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Scien i ic RepoR s | 7: 1597 | DOI:10.1038/s41598-017-01716-1
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Re inal Degene a ion In A Mouse
Model O CLN5 Disease Is Associa ed
Wi h Comp omised Au ophagy
Hen i Leinonen 1,4, Vel a Keksa-Golds eine1, Syman as Ragauskas2, Philip Kohlmann1,
Yaju inde Singh1, Eka e ina Sa chenko1, Jooseppi Pu anen2, Ta ja Malm1, Gied ius
Kalesnykas2,3, Ja i Kois inaho1, Heikki Tanila1 & Ka ja M. Kanninen1
The Finnish a ian o la e in an ile neu onal ce oid lipo uscinosis (CLN5 disease) belongs o a
amily o neu onal ce oid lipo uscinosis (NCLs) diseases. Vision loss is among he i s clinical signs
in childhood o ms o NCLs. Mu a ions in CLN5 unde lie CLN5 disease. The aim o his s udy was o
cha ac e ize how he lack o no mal unc ionali y o he CLN5 p o ein a ec s he mouse e ina. Sco opic
elec o e inog aphy (ERG) showed a diminished c-wa e ampli ude in he CLN5 de icien mice al eady a
1 mon h o age, indica i e o pa hological e en s in he e inal pigmen ed epi helium. A- and b-wa es
showed p og essi e impai men la e om 2 and 3 mon hs o age onwa ds, espec i ely. S uc u al and
immunohis ochemical (IHC) analyses showed p e e en ial damage o pho o ecep o s, accumula ion
o au o luo escen s o age ma e ial, apop osis o pho o ecep o s, and s ong in lamma ion in he
CLN5 de icien mice e inas. Inc eased le els o au ophagy-associa ed p o eins Beclin-1 and P62,
and inc eased LC3b-II/LC3b-I a io, we e de ec ed by Wes e n blo ing om whole e inal ex ac s.
Pho opic ERG, isual e oked po en ials, IHC and cell coun ing indica ed ela i ely long su i ing cone
pho o ecep o s compa ed o ods. In conclusion, CLN5 de icien mice de elop ea ly ision loss ha
e lec s he condi ion epo ed in clinical childhood o ms o NCLs. The ision loss in CLN5 de icien mice
is p ima ily caused by pho o ecep o degene a ion.
The neu onal ce oid lipo uscinoses (NCLs), also known as Ba en disease, a e he mos common neu odegen-
e a i e disease g oup among child en. The o e all incidence o NCLs in he USA is es ima ed o be 1:12 500,
and ce ain o ms o NCL a e ela i ely equen in No he n Eu ope1. As he NCLs a e di icul o diagnose he
incidence may be e en highe . This g oup o diso de s is associa ed wi h mu a ions in a leas 13 di e en genes2,
causa i e o se e al dis inc NCL disease sub ypes called he CLNs (CLN1-CLN8 and CLN10-CLN14). The cellu-
la localiza ion o all p o ein p oduc s o NCL associa ed genes is no well es ablished (e.g. CLN3), bu p incipally
ep esen soluble lysosomal enzymes, poly opic memb ane p o eins localized in lysosomes o in he endoplas-
mic e iculum (ER), o synap ic esicle associa ed p o eins3. Al hough he p o ein p oduc s ha e di e en ial
unc ions and in acellula loca ions, all sub ypes o NCL gene ally (i.e. childhood o ms), bu no en i ely (i.e.
adul hood o ms), lead o simila clinical symp oms: ision loss, mo o and men al de e io a ion, spon aneous
seizu es, and p ema u e dea h1. Vision loss is o en among he i s de ec able clinical signs in childhood o ms o
NCLs and oph halmological examina ions can be used o aid in he clinical diagnosis o he disease4. A cha ac-
e is ic pa hological ea u e o he NCLs is he accumula ion o au o luo escen s o age ma e ial, lipo uscin-like
ce oids, in o lysosomes5. Acco ding o cu en opinion, accumula ion o lysosomal s o age ma e ial may no be
he p ima y cause o NCL pa hology and he exac disease mechanisms emain un esol ed6. To da e, he e is no
d ug egula o y agency (e.g. FDA, EMA) app o ed disease modi ying pha maco he apies a ailable o NCLs7.
Howe e , se e al phase I-II clinical ials using gene, s em cell o enzyme eplacemen he apies a e ongoing (see
e.g. NCT00151216, NCT01414985, NCT02725580, NCT02678689, NCT01586455; ClinicalT ials.go ).
1A.I. Vi anen Ins i u e o Molecula Sciences, Uni e si y o Eas e n Finland, Kuopio, Finland. 2Expe imen ica
L d., Kuopio, Finland. 3Resea ch and De elopmen Cen e o Oph halmic Inno a ions (SILK), Depa men o
Oph halmology, Uni e si y o Tampe e, Tampe e, Finland. 4P esen add ess: Depa men o Pha macology, School
o Medicine, Case Wes e n Rese e Uni e si y, 10900 Euclid A enue, Cle eland, OH, 44106, USA. Co espondence
and eques s o ma e ials should be add essed o H.L. (email: hen [email protected]) o K.M.K. (email: ka ja.
