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EuropeanReviewforMedicalandPharmacologicalSciences

Eur opean Rev iewfor Med icaland Pharmacol ogical Sci ences 2015;19:4906-4919

2015;19:4906-4919

Eur opean Rev iewfor Med icaland Pharmacol ogical Sci ences 2015;19:4906-4919

Purified Cannabidiol, the main non-psychotropic component of Cannabissativa, alone, counteracts neuronal apoptosis in experimental multiple sclerosis

S. GIACOPPO 1 , T. SOUNDARA RAJAN 1 , M. GALUPPO 1 , F. POLLASTRO 2 , G. GRASSI 3 , P. BRAMANTI 1 , E. MAZZON 1

1 Experimental Neurology Laboratory, IRCCS Centre Neurolesi “Bonino-Pulejo”, Messina, Italy 2 Department of Pharmaceutical Sciences, University of Eastern Piedmont, Novara, Italy 3 Council for Research and Experimentation in Agriculture - Research Centre for Industrial Crops (CRA-CIN), Rovigo, Italy.

Abstract. – OBJECTIVE: Multiple Sclerosis

(MS) is a global concern disease leading to a progressive, chronic and demyelinating condi- tion,affectingthecentralnervoussystem(CNS). Thepathologyhasaninflammatory/autoimmune

origin;nevertheless,neuronalcelldeathmecha-

nismsarenottobeunderestimated.Thepresent study was designed to test the effects of in- traperitoneal administration of cannabidiol (CBD), the main non-psychotropic cannabinoid of Cannabis sativa (CS),inanexperimentalmod- elofMS.Theaimistoevaluatethecapabilityof

CBDadministrationtothwartthecascadeofme-

diatorsinvolvedinMS-inducedapoptosis. MATERIALS AND METHODS: Experimental Autoimmune Encephalomyelitis (EAE) was in- duced by immunization with myelin oligoden- droglial glycoprotein (MOG) 35-55 peptide in mice. Afterimmunization,micewereobserveddailyfor signsofEAEandweightloss.Diseasesignswere evaluatedusingastandardizedscoringsystem. RESULTS: Immunohistochemical and Western blot assessments of key apoptotic markers re- veal that CBD treatment is able to avoid Fas pathway activation, phospho-ERK p42/44 and cleaved caspase-3 triggering as well as alter-

ations in mitochondrial permeability due to Bax/Bcl-2 unbalance. Moreover, CBD interferes with p53-p21 axis activation. As results, the ab- senceoftissueapobodyformationinspinalcord

tissuesofEAE-micetreatedwithCBDwasestab-

lished. Most of therapeutic properties of CS are

currentlyascribedtothepsychotropiceffectsof

phenylterpenoiddelta-9tetrahydrocannabinol.

CONCLUSIONS: We have demonstrated that, alone, purified CBD possesses an anti-apoptot- ic power against the neurodegenerative processes underlying MS development. This represents an interesting new profile of CBD thatcouldleadtoitsintroductionintheclinical managementofMS.

K e y W o r ds :

Apoptosis, Experimental multiple sclerosis, Non-psy- chotropic cannabinoid, Cannabidiol, Mitochondrial per- meability.

Introduction

Multiplesclerosis(MS)isademyelinating

diseasemostlyofautoimmuneoriginthataf-

fectsanddamagesCNS,leadingtoadisabling condition 1 . In particular, myelin sheath loss causes a severe impairment of nerve signal transmission between the brain and spinal cord 2 .Actually,2.3millionindividualsareliv- ingwithMS.Accordingtodatareleasedby NationalMultipleSclerosisSociety,published in 2013 and relative to the year 2009, there

wereabout10.400cases/yearwithanincidence

of3.6women/100.000and2.0men/100.000 3 . Currently,pharmacologicalmanagementofMS

isinrelationshipwiththecourseofthepathol-

ogy.Forthetreatmentoftheso-classifiedre-

lapsing-remittingMS(RRMS),todatedisease modifyingtherapies(DMT)areadopted.They consistofimmunomodulatorydrugs,primarily belongingtoIFN-β class,specificallyIFN-β 1a (Avonex ® ,Rebif ® )andIFN-β 1b(Betaseron ® , Extavia ® ).Recenttrialsareaimedatthepossi- bleintroductionofteriflunomide(Aubagio ® ) anddimethylfumarate(Tecfidera ® )lookingat theirefficacyincomparisonwithanotherdrug prescribed for the treatment of RRMS, the glatirameracetate(Copaxone ® ) 4 ,asynthetic analogueofmyelinbasicprotein 5 .Whenhighly

