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Resea ch Pape
Loss o NRF-2 and PGC-1αgenes leads o e inal pigmen epi helium damage
esembling d y age- ela ed macula degene a ion
Szabolcs Felszeghy
a,b,1
, Johanna Vii i
c,1
, Jussi J. Pa e no
c,d,1
, Juha M.T. Hy inen
c
, Ali Koskela
c
,
Mei Chen
e
, Hen i Leinonen
, Heikki Tanila
, Niko Ki inen
c,d
, A o Kois inen
g
, Elisa To opainen
h
,
Ma ialau a Amadio
i
, Ad ian Smedowski
j
, Mika Reinisalo
c,h
, Ma eusz Winia czyk
k,l
,
Je zy Mackiewicz
l
, Maija Mu ikainen
, Anna-Kaisa Ruo salainen
, Mikko Ke unen
,
Kimmo Joki a si
, Debasish Sinha
m
, Ka i Kinnunen
d
, Go an Pe o ski
n
, Janusz Blasiak°,
Gei Bjø køy
p
, A i Koskelainen
q
, Heli Sko man
, A o U i
h,s
, An e o Salminen
, Ram Kannan
u
,
Debo ah A. Fe ing on
, Heping Xu
e
, Anna-Liisa Le onen
, Pasi Ta i
, Anu Kauppinen
h
,
Kai Kaa ni an a
c,d,⁎
a
Ins i u e o Den is y, Uni e si y o Eas e n Finland, Kuopio, Finland
b
Ins i u e o Biomedicine, Uni e si y o Eas e n Finland, Kuopio, Finland
c
Depa men o Oph halmology, Uni e si y o Eas e n Finland, Kuopio, Finland
d
Depa men o Oph halmology, Kuopio Uni e si y Hospi al, Kuopio, Finland
e
The Wellcome-Wol son Ins i u e o Expe imen al Medicine Queen's Uni e si y Bel as , Bel as , UK
A.I. Vi anen Ins i u e o Molecula Sciences, Uni e si y o Eas e n Finland, Kuopio, Finland
g
SIB Labs, Uni e si y o Eas e n Finland, Kuopio, Finland
h
School o Pha macy, Facul y o Heal h Sciences, Uni e si y o Eas e n Finland, Kuopio, Finland
i
Depa men o D ug Sciences, Sec ion o Pha macology, Uni e si y o Pa ia, Pa ia, I aly
j
Chai and Depa men o Physiology, School o Medicine in Ka owice, Medical Uni e si y o Silesia, Ka owice, Poland
k
Depa men o Epizoo iology, Uni e si y o Li e Sciences o Lublin, Poland
l
Depa men o Vi eo e inal Su ge y, Medical Uni e si y o Lublin, Poland
m
The Wilme Eye Ins i u e, The Johns Hopkins Uni e si y School o Medicine, Bal imo e, Ma yland, USA
n
Cen e o Eye Resea ch, Depa men o Oph halmology, Oslo Uni e si y Hospi al, Uni e si y o Oslo, Oslo, No way
Depa men o Molecula Gene ics, Uni e si y o Lodz, Lodz, Poland
p
Cen e o Molecula Inflamma ion Resea ch and Depa men o Cance Resea ch and Molecula Medicine; No wegian Uni e si y o Science and Technology and
Depa men o Technology; Uni e si y College o Sø -T øndelag, T ondheim, No way
q
Depa men o Neu oscience and Biomedical Enginee ing, Aal o Uni e si y School o Science, Aal o, Finland
Facul y o Medicine and Li e Sciences, BioMediTech Ins i u e, Uni e si y o Tampe e, Tampe e, Finland
s
Cen e o D ug Resea ch, Di ision o Pha maceu ical Biosciences, Uni e si y o Helsinki, Helsinki, Finland
Depa men o Neu ology, Uni e si y o Eas e n Finland, Kuopio, Finland
u
A nold and Mabel Beckman Macula Resea ch Cen e , Doheny Eye Ins i u e, Los Angeles, CA, USA
Depa men o Oph halmology and Visual Neu osciences, Uni e si y o Minneso a, Minneapolis, USA
ARTICLE INFO
Keywo ds:
Aging
Au ophagy
ABSTRACT
Age- ela ed macula degene a ion (AMD) is a mul i- ac o ial disease ha is he leading cause o i e e sible and
se e e ision loss in he de eloped coun ies. I has been sugges ed ha he pa hogenesis o d y AMD in ol es
impai ed p o ein deg ada ion in e inal pigmen epi helial cells (RPE). RPE cells a e cons an ly exposed o
h ps://doi.o g/10.1016/j. edox.2018.09.011
Recei ed 13 July 2018; Recei ed in e ised o m 6 Sep embe 2018; Accep ed 13 Sep embe 2018
Abb e ia ions: ER, endoplasmic e iculum; AMD, age- ela ed macula degene a ion; RPE, e inal pigmen epi helium; PUFAs, polyunsa u a ed a y acids; ROS,
eac i e oxygen species; NRF-2, nuclea ac o -e y h oid 2- ela ed ac o -2; ARE, an ioxidan esponse elemen ; Keap1, kelch-like ECH-associa ed p o ein 1; PGC-1,
pe oxisome p oli e a o -ac i a ed ecep o gamma coac i a o -1; PRC, PGC-1- ela ed coac i a o ; SOD2, supe oxide dismu ase 2; TRX1, hio edoxin; AMPK, AMP-
ac i a ed p o ein kinase; SIRT1, si uin 1; UPS, ubiqui in-p o easome sys em; p62/SQSTM1, seques osome 1; LC3, mic o ubule-associa ed p o ein 1A/1B ligh chain
3; dKO, double knockou ; KO, knockou ; WT, wild ype; 4-HNE, 4-hyd oxynonenal); ROI, egion o in e es ; TEM, ansmission elec on mic oscopy; ATF4, ac i a ing
ansc ip ion ac o 4; GRP78, glucose- egula ed p o ein 78; Iba-1, ionized calcium binding adap o molecule 1; ERG, elec o e inog aphy; Rd8, e inal degene a ion
8; MRI, magne ic esonance imaging; m DNA, mi ochond ial DNA
⁎
Co espondence o: Depa men o Oph halmology, Uni e si y o Eas e n Finland and Kuopio Uni e si y Hospi al, Kuopio, Finland.
1
Equal con ibu ion.
E-mail add ess: kai.kaa ni an a@ue .fi(K. Kaa ni an a).
Redox Biology 20 (2019) 1–12
A ailable online 14 Sep embe 2018
2213-2317/ © 2018 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license
(h p://c ea i ecommons.o g/licenses/BY-NC-ND/4.0/).
