ER Stress in Retinal Degeneration in S334ter Rho Rats
Vishal M. Shinde1., Olga S. Sizova1., Jonathan H. Lin2, Matthew M. LaVail3, Marina S. Gorbatyuk1*
1Department of Cell Biology and Anatomy, University of North Texas Health Science Center, North Texas Eye Research Institute, Fort Worth, Texas, United States of
America, 2Department of Pathology, University of California San Diego, San Diego, California, United States of America, 3Department of Ophthalmology, Beckman Vision
Center, University of California San Francisco, San Francisco, California, United States of America
The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a
model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The
Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H
Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR
is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of
S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21
stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12–
P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that
was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression
of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably
compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of
S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in
S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells.
Citation: Shinde VM, Sizova OS, Lin JH, LaVail MM, Gorbatyuk MS (2012) ER Stress in Retinal Degeneration in S334ter Rho Rats. PLoS ONE 7(3): e33266.
Editor: Demetrios Vavvas, Massachusetts Eye & Ear Infirmary - Harvard Medical School, United States of America
Received November 10, 2011; Accepted February 6, 2012; Published March 14, 2012
Copyright: ? 2012 Shinde et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Foundation Fighting Blindness TA-GT-0409-0508NTERI; National Institutes of Health (NIH) grant 1 RO1 EY020905-1;
Hope for Vision; NIH grants EY001919, EY006842, EY002162, EY018313, and EY020846; and a Research to Prevent Blindness grant. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Retinitis pigmentosa (RP) is an inherited retinal disorder that is
caused by the progressive loss of rod and cone photoreceptors with
clinical hallmarks that include sensitivity to dim light, abnormal
visual function and characteristic bone spicule deposits of pigment
in the retina . This disease affects approximately 1 in 3200, and
an estimated 1.5 million people are affected worldwide. The
autosomal dominant form of RP (ADRP) is associated with
mutations in at least 14 different genes; however, mutations in the
rhodopsin gene (RHO, OMIM 180380, accession ID U49742) are
the most prevalent mutation identified to date resulting in 30% to
40% of all ADRP cases [2,3].
S334ter rhodopsin (Rho) rats (line 4) express rhodopsin gene
containing an early termination codon at residue 334 and is a
model of a number of Rho truncation mutations in human RP
dopsin_Mutation). This mutation results in the expression of a
rhodopsin protein that lacks the 15 C-terminal amino acids that
are involved in rhodopsin trafficking to the photoreceptor outer
segments and in the deactivation of the rhodopsin protein after
light absorption. A previous study conducted using this rat model
demonstrated that the nature of the rhodopsin sorting defect, but
not the constitutive activation of the phototransduction cascade,
contributes significantly to apoptosis by interfering with the
normal cellular machinery in the post-Golgi transport pathway
or in the plasma membrane . The study also revealed a
correlation between the severity of mis-sorting of the truncated
rhodopsin protein and the rate of cell death in these animals.
Although the primary cause of degeneration in the S334ter-4 Rho
photoreceptors has been identified, the precise mechanism
responsible for triggering the apoptotic cascade remains unknown.
The apoptotic death of photoreceptor cells is the cornerstone of
the pathophysiological process in RP [5,6]. Although the role of
caspases in the execution of apoptosis in retinal degeneration has
been demonstrated, the process has not been fully investigated.
Moreover, conflicting data has been published regarding the pro-
apoptotic cellular signals that lead to the deterioration of
photoreceptor cells. These studies indicate that diverse cellular
pathways are involved in the demise of photoreceptor cells; for
example, two independent caspase activation pathways (the
cellular stress response and the death receptor pathway) have
been identified in the rd mouse model . The Bid protein, which
is activated by caspase-8, and phosphorylated p38 MAPK play a
key role in the cross- talk between these two activation pathways
resulting in the release of cytochrome C and the activation of
casape-3 in these animals. However, a separate study of the rd
mouse suggests that cell death occurs through a caspase-
independent pathway, and the DNA cleavage originates indepen-
dently of caspase-9, -8, -7, -3, and -2 activation and cytochrome C
release . Another study demonstrates that in S334ter-4 Rho
retinas, apoptosis contributes to the progression of retinal
PLoS ONE | www.plosone.org1March 2012 | Volume 7 | Issue 3 | e33266
degeneration in these animals [9,10]. Moreover, caspase-depen-
dent signaling involving the activation of caspases-3 and -9 and
cytochrome C leakage accompanies the activation of calpains and
poly (ADP-ribose) polymerase (PARP) and the down-regulation of
Recently, it has been proposed that the death of RP
photoreceptors may involve multiple mechanisms, including
caspase-dependent and caspase-independent pathways. Due to
their energy production and calcium homeostasis properties and
their ability to compartmentalize cell death activators, mitochon-
dria play a central regulatory role in this process [11,12]. In
addition, the activation and translocation of AIF (Apoptosis-
inducing factor) from the mitochondria and the translocation of
caspase-12 (ER stress-associated caspase) to the nucleus in dying
photoreceptors suggests that there is a link between the
mitochondrial caspase-independent pathway and the endoplasmic
reticulum (ER) stress signal in the cytoplasm . These studies
suggest that the ER stress response is involved in the pathological
events of ADRP photoreceptors. Therefore, it will be important to
determine if mislocalized truncated proteins, as opposed to
misfolded truncated proteins, are involved in the retinal pathology
of S334ter-4 Rho rats.
Recently, many studies have examined the involvement of the
ER stress response or the unfolded protein response (UPR) in
retinal degeneration [13,14,15,16,17]. In a previous study, we
suggested that the ER stress response is involved in ADRP
progression in P23H Rho-3 transgenic rats and that over-
expression of the Bip/Grp78 protein reprograms the ER stress
signal and protects the P23H Rho photoreceptors from degener-
ation . Although the investigation of the ER stress response in
P23H Rho photoreceptors in this ADRP model is not complete,
the activation of the ER stress response in the S334ter-4 Rho rat
model has never been analyzed in detail. Therefore, we
investigated how the UPR contributes to the degeneration of
S334ter-4 Rho photoreceptors. Our interest has been fueled by a
previous study in S334ter-4 Rho rats demonstrating that the
truncated S334ter-4 rhodopsin protein is retained in the cytoplasm
or is associated with the cell membrane , suggesting that the
accumulation of truncated rhodopsin in the cytoplasm may
overwhelm the protein degradation system. In addition, we are
interested to determine if the activation of the ER stress signal in
this model initiates apoptosis and if there is a dynamic link
between the activation of the UPR and the UPR-associated and
Therefore, the purpose of this investigation was to determine
whether the ER stress signaling is activated upon S334ter-4 Rho
photoreceptor degeneration and to demonstrate that additional
cellular pathways, such as mitochondria-induced apoptosis, occur
in S334-4ter Rho photoreceptors to initiate the collapse of
S334ter-4 Rho photoreceptors during the progression of ADRP.
