Oleoylethanolamide Delays the Dysfunction and Death of Purkinje Cells and Ameliorates Behavioral Defects in a Mouse Model of Cerebellar Neurodegeneration.

h ps://doi.o g/10.1007/s13311-021-01044-3 ORIGINAL ARTICLE Oleoyle hanolamide Delays heDys unc ion andDea h o Pu kinje Cells andAmelio a es Beha io al De ec s inaMouse Model o Ce ebella Neu odegene a ion Es e Pé ez‑Ma ín1,2 · Rod igoMuñoz‑Cas añeda1,2· Ma ie‑JoMou in3 · Ca meloA.Á ila‑Za za2,4 · JoséM.Muñoz‑Cas añeda5 · Ca losDelPila 1,2 · JoséR.Alonso1,2,6 · AnnieAnd ieux3 · Da idDíaz1,2 · Edua doWe uaga1,2 Accep ed: 16 Ma ch 2021 © The Ame ican Socie y o Expe imen al Neu oThe apeu ics, Inc. 2021 Abs ac Oleoyle hanolamide (OEA) is an endocannabinoid ha has been p oposed o p e en neu onal damage and neu oin lam- ma ion. In his s udy, we e alua ed he e ec s o OEA on he dis up ion o bo h ce ebella s uc u e and physiology and on he beha io o Pu kinje cell degene a ion (PCD) mu an mice. These mice exhibi ce ebella degene a ion, displaying mic o ubule al e a ions ha igge he selec i e loss o Pu kinje cells and consequen beha io al impai men s. The e ec s o di e en doses (1, 5, and 10mg/kg, i.p.) and adminis a ion schedules (ch onic and acu e) o OEA we e assessed a he beha io al, his ological, cellula , and molecula le els o de e mine he mos e ec i e OEA ea men egimen. Ou in i o esul s demons a ed ha OEA ea men p io o he onse o he p eneu odegene a i e phase p e en ed mo phological al e a ions in Pu kinje neu ons ( he soma a and dend i ic a bo s) and dec eased Pu kinje cell dea h. This e ec ollowed an in e ed U-shaped ime- esponse cu e, wi h acu e adminis a ion on pos na al day 12 (10mg/kg, i.p.) being he mos e ec- i e ea men egimen es ed. Indeed, PCD mice ha ecei ed his speci ic OEA ea men egimen showed imp o emen s in mo o , cogni i e and social unc ions, which we e impai ed in hese mice. Mo eo e , hese in i o neu op o ec i e e ec s o OEA we e media ed by he PPARα ecep o , as p e ea men wi h he PPARα an agonis GW6471 (2.5mg/kg, i.p.) abolished hem. Finally, ou in i o esul s sugges ed ha he molecula e ec o OEA was ela ed o mic o ubule s abili y and s uc u e since OEA adminis a ion no malized some al e a ions in mic o ubule ea u es in PCD-like cells. These indings p o ide s ong e idence suppo ing he use o OEA as a pha macological agen o limi se e e ce ebella neu odegene a i e p ocesses. Keywo ds Ce ebellum· Endocannabinoid· Neu odegene a ion· Neu op o ec ion· OEA· Pu kinje cell In oduc ion Mic o ubule de ec s a e known o be he basis o se - e al b ain diseases [1–9], e en causing he dea h o neu- al popula ions [10–13]. Recen ly, we showed ha excess polyglu amyla ion induces al e a ions in bo h he dynamics and s uc u e o mic o ubules in Pu kinje cell degene a ion (PCD) mice [9, 10, 13, 14]. These animals ha bo a mu a ion in he Nna1/Ccp1 gene, which encodes ca boxypep idase 1 (CCP1), an enzyme esponsible o he deglu amyla ion o mic o ubules. A lack o ac i i y o his enzyme igge s These au ho s Es e Pé ez-Ma ín and Rod igo Muñoz-Cas añeda con ibu ed equally o his wo k. * Da id Díaz [email p o ec ed] * Edua do We uaga [email p o ec ed] 1 Labo a o y o Neu onal Plas ici y andNeu o epai , Ins i u e o Neu osciences o Cas ile andLeon (INCyL), Uni e si y o Salamanca, 37007Salamanca, Spain 2 Ins i u e o Biomedical Resea ch o Salamanca (IBSAL), 37007Salamanca, Spain 3 GIN, Uni . G enoble Alpes, CNRS, CEA, G enoble Ins i u e Neu osciences, Inse m, U121638000G enoble, F ance 4 Depa men o S a is ics, Uni e si y o Salamanca, 37007Salamanca, Spain 5 Depa men o Theo e ical, A omic andOp ical Physics, Uni e si y o Valladolid, 47071Valladolid, Spain 6 Uni e sidad de Ta apacá, A ica, Chile / Published online: 7 Ap il 2021 Neu o he apeu ics (2021) 18:1748–1767 excessi e mic o ubule polyglu amyla ion and subsequen pos na al Pu kinje cell dea h [10, 13]. The neu onal degen- e a ion caused by he loss o CCP1 ac i i y is a complex p ocess in ol ing wo clea ly dis inc phases: a p eneu o- degene a i e s age om pos na al day (P) 15 o P18, which is cha ac e ized by nuclea , cy ological, and mo phological al e a ions in Pu kinje cells, and a neu odegene a i e s age om P18 onwa ds, in which Pu kinje cells die [14–16]. Pu kinje cell degene a ion a hese ages con ibu es no only o mo o dys unc ion bu also o g adual cogni i e and social impai men s in PCD mice h oughou he ce ebella degene a ion p ocess, as has been epo ed in o he mod- els in which he ce ebellum is a ec ed [14, 17–19]. Du ing p eneu odegene a ion, he explo a o y and social beha io s o PCD mu an s on C57BL/DBA backg ound a e a ec ed, showing less ea ing ime and a lack o social p e e ence compa ed o wild- ype (WT) animals [14, 20]. F om he beginning o he neu odegene a ion s age, PCD mu an s we e impai ed in mo o asks, g ooming beha io and mem- o y ecogni ion, showing lowe all la ency in he o a od es , di e ences in he ime spen in g ooming and a lack o no el y disc imina ion compa ed o he beha io o WT mice [14, 19]. This animal model ecapi ula es key ea u es o he in an ile-onse neu odegene a ion and ce ebella a o- phy obse ed in humans wi h a monogenic biallelic mu a ion in CCP1 [7, 21]. Thus, he PCD mu an mouse is a e y sui - able model o explo ing and assessing di e en he apeu ic app oaches o ea ing se e e human degene a i e diso de s. The endocannabinoid sys em plays an essen ial ole in mic o ubule- ela ed b ain diseases, and some o i s com- ponen s ha e been epo ed o exe neu op o ec i e e ec s [22–25]. Howe e , o ou knowledge, he e ha e been no s udies add essing he use o noncanonical endocannabi- noids as he apeu ic agen s o he ea men o hese neu- odegene a i e diseases. In his sense, he endocannabinoid oleoyle hanolamide (OEA) has been shown o p e en neu onal damage and neu oin lamma ion in di e