Siponimod (BAF312) prevents synaptic neurodegeneration in experimental multiple sclerosis

13 Pages·2016·3.21 MB·English

by  Antonietta Gentile

Gentileetal.JournalofNeuroinflammation (2016) 13:207
DOI10.1186/s12974-016-0686-4
RESEARCH Open Access
Siponimod (BAF312) prevents synaptic
neurodegeneration in experimental
multiple sclerosis
Antonietta Gentile1,2†, Alessandra Musella1†, Silvia Bullitta1, Diego Fresegna1,2, Francesca De Vito1,2,
Roberta Fantozzi3, Eleonora Piras4, Francesca Gargano4, Giovanna Borsellino4, Luca Battistini4, Anna Schubart5,
Georgia Mandolesi1*† and Diego Centonze2,3†
Abstract
Background: Data from multiple sclerosis (MS) and the MS rodent model, experimental autoimmune
encephalomyelitis(EAE),highlightedaninflammation-dependentsynaptopathyatthebasisoftheneurodegenerative
damagecausingirreversibledisabilityinthesedisorders.Thissynaptopathyischaracterizedbyanimbalancebetween
glutamatergicandGABAergictransmissionandhasbeenproposedtobeapotentialtherapeutictarget.
Siponimod(BAF312),aselectivesphingosine1-phosphate1,5receptormodulator,iscurrentlyunderinvestigationina
clinicaltrialinsecondaryprogressiveMSpatients.Weinvestigatedwhethersiponimod,inadditiontoitsperipheral
immunemodulation,mayexertdirectneuroprotectiveeffectsinthecentralnervoussystem(CNS)ofmicewithchronic
progressiveEAE.
Methods:Minipumpsallowingcontinuousintracerebroventricular(icv)infusionofsiponimodfor4weekswere
implantedintoC57BL/6micesubjectedtoMOG -inducedEAE.Electrophysiology,immunohistochemistry,western
35-55
blot,qPCRexperiments,andperipherallymphocytecountswereperformed.Inaddition,theeffectofsiponimodon
activatedmicrogliawasassessedinvitrotoconfirmthedirecteffectofthedrugonCNS-residentimmunecells.
Results:Siponimodadministration(0.45μg/day)inducedasignificantbeneficialeffectonEAEclinicalscoreswith
minimaleffectonperipherallymphocytecounts.SiponimodrescueddefectiveGABAergictransmissioninthestriatum
ofEAE,withoutcorrectingtheEAE-inducedalterationsofglutamatergictransmission.Weobservedasignificant
attenuationofastrogliosisandmicrogliosistogetherwithreducedlymphocyteinfiltrationinthestriatumofEAEmice
treatedwithsiponimod.Interestingly,siponimodreducedthereleaseofIL-6andRANTESfromactivatedmicroglialcells
invitro,whichmightexplainthereducedlymphocyteinfiltration.Furthermore,thelossofparvalbumin-positive(PV+)
GABAergicinterneuronstypicalofEAEbrainswasrescuedbysiponimodtreatment,providingaplausibleexplanation
oftheselectiveeffectsofthisdrugoninhibitorysynaptictransmission.
Conclusions:Altogether, our results show that siponimod has neuroprotective effects in the CNS of EAE mice,
which are likely independent of its peripheral immune effect, suggesting that this drug could be effective in
limiting neurodegenerative pathological processes in MS.
Keywords: Striatum,Synaptictransmission,GABA,Neurodegeneration,Experimentalautoimmuneencephalomyelitis,
Parvalbuminneuron
(Continuedonnextpage)
*Correspondence:[email protected]
†Equalcontributors
1LaboratoryofNeuroimmunologyandSynapticTransmission,IRCCS
FondazioneSantaLucia,CentroEuropeodiRicercasulCervello(CERC),00143
Rome,Italy
Fulllistofauthorinformationisavailableattheendofthearticle
©2016TheAuthor(s).OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Gentileetal.JournalofNeuroinflammation (2016) 13:207 Page2of13
(Continuedfrompreviouspage)
Abbreviations:BBB,Bloodbrainbarrier;BDNF,Brain-derivedneurotrophicfactor;CFA,CompleteFreund’sadjuvant;
CNS,Centralnervoussystem;DAB,Diaminobenzidine;DAPI,4′,6-Diamidino-2-phenylindole;DMSO,Dimethylsulfoxide;
dpi,Daypost-immunization;EAE,Experimentalautoimmuneencephalomyelitis;GABA,Gamma-aminobutyricacid;
GFAP,Glialfibrillaryaminoacidprotein;GM,Graymatter;IBA1,Ionizedcalciumbindingadaptorprotein-1;
icv,Intracerebroventricular;IL,Interleukin;MOG,Myelinoligodendrocyteglycoprotein;MRM,Multiplereaction
monitoring;MS,Multiplesclerosis;MSN,Mediumspinyneuron;PBS,Phosphatebuffersolution;PPMS,Primary
progressiveMS;PV,Parvalbumin;qPCR,Quantitative-polymerasechainreaction;RANTES,Regulatedonactivation,
normalTcellexpressedandsecreted;RRMS,Relapsing-remittingMS;S1P,Sphingosine-1-phosphatereceptor;
sEPSC,Spontaneousexcitatorypost-synapticcurrents;sIPSCs,Spontaneousinhibitorypost-synapticcurrents;
SPMS,SecondaryprogressiveMS;TBS,Trisbufferedsolution;TNF,Tumornecrosisfactor;WB,Westernblot
Background synaptopathy hasbeen proposed as avaluabletherapeutic
Multiple sclerosis (MS) is an inflammatory neurodegen- target[11].