viously [4,18,19], http://www.ucsfeye.net/mlavailRDratmodels.
shtml. Briefly, this is a degeneration of photoreceptors of moderate
rate compared to other rodent models. Photoreceptor degeneration
begins at about postnatal day (P) 13  and about 25% of the
photoreceptors are lost by P30, 50% by P60  and about 65% by
P120. In addition, high resolution light microscopy has demonstrat-
ed that at P4, P6, P8 and P10 the S334ter-4 retinas are
indistinguishable from age-matched wild-type controls and this is
reflected in a normal ONL thickness at P10. A very few pyknotic
nuclei are observed at P12 resulting in the ONL thinning only
beginning at P21 (LaVail, unpublished observations). Therefore, we
choose P10 as the first time point to observe the kinetics of the gene
expression involved in the UPR and UPR-associated signaling
Previous studies have demonstrated the cytoplasmic localization
of the S334ter-4 rhodopsin protein in the retina  of the
transgenic lines 3 and 5. In our study of S334ter-4 Rho, we
confirmed the mislocalization and retention of truncated rhodop-
sin in the cytoplasm of S334ter-4 Rho photoreceptors (data not
shown). Therefore, we were interested to determine if mis-
trafficking of the S334ter-4 Rho protein provokes the ER stress
signal, and if the activation of ER stress correlates with the
expression of genes that modulate the redox potential of ADRP
photoreceptors and affect homeostasis in the ER.
Expression of genes associated with oxidative stress is
elevated in S334ter-4 Rho retinas
ER homeostasis is a fragile equilibrium that is modulated by
dysregulation of the calcium or oxidative/reductive balance,
features that have previously been associated with oxidative stress.
Recently, it has been demonstrated that two processes, UPR
activation during oxidative stress [21,22] and raised oxidative
toxicity due to the involvement of ER stress, are linked .
Therefore, we analyzed the relative expression of genes that are
sensitive to an oxidative/reductive environment.
To further assess the effect of the mis-localized truncated
rhodopsin protein on oxidative stress, we examined the expression
level of the transcription of hypoxia-inducible factor a (Hif1aa),
superoxide dismutase (Sod1) and the nuclear factor kappa-light-
chain-enhancer of activated B cells (Nf-kB) genes that are known
to modulate the redox state of the cell. Figure 1 shows the relative
expression of these genes in S334ter-4 Rho and SD (Sprague
Dawley) rats on P10, P12, P15 and P21.
Our data demonstrated that Hif1a gene expression was
upregulated 1.5- and 1.9-fold on P10 and P12, respectively
(0.9360.06 in SD vs. 1.460.17 in S334ter-4 Rho and 0.6260.09
in SD vs. 1.260.14 in S334ter-4 Rho, respectively, P,0.05).
On P15, the expression level of Hif1a dropped dramatically
compared with that observed in the SD retina. The expression of
Sod1 also increased dramatically (1.4-fold) (0.6960.04 in SD vs.
1.33460.1 in S334ter-4 Rho, P,0.05), but this increase occurred
on P10. SOD1 expression subsequently declined to the level of the
SD over time. In contrast, Nf-kB gene expression increased 1.7-
fold (0.960.18 in SD vs. 1.660.2 in S334ter-4 Rho, P,0.05) only
on P15. At other time points, the Nf-kB gene expression was
comparable to that in the wild-type (WT) retinas.
Comparative analysis of ER stress and ERAD-associated
genes in S334ter-4 Rho and SD retinas
Figure 2 demonstrates the results of RT-PCR analysis of the
genes involved in the activation of ER stress signaling in S334ter-4
Rho rats. On P10 in transgenic retinas, calnexin (Cnx) and ATF4
gene expression was significantly upregulated 1.6- and 1.5-fold,
respectively (0.960.05 in SD vs. 1.460.2 in S334ter-4 Rho and
0.960.04 in SD vs. 1.360.1 in S334ter-4 Rho, respectively,
P,0.05 in both cases). Furthermore, Ero1 gene expression was
downregulated 2-fold (1.260.18 in SD vs. 0.660.18 in S334ter-4
Rho, P,0.05). On P12, the number of genes involved in the ER
stress response was extended and included the following: Cnx (2.1-
fold increase; 0.6260.09 in SD vs. 1.360.16 in S334ter-4 Rho,
P,0.05), Hsp40/Dnajc10 (1.8-fold increase; 0.6560.1 I SD vs.
1.2160.18 in S334ter-4 Rho, P,0.05), Ero1 (1.8-fold increase;
0.860.13 in SD vs. 1.560.2 in S334ter-4 Rho), Bip (1.7-fold
ER Stress and S334ter Rhodopsin
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increase; 0.760.01 in SD vs. 1.260.1 in S334ter-4 Rho, P,0.05),
eiF2a (1.9-fold increase; 0.8060.13 in SD vs. 1.50660.2 in
S334ter-4 Rho, P,0.01), Xbp1 (1.6-fold increase; 0.7260.08 in
SD vs. 1.1760.13 in transgenic rats, P,0.05), Atf6 (1.8-fold
increase; 0.6460.09 in SD vs. 1.1560.13 in S334ter-4 Rho,
P,0.05) and Chop (1.5-fold increase; 0.860.05 in SD vs.
1.360.12 in S334ter-4 Rho, P,0.05). On P15, the expression of
Cnx, Ero1, eIF2a and Atf4 dropped to different extents, and
although in some cases, the expression level of these genes was
higher than in the SD retinas, the relative expression of Chop
protein was consistently 1.3-fold higher than in the SD retinas. On
P15, the relative expression of the following genes was significantly
elevated: Hsp40/Dnajc10 (2-fold increase; 0.660.12 in SD vs.
1.260.22 in S334ter-4 Rho, P,0.05), Bip (2-fold increase;
0.6560.12 in SD vs. 1.3260.16 in S334ter-4 Rho, P,0.01),
Xbp1 (1.5-fold increase; 0.960.16 in SD vs. 1.3660.17 in
S334ter-4 Rho, P,0.05), Atf6 (greater than 2-fold increase;
0.6060.17 in SD vs. 1.2260.073 in transgenic rats, P,0.01). On
P18, the expression levels of all genes analyzed dropped to the
level of the SD retinas. The exception to this finding was the Chop
gene, which exhibited upregulated expression in P18 retinas, and
the Xbp1 gene, which was significantly downregulated in P18
S334ter-4 Rho retinas. Therefore, the data confirmed that the
expression of ER stress-related genes, the eiF2a and Atf4 genes
(the PERK pathway), the Atf6 gene (the ATF6 pathway) and the
Xbp1 gene (the IRE1 pathway) were upregulated in P12–15
S334ter-4 Rho retinas.
