Pe ei oe al. Cell & Bioscience (2024) 14:5
h ps://doi.o g/10.1186/s13578-023-01186-1
RESEARCH Open Access
© The Au ho (s) 2023. Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which
pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he
o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons licence, and indica e i changes we e made. The images o
o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons licence, unless indica ed o he wise in a c edi line
o he ma e ial. I ma e ial is no included in he a icle’s C ea i e Commons licence and you in ended use is no pe mi ed by s a u o y
egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . To iew a copy o his
licence, isi h p:// c ea i eco mmons. o g/ licen ses/ by/4. 0/. The C ea i e Commons Public Domain Dedica ion wai e (h p:// c ea i eco
mmons. o g/ publi cdoma in/ ze o/1. 0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed in a c edi line o he da a.
Cell & Bioscience
Mülle glial cells loca ed in hepe iphe al
e ina a e mo e suscep ible ohigh p essu e:
implica ions o glaucoma
Xand a Pe ei o1,3* , Noelia Ruza a1, Mikel Azka go a2, Félix Elo za2, A an xa Ace a1,
An ónio F ancisco Amb ósio3,4,5,6, Ana Raquel San iago3,4,5,6 and Elena Vecino1*
Abs ac
Backg ound Glaucoma, a p og essi e neu odegene a i e disease, is a leading cause o i e e sible ision loss
wo ldwide. This s udy aims o elucida e he c i ical ole o Mülle glia (MG) in he con ex o e inal ganglion cell (RGC)
dea h, pa icula ly ocusing on he in luence o pe iphe al MG sensi i i y o high p essu e (HP).
Me hods Co-cul u es o po cine RGCs wi h MG we e isola ed om bo h he cen al and pe iphe al egions o pig
e inas and subjec ed o bo h no mal and HP condi ions. Mass spec ome y analysis o he MG-condi ioned medium
was conduc ed o iden i y he p o eins eleased by MG unde all condi ions.
Resul s Pe iphe al MG we e ound o sec e e a highe quan i y o neu op o ec i e ac o s, e ec i ely p omo ing
RGC su i al unde no mal physiological condi ions. Howe e , unde HP condi ions, co-cul u es wi h pe iphe al MG
exhibi ed impai ed RGC su i al. Mo eo e , unde HP condi ions, pe iphe al MG signi ican ly up egula ed he sec e-
ion o p o eins associa ed wi h apop osis, oxida i e s ess, and in lamma ion.
Conclusions This s udy p o ides obus e idence sugges ing he in ol emen o MG in RGC dea h in glaucoma, hus
pa ing he way o u u e he apeu ic in es iga ions.
Keywo ds Mülle glia, Re inal ganglion cells, Glaucoma, Re ina, Neu odegene a ion
Backg ound
Re inal ganglion cells (RGCs) a e he neu ons ha
ansmi isual in o ma ion om he eye o he b ain and
hus, hei su i al is c i ical o ision. These neu ons
a e e y sensi i e o insul s, and damage o RGC axons
in he op ic ne e may lead o apid RGC dea h in acu e
diseases like ischemic op ic neu opa hy o op ic neu i is,
o in ch onic diseases like glaucoma [1]. Di e en RGC
sub ypes espond dis inc ly o s imuli and insul s,
and no as a single en i y, such ha hei ype-speci ic
ulne abili y has been s udied ex ensi ely. As such, ea ly
unc ional al e a ions o ce ain sub ypes o RGCs ha e
been p oposed [2–5]. In glaucoma, he e is a g ea e loss
o la ge RGCs in he pe iphe al e ina, which also occu s
*Co espondence:
Xand a Pe ei o
xand a.pe ei [email protected]
Elena Vecino
[email p o ec ed]
1 Expe imen al Oph halmo-Biology G oup, Depa men o Cell Biology
and His ology, Uni e si y o he Basque Coun y UPV/EHU, 48940 Leioa,
Spain
2 P o eomics Pla o m, CIC bioGUNE, Basque Resea ch and Technology
Alliance (BRTA), CIBERehdP o eoRed-ISCIII, Bizkaia Science and
Technology Pa k, 48160 De io, Spain
3 Facul y o Medicine, Coimb a Ins i u e o Clinical and Biomedical
Resea ch (iCBR), Uni e si y o Coimb a, Coimb a, Po ugal
4 Cen e o Inno a i e Biomedicine and Bio echnology (CIBB), Uni e si y
o Coimb a, Coimb a, Po ugal
5 Clinical Academic Cen e o Coimb a (CACC), Coimb a, Po ugal
6 Associa ion o Inno a ion and Biomedical Resea ch on Ligh and Image
(AIBILI), Coimb a, Po ugal
Page 2 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
in animal models o glaucoma ha esemble he ea u es
desc ibed in glaucoma pa ien s [6, 7].
Mülle Glia (MG) a e he p incipal mac oglial cells in
he e ina. They a e adially o ien ed and span he en i e
hickness o he e ina. These cells a e esponsible o he
homeos a ic and me abolic suppo o e inal neu ons,
making hem essen ial o neu onal su i al [8]. Reac i e
MG can main ain ex acellula homeos asis du ing e inal
insul o damage, he eby p o ec ing e inal neu ons [9].
These neu op o ec i e e ec s o MG may be media ed by
di e se mechanisms, enhancing physiological unc ions
like glu ama e o po assium up ake. Mo eo e , neu onal
su i al can be u he p ese ed h ough he elease o
neu op o ec i e ac o s by MG [10–12].
MG also ep esen a he e ogeneous popula ion, in
which some cells pa icipa e mo e closely in e inal
epai han o he s. Al hough li le is known abou he
he e ogenei y o MG, only one subse o MG exp ess he
Chx10 ansc ip ion ac o [13], while 30% o MG exp ess
class II MHC an igen in i o, sugges ing ha hey migh
be in ol ed in immune eac ions [14]. In e ms o hei
dis ibu ion in he e ina, MG mo phology a ies in
ela ion o e inal opog aphy [15]. The cen al e ina
is hicke han he pe iphe y, wi h a g ea e densi y o
neu ons and MG. Mo eo e , MG om he cen al e inal
a e longe and hinne han hose in he pe iphe y,
and hey ha e a smalle olume bu a highe su ace o
olume a io [16]. In he chick e ina, he egion in which
p oli e a ing MG accumula e in esponse o e inal
damage becomes mainly con ined o he pe iphe y [17].
Two ypes o MG we e dis inguished in he chick e ina,
e e ed o as ype I cells wi h nume ous hin p ocesses,
and ype II cells wi h ewe and hicke p ocesses [18,
19]. When cha ac e ized u he , he ype I cells we e
seen o be in he majo i y ac oss he e ina, whe eas
ype II MG we e mainly ound in he pe iphe al e ina
[20]. Mo eo e , while mammalian MG lose he capaci y
o p oli e a e and egene a e, unlike o he e eb a es
as ish, pe iphe al MG exp ess p o eins cha ac e is ic o
s em cells a e damage, such as CD44 [21] and he neu al
p ogeni o ma ke , nes in [22]. In he human e ina, he
mic oen i onmen o he cen al e ina no only has
speci ic cha ac e is ics wi h espec o he pe iphe y
bu also, i is mo e suscep ible o ce ain diseases [23].
Phosphoglyce a e dehyd ogenase, he a e-limi ing
enzyme in se ine syn hesis, is exp essed mo e in MG
localized in he macula han in pe iphe al MG, sugges ing
highe suscep ibili y o macula cells o oxida i e s ess
han pe iphe al cells [24]. Macula MG also exp ess mo e
aquapo in-4 han pe iphe al MG [25]. Fu he mo e, in a
mouse model o lase -induced glaucoma, he s onges
ac i a ion o mac oglial cells was in he in e media e and
pe iphe al e ina ela i e o he cen al e ina [26].
MG isola ed om he cen al o pe iphe al e ina
consis en ly espond in a di e en manne o he
condi ions o which hey a e exposed, indica ing
molecula di e ences be ween he MG om hese
wo di e en loca ions [24]. The di e ences in p o ein
exp ession among MG a dis inc e inal loca ions may
indica e ha hey ul ill di e en unc ions, al hough his
s ill needs o be ex ensi ely s udied.
MG a e conside ed he p incipal e inal senso ha
can espond p omp ly o mechanical changes [27]. The
he e ogenei y o MGs and hei abili y o sense p essu e
could explain he di e ences in suscep ibili y o dea h o
pe iphe al RGCs in glaucoma. As such, MGs may play an
impo an ole and could o e insigh s in o he po en ial
causes o he onse o glaucoma ous damage. I is impo -
an o know i he MG loca ed in he e inal a ea whe e
he RGCs die i s in glaucoma beha e di e en ly o hose
in o he e inal a eas and i hey a e also mo e ulne able
o changes in in aocula p essu e (IOP). To add ess his
issue and based on ou expe ience in handling MG and
RGCs in cul u e, we i s s udied, using co-cul u es, he
in e ac ion o MG isola ed om he pe iphe al and cen al
e ina wi h RGCs, ocusing on he su i al o hese cells.