4906

Corresponding Author: Emanuela Mazzon, Ph.D; e-mail: emazzon.irccs@gmail.com

CBD counteracts apoptosis in experimental model of MS

activeMSpatientspresentmoreandmorere- mayresultfromTHCconsumption.Ultimately,

thisisnothingmorethanthepricethatCBD,

themajornon-psychoactivecomponent,pays

(Tysabri ® ),aselectiveadhesion-moleculein-

perimentalstudieshaveshownthatCBDpos-

grin very late antigen-4 (VLA-4), used as

monotherapytodelayprogressionofdisabili-

aloneandwideexperimentalevidencesdemon-

stratedthatisolatingnon-psychotropiccom-

poundsbyTHCcomponentprovidebeneficial

effectsfortherapeuticuse,mostlyforCNSdis-

orders 16 .Actually,allcannabinoidshaveawide

antioxidant and neuroprotective action 17 . In

leastMSsymptomsthatarebasedontheaf- particular,antitumoralactivityofCBDmediat-

edbythetriggeringofapoptoticmechanismsis

not so overly declared, but not even a mys- tery 18 .CBDexertsitseffectsviaboththeinter-

actionwithcannabinoidsreceptor1and2(CB1

andCB2receptors,canonicalcannabinoidre-

functioningofthearmsandlegs);alteredsensi-

independentchannelsandbybindingwithvari-

ousnon-cannabinoidreceptors(suchasPPARs,

disturbances; neurological bladder (inconti-

nenceandconstipation);aswellasneuropsy- (TRPV1),GPR55,GPR18,GPR119and5-hy-

chological symptoms (memory loss, depres- sion).Moreover,asenseoflossofidentityis thedirectconsequenceofphysicalchangesand functionallimitations 11 . Medicinal plants are the most ancient re-

sourceofthehistoryinthetreatmentofvarious diseases 12 .Anexampleisgivenby C a n n a b i s s a t i v a ( C S ) rich in terpenophenolic con- stituents 12 . C S therapeuticuseisacontroversial open question which goes far beyond mere campaignonthelegalizationofmarijuana 13 .In

thisregarditisnoteworthytoconsiderthere-

centintroductionofSativex ® ,acannabinoid

oromucosalspraycontaininga1:1ratioofthe

phenylterpenoiddelta-9tetrahydrocannabinol

intodelineatingaclearerprofileofthecom-

poundsothatitsuseshouldbeextendedtothis

aswellasotherpathologieswithsimilarsymp-

tivity of the limbs; neurological symptoms (vertigo,pinsandneedles,neuralgiaandvisual

ceptorpathway)andactivatingotherreceptor-

variety,canrequireamultidisciplinaryman-

fectedCNSareaandthat,fortheirpluralityand

remainingintheshadowofTHC.Severalex-

hibitormonoclonalantibodyagainsttheinte-

lapseepisodes,thefirstlinedrugindicatedis the recombinant humanized natalizumab

sessesmanyproperties 15 ,oftenwronglyattrib- uted by collective imagination just to THC

ty 6 .Sadly,theriskofcontractingtheJohnCun-

ninghamvirus(JCV)ishigh,especiallyafter

longtreatments 7 .Inthisscenario,thenewfron- tiersofthepharmacologylookattheuseof complementaryandalternativemedicine 8,9 ,to counteract,ifnotthediseaseprogression,at

agement 10 .Overall,symptomscanincludemo- tor control deficit (spasms and spasticity, weakness,impairedcoordination,balanceand

transient receptor potential vanilloid type 1

droxytryptamine receptor subtype 1A (5- HT1A) 19 . Inourstudy,wedecidedtoinvestigatenotin

thewakeofotherauthors 20 ,thewellknownand assessedanti-inflammatoryeffectofCBD 21,22 , butratheritscapabilitytoavoidprogrammed celldeathinthespinalcordofanimalaffected byexperimentalautoimmuneencephalomyelitis

(EAE),amodelinducedbyMyelinOligoden-

drocyteGlycoprotein(MOG) 35-55 peptideinjec- tionandvalidatedtoreproducehumanMSin mice 23 .ByexaminingthisprofileofCBD,we stronglyhopetoprovidenewevidencesabout theefficacyofthemoleculeandtocontribute

toms.

Materials and Methods

PlantMaterial

(THC)andcannabidiol(CBD),thetwomajor components of C S 14 , for the management of symptomatic treatment of chronic pain and spasticity.Despitethiscombinationhasbeen approvedandinthecurrentstateintroducedin severalcountriesunderthisformulation,over

the years the point of view of the scientific community regarding THC and CBD is changed.Inparticular,thedichotomybetween psychotropicandnon-psychotropiceffectshave beenstressedandthemostferventsupporters fortheintroductionof C S inclinicalpractice

lookatthebeneficialphytochemicalsproper- dancewiththeirlegalstatus(Authorization

nipulationofcannabinoidswasdoneinaccor-

lectedinNovember2013.Theisolationandma-

cultivationatCRA-CIN,Rovigo(Italy),wascol-

C a n n a b i s s a t i v a L,derivedfromgreenhouse

tiesthatcouldderivebyCBDisolationrather

thantosidepossiblehallucinogeniceffectsthat

SP/10623/05/2013oftheMinistryofHealth,

Rome,Italy).

4907

S. Giacoppo, T. Soundara Rajan, M. Galuppo, F. Pollastro, G. Grassi, P. Bramanti, E. Mazzon

ExtractionandIsolationofCBD

PureCBD(>99%)wasisolatedfromanItalian

ExperimentalDesign

Micewererandomlyallocatedintothefollow-

varietyofindustrialhemp(Carmagnola)accord- inggroups(n=45totalanimals):

ingtoastandardizedmethodofthecannabinoid purification 24 toavoidanytraceofTHCthatcould interfereinthetrialorcauselegallimitation.