We also examined ERAD (ER associated degradation) genes,
such as Derl1 and Hrd1. Derl1 participates in the retrotransloca-
tion of misfolded proteins into the cytosol where they are
ubiquitinated and degraded by the proteasome. The Hrd1 gene
is an E3 ubiquitin-protein ligase and transfers ubiquitin specifically
from endoplasmic reticulum-associated UBC1 and UBC7 E2
ligases to substrates, thereby promoting their degradation. The
qRT-PCR analysis of these genes in SD and S334ter-4 Rho
retinas (Figure 2) showed that both genes were transiently
activated on P10 and P12. Derl1 expression increased 1.6-fold
on P10 (0.960.03 in SD vs. 1.460.2 in S334ter-4 Rho, P,0.01)
and subsequently decreased slightly over time. The expression of
Hrd1 increased significantly (1.6-fold) on P12 (0.7460.07 in SD
vs. 1.2460.17 in S334ter-4, P,0.05). We also examined the
expression of the Edem1 and Edem2 genes and determined that
there was no significant difference in the expression levels in
S334ter-4 Rho retinas compared with control retinas.
We analyzed levels of the main UPR markers such as BiP,
CHOP, phosphorylated protein kinase RNA-like endoplasmic
reticulum kinase (pPerk), phosphorylated eIF2a (peIF2a), active
ATF6 proteins and the Ire-mediated unconventional the Xbp1
mRNA splicing in S334ter-4 Rho rats (Figure 3A, B and C) using
western blot analysis and a semi-quantitative RT-PCR and
determined that the production of the BiP protein increased 1.5-
fold in P12 S334ter-4 Rho retinas compared with SD retinas
(0.04760.008 vs. 0.07160.005, respectively; P=0.01. However,
by P15 the difference between groups was not detected. The
CHOP protein was also dramatically over-expressed (3.5-fold)
(0.01660.005 in SD vs. 0.6060.002 S334ter-4 Rho, P=0.0003)
on P15 in S334ter-4 Rho retinas. The full length of pAtf6 protein
(90 kD) (the Atf6 pathway) was significantly elevated in S334ter-4
Rho retina by 2.7-fold (0.001760.0011 in SD vs. 0.004860.0007
in S334ter-4 Rho, P=0.04). The N-terminal of the full-length of
Atf6, cleaved pAtf6 was elevated by 1.93-fold and was 0.1860.004
in SD vs. 0.03560.002, P=0.004. We also observed that the
peIF2a protein was significantly increased in S334ter-4 Rho retina
and was 0.00160.0005 in S334ter-4 Rho vs 0.00260.0004,
P=0.0009.The hallmark of the IREI pathway, the spliced Xbp1
protein was detected in S334ter-4 retina. Its level was a 4.5-fold
higher in transgenic retina compared to SD and was 0.02260.003
in SD and 0.160.01, P=0.001 in S334ter-4 rats. Unspliced Xbp1
protein was increased to a lesser extent (2.3-fold) in S334ter-4 rats
and was 0.04960.008 in SD and 0.1360.001, P=0.03 in S334ter-
4. Images of western blots are shown in Fig. 3 C.
The spliced form of the Xbp1 mRNA was increased in S334ter-
4 Rho rats as well. The ratio of spliced Xbp1 to normalized
unspliced Xbp1 mRNA was 1.45 (0.2260.0006 in SD vs.
0.03360.031 in S334ter-4 Rho retina, P=0.025). An image of
an agarose gel loaded with RT-PCR product obtained with Xbp1
specific primers is shown in Fig. 3B.
Autophagy is involved in retinal degeneration of S334ter-
4 Rho photoreceptors
Knowing that the ERAD genes Derl1 and Hrd1 are transiently
upregulated in P10 and P12 retinas, we became interested in
investigating the activity of another protein degradation system,
autophagy. We examined the relative expression of autophagy
hallmark genes, such as Atg5 and Atg7, which are involved in
autophagosome formation, and the lysosomal-associated mem-
brane protein 2 (Lamp2). Figure 4 demonstrates that in developing
S334ter-4 Rho retinas, the relative expression of Atg5 and Atg7
was not significantly different from that observed in the SD
samples. However, the relative expression of the Lamp2 gene was
transiently upregulated 1.8-fold on P10 in S334ter-4 Rho retinas
Figure 1. Relative expression of the oxygen stress-induced
Hif1a, Sod1 and Nf-kB genes in S334ter-4 Rho retinas at
different ages. Relative gene expression in S334ter-4 Rho retina was
measured on P10, P12, P15, P18 and P21 and a fold change was
expressed as a ratio of S334ter-4Rho relative expression to SD relative
expression. Expression of the Hif1a and Sod1 genes was significantly
induced in P10 S334ter-4 Rho compared to SD retinas (1.5- and 1.4-fold,
respectively, P,0.05, *). On P12, increased Hif1a expression was still
evident (1.9-fold, P,0.05,*); however, the Sod1 expression was
decreased compared to P10. When compared with Hif1a and Sod1
mRNA, the relative accumulation of Nf-kB mRNA in S334ter-4 Rho
retinas exhibited alternate dynamics: a 1.7-fold increase in Nf-kB
expression was observed on P15 only (P,0.05,*), whereas the Hif1a and
Sod1 expression was diminished on P15.
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(0.8660.07 in SD vs. 1.560.16 in S334ter-4 Rho, P,0.01). The
expression of this gene subsequently decreased with time. The
results of the comparative qRT-PCR analyses are shown in
S334ter-4 Rho rats exhibit elevated levels of pro-
apoptotic gene expression during retinal development
The ADRP photoreceptors degenerate and die through the
process of apoptosis. Therefore, we examined the expression of
pro-apoptotic genes belonging to the Bcl-2 family: the BH3-only
interacting domain death agonist (BID), the Bcl-2-interacting killer
protein (Bik), the Bcl2-like 11 apoptosis facilitator (Bim), Noxa and
Puma. The results of this analysis are presented in Figure 5. We
determined that the relative expression of Bik was significantly
upregulated 2.3-fold in P10 S334ter-4 Rho retinas compared with
P10 SD retinas (1.2460.14 in SD vs. 2.7360.3 in S334ter-4 Rho,
P,0.001). On P10, we also observed an increase in the relative
expression of the Bim protein (1.7-fold; 0.8360.05 in SD vs.
1.4060.21 in transgenic retina, P,0.05). At the next two time
points, Bim expression was upregulated 1.34-fold compared with
controls (0.6960.15 in SD vs 0.5160.10 in S334ter-4 Rho,
P.0.05) on P12 and 1.8-fold compared with controls on P15
(0.8660.18 in SD vs. 1.5460.11 in S334ter-4 Rho, P,0.01). The
Noxa gene expression increased significantly in P12 and P15
S334ter-4 Rho retinas and was 2.8-fold and greater than 2-fold
higher compared to that in age-matched SD retinas, respectively
(0.8260.12 in SD vs. 2.3460.36 in S334ter-4 Rho at P12,
P,0.0001 and 0.8860.24 in SD vs. 1.8760.19 in S334ter-4 Rho
at P15, P,0.01). On P15, the relative expression of Bid and Puma
in S334ter-4 Rho retinas were increased 2.4- and 2.3-fold,
respectively, compared to that in SD retinas (0.6860.11 in SD
vs. 1.6260.23 in S334ter-4 Rho, P,0.01 for Bid and 1.0060.05
in SD vs. 2.3060.5 in S334ter-4 Rho, P,0.001 for Puma).