Subsequen ly, we examined he e ec o HP on hese co-
cul u es using cen al and pe iphe al MG, ocusing on
he suscep ibili y o dea h o bo h MG and RGC. Finally,
we compa ed he p o eome o MG condi ioned medium
in he expe imen al condi ions es ed, exposu e o no o
HP, o iden i y p o eins sec e ed by MG om he cen al
and pe iphe al e ina. Thus, he e we examined he possible
di e en p o ec i e e ec o MG loca ed in he cen al o
pe iphe al e ina on RGCs, iden i ying p o eins ha may
be in ol ed in hese e en s. The da a ob ained led us o
sugges ha pe iphe al MG may be in ol ed in he ini ia-
ion o glaucoma ous damage.
Me hods
Animals
This s udy was ca ied ou in s ic acco dance wi h he
Guidelines o he Ca e and Use o Labo a o y Animals.
All he expe imen al p o ocols complied wi h he Eu o-
pean (2010/63/UE) and Spanish (RD53/2013) egula ions
o he p o ec ion o expe imen al animals, and hey we e
app o ed by he E hical Commi ee o Animal Wel a e a
he Uni e si y o Basque coun y. All animal expe imen a-
ion adhe ed o he ARVO S a emen o he Use o Ani-
mals in Oph halmic and Vision Resea ch.
Fo MG cul u es, adul po cine eyes we e ob ained
om a local aba oi s and anspo ed o he labo a-
o y in cold CO2-independen medium (Li e Technolo-
gies, Ca lsbad, CA, USA) con aining 0.1% gen amicin
(Li e Technologies, Ca lsbad, CA, USA). Fo pu e
RGC cul u es, eyes we e ob ained om adul emale
Page 3 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
Sp ague–Dawley a s (200–250 g) housed on a 12 h
ligh –da k cycle wi h adlibi um access o ood and wa e ,
and hey we e sac i iced humanely by exposu e o CO2.
Po cine e inal Mülle glia cul u es
MG cul u es we e p epa ed as desc ibed p e iously
[28]. Fi s , dissec ing adul po cine eyes wi hin 2 h
o enuclea ion. B ie ly, he majo blood essels we e
emo ed and he e ina was washed in CO2-independen
medium. The e inas we e dissec ed ou and wo di e en
a eas o he e ina we e ob ained wi h an 8mm diame e
dissec ing ephine (Biomedical Resea ch Ins umen s,
Sil e Sp ing, MD, USA): cen al and pe iphe al. The
e inal issue was dissocia ed o 30min a 37°C in 0.2%
ac i a ed papain (Wo hing on, Lakewood, NJ, USA)
wi h 10% DNAse I (Wo hing on, Lakewood, NJ, USA).
Enzyme ac i i y was s opped by he addi ion o MG
medium (see below) and DNase I. The issue was hen
disagg ega ed by gen le i u a ion using pipe e ips o
dec easing diame e .
Pu i ied MG we e cul u ed in DMEM (Li e
Technologies, Ca lsbad, CA, USA) wi h 10% e al
bo ine se um (FBS: Li e Technologies, Ca lsbad, CA,
USA). MG and RGCs we e co-cul u ed in Neu obasal
A medium (NBA: Li e Technologies, Ca lsbad, CA,
USA) supplemen ed wi h 10% FBS and 2% B27 (Li e
Technologies, Ca lsbad, CA, USA). In addi ion, 1%
l-glu amine (2 mM: Li e Technologies, Ca lsbad, CA,
USA) and 0.1% gen amicin (50mg/mL: Li e Technologies,
Ca lsbad, CA, USA) we e added o he cul u e media o
pu i ied MG and o co-cul u es.
Dissocia ed cells we e pelle ed by cen i uga ion
(1200 pm, 5 min), esuspended in MG medium and
pla ed on poly-l-lysine (100μg/mL: Sigma-Ald ich, S .
Louis, MO, USA) and laminin (10μg/mL: Sigma-Ald ich,
S . Louis, MO, USA) coa ed 13 mm diame e glass
co e slips in 24-well pla es. The cells we e main ained
in a humidi ied incuba o a 37°C in an a mosphe e o
5% CO2. Cells in suspension we e emo ed a e 24h
by changing he medium. Fo main enance, hal o he
medium was eplaced e e y 2–3days.
Condi ioned medium (CM) om MG cul u es was col-
lec ed when he cul u es eached con luence a day 7.
Fi s ca e ully washing he wells h ee imes wi h NBA
medium, and subsequen ly adding esh NBA medium
o each well o 3h be o e he medium was changed
o elimina e he es o he FBS and B27. F esh NBA
medium was added and condi ioned o e 2days be o e i
was collec ed s e ilized by passing h ough a 0.22μm il-
e and ozen in aliquo s a − 20°C. Finally, he MG we e
ixed o 10min wi h me hanol a − 20°C. A leas h ee
eplica es o each cul u e we e se -up and he p ocedu e
was pe o med a leas in iplica e.
Pu i ied RGC cul u es
RGC cul u es we e p epa ed as desc ibed p e iously
[29] om a mixed suspension o e inal cells ob ained
om pig o a e inas. Pig RGCs we e ob ained om
he cen e and pe iphe y o he e ina, and a RGCs
we e ob ained om he en i e e ina. Re inal issue was
dissocia ed enzyma ically using he Papain Dissocia ion
Ki (Wo hing on Biochemical, Lakewood, NJ, USA),
ollowing he manu ac u e ’s ins uc ions, namely
diges ing he issue o 90min a 37°C in 0.2% ac i a ed
papain wi h 10% DNAse I. A e gen le i u a ion using
pipe e ips o dec easing diame e o disagg ega e he
issue, and a e using a densi y g adien , he pu i ied
RGCs we e pla ed on 13 mm poly-l-lysine (100 μg/
mL, Sigma-Ald ich, S . Louis, MO, USA) and laminin
(10μg/mL: Sigma-Ald ich, S . Louis, MO, USA) coa ed
glass co e slips in 24‐well pla es. The pig and a cells
we e hen seeded a 1 × 105 iable cells pe well and
he cul u es we e main ained in Neu obasal A medium
(Li e Technologies, Ca lsbad, AC, USA) supplemen ed
wi h 2% B27, 1% l-glu amine (2mm: Li e Technologies,
Ca lsbad, CA, USA) and 0.1% gen amicin (50mg/mL:
Li e Technologies, Ca lsbad, CA, USA).
Ra RGCs we e seeded on con luen MG cul u es
om he cen e o pe iphe y pig e ina. In addi ion,
RGCs we e also cul i a ed adding ei he CM ob ained
om cul u es o cen al o pe iphe al MG, collec ed in
NBA plus NBA/B27 (1:1). As con ol, a RGCs we e
cul u ed in NBA/B27. All he media used con ained
1% l-glu amine and 0.1% gen amicin. RGC cul u es
we e main ained o 6days a 37°C in a humidi ied
a mosphe e con aining 5% CO2, and he medium was
changed e e y 3days. Finally, he RGCs we e ixed o
10min wi h me hanol a − 20°C on day 6.
A leas ou we e pe o med o each analysis
desc ibed, epea ing each independen expe imen a
minimum o h ee imes.
Cul u es exposed ohigh p essu e
Cul u es we e subjec ed o high p essu e (HP) using a
cus om-made humidi ied p essu e chambe equipped
wi h a p essu e gauge and a p essu e egula o as
desc ibed p e iously wi h mino changes [30]. This
se -up as allowed a cons an p essu e o be main ained
wi h an ai mix u e o 95% ai , 5% CO2 and 2%O2. The
p essu e chambe was placed in an o en a 37°C and
he cul u es we e main ained unde HP o 72h. The
magni ude o p essu e ele a ion (70 mmHg abo e
a mosphe ic p essu e) was chosen in acco dance
wi h p e ious s udies [31]. Con ol cul u es we e kep
a no mal a mosphe ic p essu e in a s anda d cell
incuba o , and a leas h ee expe imen al eplica es
Page 4 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
we e analyzed, pe o med in iplica e independen
expe imen s.
Immunocy ochemis y
A e ixa ion in me hanol and washing wi h PBS
(phospha e bu e ed saline, pH 7.0), he binding o non-
speci ic an igens was blocked wi h blocking bu e (3%
BSA and 0.1% T i on X-100 in PBS) be o e incuba ing
he cells o e nigh a 4°C wi h he p ima y an ibodies
(see Table1) dilu ed in blocking bu e . A e washing,
he binding o hese an ibodies we e de ec ed wi h Alexa
Fluo 555 o 488 conjuga ed goa an i-mouse and goa
an i- abbi an ibodies (dilu ed 1:1000: Li e Technologies,
Ca lsbad, CA, USA). Cells we e inally coun e s ained
wi h he nuclea ma ke DAPI (dilu ed 1:10,000: Li e
Technologies, Ca lsbad, CA, USA). A e washing, he
co e slips we e moun ed using Fluo -sa e Reagen
(Sigma-Ald ich, S . Louis, MO, USA).