erative disease of the central nervous system (CNS), af- Severaldrugsareunderinvestigationfortheirneuropro-
fecting young adults. Clinically, two main forms of MS tectiveeffectsinprogressiveMS.Inparticular,anongoing
can be distinguished, the relapsing-remitting (RRMS) and phase-III clinical trial is currently conducted with the
the progressive, where the latter is described as the grad- sphingosine-1-phosphate receptor (S1P) modulator,
ualprogressionofclinicaldisabilityinapatienteitherwith siponimod(BAF312)[17].Siponimodisanext-generation
a preceding relapsing course (secondary progressive MS S1P modulator selective for S1P1 and S1P5 that showed
(SPMS))orwithoutaprecedingrelapsingcourse(primary good safety and tolerability in humans. This compound
progressiveMS(PPMS))[1]. has recently successfully passed a phase-II clinical trial in
Such phenotypic distinction seems to rely on predom- RRMS[18].
inance of distinct pathogenic mechanisms that appear Comparedtofingolimod,anS1Pmodulatorselectivefor
different, though poorly understood, throughout RRMS, S1P1, S1P3, S1P4, and S1P5, the half-life of siponimod is
PPMS, and SPMS [2]. In general, although inflammation shorter, allowing recoveryof peripherallymphocyte count
has a recognized pivotal role in triggering the cascade of toanormalrangewithin1weekaftertreatmentcessation.
events leading to both white and gray matter (GM) The risk of bradycardia is mitigated using a dose titration
damage, anti-inflammatory or immunomodulatory drugs schemeduringtreatmentinitiation[19,20].
havebeenfoundsuccessfulonlyinRRMS[3],withthere- Most of the effects of siponomid are attributed to S1P1
centexceptionofocrelizumab,whichwasfoundtoreduce expressedonlymphocytes,therebypreventinglymphocyte
disability progression also in PPMS [4]. A forthcoming trafficking into the brain, but both S1P1 and S1P5 are
challenge is therefore to identify drugs able to interfere widely expressed in brain-resident cells, like neurons,
with the mechanisms leading to neurodegeneration in microglia,astroglia,andoligodendrocytes[21].
PPMSandinSPMS. The aim of the present study was therefore to assess
GM pathology in MS is increasingly recognized to the central effects of siponimod in mice with myelin
contribute to the progression of the disease. In fact, GM oligodendrocyte glycoprotein peptide 35-55 (MOG )-
35-55
damage occurs early in the disease course, isindependent induced EAE, a model of progressive MS, to provide a
of demyelination, and is associated with neurological and substrate of the putative neuroprotective effects of this
neuropsychological disability [5]. Several studies in ex- drug.
perimental autoimmune encephalomyelitis (EAE), the
murinemodelofMS,haveunderscoredaninflammation- Methods
dependent synaptopathy affecting different brain struc- EAEmodel
tures (striatum, hippocampus, cerebellum) and occurring EAE was induced as described previously [7, 20]. Mice
independently of demyelination [6–10]. In such brain were injected subcutaneously at the flanks with 200 μg
areas of EAE mice, the excitatory glutamatergic trans- of MOG emulsion to induce EAE by active
35-55
mission is potentiated, while the GABAergic inhibitory immunization. The emulsion was prepared under ster-
transmission is reduced: this imbalance in synaptic ile conditions using MOG (85 % purity; Espikem)
35-55
transmission leads to excitotoxicity and subsequently to in 300 μl of complete Freund’s adjuvant (CFA; Difco)
neurodegeneration [11]. Of note, glutamate receptor an- containing Mycobacterium tuberculosis (8 mg/ml, strain
tagonistsandgamma-aminobutyricacid(GABA)agonists H37Ra; Difco) and emulsified with phosphate buffer
exertbeneficialeffectsinbothEAEandMS[12–16].This solution (PBS). All animals were injected with 500 ng
Gentileetal.JournalofNeuroinflammation (2016) 13:207 Page3of13
of pertussis toxin (Sigma) intravenously on the day of absolute cell counts for all samples without the use of
immunization and 2 days later. Control animals received beads.
thesametreatmentas EAEmicewithouttheimmunogen
MOG peptide, including complete CFA and pertussis DeterminationofsiponimodinmousebloodbyLC-MS
toxin (referred to as hereafter as “CFA”). Animals were For quantitative determination, 10 spiked samples from
scored daily for clinical symptoms of EAE according to 0.5upto10,000ng/mlwerepreparedinthesamematrix.
the following scale: 0=no clinical signs, 1=flaccid tail, Proteinswereremoved byprotein precipitationbyadding
2=hindlimb weakness, 3=hindlimb paresis, 4=complete an organic solvent mixture. The organic layer was evapo-
bilateral hindlimb paralysis, and 5=death due to EAE; rated to dryness, and the residue was re-dissolved in
intermediate clinical signs were scored by adding 0.5 HPLCbufferB,containing5mMammoniumformate.