N a i v e g r o u p (n=10): mice did not receive

(MOG) 35-55 orothertreatment .

EA E g ro u p (n=20):micesubjectedtoEAEthat

receivedonlythevehicleofthepharmacologi-

caltreatment(1:1:8EtOH:Tween20:saline);

E A E + C B D t r e a t m e n t g r o u p (n=15):starting fromtheoccurringoffirstsignsofdisease (14 th dayonset),EAEmiceweredailysubject-

edtoCBDtreatment(10mg/kgi.p.);

C B D - v e h i c l e c o n t r o l g r o u p (n=10)micethat didnotreceive(MOG )35-55 butonlyCBDvehi- cleascontrolofthetreatment. Atthe28 th dayfromEAE-induction,animals

wereeuthanizedwithi.p.ofTanax(5ml/kgbody

weight).Inaddition,spinalcordtissueswere

sampledandprocessedinordertoevaluatepara-

ClinicalDiseaseScore

Animals

MaleC57BL/6mice(HarlanMilan,Italy)12

weeksofageandweighing20-25gwerehoused

inindividuallyventilatedcageswithfoodand water a d lib itu m .Theroomwasmaintainedata

constanttemperatureandhumidityona12h/12

hlight/darkcycle.

EthicalApproval

Thisstudywascarriedoutinstrictaccordance

withtherecommendationsintheguideforthe

careanduseoflaboratoryanimalsofItalianNa-

tionalInstitutesofHealth.Theprotocolwasap- metersofdisease.

provedbytheMinistryofHealth“GeneralDi-

rectionofanimalhealthandveterinarydrug”

(Authorization150/2014-B28/03/2014).Inpar- Thefirstmeasurementofclinicaldiseasescore

wastakenonthedayofEAE-induction(dayze-

ro),thesecondafter4daysandallsubsequent

measurementswererecordedevery48hoursun-

(D.M.116/92)aswellaswiththeEECregula-

tions(O.J.ofE.C.L358/112/18/1986).Inaddi-

tion,minimizednumberofanimalswereusedfor

2=completeflaccidtail;3=hindlimbhypotonia;

4=partialhindlimbparalysis;5=completehind

limbparalysis;6=moribundordeadanimal.Ani-

InductionofExperimentalAutoimmune

Encephalomyelitis(EAE)

Afteranaesthesia,inducedwithananaesthet- animalsuffering.Themeasureofclinicaldisease

scorehasbeenexpressedasmean±SEMofall

lazine(10ml/kg,i.p.),EAEwasactivelyin- measurementsofeachexperimentalgroup.

ducedusing(MOG) 35-55 peptide(MEVGWYR- SPFSRVVHLYRNGK;%peakareabyHPLC

BodyWeightEvaluation Thefirstmeasurementofbodyweightwastaken onthedayofEAE-induction(dayzero),thesecond

subcutaneouslywith300µl/flankoftheemul- after4daysandallsubsequentmeasurementswere

sionconsistingof300µgof(MOG) 35-55 inPBS

variationofbodyweighthasbeenexpressedcom-

≥95,AnaSpec,EGTCorporateHeadquarters,

Fremont, CA, USA). Mice were immunized

ic cocktail composed of tiletamine plus xy-

malswithascore≥5weresacrificedtoavoid

theexperimentandtheirsuffering.

forexperimentalandotherscientificpurposes

tionalregulationsonprotectionofanimalsused

ticular,animalcarewasincompliancewithna-

tilsacrifice.Briefly,thesignsofEAEwere scoredusingastandardizedscoringsystem 25 as follows: 0=no signs; 1=partial flaccid tail;

recordedevery48hoursuntilsacrifice.Thedaily

mixedwithanequalvolumeofCompleteFre-

und’sAdjuvant(CFA)containing300µgheat- paredtodayofEAEinduction(dayzero),alsothe

killed M y c o b a c t e r i u m t u b e r c u l o s i s H37Ra

(DifcoLaboratories,Sparks,MD,USA).Im- animalsforeachexperimentalgroup.

mediatelyafter(MOG) 35-55 injection,theani- malsreceived100µlof B o r d e t e l l a p e r t u s s i s toxin (Sigma-Aldrich, Milan, Italy) (500

sivenesstoamechanicalstimulus.Eachanimal

followsacourseofprogressivedegeneration,

ofthehindpawwithseveralsecondsbetweenap-

withvisiblesignsofpathologyconsistingof

Thetestwasaimedtoassessmice’srespon-

ng/100µl,i.p),repeated48hlater.Thedisease

valuedayhasbeenexpressedasmean±SEMofall

NeedleTest

wassubjectedto3stimuliontheplantarsurface

flaccidityofthetailandlossofmotionofthe

hindlegs.

plicationsofneedle.Apositiveresponseisapaw

withdrawalfromthestimulus.Themaximum

4908

CBD counteracts apoptosis in experimental model of MS

scorehasavalueof6.Inspecific,7testswere

performedevery48hoursintwoweeksfromthe

firstadministrationofCBD.Thevaluesareex- tablets(RocheAppliedScience,Monza,Italy),and

pressedasmean±SEMofeachgroup.

min.Thehomogenateswerechilledonicefor15

Immunohistochemical(IHC)Evaluation

Afterdeparaffinizationwithxylene,sections

ofspinalcordsampleswerehydrated.Detection

ofFas-Ligand,Bax,Bcl-2wascarriedoutafter

boilingincitratebuffer0.01MpH6for4min.