Quan i ica ion o RGCs andMG
RGCs we e obse ed in an epi luo escence mic oscope
(Zeiss, Jena, Ge many) coupled o a digi al came a (Zeiss
Axiocam MRM, Zeiss, Jena, Ge many). All images we e
ob ained unde he same condi ions o in ensi y and
exposu e ime. A leas h ee co e slips we e analyzed
o each expe imen al condi ion and om a minimum
o h ee independen expe imen s. The densi y o he
RGC cul u es was quan i ied. In addi ion, he RGCs we e
classi ied as: (1) cells wi h no neu i es; (2) cells wi h a
longes neu i e < 50μm; (3) cells wi h he longes neu-
i e be ween 50 and 200μm; and (4) cells wi h neu i es
longe han 200μm. The o al numbe o RGCs su i ing
in each condi ion was coun ed. The MG p esen in he
cul u es we e also analyzed and coun ed in images. Semi-
au oma ic Zen so wa e (Zeiss, Jena, Ge many) was used
o coun he numbe o nuclei s ained wi h DAPI, aking
in o conside a ion he limi s o he axis o he MG nuclei
o ob ain mo e accu a e measu emen s. As such, we used
a mac o designed o speci ically measu e he limi s o he
axes (55–70μm), which was co ec ed manually o each
image.
Mass spec ome y analysis o heCM andda a p ocessing
The p o eomic analysis o he CM ob ained o he cen al
and pe iphe al MG cul u es unde con ol and HP con-
di ions was ca ied ou a he CIC bioGUNE P o eom-
ics Se ice (De io, Bizkaia, Spain), using he Fil e Aided
Sample P epa a ion (FASP) p o ocol [32], wi h mino
modi ica ions. A e solu ion diges ion, he p o eins we e
ex ac ed in a sample con aining 7M u ea, 2 M Thio-
u ea, 4% CHAPS and 5mM DTT. T ypsin was added a
a ypsin: p o ein a io o 1:20, and he mix u e was incu-
ba ed o e nigh a 37°C, d ied in a RVC2 25 speed ac
concen a o (Ch is , Os e ode am Ha z, Ge many), and
esuspended in 0.1% o maldehyde (FA). Pep ides we e
desal ed and esuspended in 0.1% FA using C18 s age
ips (Me ck Millipo e, Bu ling on, MA, USA). The sam-
ples we e analyzed in a imsTOF P o wi h PASEF (B uke ,
Bille ica, MA, USA) appa a us coupled online o a E osep
ONE liquid ch oma og aph (E osep Biosys ems, Odense,
Denma k), loading 200ng di ec ly on o he E osep ONE
and employing a 60 samples-pe -day p o ocol.
P o ein iden i ica ion and quan i ica ion was ca -
ied ou using PEAKS X so wa e (Bioin o ma ics solu-
ions, Wa e loo, Canada), ca ying ou sea ches agains
a da abase consis ing o Sus sc o a en ies om UniP o
(h ps:// www. unip o . o g/), wi h p ecu so and agmen
ole ances o 20ppm and 0.05Da. Only p o eins iden i-
ied wi h a leas wo pep ides a a False De ec ion Ra e
(FDR) o 1% we e conside ed o u he analysis. P o ein
abundances in e ed om PEAKS we e loaded on o he
Pe seus pla o m, log2 ans o med and impu ed be o e
analyzing wi h a S uden ’s - es .
P o eins ha we e conside ed signi ican ly di e en
be ween he g oups we e hose wi h a p- alue < 0.05,
and also hose ha exceeded ha alue, up o a p- alue
o 0.1, bu wi h a old change > 2 o each compa ison
analyzed. The p o eins lis ed we e o de ed acco ding o
he old change ob ained, and he p o eins selec ed, ou
o a o al o 893 p o eins, we e ca ego ized based on hei
unc ions a ibu ed in he UniP o da abase. In addi ion,
o some p o eins o pa icula in e es in his s udy, an
in silico analysis was ca ied ou using he eely a ailable
STRING (Sea ch Tool o he Re ie al o In e ac ing
Genes/P o eins) da abase (h ps:// s ing- db. o g/). The
numbe o p o ein–p o ein in e ac ions egis e ed in
he da abase was de e mined o he p o eins ha we e
di e en ially o e exp essed. Fo isualiza ion, a diag am
was assembled linking he p o eins depic ed by nodes
Table 1 P ima y an ibodies used
αSMA α-smoo h muscle ac in, TRPV4 ansien ecep o po en ial ca ion channel
sub amily V membe 4
An igen Ta ge Hos Dilu ion Supplie
α-SMA Dedi e en ia ion Mouse 1:1000 Abcam
β-III-Tubulin RGCs Rabbi 1:2000 P omega
β-Ca enin Dedi e en ia ion Rabbi 1:2000 Abcam
CD-133 Dedi e en ia ion Rabbi 1:200 Abcam
Nes in Dedi e en ia ion Rabbi 1:500 Sigma
OCT4 Dedi e en ia ion Rabbi 1:200 Abcam
p75NTR Mülle glia Rabbi 1:2000 Abcam
Piezo1 Mechanosenso Rabbi 1:100 Li e Technologies
T p 4 Mechanosenso Rabbi 1:500 Li e Technologies
Vimen in Mülle glia Mouse 1:10,000 Dako
Page 5 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
based on ecognized connec ions wi h he p o eins
iden i ied.
S a is ical analysis
The expe imen al p ocedu es we e eplica ed a leas
h ee imes o ensu e he eliabili y and consis ency o
he indings. The cell densi y was de ined as he mean
numbe o cells pe cm2, and he mean and s anda d
e o o mean (SEM) a e p esen ed o each condi ion.
S a is ical analyses we e ca ied ou using he IBM SPSS
S a is ical so wa e .24-0. The da a om he di e en
expe imen al condi ions we e compa ed using he non-
pa ame ic Mann–Whi ney U es . When mo e han wo
independen g oups we e compa ed, a K uskal–Wallis
non-pa ame ic es was used, and i he K uskal–Wallis
es was signi ican , a pos -hoc Dunn es was pe o med
in o de o de e mine which g oups di e om he
o he s. Di e ences we e conside ed signi ican o all
es s a a p- alue < 0.05.
Resul s
The su i al o pig RGCs om he cen e o om he
pe iphe y o he e ina was analyzed in pu i ied cul u es.
Cul u ed RGCs om he cen al e ina su i ed signi i-
can ly be e (173 ± 27 RGCs/cm2) han hose om he
pe iphe al e ina (87 ± 11 RGCs/cm2, Fig.1A). When he
e ec o MG, isola ed om he cen e o pe iphe y o he
pig e ina, on RGC su i al was analyzed in co-cul u es,
he su i al o pe iphe al RGCs and MG (3389 ± 471 and
61,876 ± 10,509 cells/cm2, espec i ely) was signi ican ly
inc eased compa ing o co-cul u es wi h MG and RGCs
isola ed om he cen al e ina (1566 ± 484 RGCs/cm2
and 33,232 ± 3181 MG/cm2: Fig.1B). To con i m he mo e
obus neu op o ec i e e ec o he MG isola ed om
he pe iphe al e ina, pu i ied a RGCs we e seeded on o
a monolaye o MG om he cen al and pe iphe al e -
ina al eady cul u ed o 7days in i o (DIV). The su -
i al o he a RGCs inc eased signi ican ly (1914 ± 176
RGCs/cm2) when hey we e seeded on pe iphe al MG
a he han on cen al e inal MG (987 ± 139 RGCs/cm2)
a e 6days in co-cul u e (Fig.1C). To assess whe he ac-
o s sec e ed by pe iphe al MG may ha e he same e ec
on RGC su i al as MG monolaye s, a RGCs we e
cul u ed in CM om he cen al and pe iphe al MG.
RGCs we e cul u ed in NBA/B27 (con ol) o in NBA/
B27 medium:CM (1:1) ob ained om ei he cen al o
pe iphe al MG in cul u e. Mo e RGCs su i ed when
he cells we e main ained in CM om pe iphe al MG
(249 ± 48 RGCs/cm2) han in he CM om cen al e inal
MG (114 ± 25 RGCs/cm2), o con ol RGCs wi hou CM
(71 ± 2 RGCs/cm2) (Fig.1D).
The RGC neu i e leng h was assessed o de e mine
whe he he di e en CM a ec ed hese pa ame e s
(Fig.1D). When RGCs we e main ained in CM sec e ed
by pe iphe al MG, he e we e mo e RGCs wi h sho
neu i es (< 50μm, 147 ± 32 RGCs/cm2) han when RGCs
we e main ained in CM by MG om he cen al e ina
(61 ± 13 RGCs wi h sho neu i es/cm2).
To de e mine i he cen al o pe iphe al MG beha e
di e en ly in cul u e, he exp ession o ce ain s em
cell-like ma ke s was analyzed in pu i ied MG cul u es:
imen in (MG speci ic ma ke ), β-III-Tubulin (neu-
al ma ke ), α-SMA (dedi e en ia ion ma ke ), CD133
(glial s em cell ma ke ), OCT4 (impo an in MG ep o-
g amming du ing e inal egene a ion in zeb a ish),
nes in (ma ke o glial and neu onal p ogeni o s), and
β-ca enin (s em cell-like ma ke ). Al hough he MG spe-
ci ic ma ke imen in was exp essed s ongly in bo h
ypes o cul u es, β-III-Tubulin, α-SMA, CD133, OCT4,
nes in, and β-ca enin we e exp essed mo e by MG om
he pe iphe al e ina han by cen al MG. Hence, pe iph-
e al MG appea o be mo e dedi e en ia ed owa ds a
s em cell-like pheno ype (Fig.2).
