Planagumà J, Leypoldt F, Mannara F, Gutiérrez-Cuesta J, Martín-García E, Aguilar E, Titulaer MJ

16 Pages·2014·2.18 MB·English

doi:10.1093/brain/awu310 BRAIN2015:138;94–109 | 94
Human N-methyl D-aspartate receptor
antibodies alter memory and behaviour in mice
Jesu´s Planaguma`,1,2,* Frank Leypoldt,1,3,* Francesco Mannara,1,4,* Javier Gutie´rrez-Cuesta,4
Elena Mart´ın-Garc´ıa,4 Esther Aguilar,1 Maarten J. Titulaer,5 Mar Petit-Pedrol,1 Ankit Jain,6
Rita Balice-Gordon,6 Melike Lakadamyali,2 Francesc Graus,1,7 Rafael Maldonado4 and
Josep Dalmau1,8,9
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SeeHonnorat(doi:10.1093/brain/awu342) fora scientificcommentary onthis article. w
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*Theseauthors contributed equallytothis work. lo
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Anti-N-methyl D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder that associates with prominent fro
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memory and behavioural deficits. Patients’ antibodies react with the N-terminal domain of the GluN1 (previously known as NR1) h
subunit ofNMDARcausing inculturedneuronsa selectiveandreversibleinternalizationofcell-surfacereceptors.Theseeffectsand ttp://b
the frequent response to immunotherapy have suggested an antibody-mediated pathogenesis, but to date there is no animal model ra
in
showing that patients’ antibodies cause memory and behavioural deficits. To develop such a model, C57BL6/J mice underwent .o
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placement of ventricular catheters connected to osmotic pumps that delivered a continuous infusion of patients’ or control cere- fo
rd
brospinalfluid(flowrate0.25ml/h,14days).Duringandaftertheinfusionperiodstandardizedtestswereapplied,includingtasksto jo
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assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviours (sucrose preference test), rn
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depressive-like behaviours (tail suspension, forced swimming tests), anxiety (black and white, elevated plus maze tests), aggressive- .o
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ness (resident-intruder test), and locomotor activity (horizontal and vertical). Animals sacrificed at Days 5, 13, 18, 26 and 46 were b/
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examined for brain-bound antibodies and the antibody effects on total and synaptic NMDAR clusters and protein concentration g
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using confocal microscopy and immunoblot analysis. These experiments showed that animals infused with patients’ cerebrospinal s
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fluid, but not control cerebrospinal fluid, developed progressive memory deficits, and anhedonic and depressive-like behaviours, n
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withoutaffectingotherbehaviouralorlocomotortasks.MemorydeficitsgraduallyworseneduntilDay18(4daysaftertheinfusion ov
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stopped) and all symptoms resolved over the next week. Accompanying brain tissue studies showed progressive increase of brain- m
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bound human antibodies, predominantly in the hippocampus (maximal on Days 13–18), that after acid extraction and character- r 1
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izationwithGluN1-expressinghumanembryonickidneycellswereconfirmedtobeagainsttheNMDAR.Confocalmicroscopyand , 2
0
immunoblot analysis of the hippocampus showed progressive decrease of the density of total and synaptic NMDAR clusters and 15
total NMDAR protein concentration (maximal on Day 18), without affecting the post-synaptic density protein 95 (PSD95) and
a-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid(AMPA)receptors.Theseeffectsoccurredinparallelwithmemoryandother
behaviouraldeficitsandgraduallyimprovedafterDay18,withreversibilityofsymptomsaccompaniedbyadecreaseofbrain-bound
antibodies and restoration of NMDAR levels. Overall, these findings establish a link between memory and behavioural deficits and
antibody-mediatedreductionofNMDAR,providethebiologicalbasisbywhichremovalofantibodiesandantibody-producingcells
improve neurological function, and offer a model for testing experimental therapies in this and similar disorders.
1 Institut d’Investigacions Biome`diquesAugust Pi i Sunyer(IDIBAPS), Hospital Cl´ınic, Universitat deBarcelona, Barcelona, Spain
2 ICFO-Institut deCie`nciesFoto`niques,Barcelona, Spain
3 Institute ofClinical Chemistry, Neuroimmunology Unit,University MedicalCentre Schleswig-Holstein CampusLu¨beck,
Germany
4 Laboratori deNeurofarmacologia, Facultat deCie`ncies delaSalut idela Vida,Universitat PompeuFabra, Barcelona, Spain
5 Department ofNeurology, ErasmusMedical Centre, Rotterdam, TheNetherlands
ReceivedMay6,2014.RevisedAugust11,2014.AcceptedSeptember8,2014.AdvanceAccesspublicationNovember12,2014
(cid:2)TheAuthor(2014).PublishedbyOxfordUniversityPressonbehalfoftheGuarantorsofBrain.Allrightsreserved.