[15, 22]. For each animal, the onset day was recorded Aliquots of 2 μl were directly injected on a Agilent
as the day post-immunization (dpi) when it showed the Eclipse Plus, RRHD 2.1×50 mm reversed-phase column
first clinical manifestations. with 1.8-μm particles,andkeptat 40°C.
Experiments were carried out in accordance with In- For separation, a linear gradient from 50 to 100 % B
ternal Institutional Review Committee, the European Dir- within1.7minwasused.SolventAwas0.2%formicacid
ective 2010/63/EU and the European Recommendations in water and solvent B 0.2 % formic acid in acetonitrile.
526/2007, and the Italian D.Lgs26/2014. All the efforts Theflowwaskeptat500μl/minduringthewholecycle.
were made to minimize the number of animals utilized For detection, the column effluent was guided directly
andtheirsuffering. to the electrospray source of the Agilent 6490 triple
quadrupole MS with parameters optimized for siponi-
Siponimodformulationforminipumpandsurgery mod. Compound and a structure-related internal stand-
Siponimod (Novartis Pharma AG) was dissolved in a ard were detected as their [MH]+ ions with the multiple
solutioncontaining10%Solutol/KolliphorHS15(BASF reaction monitoring (MRM) transition 517.3→159.0,
PharmaSolutions)—finalpHrangebetween6and7—ata 416.1 for siponimod. For data processing, the compound
final concentration of 2 mg/ml. This preparation allowed to internal standard ratio of the extracted ion chromato-
stabilityofthedrugforupto6weeksat37°C. gramswasused.
One week before immunization, mice were implanted The calculation was based on a second order fitted
with subcutaneous osmotic minipumps allowing continu- and 1/x weighted calibration curve (r2=0.9987). The ac-
ous intracerebroventricular (icv) infusion of either vehicle curacy of the 10individually prepared calibrationsamples
or siponimod for 4 weeks (three sets of immunizations) wasbetterthan15%.Therecoveryofthecompoundswas
[8,22].Differentsiponimoddosagesweretested:4.5,0.45, 94.3%(RSD=1.3%).Theprecisionofthecontrolsamples
and0.225μg/day. (n=3), distributed over the whole series, was better than
RSD=1.8 %. The LOQ for siponimod was found to be
Tcellabsolutecount 0.5ng/ml,basedonthelowestcalibrationsample.
T cell absolute count was performed on blood samples
keptfrom themandibular vein ofthemouse. Electrophysiology
For the phenotypic characterization of cell populations, Mice were killed by cervical dislocation, and corticos-
the following antibodies were used: CD8-FITC (Miltenyi triatal coronal slices (200 μm) were prepared from fresh
Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 tissue blocks of the brain with the use of a vibratome
(Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), [6, 15]. Single slices were then transferred to a record-
CD45R(B220)-Violblu(MiltenyBiotec),NK1.1-PE(Milteny ing chamber and submerged in a continuously flowing
Biotec). oxygenated artificial cerebrospinal fluid (ACSF) (34 °C,
At predetermined optimal concentrations, 100 μl of 2–3 ml/min) gassed with 95 % O –5 % CO . The com-
2 2
blood was stained by incubation with the antibodies. position of the control ACSF was (in mM) as follows:
Fifty microliters ofCountBrightAbsolute Counting Beads 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4
(Molecular Probes) was added, and, following lysis of red CaCl2, 11 Glucose, 25 NaHCO3. Whole-cell patch clamp
blood cells, cells were acquired on a CyAn Cytometer recordings were made with borosilicate glass pipettes
(BeckmanCoulter).Bycomparingtheratioofbeadevents (1.8 mm o.d.; 4–8 MΩ), in voltage-clamp mode, at the
to cell events, absolute numbers of cells in the sample holdingpotential(HP)of−80mV.Spontaneousexcitatory
werecalculated. andinhibitorypost-synapticcurrents(EPSCs,IPSCs)were
Some experiments were performed by acquiring the recorded from medium spiny neurons (MSNs) as in
stained blood samples on the CytoFLEX cytometer Centonze et al. [15] and Rossi et al. [6]. Siponimod
(Coulter), equipped with a volumetric sample injection (1 μM) was added to the bath solution for 1 h, before re-
module, which enables volumetric sampling and provides cording. Synaptic events were stored by using P-CLAMP
Gentileetal.JournalofNeuroinflammation (2016) 13:207 Page4of13
9.2(AxonInstruments)andanalyzedofflineonapersonal assays (ID Mm00599684_g1 and ID Mm00607939;
computerwithMiniAnalysis5.1(Synaptosoft,Leonia,NJ, Applied Biosystem).