The analysis o he p o eome o CM ob ained om
he cen al and pe iphe al MG was pe o med, iden i-
ying he exp ession o mo e s em cell-like ma ke s in
he CM o pe iphe al MG, and also mo e neu op o ec-
i e molecules. The p o eome o he MG CMs con ained
di e en ially exp essed p o eins (DEPs), wi h 22 mo e
DEPs in he cen al MG CM and 113 p o eins mo e
s ongly exp essed in he pe iphe al CM. All p o eins
mo e s ongly exp essed in he CM o pe iphe al MG,
along wi h hei associa ed unc ions: “Cy oskele on, cell
adhesion and cell shape”; “In lamma ion and immune
esponse”; “Su i al and homeos asis”; “Neu op o ec ion
and neu i e ou g ow h”; “P oli e a ion”; “Ubiqui ina ion”;
“Angiogenesis”; “Dedi e en ia ion”; “T anspo ”; “Apop-
osis and p oli e a ion inhibi ion”; “Oxida i e s ess and
s ess esponse”; and “O he unc ions” (Table2). When
he p o eins so ed by unc ion we e compa ed be ween
cen al and pe iphe al CM (Fig.3A), he p opo ion o
p o eins in he pe iphe al CM ela ed o “P oli e a ion”
and “Cy oskele on, cell adhesion, cell shape” we e 14.2%
and 24.8% o o al p o eins iden i ied, espec i ely, while
in cen al CM only 4.6% o p o eins we e ela ed o hese
unc ions. Ano he in e es ing unc ion o he p esen
s udy is “Neu op o ec ion and neu i e ou g ow h” and
as expec ed, he p o eins ela ed o his ole we e mo e
s ongly exp essed in he pe iphe al CM, ep esen ing
13.3% o he p o eins. Ano he unc ion wo h high-
ligh ing is “Dedi e en ia ion” and al hough he p opo -
ion o hese p o eins was no e y high (3.5%), hey
only appea ed in he pe iphe al CM, consis en wi h he
exp ession o he s em cell-like ma ke s de ec ed (Fig.2).
A STRING analysis was also pe o med on some o he
p o eins om he “P oli e a ion”, “Neu op o ec ion and
Page 6 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
neu i e ou g ow h” and “Dedi e en ia ion” ca ego ies
iden i ied in ou p o eomic analysis. Again, some o he
p o eins iden i ied in he STRING analysis we e mo e
s ongly ep esen ed in he pe iphe al CM (Fig.3B).
The e ec o HP on RGC and MG su i al was ana-
lyzed in co-cul u es om he cen al and pe iphe al e -
ina. A e 72h o HP, cen al RGC su i al dec eased o
65.5 ± 8.0%. Su p isingly, he su i al o pe iphe al RGCs
was mo e s ongly a ec ed, dec easing o 41.2 ± 5.3%
ela i e o he con ol. The same applied o he MG, wi h
82.7 ± 15. % o he cen al MG su i ing a e 72h o HP,
and dec easing o 69.6 ± 13.6% when he MG we e om
he pe iphe y o he e ina (Fig.4).
To con i m he e ec o HP on he suscep ibili y o
pe iphe al MG, pu i ied con ol a RGCs we e seeded
on a MG monolaye om he cen al o pe iphe al pig
e ina a 7 DIV, and exposed o HP o 72h. The su i al
o he a RGCs, compa ing o he con ol, dec eased
Fig. 1 A The su i al o cen al o pe iphe al pig RGCs in pu i ied cul u es. Images o pu i ied RGC cul u es om he cen al and he pe iphe al
e ina. The same numbe o RGCs we e seeded in bo h cases. His og am o he numbe o RGCs in he cul u es om each e inal egion. B E ec
o MG om he cen al and pe iphe al e ina on he su i al o RGCs om he cen al and pe iphe al e ina, espec i ely, in co-cul u es. Images
o co-cul u es o pig RGCs and MG om he cen al and pe iphe al e ina. The MG and RGC in he co-cul u es we e bo h isola ed om he same
a ea o he e ina (cen e and pe iphe y). Numbe o RGCs in co-cul u es om he cen e and pe iphe y o he e ina and numbe o MG
in co-cul u es om he cen e and pe iphe y o he e ina we e ep esen ed. C The su i al o a RGCs seeded on con luen pig MG isola ed
om he cen al and pe iphe al e ina. Images o a RGCs seeded on pig MG om he cen al and pe iphe al e ina. The same ini ial numbe
o a RGC we e seeded on pig MG cul u es. The numbe o a RGCs p esen on pig MG cul u es om he cen al o pe iphe al e ina. D Su i al
and neu i ogenesis o a RGCs when main ained in CM sec e ed by MG om he cen al and pe iphe al e ina. Images o a RGCs main ained
wi h condi ioned medium (CM) om cen al and pe iphe al pig MG. The same numbe o RGCs we e seeded in all condi ions. Numbe o su i ing
a RGCs main ained wi h CM sec e ed by MG om he cen al o pe iphe al pig e ina was ep esen ed. To analyze neu i ogenesis, he RGCs we e
classi ied as RGCs wi hou neu i es (blue), RGCs wi h he longes neu i e < 50 μm (o ange), RGCs wi h he longes neu i e be ween 50 and 200 μm
(g ey), and hose wi h neu i es longe han 200 μm (yellow). The numbe o RGCs in each ca ego y is shown o hose main ained in he p esence
o bo h ypes o CM. The RGCs we e labelled wi h an ibodies agains β-III-Tubulin ( ed) and he MG wi h imen in (g een). Nuclei we e s ained
wi h DAPI (blue): *p- alue < 0.05. Scale ba : 50 µm
Page 7 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
d ama ically when hey we e seeded on pe iphe al MG
unde condi ions o HP (16.4 ± 4.5% RGCs) as opposed o
MG om he cen al e ina (52.6 ± 30.1% RGCs) (Fig.5).
Ha ing obse ed dis inc suscep ibili y o bo h RGCs
and MG o HP depending on hei posi ion in he e -
ina, cen al o pe iphe al, we analyzed he exp ession o
wo common p essu e ecep o s, PIEZO1 and TRPV4
in pu i ied MG cul u es unde con ol condi ions and
a e exposu e o HP o 72h. A 7 DIV, bo h cen al
and pe iphe al MG exp ess PIEZO1 in esponse o HP
(Fig.6E, G). Likewise, TRPV4 is also mo e exp essed in
cen al and pe iphe al MG when hey a e exposed o
HP. In addi ion, in con ol condi ion, is mo e s ongly
exp essed in pe iphe al MG han in he cen al MG
(Fig.6F, H).
The p o eome o he CM ob ained om he cen al
and pe iphe al pig MG exposed o HP and unde con-
ol condi ion was compa ed. When he CM om cen-
al MG exposed o HP was compa ed o con ol, 18
p o eins we e mo e signi ican ly exp essed in he con ol
CM while 75 p o eins we e signi ican ly mo e exp essed
in he EH CM. When he p o eins we e so ed by unc-
ion, he e we e mo e p o eins ela ed o “Oxida i e
s ess, s ess esponse” (11.8%) and “Su i al, Homeos a-
sis” (17.1%) in he EH CM compa ing o he con ol CM,
in which he pe cen age o p o eins ela ed o “Oxida i e
Fig. 2 Immunolabelling o imen in, CD133, OCT4, nes in, α-SMA, β-ca enin and β-III Tubulin in pu e cul u es o MG isola ed om he cen al
and pe iphe al pig e ina. Images o cul u ed MG isola ed om he cen e (A, C, E, G, I) o pe iphe y (B, D, F, H, J) o he e ina. The cells we e
labelled wi h an ibodies agains CD133 (A, B, ed), OCT4 (C, D, ed), nes in (E, F, ed), β-ca enin (G, H, ed), β-III Tubulin (I, J, ed), α-SMA (E, F, g een),
o imen in (A–J, g een). Nuclei we e s ained wi h DAPI (blue). No e he inc ease in he exp ession o all s em cell-like ma ke s and β-III-Tubulin
in he pe iphe al MG. Scale ba : 50 µm
Page 8 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
Table 2 P o eins mo e s ongly ep esen ed in he CM om pe iphe al MG compa ing o he cen al MG, and hei unc ions
Gene symbol En ez gene name Fold change P- alueBiological unc ions
SFRP2Sec e ed izzled ela ed p o ein 2 49.99 0.049 P oli e a ion, Dedi e en ia ion
PLOD2P ocollagen-lysine 5-dioxygenase 28.82 0.0010 Cy oskele on, cell adhesion, cell shape
COL11A1Collagen ype XI alpha 1 chain 23.55 0.004 Cy oskele on,cell adhesion, cell shape
CLUClus e in 22.78 0.0003 In lamma ion, Immune esponse,
Su i al, Homeos asis