kanninen@ue . i)
Recei ed: 25 Augus 2016
Accep ed: 4 Ap il 2017
Published: xx xx xxxx
OPEN
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Scien i ic RepoR s | 7: 1597 | DOI:10.1038/s41598-017-01716-1
Se e al mouse models ha e been de eloped ha ecapi ula e di e en o ms o human NCLs8. Wi h ega d
o he e ina, he majo i y o he mouse models es ed so a show pa hology p ima ily a he ou e e ina whe e
he pho o ecep o s eside9–13. CLN6 de icien mice display he mos obus e inal pheno ype wi hin he assessed
NCL mouse models o da e. In hese mice, he e inopa hy begins b ie ly a e eye opening and is cha ac e ized by
ea ly onse pho o ecep o dea h and e inal in lamma ion9, 12. In ano he NCL mouse model, Wa e-Shap on e
al. (2015) ecen ly showed de ec s in phagosome p ocessing and au ophagy in he e inal pigmen ed epi helium
(RPE)14. Se e al esea ch g oups ha e ea lie shown comp omised au ophagy in o he o gans o NCL mouse
models15–18. In addi ion, NCLs na u ally a ec some b eeds o sheep and dogs which ha e been ha nessed as la ge
animal models o NCLs19–24. Wi h espec o ision, Dalma ian dogs, English Se e s, Tibe an Te ie s and Bo de
Collies a ec ed wi h NCL do no well esemble childhood o m o NCLs as he p og ession o e inopa hy in
hese models is slow o e en absen 20–22. Ins ead, New Hampshi e Sheep, Polish Owcza ek Nizinny (PON) dogs
and Dachshunds dogs a ec ed wi h NCL do mani es a he s ong e inopa hy20, 23, 24. As in mice, also in o ine
and canine models o NCLs he pho o ecep o s seem o be p ima ily a ec ed20, 21, 24–26. Acco dingly, he e inal
degene a ion in mos o ms o human NCLs is belie ed o s a a he ou e segmen s o pho o ecep o s5.
Ca e ul cha ac e iza ion o e inopa hy in di e en NCL sub ypes is wa an ed since i may e en ually answe
he ques ion o whe he e inopa hy in se e al dis inc o ms o NCLs ha e a common mechanism, and whe he
his mechanism sha es a link o b ain pa hology in NCLs. Indeed, he supe io accessibili y o he e ina o
expe imen al modula ion and imaging makes he e ina an a ac i e window o he b ain27. P og essi e ision
loss has been desc ibed p e iously in CLN5 de icien mice based on a simple beha io al es 28. In his s udy, we
cha ac e ized he e inal pheno ype o CLN5 de icien mice ho oughly o he i s ime.
Resul s
Ea ly od-media ed unc ional de ici s in CLN5 de icien mouse e inas. Sco opic elec o e ino-
g am (ERG) a- and b-wa e ampli udes, depic ing od- unc ion, we e no mal a 1 mon h o age in CLN5 de icien
mice. Ins ead, he c-wa e ampli ude was al eady smalle in CLN5 de icien han in con ol mice (F1,72 = 64.61,
p < 0.001, Fig.1A,C). The a-wa e appea ed educed a 2 mon hs o age eaching s a is ical signi icance a 3
mon hs o age (F1,79 = 56.7, p < 0.001, Fig.1D). The dec ease in sco opic b-wa e ampli ude s a ed a 3 mon hs
o age (F6,235 = 34.9, p < 0.001; Fig.1B). B-wa e/a-wa e ampli ude a io ended o be highe in CLN5 de icien
mice o e he cou se o he s udy (F1,73 = 16.2, p < 0.001; Fig.1E). Re inal unc ion unde pho opic condi ions,
depic ing cone- unc ion, emained simila in con ol and CLN5 de icien mice o se e al mon hs, bu e en u-
ally also he pho opic ERG b-wa e ampli ude s a ed o decline in CLN5 de icien mice a he age o 5 mon hs
(Fig.1F,G), al hough he di e ence be ween g oups eached s a is ical signi icance only a 6 mon hs o age (in e -
ac ion: F5,79 = 6.3, p < 0.001). Co espondingly, medium- o-long (M/L) wa e leng h cone densi y and mac oscopic
appea ance o M/L cone ou e segmen s (COS) seemed no mal in CLN5 de icien mice a 3 mon hs o age, whe eas
a 6 mon hs he cone densi y s a ed o show a end owa ds decline and COS appea ed sh unken (F1,15 = 4.1,
p = 0.06; Supplemen a y Fig.1). Visual acui y as de e mined by he VEP echnique was simila be ween he geno-
ypes a 2 mon hs o age, bu showed a end owa ds decline in CLN5 de icien mice a 6 mon hs o age (in e ac ion:
F1,20 = 3.00, p = 0.10, Fig.1I). The VEP implici ime emained no mal in CLN5 de icien mice upon aging (quan i-
a i e da a no shown, bu see ep esen a i e a e aged VEP wa e o m in Fig.1H), indica ing uninju ed op ic ne es.
Ou e nuclea laye hickness ollows he ime sequence o od dys unc ion. Ana omical inspec-
ion o e inal sec ions con i med ha he ou e nuclea laye (ONL), whe e pho o ecep o s eside, was p ima ily
a ec ed in CLN5 de icien mice (Fig.2A). The ONL hickness appea ed educed in CLN5 de icien mice al eady
a he age o 1 mon h (mean di e ence be ween CLN5 de icien and con ol samples was 6.19 µm). A he age
o 3 mon hs, he ONL was d as ically hinned in CLN5 de icien compa ed o he con ol mice (F5,25 = 31.75,
p < 0.001; Fig.2A,B). The mo phology o pho o ecep o ou e segmen s (POS) in CLN5 de icien mice looked
ela i ely no mal un il 3 mon hs o age, bu was clea ly damaged a 6 mon hs and e en u he a 9 mon hs o
age. In addi ion, pho o ecep o inne segmen s we e clea ly damaged in 9-mon h-old CLN5 de icien mice. In
con as , he inne e inal compa men s, i.e. in e neu on and ganglion cell laye s, showed a no mal appea ance
a all age poin s s udied (Fig.2A). Howe e , inne nuclea laye (INL) also became hinne in CLN5 de icien mice
e en ually a he age o 6 mon hs (F5,24 = 4.96, p < 0.05; Fig.2B). Likewise, he inne plexi o m laye (IPL) showed
a slow p og essi e dec ease in CLN5 de icien mice (F5,23 = 2.88, p < 0.05).
Accumula ion o au o luo escen s o age ma ial, al e ed au ophagy, apop o ic pho o ecep o
cells, and s ong in lamma ion in CLN5 de icien mouse e inas. NCLs a e cha ac e ized by accu-
mula ion o au o luo escen s o age ma e ial. We inspec ed e inal c oss-sec ions and whole moun s by luo es-
cence mic oscope and obse ed inc ease in au o luo escence (AF) in CLN5 de icien e inas compa ed o con ols
(Fig.3 and Supplemen a y Fig.2). Di use AF seemed inc eased h oughou he CLN5 de icien e inas (Fig.3).
