GABAB receptor agonist baclofen promotes central nervous system remyelination

ORIGINAL RESEARCH ARTICLE
GABA
B
ecep o agonis baclo en p omo es cen al ne ous
sys em emyelina ion
Ma i Paz Se ano-Regal
1,2
| Lau a Bay
on-Co de o
1,2,3
|
Juan Ca los Cha a Ven u a
1,2,3
| Blanca I. Ochoa-Bueno
1,2
| Vanja Tepa ce ic
1
|
Ca los Ma u e
1,2,3
| Ma ía Vic o ia Sánchez-G
omez
1,2,3
1
Labo a o y o Neu obiology, Achuca o
Basque Cen e o Neu oscience, Leioa, Spain
2
Depa men o Neu osciences, Uni e si y o
he Basque Coun y (UPV/EHU), Leioa, Spain
3
Cen o de In es igaci
on Biomédica en Red de
En e medades Neu odegene a i as
(CIBERNED), Leioa, Spain
Co espondence
Ma ía Vic o ia Sánchez-G
omez and Ca los
Ma u e, Depa men o Neu osciences,
Uni e si y o he Basque Coun y (UPV/EHU),
Ba io Sa iena s/n, 48940, Leioa, Spain.
Email: [email p o ec ed]us and ca los.
[email p o ec ed]
P esen add ess
Ma i Paz Se ano-Regal, G upo de
Neu oinmuno-Repa aci
on, Hospi al Nacional
de Pa apléjicos-SESCAM, Toledo, Spain.
Funding in o ma ion
CIBERNED, G an /Awa d Numbe :
CB06/05/0076; Basque Go e nmen ,
G an /Awa d Numbe s: IT1203-19, IT702-13;
Minis y o Economy and Compe i i eness,
Go e nmen o Spain, G an /Awa d Numbe s:
SAF2015-74332-JIN, PID2019-109724RB-
I00, SAF2016-75292-R, SAF2013-45084-R
Abs ac
P omo ing emyelina ion is conside ed as a po en ial neu o epai s a egy o p e en /
limi he de elopmen o pe manen neu ological disabili y in pa ien s wi h mul iple scle-
osis (MS). To his end, a numbe o clinical ials a e in es iga ing he po en ial o exis ing
d ugs o enhance oligodend ocy e p ogeni o cell (OPC) di e en ia ion, a p ocess ha
ails in ch onic MS lesions. We p e iously epo ed ha oligodend oglia exp ess GABA
B
ecep o s (GABA
B
Rs) bo h in i o and in i o,and ha GABA
B
R-media ed signaling
enhances OPC di e en ia ion and myelin p o ein exp ession in i o. Ou goal he e was
o e alua e he p o- emyelina ing po en ial o GABA
B
R agonis baclo en (Bac), a clinically
app o ed d ug o ea spas ici y in pa ien s wi h MS. We i s demons a ed ha Bac
inc eases myelin p o ein p oduc ion in lysoleci hin (LPC)- ea ed ce ebella slices. Impo -
an ly, Bac adminis a ion o adul mice ollowing induc ion o demyelina ion by LPC
injec ion in he spinal co d esul ed in enhanced OPC di e en ia ion and emyelina ion.
Thus, ou esul s sugges ha Bac epu posing should be conside ed as a po en ial he a-
peu ic s a egy o s imula e emyelina ion in pa ien s wi h MS.
KEYWORDS
baclo en, GABA
B
ecep o , mul iple scle osis, myelin, oligodend ocy e, emyelina ion
1|INTRODUCTION
Mul iple scle osis (MS) is a ch onic in lamma o y disease o he cen al
ne ous sys em (CNS) cha ac e ized by dissemina ed demyelina ion
(Dend ou e al., 2015). As a consequence o in lamma o y demyelin-
a ion, ac ion po en ial conduc ion is dis up ed and axons a e dep i ed
om me abolic and ophic suppo , which leads o axonal loss (Lee
e al., 2012; Saab e al., 2016), he main co ela e o pe manen dis-
abili y in pa ien s wi h MS (T app e al., 1999). The majo i y o cu -
en ly a ailable ea men s o MS a ge CNS in lamma ion and
associa ed elapses, bu do no p e en long- e m disabili y (K eme ,
Akke mann, e al., 2019). Thus, he de elopmen o he apies o p e-
en axonal and neu onal loss emains an unme he apeu ic need o
pa ien s wi h MS (Lube zki e al., 2020). Remyelina ion is he spon a-
neous egene a ion o myelin ha p e en s axonal degene a ion bo h
in animal models (I ine & Blakemo e, 2008; Mei e al., 2016) and
Ma i Paz Se ano-Regal and Lau a Bay
on-Co de o con ibu ed equally o his wo k.
Recei ed: 27 Feb ua y 2022 Re ised: 25 July 2022 Accep ed: 3 Augus 2022
DOI: 10.1002/glia.24262
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s use and dis ibu ion in any
medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica ions o adap a ions a e made.
© 2022 The Au ho s. GLIA published by Wiley Pe iodicals LLC.
