Early apoptosis of rod photoreceptors in Rpe65(-/-) mice is associated with the upregulated expression of lysosomal-mediated autophagic genes.

Abstract

RPE65-related Leber's congenital amaurosis (LCA) is a rod-cone dystrophy whose clinical outcome is mainly attributed to the loss of rod photoreceptors followed by cone degeneration. Pathogenesis in Rpe65(-/-) mice is characterized by a slow and progressive degeneration of rods dependent on the constitutive activation of unliganded opsin. We previously reported that this opsin-mediated apoptosis of rods was dependent on Bcl-2-apoptotic pathway and Bax-induced pro-death activity. In this study, we report early initial apoptosis in the newly differentiated retina of Rpe65(-/-) mice. Apoptotic photoreceptors were identified as rods and resulted from pathological phototransduction signaling. This wave of early apoptosis triggered Bcl-2-related pathway and Bax apoptotic activity, while activation of the caspases was not induced. Following cellular stress, multiple signaling pathways are initiated which either commit cells to death or trigger pro-survival responses including autophagy. We report that Bcl-2-related early rod apoptosis was associated with the upregulation of autophagy markers including chaperone-mediated autophagy (CMA) substrate receptor LAMP-2 and lysosomal hydrolases Cathepsin S and Lysozyme. This suggests that lysosomal-mediated autophagy may be triggered in response to early rod apoptosis in Rpe65-LCA disease. These results highlight that Rpe65-related primary stress induces early signaling events, which trigger Bax-induced-apoptotic pathway and autophagy-mediated cellular response. These events may determine retinal cell fate, progression and severity of the disease.

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Early apoptosis of rod photoreceptors in Rpe65?/?mice is associated with the
upregulated expression of lysosomal-mediated autophagic genes
Sylviane Métraillera, Daniel F. Schordereta,b,c, Sandra Cotteta,b,*
aIRO, Institute for Research in Ophthalmology, Sion, Switzerland
bDepartment of Ophthalmology, University of Lausanne, Lausanne, Switzerland
cSchool of Life Sciences, Federal Institute of Technology (EPFL), Lausanne, Switzerland
a r t i c l e i n f o
Article history:
Received 2 August 2011
Accepted in revised form 23 December 2011
Available online 28 December 2011
Keywords:
retina
rod
degeneration
apoptosis
autophagy
survival
a b s t r a c t
RPE65-related Leber’s congenital amaurosis (LCA) is a rod-cone dystrophy whose clinical outcome is
mainly attributed to the loss of rod photoreceptors followed by cone degeneration. Pathogenesis in
Rpe65?/?mice is characterized by a slow and progressive degeneration of rods dependent on the
constitutive activation of unliganded opsin. We previously reported that this opsin-mediated apoptosis
of rods was dependent on Bcl-2-apoptotic pathway and Bax-induced pro-death activity. In this study,
we report early initial apoptosis in the newly differentiated retina of Rpe65?/?mice. Apoptotic
photoreceptors were identified as rods and resulted from pathological phototransduction signaling.
This wave of early apoptosis triggered Bcl-2-related pathway and Bax apoptotic activity, while acti-
vation of the caspases was not induced. Following cellular stress, multiple signaling pathways are
initiated which either commit cells to death or trigger pro-survival responses including autophagy. We
report that Bcl-2-related early rod apoptosis was associated with the upregulation of autophagy
markers including chaperone-mediated autophagy (CMA) substrate receptor LAMP-2 and lysosomal
hydrolases Cathepsin S and Lysozyme. This suggests that lysosomal-mediated autophagy may be
triggered in response to early rod apoptosis in Rpe65-LCA disease. These results highlight that Rpe65-
related primary stress induces early signaling events, which trigger Bax-induced-apoptotic pathway
and autophagy-mediated cellular response. These events may determine retinal cell fate, progression
and severity of the disease.
? 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Leber’s congenital amaurosis (LCA) gathers a group of inherited
retinal dystrophies characterized by severe retinal defects with
progressive degeneration of the structured retina. LCA2, caused by
mutations in the gene encoding the retinal pigment epithelium
(RPE)-specific protein 65 kDa (RPE65), accounts for about 10% of
LCA (El Matri et al., 2006). Pathogenesis in LCA2 is characterized by
early and severe rod-cone dystrophy (Jacobson et al., 2005) and its
clinical outcome has mainly been attributed to the loss of rod and
cone photoreceptors (Jacobson et al., 2007, 2008). The retinoid
isomerase RPE65 directs the synthesis of the 11-cis retinol neces-
sary for light-induced phototransduction mediated by rod and cone
opsins (Jin et al., 2005; Moiseyev et al., 2005).
Rpe65?/?mice exhibit defects in retinal morphology, function
and biochemistry resembling those seen in human LCA2 (Galvin
et al., 2005; Redmond et al., 1998). Rpe65?/?mice lack 11-cis
retinal chromophore (Redmond et al., 1998; Seeliger et al., 2001)
and unliganded rod opsin triggers light-independent, constitutive
activation of residual phototransduction cascade leading to
photoreceptor cell death (Woodruff et al., 2003).