AF agg ega es accumula ed as agg ega es pa icula ly in o he ou e plexi o m laye (Supplemen a y Fig.2).
By Wes e n blo ing we de ec ed ma kedly inc eased le els o au ophagy-associa ed p o eins Beclin1 and P62
in 6-mon h-old CLN5 de icien mouse e inas ela i e o sex and age-ma ched con ol e inas (Beclin-1, 1.6- old
inc ease, 14 = 4.98, p < 0.001; P62, 2.3- old inc ease, 14 = 5.71, p < 0.001; Fig.4). Con e sion o LC3b-I o
LC3b-II was also inc eased in CLN5 de icien e inal samples (LC3b-II/LC3b-I a io, 1.3- old inc ease, 18 = 2.11,
p < 0.05). The le el o he lysosomal memb ane p o ein LAMP1 was educed in CLN5 de icien e inal samples
when compa ed o con ols (LAMP1, 0.6- old educ ion, 14 = 3.47, p < 0.01).
Apop o ic cells we e abundan in he ONL in CLN5 de icien mice al eady a 1-mon h o age (Fig.5). The el-
a i e coun o TUNEL posi i e cells appea ed simila in all ages in es iga ed (1–9 mon hs) when he p og essi e
loss o pho o ecep o s was aken in o accoun (Supplemen a y Fig.3). The TUNEL posi i e cells we e i ually
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Scien i ic RepoR s | 7: 1597 | DOI:10.1038/s41598-017-01716-1
es ic ed o he ONL. No posi i e TUNEL cells we e seen in he ONL o con ol mouse samples a any age; how-
e e , he e we e some spo adic TUNEL cells in he RGCL (da a no shown).
We de ec ed signs o mic oglia in il a ion and ac i a ion in he CLN5 de icien e inal whole moun sam-
ples labeled wi h an i-Iba1 and an i-CD68 an ibodies al eady a he age o 1 mon h (Fig.6). CD68 is a ma ke
o ac i a ed mic oglia, whe eas Iba1 can be used o de ec bo h es ing and ac i a ed mic oglia29. Iba1 and
CD68-posi i e cell numbe s seemed o inc ease concomi an ly wi h inc eased mic oglial cell body size. The
appea ance o he mic oglia began o be less b anched and mo e amoeboid (see 63 x images in Fig.6). Posi i e
CD68 s aining co-localized wi h he mic oglia cell body and he s aining appea ed mo e in ense in cells ha ing
amoeboid mo phology. The change o mic oglia cell mo phology om ami ied o amoeboid o m seemed o be
p og essi e wi h age in CLN5 de icien mice (Fig.6, igh -down co ne ). Few mic oglia wi h amoeboid appea -
ance we e de ec ed in con ol samples (Fig.6).
Glial ib illa y acidic p o ein (GFAP) labeling in he e ina e eals as ocy es and ac i a ed Mülle glia cells. In
CLN5 de icien e inal c oss-sec ions, he GFAP s aining was up egula ed bo h in e inal as ocy es and Mülle
cells al eady a he age o 1 mon h (Fig.7). The in ensi y o GFAP s aining con inued o inc ease un il 3 mon hs o
age as shown bo h in e inal slice samples and whole moun s.
Discussion
The i s symp oms o CLN5 disease a e usually mo o clumsiness and isual impai men 28, ye p ecise expe i-
men al da a cha ac e izing he e inopa hy in CLN5 disease a e lacking. Howe e , oph halmological examina ion
has e ealed signs o macula dys ophy and op ic ne e a ophy30. In addi ion, he ERG esponse is known o
become abno mal a an ea ly age, and unde ec able by he age o 7–9.5 yea s31. In CLN5 de icien mice, abno mal
accumula ion o au o luo escen s o age ma e ial in o he e inas has been epo ed, as well as isual impai men
by a simple beha io al es 28. In his s udy, we ho oughly cha ac e ized he e inopa hy in CLN5 de icien mice.
Figu e 1. P og essi e decline in e inal unc ion and co ical isual acui y in CLN5 de icien mice. (A) g oup
a e aged band- il e ed (70–150 Hz) sco opic ERG wa e o ms (a log 0) in 1-mon h-old con ol and CLN5
de icien mice (g oups sizes: con ol 1 mon h, n = 12; con ol 2 mon h, n = 10, con ol 3–6 mon h, n = 6;
CLN5 1 mon h, n = 9; CLN5 2–6 mon h, n = 6–8). (B) sco opic b-wa e ampli udes o e age. No e ha all
age poin s a e no shown o cla i y o p esen a ion. The maximum s imulus in ensi y (log 0) yielded ~4500
pho oisome iza ions pe od. (C,D) sco opic c- and a-wa e ampli udes (a log 0) in con ol and CLN5 de icien
mice o e age, espec i ely. (E) sco opic b-wa e/a-wa e ampli ude a io a log 0 lash. (F) g oup a e aged band-
il e ed (70–150 Hz) pho opic ERG wa e o ms (a log 0) in 1-mon h-old con ol and 1-mon h, 2-mon h and
6-mon h-old CLN5 de icien mice. (G) pho opic b-wa e ampli udes (a log 0) in con ol and CLN5 de icien
mice o e age. (H) Rep esen a i e g oup a e aged VEP wa e o ms in esponse o middle size (0.192 CPD)
pa e n s imulus in 6-mon h-old mice. (I) Co ical isual acui y as de e mined by pa e n VEPs (n = 11 pe
geno ype). Bon e oni mul iple compa ison es : *p < 0.05, **p < 0.01, ***p < 0.001.