USA) software. Offline analysis was performed on spon-
taneoussynapticeventsrecordedduringfixedtimeepochs Immunofluorescenceandconfocalmicroscopy
(1–2 min, three to five samplings), sampled every 5 or Micefromtwodifferentimmunizationexperimentswere
10 min. One to six cells per animal were recorded, and deeply anesthetized and intracardially perfused with
fouranimalsweresacrificedforeachexperimentalgroup. ice-cold 4 % paraformaldehyde. Brains were post-fixed
for 2 h and equilibrated with 30 % sucrose at least one
Westernblot overnight. Immunofluorescence was performed as in
Animals were killed by cervical dislocation at 24 dpi. Mandolesi et al. [8] and Gentile et al. [22]. The following
Striata were quicklyremoved, snap frozen indry ice, and primary antibodies were used: rabbit anti-Iba1 (1:750;
homogenized as in Mandolesi et al. [8] and Gentile et al. WakoChemicals,USA),rabbitanti-GFAP(1:500;DAKO),
[22]. Soon after blocking with 5 % milk in Tris buffered rat anti-CD3 (1:300; AbD Serotec). Secondary anti-
solution (TBS), the membrane was incubated for 1 h bodies used are as follows: Alexa Fluor-488 (1:200; Life
withmouseanti-β-actinprimaryantibody(1:20,000;Sigma- Technologies) and Cy3-conjugated donkey anti-rabbit or
Aldrich) followed by anti-mouse IgG HRP (1:10,000, GE anti-rat(1:200;JacksonImmunoResearch).4′,6-diamidino-
Healthcare, formerly Amersham Biosciences) secondary 2-phenylindole (DAPI; 0.01 mg/ml) was used to visualize
antibody. Immunodetection was performed by ECL re- nuclei. All images were acquired using a LSM7 Zeiss
agent (Amersham) and membrane was exposed to film confocal laser-scanner microscope (Zeiss, Göttingen,
(Amersham). Next, a mouse anti-glial fibrillary amino Germany)andprocessedbyNHIImageJsoftware[8,22].
acid protein (GFAP, 1:2000, Immunological Science, over
night) primary antibody was used in combination with Immunohistochemistry
anti-mouse IgG HRP (1:4000 for GFAP; GE Healthcare, A section every third (for a total of eight sections per
formerly Amersham Biosciences) secondary antibody. animal)throughouttherostro-caudalextentofthestriatum
Immunodetection was performed as for β-actin. Densito- was selected for staining and examination. Freshly cut
metric analysis of bands was performed by NIH ImageJ 30-μm serial sections, after blocking of nonspecific
software (http://rsb.info.nih.gov/ij/). Western blot (WB) staining with 10 % normal donkey serum, were incubated
results are presented as data normalized to control CFA overnightwitharabbitpolyclonalantibodyagainstparval-
values. bumin (PV) (1:1000;ImmunologicalScience),thenwitha
biotinylated rabbit secondary antibody (1:500; Vector
RNAextractionandquantitativereal-timePCR Laboratories, Burlingame, CA, USA) for 1 h, and finally
Striata were dissected in RNAse-free conditions. Total withExtravidin(1:1000;SIGMA)for1h.Reactionwasde-
RNA was extracted according to the standard miRNeasy veloped in a freshly prepared diaminobenzidine (DAB)/
Micro kit protocol (Qiagen). Next, 350 ng of total RNA H O (DAB tablet, SIGMA). Sections were mounted on
2 2
was reverse-transcribed using High-Capacity cDNA Re- glassslidesandcoverslippedunderEukitt.
verse Transcription Kit (Applied Biosystem), and 10 ng
of complementary DNA (cDNA) was amplified with Stereology
SensiMix SYBR Hi-Rox Kit (Bioline; Meridian Life Sci- Quantitative observations were limited to the dorsal
ence) in triplicate using the Applied Biosystem 7900HT striatum of the left hemisphere. Using the Stereo Inves-
Fast Real-Time PCR system. Messenger RNA (mRNA) tigatorSystem(MicroBrightFieldEuropee.K.,Magdeburg,
relative quantification was performed using the compara- Germany) composed of a Zeiss Axioimager.M2 micro-
tive cycle threshold (2−ΔΔCt) method. β-actin was used as scope and MicroBrightField’s Stereo Investigator software
endogenouscontrol. package, an optical fractionator, stereological design was
Primer sequences: applied to obtain unbiased estimates of total PV+ cells.
Ionized calcium binding adaptor protein-1 (Iba-1, Samplinggridsandmagnificationswereadjustedtoobtain
NM_019467): 5′-GACAGACTGCCAGCCTAAGACAA-3′ arelativelyconstantnumberofcellssampledandacoeffi-
(sense), 5′-CATTCGCTTCAAGGACATAATATCG-3′ cient of error (CE Gunderson) of ≤0.1. A tri-dimensional
(antisense); optical dissector counting probe (x, y, z dimension of
β-ACTIN (NM_007393): 5′-CCTAGCACCATGAAG 200×200×25μm,respectively)wasapplied.Countswere
ATCAAGATCA-3′ (sense), 5′-AAGCCATGCCAATGT performedusinga×20objective.