Photoreceptor apoptosis has been described as a final outcome
in retinal degeneration, although the molecular pathways trig-
gering retinal cell death still remain unclear (Doonan and Cotter,
2004; Marigo, 2007). Loss of retinal cells in many ocular dystro-
phies has been shown to rely on caspase-dependent or -indepen-
dent apoptosis mediated by the Bcl-2 family of proteins (Cottet and
Schorderet, 2009). We previously showed in Rpe65?/?mice that
constitutive opsin-induced photoreceptor apoptosis depended on
Bcl-2-related pathway and Bax pro-death activity (Cottet et al.,
2006; Cottet and Schorderet, 2008; Hamann et al., 2009). Auto-
phagy has been reported to prevent retinal cell death in Drosophila
(Wang et al., 2009) and to contribute to neuroprotection in retinal
* Corresponding author. IRO, Institute for Research in Ophthalmology, Avenue
Grand-Champsec 64, 1950 Sion, Switzerland. Tel.: þ41 (0) 27 205 79 06; fax: þ41
(0) 27 205 79 01.
E-mail address: sandra.cottet@irovision.ch (S. Cottet).
Contents lists available at SciVerse ScienceDirect
Experimental Eye Research
journal homepage: www.elsevier.com/locate/yexer
0014-4835/$ e see front matter ? 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2011.12.019
Experimental Eye Research 96 (2012) 70e81

Page 2

ganglion cells (Kim et al., 2008; Wu et al., 2006), indicating that
autophagy may act as a survival pathway in retinal dystrophies.
Similarly, autophagy has been shown to be involved in cone (Punzo
et al., 2009) and rod (Kunchithapautham et al., 2011) survival in
mice. Chaperone-mediated autophagy (CMA) is essential for
neuronal survival and is involved in cellular stress response
through selective targeting of cytosolic proteins for lysosomal
degradation (Cuervo, 2010; Dice, 2007). The CMA substrate
receptor LAMP-2A is required for the binding of the targeted
proteins and their translocation to the lysosomal lumen for further
degradation by lysosomal hydrolases. Increased transcriptional
(Kiffin et al., 2004) and post-transcriptional (Cuervo and Dice,
2000; Kiffin et al., 2004) expression of LAMP-2A has been
observed following CMA activation.
Here we report an early apoptosis of rods in Rpe65-LCA mice
resulting from pathological opsin signaling. Initial degeneration of
rodstriggeredBcl-2-relatedpathwayandBax-dependentapoptosis,
but did not rely on caspase activation. In response to early initial
apoptosis,lysosomal-mediatedautophagywasinducedthroughthe
upregulation of LAMP-2 and lysosomal hydrolases Cathepsin S and
Lysozyme, while macroautophagy was not involved in the patho-
genesis of the disease. Altogether these results indicate that LAMP-
2-mediated CMA may be induced in response to Bcl-2-related early
rod apoptosis and, in turn, may act as a survival response against
retinal degeneration in RPE65-LCA disease.
2. Materials and methods
2.1. Mouse lines and genotyping
These studies adhered to the Association for Research in Vision
and Ophthalmology (ARVO) statement for the use of animals in
ophthalmic and vision research and were approved (permit
number VS13) by the Veterinary service of the State of Valais
(Switzerland). Animals were kept in a 12-h light/12-h dark cycle
with unlimited access to food and water. Wild-type C57BL/6 mice
(wt) were purchased from Charles River Laboratories (Les Oncins,
France). Rpe65?/?mice are on a C57BL/6 genetic background (from
Dr T.M. Redmond, National Institutes of Health, Bethesda, USA)
(Redmond et al., 1998) and Gnat1?/?(from Dr. J. Lem, Tufts-New
England Medical Center, Boston, USA) are on a BALB/c genetic
background (Calvert et al., 2000). Rpe65?/?/Gnat1?/?double knock-
out mice were generated by crossbreeding Rpe65?/?mice with
Gnat1?/?mice. Bax?/?mice are homozygous for the Baxtm1Sjk
mutation (Charles River Laboratories). The targeted disruption of
the Bax gene was performed in a 129-derived RW-G ES cell line and
has been backcrossed 8 generations to C57BL/6. Bax?/?/Rpe65?/?
double knock-out mice were generated as previously described
(Hamann et al., 2009). Genotyping of the mice was determined by
PCR analysis with genomic DNA isolated from tail tissue. Rpe65-
and Gnat1-specific PCRgenotypings weredetermined aspreviously
described (Calvert et al., 2000; Redmond et al., 1998) and Bax-
specific PCR genotyping as recommended by The Jackson Labora-
tory (http://jaxmice.jax.org).
2.2. Tissue isolation and RNA extraction
Age-matched animals were killed by cervical dislocation.
Retinas from each mouse strain were dissected under a microscope
to exclude extra-retinal tissues, and were quickly isolated in RNA-
later (Ambion, Huntingdon, United Kingdom) before being trans-
ferred in TRIzol (Invitrogen, Basel, Switzerland) and stored
at ?80?C until RNA extraction. Total RNA was extracted according
to manufacturer’s instructions and the amount of total RNA was
determined by Ribogreen assay (Invitrogen).