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Scien i ic RepoR s | 7: 1597 | DOI:10.1038/s41598-017-01716-1
In measu emen s o e inal unc ion, we ound ha he sco opic c-wa e o ERG was selec i ely dimin-
ished in ampli ude in CLN5 de icien mice al eady a 1 mon h o age. The ERG c-wa e is la gely gene a ed by a
ligh -induced decline in sub e inal po assium ion concen a ion and subsequen hype pola iza ion o he api-
cal memb ane o he RPE32. I has been p oposed ha pa hological changes in he RPE can be in es iga ed by
eco ding he c-wa e33–35. Howe e , he diagnos ic use ulness o c-wa e emains ques ionable as e lec ed by
i s ela i ely low u ili y. Mo eo e , eco ding o he c-wa e is echnically challenging and o en leads o a iable
esul s, as was he case in he p esen s udy. Rega dless o he p oblems ela ed o he c-wa e analysis, ou da a
oge he wi h p e ious da a om canine and o ine models o NCL36–38 indica e ha he c-wa e o ERG migh
be he mos sensi i e ERG componen in de ec ing he i s signs o NCL ela ed e inal changes. I emains an
open ques ion, howe e , whe he s uc u al changes in he RPE causes hese de ec s. In ou analysis, he c-wa e
ampli ude ended o dec ease only diminu i ely wi h age in CLN5 de icien mice, and his end owa ds decline
could be a ibu ed o p og essi e impai men in od pho o ecep o unc ion35. Mo eo e , he appea ance o RPE
seemed no mal a ligh mic oscope le el in he ea ly s ages o he disease. Ne e heless, he c-wa e could ha e
some diagnos ic u ili y in NCLs.
Func ional changes a ising di ec ly om he neu onal e ina occu ed la e in he disease p og ession han
he obse ed change in he c-wa e ampli ude. We obse ed a end owa ds decline in od-dominan , sco opic
a- and b-wa e ampli udes a 2 mon hs o age and he geno ype di e ence became signi ican a 3 mon hs o age.
The ERG a- and wa e e lec he ac i i y o pho o ecep o s and bipola cells, espec i ely39, 40. B-wa e/a-wa e
ampli ude a io was highe in CLN5 de icien mice compa ed o con ol mice o e he cou se o he s udy indica -
ing ha he pa hology impai ed he unc ion o pho o ecep o s bu no inne e inal cells41. In ac , he inc ease in
b-wa e/a-wa e ampli ude implies ha he inne e ina inc eased i s sensi i i y in esponse o a loss o inpu om
pho o ecep o s. Simila sensi i i y inc ease in he inne e ina has been obse ed in mild- o-in e media e le el
Figu e 2. Robus pho o ecep o dea h in CLN5 de icien mice. (A) Rep esen a i e H&E s ained e inal sec ions.
A 1 mon h o age CLN5 de icien e ina ep esen s comple ely no mal mo phology. Also a 3 mon h o age he
CLN5 de icien e ina has no mal appea ance bu he ONL is d as ically hinned. The pa hology p og essed so
ha he POS s a ed o become diso ganized and disappea ed, and a he la e s a e o disease a 9 mon hs o age
e en PIS died ou . The inne e inal compa men s emained no mal appea ance o e he cou se o he ollow
up. (B) S a is ical analysis o ONL, INL and IPL hicknesses (con ol 1 mon h, n = 4–5; con ol 9 mon h, n = 4;
CLN5 1 mon h, n = 4; CLN5 3 mon h, n = 5; CLN5 6 mon h, n = 6–7; CLN5 9 mon h, n = 6) showed ha ONL
was p ima ily a ec ed in CLN5 de icien mice e inas wi h ea ly onse whe eas INL and IPL showed slowly
p og essi e hinning. Tukey’s pos hoc es : *p < 0.05, **p < 0.01, ***p < 0.001. RGC, e inal ganglion cells;
IPL, inne plexi o m laye ; INL, inne nuclea laye ; OPL, ou e plexi o m laye ; ONL, ou e nuclea laye ; PIS,
pho o ecep o inne segmen s; POS, pho o ecep o ou e segmen s.
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Scien i ic RepoR s | 7: 1597 | DOI:10.1038/s41598-017-01716-1
pho o ecep o degene a ions induced by b igh ligh exposu e in mice and a s41–43. The inc ease in inne e ina
sensi i i y migh esul om synap ic s eng hening be ween he emaining pho o ecep o s and inne e inal
cells42. Howe e , i is well es ablished ha he inne e ina unde goes nega i e emodeling in ad anced pho o-
ecep o degene a ions, which may lead o deple ion o RGCL and INL neu ons among se e al o he pa holog-
ical changes44, 45. Indeed, we obse ed a s a is ically signi ican hinning o INL and IPL in CLN5 de icien mice
occu ing in an ad anced s age o e inopa hy. Howe e , his change was minuscule compa ed o he a e o
pho o ecep o s. I is no able he e ha al hough AF s o age ma e ial accumula es in many pa s o he e ina28 only
pho o ecep o s expe ienced p onounced des uc ion. Indeed, he causali y o AF s o age ma e ial localiza ion
and issue damage ha e been i ually con i med be inconsequen ial in NCLs6.
De e io a ion o cone s uc u e and unc ion occu ed se e al mon hs la e han od unc ion and hinning o
he ONL. No ably, whe eas 97% o he mouse pho o ecep o s a e ods and he cones comp ise only 3%29, hinning
Figu e 3. Inc eased au o luo escence (AF) in CLN5 de icien mouse e inas. Images we e cap u ed wi h
a luo escen mic oscope using au o-exposu e. CLN5 de icien e inas seem o display s onge di use AF
h oughou he e ina as compa ed o wild- ype con ol. AF agg ega es s a ed o appea om 3 mon h o age
onwa ds pa icula ly in o he OPL (a ows), which is mo e ob ious in Supplemen a y Fig.2. RGCL, e inal
ganglion cell laye ; IPL, inne plexi o m laye ; INL, inne nuclea laye ; OPL, ou e plexi o m laye ; ONL, ou e
nuclea laye ; PIS, pho o ecep o inne segmen s; POS, pho o ecep o ou e segmen s; RPE, e inal pigmen ed
epi helium.
Figu e 4. Al e ed au ophagy in 6-mon h-old CLN5 de icien mouse e inas. (A) Rep esen a i e immunoblo s.