The apoptotic protease activating factor 1 (APAF1) also induces
apoptosis. Therefore, we analyzed Apaf1 gene expression during
the progression of ADRP and determined that its expression was
upregulated 1.9-fold in P12 transgenic retinas (0.6260.01 in SD
vs. 1.1760.10 in S334ter-4 Rho, P,0.01) (Figure 5). This
upregulation leads to the activation of caspase-dependent
apoptosis, which was confirmed by the analysis of caspase-3 and
-7 expression levels. The caspase-3 and -7 proteins are executioner
caspases. We determined that the expression of caspase-3 was
significantly upregulated 1.9-, 2.17- and 1.8-fold in P12, P15 and
P18 S334ter-4 Rho retinas, respectively (0.760.1 in SD vs.
1.3360.2 in S334ter-4 Rho, P,0.01, 0.6960.21 in SD vs.
1.560.15 in S334ter-4 Rho, P,0.01 and 0.9060.10 in SD vs.
1.6360.22 in S334ter-4 Rho, P,0.05, respectively). Caspase-7
gene expression was upregulated 1.73-fold and almost 2-fold on
P10 and P15, respectively (0.8260.04 in SD vs. 1.4260.15 in
S334ter-4 Rho, P,0.05 and 0.9960.07 in SD and 1.9260.43,
P,0.01 in transgenic rats, respectively).
Mitogen-activated protein kinases 1 (Erk2) and 8 (Jnk) are
involved in retinal degeneration in S334ter-4 Rho rats
A number of mitogen-activated kinases, such as Mapk8 (c-Jun
N-terminal kinase 1) and Mapk14 (p38), play an important role in
Figure 2. Relative expression of ER stress- and ERAD-related genes in S334ter-4 Rho retinas. The UPR gene expression was altered in
S334ter-4 RHO retinas. Relative gene expression in S334tr Rho retina was measured on P10, P12, P15, P18 and P21 and a fold change was expressed
as a ratio of S334ter-4Rho relative expression to SD relative expression. On P10, there was a significant reduction in the Ero1 gene expression
suggesting that the ER homeostasis in S334ter-4 Rho retinas is imbalanced. The expression of this gene is decreased 2-fold (P,0.05, *) in S334ter-4
Rho retinas compared with SD retinas on P10. The increased expression of Calnexin (Cnx) and Atf4 genes are the first ER stress markers that respond
to ER disturbance. The expression of these genes was increased 1.6- and 1.5-fold, respectively, (P,0.05, * in each case) on P10. On P12, the expression
of other UPR upstream and downstream markers, such as Hsp40/Dnajc10, Ero1, Bip, eIf2a, Xbp1, Atf6 and Chop, were detected, which was indicated
by relative increases of 1.8-, 1.8-, 1.5-, 1.9-, 1.6-, 1.8- and 1.6-fold, respectively, (P,0.05, * in each case with the exception of eIF2a where P,0.01, **).
On P15, the Cnx, Ero1, eIf2a, Atf4 and Chop genes were expressed to a lesser extent or there was no significant difference in their expression levels in
S334ter-4 Rho retinas compared with SD retinas. However, the Hsp40/Dnajc10, Bip, Xbp1 and ATf6 mRNAs were significantly induced on P15. Their
relative expressions were 2-, 2-, 1.5- and 2-fold higher in S334ter-4 Rho retinas compared with the WT group (P,0.01 for Bip and Atf6, and P,0.05 for
Hsp40/Dnajc10 and Xbp1). On P21, the expression of all genes was insignificant in the S334ter-4 Rho retinas compared with the SD retinas. Derl1 and
Hrd1 gene expression was upregulated 1.5- and 1.6-fold on p10 and p12, respectively (P,0.05 in each case).
ER Stress and S334ter Rhodopsin
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Figure 3. The ER stress markers BIP and CHOP proteins in retinas from S334ter-4 Rho rats. A: Ratios of normalized S334ter-4 Rho BiP,
CHOP, pATF6 (90), pATF6 (50), peIF2a ,spliced Xbp1(sXbp1), unsliced Xbp1 (uXbp1), cleaved caspase-12 proteins to corresponding proteins in SD
CHOP were used to register the alteration of protein expression. Normalization of all proteins was done by detecting b-Actin protein The ER stress
markers BIP protein was a 1.5-fold upregulated in P12 S334ter-4 Rho retinas compared with SD retinas (0.04760.008 vs. 0.07160.005, respectively;
P=0.01). In P15, the level of BiP protein was significantly reduced and was not distinguishable from SD. The Chop protein was also dramatically over-
expressed (3.5-fold) (0.01660.005 in SD vs. 0.6060.002 S334ter-4 Rho, P=0.0003) on P15 in S334ter-4 Rho retinas. The full length of pAtf6 protein
(90 kD) (the Atf6 pathway) was significantly elevated in S334ter-4 Rho retina by 2.7-fold (0.001760.0011 in SD vs. 0.004860.0007 in S334ter-4 Rho,
P=0.04). The cleaved pAtf6 (50) was significantly elevated by 1.93-fold inS334ter-4 Rho retina (0.1860.004 in SD vs. 0.03560.002, P=0.004). The
peIF2a protein was also significantly increased in S334ter-4 Rho retina and was 0.00160.0005 in S334ter-4 Rho vs 0.00260.0004, P=0.0009. The full
ER Stress and S334ter Rhodopsin
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the ER stress response. Therefore, we included these and other
members of the MAPK gene family, such as the extracellular
signal-regulated kinases Mapk1 (Erk2) and Mapk3 (Erk1), in our
study. RNA analysis of P10–P21 transgenic retinas demonstrated
no significant difference in the relative expression of the Mapk3
and Mapk14 genes compared to control. In contrast, the
expression of Mapk1 and Mapk8 was elevated by 1.7- and 2.3-
fold, respectively, in the transgenic retinas (0.8860.06 in SD vs.