APOE Apolipop o ein E 19.98 0.005 Neu op o ec ion, Neu i e Ou g ow h,
Su i al, Homeos asis
LMNB1Lamin B1 18.39 0.010 Cy oskele on, cell adhesion, cell shape
ATP5PB ATP syn hase F(0) complex subuni B1
mi ochond ial15.67 0.041 T anspo
DKK3Dickkop WNT signalling pa hway
inhibi o 314.77 0.011 Dedi e en ia ion
CRYAB Alpha-c ys allin B chain 13.28 0.05 Su i al, Homeos asis
EFEMP1 EGF con aining ibulin ex acellula ma ix
p o ein 110.88 0.016 Cy oskele on, cell adhesion, cell shape,
P oli e a ion
CLN5 Ce oid-lipo uscinosis neu onal p o ein 5 10.75 0.034 Neu op o ec ion, Neu i e Ou g ow h
GLRX3Glu a edoxin 3 10.70 0.06 Su i al, Homeos asis, P oli e a ion
SCIN Adse e in 10.17 0.08 Apop osis, P oli e a ion inhibi ion,
Dedi e en ia ion
EFEMP2 EGF con aining ibulin ex acellula ma ix
p o ein 210.12 0.009 Cy oskele on, cell adhesion, cell shape
COX5ACy och omec oxidase polypep ide Va 10.01 0.06 Oxida i e S ess
HSPA13 Hea shock 70 kDa p o ein 13 9.57 0.05 Ubiqui ina ion
ESM1 Endo helial cell speci ic molecule 19.040.042 Angiogenesis
LPLLipop o ein lipase 8.85 0.05 In lamma ion, Immune esponse
DAG1 Dys oglycan 8.54 0.013 Su i al, Homeos asis, Cy oskele on,
cell adhesion, cell shape
SPARCSec e ed p o ein acidic and cys eine ich 8.45 0.007
Cy oskele on, cell adhesion, cell shape,
P oli e a ion, In lamma ion, Immune
esponse
MYH11Myosin-11 8.26 0.09 Cy oskele on, cell adhesion, cell shape
MSTN G ow h/di e en ia ion ac o 87.430.025 In lamma ion, Immune esponse
APLP2Amyloid be a p ecu so like p o ein 27.390.015 Su i al, Homeos asis
C1QC Complemen C1q C chain7.190.035 In lamma ion, Immune esponse
SDC2 Syndecan 27.150.0009
Cy oskele on, cell adhesion, cell shape,
Neu op o ec ion, Neu i e Ou g ow h,
P oli e a ion
C5 Complemen C5a anaphyla oxin 6.84 0.05 In lamma ion, Immune esponse,
Angiogenesis
NRCAMNeu onal cell adhesion molecule 6.74 0.010 Neu op o ec ion, Neu i e Ou g ow h
FBLN2Fibulin 26.670.07Cy oskele on, cell adhesion, cell shape
APP Amyloid-be a A4 p o ein6.500.08
Neu op o ec ion, Neu i e Ou g ow h,
Oxida i e S ess, P oli e a ion,
In lamma ion, Immune esponse
TUFM Elonga ion ac o Tu 6.32 0.023 In lamma ion, Immune esponse
PSME1 P o easome ac i a o complex subuni 16.300.019 In lamma ion, Immune esponse
CFIComplemen ac o I iso o m 1
p ep op o ein6.22 0.05 In lamma ion, Immune esponse
SETP o ein SET6.130.015 Neu op o ec ion, Neu i e Ou g ow h
PTK7 Inac i e y osine-p o ein kinase 76.120.014 Dedi e en ia ion, Neu op o ec ion,
Neu i e Ou g ow h
FTLFe i in ligh chain ( agmen ) 5.93 0.06 Su i al, Homeos asis
TIMP2Me allop o einase inhibi o 25.680.05In lamma ion, Immune esponse
SDHA Succina e dehyd ogenase [ubiquinone]
la op o ein subuni mi ochond ial5.65 0.020 Oxida i e S ess
FN1Fib onec in 15.550.010 Cy oskele on, cell adhesion, cell shape,
Angiogenesis
CDH2 Cadhe in-2 5.54 0.015 Cy oskele on, cell adhesion, cell shape
HSP90AB1 HATPase_c domain-con aining p o ein5.180.07
Neu op o ec ion, Neu i e Ou g ow h,
Su i al, Homeos asis, In lamma ion,
Immune esponse
ISLR Immunoglobulin supe amily con aining
leucine ich epea 5.14 0.0007 Neu op o ec ion, Neu i e Ou g ow h
COL5A1 Collagen ype V alpha 1 chain5.130.011 Cy oskele on, cell adhesion, cell shape
Page 9 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
Table 2 (con inued)
CDH11Cadhe in 11 4.90 0.0002 Cy oskele on, cell adhesion, cell shape
SPARCL1SPARC like 14.890.06Cy oskele on, cell adhesion, cell shape
TAGLN2 T ansgelin 24.770.010 Cy oskele on, cell adhesion, cell shape
OTUB1Ubiqui in hioes e ase4.750.004 In lamma ion, Immune esponse,
Ubiqui ina ion
HNRNPU He e ogeneous nuclea ibonucleop o ein U4.640.09P oli e a ion
FMOD Fib omodulin 4.54 0.004 Cy oskele on, cell adhesion, cell shape
ARSB A ylsul a ase B4.530.05Neu op o ec ion, Neu i e Ou g ow h
ANXA7Annexin 4.46 0.09 Angiogenesis, T anspo
CLIC4Chlo ide in acellula channel p o ein4.440.047 P oli e a ion
FSTL1Follis a in-like p o ein 14.380.016
Angiogenesis, In lamma ion, Immune
esponse, P oli e a ion, Su i al,
Homeos asis
RDXRadixin 4.27 0.06 P oli e a ion
PHBP ohibi in4.260.06Apop osis, P oli e a ion inhibi ion
RPS4X 40S ibosomal p o ein S4 4.23 0.08 Cy oskele on, cell adhesion, cell shape
ANXA2Annexin A2 4.22 0.09 Cy oskele on, cell adhesion, cell shape,
Neu op o ec ion, Neu i e Ou g ow h
IL4I1Amine oxidase4.160.027 Oxida i e S ess, In lamma ion,
Immune esponse, P oli e a ion
PCMT1P o ein-L-isoaspa a e (D-aspa a e) O-
me hyl ans e ase 4.12 0.012 Su i al, Homeos asis
RNH1 Ribonuclease inhibi o 4.040.07Angiogenesis
LAMB2Laminin subuni be a 23.940.010 Cy oskele on, cell adhesion, cell shape,
Neu op o ec ion, Neu i e Ou g ow h
PGD6-Phosphoglucona e dehyd ogenase
deca boxyla ing 3.91 0.045 Oxida i e S ess
MDH2 Mala e dehyd ogenase mi ochond ial3.910.07Oxida i e S ess
SERPINE1 Plasminogen ac i a o inhibi o 13.810.06In lamma ion, Immune esponse,
Angiogenesis
LTBP1La en ans o ming g ow h ac o be a
binding p o ein 13.61 0.043 P oli e a ion
CDH6 Cadhe in-6 3.59 0.025 Cy oskele on, cell adhesion, cell shape
GAPDHGlyce aldehyde-3-phospha e
dehyd ogenase 3.24 0.09 Apop osis, P oli e a ion inhibi ion
PDGFRB Pla ele -de i ed g ow h ac o ecep o be a 3.12 0.09
Angiogenesis, In lamma ion, Immune
esponse, P oli e a ion, Su i al,
Homeos asis
CAP1 Adenylyl cyclase-associa ed p o ein3.100.08Cy oskele on, cell adhesion, cell shape
SPON1 F-spondin3.100.045 Cy oskele on, cell adhesion, cell shape,
Neu op o ec ion, Neu i e Ou g ow h
DNASE2 Deoxy ibonuclease-2-alpha 2.97 0.06 Apop osis, P oli e a ion inhibi ion
CST3 Cys a in C2.920.040 Su i al, Homeos asis
BMP1 Me alloendopep idase 2.77 0.10 Cy oskele on, cell adhesion, cell shape
WDR1 WD epea domain 12.700.09Cy oskele on, cell adhesion, cell shape,
P oli e a ion
DPYSL2Dihyd opy imidinase- ela ed p o ein 22.590.030 Neu op o ec ion, Neu i e Ou g ow h
CKAP4Cy oskele on associa ed p o ein 42.370.09P oli e a ion
COL18A1Collagen ype XVIII alpha 1 chain2.360.09Angiogenesis, P oli e a ion,
Cy oskele on, cell adhesion, cell shape
RO60 Ro60 Y RNA binding p o ein2.190.032 In lamma ion, Immune esponse
ACTB Ac in cy oplasmic 12.190.06Neu op o ec ion, Neu i e Ou g ow h,
Su i al, Homeos asis
LAMC1Laminin subuni gamma 12.100.004 Cy oskele on, cell adhesion, cell shape
AKR1A1 Aldo-ke o educ ase amily 1 membe A1 2.08 0.08 Oxida i e S ess
VTNVi onec in 1.82 0.027 Cy oskele on, cell adhesion, cell shape
HTRA1Se ine p o ease HTRA11.810.013 Apop osis, P oli e a ion inhibi ion
F2 P o h ombin1.490.015 P oli e a ion
The p o eins men ioned in he able a e a selec ion o mo e han 890 p o eins ob ained in he p o eomic analysis. P o eins conside ed signi ican ly di e en be ween
g oups we e hose wi h a p- alue less han 0.05 and ha exceeded ha alue bu had a > wo old change in each compa ison analyzed, up o a p- alue o 0.1.
P o eins lis ed we e o de ed by he old change obse ed
Page 16 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
wi h inc eased β-ca enin exp ession by pe iphe al MGs
in i o. The STRING analysis highligh s p o ein–
p o ein in e ac ion ne wo ks, which is impo an o
he sys em-le el unde s anding o cellula p ocesses.