Glia. 2022;1–15. wileyonlinelib a y.com/jou nal/glia 1
pa ien s wi h MS (Ko nek e al., 2000). Howe e , in mos pa ien s, he
e iciency o his p ocess dec eases signi ican ly wi h age and disease
p og ession (F anklin and ench-Cons an , 2017). The e o e, he
de elopmen o no el ea men s ha enhance emyelina ion is a
majo goal o cu en MS esea ch, and includes he epu posing o
exis ing d ugs (K eme , Gö le, e al., 2019).
A block in OPC di e en ia ion (Ko e e al., 2006;Kuhlmann
e al., 2008) and lack o myelin shea h o ma ion by su i ing ma u e oligo-
dend ocy es (OLs) ha e been poin ed ou as impo an con ibu o s o
emyelina ion ailu e in MS (Duncan e al., 2018; Yeung e al., 2019;Heb
e al., 2020;F ankline al.,2021). The e o e, p omo ing OPC di e en ia ion
and imp o ing OL myelina ion capaci y a e po en ial s a egies o enhanc-
ing myelin epai and p e en ing neu odegene a ionin hisdisease.
Neu o ansmi e s a e impo an media o s o OPC-neu on com-
munica ion wi h a clea in luence on OPC beha io (Dome cq
e al., 2010; Fannon e al., 2015; Hamil on e al., 2017; Li e al., 2013;
Se ano-Regal, Luengas-Escuza, e al., 2020; Zonouzi e al., 2015). As
OPCs ecei e bo h exci a o y and inhibi o y synap ic inpu s, media ed
by glu ama e and GABA (Be gles e al., 2000; Ká ad
o i e al., 2008;
Kukley e al., 2008; Lin & Be gles, 2004), hese molecules ha e been
iden i ied as key egula o s o oligodend oglial ma u a ion and myeli-
na ion (Bai e al., 2021; Fannon e al., 2015; Gau ie e al., 2015;
Se ano-Regal, Bay
on-Co de o, e al., 2020).
Rega ding myelin epai , GABAe gic signaling h ough GABA
A
Rs has
been associa ed wi h emyelina ion a e ocal demyelina ion in he a
co pus callosum (Kalakh & Mouiha e, 2019), as well as in he caudal ce e-
bella peduncle (Cisne os-Mejo ado e al., 2020). GABA
B
Rs ha e also
been sugges ed as impo an modula o s o myelina ion gi en ha
GABA
B
R an agonism inc eased OPC p oli e a ion while dec easing hei
ma u a ion and he p oduc ion o myelin- ela ed p o eins in he de elop-
ing a cingulum (Pudasaini e al., 2022). Howe e , he ole o oligoden-
d oglial GABA
B
Rs in myelin egene a ion emains o be in es iga ed.
Baclo en (Bac), he bes known GABA
B
R agonis , is cu en ly used
as a he apeu ic agen o spas ici y in MS, and can be adminis e ed
ei he in a hecally o o ally because i c osses he blood–b ain ba ie
(E zgaa d e al., 2017). We p e iously epo ed ha GABA
B
R ac i a-
ion by Bac p omo es di e en ia ion and myelin p o ein exp ession in
a co ical OPC (Se ano-Regal, Luengas-Escuza, e al., 2020). He e,
we in es iga ed whe he Bac modula es emyelina ion in lysoleci hin
(LPC)-demyelina ed o gano ypic ce ebella slices as well as in LPC spi-
nal co d lesions in adul mice. Ou esul s demons a e ha Bac s imu-
la es myelin p o ein p oduc ion ex i o and enhances emyelina ion
in i o, which sugges s ha his d ug may also be a use ul he apeu ic
agen o s imula e emyelina ion.
2|MATERIALS AND METHODS
2.1 |Animals
All expe imen s we e conduc ed wi h he app o al o he e hical com-
mi ee o he Uni e si y o he Basque Coun y (UPV/EHU). Animals
we e handled in acco dance wi h he Eu opean Di ec i e 2010/63/
EU and we e housed unde s anda d condi ions wi h a 12 h ligh –da k
cycle and ad libi um access o ood and wa e . All possible e o s
we e made o minimize animal su e ing and he numbe o animals
used. Sp ague Dawley a s, C57BL/6 mice, and ansgenic mice
exp essing luo escence epo e DsRed unde he con ol o he glial-
speci ic p o eolipid p o ein p omo e (PLP-DsRed; Hi linge
e al., 2005), gene ously p o ided by P o . D . F. Ki chho (Uni e si y
o Saa land, Hombu g, Ge many), we e used in his s udy.