ForPermissions,pleaseemail:[email protected]
Mouse model of anti-NMDA receptor encephalitis BRAIN 2015: 138; 94–109 | 95
6 Department ofNeuroscience, University ofPennsylvania, PA, USA
7 Servei deNeurologia, Hospital Cl´ınic, Universitat deBarcelona, Barcelona, Spain
8 Department ofNeurology, University ofPennsylvania, Philadelphia,PA, USA
9 Institucio´ Catalana deRecerca i EstudisAvanc¸ats(ICREA), Barcelona, Spain
Correspondence to: JosepDalmau, MD, PhD,
IDIBAPS-Hospital Cl´ınic,
Universitat deBarcelona, Department ofNeurology,
c/Villarroel 170, 08036,Barcelona, Spain
E-mail: [email protected]
Keywords: animal model; anti-NMDAR encephalitis; antibodies; pathogenesis; mechanism
Abbreviations: AMPAR=a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidreceptor; NMDAR=N-methyl D-aspartate
receptor; PSD95=post-synaptic density protein 95
Introduction immunotherapy (accompanied by removal of an underlying
tumour, usually an ovarian teratoma, when appropriate),
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Memory, learning, and behaviour depend on the proper or sometimes spontaneously (Iizuka et al., 2008; Titulaer o
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function of the excitatory glutamate N-methyl D-aspartate et al., 2013). nlo
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receptor (NMDAR) and a-amino-3-hydroxy-5-methyl- Investigations on the potential pathogenic role of patients’ de
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4-isoxazolepropionic acid receptor (AMPAR) and underly- antibodiesusingculturedneuronsshowedthattheantibodies fro
ing mechanisms of synaptic plasticity (Lau and Zukin, causedcrosslinkingandselectiveinternalizationofNMDARs m
h
2007; Shepherd and Huganir, 2007). The critical role of thatcorrelatedwiththeantibodytitres,andtheseeffectswere ttp
NMDAR in these functions has been shown in animal reversibleafterremovingtheantibodies(Hughesetal.,2010; ://b
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models in which the NMDAR are altered genetically Mikasovaetal.,2012).Incontrast,patients’antibodiesdidnot in
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(Mohnetal.,1999;Belforteetal.,2010)orpharmacologic- alterthelocalizationorexpressionofothersynapticproteins, x
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ally (Jentsch and Roth, 1999; Mouri et al., 2007). In numberofsynapses,dendriticspines,dendriticcomplexity,or rd
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humans this evidence comes from more indirect cellsurvival(Hughesetal.,2010).Inparallelexperiments,the urn
observations such as studies investigating the effects of density of NMDAR was also significantly reduced in the als
phencyclidine or ketamine (non-competitive antagonists of hippocampus of rats infused with patients’ antibodies, a .org
NMDAR that cause psychosis) (Weiner et al., 2000; finding comparable to that observed in the hippocampus of by/
Gunduz-Bruce, 2009), and brain tissue studies of patients autopsiedpatients(Hughesetal.,2010).Overall,thesestudies gu
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with schizophrenia or Alzheimer’s disease in which several suggestedanantibody-mediatedpathogenesis,butthedemon- st o
molecular pathways that modulate glutamate receptor traf- stration that patients’ antibodies caused symptoms remained n N
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ficking or function are affected (Snyder et al., 2005; Hahn pending.Modellingsymptomsandshowingthatthesecorrel- v
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et al., 2006). In 2007 we identified a novel disorder (anti- ate with antibody-mediated reduction of NMDAR would b
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NMDAR encephalitis) that occurs with highly specific anti- prove the pathogenicity of patients’ antibodies, support the r 1
2
bodies against extracellular epitopes located at the amino use of treatments directed toward decreasing the levels of , 2
0
terminal domain of the GluN1 subunit of NMDAR antibodiesorantibody-producingcells,andhelptoinvestigate 15
(Dalmau et al., 2007; Gleichman et al., 2012). The result- experimentaltherapiesinthisandsimilardisorders.Wereport
ing syndrome resembles the spectrum of symptoms that heresuchamodelusingcontinuous14-daycerebroventricular
occurs in genetic or pharmacologic models of NMDAR infusionofpatients’CSFinmice.Theaimsweretodetermine
hypofunction, including memory loss and neuropsychiatric (i) if patients’ antibodies altered memory and behaviour; (ii)
alterations ranging from psychosis to coma (Dalmau et al., whethermicesymptomscorrelatedwithbrainantibody-bind-
2008;Iranietal.,2010;Viaccozetal.,2014).Regardlessof ing and reduction of NMDAR; and (iii) whether the clinical
the type of presentation, most patients develop severe prob- and molecular alterations recovered after stopping the anti-
lems forming new memories and amnesia of the disease. bodyinfusion.
Symptoms are usually accompanied by systemic and intra-
thecal synthesis of antibodies, the latter likely produced by Materials and methods
plasma cells contained in brain inflammatory infiltrates
(Dalmau et al., 2008; Martinez-Hernandez et al., 2011).
Animals
These long-lived plasma cells and persistent antibody syn-
thesis may explain the lengthy symptoms of most patients
MaleC57BL6/Jmice(CharlesRiver),8–10weeksold(25–30g)
(average hospitalization (cid:2)3 months) (Dalmau et al., were housed in cages of five until 1 week before surgery when
2008). Yet, despite the severity and duration of the disease, they were housed individually. The room was maintained at a
80% of the patients have substantial recovery after controlled temperature (21(cid:3)1(cid:4)C) and humidity (55(cid:3)10%)
96 | BRAIN 2015: 138; 94–109 J. Planaguma` et al.
withilluminationat12-hcycles;foodandwaterwereavailable and Goodwin, 1980; Handley and Mithani, 1984; Steru et al.,
ad libitum. All experiments were performed during the light 1985; Konig et al., 1996; Caille et al., 1999; Strekalova et al.,
phase, and animals were habituated to the experimental room 2006; Taglialatela et al.,2009; Ennaceur, 2010)and following
for 1 week before starting the tests. All procedures were the schedule summarized in Fig. 1D. The tasks were aimed to
conducted in accordance with standard ethical guidelines assess memory (novel object recognition in open field and
(European Communities Directive 86/609/EU) and approved V-maze), anhedonic behaviours (sucrose preference test),
by the local ethical committees: Comite` E`tic d’Experimentacio´ depressive-like behaviours (tail suspension, and forced swim-
Animal, Institut Municipal d’Assiste`ncia Sanita`ria (Universitat ming tests), anxiety (black and white and elevated plus maze
Pompeu Fabra), and Institutional Animal Care and Use tests), aggressiveness (resident-intruder test) and locomotor ac-
Committee(UniversityofPennsylvania). tivity (horizontal and vertical activity assessment). A brief de-
scription of each task is provided in the Supplementary
material.
Patients’ CSF samples
CSF from 25 patients with high titre NMDAR antibodies (all Brain tissue processing
41:320)werepooledandusedforcerebroventricularinfusion.