Endogenousperoxidasewasquenchedwith0.3%

(v/v)hydrogenperoxidein60%(v/v)methanol

for30min.Nonspecificadsorptionwasmini- thenwerecentrifugedfor30minat15.000gat

mizedbyincubatingthesectionin2%(v/v)nor- 4°C.Then,supernatantcontainingnuclearextract

malgoatseruminPBSfor20min.

mMNaCl,10mMTris-HCl,pH7.4,1mMEG-

TA,1mMEDTAproteaseinhibitors(Roche),and

mMEGTA,2mMEDTA,5mMNaN 3 ,10mM2-

mercaptoethanol,50mMNaF,proteaseinhibitor

theywerehomogenizedatthehighestsettingfor2

minandthencentrifugedat1000gfor10minat

4°C,andthesupernatant(cytosolextract)wascol-

lectedtoevaluatecontentofcitoplasmaticproteins.

Thepelletsweresuspendedinthesuppliedcom-

pletelysisbuffercontaining1%TritonX-100,150

wascollectedtoevaluatethecontentofnuclear

Sectionswereincubatedovernightwith:

• Anti-FAS-Ligandpolyclonalantibody(1:100

inPBSv/v;Abcam,Cambridge,MA,USA)

• Anti-Baxpolyclonalantibody(1:100inPBS

v/v;SantaCruzBiotechnology,Inc.,Santa Cruz,CA,USA);

proteins.Supernatantswerestoredat-80°Cuntil

use.Proteinconcentrationinhomogenatewasesti-

matedbyBio-RadProteinAssay(Bio-Rad,Seg-

rate,Italy)usingbovineserumalbumine(BSA)as

standard,and20µgofcytosolandnuclearextract

fromeachsamplewereanalyzed.

• Anti-Bcl-2polyclonalantibody(1:100inPBS

fate-polyacrylamideminigelsandtransferredonto

PVDFmembranes(Immobilon-PTransfermem-

wereblockedbysequentialincubationfor15

nonfatdriedmilk(PM)for45minatroomtemper-

respectively.SectionswerewashedwithPBS

andincubatedwithsecondaryantibody.Specific labellingwasdetectedwithabiotin-conjugated goatanti-rabbitIgGandavidin-biotinperoxidase complex (Vectastain ABC kit, Vector, Burlingame,CA,USA).Theimmunostaining (positivityinbrowncolor)wasdevelopedwith

peroxidasesubstratekitDAB(VectorLaborato- Abcam)in1xphosphatebufferedsaline(PBS),5%

ries,Inc.),whilethecounterstainingwithMay- (w/v)nonfatdriedmilk,0.1%Tween-20(PMT).

er’s hemalum solution (blue background).

cleaved-caspase3(1:500;CellSignalingTechnolo-

gy),caspase-3(1:500;CellSignalingTechnology),

USA),ERK2(1:2000;CellSignalingTechnology),

(1:1000;CellSignalingTechnology,Boston,MA,

minwithbiotinandavidin(DBA,Milan,Italy),

brane,Millipore),blockedwithPBScontaining5%

Proteinswereseparatedonsodiumdodecylsul-

v/v;SantaCruzBiotechnology,Inc); Endogenousbiotinoravidinbindingsites

ature,andsubsequentlyprobedat4°Covernight

withspecificantibodiesforphospho-ERKp42/44

p21(1:1000;MerckMillipore)andp53(1:2000;

HRP-conjugatedgoatanti-rabbitIgGwasincubated as secondary antibody (1:2000; Santa Cruz

BiotechnologyInc.,SantaCruz,CA,USA)for1h

antibody(nosecondary)orwithonlythesec- atroomtemperature.Toascertainthatblotswere

loadedwithequalamountsofproteinlysates,they

positivestainingwasfoundinthesections,indi- werealsoincubatedwithantibodyforGAPDH

HRPConjugated(1:1000;CellSignalingTechnol-

alltheexperimentscarriedout.

ogy)andbeta-actin(1:1000;SantaCruzBiotech-

Allsectionswereobtainedusinglightmi- nology,Inc).Therelativeexpressionofprotein

croscopy(LeicaDM2000combinedwithLeica

ICC50HDcamera,Wetzlar,Germany).Leica

HRPSubstrates,Millipore,Billerica,MA,USA)

imagecomputerprogramtoacquireIHCpictures.

withChemiDoc™MPSystem(Bio-Rad)anda

ApplicationSuiteV4.2.0softwarewasusedas

bandshasbeenvisualizedusinganenhanced

ondaryantibody(noprimary).Inthesecasesno

tionswerealsoincubatedwithonlytheprimary

Toverifythebindingspecificity,somesec-

catingthattheimmunoreactionwaspositivein

chemiluminescencesystem(LuminataWestern

andproteinbandswereacquiredandquantified

WesternBlotAnalysis

computerprogram(ImageJsoftware)respectively.