2.3. Real-time PCR analysis
The equivalent of 50 ng of original total RNA was used for PCR
amplification using the 2? brilliant SYBR Green QPCR Master Mix
(Roche, Rotkreuz, Switzerland) with either 250 nM (Bax, Beclin-1,
Cathepsin S), 400 nM ribosomal protein L8 (Rl8), 500 nM (Bmf,
Bak, Bcl-2, Bcl-xL,, Caspase-7, Cathepsin B, Lamp-1, Lamp-2) or
1000 nM (Bad, Lysozyme) forward and reverse primer pairs. Real-
time PCR was performed in triplicate in a Mx3000PTM system
(Agilent Technologies, Waldbronn, Germany) with the following
cycling conditions: 40 cycles of denaturation at 95?C for 30 s,
annealing at 55?C (60?C for Bcl-2, Beclin-1, Cathepsin B) for 30 s,
and extension at 72?C for 60 s. Quantitative values were obtained
by the cycle number (Ct value) reflecting the point at which fluo-
rescence starts to increase above background at a fixed threshold
level. Values obtained for the target genes were normalized with
the housekeeping gene Rl8. For primer sequences, see Table S1.
2.4. Western blot analysis
Retinas were dissected and lysed in 100 ml RIPA buffer with
proteaseandphosphataseinhibitors.Equivalentamountsofprotein,
as determined by the Bradford protein assay (Bio-Rad, Life Science,
Reinach, Switzerland), were resolved on 12% SDS-PAGE gels fol-
lowed by transfer on PVDF membrane (Whatman/Schleicher &
Schuell, Sanford, UK). Membranes were blocked in 5% non-fat dried
milk before being immunoassayed using rabbit antibodies against
Caspase-7 (9492, 1/200), LC3-I/-II (2775, 1/10000), mTOR (2972, 1/
20000) and phosphorylated mTOR (PmTOR, 2971s, 1/10000) from
Cell Signaling Technology (Allschwil, Switzerland). Tubulin was
detected using mouse anti-a-tubulin (T6074,1/400000) from Sigma
(Buchs, Switzerland). HeLa human cervical cancer cells treated or
not with 1 mM staurosporine (STS) for 3 h were used as positive
control for cleaved Caspase-7.
2.5. Caspase assay
Caspase colorimetric assay (LubioScience GmbH, Lucerne,
Switzerland) was performed according to manufacturer’s instruc-
tion. Briefly, 50e150 mg of proteins from retina cell extracts were
incubated with 200 mM of Caspase-3/-7-specific DEVD-pNA
substrate for 1 h at 37?C. Spectrophotometric detection of the
chromophore p-nitroanilide (pNA) liberated after caspase cleavage
was quantified using a microtiter plate reader at 400-/405-nm.
661 W mouse photoreceptor cell line treated or not with 200 nM
STS for 16 h was used as positive control for caspase activity. 661 W
cells were generously provided by Dr. M. Al-Ubaidi (University of
Oklahoma, Oklahoma city, USA) (Tan et al., 2004).
2.6. Immunohistochemistry staining
Eyes were fixed in 4% paraformaldehyde (PFA)/phosphate-
buffered saline (PBS) for 45 min, followed by cryoprotection in 30%
sucrose/PBS. Ten mm-embedded frozen sections were further pro-
cessed for immunohistochemistry. Briefly, frozen retina sections
were blocked in PBS with 2% normal goat serum (Sigma) and 0.2%
Triton X-100 (Sigma) for 1 h at room temperature (RT) and incu-
bated with primary antibodies in the blocking buffer overnight at
4?C. Sections were blocked again in blocking buffer for 30 min at RT
before being incubated with fluorochrome-conjugated secondary
antibody for 1 h at RT. Incubation with secondary antibody alone
was used as a negative control. Species and dilutions of the anti-
bodies used wereas follows: rabbit anti-rhodopin 1D4 (1/100; from
Dr R. Molday, USA),rabbit anti-cone arrestin (LUMIJ,1/100; fromDr.
C. Craft, USA) (Zhu et al., 2003, 2002) and Alexa Fluor 488 goat anti-
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rabbit IgG (1/500; Invitrogen). Following 3 washes in PBS, sections
were mounted in Citifluor AF1 (Citifluor Ltd, London, United
Kingdom).Tissuesectionswere
(Invitrogen) to identity retinal cell layers.
counterstained withDAPI
2.7. Terminal dUTP Nick end-labeling (TUNEL) of fragmented DNA
DNA strand breaks in retinal cell nuclei were detected by TUNEL
assay on retina sections. Enucleated eyes were fixed in 4% PFA/PBS
for 45 min, followed bycryoprotection in 30% sucrose/PBS. Ten mm-
embedded frozen sections were further processed for TUNEL
staining with terminal deoxynucleotidyl transferase (TdT) and TMR
nucleotides to detect red fluorescence staining of apoptotic
photoreceptor nuclei according to manufacturer’s instructions
(Roche). Retina sections were further counterstained with DAPI
(Invitrogen) to identify the retinal cell layers, followed by three
washes in PBS, before to be mounted in Citifluor AF1 (Citifluor). In
quantitative experiments, apoptotic cells were counted in three
retina sections per eye and time-point, and the resulting numbers
from each experiment (n ¼ 4) were averaged.
