR dendriticcomplexity cortical development and microglial activation infetal and postnatal brains We also determined whether miceexposed in utero to patientsrsquo IgG had impairment of memorybehavior and hippocampal long-term plasticity and whetherthe antibody effects persisted or resolved from newborn toadulthood stages

MethodsMost of the methods and techniques used here have beenpreviously reported1118 and are described in appendix e-1(linkslwwcomNXIA341)

Human serum samples IgG purificationand immunoabsorptionIgG was isolated by ammonium sulfate precipitation fromserum of 7 patients with anti-NMDAR encephalitis and 7healthy blood donors All patients with anti-NMDAR

encephalitis were women (median age 20 years range 16ndash26years) with anti-NMDAR encephalitis The presence ofNMDAR antibodies (NMDAR-abs) was determined with ratbrain immunohistochemistry and a cell-based assay (CBA)1

To rule out the presence of other antibodies pooled IgG frompatients was immunoabsorbed with HEK293T cells express-ing GluN12B and the reactivity determined with brain im-munohistochemistry CBA of GluN12B and live neuronsshowing with all 3 techniques abrogation of reactivity1018 Weconfirmed that immunoabsorbed patientsrsquo IgG no longerdecreased the levels of NMDARs after 12-hour incubation asreported19

Animals infusion of IgG tissue processing anddetermination of antibodies in blood frompregnant mice and fetusesAnimal care and processing of brain of fetuses and offspringwere performed as reported11 Overall 54 pregnant C57BL6J mice 165 fetuses and 187 pups were used for behavioralelectrophysiologic morphologic and synaptic brain studies

Pooled IgG (800 μg) from patients or controls was injectedvia tail vein to pregnant mice on days 14 15 and 16 (E14E15 and E16) of gestation (figure 1A) These experimentswere planned according to the window of time in which theneonatal receptor (FcRn) which allows IgG transplacentaltransfer is expressed in placental tissue and the immaturefetal blood-brain barrier (BBB) does not restrict the crossingof IgG (by day E16 the BBB becomes more restrictive)20

The presence of human NMDAR-abs in blood from pregnantmice and fetuses was demonstrated with immunolabeling oflive rat hippocampal neurons in culture and conventionalCBA1 Antibody titers were obtained by serial sample dilutionusing CBA

Determination of human IgG NMDAR clustersand cortical plate thickness in fetal brain tissueTo determine whether human NMDAR-abs injected topregnant mice reached the brain of fetuses 5-μm-thick fetalbrain sections were immunostained for human IgG usingAlexa Fluor 488 goat anti-human IgG as above Quantifica-tion of fluorescence intensity was performed using the publicdomain Fiji ImageJ software (fijiscFiji)8 Confirmation thatpatientsrsquo IgG was bound to NMDAR was done byimmunoprecipitation18

The effects of patientsrsquo antibodies on total cell-surface andsynaptic NMDAR clusters and postsynaptic density protein95 (PSD95) were examined using immunohistochemistry

GlossaryBBB = blood-brain barrier CBA = cell-based assay fEPSP = field excitatory postsynaptic potential GEE = generalizedestimated equation GLM = generalized linear model IgG = immunoglobulin G LTP = long-term potentiation NMDAR =NMDA receptor NMDAR-ab = NMDAR antibody NOL = novel object location PD = postnatal day PPI = prepulseinhibition PSD95 = postsynaptic density protein 95




















































































Appendix Authors





MarijaRadosevicPhD



AnikaPupak MSc



VeronicaBrito PhD



Jose RıosMSc



EstherAguilar BS



EstibalizMaudesMSc











FrancescoMannaraPhD









Silvia GinesPhD



JesusPlanagumaPhD



JosepDalmauMD PhD












































Reprints






















































































Appendix Authors





MarijaRadosevicPhD



AnikaPupak MSc



VeronicaBrito PhD



Jose RıosMSc



EstherAguilar BS



EstibalizMaudesMSc











FrancescoMannaraPhD









Silvia GinesPhD



JesusPlanagumaPhD



JosepDalmauMD PhD












































Reprints












































Reprints




placental transfer of nmdar antibodies causes reversible

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