(B) LC3-I o LC-II con e sion was inc eased 1.3- old in CLN5 de icien e inas (n = 10 pe geno ype). (C,D)
P62 le el was inc eased 2.3- old, and Beclin-1 le el 1.6- old in CLN5 de icien e inas (n = 7 and 8 pe geno ype,
espec i ely). (E) LAMP1 le el was dec eased in CLN5 de icien e inas and was 0.6- old lowe when compa ed
o con ol le els (n = 8 pe geno ype). The p o ein le el a ios in CLN5 de icien samples we e compa ed o
no malized con ol alues in all analyses (B–E). *p < 0.05, **p < 0.01, p < 0.001; - es .

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o ONL esul s om loss o od nuclei a he han cone nuclei. In ag eemen wi h he pho opic ERG esponse, he
isual acui y de i ed om pa e n VEP esponses in pho opic condi ions was compa able be ween CLN5 de i-
cien and con ol mice a 2 mon hs o age, bu seemed declined in CLN5 de icien mice a 6 mon hs o age. This
is somewha consis en wi h beha io ally assessed isual decline in CLN5 de icien mice ha s a ed a a ound
4–5 mon hs o age28. Mo eo e , on Schan z e al. showed ma ked neu onal loss in he p ima y isual co ex in
CLN5 de icien mice a 4 mon h o age46. Howe e , as he p og ession o decline in pho opic isual acui y was
Figu e 5. Apop o ic pho o ecep o nuclei in CLN5 de icien mice e inas. Rep esen a i e luo escen z-s ack
images o TUNEL s aining (g een) wi h DAPI coun e s ain (blue, cell nuclei). A 1 mon h o age apop o ic cells
we e equen in CLN5 de icien mice samples bu he amoun dec eased wi h diminishing amoun o su i ing
pho o ecep o s (see Supplemen a y Fig.3). Apop o ic cells we e es ic ed o he ONL. RGCL, e inal gangion
cell laye ; INL, inne nuclea laye ; ONL, ou e nuclea laye .
Figu e 6. Ea ly mic oglial in il a ion and ac i a ion in CLN5 de icien mice e inas. Layou o ep esen a i e
z-s ack images aken a he icini y o ONL a e shown o con ol and di e en aged CLN5 de icien e inal
whole moun samples ( ows, ho izon al). CD68 ( ed, mac ophage ma ke ) and Iba1 posi i e (g een, mic oglia
ma ke ) cells a e shown wi h wo di e en magni ica ions e ically in columns. Al eady a 1 mon h o age he
CLN5 de icien e inas showed ema kably inc eased CD68 and Iba1 posi i i y and mic oglia cell mo phology
was changing om ami ied o m (inac i e mic oglia) o a mo e amoeboid (ac i e mic oglia) o m. This
pa hological p ocess p og essed so ha a 9 mon h o age e y ew Iba1 posi i e cells had ami ied appea ance.
CD68 posi i i y was localized o he mic oglial cell body. Down- igh co ne : amoeboid – ami ied cell
mo phology a io was es ima ed by manual coun ing om Iba1 posi i e cells a 8 e inal loca ions and a e aged
o illus a ion.
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modes in ou s udy, and he la ency o he VEP esponse emained no mal, he ision loss in CLN5 de icien
mice p obably has a pu ely e inal o igin. Howe e , neu onal loss in he p ima y isual co ex o CLN5 de icien
mice as shown by Von Schan z e al.46 may be in pa explained by dec eased inpu om e inal od pa hway ha
s a s a a younge age.
The p ima y damage o ods o e cones is common o e ini is pigmen osa, age- ela ed macula degene a ion
(AMD), and ligh -induced oxici y47, 48. Rods a e mo e sensi i e o ligh -induced damage han cones in pa due
o g ea e accumula ion o a oxic isual cycle-associa ed byp oduc , all- ans- e inal, in ods48. In addi ion, he
unc ion o ods depends en i ely on he ou pu o 11-cis- e inal om he canonical isual cycle whe e pho o e-
cep o s and RPE in e play49. Cone pho o ecep o s also use 11-cis- e inal om he ‘ od cycle’ bu may also u ilize
ano he RPE independen , ye poo ly known, Mülle cell-based ‘cone cycle’. Mo eo e , a ecen s udy showed
ha li ing ods a he han cones a e p e e ably phagocy ized by ac i a ed mic oglia in e inal degene a ion50.
Collec i ely, ou unc ional assessmen sugges ed ha he RPE- od pho o ecep o in e ace is he i s e inal
egion a ec ed in he CLN5 de icien mice e inas. This is ollowed by loss o od pho o ecep o nuclei, whe eas
cones, inne e inal neu ons and pho opic ision seem o be ela i ely long p ese ed. Co espondingly, p e e en-
ial loss o signal om ods o e cones has been de ec ed in Sou h Hampshi e sheep, Tibe an Te ie dogs, PON
dogs and CLN7 mouse model o NCLs21, 51, 52, well as in pa ien s wi h ju enile ype o NCLs4, 53.