en ani- mal models o b ain disease and men al diso de s [26–34]. Mo eo e , he binding o OEA o pe oxisome p oli e a o - ac i a ed ecep o -alpha (PPARα) [35–38] igge s he exp ession o di e en mic o ubule-associa ed p o eins ha may in luence he s abili y o mic o ubules, p e en ing neu- ons om degene a ing [39]. Thus, OEA has eme ged as a p omising he apeu ic agen o p e en ing mic o ubule- ela ed neu odegene a ion and e e sing subsequen neu- obeha io al impai men s. The e o e, he objec i e o his wo k was o examine he e ec o OEA on ce ebella degene a ion and neu obeha - io al de ec s in PCD mice. To his end, we analyzed he gene al ce ebella s uc u e and he mo phology and su i al o Pu kinje cells a e adminis e ing OEA a di e en dos- ages o PCD mice in i o. Then, we explo ed he e ec o OEA on mo o , cogni i e and social unc ions, which a e impai ed in his animal model. Addi ionally, we assessed he in ol emen o PPARα ecep o s in he e ec s o OEA obse ed in i o. Finally, we employed an addi ional in i o model o iden i y he cellula mechanisms igge ed by OEA, which could no be p ope ly analyzed in i o. To his end, we examined mic o ubule dynamics and s uc u e in Ccp1- KO cells by exposing hem o di e en doses o OEA. Ma e ials andMe hods Animals Mice we e housed unde a 12-h/12-h ligh /da k cycle a a cons an oom empe a u e and humidi y and p o ided ad libi um access o wa e and special oden chow a he Animal Facili ies o he Uni e si y o Salamanca (Sala- manca, Spain) o Joseph Fou ie Uni e si y (G enoble, F ance). All animal p ocedu es we e app o ed by Bioe hics Commi ees o bo h he Uni e si y o Salamanca and Joseph Fou ie Uni e si y and we e pe o med in acco dance wi h he guidelines es ablished by Eu opean (2010/63/UE) and na ional legisla ions (Spanish RD53/2013 and Law 32/2007; F ench pe mi no. 38 07 11). All e o s we e made o mini- mize animal su e ing and o use he ewes animals equi ed o p oduce s a is ically ele an esul s. WT and mu an pcd1J (PCD) mice on a C57BL/DBA backg ound we e ob ained om Jackson Labs and employed o all he in i o p ocedu es, and WT and Ccp1-KO mouse emb yos we e used o isola e cells o he in i o expe i- men s. Ccp1-KO mice a e conside ed equi alen o he s anda d PCD mouse model since like PCD mice, hey lack he exp ession o Ccp1 mRNA [10, 14]. The p ocedu es o Ccp1-KO-de i ed cell cul u e and subsequen in i o analy- ses (see below) we e s anda dized a Joseph Fou ie Uni e - si y o ensu e he easibili y o hese expe imen s. The in i o o in i o s udies a e desc ibed sepa a ely below. In i o S udies In i o Expe imen al Design To acili a e he p esen a ion o he in i o p ocedu es pe - o med in his s udy, we p o ide a summa y o he wo k low, expe imen al g oups and sample size o each se o expe i- men s below. 1. Be o e he s a o he in i o OEA expe imen s, he exp ession o he endocannabinoid PPARα ecep o in WT and PCD mice du ing pos na al de elopmen pe iod was measu ed since PPARα is conside ed he main ecep o h ough which OEA exe s i s ac ion [33]. Oleoyle hanolamide Delays he Dys unc ion and Dea h o Pu kinje Cells and Amelio a es… 1749 Animals we e so ed on he basis o geno ype (WT o PCD) and age a analysis (P7, P15, P17, P22, o P30; n = 4 animals pe g oup). 2. To assess he e ec o OEA ea men a he his ologi- cal le el, a di e en se o animals was employed and analyzed a P30. The expe imen s we e di ided in o wo phases, as depic ed in Fig.1. In he i s se o expe i- men s (Fig.1, Phase I) we aimed o de e mine he bes d ug dose (0, 1, 5, o 10mg/kg) and schedule o admin- is a ion (ch onically om P7 o P21 o acu ely a P14 o P16, which is one day be o e o one day a e he p eneu odegene a i e p ocess begins, espec i ely; n = 4 animals pe g oup) o iden i y he op imal he apeu ic dosage o OEA. Based on he esul s ob ained, we pe - o med a second expe imen (Fig.1, Phase II) in which we e ined he c i ical he apeu ic window o OEA by acu ely adminis e ing he mos e ec i e dose (10mg/ kg, see esul s) o OEA a h ee di e en ages: P14, P12 and P10 (i.e., one, h ee and i e days be o e he onse o p eneu odegene a ion in PCD mice, espec i ely; n = 7 animals pe g oup). We employed un ea ed PCD mice as con ols in bo h expe imen s (n = 4–7 animals). 3. To e i y whe he he neu op o ec i e e ec o OEA in PCD mice also ansla es o an imp o emen in beha io , WT and un ea ed and ea ed PCD mice (n = 8–9 ani- mals pe g oup) we e subjec ed a wide se o beha io al asks (desc ibed in de ail la e ). To educe he numbe o animals, only he mos e ec i e OEA ea men egimen was used based on he his ological analyses desc ibed abo e. Mo eo e , he g oups o animals subjec ed o beha io al es s we e also employed o his ological e alua ion o he long- e m e ec o OEA ea men a P40. 4. Finally, o con i m he di ec in ol emen o PPARα ecep o s in he obse ed in i o OEA e ec s a bo h he his ological and beha io al le els, an addi ional g oup o PCD mice was p e ea ed wi h he PPARα an agonis GW6471 and analyzed a P30 (n = 7 animals). The de ailed in i o expe imen al me hodology is desc ibed below. D ug Adminis a ion Bo h OEA (Calbiochem, San Diego, USA) and he PPARα an agonis GW6471 (Toc is Bioscience, B is ol, UK) we e eshly p epa ed on he day o adminis a ion o a oid d ug deg ada ion. Bo h compounds we e dissol ed sepa a ely in 100% e hanol / and dilu ed in H2O Elix. OEA was admin- is e ed by in ape i oneal injec ion (i.p.) a a dose o 1, 5 o 10mg/kg b.w. acco ding o p e ious s udies [26, 28, 29]. When GW6471 was equi ed, i was adminis e ed i.p. a a dose o 2.5mg/kg b.w. 15min be o e OEA ea men . PCD con ol animals we e injec ed i.p. wi h 0.9% NaCl w/ . All animals we e ea ed in he mo ning. The body weigh o each animal was moni o ed h oughou he en i e expe imen because OEA has ano exigenic side e ec s [35, 38, 40]. Tissue Ex ac ion andP epa a ion