This analysis is based on physical in e ac ions and on
he in e ac ions o di e en molecula pa hways. The
STRING analyses ep esen ed he e ocused on p o eins
ha a e mo e s ongly ep esen ed in he CM o he
pe iphe al MG, highligh ing in e ac ions wi h p o eins
mainly ep esen ed in his CM and suppo ing he esul s
ob ained in he p o eomic analysis. These signaling
pa hways may unde lie he di e ences be ween he wo
ypes o MG and hei e ec on RGC su i al.
Fu he mo e, ele a ed IOP is he main isk ac o o
he onse and p og ession o glaucoma. Al hough he e
has been conside able esea ch in he ield o glaucoma,
he pa hological mechanisms unde lying he disease
onse and de elopmen a e s ill no ully unde s ood.
Neu onal degene a ion in glaucoma migh be due o
a combina ion o ac o s, among which he RGC and
MG in e ac ions. The e ec o HP mimics he e ec o
ele a ed IOP in he e ina [59]. In p ima y cul u es o
MG and RGCs exposed o HP, he cell dea h is enhanced
[60]. Mo eo e , e inal as ocy es and mic oglia ha e a
di e en ial e ec on he p essu e-induced dea h o RGCs
[61]. The ob ained esul s demons a e ha pe iphe al
and cen al MG ha e di e en suscep ibili y o HP, and
his impac s on he ype o sec e ed ac o s, consequen ly
a ec ing RGC su i al.
I is known ha MG a e senso s o p essu e wi hin he
e ina, as demons a ed in i o [27] and in i o [62]. We
s udied how condi ions o HP migh al e he exp ession
o he p essu e ecep o channels TRPV4 and Piezo1,
demons a ing ha bo h ecep o s we e o e exp essed in
MG exposed o HP. Piezo p o eins play impo an oles
in ouch sensing p essu e, espi a ion, angiogenesis and
s em cell di e en ia ion, and hei ac i a ion inc eases
calcium in lux aising he in acellula calcium ion con-
cen a ion ([Ca2+]i), which may igge apop osis [63].
The e o e, changes in cell s i ness and p essu e ac ing
h ough Piezo1 mechanosensi i e channels could con-
ibu e o neu odegene a ion [64]. TRPV4 ac i a ion can
also p o oke an inc ease in [Ca2+]i and con inued chan-
nel ac i a ion induces MG gliosis in he mouse e ina, as
well as apop osis in cul u ed mouse RGCs and adul po -
cine RGCs [65–67], which is in acco dance wi h he da a
p esen ed he e. Indeed, he TRPV4 exp ession in pe iph-
e al MG could explain why hese glial cells a e mo e sus-
cep ible o HP.
The p o eomic analyses o he cen al and pe iph-
e al CM om con ol and HP condi ions iden i ied a
se ies o DEPs. When hese we e so ed by unc ion, an
inc ease o p o eins ela ed o “Oxida i e s ess and s ess
esponse”, “In lamma ion and immune esponse” and
“Apop osis and inhibi ion o p oli e a ion” was e iden
in he CM om he pe iphe al MG exposed o HP. How-
e e , in he CM o cen al MG he e was an inc ease o
he p o eins ela ed o “Su i al and homeos asis”. These
esul s could explain why cen al MG and RGCs a e mo e
esis an o HP han he pe iphe al cells. Among he p o-
eins o e exp essed in pe iphe al MG unde exposed
o HP as opposed o he con ol pe iphe al MG a e:
ACTN4, a membe o he ac in binding p o ein amily
ha in e ac s wi h DNaseY and media es DNA agmen-
a ion du ing apop osis [68]; Epha2, om he Eph ecep-
o y osine kinase (RTK) amily, he la ges g oup o
y osine kinases in he genome [69], an RTK egula ed by
p53 p o eins ha induces apop osis [70]; FABP3, membe
o a amily o binding p o eins ha inhibi s p oli e a ion
and p omo es apop osis when o e exp essed [71]; TXN
is a key elemen in he elimina ion o eac i e oxygen spe-
cies [72]; and PSMA4 ha in e ac s wi h p o eins wi h a
s ong immune esponse [73]. The inc ease in he p o-
po ion o p o eins ela ed o hese unc ions sugges s
ha he pe iphe al MG a e mo e sensi i e o HP, and
consequen ly hey nega i ely a ec he su i al o RGCs
and o hemsel es. In addi ion, a STRING analysis o se -
e al o hese p o eins show ha hey in e ac wi h p o-
eins also p esen in he CM o pe iphe al MG exposed
o HP, sugges ing ha hese pa hways may be in ol ed
in he inc eased suscep ibili y o MG o HP and conse-
quen ly dec ease RGC su i al.
Conclusion
This s udy (summa ized in Fig.8), demons a es a clea
he e ogenei y be ween MG om he pe iphe y and cen-
al e ina, based on hei beha io and sec e ion o spe-
ci ic ac o s in i o. In con ol cul u es, pe iphe al MG
is mo e neu op o ec i e o RGCs, which could be due o
being in a mo e dedi e en ia ed s a e. Howe e , pe iph-
e al MG a e mo e suscep ible o p essu e, which causes
he sec e ion o p o eins ela ed o apop osis, oxida i e
s ess and in lamma ion, which may be implica ed in
RGC dea h a he ea ly s ages o glaucoma. Be e unde -
s anding he di e en ac o s sec e ed by subpopula-
ions o MG could iden i y po en ial he apeu ic a ge s
o enhance e inal neu op o ec ion and con i m he ole
o MG in he dea h o RGCs. We conclude ha MG a e
impo an senso s o p essu e changes in he eye, and
hey can in luence he su i al o hei neighbo ing RGCs
by sec e ing di e en p o eins.
Page 17 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
Abb e ia ions
RGCs Re inal ganglion cells
MG Mülle glia
HP High p essu e
IOP In aocula p essu e
FBS Fe al bo ine se um
NBA Neu obasal A
CM Condi ioned medium
PBS Phospha e bu e ed saline
BSA Bo ine se um albumin
DAPI 4′,6-Diamidino-2-phenylindole
αSMA α-Smoo h muscle ac in
OCT4 Oc ame -binding ansc ip ion ac o 4
p75NTR P75 neu o ophin ecep o
TRPV4 T ansien ecep o po en ial ca ion channel sub amily V membe 4
FASP Fil e aided sample p epa a ion
DTT DL-Di hio h ei ol
FA Fo maldehyde
FDR False de ec ion a e
STRING Sea ch ool o he e ie al o in e ac ing genes/p o eins
SEM S anda d e o o mean
DIV Days in i o
DEP Di e en ially exp essed p o ein
DKK Dickkop
RTK Recep o y osine kinase
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. 1186/ s13578- 023- 01186-1.
Addi ional ile1: Comple e p o eomic analysis and compa isons o Mül-
le glia Condi ioned Media (CM).
Acknowledgemen s
We would like o exp ess ou since e g a i ude o S e anie M. Hauck o hei
in aluable con ibu ion in he p elimina y analysis o he p o eomic s udy.
Au ho con ibu ions
Concep ualiza ion: XP, EV. Me hodology: XP, NR, MA, FE. In es iga ion: XP,
NR, MA, FE, AA, AFA, ARS, EV. Visualiza ion: XP, EV. Supe ision: EV, AFA, ARS.
W i ing—o iginal d a : XP. W i ing— e iew and edi ing: XP, NR, AA, AFA, ARS,
EV.
Funding
This esea ch was suppo ed by: G upos Consolidados Gobie no Vasco
IT1510-22 (EV), ELKARTEK KK-2019/00086 (EV), MINECO-Re os PID2019-
111139RB-I00 (EV), PIBA 2020_1_0026 (EV), Gobie no Vasco pos doc o al g an
POS_2022_2_0007 (XP). UPV/EHU pos doc o al g an (NR).
A ailabili y o da a and ma e ials
All da ase s gene a ed a e included in his a icle.
Decla a ions
E hics app o al and consen o pa icipa e
This s udy does no con ain any human da a. All he expe imen al p o ocols
complied wi h he Eu opean (2010/63/UE) and Spanish (RD53/2013)
egula ions o he p o ec ion o expe imen al animals, and hey we e
app o ed by he E hical Commi ee o Animal Wel a e a he Uni e si y o
Basque coun y.
Consen o publica ion
All au ho s ag eed o publish.
Compe ing in e es s
All au ho s decla e no compe ing in e es s.
Fig. 8 G aphical o e iew. Visual summa y illus a ing he key indings and main conclusions o he s udy
Page 18 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
Recei ed: 20 Sep embe 2023 Accep ed: 13 Decembe 2023
Re e ences
1. Goldbe g JL, Co edo RG. Re inal ganglion cell li e and dea h—
mechanism and implica ions o oph halmology. Eu Oph halmic Re .
2009;3:109–12.
2. Ou Y, Jo RE, Ullian EM, Wong RO, Della San ina L. Selec i e ulne abili y
o speci ic e inal ganglion cell ypes and synapses a e ansien ocula
hype ension. J Neu osci. 2016;36:9240–52.
3. Zhang N, He X, Xing Y, Yang N. Di e en ial suscep ibili y o e inal gan-
glion cell sub ypes agains neu odegene a i e diseases. G ae es A ch Clin
Exp Oph halmol. 2022;260:1807–21.