Fig. 1. Early photoreceptor apoptosis was initiated in Rpe65-deficient mice. A. TUNEL assay (in red) in retina sections (400X) was performed in wt and Rpe65?/?mice from post-
natal days (P)7 to P25. DAPI counterstaining of the nuclei (in blue) was used to identify the retinal cell layers. B. Counting of TUNEL-positive apoptotic cells in the ONL of wt (gray
triangle) and Rpe65?/?(black square) retinas showed that photoreceptor apoptosis in Rpe65?/?mice appeared at P13, peaked between P16 and P19, and quickly decreased from P19
to P25. Two-way ANOVA analysis indicated that the number of apoptotic cells was significantly different between genotypes for all time-points (p < 0.0001). Data are the mean ? SE
of three independent experiments. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
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2.8. Imaging
Images were viewed under a fluorescence microscope equip-
ped with a digital camera (Olympus BX61; Olympus, Lausanne,
Switzerland) using appropriate filters.
2.9. Statistical analysis
All results were expressed as means ? SE of the indicated
number of experiments. Statistical significance was either calcu-
lated with the t-test or the ANOVA test followed by Bonferroni post
test adjustment.
3. Results
3.1. Early initial apoptosis of rod photoreceptors in Rpe65?/?mice
Rod degeneration in Rpe65?/?mice has been described as a slow
and progressive disease. Previous studies reported that the loss of
rods, characterized by decreased outer nuclear layer (ONL) thick-
ness, was not significant before 2 months of age and further pro-
gressed slowly during the course of the disease (Hamann et al.,
2009; Redmond et al., 1998; Woodruff et al., 2003). However, we
observed in this study that a strong early apoptotic response was
initiated in the newly differentiated Rpe65?/?retinas. Early retinal
cell death was assessed by TUNEL assay (Fig. 1A), showing that
photoreceptor apoptosis in the ONL of Rpe65?/?mice appeared at
post-natal day (P)13, peaked between P16 and P19, and quickly
decreased from P19 to P25 (Fig. 1B). This early apoptotic response
did not lead to a detectable decrease in the ONL thickness.
To further ascertain the identity of the degenerating photore-
ceptors during early apoptosis, we performed TUNEL assay
combinedwithrod-andcone-specificimmunohistologicalanalyses.
As depicted in Fig. 2, TUNEL-positive nuclei in retina from P15
Rpe65?/?miceco-localizedwithrhodopsin-labeledrods(upperright
panel; arrowheads) but not with cone arrestin-labeled cones (lower
right panel; arrows), demonstrating that initial apoptotic photore-
ceptors are rods and not cones.
Taken together these results show that sustained but transient
rod apoptosis is triggered early in newly differentiated retina in
Rpe65-LCA disease.
3.2. Rpe65-dependent early rod apoptosis was triggered by
pathological opsin activity
To assess whether early rod apoptosis was triggered by patho-
logical activity of unliganded opsin, TUNEL assay was performed in
P15 wt, Rpe65?/?and double knock-out Rpe65?/?/Gnat1?/?mice
(Fig. 3). In contrast to Rpe65?/?retina, apoptosis of rods was pre-
vented in phototransduction-deficient Rpe65?/?/Gnat1?/?retina in
which phototransduction signaling was blocked in the absence of
functional rod transducin (Gnat1) (Woodruff et al., 2003).
During post-natal development of the retina, retinal cells
undergo naturally occurring cell death to proceed with terminal
differentiation of the retinal layers. To further exclude thatearly rod
apoptosis was due to altered physiological apoptosis, TUNEL-
positive apoptotic cells were quantified in wt, Rpe65?/?and
Rpe65?/?/Gnat1?/?mice from P7 to P13. In accordance with several
reports (Donovan et al., 2006; Mervin and Stone, 2002; Péquignot
et al., 2003; Portera-Cailliau et al., 1994; Young, 1984), we
observed that physiological apoptosis triggered from the second
week after birth essentially occurred within the inner nuclear layer
(INL) (Fig. 1). Although some variation in the number of TUNEL-
positive cells in the INL was observed at P7, no shift or delay in
the course of physiological apoptosis was observed among all
mouse strains from P7 to P13 (Fig. 4). Altogether these results
indicate that naturally occurring cell death proceeds normally in
the differentiating Rpe65-deficient retinas and that early rod
apoptosis is triggered by pathological opsin activity.
Fig. 2. Immunohistological analysis identified early apoptotic photoreceptors as rods. Combined TUNEL assay (in red) and immunohistological analyses (in green) using rod-specific
rhodopsin (Rho/TUNEL/DAPI panels) or cone-specific arrestin antibodies (CArr/TUNEL/DAPI panels) were performed in retina sections (600X) from P15 wt and Rpe65?/?mice. TUNEL-
positive apoptotic nuclei co-localized with rods (arrowheads) but not with cones (arrows). DAPI counterstaining (in blue) was used to label the nuclei within the ONL. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.3. Early apoptosis of rod photoreceptors depended on Bax-
induced apoptotic pathway
To ascertain whether activation of the Bcl-2-apoptotic pathway
was associated with early rod apoptosis, we first investigated
gene regulation of the Bcl-2-related members by quantitative PCR
(qPCR) analysis in wt, Rpe65?/?, Bax?/?and double knock-out
Rpe65?/?/Bax?/?mice at P15 and P30 (Fig. 5A). Increased expres-
sion of the pro-apoptotic effector Bax and Bak observed in Rpe65?/?