Animals we e deeply anes he ized a P7, P15, P17, P22, P30, o P40 (depending on he expe imen ) wi h 10µl/g b.w. chlo al hyd a e (P olabo, Fon enay-sous-Bois, F ance) and in aca dially pe used wi h 0.9% NaCl w/ ollowed by 5ml/g b.w. modi ied Somogyi’s ixa i e consis ing o 4% Fig. 1 The expe imen al in i o pa adigm. The imeline de ails he wo se s o expe imen s o he OEA ea men , he ime poin s o beha io al es ing and issue collec ion, and hei ela ionship wi h he neu odegene a i e p ocess o he PCD mouse E. Pé ez-Ma ín e al. 1750 pa a o maldehyde w/ . Ce ebella we e sec ioned sagi ally using a eezing-sliding mic o ome (Leica Jung SM 2000, Nussloch, Ge many; 30µm hick) and washed in phospha e- bu e ed saline (PBS), pH 7.4. Immuno luo escence F ee- loa ing sec ions we e incuba ed a 4°C o 72h unde con inuous o a ion in 0.2% T i on X-100 / , 5% no mal se um / and he ollowing p ima y an ibodies dilu ed in PBS: guinea pig an i-CB1 (1:1,000; F on ie Ins i u e, Hok- kaido, Japan), abbi an i-PPARα (1:100; Pie ce An ibod- ies, Rock o d, IL, USA), and mouse an i-calbindin D-28k (Cb28k; 1:1000; Swan , Bellinzona, Swi ze land). The ollowing p ima y an ibodies we e employed o comple- men a y analyses: goa an i-CB2 (1:500, San a C uz Bio- echnology, Dallas, TX, USA) and mouse an i-S100 (1:500; Sigma-Ald ich, S . Louis, MO, USA). Di e en combina- ions o hese an ibodies we e used o each expe imen . Then, he sec ions we e incuba ed wi h app op ia e sec- onda y an ibodies conjuga ed o Cy2, Cy3, o Cy5 (1:500; Jackson Labo a o ies, Wes G o e, PA, USA) o 2h a oom empe a u e and coun e s ained wi h DAPI (1:10,000; Sigma-Ald ich) o iden i y he cell nuclei. Fo all his ologi- cal analyses, ou equidis an e mis pa asagi al sec ions we e assessed o each mouse. Quan i ica ion o  heEndocannabinoid PPARα The pe cen age o Pu kinje cells exp essing PPARα in pa - asagi al sec ions o he e mis du ing pos na al ce ebella de elopmen (P7, P15, P17, P22, and P30) was analyzed by immunos aining o bo h PPARα and Cb28k. Only Pu kinje cells wi h clea Cb28k+ soma a and comple e nuclei s ained wi h DAPI we e coun ed o a oid biases. Addi ionally, changes in he pe cen age o hese cells exp essing PPARα in PCD mice we e e alua ed a e OEA ea men a P30. Densi y andMo phological Analyses o Pu kinje Cells Bo h analyses we e pe o med on pa asagi al sec ions o he e mis immunos ained o Cb28k. Only Pu kinje cells wi h clea Cb28k+ soma a and dend i ic a bo s and com- ple e nuclei s ained wi h DAPI we e analyzed o a oid planime ic biases. Pu kinje cell su i al was exp essed as he Pu kinje cell linea densi y, which was calcula ed as he numbe o Pu kinje cell soma a pe mm o he Pu kinje cell laye ( om lobule I o lobule X o he e mis) in each sec ion. Then, he ollowing mo phological pa ame e s we e analyzed: (1) soma a ea, (2) leng h o Pu kinje cell dend i ic a bo s (i.e., molecula laye hickness), and (3) leng h and (4) wid h o he p ima y dend i e. These analy- ses we e pe o med a bo h P30 and P40 and ca ied ou using Neu olucida (MBF Bioscience, Willis on, Ve mon , USA) and ImageJ (NIH, USA) so wa e, as p e iously desc ibed [14]. Beha io al Analyses A ba e y o es s o beha io s associa ed wi h ce ebel- la unc ion was pe o med a P15, P17, P22, P30, and P40 be ween 9:00 am and 1:00 pm. Video eco dings we e ob ained and me iculously analyzed by a esea che blinded o he expe imen al condi ions (E P-M). A e each ial, he a ious appa a uses and objec s we e cleaned wi h 70% e hanol / . The o a od es (Panlab, Ba celona, Spain) was used o assess mo o coo dina ion as p e iously desc ibed [14]. The od accele a ed a a a e o 0.06 pm/s om 4 o 40 pm o 10min ( od diame e = 30mm). The la ency o all o was measu ed in se en ials pe day wi h 20-min in e als be ween ials. The mean la ency was calcula ed o each mouse on each day o he ask. Home-cage beha io analysis was used o cha ac e ize gene al beha io [14]. A e 10min o habi ua ion, g oom- ing ime (inna e s e eo yped beha io ), ea ing ime (en i- onmen al explo a o y beha io ), ime spen mo ing (gene al mo emen ), ime in ac i i y and es ing ime we e analyzed. Each animal was assigned o an indi idual home cage o a oid he in luence o odo emi ed om he o he animals. The no el objec ecogni ion (NOR) es was pe o med o e alua e long- e m objec ecogni ion memo y in oden s [41,42]. On he i s day (P15), wo iden ical objec s placed in opposi e co ne s o a cage we e p esen ed o a single ani- mal o 10min. Then, in each o he ollowing sessions (a P17, P22, P30, and P40), he animals we e e u ned o he cage, which con ained he amilia objec and a no el one. All objec s we e made o plas ic and we e o di e en colo s and shapes bu o simila size. Two di e en measu es o disc imina o y beha io we e analyzed. The i s measu e was he pe cen age o ime explo ing he no el (TN) and amilia (TF) objec s, which was calcula ed as ollows: The second measu e was he disc imina o y index (DI), which was calcula ed as ollows: The DI can a y be ween +1 and −1, wi h a posi i e sco e indica ing mo e ime spen explo ing he no el objec , a nega i e sco e indica ing mo e ime spen explo ing he amilia objec , and ze o indica ing a lack o p e e ence [43]. % TN= T N T N +T F ,%TF= T F T N +T F . DI = T N −T F T N +T F . Oleoyle hanolamide Delays he Dys unc ion and Dea h o Pu kinje Cells and Amelio a es… 1751 The h ee-chambe ed social p e e ence es was pe - o med o assess sociabili y in a whi e Plexiglas box (50 × 29cm) di ided in o h ee connec ed chambe s [14, 44]. A e 10min o habi ua ion, each mouse was placed in he h ee-chambe ed box con aining ei he a mouse o he same age and sex o an objec , bo h o which co e ed by a simila d illed pencil cup and placed in each o he la e al ooms o he box. The esul s we e exp essed as wo di e - en measu es o sociabili y and p e e