4. Vande Wall KB, Lu B, Al a o JS, Allsop AR, Ca AS, Wang S, Meye JS. Di e -
en ial suscep ibili y o e inal ganglion cell sub ypes in acu e and ch onic
models o inju y and disease. Sci Rep. 2020;10:17359.
5. Duan X, Qiao M, Bei F, Kim IJ, He Z, Sanes JR. Sub ype-speci ic egene a-
ion o e inal ganglion cells ollowing axo omy: e ec s o os eopon in
and mTOR signaling. Neu on. 2015;85:1244–56.
6. Ruiz-Ede a J, Ga cia M, He nandez M, U cola H, He nandez-Ba bachano E,
A aiz J, Vecino E. The pig eye as a no el model o glaucoma. Exp Eye Res.
2005;81:561–9.
7. Laquis S, Chaudha y P, Sha ma SC. The pa e ns o e inal ganglion cell
dea h in hype ensi e eyes. B ain Res. 1998;784:100–4.
8. Reichenbach A, B ingmann A. Glia o he human e ina. Glia.
2020;68:768–96.
9. B ingmann A, Iandie I, Pannicke T, Wu m A, Hollbo n M, Wiedemann P,
Osbo ne NN, Reichenbach A. Cellula signaling and ac o s in ol ed in
Mülle cell gliosis: neu op o ec i e and de imen al e ec s. P og Re in Eye
Res. 2009;28:423–51.
10. Ruza a N, Pe ei o X, Leppe MF, Hauck SM, Vecino E. A p o eomics
app oach o iden i y candida e p o eins sec e ed by mulle glia ha
p o ec ganglion cells in he e ina. P o eomics. 2018;18: e1700321.
11. Ga cia M, Fo s e V, Hicks D, Vecino E. E ec s o mulle glia on cell su i al
and neu i ogenesis in adul po cine e ina in i o. In es Oph halmol Vis
Sci. 2002;43:3735–43.
12. Pe ei o X, Mil ne AM, La To e A, Vecino E. E ec s o adul Mulle cells
and hei condi ioned media on he su i al o s em cell-de i ed e inal
ganglion cells. Cells. 2020;9:1759.
13. Rowan S, Cepko CL. Gene ic analysis o he homeodomain ansc ip ion
ac o Chx10 in he e ina using a no el mul i unc ional BAC ansgenic
mouse epo e . De Biol. 2004;271:388–402.
14. Robe ge FG, Caspi RR, Chan CC, Kuwaba a T, Nussenbla RB. Long- e m
cul u e o Mulle cells om adul a s in he p esence o ac i a ed lym-
phocy es/monocy es p oduc s. Cu Eye Res. 1985;4:975–82.
15. Reichenbach A. Quan i a i e and quali a i e mo phology o abbi e inal
glia. A ligh mic oscopical s udy on cells bo h in si u and isola ed by
papaine. J Hi n o sch. 1987;28:213–20.
16. Reichenbach A, B ingmann A. Re inal glia. San Ra ael: Mo gan & Claypool
Publishe s; 2015.
17. Fische AJ, Reh TA. Po en ial o Mulle glia o become neu ogenic e inal
p ogeni o cells. Glia. 2003;43:70–6.
18. P ada FA, Magalhaes MM, Coimb a A, Genis-Gal ez JM. Mo phological
di e en ia ion o he Mulle cell: Golgi and elec on mic oscopy s udy in
he chick e ina. J Mo phol. 1989;201:11–22.
19. P ada FA, Espina A, Chmielewski CE, Do ado ME, Genis-Gal ez JM.
Regional adap a ion o Mulle cells in he chick e ina. A Golgi and elec-
on mic oscopical s udy. His ol His opa hol. 1989;4:309–15.
20. Aneza y L, Medina JI, Sanchez-Noguei o J, Lopez-Galla do M, P ada C.
Shape di e si y among chick e ina Mulle cells and hei pos na al di -
e en ia ion. J Comp Neu ol. 2001;438:32–49.
21. Too LK, G acie G, Hasic E, Iwaku a JH, Che epano S. Adul human e inal
Mulle glia display dis inc pe iphe al and macula exp ession o CD117
and CD44 s em cell-associa ed p o eins. Ac a His ochem. 2017;119:142–9.
22. Suga A, Sadamo o K, Fujii M, Mandai M, Takahashi M. P oli e a ion po en-
ial o Mulle glia a e e inal damage a ies be ween mouse s ains. PLoS
ONE. 2014;9: e94556.
23. Kaplan L, D exle C, P alle AM, B enna S, Wunde lich KA, Dimi acopou-
los A, Me l-Pham J, Pe ez MT, Schlo ze -Sch eha d U, Enzmann V, e al.
Re inal egions shape human and mu ine Mulle cell p o eome p o ile
and unc ionali y. Glia. 2023;71:391–414.
24. Zhang T, Zhu L, Madigan MC, Liu W, Shen W, Che epano S, Zhou F,
Zeng S, Du J, Gillies MC. Human macula Mulle cells ely mo e on se ine
biosyn hesis o comba oxida i e s ess han hose om he pe iphe y.
Eli e. 2019;8: e43598.
25. Da uich A, Ma e A, Moulin A, Kowalczuk L, Nicolas M, Sellam A, Ro hs-
child PR, Om i S, Gelize E, Jone L, e al. Mechanisms o macula edema:
beyond he su ace. P og Re in Eye Res. 2018;63:20–68.
26. Fe nandez-Alba al JA, de Hoz R, Ma amo os JA, Chen L, Lopez-Cuenca
I, Salob a -Ga cia E, Sanchez-Puebla L, Rami ez JM, T i ino A, Salaza JJ,
Rami ez AI. Re inal changes in as ocy es and Mulle glia in a mouse
model o lase -induced glaucoma: a ime-cou se s udy. Biomedicines.
2022;10:939.
27. Lindq is N, Liu Q, Zajadacz J, F anze K, Reichenbach A. Re inal glial (Mul-
le ) cells: sensing and esponding o issue s e ch. In es Oph halmol Vis
Sci. 2010;51:1683–90.
28. Pe ei o X, Ruza a N, Ace a A, U cola A, Vecino E. Op imiza ion o a me hod
o isola e and cul u e adul po cine, a s and mice Mulle glia in o de o
s udy e inal diseases. F on Cell Neu osci. 2020;14:7.
29. Vecino E, Helle JP, Veiga-C espo P, Ma in KR, Fawce J. In luence
o di e en ex acellula ma ix componen s on he exp ession o
in eg ins and egene a ion o adul e inal ganglion cells. PLoS One.
2015;10(5):e0125250.
30. Madei a MH, Boia R, El as F, Ma ins T, Cunha RA, Amb osio AF, San iago
AR. Selec i e A2A ecep o an agonis p e en s mic oglia-media ed neu-
oin lamma ion and p o ec s e inal ganglion cells om high in aocula
p essu e-induced ansien ischemic inju y. T ansl Res. 2016;169:112–28.
31. Sapping on RM, Calkins DJ. P essu e-induced egula ion o IL-6 in e inal
glial cells: in ol emen o he ubiqui in/p o easome pa hway and NFkap-
paB. In es Oph halmol Vis Sci. 2006;47:3860–9.
32. Wisniewski JR, Zougman A, Naga aj N, Mann M. Uni e sal sample p epa-
a ion me hod o p o eome analysis. Na Me hods. 2009;6:359–62.
33. Hayashi H, Takagi N. Endogenous neu op o ec i e molecules and
hei mechanisms in he cen al ne ous sys em. Biol Pha m Bull.
2015;38:1104–8.
34. Newman E, Reichenbach A. The Mulle cell: a unc ional elemen o he
e ina. T ends Neu osci. 1996;19:307–12.
35. Ga ca M, Ruiz-Ede a J, He nandez-Ba bachano H, Vecino E. Topog aphy
o pig e inal ganglion cells. J Comp Neu ol. 2005;486:361–72.
36. Ga cia M, Fo s e V, Hicks D, Vecino E. In i o exp ession o neu o ophins
and neu o ophin ecep o s is conse ed in adul po cine e ina in i o.
In es Oph halmol Vis Sci. 2003;44:4532–41.
37. Pe ei o X, Ruza a N, Ace a A, Fonollosa A, Rod iguez FD, Vecino E. Dexa-
me hasone p o ec s e inal ganglion cells bu no Mulle glia agains
hype glycemia in i o. PLoS ONE. 2018;13: e0207913.
38. Ruza a N, Pe ei o X, Fonollosa A, A aiz J, Ace a A, Vecino E. Plasma ich
in g ow h ac o s (PRGF) inc eases he numbe o e inal Mulle glia in
cul u e bu no he su i al o e inal neu ons. F on Pha macol. 2021;12:
606275.
39. Vecino E, Ga cia-B inon J, Velasco A, Caminos E, La a J. Calbindin D-28K
dis ibu ion in he e ina o he de eloping ou (Salmo a io L.). Neu osci
Le . 1993;152:91–5.
40. Ma inez-Na a e e GC, Angulo A, Ma in-Nie o J, Cuenca N. G adual
mo phogenesis o e inal neu ons in he pe iphe al e inal ma gin o
adul monkeys and humans. J Comp Neu ol. 2008;511:557–80.
41. Hauck SM, Suppmann S, Ue ing M. P o eomic p o iling o p ima y e inal
Mulle glia cells e eals a shi in exp ession pa e ns upon adap a ion o
in i o condi ions. Glia. 2003;44:251–63.