mice was inhibited in Rpe65?/?/Bax?/?mice. Transcriptional upre-
gulation of the BH3-only Bmf and Bad was observed in Rpe65?/?
mice at both ages. At P30, increased Bmf mRNA was also visible in
Rpe65?/?/Bax?/?mice and increased Bad mRNA was observed in
both Bax-deficient mice. These results suggest that Bmf and Bad are
not directly involved in Bax-dependent rod apoptosis. We cannot
exclude that altered expression of the BH3-only members is due to
a compensatory effect in the absence of Bax. In contrast, expression
of the anti-apoptoticmembers Bcl-2 and Bcl-XLwas downregulated
in Rpe65?/?and Rpe65?/?/Bax?/?retinasfromP15 toP30, indicating
that altered equilibrium between anti- and pro-apoptotic Bcl-2
memberstookplaceearlyinthepathogenesisofRpe65-LCAdisease.
We furtheraddressedthe directroleof Bax in earlyrodapoptosisby
TUNEL analysis. As shown in Fig. 5B and C, Rpe65-dependent rod
apoptosisobservedinRpe65?/?miceatP15andP30wascompletely
abolished in Rpe65?/?/Bax?/?mice. As previously reported by
Péquignot et al. (Péquignot et al., 2003), we also observed that
photoreceptor programmed cell death at P15 was inhibited in Bax-
deficient retinas as compared with wt retinas (Fig. 5C).
Altogether these results indicate that early pathological
apoptosis of the rods is associated with altered regulation of the
Bcl-2-related apoptotic pathway and is dependent on Bax pro-
death activity.
Fig. 3. Rpe65-dependent early rod apoptosis was triggered by pathological opsin activity. TUNEL assay (A) and counting (B) of TUNEL-positive apoptotic cells in the ONL showed that
early apoptosis of rods was prevented in P15 phototransduction-deficient Rpe65?/?/Gnat1?/?mice as compared in wt and Rpe65?/?mice (* p < 0.001 by one-way ANOVA for Rpe65?/
?versus wt and Rpe65?/?/Gnat1?/?). Data are the mean ? SE of three independent experiments. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Fig. 4. Rpe65-dependent early apoptosis was not due to altered naturally occurring cell
death during retinal development. TUNEL-positive apoptotic nuclei were counted
within the INL of wt, Rpe65?/?and Rpe65?/?/Gnat1?/?mice from P7 to P13. Although
some variation in the number of TUNEL-positive cells was shown at P7, no shift in the
kinetics of programmed cell death was observed in the developing retina of all mouse
strains. (* p < 0.05 by one-way ANOVA for Rpe65?/?versus Rpe65?/?/Gnat1?/?at P7).
Data are the mean ? SE of four independent experiments.
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3.4. Bax-dependent apoptosis of the rods did not rely on caspase
activation
In response to various stresses, Bax/Bak-dependent mitochon-
drial disruption may in turn activate caspase-dependent or
caspase-independent apoptosis.
Inaninitialgeneprofilingexperiment,wereportedthatCaspase-
7mRNAwasdownregulatedinRpe65?/?mice(Cottetetal.,2006).To
addresstheroleof caspasesinrodapoptosis, wefirstinvestigatedin
phototransduction-deficient
Rpe65?/?/Gnat1?/?
Rpe65-mediated altered regulation of Caspase-7 was dependent on
constitutive opsinactivity.As reportedinFig. 6A,downregulation of
Caspase-7 still persisted in Rpe65?/?/Gnat1?/?mice, indicating that
altered caspase expression was independent of pathological opsin
activity.
We further investigated whether caspase activity was triggered
in diseased retinas. Activation of Caspase-7 was first assessed in wt
and Rpe65?/?mice by Western blot analysis using a Caspase-7
antibody that recognizes both the 35 kDa full length and the
active 20 kDa cleaved protein (Fig. 6B). At all ages, only the full-
length inactive protein could be detected in healthy and diseased
retinas, while cleaved Caspase-7 was shown in staurosporine-
treated HeLa cells used as control. No significant decrease in the
level of Caspase-7 protein could be observed in Rpe65?/?mice
compared to wt mice, as it was the case at the transcript level. This
might reflect additional post-transcriptional regulation of the
protein stability. Caspase activation was also addressed by
measuring the cleavage activity of Caspase-3/-7 that both recognize
the substrate amino acid sequence DEVD (Fig. 6C). No increase in
caspase activity was detected in Rpe65?/?retinas, whereas induced
activity was shown in staurosporine-treated 661 W cells used as
control (al-Ubaidi et al., 1992).