T
Degene a ion
Oxida i e s ess
P o ein agg ega ion
P o easome
oxida i e s ess ha may lead o he accumula ion o damaged cellula p o eins, DNA and lipids and e oke issue
de e io a ion du ing he aging p ocess. The ubiqui in-p o easome pa hway and he lysosomal/au ophagosomal
pa hway a e he wo majo p o eoly ic sys ems in euka yo ic cells. NRF-2 (nuclea ac o -e y h oid 2- ela ed
ac o -2) and PGC-1α(pe oxisome p oli e a o -ac i a ed ecep o gamma coac i a o -1 alpha) a e mas e
ansc ip ion ac o s in he egula ion o cellula de oxifica ion. We in es iga ed he ole o NRF-2 and PGC-1αin
he egula ion o RPE cell s uc u e and unc ion by using global double knockou (dKO) mice. The NRF-2/PGC-
1αdKO mice exhibi ed significan age-dependen RPE degene a ion, accumula ion o he oxida i e s ess
ma ke , 4-HNE (4-hyd oxynonenal), he endoplasmic e iculum s ess ma ke s GRP78 (glucose- egula ed p o ein
78) and ATF4 (ac i a ing ansc ip ion ac o 4), and damaged mi ochond ia. Mo eo e , le els o p o ein ubi-
qui ina ion and au ophagy ma ke s p62/SQSTM1 (seques osome 1), Beclin-1 and LC3B (mic o ubule associa ed
p o ein 1 ligh chain 3 be a) we e significan ly inc eased oge he wi h he Iba-1 (ionized calcium binding
adap o molecule 1) mononuclea phagocy e ma ke and an enla gemen o RPE size. These his opa hological
changes o RPE we e accompanied by pho o ecep o dysmo phology and ision loss as e ealed by elec o-
e inog aphy. Consequen ly, hese no el findings sugges ha he NRF-2/PGC-1αdKO mouse is a aluable model
o in es iga ing he ole o p o easomal and au ophagy clea ance in he RPE and in he de elopmen o d y
AMD.
1. In oduc ion
Ch onic oxida i e and endoplasmic e iculum (ER) s ess, impai ed
au ophagy, mi ochond ial dys unc ion and inflamma ion a e s ongly
linked o age- ela ed macula degene a ion (AMD) [1–6]. One clinical
hallma k o AMD is he degene a ion o e inal pigmen epi helial (RPE)
cells, a p ocess ha associa es wi h he accumula ion o oxida i e
s ess–de i ed lysosomal lipo uscin, impai ing lysosomal deg ada ion,
and he p esence o ex acellula p o ein/lipid deposi s (d usen) be-
ween he basal lamina o he RPE and he inne collagenous laye o
he B uch's memb ane [7–11]. AMD can be subdi ided in o d y and we
o ms wi h 80% and 20% p e alences, espec i ely. The e a e no e -
ec i e ea men s o d y AMD.
The quiescen RPE cells in he macula a e cons an ly subjec ed o
high oxida i e s ess due o he diu nal diges ion o high concen a ions
o polyunsa u a ed a y acids (PUFAs) o igina ing om he phagocy-
osis o he e inal ou e segmen s, exposu e o he oxygen- ich cho -
iocapilla y ci cula ion, and he ligh -induced p oduc ion o eac i e
oxygen species (ROS) by cellula pigmen g anules [7]. The mi-
ochond ia also p oduce ROS as a by-p oduc o espi a ion. Du ing he
aging o RPE cells, he cellula capaci y o neu alize ROS diminishes
due o a dec eased p oduc ion o ROS-sca enge s and o he an i-
oxidan s [12,13].
In RPE cells, he NRF-2/ARE (nuclea ac o -e y h oid 2- ela ed
ac o -2/an ioxidan esponse elemen ) pa hway plays he majo ole in
he oxida i e s ess egula ion [14,15]. In esponse o oxida i e s ess,
he NRF-2 is eleased om NRF-2-Keap1 (kelch-like ECH-associa ed
p o ein 1) complex allowing he ansloca ion o NRF-2 om cy osol
in o he nucleus and he exp ession o se e al an ioxidan de ense
sys em and au ophagy - ela ed genes and p o easomal subuni s
[16–20]. Mice models wi h NRF-2 deficiency ha e been shown o be
mo e suscep ible o oxida i e damage and p o ein agg ega ion han WT
(wild ype) mice and o nega i ely egula e au ophagy- ela ed genes
[21,22].
The pe oxisome p oli e a o -ac i a ed ecep o gamma coac i a o -
1 (PGC-1) amily, consis ing o PGC-1α, PGC-1βand PRC (PGC-1- e-
la ed coac i a o ), egula es a ne wo k go e ning he ansc ip ional
con ol o mi ochond ial biogenesis and espi a o y unc ion as well as
a ge ing mi ochond ial an ioxidan de ense sys em [23–25]. The ole
o PGC-1αis o se e as a swi ch be ween mi ochond ial biogenesis and
oxida i e damage by con olling he mi ochond ial le els o ROS. Loss-
o - unc ion s udies o PGC-1αha e shown bu s o ROS and an inc ease
in mi ochond ial damage and deg ada ion whe eas gain-o - unc ion
enhanced mi ochond ial biogenesis and he exp ession o mi ochon-
d ial an ioxidan de ense sys em- ela ed genes, such as SOD2 (supe -
oxide dismu ase 2) and TRX1 ( hio edoxin) [23,25]. Howe e , bo h
pa hways, mi ochond ial biogenesis and ROS con ol, s i e o he
p ese a ion o mi ochond ial homeos asis. Pos - ansla ional
modifica ion o he ene gy senso s, AMPK (AMP-ac i a ed p o ein ki-
nase) and SIRT1 (si uin 1) ha induce au ophagy, a e known o eg-
ula e PGC-1αac i i ies [26–28]. In RPE cells, PGC-1αhas been shown
o d i e mi ochond ial biogenesis as well as ac i a ing he an ioxidan
de ense sys em [29,30].