Blotsarerepresentativeofthreeseparateand

iceusingice-coldreagents.Inbrief,spinalcordtis- reproducibleexperiments.Thestatisticalanalysis

suesweresuspendedinextractionbuffercontain- wascarriedoutonthreerepeatedblotsper-

ing0.32Msucrose,10mMTris-HCl,pH7.4,1

Alltheextractionprocedureswereperformedon

formedonseparateexperiments.

4909

S. Giacoppo, T. Soundara Rajan, M. Galuppo, F. Pollastro, G. Grassi, P. Bramanti, E. Mazzon

TUNELAssay

TotestwhetherinEAEmodelspinalcord

pawsensibilityinEAE-affectedmicewithsig-

nificantscorelevels.

wasassociatedwithcelldeathbyapoptosis,we

measuredTUNEL-likestainingintheperile- CBDTreatmentisAbletoModulate

sionalspinalcordtissue.TUNELassaywas

conductedbyusingaTUNELdetectionkitac- FasPathwayModulation

To evaluate upstream-enhanced apoptotic mechanismswelookedatprocessesmediated bythesignalcascadetriggeredbyFAS-ligand. IHClocalizationofspinalcordsectionsfor

withPBS.Endogenousperoxidasewasinacti- FAS-Ligandhasshownamarkedimmunoposi-

15minatroomtemperatureandthenwashed

wereincubatedwith15mg/mlproteinaseKfor

cording to the manufacturer’s instruction (Apotag,HRPkitDBA,Milan,Italy).Sections

ApoptoticPathwayTriggeringvia

tivityinuntreatedEAEmice(Figure3c)com-

paringtoanimalthatunderthesameconditions

immersedinterminaldeoxynucleotidyltrans- received the intraperitoneal CBD treatment

(Figure3d).Naïvemice(Figure3b)andmice

thatwereadministratedwiththeCBD-vehicle

alone(Figure3a)resultednegativeforFAS-

ligand. Moreover, statistical analysis estab- lished significant differences between EAE groupandallotherexperimentalgroups(see

densitometricanalysis,Figure3e).

(DAB)andcontrostainedwithnuclearfastblue.

signalswerevisualizedwithdiaminobenzidine

radishperoxidase-conjugatedantibody,andthe

roomtemperaturefor30minwithanti-horse-

washedwithPBS.Sectionswereincubatedat

ahumidatmosphereat37°Cfor1h,andthen

ferase(TdT)buffercontainingdeoxynucleotidyl transferaseandbiotinylateddUTP,incubatedin

vatedby3%H 2 O 2 for5minatroomtempera- tureandthenwashedwithPBS.Sectionswere

StatisticalAnalysis

GraphPadPrismversion6.0program(Graph-

PadSoftware,LaJolla,CA,USA)wasusedfor

statisticalanalysisofthedata.Theresultswere

statisticallyanalyzedusingone-wayANOVA

followedbyaBonferroni p o s t h o c testformulti- plecomparisons.A p valuelessthanorequalto

0.05wasconsideredsignificant.Resultsareex-

pressedasthemean±SEMofnexperiments.

Results

CBDTreatmentAmelioratesGeneral

WellnessinEAE-AffectedMice

Bodyweightmeasurement(Figure1a)aswell

CBDTreatmentControlsCascadeof MediatorsInvolvedinApoptosis Western blot analysis for phospho-ERK

p42/44revealedthatMitogen-ActivatedProtein

Kinases ( MAPK)signalingpathwayisstrongly activatedfollowingEAE-inductionwhileCBD

treatmentreducestheexpressionlevelsofthis

marker(Figure4a,seedensitometricanalysisb).

SinceERKactivityhasbeenclearlyandwide-

lyimplicatedinthereleasingofclassicalmarkers

ofapoptosisexecution,suchaseffectorcaspase-

3 26 ,weevaluatedthedegreeofcaspase3activa-

tionviadetectionofcleaved-caspase3expres-

sionlevels.TreatmentwithCBDinhibitsthis

markerthat,conversely,resultshighlyexpressed

inuntreatedEAEmice(Figure4c,seedensito-

metricanalysisd).

asClinicalDiseaseScore(Figure1b)evaluation

wereassessedasparametersofdisease.Inboth

cases,CBD-treatedEAE-affectedmiceshowa

trendofrecoveryovertimecomparedtountreat- SpinalcordtissuessampledbyuntreatedEAE

edEAEmice,inparticularfollowingthedisease

onset(fifthmeasurement)anduntilsacrifice.

MicebelongingtovehicleCBDandnaivegroup

haveanormalincreaseinbodyweightaswellas

absenceofmotordeficit.

stainingforBcl-2leadingtobelievethatmecha-

nismofmitochondrialalteredpermeabilityare

primedintheseanimals(Figures5cand6c,re-

spectively).Conversely,intraperitonealadminis-

trationofCBDinhibitstheabovecitedalter-

ations.Infact,IHClocalizationforBax/Bcl-2re-

vealsthatinCBD-treatedEAEmicethesemark-

NeedletesttoEvaluatepawSensibility

micedisplayhighpositivityforBaxandnegative

CBDTreatmentAvoidsBax/Bcl-2

Unbalance.