In summary, these results indicate that caspase activation is not
involved in Bax-dependent apoptosis of the rods in Rpe65?/?mice.
micewhether
3.5. Lysosomal-mediated autophagic pathway was induced in
response to early retinal apoptosis
We further reasoned that the decrease in rod apoptosis
observed in Rpe65?/?mice from P15 to P30 might be explained
either by the downregulation of triggered death pathway or by the
activation of pro-survival pathway in response to Rpe65-LCA retinal
degeneration. Autophagy acts as a survival pathwayagainst cellular
stress through lysosomal degradation of cytoplasmic content
(Eskelinen and Saftig, 2009; Levine and Kroemer, 2008) and has
been involved in a protective response in retinal degeneration (Kim
et al., 2008; Wang et al., 2009; Wu et al., 2006). We thus addressed
the question whether such a protective signal might be activated in
response to early Rpe65-related retinal apoptosis.
Regulated expression of autophagic markers during early
apoptosis from P15 to P30 was investigated by qPCR (Fig. 7A).
Autophagic markers including the lysosomal hydrolases Cathepsin
S and Lysozyme, as well as the CMA substrate receptor LAMP-2
were significantly upregulated as early as at P15 and further
increased from P15 to P30 in Rpe65?/?mice. In contrast, expression
of Cathepsin B and the lysosomal marker LAMP-1 remained
unchanged. We further investigated whether induction of the
autophagic response was dependent on constitutive opsin activa-
tion (Fig. 7B). Upregulation of Cathepsin S, Lysozyme and LAMP-2
was maintained in phototransduction-deficient Rpe65?/?/Gnat1?/
?mice. At P30, Cathepsin S transcript was significantly increased in
Rpe65?/?/Gnat1?/?mice compared to wt mice, but around 2-fold
less than in Rpe65?/?mice. Altogether these data suggest that
lysosomal autophagic response is triggered in response to a cellular
stress independent of constitutive opsin signaling activity.
3.6. Macroautophagy was not activated in Rpe65-LCA disease
As lysosomal hydrolases and LAMP-2 are involved in the
degradation of autophagosomes through fusionwith lysosomes, we
also investigated whether a macroautophagic response was acti-
vated in P15 and P30 Rpe65?/?mice.
Atg6/Beclin-1 and Atg8/LC3 play a central role in the regulation
of macroautophagyandthe
(Eskelinen and Saftig, 2009). As assessed by qPCR, expression of
Beclin-1 was not significantly modified in Rpe65?/?
(Fig. 8A). Furthermore, Western blot analysis did not show signifi-
cant increase in lipidated LC3-II, the activated form of the protein
associated with the autophagosome membrane (Fig. 8B). Similarly
the mTOR pathway, known as a negative regulator of macro-
autophagy (Levine and Kroemer, 2008), was not involved as
reflected by unchanged levels of phosphorylated mTOR (PmTOR)
(Fig. 8C). Therefore, these results show that macroautophagy is not
induced in response to early rod apoptosis in Rpe65-LCA disease.
formationof autophagosomes
retinas
4. Discussion
In this study, we report for the first time an early wave of
apoptosis of rods in Rpe65-LCA mice. This is in contrast with the
current view of Rpe65-mediated rod cell death as a slow and
progressive degeneration (Hamann et al., 2009; Redmond et al.,
1998; Rohrer et al., 2003; Woodruff et al., 2003; Wrigstad et al.,
1994). Nevertheless in human LCA2, severely impaired vision is
already diagnosed at birth or in the first few months of life,
although the retina of newbornpatients appears anatomically fairly
normal. This supports the idea that structural alterations of the
retina occur well after functional dysfunction of the photo-
transduction signaling. However, a prenatal study in 33-week
RPE65-LC2 retina reported loss of photoreceptors associated with
reduced level of rhodopsin and shortening of rod outer segments
(OS) (Porto et al., 2002). This suggests that early initial apoptosis is
also triggered in human LCA, corroborating early rod degeneration
observed in the present study in Rpe65?/?mice. Early initial
apoptosis has also been described in other mouse models of
Retinitis Pigmentosa (RP). In rd/rd mice, a single apoptotic wave
triggers rod cell death from P10, peaking by P14 and resulting in
complete loss of photoreceptors before 1 month of age (Lohr et al.,
2006; Portera-Cailliau et al., 1994). This contrasts with the patho-
genesis observed in rds/rds mice which fail to develop normal OS
structure (Hughes et al., 2004). Although photoreceptor apoptosis
begins around P10 and peaks at P18, retinal degeneration further
progresses but much more slowly, with all rods lost at approxi-
mately 1 year of age (Hawkins et al., 1985; Portera-Cailliau et al.,
1994; Sanyal et al., 1980; Yang et al., 2007).