1.560.20 in S334ter-4 Rho and 0.6860.07 in SD vs. 1.6060.3 in
S334ter-4 Rho, respectively; P,0.01 in each case) on P10 and
their expression subsequently decreased over time (Figure 6).
pTen/Akt1 signaling is involved in retinal degeneration in
S334ter-4 Rho rats
A recent study by Hu and colleagues demonstrated a critical
role for the endogenous Akt and MEK1/ERK pathways in
counteracting ER stress and proposed that the endogenous Akt/
IAPs and MEK/ERKs control cell survival by resisting ER stress-
induced cell death signaling . Figure 7 shows the progressive
changes in Akt1/2 gene expression in S334ter-4 Rho rats during
retinal degeneration. On P12, we observed the elevation of the
tumor-suppressor gene pTen that catalyzes the dephosphorylation
of the Akt gene leading to the inhibition of the Akt signaling
pathway. An approximately 1.65-fold increase in the relative
expression of the Pten gene (0.8060.13 in SD vs.1.5260.2 in
S334ter-4 Rho, P,0.05) was observed. Subsequently, pTen
expression decreased to the level of the WT samples. In contrast,
the expression of the Akt1/2 genes increased after P12 and
reached a peak on P15 in S334ter-4 Rho rats. The Akt1 gene
expression increased by 1.65-fold (0.8460.15 in SD vs. 1.3960.13
in S334ter-4 Rho rats, P,0.05) and the Akt2 gene expression
increased 1.7-fold (1.03243860.17 in SD vs. 1.7160.15 in
S334ter-4 Rho, P,0.01).
ER-mitochondrial cross-talk in S334ter-4 Rho retina
detected by the elevated calpain activity and the
cytosolic release of apoptotic inducing factor 1
The physical and functional interactions between the ER and
the mitochondria occur throughout their networks. The molecular
foundations of this cross-talk are diverse, and Ca2+is an important
signal that these organelles use to communicate. A recent study by
Bravo et al.  demonstrated that during the adaptive phase of
ER stress mitochondrial events are already underway before the
appearance of cell death. The ER-induced Ca2+release may
facilitate the permeabilization of the mitochondrial membrane
through the activation of the mitochondrial permeability transition
(MPT) pore. In addition, Ca2+release from the ER activates Ca2+-
sensitive cytosolic enzymes, which may control the distribution
and activity of Bcl-2 proteins and calpains or modulate the
expression of apoptosis regulatory proteins.
Therefore, we investigated calpain activation in this study.
Figure 8 demonstrates that compared with SD there is a 1.4-fold
and a greater than 2-fold increase in activated calpains on P15 and
P30 in S334ter-4 Rho retinas (2.2460.09 in SD vs. 3.160.127 in
S334ter-4 Rho, P,0.01 and 1.28760.27 in SD vs. 2.760.04 in
S334ter-4 Rho, P,0.05, respectively). To further understand if the
activation of calpains triggers the MPT pore in S334ter-4 Rho
retinas resulting in mitochondria-associated apoptosis, in P15
retinas we analyzed the mitochondrial release of the Apoptotic
Inducing Factor 1 (AIF1) (Figure 9) and cytochrome C, which form
an apoptosome with caspase-9 and apoptotic peptidase activating
factor 1 (Apaf1) to activate caspase-induced apoptosis. In S334ter-
4 Rho retina, we observed a 3-fold increase in the cytosolic AIF1
compared to SD. However, in P15 S334ter-4 Rho retina we did
not detect any difference in the levels of cytochrome C compared
to SD. In contrast, the relative expression of Apaf1 was
upregulated (Figure 5).
Activation of calpains provokes cleavage of caspase-12.
Therefore, we analyzed caspase-12 activity in transgenic retina
and found that the level of cleaved caspase-12 was elevated in
S334ter-4 Rho rats on P15 compared to control over 2-fold and
was 0.0560.007 in SD vs. 0.1260.02 in S334ter-4 Rho, P=0.014
on P15. However, further analysis of S334ter-4 Rho retina by
length of pAtf6 protein (90 kD) (the Atf6 pathway) was significantly elevated in S334ter-4 Rho retina by 2.7-fold (0.001760.0011 in SD vs.
0.004860.0007 in S334ter-4 Rho, P=0.04). The N-terminal of the full-length of Atf6, cleaved pAtf6 was elevated by 1.93-fold and was 0.1860.004 in
SD vs. 0.03560.002, P=0.004. We also observed that the peIF2a protein was significantly increased in S334ter-4 Rho retina and was 0.00160.0005 in
S334ter-4 Rho vs 0.00260.0004, P=0.0009. The spliced Xbp1 protein was detected in S334ter-4 retina. Its level was a 4.5-fold higher in transgenic
retina compared to SD and was 0.02260.003 in SD and 0.160.01, P=0.001 in S334ter-4 rats. Unspliced Xbp1 protein was increased to a lesser extent
(2.3-fold) in S334ter-4 rats and was 0.04960.008 in SD and 0.1360.001, P=0.034 in S334ter-4. Increase in active caspase-12 was observed in S334ter-4
Rho retina. The level of cleaved caspase-12 (20 kD) was elevated in S334ter-4 Rho rats on P15 compared to control over 2-fold and was 0.0560.007 in
SD vs. 0.1260.02 in S334ter-4 Rho, P=0.014 on P15. B: Upper panel: Quantification of spliced form of the Xbp1 mRNA (the IRE signaling) detected by
RT-PCR reaction. We observed 1.45-fold increased in the spliced form of Xbp1 mRNA in S334ter-4 Rho retina. The ratio of spliced Xbp1 to normalized
unspliced Xbp1 mRNA was 0.2260.0006 in SD vs. 0.03360.031 in S334ter-4 Rho retina, P=0.025). Image of the agarose gel loaded with RT-PCR
product obtained with Xbp1 specific primers is shown in a lower panel. C: Images of western blots treated with anti-Actin, Bip, CHOP, peIf2a, pATF6,
Xbp1 antibodies and detected with secondary antibodies and infrared imaging scanner are presented.
Figure 4. Relative expression of Lamp2 in S334ter-4 Rho
retinas. Relative gene expression in S334tr Rho retina was measured
on P10, P12, P15, P18 and P21 and a fold change was expressed as a
ratio of S334ter-4Rho relative expression to SD relative expression. The
Lamp2 gene expression was upregulated in p10-p15 S334ter-4 Rho
retinas. In p10, the relative expression of this gene increased 1.7-fold in
S334ter-4 Rho retinas compared to that in SD retinas (P,0.001), and it
subsequently declined in a time-dependent manner.
ER Stress and S334ter Rhodopsin
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western blot and caspase-12 fluorometric activity assay showed
that the activity of ER-associated caspase-12 was declined in a
time-dependant manner (data not shown). Comparison of the level
of active protein kinase C (PKC), that has been earlier reported to
inhibit the caspase-12 activity , did not reveal a difference
Knowing that calpains trigger the cleavage of AIF1 in the
mitochondria resulting in its translocation to the cytoplasm, we
analyzed the cleaved AIF1 protein (figure 9). There was a 6.5-fold
increase in the levels of cleaved Aif1 in the cytoplasmic fraction of
S334ter-4 Rho compared to SD photoreceptors on P15
(0.03860.01 in SD vs. 0.2560.05 in S334ter-4 Rho, P=0.004).