A high ROS p oduc ion combined wi h impai ed an ioxidan sys-
ems esul s in de imen al p o ein agg ega ion [8,31]. The ubiqui in-
p o easome sys em (UPS) and he lysosomal/au ophagosomal de-
g ada ion sys em sha e he majo esponsibili y o main aining cellula
p o eos asis [8,32]. Bo h sys ems ecognize and ac i ely selec he
ma e ial des ined o deg ada ion and ecycling in cells. The p62/
SQSTM1 (seques osome 1) has a mul i ac o ial ole since i ac s as a
b idge shu ling p o eins be ween UPS and au ophagy clea ance as well
as egula ing he an ioxidan esponse in RPE cells du ing oxida i e
s ess [9,33,34]. P o eins assigned o he deg ada ion a e o en ubi-
qui ina ed and di ec ed o he p o easomal deg ada ion. Howe e , he
a e o p o ein deg ada ion by p o easomes is limi ed, and when UPS is
o e whelmed, o example unde hea y oxida i e s ess, p o eins o m
agg ega es which canno be emo ed by p o easomes. Au ophagy is
specialized o emo ing p o ein agg ega es and la ge cell o ganelles,
such as mi ochond ia. p62/SQSTM1 ecognizes ubiqui ina ed pe i-
nuclea p o ein agg ega es and he p62/SQSTM1 agged ma e ial is
hen isola ed om he cy osol in p62/SQSTM1-LC3 (mic o ubule-as-
socia ed p o ein 1A/1B ligh chain 3) in e ac ion-guided au ophago-
some o ma ion [35]. In he final s ep o he au ophagy p ocess, a ly-
sosome is used o he au ophagosome esul ing in he o ma ion o he
au olysosome ollowed by he deg ada ion o i s con en s including
ubiqui in, p62/SQSTM1 and LC3. The eby, hese p o eins can be
exploi ed as bioma ke s o p o ein agg ega ion and au ophagy ac i i y
[35,36]. Mo eo e , p62/SQSTM1 in e ac s wi h he NRF-2/ARE
pa hway by dis up ing he NRF-2-Keap1 complex leading o nuclea
localiza ion o NRF-2 [37].
The la ge and inc easing numbe o indi iduals affec ed by AMD
means ha his ep esen s an u gen global heal h eme gency equi ing
effec i e p ima y p e en ion s a egies as well as imp o emen s in he
cu en ea men s o his disease. The e o e, new s udies con ibu ing
o a be e unde s anding o he mechanisms egula ing an ioxidan
de ense sys em, p o eos asis, and mi ochond ial unc ion may lead o
b eak h oughs in AMD esea ch. Since he NRF-2 and PGC-1α an-
sc ip ion ac o s ha e a key ole in cellula de oxifica ion and mi-
ochond ial unc ion in esponse o oxida i e s ess, we gene a ed and
cha ac e ized he NRF-2/PGC-1αdouble knockou (dKO) mouse model
o s udy e inal changes ha occu in deg ada i e pa hways and
documen ea u es p e iously associa ed wi h AMD.
S. Felszeghy e al. Redox Biology 20 (2019) 1–12
2
2. Ma e ial & me hods
2.1. Animals
All animal p o ocols we e app o ed by he Animal Expe imen
Boa d o Finland and conduc ed in compliance wi h he Eu opean
Communi y Council Di ec i es 2010/63/EU and ARVO s a emen o
he Use o Animals in Oph halmic and Vision Resea ch. Mice we e
g oup-housed in he Labo a o y Animal Cen e o Uni e si y o Eas e n
Finland, Kuopio, Finland on a 12-h ligh /da k cycle and gi en ood and
wa e ad libi um.
The mice s ains deficien in NRF-2 o PGC-1α(C57BL/6J back-
g ound) used in his s udy ha e been desc ibed ea lie [38,39].To
ob ain mice lacking bo h NRF-2 and PGC-1α, homozygous-null NRF-2
and PGC-1αs ains we e c ossed fi s , and he esul an double he -
e ozygo es we e c ossed oge he . Female and male KO (knockou ) and
dKO mice a six weeks, h ee mon hs and one yea o age we e used o
his s udy, oge he wi h age-ma ched WT (wild ype) con ols.
2.2. The geno yping o NRF-2 and PGC-1αknockou mice
The genomic mouse DNA was ex ac ed om small ea samples by
hea ing he issues o 1 h a 95 °C in 100 μl o 25 mM NaOH + 0.2 mM
EDTA. The solu ions we e neu alized by adding 100 μl o 40 mM T is-
HCl pH 7.5, and cen i uged o 10 min a 2000 pm. The uppe hal es
o he supe na an s (100 μl) we e collec ed o be used di ec ly as em-
pla es in he PCR eac ions.
The geno ypes o PGC-1αwe e de ec ed wi h a 4-p ime PCR. The
p ime sequences we e: WTA,5′-CCA GTT TCT TCA TTG GTG TG; WTB,
5′-ACC TGT CTT TGC CTA TGA TTC; KOA,5′-TCC AGT AGG CAG AGA
TTT ATG AC; KOB,5′-CCA ACT GTC TAT AAT TCC AGT TC. The NRF-2
geno ypes we e s udied wi h he ollowing h ee p ime s: LacZ,5′-GCG
GAT TGA CCG TAA TGG CAT AGG; N 2–5′,5′-TGG ACG GGA CTA TTG
AAG GCT G; N 2–3’, GCC GCC TTT TCA GTA GAT GGA CG.
Bo h eac ions included 4 μl o DNA ex ac ed om mouse ea s,
eac ion buffe , 100 μM o each dNTPs, 1.5 mM o MgCl
2,
1μM o each
p ime s, and 1.2 U o D eamTaq DNA polyme ase (The mo Fishe
Scien ific Wal ham, MA USA), in a olume o 30 μl. Fo he PGC-1α,
samples we e dena u ed a 95 °C o 5 min, ollowed by 39 cycles a
95 °C o 30 s, 58 °C o 30 s, 72 °C o 30 s, and a final ex ension a 72 °C
o 7 min. Fo he NRF-2 geno yping, he eac ion condi ions we e:
dena u a ion a 95 °C o 5 min, ollowed by 35 cycles a 95 °C o 30 s,
a 59 °C o 30 s, and a 72 °C o 45 s, and a final ex ension a 72 °C o
7 min. All PCR p oduc s we e sepa a ed using 1% aga ose (Seakem LE,
Camb ex) gel elec opho esis in s anda d T is-ace a e-EDTA buffe , i-
sualized in UV ligh wi h e hidium b omide, and pho og aphed. The
amplicon sizes he o wild ype allele o PGC-1αa e 600 bp and o he
KO allele 400 bp. Thei sizes o he NRF-2 alleles a e 700 and 400 bp,
espec i ely (Supplemen a y Fig. 1).