ence. The i s was he pe cen age o ime spen in e ac ing wi h he animal (TA) and he objec (TO), which was calcula ed as ollows: Analogous o he DI, he sociabili y index (SI) was cal- cula ed as ollows: In his case, he SI can also a y be ween +1 and −1, wi h a posi i e sco e indica ing a p e e ence o social in e ac ion, a nega i e sco e indica ing an a oidance o social in e ac ion and ze o indica ing he lack o any p e e ence conce ning socializa ion. In i o S udy Cell Cul u e andT ans ec ion To s udy whe he he e ec o OEA is ela ed o changes in mic o ubule s abili y, which is a ec ed in PCD cells, we employed a s anda dized in i o sys em based on mouse emb yonic ib oblas s (MEFs) since his app oach allows isualiza ion o indi idual mic o ubules and measu emen o pa ame e s ela ed o hei mo phology and dynamics [14]. The cell cul u e and ans ec ion echniques equi ed o hese analyses comp omise he iabili y o PCD-de i ed neu ons and hus he abili y o ob ain eliable da a [14]. Fo his eason, Ccp1-KO MEFs we e employed o hese analy- ses. The animal sou ce o hese cells is conside ed equi a- len o PCD mice since hey also lack mRNA exp ession o Ccp1 [10, 14]. All he p ocedu es we e s anda dized in hese PCD-like cells a Joseph Fou ie Uni e si y o ensu e he easibili y and eliabili y o hese expe imen s. MEFs om he b ains o 13.5-day-old WT and Ccp1-KO mouse emb yos we e isola ed ollowing s anda d p ocedu es as p e- iously desc ibed [14,45, 46]. B ie ly, MEFs we e cul u ed in Dulbecco’s modi ied Eagle’s medium (DMEM; Li e Tech- nologies, Gibco) supplemen ed wi h 10% e al bo ine se um (FBS) un il hey eached 80% con luence. A e wa ds, o % TA= T A T A +T O ,%TO= T O T A +T O . SI = T A −T O T A +T O . isualize mic o ubules, MEFs we e ans ec ed wi h di e - en plasmids; Nucleo ec o ™ Ki s o MEFs (Amaxa Bio- sys ems) we e used o ans ec cells wi h GFP-EB3 o isu- alize mic o ubule plus-ends in g een (p o ided by N. Galja , E asmus Medical Cen e , Ro e dam, The Ne he lands) and m-che y α- ubulin o isualize he en i e mic o ubule s uc- u e in ed (p o ided by F. Saudou, Cu ie Ins i u e, Pa is, F ance) as p e iously desc ibed [14]. Cell Cul u e T ea men s Cells we e di ided in o expe imen al g oups (n = 3 emb yos pe g oup) on he basis o geno ype (WT o PCD) and he concen a ion o OEA adminis e ed (0, 0.1, 0.5, o 1.0µM) based on p e ious wo ks [26]. OEA was dissol ed in 100% DMSO and s o ed a − 80°C. On he day o he expe imen , OEA was dilu ed in cul u e medium (DMEM + 10% FBS) a he app op ia e concen a ion and added o he cells. Con- ol (un ea ed) cells om WT and PCD animals we e also cul u ed wi h he same concen a ion o DMSO o a oid possible con ounding e ec s. Analysis o Mic o ubule S uc u e andDynamics Mic o ubule dynamics we e analyzed using images om ime-lapse ideos o GFP-EB3 s aining and plusTipT acke so wa e [47]. Time-lapse ideos o ans ec ed MEFs we e cap u ed wi h an in e ed mic oscope (Axio- e 200M; Ca l Zeiss, Inc., Obe kochen, Ge many) and a × 100 NA 1.3 Plan-Neo lua oil objec i e ha was con olled wi h Me aMo ph so wa e (MDS Analy ical Technologies, CA, USA). Images we e cap u ed wi h a cha ge-coupled de ice came a (CoolSNAP HQ; Rope Scien i ic, Vianen, The Ne he lands) e e y 3 s o 5min. The pa ame e s ha we e analyzed we e g ow h and sh inking speed; g ow h and sh inking mean leng h; pe cen age o ime in pause, g owing, and sh inking; equency o ca as ophes (changes om g owing o sh inking); equency o escues (changes om sh inking o g owing); mic o ubule cu a u e (s a ics); and mic o ubule ajec o y cu a u e (mo emen ) based on p e ious s udies [14,46,47]. B ie ly, pa ame e s ela ed o g ow h and sh inking p ocesses we e analyzed using he comple e sequence images ime-lapse ideos o GFP-EB3 (labels mic o ubule plus-ends) s aining, whe eas o s a ic mic o ubule cu a u e analysis, he i s image o each ideo ime-lapse ideo o m-che y α- ubulin (labels he en i e mic o ubule) s aining was employed. Addi ionally, o GFP- EB3 labeling, we ob ained he maximum p ojec ion o he en i e ime-lapse ideo, allowing us o econs uc he mo e- men ajec o y cu a u es o he mic o ubule plus-ends as p e iously desc ibed [14]. E. Pé ez-Ma ín e al. 1752 S a is ical Analysis The esul s a e exp essed as he means ± s anda d e o s o he mean (SEMs). Homoscedas ici y and no mali y we e checked p io o all o he s a is ical analyses (Kolmogo- o –Smi no ’s and Le ene’s es s). Due o he complexi y and a iabili y o he condi ions analyzed in he in i o and in i o expe imen s and o acili a e comp ehension, each s a is ical es and he a iables compa ed a e p o ided wi h he esul s o each co esponding expe imen . All analyses we e pe o med using SPSS so wa e o Windows V25 (IBM, NY, USA). Resul s Exp ession o Endocannabinoid Recep o s inPCD Mice Be o e s a ing he in i o OEA expe imen , we analyzed he exp ession o componen s o he endocannabinoid sys- em h oughou pos na al ce ebella de elopmen , ocusing p ima ily on PPARα since i is conside ed he main a - ge h ough OEA exe s i s ac ions [35]. On he one hand, PPARα was de ec ed and quan i a i ely analyzed in he h ee ce ebella laye s o he ce ebellum in bo h geno ypes a all analyzed (Fig.2a–c). Howe e , S uden ’s es showed ha he pe cen age o Pu kinje cells exp essing PPARα was sligh ly lowe in he mu an animals han in he WT animals om P15 onwa d (Fig.2d; P15, p < 0.05; P17, p < 0.05; P22, p < 0.05). On he o he hand, due o he di use exp ession and dis ibu ion o he canonical cannabinoid ecep o s CB1 and CB2 in he ce ebellum, he analysis o hei exp ession was quali a i ely pe o med. CB1 ecep o s we e exp essed in he molecula laye om P7 onwa ds and in he baske cells o “Pinceaux o ma ion” om P17 onwa ds. Howe e , no quali a i e di e ences in CB1 exp ession we e de ec ed be ween geno ypes a any o he ages analyzed (Fig.2c). By