2.2 |Ce ebella o gano ypic slice cul u e
Slice cul u es we e made om ce ebella o P5-P7 o P11-day-old
Sp ague Dawley a s and P11-day-old ansgenic PLP-DsRed mice
acco ding o p e iously desc ibed p ocedu es (Doussau e al., 2017;
Dusa e al., 1997; Tan e al., 2018). B ie ly, ce ebella we e cu wi h a
issue choppe (Mcllwain) in o 350 μm pa asagi al slices. Meninges
we e emo ed and slices we e pla ed on o 0.4 μm po e size Millicell
CM cul u e inse s (Millipo e), con aining 2–3 slices each. Ra slices
we e main ained in six-well pla es o 13–15 days and mice ce ebella
slices o 11 days in cul u e medium consis ing o 50% basal medium
wi h Ea le's sal (BME), 25% Hank's Balanced Sal Solu ion (HBSS),
25% inac i a ed ho se se um (all om The moFishe Scien i ic),
5 mg/ml glucose (Pan eac), 0.0025 mM
L
-glu amine (Sigma-Ald ich)
and an ibio ic-an imyco ic solu ion (100 U/ml o penicillin, 100 μg/ml
o s ep omycin and 0.25 μg/ml o ampho e icin B; The moFishe Sci-
en i ic) a 37C in a humidi ied a mosphe e wi h 5% CO
2
. Cul u e
medium was eplaced e e y 2–3 days. Slices we e ea ed wi h
GABAe gic d ugs s a ing on he day 2 in i o (Table 1). Lysoleci hin
(LPC)-induced demyelina ion expe imen s we e ca ied ou in ce ebel-
la slices om P11 animals a day 7 in i o by incuba ion o 16 h
wi h 0.5 mg/ml LPC (Sigma-Ald ich) (Bi gbaue e al., 2004). T ea -
men s we e pe o med a he same ime as he LPC-s imulus. Slices
we e ixed in cul u e inse s wi h 4% pa a o maldehyde (PFA) solu ion
in phospha e-bu e ed saline (PBS; pH 7.4) o immunochemis y o
p ocessed o wes e n blo analysis a 4 and 6 days a e ea men .
2.3 |Op ic ne e-de i ed o gano ypic slice cul u e
Cul u es we e ob ained om op ic ne es o P11-day-old ansgenic PLP-
DsRed mice. Op ic ne es oge he wi h he e ina we e ex ac ed in
TABLE 1 GABAe gic agonis s and an agonis s used in his s udy
P oduc Re e ence Supplie
Concen a ion
(in i o)
GABA A2129 Sigma-Ald ich 100 μM
Gabazine SR-95531 Sigma-Ald ich 50 μM
Baclo en 0796 Toc is
Bioscience
100 μM
Muscimol 0289 Toc is
Bioscience
100 μM
2SERRANO-REGAL ET AL.
o de o main ain issue o ganiza ion and cellula connec ions. Meninges
and esidual issue we e emo ed in supplemen ed (2 μl/ml gen amicin,
1 mg/ml bo ine se um albumin, BSA and 2 mM L-glu amine) HBSS unde
he mic oscope, and he op ic ne e- e ina uni s we e main ained
in 0.4 μmpo esizeMillicellCMcul u einse s (Millipo e), con ain-
ing one uni each. Explan s we e placed in six-well pla es o
3 days in he cul u e medium as desc ibed abo e o ce ebella
o gano ypic cul u es and in he same condi ions. To a o app op i-
a e eeding o he op ic ne e- e ina uni , 50 μl o cul u e medium we e
added di ec ly o e he issue (Azim & Bu , 2011). GABA
B
Rspeci icago-
nis baclo en (100 μM) was added o he medium immedia ely a e pla -
ing and main ained o 3 days wi h daily enewal. Op ic ne es wi hou
e ina we e ixed wi h 4% PFA in PBS and whole-moun ed on slides wi h
P olong™Gold an i ade (In i ogen).
2.4 |EdU labeling and de ec ion
5-e hynyl-20-deoxyu idine (EdU; In i ogen) (10 μM) was added o he
o gano ypic medium a day 5 in i o and le o 48 h, o label p oli -
e a ing cells. EdU was e ealed using Click-iT Alexa Fluo 647 Imaging
Ki acco ding o he manu ac u e 's ins uc ions (In i ogen).
2.5 |Demyelina ing lesion induc ion
Demyelina ing lesions we e induced in he spinal co d o 10-week-old
emale C57BL/6 mice by a s e eo axic injec ion o 0.5 μlo 1%LPC
(Sigma-Ald ich) in s e ile 0.9% NaCl solu ion, as p e iously desc ibed
(Tepa ce ic e al., 2014). Mice we e anes he ized by in ape i oneal injec-
ion (i.p.) o a solu ion o ke amine (90 mg/kg; Fa o)/xylazine (20 mg/kg;
Calie ). Bup eno phine (0.1 mg/kg; Dech a) was subcu aneously adminis-
e ed as pos ope a i e analgesic ea men . Daily i.p. injec ions o ehicle
(saline solu ion) o baclo en (8 mg/kg) we e pe o med om 5 o 16 days
pos lesion (dpl). Mice we e sac i iced a 12 o 16 dpl, and he issue was
p ocessed o immunohis ochemical (IHC) o ansmission elec on
mic oscopy (TEM) analysis, espec i ely.