CSF from 25 subjects without NMDAR antibodies (11 with To determine the effects of patients’ antibodies on mouse
normalpressurehydrocephalus and14with non-inflammatory brain, animals were sacrificed at the indicated time points
CNS disorders) were similarly pooled and used as controls. (Fig. 1D, Days 5, 13, 18, 26 and 46) with CO2. Brains were
Before loading the osmotic pumps (discussed below), the harvested, sagittally split,and transferredtoice-cold PBS.Half
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pooled CSF samples from patients and controls were dialyzed of the brain was fixed by immersion in 4% paraformaldehyde o
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(Slide-A-Lyzer 7K, Thermo) against sterile phosphate-buffered (PFA) for1hat4(cid:4)C,cryoprotected with40% sucrosefor48h n
saline (PBS) overnight at 4(cid:4)C, and the concentration of total at 4(cid:4)C, embedded with freezing media, and snap-frozen with load
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IdglG. AnllomrmicaelizreecdeitvoedthteheCSsaFmpehypsoioolleodgiCcScFoneciethnetrraftrioomn opfa2tiemngts/ iwsoapseunstaednefocrhildleisdsewctiitohnlioqfuihdipnpiotrcoagmepnu.sTahnedotcheererbhelallufm-brafoinr d from
or controls. Studies were approved by the institutional review IgG and protein extraction (see below). h
bBoioamrde`diqoufesHAouspgiutastl PCi l´ıiniScunyaenrd(IDInIsBtiAtuPtS),dU’Innviveesrtisgitaactiodnes Immunohistochemistry and quanti- ttp://bra
Barcelona. tative peroxidase staining in.o
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Surgery, placement of ventricular For determination of antibodies bound to brain tissue using rdjo
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catheters and osmotic pumps immunoperoxidase staining, 7-mm thick tissue sections were rna
sequentially incubated with 0.25% H2O2 for 10min at 4(cid:4)C, ls.o
Cerebroventricular infusion of CSF was performed using 5go%atgaonatti-sheurmumanfoIgrG15(1m:2in00a0t,rVooecmtortemlabpse)raotuverern,ibgihottiantyl4a(cid:4)teCd, brg/
osmotic pumps (model 1002, Alzet) with the following char- y
acteristics: volume 100ml, flow rate 0.25ml/h, and duration 14 and the reactivity developed using avidin-biotin-peroxidase gu
e
days. Twenty-four hours before surgery, two osmotic pumps and diaminobenzidine. Sections were mildly counterstained st o
per animal were each loaded with 100ml of patient or control with haematoxylin, and results photographed under a Leica n N
DMD108 microscope. Images were prepared creating a mask o
CSF. The pumps were then connected to a 0.28mm IM v
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(internal diameter) polyethylene tube (C314CT, PlasticsOne) for diaminobenzidine colour, converting the mask to greyscale m
b
and left overnight in sterile PBS at 37(cid:4)C. The next day, mice intensities, and inverting the pixels using Adobe Photoshop er 1
wmeixreturdeeeopflykaentaamesitnheeti(z1e0d0bmyg/kingt)rapanerditoxnyelaalziinnejec(t1io0nmgo/fkga) CbeSl6larparecgkiaognes. wHeiprepomcaanmupaallly, forountltianledco;rintetxen,ssittryiaatunmd aarneda wceerree- 2, 201
quantified in two serial sections using the public domain Fiji 5
along with subcutaneous administration of the analgesic
ImageJ software (http://fiji.sc/Fiji). Values were divided by area
meloxicam (1mg/kg). Mice were then placed in a stereotaxic
andnormalizedtothegroupwiththehighestmean(definedas
frame, and a bilateral catheter (PlasticsOne, model 3280PD-
100%, patients’ CSF treated animals sacrificed at Day 18).
2.0/SP) was inserted into the ventricles (0.02mm anterior and
1.00mm lateral from bregma, depth 0.22mm) and secured
with dental cement. Each arm of the catheter was connected Immunofluorescence and confocal
tooneosmoticpump,whichwassubcutaneouslyimplantedon
microscopy with brain tissue
the back of the mice. Appropriate ventricular placement of the
catheters was assessed in randomly selected mice injecting For determination of antibodies bound to brain tissue using
methylene blue through the catheters (Fig. 1A–C). immunofluorescence, 5mm-thick tissue sections were blocked
with 5% goat serum and 1% bovine serum albumin for
Cognitive tasks 60min at room temperature, and incubated overnight at 4(cid:4)C
with Alexa Fluor(cid:2) 488 goat anti-human IgG (A11013, diluted
Allbehaviouraltaskswereperformedbyresearchersblindedto 1:1000,MolecularProbes/LifeTechnologies).Slideswerethen
experimental conditions using standardized tests reported by mounted with ProLong(cid:2) Gold (P36930, Molecular Probes)
us (Maldonado et al., 1970; Filliol et al., 2000; Berrendero and results scanned under a LSM710 Zeiss confocal micro-
et al., 2005; Bura et al., 2007, 2010, 2013; Aso et al., 2008; scope. Sections from all animals were analysed in parallel.
Puighermanal et al., 2009; Burokas et al., 2012; Llorente- Quantification of fluorescent intensity in areas of CA1, CA3
Berzal et al., 2013) and others (Porsolt et al., 1977; Crawley and dentate gyrus was done using Fiji ImageJ software.