con as , he exp ession o CB2 seemed o inc ease in he PCD mice du ing he neu odegene a i e p ocess ( om P22) and colocalized wi h he apical adial p ocesses o he Be gmann glial cells (Supplemen a y Fig.1). Anyway, he inding ha PPARα is exp essed in he ce ebellum led us o belie e ha OEA may exe a neu op o ec i e e ec on Pu kinje cells in PCD mice in i o. OEA Adminis a ion Inc eases PPARα Exp ession inPCD Mice A e demons a ing ha PPARα is exp essed du ing pos - na al ce ebella de elopmen , we assessed he in luence o OEA adminis a ion on he exp ession o PPARα in PCD mice. One-way ANOVA ollowed by Dunne ’s pos hoc es e ealed an inc ease in he pe cen age o Pu kinje cells exp essing PPARα in PCD mice when OEA was adminis- e ed a doses o 5 and 10mg/kg, independen o he sched- ule o adminis a ion (Fig.2e, Supplemen a y Fig.2; 5mg/ kg, p < 0.05; 10mg/kg, p < 0.01). OEA P e en s Al e a ions in heMo phology o Pu kinje Cells We hen analyzed he e ec o OEA adminis a ion on p o- g essi e mo phological al e a ions in PCD Pu kinje cells. Ou i s objec i e was o de e mine he e ec i eness o di e en OEA doses (1, 5, o 10mg/kg) and adminis a- ion schedules (ch onically om P7–P21 o acu ely a P14 o P16) on he main enance o Pu kinje cell mo phology (Fig.3a–h). One-way ANOVA ollowed by Dunne ’s pos hoc es was pe o med o compa e each dose and adminis- a ion schedule s. con ol ea men . Analysis o he da a a P30 showed ha ch onic and acu e adminis a ion o bo h 5 and 10mg/kg OEA p io o he p eneu odegene a i e s age amelio a ed mo phological al e a ions in Pu kinje cells (Fig.3i–l). In con as , when OEA was adminis e ed a P16, only he highes dose, 10mg/kg, was e ec i e in alle ia ing some o he mo phological changes (Fig.3i–l). Then, we ca ied ou a second se o expe imen s o imp o e u he he he apeu ic e ec o OEA in i o. Based on he p e ious esul s and o educe animal su e ing, we adminis e ed a single injec ion o he mos e ec i e dose o OEA (10mg/kg) p io o p eneu odegene a ion. We com- pa ed he e ec o OEA adminis a ion a h ee ime poin s (P14, P12, P10, i.e., one, h ee o i e days be o e he onse o neu odegene a ion in PCD mice, espec i ely) by one-way ANOVA ollowed by Bon e oni’s pos hoc es (Fig.4). The esul s o his analysis showed ha acu e adminis a ion a Fig. 2 Exp ession o he PPARα endocannabinoid ecep o in he ce ebellum. (a, b) Sagi al sec ion o he e mis o a WT mouse (a) and con ocal image wi h idimensional p ojec ion (b) showing he co-localiza ion o PPARα (g een) in Pu kinje cells (Cb28k, ed) a P30. (c) Mic og aphs showing he exp ession o he PPARα ( ed), CB1 ecep o (g een), and Pu kinje cells (Cb28k, blue) in WT and PCD mice h oughou he pos na al ce ebella de elopmen (P7, P15, P17, P22, P30); PPARα exp ession can be obse ed in he h ee ce - ebella laye s a all pos na al ages. The CB1 ecep o is exp essed in he molecula laye om P7 onwa ds. In addi ion, om P15 onwa d, he CB1 ecep o begins o be exp essed a ound Pu kinje cell soma a (Cb28k posi i e), leading o he “Pinceaux o ma ion.” (d) Quan i i- ca ion o he pe cen age o Pu kinje cells exp essing PPARα a di e - en ime poin s o he pos na al ce ebella de elopmen ; no e ha his alue is lowe in PCD mice han in WT om P15 o P22. (e) Quan i- ica ion o he pe cen age o PCD’s Pu kinje cells exp essing PPARα a P30 a e di e en OEA dosages; OEA adminis a ion leads o an inc ease in he numbe o Pu kinje cells exp essing PPARα in he h ee di e en ea men s a doses o 5 and 10mg/kg. Da a a e ep- esen ed as mean ± SEM; n = 4 each expe imen al g oup; S uden ’s es o (d); one-way ANOVA ollowed by Dunne ’s pos hoc es o (e); *p < 0.05; **p < 0.01 ◂ Oleoyle hanolamide Delays he Dys unc ion and Dea h o Pu kinje Cells and Amelio a es… 1753 E. Pé ez-Ma ín e al. 1754 Fig. 3 In i o e ec o di e en OEA dosages on he mo phology and su i al o he Pu kinje cells o he PCD mouse analyzed a P30 ( i s se o expe imen s). (a–d) Mic og aphs o PCD ce ebella e mis slices labeled wi h calbindin (Cb28k, g een) a e di e en OEA ea men s: con ol (a), ch onic adminis a ion om P7 o P21 o 5mg/kg (b), acu e adminis a ion o 10mg/kg a P14 (c), acu e adminis a ion o 10mg/kg a P16 (d).(e–h) Mic og aphs o Pu kinje cells labeled wi h calbindin (Cb28k; ed) in PCD animals ea ed wi h di e en dosages o OEA: con ol (e), ch onic adminis a ion om P7 o P21 o 10mg/kg ( ), acu e adminis a ion o 10mg/kg a P14 (g), acu e adminis a ion o 10mg/kg a P16 (h). (i–l) Quan i- ica ion o he OEA e ec on di e en mo phological pa ame e s o PCD Pu kinje cells; no e ha he ch onic and acu e adminis a ion a P14 ha e a s onge neu op o ec i e e ec han he acu e admin- is a ion a P16 in all he pa ame e s e alua ed. (m) Quan i ica ion o PCD Pu kinje cell su i al a di e en OEA dosages; no e ha bo h ch onic adminis a ion a a dose o 5mg/kg and acu e adminis a ion a P14 a a dose o 10mg/kg p e en ed Pu kinje cell dea h. Da a a e ep esen ed as mean ± SEM; n = 4 each expe imen al g oup; one-way ANOVA ollowed by Dunne ’s pos hoc es o (i-m) * p < 0.05; **p < 0.01 Oleoyle hanolamide Delays he Dys unc ion and Dea h o Pu kinje Cells and Amelio a es… 1755 he h ee ime poin s p e en ed mo phological al e a ions a P30 (Fig.4i–l). Fu he mo e, his neu op o ec i e e ec ol- lowed an in e ed U-shaped ime- esponse cu e, wi h acu e adminis a ion a P12 being he mos e ec i e adminis a- ion ime poin o p e en ing Pu kinje cell mo phological al e a ions, as he mo phological pa ame e s eached al- ues simila o hose o WT animals (Fig.4i–l). Nex , we decided o de e mine whe he his neu op o ec i e e ec o OEA adminis e ed a P12 was main ained o e ime. How- e e , he esul s a P40 showed ha he alues o only wo o he pa ame e s analyzed (soma a ea and p ima y dend i e leng h) we e main ained (Supplemen a y Fig.3). OEA Adminis a ion Dec eases Pu kinje Cell Dea h One-way ANOVA ollowed by