2.6 |Pe usion and issue p ocessing
Mice we e eu hanized wi h ke amine/xylazine and ansca dially pe -
used wi h 2% PFA solu ion in PBS o IHC analysis o 4% glu a alde-
hyde in 0.1 M PB o TEM s udies. Fo IHC analysis, spinal co ds we e
pos - ixed wi h he same PFA solu ion, c yop o ec ed in 15% suc ose
solu ion (Pan eac) and ozen in 7% gela in (Sigma-Ald ich)/15%
suc ose solu ion in PBS. Samples we e cu using a c yos a CM3050 S
(Leica) o ob ain 12 μm- hick co onal sec ions. Fo TEM s udies, spinal
co ds we e pos ixed o e nigh , washed in 0.1 M PB, and cu in o
2 mm- hick blocks. The issue was pos ixed in 1% osmium solu ion in
0.1 M PB, dehyd a ed and embedded in epoxy esin (Sigma Ald ich).
Semi hin (1 μm- hick) and ul a hin (55 nm- hick) sec ions we e cu
wi h an ul amic o ome RMC Boeckele .
2.7 |Immunochemis y
Ce ebella slices we e washed in PBS, pe meabilized and blocked in
4% goa se um and 0.1% T i on X-100 in PBS (blocking bu e ) o 1 h
and incuba ed o e nigh a 4C wi h p ima y an ibodies (Table 2).
Slices we e washed in PBS wi h 0.1% T i on X-100 and incuba ed
wi h Alexa luo opho e-conjuga ed seconda y an ibodies (1:400; In i-
ogen) in blocking bu e o 1 h a RT. Slides wi h c yos a spinal
co d sec ions we e ai -d ied o 1 h, ehyd a ed in T is bu e saline
(TBS; 20 mM T is and 1.4 M NaCl in dH
2
O; pH 7.6) and p e- ea ed
wi h absolu e e hanol (Sha lab) o 15 min a 20C. Fo APC and
Olig2 immunos aining, an igen e ie al was pe o med by hea ing he
sec ions in low-pH e ie al bu e (Vec o Labo a o ies) o 45 s using
a mic owa e. A e washing, samples we e incuba ed in blocking
bu e solu ion (1% BSA, 5% goa se um, and 0.1% T i on X-100) o
30 min a RT, and hen wi h he p ima y an ibodies dilu ed in blocking
bu e o e nigh a 4C (Table 2). Sec ions we e washed in TBS, and
incuba ed wi h Alexa luo opho e-conjuga ed seconda y an ibodies
(1:500; In i ogen) in blocking solu ion o 1 h a RT. Cell nuclei we e
coun e s ained wi h DAPI (4 μg/ml, Sigma-Ald ich) and sec ions we e
moun ed wi h Fluo omoun -G (Sou he nBio ech).
TABLE 2 An ibodies used in his s udy o immunohis ochemis y
An ibody Hos Dilu ion ( a issue) Dilu ion (mouse issue) Supplie Re e ence
An i-GABAR
B1
Rabbi 1:200 1:200 Alomone labs AGB-001
An i-GABAR
B1
Mouse —1:200 Abcam #55051
An i-GABAR
B2
Rabbi 1:200 1:200 Alomone labs AGB-002
An i-APC (clone CC1) Mouse 1:200 1:200 Calbiochem #OP80
An i-Olig2 Mouse 1:200, 1:1000 1:500 Millipo e #MABN50
An i-MBP Chicken —1:200 Millipo e #AB9348
An i-PDGFRαRa —1:300 BD Biosciences #558774
An i-Iba1 Guinea pig —1:200 Synap ic sys ems 234,004
An i-Nkx2.2 Mouse —1:20 De elopmen al S udies
Hyb idoma Bank
#Q4818001B
An i-GFAP Rabbi 1:100 Millipo e #AB5804
SERRANO-REGAL ET AL.3
2.8 |Image acquisi ion and analysis
Images om ce ebella o gano ypic slices and op ic ne e explan s
we e acqui ed using Zeiss LSM800 and/o Leica TCS SP8 lase scan-
ning con ocal mic oscopes. Cells in ce ebella slices we e coun ed
blindly along he z-s ack using a 20objec i e in Leica TCS SP8 con-
ocal mic oscope. A leas 3 di e en ields om 2 slices pe expe i-
men we e analyzed by using LAS AF Li e so wa e (Leica). The
luo escence signal co esponding o he PLP-DsRed OLs was quan i-
ied by ImageJ so wa e and da a we e exp essed as a bi a y uni s o
luo escence o each expe imen al si ua ion. Images om spinal co d
sec ions we e collec ed using Zeiss LSM800 and/o Leica TCS SP8
con ocal mic oscope and impo ed o ImageJ so wa e. A ea lacking
myelin basic p o ein (MBP) s aining wi hin he do sal uniculus o he
spinal co d (a ea o demyelina ion) was delimi ed as egion o in e es
(ROI) and measu ed. Cells posi i e o he ma ke s o in e es we e
coun ed om a leas 3 di e en slices pe animal. Resul s a e p e-
sen ed as pe cen age o posi i e cells pe lesion a ea measu ed o pe -
cen age a ea o lesion occupied by he co esponding ma ke s. Same
se ings we e kep o all samples (con ol and ea ed) belonging o a
speci ic expe imen . All images a e shown as p ojec ions om z-
s acks. Fo TEM s udies, semi hin sec ions s ained wi h Richa dson's
Blue we e used o iden i y he lesion a ea. Ul a hin sec ions we e cu
and con as ed by incuba ion in 4% u anyl ace a e and lead ci a e
solu ion o i s isualiza ion in Philips CM200 ansmission elec on
mic oscope. Remyelina ed axons we e coun ed. Remyelina ion was
de e mined as he pe cen age o OL and Schwann cell (SC)-
emyelina ed axons wi hin he o al numbe s o axons ini ially demye-
lina ed ( hose emyelina ed + hose demyelina ed).