Early retinal apoptosis in Rpe65?/?mice prompted us to inves-
tigate the death pathways involved in the early loss of rods. We
report in this study that initial rod demise was dependent
on Bax-induced apoptotic pathway, as reflected by complete inhi-
bition of rod apoptosis in the absence of functional Bax. We also
observed that Rpe65-dependent upregulation of Bak was inhibited
in Rpe65?/?/Bax?/?mice, suggesting that Bak along with Bax may
participate in rod demise. Moreover, Rpe65-dependent down-
regulated expression of Bcl-2 in Rpe65?/?/Bax?/?mice further
indicated that protection against apoptosis could not be explained
by re-expression of the anti-apoptotic protein. In addition, early
transcriptional regulation of the Bcl-2 members supported initial
altered equilibrium between pro- and anti-apoptotic partners in
predegenerate Rpe65?/?retina. We further showed that early
apoptosis was prevented in phototransduction-deficient Rpe65?/
?/Gnat1?/?mice, demonstrating that the initial apoptotic response
resulted from constitutive opsin activity. This confirmed our
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Fig. 5. Early rod apoptosis depended on Bax-induced apoptotic pathway. Experiments were performed in P15 and P30 wt (white square), Rpe65?/?(black square), Bax?/?(dark gray
square) and double knock-out Rpe65?/?/Bax?/?(light gray square). A. QPCR analysis showed that upregulation of the pro-apoptotic effector Bax and Bak in Rpe65?/?mice
was abolished in Rpe65?/?/Bax?/?mice (* p < 0.001 by one-way ANOVA for wt versus Rpe65?/?at P15 and P30). Increased expression of the BH3-only Bmf and Bad was shown in
Rpe65?/?mice at both ages. At P30, increased Bmf mRNA was also visible in Rpe65?/?/Bax?/?mice and increased Bad mRNA was observed in both Bax-deficient mice (* p < 0.001 by
one-way ANOVA for wt versus Rpe65?/?at P15 and P30 and for wt versus Rpe65?/?/Bax?/?at P30; ** p < 0.05 by one-way ANOVA for wt versus Bax?/?at P30). Decreased
mRNA expression of the anti-apoptotic members Bcl-2 and Bcl-XLwas further observed in Rpe65-deficient retinas from P15 to P30 (** p < 0.001 by t-test for Rpe65?/?at P15 versus
Rpe65?/?at P30). Data are the mean ? SE of at least three independent experiments. B. TUNEL assay in retina sections (400X) and (C) counting of TUNEL-positive apoptotic cells in
the ONL showed that early rod apoptosis was completely abolished in Rpe65?/?/Bax?/?mice at both ages (* p < 0.001 by one-way ANOVA for Rpe65?/?/Bax?/?versus Rpe65?/?mice).
Data are the mean ? SE of three independent experiments. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
S. Métrailler et al. / Experimental Eye Research 96 (2012) 70e81
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Fig. 6. Bax-dependent apoptosis of the rods did not rely on caspase activation. A. QPCR
analysis showing that Rpe65-dependent transcriptional downregulation of Caspase-7
still persisted in phototransduction-deficient Rpe65?/?/Gnat1?/?mice (* p < 0.001 by
one-way ANOVA for Rpe65?/?versus wt and Rpe65?/?/Gnat1?/?). Data are the
mean ? SE of four independent experiments. Caspase activation was further investi-
gated by (B) Western blot analysis using a Caspase-7 antibody that detects both the
inactive 35 kDa full-length and the active 20 kDa cleaved proteins (one representative
experiment out of four was shown), as well as by (C) caspase colorimetric assay to
measure Caspase-3/-7 activity using the specific chromophore substrate DEVD-pNA.
No activation of the effector caspases could be observed in Rpe65?/?mice at all ages.
Data from caspase assay are the mean ? SE of three independent experiments.
Untreated (nt) or staurosporine (STS)-treated HeLa (1 mM for 3 h) and 661 W cells
(200 nM for 16 h) were used as controls.
Fig. 5. (continued).
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previous
pathway was triggered in adult Rpe65?/?mice during progression
of the disease (Hamann et al., 2009).
We also observed that Bax-induced retinal degeneration did not
trigger caspase-mediated death pathway, as reflected by the lack of
activation of effector Caspase-3 and -7 in diseased mice even at the
peak of early rod apoptosis. Although Caspase-3 has been involved
in photoreceptor apoptosis in several RP models (Bode and
Wolfrum, 2003; Hughes et al., 2004; Liu et al., 1999), caspase-
independent pathways may trigger retinal cell death that
involveslysosomalproteases
Kunchithapautham and Rohrer, 2007; Lohr et al., 2006), calpains
(Paquet-Durand et al., 2006; Sanges et al., 2006) and cathepsins
(Ahuja et al., 2008; Doonan et al., 2005; Rakoczy et al., 2002).
observationthatopsin-dependentBcl-2-apoptotic
(Doonanetal., 2005;
Autophagy is mainly described as a pro-survival response to
preserve homeostasis under cellular stress (Levine and Kroemer,
2008). In the present study, we report that early rod apoptosis in
Rpe65?/?mice was associated with the upregulation of autophagy
markers including CMA substrate receptor LAMP-2 and lysosomal
hydrolasesCathepsinS and
lysosomal-mediated autophagy may be triggered in response to
early rod apoptosis. Similarly, it was shown in rds/rds mice that the
activity of Cathepsin B and Lysozyme was induced after the peak of
rod apoptosis at a time when cell death occurred at a much slower
rate (Lohr et al., 2006). In summary, these results suggest that
LAMP2-mediated CMA may counteract early rod apoptosis and act
as a survival response against retinal degeneration in Rpe65-LCA
disease. CMA has been shown to be altered inpatients with Danon’s
Lysozyme. Thissuggests that
Fig. 7. Lysosomal-mediated autophagic pathway was induced in response to early rod apoptosis. Transcriptional expression of autophagy markers was investigated by qPCR in P15
and P30 wt (white square), Rpe65?/?(black square) and Rpe65?/?/Gnat1?/?(gray square) mice. A. Induced expression of the autophagic genes Cathepsin S (CathS), Lysozyme and
Lamp-2 was observed in Rpe65?/?mice compared to wt mice at both ages, whereas Cathepsin B (CathB) and Lamp-1 transcripts remained unchanged. Data are the mean ? SE of
four independent experiments. Statistical analyses by t-test were performed (* p < 0.005 and ** p < 0.001 for Rpe65?/?at P15 versus P30; # p < 0.005 and ## p < 0.0001 for wt
versus Rpe65?/?at P15 and P30). B. Rpe65-dependent upregulation of Cathepsin S, Lysozyme and Lamp-2 was maintained in phototransduction-deficient Rpe65?/?/Gnat1?/?mice.