Mouse model of anti-NMDA receptor encephalitis BRAIN 2015: 138; 94–109 | 97
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Figure1 Experimentaldesignandplacementofventricularcatheters.(A)Representativecoronalmousebrainsectionwithcatheter h
placement.Scalebar=2mm.(BandC)Coronalandsagittalmousebrainsectionsdemonstratingcerebroventriculardiffusionofmethyleneblue ttp
afterventricularinfusion.Scalebars=2mm.(D)Scheduleofcognitivetestingandanimalsacrifice.AtDay0,cathetersandosmoticpumpswere ://b
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placedandbilateralventricularinfusionofpatients’orcontrolCSFstarted.Infusionlastedfor14days.Memory[novelobjectrecognition(NOR)], in
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anhedonia[sucrosepreferencetest(ANH)],depressive-likebehaviour[tailsuspensiontest(TST)andforcedswimmingtest(FST)],anxiety[black x
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andwhitetest(BW)andelevatedplusmazetest(EPM)],aggressiveness[residentintrudertest(RI)]andlocomotoractivity(LOC)wereassessed rd
blindedtotreatmentattheindicateddays.ThenovelobjectrecognitionwasassessedinopenfieldandV-mazeparadigmsintwodifferentcohorts jou
ofmice.Animalswerehabituatedfor1to4daysbeforesurgery(baseline)tonovelobjectrecognition,anhedonia,andlocomotoractivity.Red rna
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arrowheadsindicatethedaysofsacrificeforstudiesofeffectsofantibodiesinbrain. .o
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b/
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g
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s
Background was subtracted and intensity divided by area. algorithms of Huygens Essential software (Scientific Volume t o
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Mean intensity of IgG immunostaining in animals treated Imaging). For cluster density analysis a spot detection algo- N
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with patients’ CSF and sacrificed at Day 18 was defined rithm from Imaris suite 7.6.4 (Bitplane) was used based on v
e
m
as 100%. automatic segmentation of the images to spots (Banovic b
To determine the effects of patients’ antibodies on total et al., 2010). Density of clusters was expressed as spots/mm3. er 1
2
and synaptic NMDAR clusters and PSD95, Three-dimensionalcolocalizationofclusters(e.g.NMDARand , 2
non-permeabilized 5-mm thick sections were blocked with PSD95) was done using a spot co-localization algorithm im- 01
5
5% goat serum and 1% bovine serum albumin as above, plemented in Imaris suite 7.6.4. Synaptic localization was
incubated with human CSF antibodies for 2h at room tem- defined as co-localization of NMDAR or AMPAR with
perature, washed with PBS, permeabilized with TritonTM post-synaptic PSD95. Synaptic cluster density was expressed
X-100 0.3% for 10min at room temperature, and incubated as colocalized spots/mm3. For each animal, five identical
with rabbit polyclonal antibody against PSD95 (diluted 1:250, image stacks in each hippocampal area (CA1, CA2 and den-
Clone 18258 Abcam) overnight at 4(cid:4)C. Next day, the slides tategyrus)wereacquiredandthemeandensitiescalculatedfor
were washed and incubated with the corresponding secondary total and synaptic NMDAR and AMPAR. Densities were nor-
antibodies, Alexa Fluor(cid:2) 594 goat anti-human IgG and Alexa malized to the mean density of control CSF treated animals
Fluor(cid:2) 488 goat anti-rabbit IgG (A-11014, A-11008, both (100%). For the AMPAR the antibody used was guinea pig
diluted 1:1000, Molecular Probes) for 1h at room tempera- GluA1 antibody (1:100, clone AGP-009, Alomone), and as
ture. Slides were mounted as above and results scanned with a secondary antibody Alexa Fluor(cid:2) 594 goat anti-guinea pig
confocal microscope (Zeiss LSM710) with EC-Plan IgG (A11076, 1:1000, Molecular Probes).
NEOFLUAR CS (cid:5)100/1.3 NA oil objective. Standardized The presence of apoptosis, cellular infiltrates, and comple-
z-stacks including 50 optical images were acquired from five ment was assessed in the hippocampal region (CA3) in mice
different, equallyspaced areas ofCA1,CA3and dentategyrus sacrificed on Day 18 and corresponding controls. Apoptosis
of hippocampus using sequential scanning, 1024(cid:5)1024 lat- was determined by standard terminal deoxynucleotidyl trans-
eral resolution, and Nyquist optimized z-sampling frequency. ferase mediated biotinylated UTP nick end labelling (TUNEL)
Images were deconvolved with 20 iterations using theoretical using the TACS 2TdT-Fluor in situ apoptosis detection kit
point spread functions and maximum likelihood estimation (Trevigen), and immunolabelling of cleaved caspase 3 (1:200,
98 | BRAIN 2015: 138; 94–109 J. Planaguma` et al.
#9661 Cell Signalling, Technologies) using a goat anti-rabbit separated in a 10% SDS gel electrophoresis with semi-dry
Alexa Fluor(cid:2) 488 as secondary antibody (1:1000 Molecular blotting on PVDF membranes. Membranes were blocked in
Probes).Thepresenceofcomplementwasassessedusingrabbit 5% non-fat skimmed milk and incubated overnight at 4(cid:4)C
anti-mouse C5b-9 (1:500, Abcam) and Alexa Fluor(cid:2) 488 goat with the following polyclonal rabbit antibodies: GluN1
anti-rabbit IgG (1:500, #A11008, Molecular Probes). (1:1000, Sigma-Aldrich), GluR2/3 (1:1000, Abcam), and
Immunolabelling for T and B lymphocytes was done using PSD95 (1:1000, Synaptic Systems), or a monoclonal mouse
rabbit anti-mouse CD3 (1:1000, #ab16669 Abcam) followed anti-b-actin (1:20000, Sigma-Aldrich). Membranes were incu-
by secondary antibody goat anti-rabbit Alexa Fluor(cid:2) 488 bated with secondary antibodies for 1h at room temperature
(1:1000, Molecular Probes), and rat anti-CD45R (1/10000, (anti-rabbit IgG HRP 1:1000, anti-mouse IgG HRP 1:10000)
#ab64100) followed by goat anti-rat Alexa Fluor(cid:2) 594 and analysed by enhanced chemiluminescence (all Amersham
(1/1000, #A-11007 Molecular Probes). Results were scanned GE Healthcare) on a LAS4000 (GE Healthcare). All studies
with a confocal microscope Zeiss LSM710. were done in duplicate. Analysed films were in the linear
range of exposure, digitally scanned, and signals quantified
Extraction of human IgG bound to usingFiji ImageJsoftware. Thesignal intensity ofeachantigen
was normalized to that of actin in the same lane. The mean
mice brain
intensity of signal in control CSF treated animals was defined
as 100% and all other intensities expressed in per cent relative
Under a dissection microscope (Zeiss stereomicroscope, Stemi
to this value.