The RPE mus phagocy ose 10% o he ou e ips o POS e e y day o enewal, ende ing e inal homeos asis
ex emely sensi i e o insul s in RPE’s phagocy ic and au ophagic capaci y. POS enewal also main ains he isual
cycle ( egene a ion o isual pigmen s) h ough a noncanonical o m o au ophagy54. A de ec in any phase o
he isual cycle can cause e inal degene a ion55. Wa e-Shap on and cowo ke s ecen ly showed de ec s in pho-
o ecep o phagosome p ocessing and au ophagy in he RPE o a mouse model o CLN3 disease14. They ound
ha he deg ada i e compa men s o au ophagy and phagocy osis pa hways could o m no mally in Cln3del aex1-6
mouse RPE, bu he e minal phase o au ophagic/phagocy ic p ocessing; lysosome usion-media ed deg ada-
ion and ecycling o au ophagosome and phagosome con en s, was signi ican ly impai ed. E en mo e ecen ly,
B andens ein e al. ound inc eased exp ession o LC3-II and P62 posi i e agg ega es, as well as inc eased exp es-
sion o lysosomal ca hepsins in mic oglia and neu ons om CLN7 de icien mice b ains, sugges ing impai ed lys-
osomal unc ion and au ophagy15. Fu he mo e, Thelen e al. showed inc eased le els o LC3-II and P62 in b ain
ex ac s o CLN6 de icien mice18. In line wi h he p e ious esul s, we de ec ed inc eased le els o Beclin-1 and
P62 p o eins in he e inal ex ac s o adul CLN5 de icien mice by Wes e n blo ing. Beclin-1 is c ucial o o -
ma ion o au ophagosomes and P62 ma ks poly-ubiqui ina ed p o eins o selec i e au ophagy56, 57. We also ound
a sligh inc ease in LC3II/LC3I a io in CLN5 de icien e inas, which may be a ibu able o inc eased au ophago-
some con e sion om i s cy osolic o m o he memb ane-bound o m58. Ins ead, he le el o lysosome-associa ed
memb ane-p o ein LAMP1, which is essen ial o phagosome-lysosome usion59, was dec eased. A p obable
explana ion o he inc eased le els o Beclin-1 and P62 p o eins, and LC-I o LC-II con e sion, is a compensa-
o y e ec in esponse o a blockage in he las p ocessing s ep o au ophagy, he au ophagosome-lysosome usion.
Howe e , since ou analysis was pe o med om 6-mon h-old neu al e inas, he analysis is only a sugges i e
su oga e ma ke o wha migh be ongoing in he RPE in he ea ly disease p og ess. The de ec in RPE phagocy-
osis would be bes in es iga ed by elec on mic oscopy om hin e inal sec ions, o al e na i ely om RPE cell
cul u e by Wes e n blo ing.
Simila ly o CLN6, he unc ion o he CLN5 p o ein s ill emains unknown o da e. Howe e , i has been
shown ha CLN5 is a soluble p o ein localizing o he lysosomal compa men in wild- ype samples in i o60,
whe eas CLN6 is a ansmemb ane endoplasmic e iculum (ER) p o ein2. Al hough CLN6 is e ained in he ER
Figu e 7. Mülle cell gliosis and as ogliosis in CLN5 de icien mice e inas. Uppe ow: ep esen a i e
luo escen z-s ack images a e shown om an i-GFAP (g een, mac oglia) s aining wi h a DAPI coun e s ain
(blue, cell nuclei). As ocy es localize a he e inal ne e ibe laye , abo e he RGCL, ho izon ally and a e
exp essing GFAP li le in con ol samples. Mülle cells a e e ically o ien ed in he e ina and do no exp ess
GFAP once inac i a ed. Al eady a 1 mon h o age, as ocy es and Mülle cells we e s ongly ac i a ed in CLN5
de icien mice as shown by in ense GFAP posi i i y. GFAP posi i e s aining inc eased un il 3 mon h o age.
Lowe ow: an i-GFAP s aining o e inal whole moun s. RGCL, e inal ganglion cell laye ; INL, inne nuclea
laye ; ONL, ou e nuclea laye ; GFAP, glial ib illa y acidid p o ein.
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and does no co-localize wi h lysosomal ma ke s, lysosomal deg ada ion is d as ically a ec ed in CLN6 de icien
cell lines61. Co espondingly, mu a ions in he CLN5 gene ha e been sugges ed o ha e an e ec on lysosomal
unc ion62. In e es ingly, he e inal pheno ype o CLN5 de icien mice cha ac e ized he e closely esembles ha
obse ed by o he s in CLN6 de icien mice. Ba sch e al. epo ed ha ONL hickness was no mal a eye opening
bu signi ican ly declined al eady a 1 mon h o age in CLN6 de icien mice9. The loss o ONL nuclei was accom-
panied by s ong mic oglial in il a ion and ac i a ion, as ogliosis, and Mülle cell gliosis om 1 mon h o age
onwa ds in CLN6 de icien mice e inas9, 12. Ins ead, a wo weeks o age, glial ac i a ion was no obse ed9. As
he mouse eye opening occu s a 14–15 days pos na al63, he onse o isual pa hology in CLN6 de icien mice
may coincide wi h he ac i a ion o he isual cycle. We ound clea signs o mic oglia in il a ion and ac i a ion,
Mülle cell gliosis, as ogliosis and apop o ic pho o ecep o nuclei in CLN5 de icien mice e inas al eady a
he i s ime poin s udied, a he age o 1 mon h, al hough pho o ecep o unc ion was no ye diminished as
assessed by ERG.
In conclusion, ision loss in CLN5 de icien mice seems o be p ima ily caused by pa hological e en s in he
RPE- od pho o ecep o in e ace, and subsequen dea h o od pho o ecep o s. In addi ion o he CLN5 mouse
model, indings om se e al o he animal models o NCLs imply a simila pa e n o pa hological e en s in he
e ina12, 21, 51, 52. We specula e ha he e inopa hy in se e al o ms o NCLs may be caused by ailu e o lysosomes
o deg ade phagosomes and au ophagosomes. This specula ion is based on p e ious epo s by o he s p oposing
de ec i e phagosome/au ophagosome-lysosome usion aking place in a ious issues o NCL animal models,
cell cul u es o human pa ien s14–18, and ou en a i e da a indica ing al e ed au ophagy in he CLN5 de icien
mouse e inas. The de ec in phagocy osis/au ophagy could be di ec ly caused by a c ucial NCL-associa ed p o-
ein dys unc ion, o by p o ein-p o ein in e ac ions. O no e, dis inc CLN p o eins may exe some o hei
ac ions h ough mu ual in e ac ions; o ins ance, CLN5 has been shown o in e ac wi h PPT1 (CLN1/CLN4),
TPP1 (CLN2), CLN3, CLN6 and CLN8 p o eins64, 65. F om a he apeu ic poin o iew, i seems ha ea men s
ha e ain he no mal unc ionali y o CLN p o eins by gene o s em cell he apies, o in some cases by enzyme
eplacemen he apy (e.g. CLN2), may be he bes s a egy o ea NCLs bo h in he b ain and he e ina7, 66.