EAEdevelopmentleadsuntreatedmicetoa

constantlossofpawsensibilitymeasured(Figure

ersaremoretargetedtowardsanti-apoptotic

2)sincethediseaseonsetuntilsacrifice.Con- mechanismsshowinghigherBcl-2(Figure6d)

thanBaximmunopositivity(Figure5d)aswell

versely,CBD-treatmentpreservesadegreeof

4910

CBD counteracts apoptosis in experimental model of MS

CBD counteracts apoptosis in experimental model of MS F i g u r e 1 .

F i g u r e 1 . CBD-treatedEAE-injuredmicedisplaybodyweightandmotorfunctionrecovery.Ameasureofdiseasedegreeis theevaluationofbodyweight(a)andofthedisabilityscore(b).ForbothparametersCBD-treatedmiceshowhighsignificant

recoverywhencomparedwithuntreatedEAEmice,inparticularfollowingthediseaseonset(fifthmeasurement)anduntilsac-

rifice.A p value<0.05wasconsideredsignificant.vs.NAIVE; # vs.VEHICLECTR.

asinvehicle-control(Figures6aand5a,respec- CBDRegulatesp53-p21AxisActivation

tively)andnaïvegroups(Figures6band5b,re- Aboutdownstreamnuclearmechanismsof

spectively).ForbothBaxandBcl-2,aquantita- apoptosisWesternblotanalysisrevealedthat

tiveanalysisoftheimmunopositivitywasper- thereareveryhighexpressionlevelsofthetran-

formedinallexperimentalgroupsasshowedin

Figures5eand6e,respectively.

scription factor p53 in untreated EAE mice

(Figure7a,seedensitometricanalysisb),that,

4911

S. Giacoppo, T. Soundara Rajan, M. Galuppo, F. Pollastro, G. Grassi, P. Bramanti, E. Mazzon

inturn,activatesandstimulatestheoverexpres- CBDTreatment,Interferingwiththe

sionofp21(Figure7c,seedensitometricanaly- ActivationofSeveralMediators

sisd).CBDtreatmentinEAEmicereversesthis

paneldecreasingp53andp21atlevelscompa-

rablewithvehicle-control(Figure7a,seeden- playedcellularmechanismsofapoptosisare

Finally,TUNELassayrevealedthatalldis-

ThwartsNeuronalCellDeath

sitometricanalysisbandc,seedensitometric

analysisd,respectively)andnaïvegroups(Fig- (meanof±337.5positivenuclei/field,positive

stainingcontrolFigure8e)inuntreatedEAE

densitometricanalysisdrespectively).

mice(Figure8c)that,converselyarewhollyab-

translatedinamarkedpresenceofapobodies

ure7a,seedensitometricanalysisbandc,see

ure7a,seedensitometricanalysisbandc,see F i g u r e 2.

F i g u r e 2. CBDtreatmentpreservespawsensibility.Behavioraltestaimedtoevaluatethelossofpawsensibilitycausedby

diseasedevelopment.NeedletestclearlyshowsthatCBD-treatedEAEmicekeepahigherandsignificantsensitivityofthe

lowerlimbswhencomparedwithuntreatedCBDmicethatfollowingthediseaseonset(14thfromdiseaseinduction,firsttest

measurement)displayatotallossofresponsetoanyneedlesolicitation.A p value<0.05wasconsideredsignificant. vs .CBD

IP; # vs .NAIVE;°vs .VEHICLECTR.

4912

CBD counteracts apoptosis in experimental model of MS

CBD counteracts apoptosis in experimental model of MS F i g u r e 3 .

F i g u r e 3 . CBD-treatmentinterfereswithFaspathway.IHCevaluationofapoptoticpathwaysmodulatedbyCBD-treatment

displaythatuntreatedEAEmiceresulthighlypositivetoFas-Ligandstaining(c).Conversely,sectionssampledbyCBD-treat-

edEAEmice(d),vehicleCBD(a)aswellasnaive(b)groupsresultcompletelynegativeforthismarker.Adensitometric analysisoftheresultisprovidedine.A p value*<0.05wasconsideredsignificant.(20x).

sentinCBD-treatedEAEmice(Figure8d)as

wellasinvehicle-control(Figure8a)andnaïve

groups(Figure8b).

Discussion

rodegenerativediseasewithawiderangeof

outcomeandsymptomsclassifiedasprimary,

secondary,ortertiary 27 .Inthisregard, C S isex-

perimentallydemonstratedtolimitneurodegen-

erationthatleadstoprogressivedisability 28 as

wellascannabinoidsarecommerciallyavail-

ableandcurrentlyintroducedintheclinical

practiceindifferentpercentageandformula-

tionswithTHC(i.e.Sativex ® aswellasBedro-

can ® ,Bedrobinol ® ,Bediol ® )totreatMSsymp- toms.Forexample,experimentalstudiesreveal

thatactivationofcannabinoidCB2receptors

byJWH-133,asyntheticcannabinoid,reduces

hyperalgesiainEAE-affectedmice 29 .Theseef-

fectscanbetheresultofthespecificandselec-

tiveCB1/CB2receptorsactivationbycannabi-

MSisacomplex,chronic,progressiveneu- noids(naturalandsynthetic)ornon-classicre-

ceptorligands 30 (onereceptorclass,manyago-

nists)aswellastheeffectofcannabinoidbind-

ingtothereceptorsotherthanCB1/CB2recep-

tors(oneagonistclass/manyreceptorclasses). Thepresentworkwasdesignedtodefineanew profile of CBD, the main non psychotropic compoundpresentin C S , whoseintrinsicpo- tentialasamoleculewithatherapeuticeffect hasnotyetcompletelyunderstood.Infact,itis