Dunne ’s pos hoc es was pe o med o compa e he e ec o di e en OEA doses used in he i s se o expe imen s on Pu kinje cell su - i al. The esul s showed ha ch onic adminis a ion o OEA a a dose o 5mg/kg (p < 0.05) bu no 1 o 10mg/ kg om P7-P21 had a p e en i e e ec on Pu kinje cell dea h (U-shaped esponse). OEA adminis a ion a P14 also amelio a ed his degene a ion, bu only a a dose o 10mg/ kg (p < 0.05). In con as , when OEA was adminis e ed a P16, ha is, a e p eneu odegene a ion had al eady s a ed, he ea men had no e ec on Pu kinje cell dea h (Fig.3m). Thus, simila o he indings ela ed o Pu kinje cell mo - phology, OEA seems o inc ease Pu kinje cell su i al only when i is adminis e ed p io o he onse o p eneu ode- gene a ion. As be o e, we compa ed he e ec o acu e p e en i e ea men a P14, P12, and P10 on Pu kinje cell su i al by pe o ming one-way ANOVA ollowed by Bon e oni’s pos hoc es . The esul s con i med ha acu e adminis a ion o OEA (10mg/kg) educed Pu kinje cell degene a ion when adminis e ed p io o he onse o p eneu odegene a ion a ei he P12 (p < 0.01) o P14 (p < 0.05). This e ec also ollowed an in e ed U-shaped ime- esponse cu e, wi h OEA adminis a ion a P12 being he mos e ec i e ea men (Fig.4m; p < 0.01). OEA adminis a ion a P10 had no e ec on Pu kinje cell densi y. Mo eo e , he numbe o Pu kinje cells in animals ea ed a P10 seemed o be e en lowe han ha in un ea ed PCD mice, p obably due o he dele e ious e ec s o OEA a e y ea ly s ages (Fig.4m). Indeed, he weigh o he PCD animals ea ed a P10 was signi ican ly lowe han ha o he es o he PCD animals (Supplemen a y Table1). E ec o OEA onMo o Coo dina ion Once bo h he neu op o ec i e p ope ies o OEA we e demons a ed his ologically and he mos e ec i e he a- peu ic ime window was iden i ied, we wonde ed whe he he cellula neu op o ec i e e ec s o OEA could be ans- la ed in o an imp o emen in beha io al impai men s in PCD mice. To answe his ques ion and o educe he numbe o animals employed in he s udy, we analyzed he e ec o only he mos e ec i e OEA ea men egimen (10mg/kg, i.p. a P12) on he mo o , cogni i e, and social beha io o PCD mice h oughou he en i e neu odegene a i e p ocess (a P15, P17, P22, P30, and P40). Fi s , o s udy mo o coo - dina ion, we subjec ed mice in he h ee expe imen al g oups (WT mice, PCD mice and PCD mice ea ed a P12) o he o a od es (Fig.5a). One-way epea ed-measu es ANOVA esul s e ealed ha o a od da a es iola ed sphe ici y (p Mauchly’s es < 0.01). They exhibi ed s a is ically signi i- can di e ences wi hin he ‘day o es ing’ ac o (p < 0.01) and he ‘expe imen al g oup’ ac o (p < 0.01) sepa a ely; and in he in e ac ion be ween bo h ac o s ‘expe imen al g oup’ * ‘day o es ing’ (p < 0.01), o he ou c i e ia o he mul i a ia e analysis (Pillai’s ace, Wilks’ Lambda, Ho elling’s ace and Roy’s la ges oo ). G aphic ep esen- a ion o he o a od es da a showed ha he gene al mo o pe o mance o he h ee expe imen al g oups di e ged o e ime (which can explain he in e ac ion be ween ac o s, see “Discussion”), wi h he mos signi ican di e ences being be ween he WT and PCD mice (Fig.5b). The pe o mance o he WT mice in he o a od es g adually imp o ed un il P30 and hen s abilized, p obably due o lea ning (Fig.5b). Howe e , ea ed PCD mice showed simila beha io as WT mice un il P22 bu hen g adually exhibi ed mo o beha io ha was mo e simila o ha o un ea ed PCD animals (Fig.5b). To e alua e u he he possible di e - ences be ween he h ee expe imen al g oups wi hin each day o es ing, we pe o med one-way ANOVA ollowed by Bon e oni’s pos hoc es on each day o o a od es - ing. These esul s e ealed ha he mo o beha io o WT, un ea ed PCD and ea ed PCD mice was simila a P15 (p > 0.05), bu di e ed om each o he om P17 onwa ds (Fig.5b; P17, p < 0.01; P22, p < 0.01; P30, p < 0.01; P40, p < 0.01). Fi s , he mo o coo dina ion o PCD mice (bo h un ea ed and ea ed) was a ec ed compa ed o ha o WT animals since he beginning o p eneu odegene a ion excep o P15 (un ea ed PCD mice: p < 0.01 o P17, P22, P30, and P40; ea ed PCD mice: p < 0.01 o P17, P22, P30 and P40). Howe e , PCD animals ea ed wi h OEA showed an imp o emen in he pe o mance o he o a od es a P22 and P30 compa ed o un ea ed PCD mice (Fig.5b; P22, p < 0.05; P30, p < 0.05). Un o una ely, his amelio a ion was no de ec ed a P40 when mo o beha io o bo h ea ed and un ea ed PCD we e simila and no di e ences we e de ec ed be ween hese wo expe imen al g oups (p > 0.05). These inding indica es ha al hough OEA ea men did no com- ple ely es o e he no mal mo o coo dina ion o PCD mice, i con ibu ed o imp o ing i since he mo o beha io o E. Pé ez-Ma ín e al. 1756 OEA Inc eases Mic o ubule Dynamics andRes o es Mic o ubule Shape In i o A e demons a ing ha OEA exe s i s neu op o ec i e e ec s in i o in PCD mice a bo h he his ological and beha io al le els h ough PPARα and gi en ha excess mic o ubule polyglu amyla ion in mu an cells igge s al e a ions in bo h he dynamics and s uc u e o mic o- ubules [10, 13, 14], we wonde ed whe he he molecu- la e ec o OEA could be ela ed o hese mic o ubule cha ac e is ics. To answe his ques ion, we employed an addi ional in i o MEF model ha allowed us o isualize Fig. 9 In i o e ec o OEA on WT and Ccp1-KO mic o ubule dynamics o MEFs.