2.9 |Wes e n blo
A e ea men s, ce ebella slices we e di ec ly esuspended in
sodium dodecyl sul a e sample bu e on ice o enhance he lysis
p ocess and a oid p o ein deg ada ion. Samples we e boiled a 99C
o 8 min, size-sepa a ed by sodium dodecyl sul a e polyac ilamide gel
elec opho esis (SDS-PAGE) in 4%–20% C i e ion TGX P ecas gels and
ans e ed o T ans-Blo Tu bo Midi PVDF T ans e Packs (Bio-Rad,
He cules). Memb anes we e blocked in 5% BSA (Sigma-Ald ich) in
T is-bu e ed saline/ 0.05% Tween-20 (TBS-T) and p o eins we e
de ec ed wi h speci ic p ima y an ibodies (Table 3). Memb anes we e
incuba ed wi h ho se adish pe oxidase-conjuga ed seconda y an ibodies
(1:2000; Sigma-Ald ich) and we e de eloped by using an enhanced
chemiluminescence de ec ion ki acco ding o he manu ac u e 's ins uc-
ions (Supe signal Wes Du a o Fem o; The moFishe Scien i ic). P o ein
bands we e de ec ed wi h a ChemiDocXRSImagingSys em(Bio-Rad)
and quan i ied by olume using ImageLab so wa e ( e sion 3.0; Bio-Rad).
2.10 |S a is ical analysis
All da a a e p esen ed as mean ± SEM. S a is ical analyses we e pe -
o med using G aphPad P ism s a is ical so wa e ( e sion 8.0; G aph-
Pad so wa e). Compa isons be ween mul iple expe imen al g oups
we e made using one-way analysis o a iance (ANOVA) ollowed by
Tukey's pos hoc es . Fo compa isons be ween wo g oups, we used
he wo- ailed S uden 's - es assuming equal a iance. In all
ins ances, s a is ical di e ences we e conside ed signi ican whe e
p< .05. All he images shown ep esen he da a ob ained om a
leas h ee independen expe imen s.
3|RESULTS
3.1 |Baclo en ea men inc eases myelin p o ein
le els in o gano ypic slice cul u es
We i s alida ed he ole o he GABAe gic signaling in egula ing
oligodend oglial di e en ia ion and myelina ion in o gano ypic cul-
u es ob ained om P5-P7 a s. We in es iga ed GABA
B1
and
GABA
B2
ecep o -subuni exp ession du ing myelina ion ex i o, and
ound ha oligodend oglial cells–labeled using an i-Olig2 an ibody–,
and mo e speci ically ma u e OLs–labeled using an i APC an ibody–,
exp ess he wo GABA
B
R subuni s (Figu e 1a,b), as we p e iously
obse ed in OLs in i o and in i o (Se ano-Regal, Luengas-Escuza,
e al., 2020).
Then, we we e in e es ed in e alua ing whe he GABA agonis s
could also modula e myelin-p o ein exp ession le els in ce ebella
o gano ypic cul u es. Exposu e o 100 μM GABA o 100 μM muscimol
(Mus; GABA
A
R speci ic agonis ) did no change he le els o exp es-
sion o myelin-associa ed glycop o ein (MAG), 20,30-cyclic nucleo ide-
30- phosphodies e ase (CNPase) and MBP, compa ed wi h con ol
slices (Supplemen a y Figu es S2 and S3). Howe e , ea men wi h
100 μM Bac (Figu e 1c) induced a signi ican inc ease in he exp es-
sion o MAG and MBP myelin p o eins (2.0 ± 0.34 Bac s. 1.0 ± 0.19
con ol o MAG, Figu e 1d; and 1.29 ± 0.14 Bac s. 1.0 ± 0.12 con-
ol o MBP, Figu e 1 ), oge he wi h a non-signi ican inc ease in
he exp ession o CNPase (1.23 ± 0.11 Bac s. 1.0 ± 0.14 con ol o
CNPase, Figu e 1e). This inc ease in MBP and MAG is simila o he
e ec o Bacincul u edOPCs,andbecomesab oga edin hep esenceo
GABA
B
R speci ic an agonis CGP55845 (Supplemen a y Figu e S1). To
in es iga e whe he his e ec o Bac was associa ed wi h changes in oligo-
dend oglial p oli e a ion, ce ebella o gano ypic cul u es we e exposed o
EdU o 48 h, in he absence o p esence o GABA o Bac (100 μM). Nei-
he GABA no Bac modi ied he pe cen ageo ma u eOLs(APC
+
Olig2
+
)
TABLE 3 An ibodies used in his s udy o wes e n blo analysis
An ibody Hos Dilu ion Supplie Re e ence
An i-MAG Mouse 1:500 San a C uz SC-376145
An i-CNPase Mouse 1:1000 Sigma-Ald ich #C5922
An i-MBP Mouse 1:1000 Biolegend #SMI 99
An i-GAPDH Mouse 1:1000 Millipo e #MAB374
An i-β- ubulin Mouse 1:5000 abcam AB7291
4SERRANO-REGAL ET AL.