4913

S. Giacoppo, T. Soundara Rajan, M. Galuppo, F. Pollastro, G. Grassi, P. Bramanti, E. Mazzon

M. Galuppo, F. Pollastro, G. Grassi, P. Bramanti, E. Mazzon F i g u r e

F i g u r e 4 . CBD-treatmentmodulatesphospho-ERKp42/44andcleaved-caspase3expression.Westernblotanalysistoevalu-

atephospho-ERKp42/44levelexpression(a,densitometricanalysisb)displaysthatuntreatedEAE-affectedmicehavethe

highestproteinlevels.Conversely,CBD-treatedEAEmice,vehicleCBDaswellasnaivegroupsshowsignificantlowerphos-

pho-ERKp42/44expressionlevels.ERKp42wasusedtonormalizethesignal.A p value*<0.05wasconsideredsignificant.

Parallel,cleaved-caspase3detection(c,densitometricanalysisd)revealsthattissuehomogenatessampledbyuntreatedEAE-

affectedmicecontainhighlevelsofthismarker.Conversely,CBD-treatedEAEmice,vehicleCBDaswellasnaivegroups showsignificantcleaved-caspase3lowerexpressionlevels.Caspase3wasusedtonormalizethesignal.A p value*<0.05 wasconsideredsignificant.

wellknownthatCBDexertsanti-inflammatory

properties,reducingthegenerationofpro-in- hasbeenhereprovidedbyassessmentofFas-

Liganddetection.Thisstepwasconsideredby

usasthefirstevidenceaboutthepossibleanti-

theless,todatelittleornothinghasbeendeep- apoptoticpowerofCBDthathasprovedableto

enedaboutitsanti-apoptoticpower.Inparticu- avoidthereleasingofthiskey-mediator.More-

lar,ourstudywasaimedtohighlighttheseun- over,sincetheFaspathwayiscloselylinkedto

derestimatedpropertiesofCBD.Inthisper- theintrinsicpathwayofapoptosis,itwasinter-

spective,ourpresentdataareencouraging.

estingtoinvestigatetheBcl-2familyproteins,

Verifiedthehighpresenceofapobodiesfol- regulatingtheintegrityoftheoutermitochon-

lowingEAEdegeneration,wehavefurtherin- drialmembranepermeability 32 .Mitochondria

arethepowerhouseofallcellsandtheirin-

tegrityisthecorefortheregulationofcellsur-

vival 33 ,directingcellbalancetowardssurvival

ordeathsignals.Moreover,Rimmermanetal 34 showedthatCBDdirectlymodulatestheouter mitochondrialmembranechannelasanagonist of voltage-dependent anion channel 1

(VDAC1),involvedinfunctioningofcellener-

vestigatedtheprocessesleadingtoCBDneu-

flammatorycytokines(suchasTNF-α andIL-

rosis 31 .AnindirectmeasureofFasactivation

1β)aswellasreactingoxygenspecies 19 ,never-

ronal protection. The mechanism by which CBDcounteractsneuronaldeathisnotfully

known.However,byevaluatingthemainmedi-

atorsofbothintrinsicandextrinsicapoptotic

pathway,wecansummarizewithsomeconfi-

dence that CBD can interfere, modulating them,processesunderlyingtheprogrammed

celldeathmechanism.Fascellsurfacerecep-

tor,belongingtoTNFreceptorsuperfamily,

hasbeenwidelyrecognizedtohavearolein

transducingapoptoticsignalsinmultiplescle-

gy,metabolichomeostasisandapoptosis. Clinicalstudies 35 showedthatRRMSpatients

displayaberrantexpressionanalysisofapopto-

sis-relatedgenes.Nevertheless,ourachievedre-

4914

CBD counteracts apoptosis in experimental model of MS

CBD counteracts apoptosis in experimental model of MS F i g u r e 5 .

F i g u r e 5 . CBD-treatmentcounteractsalterationofmitochondrialpermeabilityavoidingBaxoverexpression.IHCstainingfor BaxlocalizationdisplaysahighimmunopositivityinsectionssampledbyuntreatedEAE-affectedmice(c),whencompare

withCBD-treatedEAEmice(d),vehicleCBD(a)aswellasnaivegroups(b).Adensitometricanalysisoftheresultisprovid-

edine.A p value * <0.05wasconsideredsignificant.(20x).

sultsindicatethatmitochondrialmaintenance

waspreservedbyCBDtreatedEAE-micewitha

greatertendencytoreleaseanti-apoptotic(Bcl-2)

thanpro-apoptoticfactors(Bax).Parallel,look-

ingattheextrinsicpathwayofapoptosis,were-

ceivedafurtherconfirmaboutthepreservation ofcellsurvivalmediatedbyCBDadministration. Inthis,acentralroleiscoveredbythelackingof

cleaved-caspase3activation,aclassichallmark

ofapoptosis-inductionofparticularimportance foroligodendrocytedeathinmultiplesclerosis 36 .