Time-dependent decline of Crx and Nrl transcription
factors in the S334ter-4 Rhophotoreceptor
To estimate the massive loss of photoreceptor cells in S334ter-4
Rho retinas, we analyzed the relative expression of the NRL and
CNX photoreceptor-specific transcription factors and determined
that their expression patterns were altered during the progression
of ADRP (Figure 10). The expression of the CRX gene was slightly
higher in P10 and P12 S334ter-4 Rho retinas compared with SD
retinas (0.9560.04 in SD vs. 1.260.17 in S334ter-4 RHO,
P.0.05 and 0.5860.1 in SD vs. 0.7060.12in S334ter-4 Rho,
respectively). The expression of Crx decreased with time and was
significantly lower in S334ter-4 Rho P21 retinas compared with
SD retinas (1.1060.11 in SD vs. 0.5560.02 in S334ter-4 Rho,
P,0.05). The levels of Nrl gene expression did not differ from WT
levels during P10-P12. However, by P21, Nrl expression decreased
dramatically in the S334ter-4 Rho retinas (1.0060.21 in SD vs.
0.2360.02 in S334ter-4 Rho, P,0.05).
Figure 5. Relative expression of pro-apoptotic genes involved in S334ter-4 Rho retinas. Relative gene expression in S334ter-4 Rho retina
was measured on P10, P12, P15, P18 and P21 and a fold change was expressed as a ratio of S334ter-4 Rho relative expression to SD relative
expression. The expression of BH3-only proteins, BID, Bim, Bik, Noxa and Puma was upregulated with different patterns in S334ter-4 Rho retinas. On
P10, the expression of Bik, Bim and Bid genes increased 2.3- (P,0.001) 1.7- (P,0.05) and 1.6-fold (P,0.05), respectively. The expression of Bim was
elevated until p15 and dropped 0.6-fold to WT levels (P,0.05) on P21. On P12, the Noxa gene was elevated 2.8-fold (P,0.0001) and remained
upregulated until p15 (2-fold increase, P,0.01) and its expression subsequently dropped. On P15, the Puma gene appears to be upregulated 2.2-fold
in S334ter-4 Rho retinas when compared to SD retinas (P,0.001). The Apaf1 gene expression was increased in P12 and in P15 retinas. A significant
1.9-fold upregulation Apaf1 expression was detected on P12 (P,0.01). The relative expression of caspase-3 was significantly upregulated during the
progression of ADRP. On P12, P15 and P18, caspase-3 expression in S334ter-4 Rho retinas was elevated 1.9-fold (P,0.01), 2.2-fold (P,0.01) and 1.8-
fold (P,0.05), respectively. compared to SD retinas. The analysis of caspase-7 gene expression demonstrates a 1.7-fold (P,0.05) and 2-fold (P,0.01)
increase in caspase-7 mRNA on P10 and P15, respectively.
Figure 6. Relative expression of the MAPK1 (Erk2) and MAPK8
(JNK) genes in S334ter-4 Rho retinas. Relative gene expression in
S334ter-4 Rho retina was measured on P10, P12, P15, P18 and P21 and a
fold change was expressed as a ratio of S334ter-4Rho relative
expression to SD relative expression. On P10, the Mapk3 (Erk1) and
Mapk14 (p38) gene expression was upregulated 1.7- and 2.3-fold in
S334ter-4 Rho retinas, respectively (P,0.01 for both). Starting on P12,
their expression decreased with time.
ER Stress and S334ter Rhodopsin
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Although retinal degeneration has many etiologies, they all lead
eventually to photoreceptor cell death and blindness . A
number of investigators have dedicated their research to the
elucidation of the mechanism of retinal degeneration in ADRP,
and some of these studies have focused on ADRP progression in
S334ter-4 Rho rats. For example, a recent publication by Kaur
et al. highlighted the contribution of mitochondria-associated
apoptosis  in which cytochrome C release plays an important
role, and the contribution of non-apoptotic cell death, which
involves calpain and PARP activation, to photoreceptor cell death
and the degeneration of S334ter-4 Rho retinas. Despite the
progression in our understanding of the cellular mechanisms
leading to the decline of S334ter-4 Rho photoreceptors, the
essential link that connects ER stress, UPR, and calpain activation
with mitochondria-induced apoptosis has not been elucidated. In
this study, we present a detailed investigation of the mechanisms of
ER stress that lead to the activation of the UPR and apoptosis in
retinas expressing mis-localized S334ter rhodopsin. We examine
the status of the ER stress response and its correlation with pro-
apoptotic gene expression over time (P10, P12, P15 and P21) and
dissect the cellular mechanism of retinal degeneration during the
development of a normal retina. Although gene expression during
the maturation of WT retina is altered [28,29], in this study, we
focus on the relationship between gene expression and the
progression of ADRP in transgenic rats expressing truncated
The UPR is a conserved, adaptive cellular program that is
activated in response to the accumulation of misfolded proteins in
the ER . Homeostasis in the ER may be compromised by a
variety of stimuli including disturbances in redox regulation,
calcium regulation, glucose deprivation, and viral infection.
Oxidative protein folding depends upon the maintenance of
adequate oxidizing conditions within the ER lumen and is
Figure 7. Relative expression of pTten, Akt1 and Akt2 genes in
S334ter-4 Rho retinas. Relative gene expression in S334ter-4 Rho
retina was measured on P10, P12, P15, P18 and P21 and a fold change
was expressed as a ratio of S334ter-4Rho relative expression to SD
relative expression. An approximate 1.65-fold increase in the relative
expression of the pTen gene was observed (0.8060.13 vs. 1.560.2,
P,0.05) in P12 transgenic retinas. Subsequently, the expression of this
gene decreased to WT levels. In contrast, the expression of the Akt1 and
2 genes increased after P12 and reached a peak on P15. The Akt1 gene
expression was 0.8460.15 in SD retinas vs. 1.4060.13 in S334ter-4 Rho
retinas, P,0.05. The Akt2 gene expression was 1.0360.17 in SD vs.
1.7060.15 in P15 S334ter-4 Rho, P,0.01.
Figure 8. Activation of calpains in S334ter-4 Rho retinas. In P15
and P30 S334ter-4 Rho retinas, we observed an activation of calpains in
the cytosol measured by the fluorescence intensity emitted from the
calpain substrate Ac-LLY AFC. On P15 and P30, there was a 1.4- and a
greater than 2-fold increase in activated calpains in S334ter-4 Rho
retinas compared with SD retinas, respectively (2.2460.09 in SD vs.
3.160.127 in S334ter-4 Rho, P,0.01 and 1.28760.27 in SD vs. 2.760.04
in S334ter-4 Rho, P,0.05, respectively).
Figure 9. Release of AIF1 from S334ter-4 Rho mitochondria.
Analysis of isolated cytosolic fractions of P15 S334ter-4 Rho retinas
determined that the concentration of the cleaved form of Aif1 (48 kD)
was 6.5-fold higher compared with the WT cytosolic samples
(0.03860.01 in SD vs. 0.2560.05 in S334ter-4 Rho, P=0.004). Images
of western blot detecting the AIF1 and COXIV proteins are shown.
Detection of COXIV protein was observed only in SD (Msd) and S334ter-
4 (MS s334ter-4) retinal mitochondrial fractions.
