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 .
Oleoyle hanolamide Delays he Dys unc ion and Dea h o Pu kinje Cells and Amelio a es… 1765
Funding This wo k was suppo ed by he Minis y o Economy, Indus-
y and Compe i i eness (MINECO) (SAF2016-79668-R o EW),
he Minis y o Science and Inno a ion (PID2019-106943RB-I00 o
EW), he Minis y o Science and Inno a ion/Uni e si ies (MICINN/
MIU) (FPU16/04259 o EPM; FPU14/02963 o CdP), he Regional
Go e nmen o Cas ile and Leon (SA178U13 o EW), he Cen e o
Regene a i e Medicine and Cell The apy o Cas ile and Leon (EW),
he Uni e si y o Salamanca (EW), Inse m, Uni e si y G enoble Alpes,
CNRS, CEA, La Ligue Con e le Cance Comi é de l’Isè e (MJM), and
Fonda ion F ance Alzheime ( o MJM).
Decla a ions
Con lic o In e es The au ho s decla e ha hey ha e no compe ing
in e es s.
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