Thisdatahasbeenanobjectofstudyforother

authorsinapaperwheretheydidnotattribute

thecaspase3cleavagetotheinvolvementof

CB1,CB2,TRPV1orPPARγ receptors,but rathertoandactivityindependentbyclassical andalternativecannabinoidreceptors 37 .CBDhas demonstratedtoavoidupstreamtriggeringof

MAPKpathway,serine/threonineproteinkinas-

es,whichplaypivotalrolesregulatingmanycell

functionsindifferentcelltypes 38 .Finally,wein-

vestigatedthepossibleroleofp53,animportant

transcriptionfactorofgenessuchasp21,strong-

lyinvolvedinprocessesbothofcellproliferation anddeath 39 .Corroboratingp53rolepromoting

p21transactivation,EAE-affectedmicedisplay

4915

S. Giacoppo, T. Soundara Rajan, M. Galuppo, F. Pollastro, G. Grassi, P. Bramanti, E. Mazzon

M. Galuppo, F. Pollastro, G. Grassi, P. Bramanti, E. Mazzon F i g u r e

F i g u r e 6 . CBD-treatmentcounteractsalterationofmitochondrialpermeabilityavoidingBcl-2unbalance.IHClocalizationfor

Bcl-2displaysanegativestaininginsectionssampledbyuntreatedEAE-affectedmice(c).Conversely,CBD-treatedEAE

mice(d),vehicleCBD(a)aswellasnaivegroups(b)resultpositiveforthismarker.Adensitometricanalysisoftheresultis providedine.A p value * <0.05wasconsideredsignificant.(20x).

highexpressionlevelsofthesemarkersthat,con-

versely,resultdownregulatedfollowingCBD

treatment.

tureabouttheoverallroleofcannabinoidsinthe treatmentofneurodegenerativediseases 19,40 .In

ourview,consideringitsevidenttherapeuticben-

efits,thebarriers,largelymental,abouttheintro-

ductionof C S ,andespeciallyofCBDformula- tion,inclinicalpractice,shouldbetorndown. Ofcourse,furtherstudiesshouldbeperformed toisolateandcharacterizethenon-psychotropic componentoftheplant,amongallmostlythe CBD,that,asweshowedinthepresentwork,

alone,possessesveryinterestingpharmacologi-

calpropertiesintocounteractingthecascadeof

Conclusions

SummarizingtheCBDactivityontheapoptot-

icpathway,westronglyhopetohaveprovideda,

shortbutsignificantroundupofevidencesthat

areusefultobettercharacterizetheefficacyas

wellasthemolecularpathwaysmodulatedbythe

molecule.Currently,thereisanextendedlitera- mediatorsleadingtoneuronalcelldeathinMS.

4916

CBD counteracts apoptosis in experimental model of MS

CBD counteracts apoptosis in experimental model of MS F i g u r e 7 .

F i g u r e 7 . CBD-treatmentinhibitsp53/p21activation.Westernblotanalysistoevaluatep53levelexpression(a,densitometric

analysisb)displaysthatuntreatedEAE-affectedmicehavethehighestproteinlevels.Conversely,CBD-treatedEAEmice,ve-

hicleCBDaswellasnaivegroupsshowsignificantlowerp53expressionlevels.A p value*<0.05wasconsideredsignificant.

Parallel,p21detection(c,densitometricanalysisd)revealsthattissuehomogenatessampledbyuntreatedEAE-affectedmice

containhighlevelsofthismarker.Conversely,CBD-treatedEAEmiceshowsignificantlowerexpressionlevels.VehicleCBD aswellasnaivegroupshavetotallynegativep21expressionlevels.A p value*<0.05wasconsideredsignificant.

F
F

i gu r e 8 . CBDavoidstheformationofneuronalapobodies.

TUNELassayrevealspresenceofapobodies(positivestain-

ingcontrole)inuntreatedEAEmice(c)that,conversely,are

whollyabsentinCBD-treatedEAEmice(d)aswellasinve-

hicle-CBD(a)andnaivegroups(b).(20x).

4917

S. Giacoppo, T. Soundara Rajan, M. Galuppo, F. Pollastro, G. Grassi, P. Bramanti, E. Mazzon

––––––––––––––––––––

Acknowledgements

AuthorswouldliketothankProf.AppendinoGiovanni

(UniversityofEasternPiedmontAmedeoAvogadro,No-

vara,Italy),forhistechnicalassistanceandpreciouscontri-

butionduringmanuscriptdrafting.

Theresearchwassupportedbycurrentresearchfunds2014

ofIRCCS“CentroNeurolesiBonino-Pulejo”,Messina,Italy.

–––––––––––––––––-––––

Conflict of Interest

TheAuthorsdeclarethattheyhavenoconflictofinterests.

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