(a) Analyses o WT and Ccp1-KO mic o ubule g ow h and sh inking a es; OEA adminis a ion a ec s bo h pa ame- e s and expe imen al g oups in a dose-dependen manne , al hough a highe OEA concen a ion is equi ed in Ccp1-KO cells. (b) Analyses o WT and Ccp1-KO s a ic mic o ubule cu a u e; OEA adminis a- ion dec eased Ccp1-KO mic o ubule cu a u e bu did no a ec WT cu a u e. (c) Analyses o WT and Ccp1-KO mic o ubule ajec o y cu a u e; OEA did no a ec ajec o y cu a u e in any o he wo geno ypes. (d, e) Examples o WT and Ccp1-KO mic o ubule cu - a u e (d) and ajec o y (e) in con ol and OEA-adminis e ed cells (1.0 μM) and he co esponding g aphical ep esen a ion. Da a a e ep esen ed as mean ± SEM; n = 3 emb yos pe expe imen al g oup; a.u. = a bi a y uni s; one-way ANOVA ollowed by Dunne ’s pos hoc es o (a–c); *p < 0.05 Oleoyle hanolamide Delays he Dys unc ion and Dea h o Pu kinje Cells and Amelio a es… 1763 indi idual mic o ubules since hese analyses canno be p ope ly pe o med in i o due o he s uc u al complex- i y and highly b anched dend i ic ees o Pu kinje neu ons. One-way ANOVA ollowed by Dunne ’s pos hoc es was employed o compa e he e ec o he di e en OEA doses s. con ol ea men (un ea ed WT and un ea ed Ccp1-KO cells) sepa a ely o each geno ype. The esul s showed ha OEA modi ied he dynamics and s uc u e o mic o ubules in cells o bo h geno ypes (Fig.9). Rega ding mic o ubule dynamics, OEA inc eased he g ow h and sh inkage a es o bo h WT (p < 0.05) and Ccp1-KO (p < 0.05) mic o ubules (Fig.9a). Howe e , he OEA concen a ion equi ed o a signi ican e ec was highe in Ccp1-KO cells (1.0µM) han in WT cells (0.1 and 0.5µM). Simila ly, he mean g ow h and sh inkage leng hs we e also inc eased in cells o bo h geno ypes a e OEA ea men , and he same OEA concen- a ion was equi ed (Supplemen a y Table2). In con as , WT mic o ubules unde wen a dec ease in he pe cen age o g owing ime (p < 0.05) and an inc ease in he pe cen - age o ime in pause (p < 0.05), whe eas no e ec on hese pa ame e s was seen o Ccp1-KO mic o ubules (Supple- men a y Table2). Rega ding mic o ubule s uc u e, OEA did no a ec he s a ic cu a u e o WT mic o ubules a any concen a ion (Fig.9b). Con e sely, OEA dec eased Ccp1-KO mic o ubule cu a u e a all he concen a ions es ed (Fig.9b; p < 0.05 o all analyses), making he mic o- ubules appea simila o WT mic o ubules. Las , OEA did no seem o a ec he cu a u e o he mic o ubule ajec o y in cells o any geno ype (Fig.9c). These indings sugges ha he molecula e ec o OEA may be ela ed o mic o- ubule dynamics and s uc u e since OEA in luenced hese mic o ubule ea u es in Ccp1-KO (PCD-like) cells. Discussion In he p esen wo k, we explo ed he neu op o ec i e e ec s o he endocannabinoid OEA and i s in luence on he ce - ebella in eg i y and beha io o PCD mice on C57BL/ DBA backg ound s ain. O e all, OEA delayed Pu kinje cell degene a ion and e e sed beha io al impai men h ough he endocannabinoid ecep o PPARα in his model o ce - ebella neu odegene a ion. PPARα is conside ed he main a ge o OEA [35–38]. I s exp ession was de ec ed in he h ee laye s o he ce ebel- lum in mice o bo h geno ypes. Howe e , in he Pu kinje cells o PCD mice, PPARα ecep o exp ession dec eased om P15 o P22, indica ing ha he pcd mu a ion a ec s he exp ession o elemen s o he endocannabinoid sys em in bo h he p eneu odegene a i e and neu odegene a i e s ages. Changes in PPARα exp ession may be e lec plas- ici y o he ce ebellum in an a emp o p e en neu onal dea h, which is in acco dance wi h p e ious s udies [26, 28, 29, 33]. Ne e heless, he de ec ion o PPARα exp ession in Pu kinje cells led us o design an expe imen o assess he e ec s o OEA in i o. Indeed, he adminis a ion o OEA o PCD animals inc eases he exp ession o PPARα, as p e iously desc ibed [32]. This inding may be ela ed o plas ici y o Pu kinje cells aimed a p e en ing neu onal dea h, which is consis en wi h he gene al neu op o ec i e e ec s exe ed by OEA ha we obse ed in PCD mice. In con as , he exp ession o he CB1 and CB2 ecep o s was no a ec ed. Al hough a pu a i e e ec o OEA on hese ecep o s canno be excluded, he ac i i y o OEA is con- side ed o be linked o PPARα [33–35, 37], hus suppo ing ou hypo hesis (see also he la e discussion o he use o he PPARα an agonis GW6471). OEA al e ed he in i o dynamics and s uc u e o mic o- ubules in cells o bo h geno ypes, causing mic o ubules om PCD-like cells o be simila o hose om WT cells. Since OEA no malizes some o he mic o ubule cha ac e - is ics impai ed in he mic o ubules o PCD-like cells, we specula e ha he p e en i e e ec o OEA on Pu kinje cell mo phology al e a ions may be ela ed o i s c ucial e ec s on cy oskele al s uc u es wi hin hese cells. P e ious ind- ings ha e shown ha he binding o OEA o PPARα igge s he exp ession o di e en p o eins (i.e., MAP-2 and GAP- 43) ha a e ela ed o mic o ubule s abili y and s uc u e and neu onal g ow h [37]. Thus, he e-es ablishmen o PCD mic o ubule shape by OEA may e lec he main enance o he mo phology o Pu kinje cell dend i ic a bo s and he p e en ion o Pu kinje cell dea h in PCD mice. The obse ed e ec s o OEA in i o a e in line wi h p e ious esul s, bo h in ela ion o i s pha macokine ics and e ec i e ime win- dow [24, 27, 29, 31]. On he one hand, OEA adminis a ion p e en s Pu kinje cell mo phological de ec s and dec eases cell loss when i is adminis e ed p io o p eneu odegene a- ion (i.e., ch onic adminis a ion om P7 o P21 and acu e adminis a ion a P14, P12 and P10). On he o he hand, his neu op o ec i e e ec ollows an in e ed U-shaped ime- esponse cu e, wi h acu e adminis a ion a P12 being he mos e ec i e ea men , especially ega ding dend i ic a bo- iza ion main enance. Speci ically, abno mali ies in neu al a bo iza ion a e associa ed wi h nume ous neu ological dis- o de s, such as schizoph enia, epilepsy, Alzheime ’s disease and au ism spec um diso de [48–51,52]. Hence, ou indings highligh he po en ial he apeu ic use o OEA as a neu o- p o ec i e d ug, no only o ce ebella diseases. Al hough he ce ebellum has long been consid- e ed o be a pu ely mo o s uc u e, ecen s udies ha e e ealed ha i also plays an impo an ole in nonmo- o unc ions such as cogni i e and a ec i e beha io [14, 17, 18], among o he s. A p e ious s udy conduc ed in ou labo a o y demons a ed ha ce ebella degene a- ion a an ea ly age in PCD mice leads o mo o , cog- ni i e and social