2000), the hippocampus and cerebellum were isolated,
weighed, snap-frozen, and stored at (cid:6)80(cid:4)C. Tissue (10mg) D
was homogenized in 0.5ml ice-cold PBS with protease inhibi- Statistics own
tors (Sigma-Aldrich) and centrifuged at 16000g for 5min. All lo
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stepswereperformedat4(cid:4)C.Washingwasrepeatedfourtimes Behavioural tests were analysed using repeated measures two- de
d
toremoveunboundIgG.Thelastwashwasdonein100mland way ANOVA for tests with multiple time points (novel object fro
thesupernatantsavedaspre-extractionfraction.Toextractthe m
specifically bound antibodies, the pellet was solubilized for rmecootogrniaticotinv,itsyu)c,rionsdeepperenfdeerenntcseamtepstl,erte-stiedsetsntf-oinrttreusdtserwtietsht,slioncgole- http
5min in acid (86ml 0.1 M Na-citrate buffer pH 2.7), centri- time points (forced swimming test, black and white test, ele- ://b
fuged at 16000g for 5min, and the supernatant neutralized vated plus maze test) or by Mann Whitney-U for skewed dis- rain
with 14ml 1.5 M Tris pH 8.8, and used to determine the tributions (tail suspension test). Non-normally distributed .ox
presence of NMDAR (GluN1) antibodies (see below). parameters were log-transformed (black and white test, ele- ford
vated plus maze test). Significance of NMDAR antibody titre jo
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Immunofluorescence with HEK293 in acid-extracted IgG fractions was calculated using the rn
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cells expressing GluN1 Kruskal-Wallis test and Dunn’s post hoc test compared to ls.o
titresatDay46.HumanIgGintensity,confocalclusterdensity brg/
The presence of GluN1 antibodies in IgG extracts from brain and immunoblot data (GluN1, PSD95) from different time y g
was determined using a HEK293 cell-based assay expressing points or regions were analysed using two-way ANOVA ue
s
GluN1, as reported (Dalmau et al., 2008). After fixation with with Sidak-Holm post hoc testing to calculate multiplicity-ad- t o
n
4% paraformaldehyde and permeabilization with 0.3% justed P-values. Confocal cluster density in the different hip- N
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TritonTM X-100, cells were blocked with 1% bovine serum pocampal subregions (CA1, CA3, dentate gyrus) were not ve
m
albumin for 90min, and incubated with undiluted acid- significantly different and were analysed pooled. All experi- b
e
extracted IgG or pre-extraction fraction from brain of infused ments were assessed visually for outliers (e.g. one animal r 1
mice, at 4(cid:4)C overnight. The next day, cells were washed and with very different results from the other animals at the 2, 2
incubated with a mouse monoclonal antibody against a non- same time point), but none were identified, so measurements 01
5
competing GluN1 epitope located at amino acid 660-811 were pooled per time point and treatment (patient or control
(1:20000;cloneMAB363,Millipore)for1hatroomtempera- CSF). For confocal AMPAR cluster density measured at single
ture, followed by the corresponding Alexa Fluor(cid:2) secondary timepoints,independentsamplet-testswereused.AP-valueof
antibodies (A11013, A11032, both diluted 1:1000, Molecular 50.05 was considered significant in post hoc testing after cor-
Probes) for1hat room temperature. Thetitre ofpositive sam- rection for multiple testing (Sidak-Holm). In the two-way
ples was calculated by serial dilutions until the reactivity was ANOVA the cut-off for interaction between two factors was
no longer visible. Results were photographed under a fluores- set at 0.10; if the P-value for interaction was 50.10, the ef-
cence microscope using Zeiss Axiovision software. fects of treatment were considered for the separate time points
(post hoc analysis). All tests were done using GraphPad Prism
Immunoblot analyses (Version 6).
Total protein from hippocampus and cerebellum was obtained
by dissecting these regions from 20-mm thick paraformalde-
Results
hyde-fixed sagittal mouse brain sections on glass slides at
4(cid:4)C under a Zeiss stereomicroscope (Stemi 2000). Two con-
One-hundred and eleven mice were included in the studies,
secutive sections of isolated hippocampus or cerebellum were
then transferred to an Eppendorf tube in PBS supplemented 56 for cognitive and behavioural tests, and 55 for assess-
with protease inhibitors. Loading buffer (RotiLoad) was ment of antibody binding to brain and the effects on total
added, the solubilized tissue boiled for 5min, and the proteins and synaptic NMDAR (Fig. 1).
Mouse model of anti-NMDA receptor encephalitis BRAIN 2015: 138; 94–109 | 99
Cerebroventricular infusion of patients’ CSF or a monoclonal antibody against GluN1
(Dalmau et al., 2008). Upon quantification of immunos-
patients’ CSF alters memory and
taining, the maximal antibody binding was identified in
behaviour in mice
mice sacrificed on Day 18, which had received 14 days of
CSF infusion, compared with mice sacrificed on Days 5 or
The most robust effect during the 14-day infusion of
13 (Fig. 4B and C). In animals sacrificed on Days 26 and
patients’ CSF was on the novel object recognition test in
46 the presence of IgG immunostaining progressively
both the open field and V-maze paradigms (Fig. 2A and B).
decreased. In frontal cortex the dynamics of IgG binding
Compared with animals infused with control CSF, those
were similar to those of the hippocampus (Supplementary
infused with patients’ CSF showed a progressive decrease
Fig. 1), but the amount of IgG was substantially less; in
of the object recognition index, indicative of a memory
otherbrainregionssuchasthecerebellumandstriatum,the
deficit (Bura et al., 2007; Puighermanal et al., 2009;
IgG immunostaining was sparse and not significantly dif-
Taglialatela et al., 2009). The memory deficit became
ferent between animals infused with patients’ CSF or con-
significant on Day 10 and was maximal on Day 18 (4
trol CSF (data not shown).