2.3. Immunomapping o de ec ion o p o ein agg ega ion, au ophagy and
oxida i e s ess ma ke s: Ubiqui in, p62/SQSTM1, Beclin-1, LC3 and 4-
HNE
The mice we e sac ificed wi h ce ical disloca ion and he eyes we e
immedia ely ca e ully enuclea ed and placed in PBS (pH 7.4). Be o e
dehyd a ing in g aded se ies o e hanol and embedding in pa affin, he
eyes we e fixed in 4% pa a o maldehyde in 0.1 M phospha e buffe o
24–48 h. Fi e μm hick pa asagi al se ial sec ions we e cu om em-
bedded blocks wi h a mic o ome (SM2000 R, Leica, Heidelbe g,
Ge many). A e a andom selec ion o slides om each o he in-
di idual specimens, he sec ions we e dewaxed and ehyd a ed and he
labeling o he e ina was pe o med acco ding o p e iously published
me hods, wi h mino modifica ions [36]. B iefly, he sec ions we e
ea ed wi h blocking solu ion (#IHC-101B Be hyl labo a o ies, Mon-
gome y, TX, USA) o 30 min a oom empe a u e o p e en he
possibili y o non-specific binding o he p ima y an ibodies; he sam-
ples we e hen immunos ained wi h diffe en p ima y an ibodies as
ollows: an i-ubiqui in (1/200; Dako, Glos up, Denma k), an i-
SQSTM1/p62 (1/100; Abcam, Camb idge, UK), an i-Beclin 1 (1/160;
No us Biologicals, CO, USA), an i 4-HNE (4-hyd oxynonenal) (1/200;
Li eSpan BioSciences, Inc, Sea le, WA, USA), an i LC3B (1/200; Cell
Signaling Technology, Dan e s, MA, USA) o e nigh , a 4 °C. A e
insing he p ima y an ibodies in TBS, he samples we e hen incuba ed
wi h a seconda y an ibody an i mouse Alexa Fluo 594 (1/500; The mo
Fishe Scien ific Wal ham, MA USA) o 3 h a oom empe a u e and
insed wi h TBS. A fluo escen nuclea ma ke DAPI (4′,6-diamidino-2-
phenylindole dihyd ochlo ide, #D9542, Sigma, S . Louis, MO, USA)
dilu ed 1:10 000 in TBS was added and incuba ed o 30 min a oom
empe a u e. The sec ions we e insed wi h TBS and co e ed wi h
Mowiol moun ing media. Immunofluo escence was e alua ed in-
dependen ly o andomly selec ed sec ions by h ee esea che s and no
signal was eco ded om echnical nega i e con ols (TBS ins ead o
p ima y an ibody). A e he s aining, he samples we e analyzed as
desc ibed abo e, pho omic og aphs we e aken in a Zeiss AX10 Image
A2 (Zeiss, Gö ingen, Ge many) mic oscope. Images we e cap u ed se-
quen ially om he g een, ed, and a - ed channels on slices using a
63× oil imme sion objec i e (NA:1.42, Plan Apoch oma ). The mi-
c oscope se ings we e iden ical o all scans and kep cons an du ing
imaging. Rep esen a i e scans we e aken wi h a Jenop ik P ogRes C5
(Zeiss, Gö ingen, Ge many) digi al came a moun ed on o he mic o-
scope. In all imaging p ocedu es, gamma adjus men was made on he
whole image in o de o main ain app op ia e con as and images we e
p ocessed using Adobe Pho oshop o documen a ion. The con ol sec-
ions o igina ed om he same eyes and he alidi y o he sequen ial
s aining was hen e ified wi h he same p o ocol as desc ibed abo e
excep ha one o he p ima y an ibodies was omi ed. The nega i e
con ol samples displayed only a sub-minimal au ofluo escence signal.
2.4. Spec al imaging analysis
The immunohis ochemical esul s we e examined by h ee in-
dependen obse e s sea ching o immuno eac i e RPE cells. The di -
e en ma ke s dis ibu ion pa e n we e semi-quan i a i ely analyzed
by compu e imaging as ollows. High powe iew RGB 8-bi images o
single RPE cells we e gene a ed wi h a Jenop ik P ogRes C5 as i de-
sc ibed abo e in fluo escen mode, using a 63× objec i e lens. Blue
pseudocolo o he nuclei was assigned by he in es iga o s o help
segmen a ion p ocesses o RPE cell nuclei, espec i ely. Fo quan i a-
ion o he diffe en p o ein agg ega ion and au ophagy ela ed
(Ubiqui in, p62/SQSTM1, Beclin-1, LC3B) ma ke exp ession and oxi-
da i e s ess ma ke (4-HNE), 10 RPE cells pe animal o in e es (n = 3
WT and n = 3 NRF-2/PGC-1αdKO) we e manually designa ed as e-
gions o in e es (ROI). Special ca e was aken o selec wo e-
p esen a i e a eas pe sec ion o collec ing RPEs om each indi idual
samples close o he icini y o op ic ne e. Sampling o RPE image
analysis was pe o med wi hou ex e nal knowledge on samples geno-
ype o be able o compa e wi hou influences om es e s' p e e ences
o expec a ions. A spec al imaging con aining he cha ac e is ic wa-
eleng h emission o Alexa Fluo 594 was c ea ed by sampling pu e
RED spec a om RGB images; hese we e con e ed o black and whi e
images, i.e. only he posi i e signal o in e es was analyzed u he . Via
his g ayscale con e sion, he o iginal, ue RED colo spec al in-
o ma ion was au oma ically con e ed in o one g ayscale image. This
p ocess is based on he calcula ion o he a e age in ensi y pe channel,
he e o e one can keep he o iginal s aining in ensi y in o ma ion.
Fu he mo e, his con e sion makes i possible o ocus only on he RED
channel o in e es . Using he open sou ce Image J so wa e (h p//
:imagej.nih.go ; NIH, USA), we quan ified he immunos aining in he
ROI, he so wa e measu ed he amoun o a ge signal (in ensi y o B&
W pseudo-colo ) wi hin each ROI, by measu ing he densi y pe pixel
and e u ning a nominal alue be ween 0 ( he limi o noise le el i.e.
S. Felszeghy e al. Redox Biology 20 (2019) 1–12
3
da a poin om a blank sample /=nega i e immunohis ochemical e-
ac ion/ was de e mined below 20) and 255 (whi e maximal s aining)
o ha pixel. The in eg a ed a e age g ay le el used in ou u he
analysis was he sum o all he sepa a e pixels in he examined a ea.
Final da a we e displayed as a e age g ay alue/cell. Wi h his se up
desc ibed b iefly abo e, we belie e ha he isk o in e p e a ion bias is
a oided. Howe e , ob aining accu a e esul s wi h his semi-quan i a-
i e-immunohis ochemical analysis depends upon he specifici y o he
an ibodies used.
2.5. TEM analysis
P io o ansmission elec on mic oscopy (TEM), he mice eyes
we e p e-fixed wi h 3% pa a o maldehyde in 0.075 M cacodyla e buffe
(pH 7.2) wi h 1% glu a aldehyde o 5–7 days a + 4 °C. A e
2 × 10 min washing in 0.075 M cacodyla e buffe , he samples we e
pos -fixed in 1% osmium e aoxide and 1.5% po assium- e ocyanide
in 0.1 M cacodyla e buffe (pH 7.2) o 2 h, and again washed wi h
cacodyla e buffe o 2 × 10 min and 5 min in dis illed wa e p io o
s anda d e hanol dehyd a ion. Subsequen ly, he samples we e in-
fil a ed and embedded in LX-112 esin (Ladd Resea ch, Willis on, VT,
USA). Polyme iza ion was ca ied ou a 60 °C o 48–72 h. One-mic on
semi- hin sec ions we e cu wi h a Reiche Ul acu E mic o ome (Leica
Mic osys ems Inc, IL, USA), s ained wi h 1% oluidine blue, and ex-
amined wi h a ligh mic oscope o find he localiza ion o in e es o
u he TEM sec ioning. Nex , he 70 nm hick sec ions (cu wi h Leica
EM UC7, We zla , Ge many) we e s ained wi h 1% u anyl ace a e o
30 min and wi h lead ci a e o 2 min. The sec ions we e examined
wi h a JEM-2100F TEM (Jeol, Tokyo, Japan) a 200 kV. Lipo uscin-like
agg ega es and au ophagic compa men s we e manually coun ed by
h ee diffe en indi iduals. Simila ly as wi h he his ological samples,
ep esen a i e a eas pe sample we e selec ed o collec ing RPEs om
each indi idual sample close o he icini y o he op ic ne e. Six cells
we e andomly selec ed om each g oup (WT and dKO mice aged 12
mon hs, n = 3) o coun ing.