TGTCTCT-3′(antisense). Total number was estimated according to the following
For CD3+ mRNA detection, 17.5 ng of the same formula:
cDNA was amplified with SensiMix II Probe (Bioline;
MeridianLifeScience)byusingTaqMangeneexpression N¼∑Q(cid:2)1=ssf (cid:2)1=asf(cid:2)1=tsf:
Gentileetal.JournalofNeuroinflammation (2016) 13:207 Page5of13
where ΣQ represents the total number of neurons lymphocytes in the blood of EAE mice compared to
counted in all optically sampled fields of the dorsal stri- EAE-vehicle mice (n=4, p<0.001; unpaired T test), the
atum, ssf is the section sampling fraction, asf is the area intermediate (0.450 μg/day; n=6) and the lowest
sampling fraction, and tsf is the thickness sampling (0.225 μg/day; n=4) dosages of siponimod induced a
fraction. slight and not significant drop in T lymphocyte counts
with respect to EAE-vehicle mice (unpaired T test; p>
CellculturesupernatantLuminexassay 0.05) (Fig. 1b). This result was consistent with siponi-
The BV2 immortalized murine microglial cell line was mod concentrations found in peripheral blood samples
pre-treated for 1 h with siponimod 0.1 μM in dimethyl (Fig. 1c): intracerebroventricular release of 4.5 μg/day
sulfoxide (DMSO), before incubation with tumor necro- siponimod (n=4) resulted in a high amount of the drug
sis factor (TNF; 200 U/ml; Milteny Biotec); control cells in the peripheral blood (169.9±31.49 nM), significantly
received equal volume of DMSO (n=3 per condition). different from that measured in the blood of mice receiv-
The 24-h conditioned media was assayed for IL-6 and ing 0.45 μg/day (n=3) and 0.225 μg/day (n=5; one-way
RANTES by Luminex assay, according to the manufac- ANOVA p<0.01; Tukey’s post hoc comparisons: 4.5 μg/
turer instructions (R&D systems). The plate was read on day vs 0.45 and 0.225 μg/day, p<0.001). The concentra-
a Luminex-200 instrument (LuminexCorp., Austin,TX). tions of siponimod detected in mice receiving 0.45 or
Concentrations were calculated by using a standard 0.225 μg/day were detectable, but low (13±4.7 nM and
5P-logistic weighted curve generated for each target and 6.6±1.5 nM for the 0.45 and 0.225 μg/day groups, re-
expressedaspicogramspermilliliter(pg/ml). spectively). These levels are not expected to have major
effects on peripheral lymphocytes in the majority of ani-
Statisticalanalysis mals as confirmed by minor effects observed on periph-
For each type of experiment, at least three mice per erallymphocytecount.
group were employed. Throughout the text, “n” refers to From a clinical point of view, such preventive and
the number of animals, except for electrophysiology, CNS-directed treatment reduced motor disability associ-
whereitmeansthenumber ofcells. Datawerepresented ated to EAE in a dose-dependent manner (Fig. 1d). The
as the mean±S.E.M. The significance level was estab- highest dose of siponimod used in this study fully inhib-
lished at p<0.05. Statistical analysis was performed using ited EAE development in treated mice compared to
unpaired Student’s T test for comparisons between two EAE-vehicle (EAE-siponimod n=5, EAE-vehicle n=9;
groups and non-parametric Mann-Whitney test, where from day 15 to 24 p<0.001, non-parametric Mann-
needed. Multiple comparisons were analyzed by one-way Whitney). This result, together with that shown in
ANOVA for independent measures followed by Tukey’s Fig. 1b, c, suggests that siponimod effects on peripheral
HSDorNewman-Keuls. lymphocytesmediateitsdisease-modifyingactivity,when
administered at this daily concentration. Interestingly,
Results whilethe0.450μg/day(n=6)andthe0.225μg/day(n=6)
Intracerebroventricularinjectionof0.45μg/daysiponimod doses exerted comparable effects on peripheral lympho-
amelioratesclinicalscoreofEAEmicewithoutaffecting cytes,onlytheformersignificantlyamelioratedthedisease
peripheralCD3+cellcount severity (p<0.05, non-parametric Mann-Whitney) in
In order to assess whether siponimod has direct neuro- the dpi range of 21–24 of the disease compared to
protective effects, the drug was delivered directly into EAE-vehicle (n=9) (Fig. 1d).
the brain by means of continuous intracerebroventricu- Bringing together these data, we decided to carry out
lar infusion, starting 1 week before the induction of the study using the 0.450 μg/day dose to better dissect
EAE. During the acute phase of the disease, several bio- theneuroprotectiveaction ofsiponimod.
chemicalandelectrophysiologicalanalyseswereperformed
onboththeperipheralandtheCNScompartment(Fig.1a, Siponimodicvtreatmentreducesgraymatter
timelineoftheexperimentaldesign). inflammationinEAEmice
First, we tested three dosages of the drug (4.50, 0.450, Activation of microglia and astrocytes are hallmarks of
and0.225μg/day)fortheirpossibleimpactonperipheral both MS and EAE [23]. Both cell populations have been
T lymphocytes: siponimod is lipophilic and therefore demonstrated to sustain inflammation within the CNS
able to cross the blood brain barrier (BBB). At 18 dpi, during autoimmune attack through antigen presentation
during the symptomatic phase of the disease, we counted and/or cytokine/chemokine secretion [24–26]. Moreover,
the total number of CD3+ lymphocytes in peripheral we previously demonstrated that microglia and astroglia
blood samples of EAE mice receiving different dosages of together with infiltrating lymphocytes play a pivotal
siponimodor vehicle:whilethehighestdoseofsiponimod role in inducing the synaptic alterations observed in
(4.5 μg/day; n=3) elicited a strong reduction of T EAE brain [8–10, 15, 27].