al e a ions [14, 19, 20], mimicking E. Pé ez-Ma ín e al. 1764 he ea u es o human neu ode elopmen al diso de s in which he ce ebellum is in ol ed [7, 18, 21]. I is impo an o conside ha impai men s in PCD beha io ake place when mo phological al e a ions in Pu kinje cells a e exace ba ed o when hese cells a e los , and no o he b ain egions o neu al cell popula ion a e a ec ed [14,53–55]. Thus, he e ec o OEA in s abiliz- ing Pu kinje cell a bo iza ion and inc easing Pu kinje cell su i al should esul in unc ional imp o emen s. Indeed, he esul s o his s udy e ealed ha OEA ame- lio a ed all he beha io al de ec s obse ed in he PCD mu an mice un il P30, which is in ag eemen wi h he his ological indings. The esul s o he o a od es show ha he gene al mo o beha io o he ea ed PCD mice was hal way be ween ha o he WT and un ea ed PCD mice om P22 o P40. Howe e , he imp o emen s obse ed in WT mice om P30 onwa d due o lea ning [56] was no de ec ed in ea ed mice. The di e gen mo o beha io ansla ed in o a di e en ajec o y cu e in he g aph would explain he in e ac ion obse ed be ween he analyzed ac o s. These indings e ealed ha he neu o- p o ec i e e ec s exe ed by OEA a bo h mo phological and cell su i al le els con ibu ed o imp o e he mo o ask pe o mance o he PCD mice al hough hey we e no enough o comple ely es o e i s mo o de ec s. Rega ding o e all beha io , ou esul s a e in ag eemen wi h p e i- ous s udies showing ha gene al ce ebella degene a ion is mani es ed as changes in he ime spen in g ooming, ea ing, explo a ion and displacemen [14, 19, 20,57,58]. In his sense, he adminis a ion o OEA led o ees ablish- ing no mal beha io al pa e ns, wi h ea ed PCD mice exhibi ing beha io simila o ha o WT animals up o P30. In ou s udy, no hypolocomo o e ec s ela ed o high doses o OEA we e obse ed [29, 40,59]. In addi ion, i was ecen ly ound ha no mal cogni i e and social abili- ies equi e no mal ce ebella ac i i y [14, 17, 18,60,61]. In his con ex , he esul s ob ained in he NOR and social p e e ence es s e ealed ha OEA p o ec s agains he impai men s obse ed in he mouse model o ce ebella degene a ion employed in his s udy. These indings a e in ag eemen wi h hose o o he s udies in which o he endocannabinoids mainly es o ed o imp o ed cogni i e unc ion in di e en mouse models o b ain damage [25, 32,62–65]. Fu he mo e, i should be no ed ha , as a as we know, ou s udy is he i s o show ha OEA has he e ec o main aining no mal social beha io in mice wi h a ec i e de ici s ela ed o ce ebella dys unc ion. O e all, OEA was obse ed o ha e a neu op o ec i e e ec a bo h he his ological and beha io al le els un il P30. Al hough he changes in wo o he i e pa ame e s measu ed we e main ained a P40 a he his ological le el (soma a ea and p ima y dend i e leng h), his was no he case o any beha io al changes. In addi ion, i should be no ed ha mo o impai men s in PCD mice a his age could be esponsible o he esul s ob ained in he memo y and he social p e e ence es s a P40; he e o e, no conclusi e esul s can be d awn. Rega dless o his, i is impo an o no e ha we managed o delay he degene a ion in PCD mice and inc ease he empo al window in which o he he apeu- ic app oaches could be employed syne gis ically o igh agains apid and agg essi e neu odegene a ion [58,66,67]. Finally, he neu op o ec i e e ec s o OEA ea men in PCD mice we e mainly media ed by PPARα since he adminis a ion o he PPARα an agonis GW6471 ully e e sed he OEA-media ed e ec s a bo h he cellula and beha io al le els. In he pa icula case o Pu kinje cell su - i al, ou esul s showed ha he inhibi ion o PPARα be o e OEA ea men no only abolished he p e en i e e ec o OEA bu also inc eased Pu kinje cell dea h. This inding may be ela ed o apop osis since PPARα inhibi ion can p o- mo e p og ammed cell dea h [68]. Al hough we canno dis- ca d a di ec o indi ec e ec on o he di e en neu al cell ypes and/o he implica ion o o he molecula in e ac ions [29,69,70], ou indings con i m he di ec in ol emen o PPARα [36–38] in he neu op o ec i e e ec s o OEA obse ed in his s udy. Indeed, hese esul s a e consis en wi h he absence o changes in he exp ession o o he clas- sical endocannabinoid ecep o s in PCD mice. In conclusion, al hough he molecula mechanism unde - lying he e ec o OEA equi es u he s udy, i s neu op o- ec i e e ec is appa en a bo h he his ological and beha - io al le els. This neu op o ec i e e ec may be ela ed o he e-es ablishmen o cy oskele al p ope ies and he ensuing main enance o he s uc u e o Pu kinje cells in he ce ebel- lum and, he e o e, imp o emen s in impai ed beha io al unc ions. These indings p o ide e idence suppo ing he clinical use o OEA as a po en ial pha macological molecule o limi ing se e e neu odegene a i e p ocesses. Supplemen a y In o ma ion The online e sion con ains supplemen- a y ma e ial a ailable a h ps:// doi. o g/ 10. 1007/ s13311- 021- 01044-3. Acknowledgemen s The au ho s would like o hank he zoo echni- cians o he G enoble Ins i u e Neu oscience (GIN) and exp ess hei g a i ude o M. J. Sánchez-Domínguez o echnical suppo . Requi ed Au ho Fo ms Disclosu e o ms p o ided by he au ho s a e a ailable wi h he online e sion o his a icle. Au ho Con ibu ion EPM, RMC, DD, MJM, AA, JRA, and EW concei ed he s udy and designed he expe imen s; EPM and RMC pe o med he in i o expe imen s; EPM pe o med and analyzed he beha io al es s; EPM, RMC, and CdP pe o med he his ological analyses; RMC and MJM pe o med he in i o expe imen s; RMC and JMMC pe o med he ma hema ical analyses o mic o ubule cu - a u e; EPM, RMC, CCAZ, JRA, DD, and EW in e p e ed he esul s; and EPM was a majo con ibu o o he w i ing o he pape and o ganiza ion and design o all he igu es. All he coau ho s e ised and app o ed he inal manusc ip . 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