2.6. Vacuole a ea ac ion analysis
The analysis echnique used in his s udy objec i ely measu ed he
mo phome ic pa ame e o he acuoles a he basal cy oplasmic si e o
RPE om TEM images, o cha ac e ize a ea ac ions o acuoles in he
WT and dKO RPE a 1 yea o age. Using he open sou ce Image J
so wa e (h p//:imagej.nih.go ; NIH, USA), he so wa e measu ed he
size o acuoles and da a we e exp essed acco ding o equal size ROI
(200 µm
2
).
2.7. De ec ion o lipo uscin-like g anules in he RPE cells
Du ing he egula dewaxing and ehyd a ion, he p ocessed WT
and dKO samples o in e es we e washed wi h 0.3% w/ Sudan Black B
(SBB, ca . no. 3545-12, EMD Chemicals, Gibbs own, NJ, USA) in 70%
e hanol a 37 °C o 20 min, insed wi h PBS 3 imes 10 min and hen he
sec ions we e moun ed wi h Mowiol moun ing Media (Sigma, S . Louis,
MO, USA) [40]. The samples we e examined in a lase scanning con-
ocal mic oscope (Zeiss AX10 Image A2, Zeiss, Gö ingen, Ge many)
wi h sequen ial scanning and de ec ion o do -like lipo uscin g anules,
espec i ely, ollowed by me ging and sa ing o he images.
2.8. Apop osis assay
TUNEL ( e minal deoxynucleo idyl ans e ase dUTP nick end la-
beling) assay was pe o med using he ApopTag®Pe oxidase in si u
Apop osis De ec ion Ki (Millipo e S.A.S., Molsheim, F ance) acco ding
o he manu ac u e 's ins uc ions in one-yea -old samples. TUNEL
s ained sec ions we e washed in PBS and coun e s ained wi h DAPI. The
sec ions we e dehyd a ed and moun ed. Posi i e and nega i e con ols
we e used o con ol s aining quali y.
Fig. 1. NRF-2/PGC-1αdKO mice display inc eased p o ein agg ega ion, au ophagy and oxida i e s ess ma ke s in RPE cells. Rep esen a i e images and
compa a i e compu e -aided densi ome ic assay ocusing on ubiqui in (whi e), p62/SQSTM1 (b own), Beclin-1 ( ed), LC3B (g een) and 4-HNE (yellow) im-
munofluo escences in RPE cells (DAPI-s ained nuclei/blue) o one-yea -old wild ype (WT), PGC-1αKO, NRF-2 KO and NRF-2/PGC-1αdKO mice. The scale ba
indica es 5 µm. The g ay le el in ensi ies o WT, PGC-1αKO, NRF-2 KO and NRF-2/PGC-1αdKO mice ep esen a e age in ensi ies. Combined esul s om h ee
diffe en animals pe geno ype and n = 30. *p < 0.001 one-way ANOVA ollowed by Games-Howell pos hoc es (n = 30). Resul s a e exp essed as means ± SD.
S. Felszeghy e al. Redox Biology 20 (2019) 1–12
4
2.9. Immunohis ochemis y o ER s ess ma ke s
One-yea -old WT and dKO e inas we e s ained in iplica e wi h
abbi an i-ATF4 (ac i a ing ansc ip ion ac o 4, Abcam Camb idge,
MA) and abbi an i-GRP78 (glucose- egula ed p o ein 78, Abcam
Camb idge, MA) an ibodies, sepa a ely. Immunohis ochemis y was
pe o med on pa affin embedded sec ions a e an igen e ie al using
an In i ogen AEC ed (Ca lsbad, CA) subs a e ki . Fo accu a e com-
pa isons, all sec ions we e s ained simul aneously and slides we e de-
eloped unde he same condi ions. Imaging was pe o med wi h he
Leica-Ape io CS digi al ScanScope (Leica Mic osys ems, Buffalo, IL) a
40× magnifica ion. When measu ing posi i e pixels, images we e di-
gi ally sc ibed using Ape io ScanScope so wa e o c ea e a ROI ha
enci cled he en i e c oss-sec ional p ofiles o he e ina on each slide.
The posi i e pixel coun algo i hm Ape io .9.1 was used o de e mine
s ained s. uns ained pixel coun s o a eas wi hin each ROI.
2.10. Fla moun and RPE size analysis
Mice eyes we e collec ed and fixed in 2% PFA o 2 h. The samples
we e hen p ocessed o he RPE/cho oid fla moun p epa a ion using a
p o ocol p e iously desc ibed [34,35]. B iefly, he an e io segmen o
he eye, lens and he neu onal e ina we e emo ed unde a dissec ing
mic oscope. Fou o fi e e ical cu s we e made om he edge o he
RPE/cho oid/scle a cup o he equa o . The samples we e hen washed
and ea ed wi h 1% i on X-100 o 2 h, ollowed by incuba ion wi h
abbi an i-mouse Iba-1 (ionized calcium binding adap o molecule 1,
Abcam, Camb idge, MA) 1:100, a 4 °C o 16 h, ollowed by FITC-
Fig. 2. T ansmission elec on mic oscopy (TEM) analysis e ealed d y AMD-like pa hology in one-yea -old NRF-2/PGC-1αdKO mice. Rep esen a i e TEM
image o WT RPE indica es no mal cellula o ganelles, while in one yea old dKO RPE loss o basal in oldings and hickened B uchs's memb ane (a owheads) we e
de ec ed (A and B). (C) Inc eased amoun o (D1) melanosomes, (D2) au olysosomes, (D3, D4) damaged mi ochond ia and (D5) lipo uscin we e de ec ed in dKO RPE.
The scale ba s indica e 2 µm. WT = wild ype; dKO = double knockou ; BM = B uch's memb ane; HM = heal hy mi ochond ia, DM = damaged mi ochond ia Da a
a e quan ified as % a io o dKO e sus WT ± SD. Thickness o B uch's memb ane (BM) mic ons ± SD. *p < 0.05, S uden 's - es (n = 18).