days after the infusion of CSF had stopped). On Day 25,
Studies with immunofluorescence and confocal micros-
the object recognition index had normalized and was simi-
copy showed that in animals infused with patients’ CSF
lartothatofanimalstreatedwithcontrolCSF(Fig.2Aand
the presence of hippocampal IgG was visible as a punctate
B). For all time-points, the total time spent exploring both
immunolabelling on the surface of neurons and neuronal D
objects (internal control) was similar in animals infused o
processes in contrast to mice infused with control CSF w
wiTthhecopnretrfoerleonrceptaotiednrtisn’kCsSwFee(tSeunpepdlewmaetnetra(rsyucTraobselep1r)e.fer- where minor amounts of IgG reactivity without preference nload
for neuronal structures were noted (Fig. 4D–G). In add- ed
ence test) was used as a measure of anhedonic behaviour. ition, the amount of human IgG bound to all selected re- fro
Mice infused with patients’ CSF and tested during the m
gions of hippocampus was significantly higher than in the h
infusion period (Day 10) had less preference for sucrose ttp
compared with mice infused with control CSF (Fig. 2C). control group (Fig. 4H). ://b
In contrast, the same mice tested 10 days after the infusion To determine if the IgG immunostaining represented rain
of CSF had stopped (Day 24) showed a preference for su- brain-bound NMDAR antibodies, IgG was extracted from .ox
several brainregionsandexaminedforreactivitywithHEK fo
crose similar to that of the control mice. The total con- rd
cells expressing GluN1. These studies showed that the IgG jo
sumption of water with and without sucrose was similar u
extracted from hippocampus of mice infused with patients’ rn
iInn baodtdhitigorno,uptws o(intteesrtnsalocfodnetrporle,ssSiuvep-plilkeme ebnethaaryvioTuarblewe1r)e. CSF reacted specifically with GluN1 (Fig. 5A). The als.org
performed. The tail suspension test, performed on Day NMDAR antibody concentration in the extracts correlated b/
12, showed that animals infused with patients’ CSF had with the duration of infusion of CSF; it increased until Day y g
u
lwointhgercopnetrriooldsCSoFf i(mFmig.ob2iDlit)y. Icnomcopnatrreadstw, i6thdtahyosseafitnefrustehde 1an3d, rdeeaccrheeadsedthaeftmerawxairmdasl(Fcoign.c5enAtraantidonC)o.nNDMaDysAR13a–n1t8i-, est on N
infusion of CSF had stopped (Day 20), no differences were bodies were also detected in IgG extracts from other brain ov
notedwiththeforcedswimmingtest(examiningimmobility regions (frontal cortex, cerebellum) but at lower concentra- em
b
in inescapable situations; Fig. 2E and Supplementary Table tion to that obtained from hippocampus (Fig. 5D). er 1
Demonstration that the extracted antibodies were specific- 2
1). Overall, these findings suggest that the infusion of , 2
ally bound to the NMDAR was provided by the lack of 0
NMDAR antibodies was associated with anhedonic and 1
5
GluN1 reactivity in the pre-extraction fractions (Fig. 5B
depressive-like behaviours.
and E). Parallel studies with tissue from animals infused
In contrast to the prominent memory deficit, along with
with control CSF did not show NMDAR antibodies
anhedonia and depressive behaviour, no significant differ-
(Supplementary Fig. 2).
ences were noted in tests of anxiety (black and white test,
elevated plus maze test), aggression (resident-intruder test)
and locomotor activity (Fig. 3A–D). Effects of patients’ antibodies on
NMDAR
Patients’ antibodies bind to NMDAR
To determine the effects of patients’ antibodies on
in mouse brain
NMDAR, we focused on the hippocampus, which was
Animals infused with patients’ CSF, but not control CSF, the region with maximal concentration of NMDAR-
had progressively increasing human IgG immunostaining bound antibodies. Compared with animals infused with
(representing IgG bound to brain) that correlated with the control CSF, those infused with patients’ CSF had on
duration of the infusion. The distribution of IgG immunos- Days 13 and 18 a significant decrease of the density of
taining predominated in regions with high density of total and synaptic hippocampal NMDAR clusters followed
NMDAR, mainly the hippocampus (Fig. 4A), resembling by a gradual recovery after Day 18 (pooled analysis of
that obtained with brain sections directly incubated with CA1, CA3 and dentate gyrus; Fig. 6A–D). No significant
100 | BRAIN 2015: 138; 94–109 J. Planaguma` et al.