among o al oligodend oglial cells, no Olig2
+
cells ha unde wen p oli e a-
ion in his ime pe iod (Olig2
+
Edu
+
)(Figu e1g–i), sugges ing ha GABA
B
R
ac i a ion p omo es myelin gene a ion by ma u e OLs wi hou a ec ing
OPC p oli e a i e capaci y. Addi ionally, we examined he e ec o Bac in
op ic ne e explan s o ansgenic PLP-DsRed mice (Figu e 1j). Quan i ica-
ion o PLP-DsRed- luo escen signal (Figu e 1k) e ealed a signi ican
inc ease in hose op ic ne es ea ed wi h Bac compa ed o con ols
(27.82 ± 2.27 o Bac s. 19.62 ± 2.59 o con ol; Figu e 1l). Toge he ,
hese esul s show ha Bac enhances myelin p o ein p oduc ion in mu ine
o gano ypic cul u es and in op ic ne e explan s, con i ming ou p e ious
obse a ions in isola ed OLs (Se ano-Regal, Luengas-Escuza, e al., 2020)in
a complex en i onmen mo e simila o physiological condi ions.
FIGURE 1 Legend on nex page.
SERRANO-REGAL ET AL.5

3.2 |GABA
B
R ac i a ion ele a es he le els o
majo myelin p o eins du ing emyelina ion ex i o
Since Bac p omo ed myelin p o ein syn hesis in o gano ypic slices, we
nex s udied he impac o GABA
B
R ac i a ion unde expe imen al
condi ions mimicking damage o myelin. P11 a -de i ed ce ebella
slices we e main ained o 7 days o allow myelina ion ex i o and
hen exposed o LPC o 16 h. In he i s se o expe imen s, slices
we e daily ea ed wi h GABA (100 μM) o Bac (100 μM) o 6 days
a e LPC exposu e (Supplemen a y Figu e S4A, B) and MAG and
MBP p o eins we e analyzed by wes e n blo . We ound ha LPC
induced a signi ican dec ease in bo h p o eins (0.99 ± 0.11 LPC
s. 1.44 ± 0.11 old con ol o MAG, and 1.00 ± 0.08 LPC s. 1.50
± 0.09 old con ol o MBP). Bac ea men pos -LPC signi ican ly
inc eased MAG le els (1.44 ± 0.11 Bac s. 0.99 ± 0.11 LPC; Supple-
men a y Figu e S4C) while MBP le els we e no a ec ed
(Supplemen a y Figu e S4D). We hen in es iga ed whe he p e ious
applica ion o hese agonis s du ing demyelina ing phase may be mo e
e ec i e in eco e ing he le els o myelin p o ein exp ession a e
exposu e o LPC. We exposed ce ebella slices o LPC concomi an ly
o GABA o Bac (100 μM) applica ion. A e LPC emo al, GABA o
Bac was main ained in he medium o 6 mo e days (Figu e 2a). Unde
his expe imen al pa adigm, we obse ed a signi ican inc ease in he
exp ession le els o bo h MAG and MBP (2.75 ± 0.35 GABA and
2.81 ± 0.26 Bac s. 1.0 ± 0.26 LPC o MAG, and 3.39 ± 0.52 GABA
and 2.82 ± 0.29 Bac s. 1.0 ± 0.37 LPC o MBP; Figu e 2b–d).
We also used immuno luo escence o in es iga e he e ec o
GABA
B
R-media ed signaling on OL di e en ia ion and myelin p o ein
p oduc ion ex i o by aking ad an age o PLP-DsRed epo e mice,
in which changes in PLP-associa ed endogenous luo escence can be
exploi ed o ack changes in PLP-exp ession. We p epa ed o gano ypic
ce ebella slices and main ained hese in cul u e o 7 days, a e which
we exposed he slices o LPC in combina ion wi h d ug ea men s o
4days. We applied GABA (100μM), Bac (100 μM), and GABA plus
gabazine (50 μM)–aGABA
A
R an agonis –, in o de o s udy he e ec
o GABA di ec ly o e GABA
B
Rs, o gabazine alone (50 μM) (Figu e 2e,
). As shown in Figu e 2g, he PLP-DsRed luo escen signal inc eased
signi ican ly in Bac- and GABA plus gabazine- ea ed slices com-
pa ed o hose exposed o LPC wi hou ea men (10.42 ± 0.65 Bac
and 20.74 ± 5.32 GABA plus gabazine s. 4.5 ± 0.38 LPC; Figu e 2g),
indica ing ha GABA
B
ecep o s imula ion in hese slices inc eases
PLP p oduc ion. O e all, hese esul s indica e ha GABA
B
Rac i a-
ion wi h Bac a o s emyelina ion ex i o in ce ebella o gano ypic
slices.