Gentileetal.JournalofNeuroinflammation (2016) 13:207 Page6of13
Fig.1Intracerebroventricularadministrationof0.45μg/daysiponimodattenuatesEAEmotordeficitswithoutinducingperipherallymphocyte
depletion.aSchematicrepresentationoftheexperimentaldesignusedinthestudy.Minipumpimplantationandsiponimod/vehiclereleasepreceded
theinductionofEAEby1week.Allexaminationswereperformedonanimalsduringthepeakofthesymptomaticphaseofthedisease18–24dpi,
bothintheperipheralbloodsamplesandinthestriatumoftheanimals.bCD3+lymphocyteswerecountedintheperipheralbloodofEAEmice
receivingvehicleordifferentsiponimoddosages:significantreductionwasobservedforthe4.5μg/daydosage.Siponimod4.5μg/dayvs
vehicle##p<0.01unpairedTtest;siponimod0.45μg/dayvsvehiclep>0.05unpairedTtest;siponimod0.225μg/dayvsvehiclep>0.05
unpairedTtest.cSiponimodconcentrationsweredeterminedinperipheralbloodsamplesofEAEmicereceivingintracerebroventricular
releaseofdifferentsiponimoddosages:thehighestconcentration(4.5μg/day)usedwassignificantlyhighercomparedtoboth0.450μg/day
and0.225μg/day,whiletherewerenostatisticallydifferencesbetweentheothertwodosages.Tukey’sposthoc***p<0.001.dRepresentative
clinicalcourseofEAEmicetreatedwiththreedifferentsiponimoddosages:EAEdiseaseprogressionisstronglyaffectedby4.5μg/daydosage
duringthesymptomaticphaseofthediseaseandsignificantlyattenuatedby0.45μg/dayconcentrationinthedpirangeof18–24.Daily
statisticalsignificancewasevaluatedbynon-parametricMann-Whitneytest
Gentileetal.JournalofNeuroinflammation (2016) 13:207 Page7of13
We investigated by immunohistochemistry the effect same slices, icv treatment with siponimod reduced the
ofsiponimodonbraininflammatoryreactionduringEAE. number of infiltrating lymphocytes compared to the
CoronalstriatalsliceswereprobedfortheGFAP,amarker icv-vehicle control group (Fig. 3a, green fluorescence).
upregulated during astrocyte activation. As depicted in Quantitative analysis of IBA1 and CD3 transcripts by
Fig. 2a, a substantial reduction of astrogliosis was ob- qPCR confirms reduced microglia activation and
servedinEAE-siponimodstriatum(greenfluorescencefor lymphocyte infiltration in the striatum of EAE mice re-
GFAP staining, DAPI counterstaining in gray) compared ceiving siponimod in comparison to EAE-vehicle (Fig. 3b;
to EAE-vehicle. Qualitative data of confocal microscopy n=4 for EAE-vehicle, n=5 for EAE-siponimod; p<0.05,
imageswereconfirmedbywesternblotanalysisofprotein unpairedTtest).
lysates from EAE-siponimod striatum compared to EAE-
vehiclestriatum:GFAPlevels,normalizedtoβ-actin,were InvitroapplicationofsiponimodreducesIL-6andRANTES
reducedby50%inthestriatumofEAEmicetreatedwith releasebyTNF-treatedmicroglialcells
siponimod (Fig. 2b, b’; n=5 for EAE-vehicle, n=5 for Microglia havebeen clearly involvedinneuronal damage
EAE-siponimod;p<0.05,unpairedTtest). and T lymphocyte recruitment in the brain, by releasing
Microgliosis was assessed by immunofluorescence and pro-inflammatory cytokines and chemokines, like IL-6
qPCR. As shown in Fig. 3a, the staining for the ionized and RANTES, which have been implicated in EAE and
calcium binding adaptor protein-1 (IBA1), marker of MSpathogenesis[28–32].