S. Felszeghy e al. Redox Biology 20 (2019) 1–12
5

conjuga ed (fluo escein iso hiocyana e) goa an i- abbi IgG (1:200,
Abcam) and Alexa Fluo 568 Phalloidin (1:100, Li e Technologies) a
oom empe a u e o 2 h. The samples we e washed and fla moun ed
on a glass slide o con ocal mic oscopy in es iga ion (Eclipse TE200-U;
Nikon UK L d.). Z-s ack con ocal images o RPE fla moun s we e e-
cons uc ed using he NIS Elemen (Nikon) so wa e. The bo de o each
RPE cell was ou lined based on he Phalloidin (F-ac in) s aining, and he
size o each RPE cell was measu ed manually using he same so wa e.
2.11. ERG eco dings
We es ed he e inal unc ion in h ee-mon h-old ( e e ed o as
“young”; WT, n = 6; dKO, n = 6) and one-yea -old mice ( e e ed o as
“old”; WT, n = 6; dKO, n = 7) by ERG (elec o e inog aphy, Tho labs
PM100D, Tho labs, NJ, USA). The ERG me hod has been desc ibed in
de ail p e iously [41]. The animals we e da k-adap ed o e nigh o a
leas 12 h. They we e anes he ized wi h isoflu ane (induc ion: con-
cen a ion 4–4.5%; main enance: concen a ion ~ 1.2%) and placed
in o a cus omized s e eo axic ame and kep wa m wi h a physiological
hea ing pad a 38 °C. The co neal ERG elec ode was a sil e wi e
cons uc ed as a semi-ci cula loop. One s ainless s eel subcu aneous
needle was a ached in o he cheek ( e e ence) and ano he in o he
lowe back (common g ound). All animal manipula ions we e pe -
o med unde a dim ed ligh and animals we e u he da k-adap ed in
a comple ely da k oom o 5 min. The da k-adap ed ligh s imula ion
was pe o med in an ascending se ies wi h espec o he s imulus
in ensi y (3.60, −2.90, −2.10, −1.65, −0.45, 0.50, 1.00, 1.60 and
2.00 log cd·s/m
2
), and he in e -s imulus in e al (ISI) was inc eased
acco dingly (2 s, 4 s, 4 s, 10 s, 10 s, 10 s, 20 s, 50 s and 50 s, espec-
i ely). Finally, a pai ed-flash pa adigm was used o isola e he cone
componen om he mixed od-cone wa e o m [42]. A s ong flash
(2.00 log cd·s/m
2
) was deli e ed o e oke ansien od sa u a ion and
400 ms la e a second p obe flash (2.00 log cd·s/m
2
) was deli e ed o
gene a e a cone-specific esponse. Rod-sa u a ion a 400 ms a e flash
was assumed based on a p e ious ex i o ERG sa u a ion es [41],
whe e he s onges flash was es ima ed o yield ~ 14,000 pho o-
isome iza ions (*R) pe mouse od. The ligh in ensi y was quan ified
wi h an ene gy me e (Tho labs PM100D, Tho labs, NJ, USA). In he
wa e o m analysis, he baseline o ERG esponse was aken as an
a e age ampli ude be ween −100 and 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
fi s nega i e deflec ion a e he s imulus onse . The b-wa e was cal-
cula ed om he a-wa e ough o he peak o he fi s majo posi i e
wa e. The ´cone´ esponse ampli ude was calcula ed be ween he la -
ges nega i i y and he majo posi i e wa e peaking a ound 40 ms a e
he p obe flash.
2.12. S a is ical analysis
All s a is ical analysis was pe o med using SPSS s a is ics so wa e
(SPSS Inc., Chicago, Ill, USA). p- alues < 0.05 we e conside ed sig-
nifican . Spec al imaging analysis: The s a is ical analysis o he da a
Fig. 3. The pa hological changes o RPE in NRF-2/PGC-1αdouble knockou (dKO) mice. (A) The ligh mic oscopic images o WT and dKO samples o he
oluidine blue-s ained epoxy sec ion om one-yea -old mouse. The whi e dashed a owheads indica e he dome-shaped ex acellula deposi s be ween he RPE and
B uch's memb ane (uppe panels). The cys ic cy oplasmic al e a ions we e obse ed in dKO samples (as e isks). (B) Excessi e accumula ion o lipo uscin-like ma e ial
(whi e a owhead) in he dKO RPE. In he inse s o Fig. B On he inse o figu e B, he yellow dashed a ows indica e he RPE laye . The scale ba indica es 10 µm.
(C) The ed a owhead indica es he ubiqui in posi i i y o d usen-like deposi s in he close icini y o B uch's memb ane. (D) Res ic ed apop osis was de ec ed
wi hin some cells in he RPE laye (g een a ow) o he dKO e ina wi h Apop ag®ki . PR: pho o ecep o laye ; RPE: e inal pigmen epi helium; BM: B uch's
memb ane. The scale ba indica es 5 µm. E (endo helial cell nucleus); N (RPE nucleus).
S. Felszeghy e al. Redox Biology 20 (2019) 1–12
6
was conduc ed wi h a one-way analysis o a iance (ANOVA), ollowed
by Games-Howell pos hoc es (n = 30). TEM analysis: The significance
o diffe ences be ween WT and dKO g oups we e analyzed wi h
S uden 's - es (n = 18). Vacuole a ea ac ion analysis: The s a is ical
analysis o he da a was conduc ed wi h a S uden 's - es (n = 25).
Immunohis ochemis y o ER s ess ma ke s: The a e age cell numbe s
om 5 diffe en egions we e calcula ed. S uden 's - es was applied o
assess s a is ical diffe ences be ween WT and dKO g oups (n = 4–7).
Fla moun and RPE size analysis: S a is ical significance was analyzed
wi h S uden 's - es (n = 4). ERG eco dings: a- and b-wa e ampli ude
and b-a a io s a is ical analyses we e pe o med wi h a wo-way e-
pea ed measu ed ANOVA using he geno ype as he be ween-subjec
ac o and s imulus in ensi y as he wi hin-subjec s ac o , ollowed by
Bon e oni pos hoc es . The cone-flash ampli ude was analyzed wi h a
egula wo-way ANOVA.
3. Resul s
We in es iga ed he effec s o NRF-2/PGC-1αdKO on mac oscopic
ana omy and age- ela ed RPE degene a ion using ligh , con ocal, and
elec on mic oscopy, as well as pe o ming a unc ional elec o-
e inog aphy analysis (Supplemen a y Figs. 2 and 3). To ensu e ha
he e we e no complica ions due o he spon aneous Rd8 ( e inal de-
gene a ion 8) ameshi mu a ion c.3481delC in C b1 (c umbs homolog
1) esul ing e inal degene a ion [43,44], sc eening o his mu a ion
was pe o med o mouse lines used in his s udy: WT, NRF-2 KO, PGC-
1αKO and NRF-2/PGC-1αdKO. Ou analysis e ealed ha hese mice
s ains we e nega i e o he Rd8 mu an allele (Supplemen a y Fig. 4).