ER Stress and S334ter Rhodopsin
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achieved with the help of ER oxydoreductase (ERO1). ERO1 and
PDI form the main pathway for protein disulfide bond formation
in the eukaryotic ER. Therefore, the modulation of ERO1 activity
is a component of a homeostatic feedback system in the ER that
allows the cell to rapidly adjust to fluctuations in the ER redox
environment and to maintain conditions that are conducive to
oxidative protein folding . We analyzed the expression of the
Ero1 gene during the development and progression of S334ter-4
Rho photoreceptors on P10, P12, P15 and P21 and determined
that there is a deficiency in Ero1 gene expression on P10, which is
followed by a rapid over-expression of the gene on P12 (nearly a 2-
fold increase) in transgenic retinas (Figure 2). After P15, Ero1
expression decreased dramatically, which reflects the demand for
enzymatic protein folding in the cell and the adjustment of the ER
to the redox potential. For example, during hypoxia, transcription
of the Ero1 gene is dramatically induced through the activation of
Hif1a [32,33], suggesting that hypoxia negatively regulates the
activity of many enzymatic pathways including Ero1. Therefore,
in our experiments, the modulation of Ero1 gene expression
suggests that there is an alteration in physiological oxygen tension
and this could be considered as a key adaptive response to a
hypoxia-induced Hif-1-mediated pathway (see below).
Recently, hypoxia-induced HIFf1 elevation has been studied in
detail. HIF1a is a pivotal regulator of the cells’ adaptation to
hypoxia and is induced by hypoxia, growth factors, and oncogenes
. In addition, the elevated expression of HIF1a under hypoxic
conditions is accompanied by the activation of ER stress genes,
such as eIF2a , and by the increased generation of reactive
oxygen species (ROS) that provide a redox signal for the induction
HIF1a . A number of investigators have proposed that retinal
hypoxic preconditioning, which leads to HIF1a induction,
morphologically and functionally protects retinal cells against
light-induced retinal degeneration [37,38]. Others have identified
HIF1a as a protein that may be required (directly or indirectly) for
the normal development of the retinal vasculature , suggesting
that hypoxia is a part of normal retinal development. In addition,
oxidative stress has been identified as an important contributor to
retinal degeneration in a number of studies . Thus, with
respect to hypoxia, it is a logical assumption that Hif1a expression
levels are also modulated in transgenic rats during retinal
development. Therefore, it is not surprising that the level of Hif1a
expression in S334ter-4 Rho retinas correlates with Ero1
transcription, increasing 1.5- and 2-fold on P10 and P12,
respectively. The expression of Hif1a has not been examined
previously in S334ter-4 Rho retinas. In this study, we demon-
strated that hypoxic conditions leading to oxygen deprivation
persisted, at least temporarily, in the developing S334ter-4 Rho
retinas. The mechanism by which the Hif1a mRNA is induced in
ADRP photoreceptors appears to be adaptive and is linked to
mechanisms that maintain a homeostasis in the photoreceptor
cells. In favor of this hypothesis, a rapid decline in Hif1a gene
expression was observed in P15 S334ter-4 Rho retinas. Additional
evidence supporting this hypothesis regarding the hypoxic status of
transgenic retina comes from the analysis of the over-expression of
the Nf-kB gene. Compared with Hif1a expression, Nf-kB over-
expression was delayed until P15. The delay in the onset of Nf-kB
over-expression may be associated at least in part with the
modulation of Sod1 gene expression, which was over-expressed on
P10. Recently, a direct link between the alternating expression
patterns of both genes has been proposed .
SOD1 is a soluble protein that acts as a scavenger of superoxide
converting it to molecular oxygen and hydrogen peroxide, and the
SOD1 gene is considered the first line of defense against oxidative
stress. The elevated expression of SOD1 has been associated with
a number of neurodegenerative disorders, such as SALS and
Alzheimer disease, suggesting that SOD1 upregulation is a
pathological phenomenon . Therefore, changes in Sod1
mRNA levels would indirectly reflect the increased accumulation
of superoxide radicals in S334ter-4 Rho retinas. Although the
Sod1 gene was over-expressed in P10 retinas, a dramatic drop in
the expression levels of this gene was subsequently observed, which
may be provoked by the induction of Nf-kB expression. The
transient over-expression of Sod1 in S334ter-4 Rho retinas may be
a result of the activation of the hydrogen peroxide-responsive
element within the Sod1 promoter by H2O2[42,43], which has
been shown to play a protective role in oxygen-deprived
dopaminergic neurons in the rat substantia nigra . Alterna-
tively, an adaptive mechanism in S334ter-4 Rho photoreceptors
that manages the stress by over-expressing a powerful antioxidant
enzyme may be involved. As discussed above, the data indicate
that the imbalance in ER homeostasis in S334ter-4 Rho retinas is
created by hypoxic preconditions that lead to the induction of
Ero1, Hif1a and Nf-kb gene expression in P10–P12. Therefore,
we next investigated if the modulation of these genes provokes the
activation of the UPR in S334ter-4 Rho retinas.
We analyzed the expression profiles of the following proteins:
the ER resident chaperone proteins, such as calnexin (Cnx),
Hsp40/Dnajc10, and Grp78/Bip; activating transcription factors
Atf4, Atf6, and Xbp1; Gadd153/Chop, eIF2a (eukaryotic
translation initiation factor 2a) and ERAD (ER-associated
degradation) genes, such as Edem1, Edem2, Derl1, Derl2, and
Hrd1 (Synovalin). The data suggest that during the development
and progression of the ADRP retina, the expression of the
majority of these ER stress gene is modulated. For example, the
expression of Cnx was dramatically increased 1.5-fold and greater
than 2-fold in P10 and P12 transgenic retinas, which paralleled the
expression pattern of the Ero1 gene. The Cnx gene is an
important component of the ER, where it is involved in the
maintenance of ER protein homeostasis and participates in the
folding and assembly of nascent glycoproteins and aids their
Figure 10. Relative expression of the Crx and Nrl transcription
factors in S334ter-4 Rho retinas. Relative gene expression in
S334ter-4 Rho retina was measured on P10, P12, P15, P18 and P21 and a
fold change was expressed as a ratio of S334ter-4Rho relative
expression to SD relative expression. The qRT-PCR analysis of S334ter-
4 Rho retinas revealed that the expression of the Nrl and Crx
transcription factors decreased in a time-dependent manner in P10-
P21 retinas. A significant reduction in the expression of both
transcription factors was observed in P21 retinas, the Crx gene
expression was reduced 55% (P,0.05), and the NRL gene expression
was downregulated 23% (P,0.05).
ER Stress and S334ter Rhodopsin
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transport out of the ER quality control system. Therefore, a high
expression of the Cnx gene reflects a high demand for protein
folding in the ER. One of these ER proteins may be an aberrant
rhodopsin, which is rescued by the over-expression of the Cnx
chaperone protein .