microglia/macrophage,ismorepronouncedinEAE-vehicle We tested the hypotheses that siponimod might reduce
than in EAE-siponimod striatum (red fluorescence, microglia activation. To this aim, we used an in vitro
counterstaining with DAPI in gray). Moreover, in the model of microglia activation. BV2 microglial cells were
Fig.2CNS-directeddeliveryofsiponimodreducesastrogliosisintheEAEstriatum.aConfocalmicroscopyimagesdepictthestriatumofEAE-vehicle
andsiponimodmicestainedfortheastrocytemarkerGFAP(greenfluorescence,counterstainedwithDAPI,gray).ExpressionofGFAPismarkedly
reducedinEAE-siponimodstriatumcomparedtoEAE-vehiclestriatum(scalebar:100μm).Theimagesarerepresentativeofstainedsections
fromthreeanimalspergroupsfromtwoimmunizations.ThepanelinbshowswesternblotcomparingEAE-siponimodandEAE-vehiclestriatal
extractsprobedforanti-GFAPantibody.Densitometricanalysisofthebandsinb’revealsreducedGFAPcontent,relativetoβ-actin,inEAE-siponimod
lysatescomparedtoEAE-vehicle.WBdataarenormalizedtoEAE-vehiclevalues.UnpairedTtestwasusedfortwo-groupanalysis.Valuesare
means±SEM,*p<0.05
Gentileetal.JournalofNeuroinflammation (2016) 13:207 Page8of13
Fig.3AttenuatedmicrogliosisandreducedlymphocyteinfiltrationcharacterizesthestriatumofEAEmicetreatedwithsiponimod.aDouble
immunostainingofcoronalstriatalsections(ingrayDAPInuclei)showingexpressionofIba1-positivemicroglia/macrophagecells(red)andofCD3
(green)inEAE-siponimodandEAE-vehiclemice.Onlyfewlymphocytescouldbedetectedinthestriatumofmicetreatedwithsiponimod,and
microgliosisisvisiblyreducedinthesameanimals.Scalebar:100μm.bThelevelsofIBA1andCD3mRNAwerequantifiedbyqRT-PCRinthe
striatumofEAE-siponimodversusEAE-vehiclemiceusingβ-actinasinternalcontrol.ThehistogramshowsthatbothIBA1andCD3mRNA
weresignificantlyreducedinthestriatumofsiponimod-treatedmice.StatisticalsignificancewasevaluatedbyunpairedTtest:*p<0.05
pre-treated with0.1 μMsiponimod priorto24-hstimula- of TNF did not alter the basal secretion of IL-6 (control
tion withTNF. By Luminex assay (Fig. 4a), we found that cells vs vehicle+siponimod 11.6±1.11 pg/ml, p>0.05
siponimod prevented the release of IL-6 induced by TNF Tukey’sposthoc).
stimulation of BV2 cells (one-way ANOVA: control cells TNF induced a strong release of RANTES in cell
12.06±0.55 pg/ml vs TNF 15.61±0.40 pg/ml, p<0.05 culture supernatant of BV2 cells (one-way ANOVA: con-
Tukey’s post hoc; TNF+siponimod 12.01±0.21 pg/ml vs trol cells 596.2±17.4 pg/ml vsTNF 1794±33.65 pg/ml,
TNF,p<0.05Tukey’sposthoc).Siponimodintheabsence p<0.001 Tukey’s post hoc; Fig. 4b). Siponimod reduced
Fig.4InvitrosiponimodapplicationtoactivatedBV2microglialcellreducesIL-6andRANTESrelease.BV2cellswerepre-treatedwithsiponimod
priortostimulationwithTNF.ThelevelsofsecretedIL-6(a)andRANTES(b)weremeasuredbyLuminexassayoncollectedmedia.Applicationof
siponimodtonon-activatedcellsdoesnotmodulatethesecretionofbothmolecules.Datawereexpressedaspicogramspermilliliter(pg/ml)
andwereanalyzedbyone-wayANOVA,followedbyTukey’sposthoctest.*p<0.05,**p<0.01,***p<0.001
Gentileetal.JournalofNeuroinflammation (2016) 13:207 Page9of13
the amount of RANTES released by TNF-stimulated 0.45 μg/day on sEPSCs, by recording striatal slices in
cells (TNF vs siponimod+TNF 1621±21.67 pg/ml, p< thedpirangeof20–24.Siponimoddidnotcorrectneither
0.01 Tukey’s post hoc; siponimod+TNF vs control p< the kinetic parameters of rise time, decay time, and half
0.01 Tukey’s post hoc) (Fig. 4b). As for IL-6, siponimod width(Fig.5a–a”)northefrequencies(Fig.5a’–a”)ofEAE
did not alter the release of RANTES by BV2 microglial sEPSC(n=8forbothgroups;p>0.05,unpairedTtestfor
cells in the absence of TNF (control vs siponimod-TNF allcomparisons).
698.4±16.44 pg/ml, p>0.05,Tukey’s post hoc). Conversely,siponimodicvtreatment significantlyame-
liorated GABAergic defects in the striatum of EAE mice,
SiponimodtreatmentrecoversGABAergictransmission by increasing GABA frequencies, which are considerably
alterationsinEAE reducedinthesymptomaticphaseofthedisease[6].Sipo-
Wenextaskedwhethersiponimodmightimprovethesyn- nimod restored normal values of frequency of spontan-
aptictransmissionalterationstypicalofEAEbrains[11],by eousinhibitorypost-synapticcurrents(sIPSCs)(Fig.5b,b’;
recording both the glutamatergic and the GABAergic EAE-vehicle 0.61±0.11 Hz, n=10; EAE-siponimod:
currents from striatal MSNs. 1.52±0.12 Hz, n=8; p<0.05, unpaired T test), suggest-
We previously demonstrated that both the presynaptic ing that siponimod might have neuroprotective effects
(frequency) and post-synaptic (kinetic) properties of the by preventing GABAergic transmission deficits.