Howe e , since NCLs also a ec pe iphe al o gans nega i ely, sys emic deli e y o he apeu ics ha c oss he
blood-b ain and blood- e ina ba ie would be op imal and wo h explo ing67. Finally, he s udy o he eye in
NCLs is pi o al no only om an oph halmological poin o iew – unde s anding he mechanisms o e inopa hy
in NCLs could help o disen angle he pa hological basis o hese de as a ing diso de s in he es o body.
Ma e ial and Me hods
Animals. We u ilized he CLN5 mouse model o la e in an ile Finnish a ian o NCL gene a ed o iginally
in Uni e si y o Helsinki28. The mouse model has been gene a ed by dis up ing exon 3 o he mouse Cln5 gene,
which esul s in a ame shi and p ema u e s op codon in exon 4 o mouse Cln5. Bo h male (CLN5, n = 24;
con ol, n = 26, in o al) and emale mice (CLN5, n = 22; con ol, n = 22, in o al) we e used. Age ma ched wild-
ype mice we e used as con ols. All he mice we e main ained on he C57BL/6JRccHsd backg ound. Animals
we e g oup-housed a 55% humidi y, 22 °C empe a u e and wi h a 12 h / 12 h da k-ligh cycle (ligh s on 7 a.m o
7 p.m). Food and wa e we e p o ided ad libi um. All expe imen s we e conduc ed in acco dance o Associa ion
o Resea ch in Vision and Oph halmology s a emen o he use o animals in esea ch and he EU di ec i e
2010/63/EU o animal well-being, using p o ocols app o ed and moni o ed by he Animal Expe imen Boa d o
Finland (ESAVI-2013-002477).
Elec o e inog aphy (ERG). The ERG me hodology ha e been desc ibed in de ail p e iously68. The ani-
mals we e da k-adap ed o e nigh o a leas 12 h, all handling o ERG eco dings we e pe o med unde dim
ed ligh , and mice we e u he da k-adap ed in comple ely da k oom o 7 min be o e s imula ion. The ligh
s imula ion in sco opic condi ions ( od-pa hway) was done in ascending se ies in espec o s imulus in en-
si y (−4.07, −3.23, −2.41, −0.90 and 0 log ela i e o max in ensi y), and he in e -s imulus in e al (ISI) was
inc eased acco dingly (5 s, 10 s, 10 s, 20 s, 50 s, espec i ely). The maximum lash yielded ~4500 pho oisome iza-
ions (*R) pe mouse od (calib a ion desc ibed in e . 69). A e sco opic eco dings a moni o in a aday cage
was u ned on o e lec g ey ligh , a 17 lux, o he animal. A e 10 min o ligh -adap a ion pho opic esponses
(cone-pa hway) we e eco ded o a single ligh -in ensi y (log 0, 2 s ISI). Ampli ude pa ame e s o sco opic a-,
b- and c-wa es, and pho opic b-wa e we e analyzed in his s udy. The baseline o ERG esponse was aken as he
a e age ampli ude be ween −100 o 0 ms be o e he s imulus onse . The a-wa e ampli ude was calcula ed om
baseline o he ough o he i s nega i e de lec ion a e he s imulus onse . The b-wa e was calcula ed om
he a-wa e ough o he peak o he i s majo posi i e wa e. The c-wa e ampli ude was calcula ed be ween he
maximum nega i i y be ween b- and c-wa es ( ail o PIII componen in o he wo ds) o he maximum peak o he
c-wa e. Dis inc coho s o animals we e es ed o un ela ed easons a 1 mon hs o age and 2–6 mon hs o age.
Visual e oked po en ials (VEPs). The VEP expe imen was pe o med in awake, head- es ained animals
ollowing me hods desc ibed ea lie 70. The expe imen s a ed wi h elec ode assembly and head-s age implan-
a ion unde iso lu ane anes hesia when animals we e 2 mon hs o age. S e eo axical coo dina es o he sc ew
elec odes abo e he p ima y isual co ex (V1) we e −3.8 mm an e iopos e io ly and −2.5 mm mediola e ally
om b egma. Two sc ews we e a ached on o he on al bone o se e as e e ence and g ound elec odes. VEP
s imuli consis ed o e ical sinusoidal g a ings displayed on a gamma-co ec ed moni o sc een wi h e e sing
con as a 1 Hz. Sweep VEP echnique was used o de e mine he isual acui y o mice ( echnique e iewed in e .
71). The VEP esponses we e eco ded a 0.024, 0.048, 0.096, 0.192, 0.384, 0.48 and 0.576 cycles pe deg ee (CPD)
o isual angle. The pa e n-VEP wa e o m comp ised o a majo posi i e wa e occu ing a a ound 55–80 ms
om he s imulus onse ollowed by a nega i e de lec ion peaking a a ound 120–170 ms (see Fig.1H). The VEP
ampli ude was calcula ed as he di e ence be ween he majo posi i e (P1) and he majo nega i e (N2) wa e. I
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he nega i e wa e was no p onounced, he VEP was calcula ed om he peak o he posi i e wa e o he base-
line. Visual acui y o each animal was de e mined by plo ing a linea eg ession line h ough he ampli udes
a di e en CPDs and ex apola ing he ol age o 0 V. The VEPs we e eco ded a 2 and 6 mon hs o age. To be
p ecise, he i s eco ding ook place a 9 weeks o age due o eco e y om su ge y bu is displayed as 2-mon h
o p esen a ion cla i y and consis ency.
Tissue collec ion. Re inal samples we e collec ed om mice pe used wi h hepa inized saline. The mice
we e deeply anes he ized by an o e dose injec ion o A e in ( ib omoe hanol 625 mg/ml in 40% e amylalco-
hol; 0.02 ml/g, i.p.) and pe used h ough he le en icle o 3 min a 20 ml/min p io o he eyes being enucle-
a ed. Fo immunohis ochemis y, one eye om each mouse was ixed in 4% pa a o maldehyde (PFA) o e nigh ,
washed in phospha e bu e ed saline (PBS, 0.1 M, pH 7.4) o e nigh , and inally embedded in o pa a in blocks.