42. Jiang SM, Zeng LP, Zeng JH, Tang L, Chen XM, Wei X. be a-III-Tubulin: a
eliable ma ke o e inal ganglion cell labeling in expe imen al models
o glaucoma. In J Oph halmol. 2015;8:643–52.
43. Da by I, Skalli O, Gabbiani G. Alpha-smoo h muscle ac in is ansien ly
exp essed by myo ib oblas s du ing expe imen al wound healing. Lab
In es . 1990;63:21–9.
44. Swain N, Thaku M, Pa hak J, Swain B. SOX2, OCT4 and NANOG: he co e
emb yonic s em cell plu ipo ency egula o s in o al ca cinogenesis. J O al
Maxillo ac Pa hol. 2020;24:368–73.
45. Liu L, Michowski W, Kolodziejczyk A, Sicinski P. The cell cycle in s em
cell p oli e a ion, plu ipo ency and di e en ia ion. Na Cell Biol.
2019;21:1060–7.
Page 19 o 19
Pe ei oe al. Cell & Bioscience (2024) 14:5
•
as , con enien online submission
•
ho ough pee e iew by expe ienced esea che s in you ield
•
apid publica ion on accep ance
•
suppo o esea ch da a, including la ge and complex da a ypes
•
gold Open Access which os e s wide collabo a ion and inc eased ci a ions
maximum isibili y o you esea ch: o e 100M websi e iews pe yea
•
A BMC, esea ch is always in p og ess.
Lea n mo e biomedcen al.com/submissions
Ready o submi you esea ch
Ready o submi you esea ch
? Choose BMC and bene i om:
? Choose BMC and bene i om:
46. Cui Y, Xu N, Xu W, Xu G. Mesenchymal s em cells a enua e hyd ogen
pe oxide-induced oxida i e s ess and enhance neu op o ec i e e ec s
in e inal ganglion cells. In Vi o Cell De Biol Anim. 2017;53:328–35.
47. Osbo ne A, Sande son J, Ma in KR. Neu op o ec i e e ec s o human
mesenchymal s em cells and pla ele -de i ed g ow h ac o on human
e inal ganglion cells. S em Cells. 2018;36:65–78.
48. Pan D, Chang X, Xu M, Zhang M, Zhang S, Wang Y, Luo X, Xu J, Yang X,
Sun X. UMSC-de i ed exosomes p omo e e inal ganglion cells su i al in
a a model o op ic ne e c ush. J Chem Neu oana . 2019;96:134–9.
49. Valen a T, Hausmann G, Basle K. The many aces and unc ions o be a-
ca enin. EMBO J. 2012;31:2714–36.
50. Liu H, Mohamed O, Du o D, Wallace VA. Cha ac e iza ion o Wn signal-
ing componen s and ac i a ion o he Wn canonical pa hway in he
mu ine e ina. De Dyn. 2003;227:323–34.
51. Pa el AK, Su apaneni K, Yi H, Nakamu a RE, Ka li SZ, Syeda S, Lee T,
Hackam AS. Ac i a ion o Wn /be a-ca enin signaling in Mulle glia p o-
ec s pho o ecep o s in a mouse model o inhe i ed e inal degene a ion.
Neu opha macology. 2015;91:1–12.
52. Sei z R, Hackl S, Seibuchne T, Tamm ER, Ohlmann A. No in media es
neu op o ec i e e ec s on e inal ganglion cells ia ac i a ion o he
Wn /be a-ca enin signaling pa hway and he induc ion o neu op o ec-
i e g ow h ac o s in Mulle cells. J Neu osci. 2010;30:5998–6010.
53. Musada GR, D o ian chiko a G, Mye C, I ano D, Bha acha ya SK,
Hackam AS. The e ec o ex insic Wn /be a-ca enin signaling in
Mulle glia on e inal ganglion cell neu i e g ow h. De Neu obiol.
2020;80:98–110.
54. Liang Y, Fan Y, Liu Y, Fan H. HNRNPU p omo es he p og ession o
hepa ocellula ca cinoma by enhancing CDK2 ansc ip ion. Exp Cell Res.
2021;409: 112898.
55. Kuai L, Peng J, Jiang Y, Zheng Z, Zhou X. Apolipop o ein E-mime ic
pep ide COG1410 enhances e inal ganglion cell su i al by a enua ing
in lamma ion and apop osis ollowing TONI. F on Neu osci. 2019;13:980.
56. Pe e ziel H, Sackmann T, S elau J, Kuhn PH, Lich en hale SF, Ma om
K, Kla A, Unsicke K. F-spondin egula es neu onal su i al h ough
ac i a ion o disabled-1 in he chicken cilia y ganglion. Mol Cell Neu osci.
2011;46:483–97.
57. Liu H, Bell K, He mann A, A nhold S, Me cieca K, Ande s F, Nagel-Wol um
K, Thanos S, P okosch V. C ys allins play a c ucial ole in glaucoma and
p omo e neu onal cell su i al in an in i o model h ough modula ing
Mulle cell sec e ion. In es Oph halmol Vis Sci. 2022;63:3.
58. Nakamu a RE, Hun e DD, Yi H, B unken WJ, Hackam AS. Iden i ica ion o
wo no el ac i i ies o he Wn signaling egula o Dickkop 3 and cha ac-
e iza ion o i s exp ession in he mouse e ina. BMC Cell Biol. 2007;8:52.
59. Ai es ID, Amb osio AF, San iago AR. Modeling human glaucoma: lessons
om he in i o models. Oph halmic Res. 2017;57:77–86.
60. Lee SJ, Duncan DS, Eche a ia FD, McLaughlin WM, Ha che JB, Sap-
ping on RM. P essu e-induced al e a ions in PEDF and PEDF-R exp ession:
implica ions o neu op o ec i e signaling in glaucoma. J Clin Exp Oph-
halmol. 2015;6:491.
61. Sapping on RM, Chan M, Calkins DJ. In e leukin-6 p o ec s e inal
ganglion cells om p essu e-induced dea h. In es Oph halmol Vis Sci.
2006;47:2932–42.
62. Jo AO, Lakk M, Rudzi is CN, K izaj D. TRPV4 and TRPC1 channels medi-
a e he esponse o ensile s ain in mouse Mulle cells. Cell Calcium.
2022;104: 102588.
63. F iemel T, G illo S, S ella SL J . Ac i a ion o piezo1 mechanosensi i e
channels in e inal ganglion cells leads o cell dea h. FASEB J. 2020;34:1–1.
64. Mo ozumi W, Inagaki S, Iwa a Y, Nakamu a S, Ha a H, Shimazawa M.
Piezo channel plays a pa in e inal ganglion cell damage. Exp Eye Res.
2020;191: 107900.
65. Ryskamp DA, Jo AO, F ye AM, Vazquez-Chona F, MacAulay N, Tho e-
son WB, K izaj D. Swelling and eicosanoid me aboli es di e en-
ially ga e TRPV4 channels in e inal neu ons and glia. J Neu osci.
2014;34:15689–700.
66. Ryskamp DA, Wi ko sky P, Ba abas P, Huang W, Koehle C, Akimo
NP, Lee SH, Chauhan S, Xing W, Ren e ia RC, e al. The polymodal ion
channel ansien ecep o po en ial anilloid 4 modula es calcium lux,
spiking a e, and apop osis o mouse e inal ganglion cells. J Neu osci.
2011;31:7089–101.
67. Li Q, Cheng Y, Zhang S, Sun X, Wu J. TRPV4-induced Mulle cell gliosis
and TNF-alpha ele a ion-media ed e inal ganglion cell apop osis in
glaucoma ous a s ia JAK2/STAT3/NF-kappaB pa hway. J Neu oin lamm.
2021;18:271.
68. Liu QY, Lei JX, LeBlanc J, Sodja C, Ly D, Cha lebois C, Walke PR, Yamada T,
Hi ohashi S, Siko ska M. Regula ion o DNaseY ac i i y by ac inin-alpha4
du ing apop osis. Cell Dea h Di e . 2004;11:645–54.
69. Gale NW, Yancopoulos GD. Eph ins and hei ecep o s: a epulsi e opic?
Cell Tissue Res. 1997;290:227–41.
70. Dohn M, Jiang J, Chen X. Recep o y osine kinase EphA2 is egula ed by
p53- amily p o eins and induces apop osis. Oncogene. 2001;20:6503–15.
71. Zhu C, Hu DL, Liu YQ, Zhang QJ, Chen FK, Kong XQ, Cao KJ, Zhang JS,
Qian LM. Fabp3 inhibi s p oli e a ion and p omo es apop osis o emb y-
onic myoca dial cells. Cell Biochem Biophys. 2011;60:259–66.
72. Ma suzawa A. Thio edoxin and edox signaling: oles o he hio edoxin
sys em in con ol o cell a e. A ch Biochem Biophys. 2017;617:101–5.
73. Bai Y, Zheng J, Cheng L, Liu Q, Zhao G, Li J, Gu Y, Xu W, Wang M, Wei Q,
Zhang R. Po en ially unc ional gene ic a ian s o VAV2 and PSMA4 in he
immune-ac i a ion pa hway and non-small cell lung cance su i al. J
Gene Med. 2022;24: e3447.
Publishe ’s No e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in
published maps and ins i u ional a ilia ions.