3.3 |Baclo en adminis a ion p omo es OPC
di e en ia ion in adul mouse CNS
We hen aimed o assess he e ec o Bac adminis a ion on CNS
emyelina ion in i o. We i s alida ed he exp ession o GABA
B
R
subuni s in OPCs in no mal spinal co d issue o adul mice
(Figu e 3a), and obse ed exp ession o bo h B1 and B2. We hen
con i med ha his exp ession was main ained a e induc ion o
demyelina ion by LPC injec ion in he do sal uniculus, bo h on eac i e
OPCs (Figu e 3b) and by newly gene a ed OLs (Figu e 3c), which sug-
ges ed hese cells could be a ge ed by Bac.
To in es iga e he e ec o Bac on emyelina ion, a 5 days pos
lesion (dpl), daily i.p. injec ions o ehicle o Bac (8 mg/kg/day) we e
ini ia ed and adminis e ed du ing 7 days. Bac adminis a ion was ini i-
a ed a 5 dpl o ensu e ha he ea men would no a ec he ex en
o demyelina ion, as demyelina ion in LPC model is compli ed by
2 dpl. OPC di e en ia ion was in es iga ed a 12 dpl, gi en ha he
peak o OPC di e en ia ion occu s du ing he second week pos
demyelina ion (Figu e 4a).
Then, we explo ed he changes induced by Bac adminis a ion in
OPC numbe s and mic oglia/mac ophage esponse a 12 dpl using
an i-PDGFRαan ibody as OPC ma ke and an i-Iba1 an ibody as
mic oglia/mac ophage ma ke (Figu e 4b). Quan i ica ion o PDGFRα
+
cells pe mm
2
(578.2 ± 117.3 Bac s. 461.3 ± 49.93 ehicle; Figu e 4c)
o he pe cen age o lesion a ea occupied by Iba-1 (60.10 ± 5.52%
Bac s. 49.87 ± 11.84% ehicle; Figu e 4d) e ealed no a ia ions
be ween con ol s Bac- ea ed mice. In addi ion, we used an i-
Nkx2.2 an ibody o label eac i e OPCs and an i-GFAP an ibody as
FIGURE 1 Baclo en inc eases myelin- ela ed p o ein syn hesis in o gano ypic cul u es wi hou al e ing he p oli e a ion a io o
oligodend oglial lineage. (a) Oligodend oglial cells, dis inguished as Olig2
+
cells ( ed), a e posi i e o GABA
B1
and GABA
B2
subuni s (g een) o
GABA
B
Rs in ce ebella slices o P5-P7 a s. (b) Ma u e oligodend ocy es (OLs), iden i ied as APC
+
cells ( ed), exp ess GABA
B1
, and GABA
B2
subuni s (g een) o GABA
B
Rs in he same p epa a ions. A ows indica e double-s ained cells and a owheads show he cell magni ied in he
co esponding inse . Scale ba s =20 μm. (c) Rep esen a i e wes e n blo image-showing exp ession o myelin-associa ed glycop o ein (MAG),
CNPase, and MBP p o eins in con ol and baclo en- ea ed ce ebella slices. Quan i ica ion o MAG (d), CNPase (e), and myelin basic p o ein
(MBP) ( ) exp ession no malized o GAPDH alues. *p< .05 and ** p< .01 e sus con ol; pai ed S uden 's - es . (g) Rep esen a i e images
showing immuno luo escence o ma u e OLs (APC
+
, ed) and o al oligodend oglial cells (Olig2
+
, g een) co-labeled wi h EdU (cyan) o iden i y
ma u e OLs (APC
+
) and Olig2
+
cells in ce ebella slices in he indica ed condi ion. A ows indica e ma u e OLs (APC
+
Olig2
+
) o newly gene a ed
oligodend oglial cells (Olig2
+
EdU
+
). Scale ba =50 μm. Quan i ica ion o (h) pe cen age o ma u e OL om o al oligodend oglial cell pool
(APC
+
Olig2
+
/Olig2
+
) and (i) densi y o newly o med oligodend oglial cells (Olig2
+
EdU
+
), in he indica ed condi ions. One-way ANOVA ollowed
by Tukey's pos - es . (j) Op ic ne e- e ina uni om P11 PLP-DsRed ansgenic mice. Scale ba =500 μm. (k) Op ic ne es explan s cul u ed in
con ol condi ions (le ) o in p esence o baclo en ( igh ) showing DsRed luo escen signal. Scale ba s =100 μm; highe magni ica ion scale
ba =315 μm. (l) Quan i ica ion o DsRed luo escen signal in con ol and ea ed op ic ne e explan s. *p< .05 e sus con ol; unpai ed
S uden 's - es . (a– ): Con ol and bac 6 slices om di e en animals. (g–i): Con ol 15, GABA 18, and bac 18 images om ce ebella slices. (j–l):
Con ol 27, bac 24 images om op ic ne e explan s
6SERRANO-REGAL ET AL.