The second eye was ixed in 4% PFA o 3 h. The co nea was cu away ollowing he o ien a ion o o a se a a and
he lense was emo ed om he eye cup. The e ina was dissec ed om he eyecup and p epa ed as whole moun
sample. Fo Wes e n blo analyses, he eyes we e quickly enuclea ed a e pe usion and placed on a pe i dish in o
ice-cold PBS d ople s. The e ina was dissec ed ou as desc ibed abo e. The esh e inal samples we e placed in
Eppendo ubes, snap- ozen wi h liquid ni ogen and s o ed a −70 °C un il u he analysis.
Wes e n blo . P o ein concen a ion in e inal issue homogena es collec ed om 6-mon h old mice was
de e mined wi h Pie ce BCA P o ein assay ki (The mo Ca , No. 23225). Equal amoun s o p o ein we e loaded on
10% SDS-PAGE gels and sepa a ed using a Mini-P o ean 3 (Bio-Rad) de ice a 200 cons an ol age. Fo immu-
noblo ing, p o eins we e ans e ed on o Hybond P memb ane (GE Heal hca e) in a Mini T ansBlo (Bio-Rad)
chambe . Memb anes we e blocked in 5% skimmed milk solu ion in PBS (con aining Tween) and incuba ed
wi h p ima y an ibodies agains P62, Beclin-1, LC3b, LAMP1 (see Table1 o spesi ica ions) and β-ac in (mouse
monoclonal, 1:5000 dilu ion, Sigma, S . Louis, MO). The blo s we e de eloped by incuba ing wi h an i- abbi
HRP-labelled seconda y an ibodies (GE Heal hca e, 1:2000) and Pie ce ECL Plus WB subs a e (The mo Ca .No.
32132), o an i-mouse Cy5-labelled an ibodies (Jackson ImmunoResea ch, 1:1000). The memb anes we e isu-
alized on S o m 860 Fluo oimage (GE Heal hca e) and quan i ied wi h ImageQuan so wa e (GE Heal hca e).
TUNEL assay. Te minal deoxynucleo idyl ans e ase dUTP nick end labeling (TUNEL) s aining was pe o med
as pe manu ac u e ’s ins uc ions (Sigma Ald ich, Ca no. 11684795910). B ie ly, he pa a in embedded sec ions
we e depa a inized in xylene and descending g ades o alcohol. Nex , an igen e ie al was pe o med in a mic owa e
o en a 750 W o 1 min ollowed by apid cooling in PBS. The es slides we e inally ea ed wi h eshly p epa ed
TUNEL eac ion mix u e ollowed by incuba ion in a humidi ied chambe a 37 °C o 1 h. The posi i e con ol was
ea ed wi h DNAase I (Sigma Ald ich, Ca no. 9003–98–9) be o e applica ion o TUNEL eac ion mix u e and he
enzyme solu ion was omi ed om he nega i e con ol. An open-sou ce imaging pla o m (h ps://imagej.nih.go /ij)
in ImageJ p og am was used o coun DAPI posi i e cells in he ONL. B ie ly, images aken wi h a 20 x magni ica ion
we e con e ed o g ay scale, s aigh ened and analyzed wi hin 6 coun ing windows measu ing app oxima ely 370 µm
in wid h. TUNEL posi i e cells we e coun ed manually online ac oss he en i e ONL using a 20 x magni ica ion.
His ology, mo phome y, immunohis ochemis y and mic oscopy. The pa a in-embedded eyes
we e cu in o 5 µm sec ions wi h a mic o ome. Fo hickness analysis o e inal laye s, e inal pano ama pic u es
we e cons uc ed om hema oxylin & eosin s ained samples. The leng h o he e ina was manually de e mined
wi h a ule ool in Zen 2012 so wa e (Zeiss, Jena, Ge many). Ma ks we e digi ally d awn o he pano ama
pic u e in o loca ions o 25%, 50% and 75% dis ance om op ic ne e head (ONH) in ela ion o he end ip o
he e ina a o a se a a o bo h sides o he e ina. The hickness o he ONL, INL and IPL we e measu ed a he
icini y o he six p ede e mined loca ions using he ule ool. The measu emen s a 25%, 50% and 75% loca ions
om bo h sides o he e ina we e inally a e aged and he a e age was used o s a is ical analysis. The samples
we e inspec ed by a luo escen ligh -mic oscope (Zeiss Image M2, Zeiss, Obe kochen, Ge many) and images
acqui ed wi h a digi al came a o ligh -mic oscope (AxioCam ERc5s, Zeiss, Obe kochen, Ge many) o luo es-
cen mic oscope (AxioCam MRm, Zeiss, Obe kochen, Ge many).
Fo immunohis ochemis y (IHC), he sec ions we e depa a inized, ehyd a ed and washed in PBS (0.1 M,
pH 7.4) o is-bu e ed saline (TBS, pH 7.6). An igen e ie al in GFAP s aining was pe o med by boiling sec-
ions in 0.05 M i-sodium ci a e dihyd a e (pH 6.0) a 90 °C o 5 min ollowed by washing 3 × 5 min in PBS/
TBS. The sec ions we e blocked in 3% bo ine se um albumin (BSA) o 10% no mal goa se um (NGS) o 1 h
An ibody Hos Sou ce Dilu ion Spesi ici y
An i-GFAP Rabbi DakoCy oma ion 1:10 000 (IHC) Mac oglial ma ke
An i-Iba1 Rabbi Wako 1:500 (IHC) Mic oglial ma ke
An i-CD68 Ra AbD Se o ec 1:1000 (IHC) Mac ophage ma ke
An i-Beclin1 Rabbi The mo Scien i ic 1:1000 (WB) Au ophagy ma ke
An i-P62 Rabbi Cell Signaling 1:1000 (WB) Au ophagy ma ke
An i-LC3b Rabbi Cell Signaling 1:1000 (WB) Au ophagy ma ke
An i-Lamp1 Rabbi Abcam 1:1000 (WB) Lysosomal ma ke
An i-M/L opsin Rabbi EMD Millipo e 1:1000 (IHC) M/L opsin ma ke
Table 1. De ails o an ibodies used in immunohis ochemis y (IHC) and Wes e n blo (WB).