Data are the mean ? SE of four independent experiments. Statistical analyses by ANOVA test were performed (** p < 0.001 for wt versus Rpe65?/?and Rpe65?/?/Gnat1?/?; * p < 0.01
for wt versus Rpe65?/?/Gnat1?/?at P30; ## p < 0.001 for Rpe65?/?versus Rpe65?/?/Gnat1?/?at P30).
S. Métrailler et al. / Experimental Eye Research 96 (2012) 70e81
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disease and LAMP-2 mutation (Nishino et al., 2000) and these
patients also suffered from retinopathy (Prall et al., 2006;
Schorderetet al., 2007; Tayloret al., 2007). Autophagy has also been
demonstrated to contribute to neuroprotection in retinal ganglion
cells (Kim et al., 2008; Wu et al., 2006) and to prevent retinal cell
death in Drosophila (Wang et al., 2009). Furthermore, it has been
shown in Drosophila NorpA mutant that stable rhodopsinearrestin
complexes fail to undergo lysosomal degradation and that impaired
lysosomal turnover of rhodopsin is involved in photoreceptor
degeneration (Chinchore et al., 2009). In Rpe65?/?mice, constitu-
tive phosphorylation of opsin apoprotein (Ablonczy et al., 2002)
and high binding affinity of rod arrestin for phosphorylated opsin
(Gurevich and Benovic,1993) have been reported. Light-dependent
translocation of arrestin to OS and redistribution of transducin to IS
are impaired in Rpe65?/?rod photoreceptors (Mendez et al., 2003).
Moreover, KFERQ-like CMA target motif (Dice, 1990) is present in
the rod-specific proteins arrestin, recoverin, rhodopsin kinase and
transducin. It is thus tempting to speculate that accumulating
arrestin or other phototransduction proteins may overwhelm
normal protein degradation turnover and trigger CMA activation.
Further experiments are needed to investigate the role of rod-
specific proteins in autophagy response and to assess whether
modulation of CMA may impact on rod survival in Rpe65-LCA
disease.
Photoreceptor cells are very sensitive to any long-term stress
resulting in perturbed phototransduction cascade, energy metab-
olism or structural cell integrity. If pro-survival responses succeed
in re-establishing homeostasis, cells will overcome stressful
Fig. 8. Macroautophagy was not activated in Rpe65-deficient mice. Activation of macroautophagy was assessed in P15 and P30 wt (white square) and Rpe65?/?(black square) mice.
A. QPCR analysis showing that Beclin-1 transcript remained unchanged in mutant retina as compared with wt retina. Data are the mean ? SE of four independent experiments. B.
Western blot analysis of soluble LC3-I and lipidated LC3-II levels in retinal cell extracts (representative immunoblot, left panel). Following densitometric analysis of LC3 and tubulin
immunoblots, tubulin-normalized LC3-II/LC3-I ratio was expressed as fold change relative to wt retina (right panel). Data are the mean ? SE of three independent experiments. C.
Western blot analysis of the amount of total mTOR and phosphorylated mTOR (PmTOR) in retinal cell extracts (representative immunoblot, left panel). Following densitometric
analysis of mTOR, PmTOR and the corresponding tubulin immunoblots, tubulin-normalized PmTOR/mTOR ratio was expressed as fold change relative to wt retina (right panel). Data
are the mean ? SE of four independent experiments.
S. Métrailler et al. / Experimental Eye Research 96 (2012) 70e81
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stimulus and survive; if not, cell death will occur despite cellular
attempts to escape their fate. Hence, interplay between survival
and death pathways may dictate mid- to long-term photoreceptor
cell fate in inherited retinal degeneration. This opens new
perspectives toward the development of therapeutic strategies in
inherited retinal diseases that may be complementary, by
combining inhibition of pro-death pathways and sustained acti-
vation of pro-survival responses.
Acknowledgments
We thank Dr. M. Al-Ubaidi for providing the 661 W cell line,
Dr. R. Molday for anti-rhodopsin antibody, and Dr. C. Craft and the
Mary D. Allen Laboratory for Vision Research (Doheny Eye Institute)
for mouse cone arrestin antibody (LUMIJ).
This research was supported by The Swiss National Science
Foundation (SNSF) Grant 31003A-138313.
Appendix. Supplementary material
Supplementary data related to this article can be found online at
doi:10.1016/j.exer.2011.12.019.
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