FIGURE 2 GABA
B
Rs modula e emyelina ion in lysoleci hin (LPC)- ea ed ce ebella o gano ypic slices. (a) Time cou se showing he
expe imen al design in LPC-induced demyelina ion in ce ebella o gano ypic cul u es ob ained om P11 a s. (b) Rep esen a i e wes e n blo
image showing in duplica es he e ec o GABA and baclo en in modula ing myelin- ela ed p o ein es o a ion in LPC- ea ed o gano ypic
cul u es ollowing he pa adigm shown in a. (c, d) Quan i ica ion o myelin-associa ed glycop o ein (MAG) (c) and myelin basic p o ein (MBP)
(d) le els in indica ed condi ions. **p< .01 and ****p< .0001 e sus con ol,
##
p< .01 and
###
p< .001 e sus LPC; one-way ANOVA ollowed by
Tukey's pos - es . (e) Rep esen a i e images o ce ebella slices om P11 PLP-DsRed ansgenic mice showing DsRed luo escence in indica ed
condi ions. Scale ba =100 μm. ( ) T ea men s we e added o he slices in conjunc ion wi h LPC o 16 h and main ained he ea e o 4 days
a e . GABA
B
Rs we e selec i ely ac i a ed wi h baclo en o wi h GABA plus he GABA
A
R an agonis gabazine. (g) Quan i ica ion o DsRed
luo escen signal in indica ed condi ions. ****p< .0001 e sus con ol,
#
p< .05 and
####
p< .001 e sus LPC; one-way ANOVA ollowed by
Tukey's pos - es . (b–d): Con ol 9, LPC 8, GABA 9, bac 8 slices om di e en animals. (e–g): Con ol 27, LPC 25, GABA 8, bac 19, Gbz
10, GABA +Gbz 10 images om op ic ne e explan s
SERRANO-REGAL ET AL.7
as ocy e ma ke (Figu e 4e). We obse ed no signi ican di e ences
in Nkx2.2
+
cells pe mm
2
(437.4 ± 46.8 Bac s. 496.5 ± 65.5 con ol;
Figu e 4 ) no in he pe cen age o lesion a ea occupied by GFAP
(21.2 ± 4.0 Bac s. 26.9 ± 2.0 con ol; Figu e 4g) be ween ehicle e -
sus Bac- ea ed animals. Finally, we in es iga ed whe he Bac admin-
is a ion accele a es OPC di e en ia ion in he lesions by analyzing
he numbe s o ma u e OLs (APC
+
Olig2
+
) ela i e o he o al numbe
o oligodend oglial cells (Olig2
+
) (Figu e 4h). The pe cen age o APC
+
among o al Olig2
+
cells was signi ican ly inc eased in Bac- ea ed
LPC-injec ed mice (41.91 ± 2.29% Bac s. 27.95 ± 1.46% ehicle;
Figu e 4i), wi hou al e ing he o al numbe s o Olig2
+
cells (928.0
± 100.7 Bac s. 814.8 ± 50.13 ehicle; Figu e 4j). These esul s dem-
ons a e ha Bac ea men p omo es di e en ia ion o OPCs in LPC-
induced demyelina ing lesions.
3.4 |Baclo en adminis a ion accele a es
emyelina ion
We nex analyzed whe he Bac adminis a ion accele a es emyelina-
ion. Vehicle and Bac injec ions we e ini ia ed a 5 dpl, and he p o-
po ion o emyelina ed axons was analyzed a 16 dpl (Figu e 5a),
gi en ha he onse o emyelina ion in he LPC model akes place a
14 dpl and is nea comple ion a 21 dpl. A his ime poin , OL
emyelina ion–iden i ied as hin myelin shea hs–was ound p edomi-
nan ly a ound lesion bo de s, while SC emyelina ion, a well-
ecognized ea u e o he LPC lesion in he spinal co d (Je e y &
Blakemo e, 1995), was obse ed in he lesion cen e . TEM analysis
e ealed ha he pe cen age o emyelina ed axons (Figu e 5b, c) was
highe ollowing Bac ea men (36.95 ± 2.18% Bac s. 22.29 ± 1.85%
FIGURE 3 GABA
B
ecep o s a e
exp essed by oligodend ocy e p ogeni o
cell (OPCs) and ma u e oligodend ocy es
om he mouse spinal co d. (a) Con ocal
images showing OPCs (Nkx2.2
+
, g ay)
exp essing GABA
B1
( ed) and GABA
B2
(g een) subuni s o GABA
B
Rs in he do sal
uniculus o he spinal co d o unlesioned
mice. (B, C) Con ocal images showing
OPCs (Nkx2.2
+
, ed) (b) and ma u e OLs
(APC
+
, ed) (c) exp essing GABA
B1
(g een,
op) and GABA
B2
(g een, bo om) subuni s
o GABA
B
Rs in he do sal uniculus o he
spinal co d o con ol lysoleci hin (LPC)-
injec ed mice. Whi e dash line indica es
lesion bo de . A owheads poin a cells
shown a highe magni ica ion in each
pho og aph. Scale ba s =50 μm. Highe
magni ica ion =10 μm
8SERRANO-REGAL ET AL.
FIGURE 4 Legend on nex page.
SERRANO-REGAL ET AL.9