3.1. NRF-2/PGC-1αgene inac i a ion e okes se e e axial skele on and eye
mo phology al e a ions
The adiog aphs e eal he abno mally small axial skele on al eady
p esen in six-week-old dKO mice (Supplemen a y Fig. 3). In compa -
ison wi h WT con ols, he dKO mice had ligh e body weigh s and
exp essed p ominen dysmo phic ea u es, such as a sho e axial ske-
le on, smalle limbs and head pa ame e s a all ages s udied
(Supplemen a y Fig. 3). The u o he dKO mice was also mo e g eyish
han in he co esponding single KOs o WT mice. The mac o-
pho og aphy and mic o-MRI (magne ic esonance imaging) e ealed a
smalle eyeball size in he dKOs.
3.2. NRF-2/PGC-1αdKO mice display inc eased p o ein agg ega ion and
oxida i e s ess ma ke s in RPE cells
Ch onic oxida i e s ess and impai ed au ophagy a e s ongly linked
o RPE degene a ion and he p o ein agg ega ion obse ed in AMD
[1,3]. The con en and localiza ion o p o ein agg ega es-conjuga ed
ma ke ubiqui in, au ophagy egula o Beclin-1, au ophagy ma ke s
p62/SQSTM1 and LC3B, and oxida i e s ess ma ke 4-HNE (p oduc o
lipid pe oxida ion) we e e alua ed in RPE cells by immunos ainings
and con ocal mic oscopy in one-yea -old WT, PGC-1αKO, NRF-2 KO
and NRF-2/PGC-1αdKO mice (Fig. 1).
Remo al o PGC-1αgene esul ed in highe le els o Beclin-1
(p < 0.001), LC3B (p < 0.001) and 4-HNE (p < 0.001) compa ed o
he WT mice. Howe e , he ma ke o p o ein agg ega ion, ubiqui in,
dec eased significan ly and he p62/SQSTM1 le els emained s able
indica ing an inc ease in oxida i e s ess bu , as expec ed, unc ional
UPS/au ophagy appea s capable o main ain mi ochond ial/p o ein
homeos asis. NRF-2 KO esul ed in highe exp ession le els o all
ma ke s s udied compa ed o he WT (p < 0.001). The ma ke o
oxida i e s ess (4-HNE) as well as au ophagy ma ke s Beclin-1 and
p62/SQSTM1 and p o ein agg ega ion ma ke ubiqui in we e sig-
nifican ly ele a ed in NRF-2 KO compa ed o PGC-1αKO mice sug-
ges ing s onge oxida i e s ess, insufficien p o easome unc ion and
au ophagic clea ance. NRF-2/PGC-1αdKO mice had he highes
Fig. 4. NRF-2/PGC-1αdKO mice display inc eased endoplasmic e iculum (ER) s ess in RPE cells. (A) Rep esen a i e ansmission elec on mic oscopy (TEM)
image o WT RPE indica es no mal cellula s uc u es and melanosomes. (B) Inc eased numbe o melanosomes, he basal lamina deposi s (a ow), he loss o basal
in oldings and ER damage (as e isks) we e de ec ed in dKO samples. (C) Inc ease o he ER acuoliza ion (% pe 200 µm2) was eco ded in dKO RPE compa ed o
aged ma ched WT samples. Inc eased immunohis ochemical s aining in ensi y o (D) GRP78 and (E) ATF4 was de ec ed in RPE laye o dKO mice compa ed o WT.
The scale ba indica es 10 µm (A, B) and 2 µm (C, D). *p < 0.05, **p < 0.01, S uden s - es . Da a a e mean ± SD (A-D n = 4–7; E n = 25).
S. Felszeghy e al. Redox Biology 20 (2019) 1–12
7
accumula ion o all ma ke s s udied sugges ing he highes deg ee
oxida i e s ess and mo e se e e de ec in he UPS/au ophagy clea -
ance.
3.3. Ul as uc u al analysis indica es an inc ease in au olysosomes,
damaged mi ochond ia and melanosomes in NRF-2/PGC-1αdKO mice
Since insufficien au ophagic clea ance and high oxida i e s ess
we e de ec ed in dKO mice, we assessed ul as uc u al changes o WT
and dKO RPE cells om he TEM images (Fig. 2). Compa ed o in ac
no mal cell s uc u es in WT RPE cells, he examina ion o dKO e-
ealed he p esence o la ge au olysomes, highe a io o damaged
mi ochond ia (p < 0.05), hicke B uch's memb ane (p < 0.05), he
loss o basal in oldings and mode a e inc ease in lipo uscin pa icles.
In e es ingly, he numbe o melanosomes was highe in dKO mice.
Mo eo e , B uch's memb ane elas ic and collagenous laye s we e no
o en obse ed, and inc eased hickness o B uch's memb ane was de-
ec ed along wi h an accumula ion o elec on dense amo phous ma-
e ial in dKO animals.
3.4. Mic o-s uc u al changes in he RPE and B uch's memb ane o NRF-2/
PGC-1αdKO mice
Ou aim was hen o e alua e whe he he NRF-2/PGC-1αdKO
issue samples showed AMD-like pa hological ea u es a he his olo-
gical le el. Analysis o he epoxy semi- hin and wax sec ions e ealed
B uch's memb ane dysmo phology, inc eased accumula ion o lipo-
uscin-like pa icles and cy oplasmic al e a ion in he dKO RPE
(Fig. 3A). D usen-like deposi s be ween RPE and B uch's memb ane
we e posi i e o ubiqui in s aining (Fig. 3B and C). The in acellula
cys ic degene a ion o RPE, nuclea deb is and apop osis (Fig. 3D) we e
obse ed in dKO RPE cells.
3.5. NRF-2/PGC-1αdKO mice RPE cells display inc eased ER s ess
ma ke s and loss o basal in oldings
The in ol emen o ER s ess was assessed by ul as uc u al ana-
lysis and s aining wi h an i-GRP78 and an i-ATF4. Ul as uc u al
analysis e ealed s a is ically significan ER acuoliza ion as a ma ke
o ER damage in dKO RPE cells compa ed o co esponding WT RPE
cells (Fig. 4A-C). The e inas om one-yea -old dKO exhibi ed mo e
in ense s aining o GRP78 (p < 0.05) and ATF4 (p < 0.01) in he
RPE laye when compa ed o he co esponding issues in WT con ols
(Fig. 4D-E).