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Figure2 InfusionofCSFfrompatientswithNMDARantibodiescausesdeficitsinmemory,anhedoniaanddepressive-like ra
behaviour.(AandB)Novelobjectrecognitionindexinopenfield(A)orV-mazeparadigms(B)inanimalstreatedwithpatients’CSF(grey in.o
x
circles)orcontrolCSF(whitecircles).Ahighindexindicatesbetterobjectrecognitionmemory.(C)Preferenceforsucrose-containingwaterin fo
animalsinfusedwithpatients’CSF(grey)orcontrolCSF(white).Lowerpercentagesindicateanhedonia.(DandE)Totaltimeofimmobilityintail- rd
jo
suspensiontestduringtheinfusionperiod(D,Day12)andinforcedswimmingtestaftertheinfusionperiod(E,Day20).Dataarepresentedas urn
mean(cid:3)SEM(median(cid:3)IQRinD).Numberofanimals:patients’CSFn=18(openfieldnovelobjectrecognitionn=8),controlCSFn=20(open als
fieldnovelobjectrecognitionn=10).Significanceoftreatmenteffectwasassessedbytwo-wayANOVA(A–C)withana-errorof0.05andpost .org
hoctestingwithSidak-Holmadjustment(asterisks),unpairedt-test(E)orMann-WhitneyUtest(D).*P50.05,***P50.001.SeeSupplementary b/
y
Table1fordetailedstatistics. g
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s
t o
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N
o
differences inbetween hippocampal subregions (CA1, CA3, hippocampus of animals infused with patients’ or control ve
m
dentate gyrus) were observed (not shown). In contrast, CSF, examined on Day 18, showed no abnormalities b
e
patients’ antibodies did not alter the density of PSD95 or (Fig. 8). r 1
2
AMPAR clusters (Fig. 6E and F). , 2
0
1
Immunoblot analysis of total protein extracted from 5
hippocampusshowedthatonDays13and18,miceinfused Discussion
with patients’ CSF had a significant decrease of total
NMDAR protein concentration compared with mice We report that passive transfer of NMDAR antibodies by
infused with control CSF (Fig. 7A and B). The magnitude continuous ventricular infusion of CSF from patients with
of this effect was greater in animals with higher concentra- anti-NMDAR encephalitis causes memory and behavioural
tion of IgG bound to hippocampus (Fig. 7C). Parallel deficits in mice, and that the effects are likely mediated by
studies examining the effect on the protein concentrations the binding of antibodies to NMDAR resulting in a specific
of PSD95 (Fig. 7A and E) and AMPAR (Fig. 7D) demon- decrease of the density of these receptors. Data from earlier
strated no significant differences between mice infused with reports showing that despite the severity and duration of
patients’ CSF or control CSF. symptoms, most patients with anti-NMDAR encephalitis
In cerebellum, no significant effects on the cluster density respond to immunotherapy (Gresa-Arribas et al., 2014),
or total protein concentration of NMDAR, PSD95 and and findings at the cellular level demonstrating that pa-
AMPAR were noted in animals infused with patients’ tients’ antibodies cause a titre-dependent decrease of syn-
CSF compared to those infused with control CSF (data aptic NMDAR receptors fulfilled most of the Witebsky’s
not shown). criteria for an antibody-mediated disease (Rose and Bona,
Immunohistochemical studies for neuronal apoptosis, 1993), but the transfer of symptoms to animals was pend-
infiltrates of T or B cells, and deposits of complement in ing. In the current study, four sets of experiments satisfy
Mouse model of anti-NMDA receptor encephalitis BRAIN 2015: 138; 94–109 | 101
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Figure3 InfusionofCSFfrompatientswithNMDARantibodiesdoesnotalterthetestsofanxiety,aggressionandlocomotor in
.o
activity.(AandB)Numberofentriesintobright/opencompartmentsduringa5minperiodinastandardblackandwhite(A,Day6)orelevated xfo
plusmazetest(B,Day14)inanimalstreatedwithpatients’CSF(filledcircles)orcontrolCSF(opencircles).(C)Numberofaggressiveevents rd
jo
overa4-minperiodinaresidentintruderparadigminbothtreatmentgroups.(D)Horizontal(solidlines)andvertical(dashedlines)movement u
rn
countovera10minperiodinbothtreatmentgroups.Dataarepresentedasmean(cid:3)SEM.Numberofanimals:patients’CSFn=18,controlCSF a
ls
n=20.StatisticalassessmentasindicatedinFig.2andSupplementaryTable1. .o
rg
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this postulate: (i) the development of symptoms in animals impairment in the visual recognition of objects or faces t o
n
infused with patients’ CSF, but not control CSF; (ii) the (e.g. physicians, nurses) (Frechette et al., 2011). Owing to N
o
demonstration that the infused antibodies reacted predom- the wide range of symptoms of the disease and lack of ve
m
inantly with brain regions with high density of NMDAR previous studies examining the distribution of brain tissue b
e
(e.g. hippocampus) and specifically recognized these recep- NMDAR-antibody binding when these antibodies are r 1
2
tors; (iii) the identification of a selective decrease of the infused intraventricularly, we used standardized memory , 2
0
1
density of total and synaptic NMDAR clusters and total and behavioural tests. The most notable effects were 5
NMDAR protein concentration without affecting PSD95, observed in the tests of memory (novel object recognition)
and that these effects correlated with the concentration of using different groups of animals in two different para-
brain-bound antibodies; and (iv) the correlation noted be- digms (open field and V-maze). While the first depends
tween the intensity of the abovementioned findings and predominantly on normal hippocampal function, the
time-course of patients’ antibody infusion, as well as be- second is dependent of perirhinal-hippocampal structures
tween the reversibility of symptoms and restoration of (Winters et al., 2004). Compared with animals infused
NMDAR levels after stopping the infusion of CSF with control CSF, those infused with patients’ CSF
antibodies. developed progressive memory deficits, which were
Approximately 75% of patients with anti-NMDAR maximal on Days 13–18 when the highest concentration
encephalitis present with mood and psychiatric alterations of brain-bound NMDAR antibodies and lowest density of
ranging from manic or depressive behaviour to psychosis, NMDAR occurred. Other paradigms affected were related
often followed by stereotyped movements, seizures, or to depressive-like behaviours (tail suspension test) and
decreased level of consciousness (Kayser et al., 2013; anhedonic behaviours (sucrose preference test). We did
Titulaer et al., 2013). Regardless of the presentation, not find significant abnormalities in the tests of aggression
most patients develop severe problems forming new mem- and anxiety, which are often present in the human disease,
ories and have amnesia of the disease. Close examination or in locomotor activity (an expected finding given that
during the phase of recovery shows, in some patients, paralysis rarely occurs in patients).
102 | BRAIN 2015: 138; 94–109 J. Planaguma` et al.