spontaneous glutamatergic currents (sEPSCs) are altered Furthermore, in order to better explain the protective
in the striatum of EAE mice during the acute phase of role exerted by siponimod on GABA synapses, we incu-
the disease [15]. Here, we first investigated the effect bated EAE slices taken during the symptomatic phase
on sEPSCs after in vivo treatment with siponimod of the disease (20–25 dpi) with siponimod (1 μM) or
Fig.5SiponimodtreatmentcorrectsGABAergicdefectsinEAEstriatum.aTheglutamatergictransmissionwasrecordedfromMSNsofEAE-vehicle
andEAE-siponimodmice.Theanalysisoftheincreasedkineticproperties(risetime,decaytime,andhalfwidth)ofsEPSCsshowedthattherewereno
differencesbetweenthetwogroups(unpairedTtest,p>0.05).a’Thefrequenciesoftheglutamatergiccurrents,increasedinEAEstriatum,werenot
correctedbysiponimod(unpairedTtest,*p>0.05).a”ElectrophysiologicaltracesrepresentativeofglutamatergiccurrentsrecordedfromMSNsof
EAE-vehicleandEAE-siponimodmice.bSiponimodrestoresnormalGABAergicfrequenciesinthestriatumofacutephaseEAEmice(unpaired
Ttest,*p<0.05).b’ElectrophysiologicaltracesrepresentativeofGABAergiccurrentsrecordedfromMSNsofEAE-vehicleandEAE-siponimod
mice.cThefrequenciesoftheGABAergiccurrents,reducedinEAEstriatum,werecompletelyrescuedafter1hofsiponimodincubationin
EAEslices(unpairedTtest,**p<0.01)
Gentileetal.JournalofNeuroinflammation (2016) 13:207 Page10of13
vehicle(DMSO)for1h.Theinvitrotreatmentwithsipo- To avoid possible interference of the damage caused
nimod was able to rescue the GABAergic impairment in- by the cannula inserted in the right lateral ventricle, we
duced by EAE (Fig. 5c). The sIPSC frequency was higher performed unbiased stereological counts in the left
in EAE-siponimod slices (1.47±0.61 Hz, n=8) in com- dorsal striatum of EAE mice. In Fig. 6a, there are repre-
parisontoEAEuntreatedslices(0.78±0.11Hz;n=8;p< sentative images of coronal slices stained with anti-
0.01, unpaired T test), reaching values indistinguishable parvalbumin antibody. This experimental approach con-
fromcontrolslices.Thesedatasupporttheideathatsipo- firmed the reduction of the total numberofPV+ neurons
nimod exerts its central protective role likely by reducing in EAE-vehicle (1885±117.7, n=5) compared to control
residentimmunecellactivation. CFA-vehicle striatum (2664±156.6, n=3) and, more
interestingly, showed a recovery induced by siponimod
SiponimodpromotesPV+interneuronsurvival (2293±115.3, n=5;one-way ANOVA p<0.01; Newman-
GABAergic interneurons, together with axon collaterals Keuls posthoc comparisons: CFA-vehicle vs EAE-vehicle,
from MSNs themselves, are the main source of GABA p<0.01; CFA-vehicle vs EAE-siponimod, p>0.05; EAE-
inputs to striatal MSNs [33, 34]. The loss of parvalbumin- vehiclevsEAE-siponimod,p<0.05;Fig.6b).
positive (PV+) GABAergic interneurons together with the These data indicate that siponimod prevents GABAergic
noxiouseffectsofpro-inflammatorycytokineshasbeenpro- interneuronlossinthestriatumofsymptomaticEAEmice.
posedasmajordeterminantofthereducedGABAergictone
in the striatum of EAE mice [6]. Therefore, we assessed Discussion
whethersiponimodcouldpreventPV+interneurondeathin Discovering drugs effective in progressive forms of MS
thestriatumofEAEmice. is a hard challenge for both researchers and physicians
Fig.6BraininfusionofsiponimodpromotesthesurvivalofPV+GABAergicinterneuronsduringthecourseofEAE.aLowmagnificationimages
(×5objective)ofcoronalstriatalsectionsfromcontrolCFA-vehicle,EAE-vehicle,andEAE-siponimodshowingareaselectedforPVcountingon
serialsections(dorsalstriatum)andDAB-immunolabeledinterneurons(scalebar:200μm):athighmagnification,thedensityofPV+cellsis
visiblyreducedinEAE-vehiclecomparedtothatinCFA-vehiclewithapartialrecoveryinEAE-siponimodslices.YellowarrowsindicatePV-labeled
cells(scalebar:20μm).bHistogramrepresentsthequantitativestereologicalcountsofPV+cellsperformedonstriatalslices:theintracerebroventricular
treatmentwithsiponimodsignificantlyincreasesthetotalnumberofPV+cellsinthedorsalleftstriatumofEAEmice.Dataareexpressedasmean±
S.E.M.StatisticalcomparisonsshowedinthegraphwerecalculatedwithNewman-Keulsposthoc:CFA-vehiclevsEAE-vehicle**p<0.01;CFA-vehiclevs
EAE-siponimodp>0.05;EAE-vehiclevsEAE-siponimod*p<0.05