3.6. Iba-1 is up egula ed and he size o RPE cell is inc eased in NRF-2/
PGC-1αdKO mice
The inna e immune sys em is ac i a ed in a numbe o degene a i e
and inflamma o y e inal diso de s, including AMD. Re inal mono-
nuclea phagocy es a e also p esen in mos mouse models o e inal
disease [45]. To e alua e whe he Iba-1 exp ession is enhanced in dKO
RPE due o inflamma o y p ocesses, Iba-1 analysis was pe o med in
RPE fla moun s. Da a in Fig. 5 show a s a is ically significan 400%
inc ease in he Iba-1 s aining in ensi y in RPE o one-yea -old NRF-2/
PGC-1αdKO e sus WT mice (p < 0.05). No ably, we also obse ed a
clea RPE size inc ease in ou dKO mice compa ed o hei WT coun-
e pa s (p < 0.01). Du ing no mal aging, an inc eased RPE cell size in
C57BL/6J mouse has been in e p e ed as a p ognosis o cell dea h [46].
3.7. NRF-2/PGC-1αdKO mice show age- ela ed isual loss and
pho o ecep o dysmo phology
Gi en he obse ed s uc u al changes no ed abo e, we subse-
quen ly assessed he in i o unc ion o od and cone pa hways by
pe o ming ERG in he WT and dKO mice. As p esen ed in Fig. 6, he
ERGs ampli udes we e lowe in dKO mice e en a a young age, bu he
diffe ence eached s a is ical significance only in b-wa e ampli udes (a-
wa e: p = 0.13; b-wa e: p < 0.05). The cone esponse, as de e mined
by he pai ed-flash pa adigm, was compa able be ween young dKO and
WT mice (Fig. 6E). Wi h age, he od- unc ion significan ly dec eased in
WT mice (a- and b-wa e: p < 0.001), while cone- unc ion emained
s able (Fig. 6B-E). G oup-wise compa ison e ealed significan diffe -
ences be ween old dKO and WT mice in da k-adap ed a- and b-wa e
ampli udes (a-wa e: p < 0.05, b-wa e: p < 0.01). The e was a clea
endency owa ds age-geno ype in e ac ion in cone-flash ampli ude
(p = 0.06), indica ing ha also cone- unc ion ends o dec ease in dKO
mice wi h age (see Fig. 6E). The a io be ween b- and a-wa e ampli-
udes emained unchanged ega dless o geno ype o age (Fig. 6D, all
g oup-wise compa isons p > 0.28), indica ing ha a pho o ecep o
de ec was he cause o he ERG ampli ude changes. Since impai ed
pho o ecep o unc ion was ound in he e ina o dKO mice, he nex
s ep was o s udy he pho o ecep o laye in mo e de ail. The pho o-
ecep o cells o dKO mice showed dec eased hickness o he ou e
nuclea laye (ONL) and diso ganiza ion in he ou e po ion o he ONL
(Fig. 6F-I). The s aining o p o ein agg ega ion, au ophagy and
Fig. 5. Inc eased immune cell infil a ion and he enla gemen o RPE
cells a e p esen in NRF-2/PGC-1αdKO mice. Rep esen a i e con ocal Iba-1
(g een) and Phalloidin ( ed) immunohis ochemical images o e inal fla
moun s samples o one-yea -old (A) WT and (B) NRF-2/PGC-1αdKO mice.
Inse s show he low magnifica ion iew om RPE laye . The scale ba indica es
20 µm. (C) The e was a 400% inc ease in Iba-1 posi i e cells in he NRF-2/PGC-
1αdKO RPE compa ed o he WT fla moun samples. (D) The RPE cell size
diffe ence in NRF-2/PGC-1αdKO and WT mice. The ho izon al black lines e-
p esen he a e age size o he RPE cell. *p < 0.05, S uden 's - es (n = 4).
S. Felszeghy e al. Redox Biology 20 (2019) 1–12
8
oxida i e s ess ma ke s and cellula mo phology o pho o ecep o s in
one-yea -old mice we e simila o hose obse ed in RPE cells
(Supplemen a y fig. 5). Elec on mic og aphs, in addi ion o RPE cells,
e ealed ocal pho o ecep o laye degene a ion in dKO mice
(Supplemen a y fig. 5).
3.8. NRF-2/PGC-1αdKO mouse model shows a d y AMD-like pheno ype
Taken oge he , ou dKO mice show pa hological signs ha a e
consis en wi h hose obse ed in pa ien s wi h d y AMD. The pa ho-
logical changes obse ed in hese dKO mice include age- ela ed RPE
degene a ion, in a- and ex acellula deposi accumula ion, B uch's
memb ane dysmo phology, immune cell in asion, damaged
mi ochond ia, impai ed au ophagy, al e ed pigmen a ion and isual
loss consis en wi h a de ec in pho o ecep o s (Fig. 7).
4. Discussion
Recen ly, impai ed p o eolysis in esponse o ch onic and o e
oxida i e s ess has been sugges ed as a key con ibu o o age- ela ed
e inal degene a ion, pa icula ly in AMD [1,8]. To s udy p o eolysis o
age- ela ed e inal degene a ion, we sough o de elop an animal model
ha would p o ide he mos obus p o ein agg ega ion in RPE cells.
P e iously, gene ic abla ion o he NRF-2 and PGC-1αhas been shown
o induce RPE degene a ion [15,30]. Ou findings e eal ha knockou
o bo h, NRF-2 and PGC-1α, esul ed in he highes inc ease o ma ke s
Fig. 6. Dysmo phology o pho o ecep o s coincides wi h impai ed e inal unc ion in NRF-2-PGC1αdKO mice. (A) Da k-adap ed ERG wa e o ms a 1.60 log cd·s/
m
2
flash. Thin lines ep esen indi idual mouse esponses and hick lines g oup-a e aged wa e o ms. (B) ERG b-wa e declined al eady in h ee-mon h-old (3 M) dKO
mice (n = 6) as compa ed o hei age-ma ched WT (n = 6) li e ma e mice, and he diffe ence became mo e significan in one-yea -old (1Y) mice (dKO: n = 7; WT:
n = 6). (C) ERG a-wa e ended o decline a bo h ages bu eached s a is ically significan le el only a olde age. (D) The a io be ween b- and a-wa e ampli udes did
no change ega dless o he geno ype o he age. (E) The double-flash pa adigm e ealed a endency owa ds age- ela ed decline in he cone-dominan unc ion in
dKO mice. (F) Thin uns ained wax sec ion, (G) DAPI s aining o ou e nuclea laye (ONL) in WT samples compa ed wi h (H,I) one-yea -old (y) dKO mice, e-
spec i ely. Pho o ecep o a ophy o he ONL ( ed e ical a ow) is seen in DAPI-labeled dKO e inas cu om sec ions loca ed 250–350 µm om he op ic ne e
head. Re inas show diso ganiza ion in he ou e po ion o he ONL (yellow as e ics). The scale ba indica es 5 µm. **p < 0.01, *p < 0.05, wo-way ANOVA.
S. Felszeghy e al. Redox Biology 20 (2019) 1–12
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