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Figure4 Animalsinfusedwithpatient’sCSFhaveaprogressiveincreaseofhumanIgGboundtohippocampus.(AandB)
ImmunostainingofhumanIgGinsagittalbrainsections(A)andhippocampus(B)ofrepresentativeanimalsinfusedwithpatients’CSF(left)and
controlCSF(right),sacrificedattheindicatedexperimentaldays.Inanimalsinfusedwithpatients’CSFthereisagradualincreaseofIgG
immunostaininguntilDay18,followedbydecreaseofimmunostaining.Scalebars:A=2mm;B=200mm.(C)Quantificationofintensityof
humanIgGimmunolabellinginhippocampusofmiceinfusedwithpatients’CSF(darkgreycolumns)andcontrolCSF(lightgreycolumns)sacrificed
attheindicatedtimepoints.(D–H)ConfocalmicroscopyanalysisofIgGboundtothehippocampusonDay18.(D)Sagittalsectionofthe
hippocampuswithareasexaminedathighermagnificationinE(arrowinCA1),F(arrowheadsinCA3)andG(asterisksindentategyrus).Note
thefinepunctateIgGimmunolabellingsurroundingneuronalbodiesinmiceinfusedwithpatients’CSF;thisimmunolabellingissimilartothat
reportedinbrainsectionsdirectlyincubatedwithpatients’antibodies,asinDalmauetal.(2008).Scalebars:D=200mm;E–G=10mm.(H)
QuantificationoftheintensityofhumanIgGimmunofluorescenceintheindicatedareasinanimalsinfusedwithpatients’CSF(darkgreycolumns)
orcontrolCSF(lightgreycolumns).Forallquantifications,meanintensityofIgGimmunostaininginthegroupwiththehighestvalue(animals
treatedwithpatients’CSFandsacrificedatDay18)wasdefinedas100%.Alldataarepresentedasmean(cid:3)SEM.Foreachtimepointfiveanimals
infusedwithpatients’CSFandfivewithcontrolCSFwereexamined.Significanceoftreatmenteffectwasassessedbytwo-wayANOVAwithan
a-errorof0.05(*)andposthoctestingwithSidak-Holmadjustment($).***,$$$P50.001;$P50.05.SeeSupplementaryTable2fordetailed
statistics.
Mouse model of anti-NMDA receptor encephalitis BRAIN 2015: 138; 94–109 | 103
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Figure5 ThehumanIgGextractedfrombrainofmiceinfusedwithpatients’CSFisspecificforNMDARs.(AandB)HEK293 ra
in
cellsexpressingtheGluN1subunitoftheNMDARimmunolabelledwithacid-extractedIgGfractions(toprowinA)orpre-extractionfractions .o
x
(toprowinB)fromhippocampusofmiceinfusedwithpatients’CSFandsacrificedontheindicateddays.ThemaximalreactivitywithGluN1- fo
rd
expressingcellswasnotedinacid-extractedIgGfractionsfromDays13and18(A);noneofthepre-extractionfractionsshowedGluN1reactivity jo
u
(B)indicatingthatthereactivityofacid-extractedfractionscorrespondstoIgGantibodiesthatwereboundtobrainNMDARreceptors.The rn
a
secondrowinAandBshowsthereactivitywithamonoclonalGluN1antibody,andthethirdrowthecolocalizationofimmunolabelling.Scale ls
.o
bars=10mm.(C)QuantificationofNMDARantibodytitreinIgG-extractedfractionsfromhippocampusofanimalstreatedwithpatients’CSF rg
(n=5micepereachtimepoint,exceptfourmiceforDay5).Solidline=median.SignificancewastestedbyKruskal-Walliswithana-errorof0.05 b/
y
(asterisks)andposthoctestingwithDunn’stest($).**,$$P50.01,***,$$$P50.001.SeeSupplementaryTable2fordetailedstatistics.(DandE) gu
e
HEK293cellsexpressingtheGluN1subunitoftheNMDARimmunolabelledwithacid-extractedIgGfractions(D)andpre-extractionfractions st o
(E)fromhippocampus(Hippo),cerebralcortex(Ctx)andcerebellum(Cb)ofmiceinfusedwithpatients’CSF(Day18).Theacid-extractedIgG n
N
fractionfromhippocampusshowedhigherlevelofNMDARantibodiesthanthoseextractedfromcerebralcortex(Ctx)andcerebellum(Cb). o
v
Scalebars=10mm.n.s=notsignificant. em
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, 2
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1
The high levels of brain-bound NMDAR antibodies at least 4 days after the ventricular infusion stops and the 5
between Days 13–18 suggests that after stopping the infu- subsequent recovery starts.
sion of patients’ CSF on Day 14, the NMDAR antibodies Although the hippocampus was the region with the
continued being distributed from mice cerebroventricular highest concentration of brain-bound NMDAR antibodies,
system to parenchyma. This distribution occurred slowly; theseantibodieswerealsoextractedfromcerebralcortexor
for example, 5 days after starting the infusion of patients’ cerebellum though at much lower levels. The higher con-
CSF the amount of NMDAR antibodies that had reached centration of antibodies and predominant decrease of
the hippocampus was very limited compared to that seen NMDAR in the hippocampus are consistent with the
on Days 13–18 (shown in Fig. 4B). Moreover, previous predominant binding of human antibodies to this brain
studies using cultured neurons treated with patients’ CSF region when sections of rodent brain are directly incubated
showed that once the antibodies bound to the NMDARs, with patients’ antibodies (Dalmau et al., 2007; Moscato
the reduction of receptors was microscopically visible in 2h et al., 2014). Additionally, because of the close spatial
but it took 12h to result in the lowest receptor density. relationship to the ventricles, the intraventricular infusion
Subsequently, there was a steady state of low NMDAR of human CSF antibodies might have contributed to the
density foraslongas theneuronswere exposedtopatients’ preferential binding to the hippocampus.
antibodies (Moscato et al., 2014). Together, these findings The correlation between the concentration of brain-
explain the progressive worsening of symptoms along with bound antibodies and selective reduction of NMDAR
continued antibody binding and decrease of NMDAR for cluster density and protein concentration was similar to