Further analysis of the ER chaperones Hsp40/Dnajc10, and
Bip demonstrated that there is increase in the levels of Hsp40.
Recently, a model of the Hsp40-mediated ERAD pathway has
been proposed . According to this model, the Hsp40 protein
accelerates the ERAD pathway by reducing the number of
incorrect disulfide bonds in misfolded glycoproteins that are
recognized by EDEM1. ERAD substrates that are released from
CNX are recruited by EDEM1 to the C-terminal cluster of
HSP40. Therefore, an increase in Cnx suggests that there is a
correlation between these synergistically working genes, which is
confirmed by our experiments (Figure 2). Similar to the expression
patterns of the Cnx gene, Hsp40 gene expression was also elevated
1.8- and 2-fold in P12 and P15 retinas, respectively (Figure 2),
whereas the expression of Edem1 as Edem2 in the transgenic
retinas (data not shown) was not significantly different from the SD
retinas. This result suggests that in S334ter-4 Rho retinas, there is
a high demand for the chaperoning assistance of Hsp40.
Another component of the ERAD pathway is the BIP (GRP78)
protein. BiP binds to the J domain of Hsp40 in an ATP-dependent
manner and transfers ERAD-targeted substrates to the retro-
translocation channel upon ATP hydrolysis . In addition to
participation in the ERAD system, BiP is an up-stream marker in
the ER stress pathway and is the first line of defense in a
compromised ER. This protein activates three independent UPR
pathways, PERK, ATF6 and IRE1. RNA analysis of the S334ter-
4 Rho and SD retina samples demonstrated that the increase in
Bip expression correlated with the expression of Cnx and Hsp40/
Dnajc10, reaching a peak on P15 with a 2-fold increase in the
S334ter-4 Rho retinas. Western blot analysis was used to confirm
the increased production of the BiP protein providing proof of the
elevation of the BiP protein in S334ter-4 Rho rats.
The PERK pathway is activated in P12 transgenic retinas,
which was evident from the upregulation of the eIf2a and Atf4
genes and elevation of peIF2a. In P15 retinas western blot analysis
demonstrated the increased production of the peIf2a protein
providing proof of activation of the PERK signaling pathways in
S334ter-4 Rho rats. In addition, expression of the ATF6 (the
ATF6 pathway) gene was increased steadily up to P15, after which
they decreased to levels that were observed in the controls.
Therefore, it is not surprising that the level of full-length pAtf6
(90 kD) and its cleaved form (pAtf6-50) in P15 S334ter-4 Rho
retina were significantly increased. The level of Xbp1 (the IRE1
pathway) was also steadily increased up to P15. However, later in
P21 retina expression of the Xbp1 gene was significantly reduced
in transgenic retinas that could be a result of a decreased catalase
expression, enhanced ROS generation, and the loss of mitochon-
drial MPTP after H2O2exposure in S334ter-4 Rho photorecep-
tors . In P15 S334ter-4 Rho retina, we observed increase in a
spliced and unspliced forms of Xbp1 protein suggesting that the
IRE pathways is activated. Despite the general decline in the
Xbp1 gene expression in P21, the splicing of the Xbp1 mRNA was
persistent in S334ter-4 Rho retina (figure 3 B).
Signaling through the PERK, ATF6 and IRE1 genes triggers
pro-apoptotic stimuli during prolonged ER stress. However, these
genes do not directly cause cell death, but they initiate the
activation of downstream molecules, such as CHOP or JNK,
which further push the cell down the path towards death. CHOP,
a downstream marker in the UPR, is a pro-apoptotic protein that
regulates the activity of genes including Bcl2, GADD34, ERO1
and TRB3 . In our experiments, we demonstrated that Chop
mRNA was elevated during ER stress in P12 S334ter-4 Rho
retinas. The increase in Chop expression suggested that the
adaptive phase of the UPR in the transgenic retinas initiated
apoptosis, causing the S334ter-4 Rho photoreceptors to self-
destruct. The over-expression of the CHOP protein was confirmed
by western blot analysis suggesting that the CHOP protein is
overproduced at transcriptional and translational levels. There-
fore, in summary, we propose that all three UPR pathways are
activated in S334ter-4 Rho retinas.
In general, the CHOP protein is post-translationally controlled
by p38 MAPK (14). Although in our study, a significant difference
in p38 expression was not observed, another MAPK 8 (JNK) was
dramatically upregulated 1.6-fold in P10 transgenic retinas. This
JNK protein is a stress-activated protein kinase that regulates
apoptosis through the induction and/or post-translational modi-
fication of BH3-only proteins and plays a central role in setting the
apoptotic cascade in motion. Evidently, in S334ter-4 Rho
photoreceptors, the upregulation of the JNK gene is associated
with the recruitment of c-JNK via the IRE1 pathway through
TRAF2-c-JNK-ASK1. In support of this hypothesis, we observed
the activation of the Ire1 pathways in P12 S334ter-4 Rho retinas.
The expression of Xbp1 in P12 transgenic retinas was higher when
compared with the control suggesting that the Xbp1 transcrip-
tional factor is required by the elevated production of JNK.
Another rationale for the increase in JNK expression is associated
with the activation of Bim, Bak and Bax proteins .
Regarding the pro-apoptotic Bax/Bak BH3-only proteins, it is
important to note that their relative expression did not change
significantly in the transgenic retinas compared with the control
retinas. This observation implies that the post-translational
phosphorylation of BAX/BAK proteins is primarily a result of
the increase in Jnk expression. In addition, in the developing WT
retina, apoptosis appears to initiate the downregulation of Bax and
Bak, which are key initiators of the caspase-dependent pathway
. The BH3-only BID protein participates in an extrinsic
apoptosis that may occur in cone photoreceptor cells . Because
this BID protein is considered a component of caspase-8-induced
apoptosis, the increase in its expression during P10-P15 may be
associated with the elevated gene expression and activation of JNK
that eventually cleaves BID into a novel form called tBID. This
observation suggests that beginning on P10, JNK may be involved
in TNF-mediated caspase-8 activation resulting in the activation of
the BID protein followed by mitochondrial-associated apoptosis
. However, further investigation is required to confirm this
In general, we determined that other members of the BH3-only
family of proteins are involved in retinal degeneration in S334ter-4
Rho rats. Thus, Bik (Bcl2-interacting killer) protein, which is a
novel death-inducing protein, is over-expressed significantly in P10
retinas. The higher demand for the Bik protein in transgenic
retinas may correlate with the changes in Bcl-xl gene expression.
Again, in our study, a modulation of Bcl-xl gene expression in
transgenic retinas was not observed. An alternative explanation for
the increase in production of the BIK protein is that the elevated
expression of the p53 gene in S334ter-4 Rho retinas promotes Bik
mRNA expression . In support of this hypothesis, we observed
an increase in relative expression of other p53-induced proteins,
such as Noxa and Puma. In P12–P15 S334ter-4 Rho retinas, the
levels of Puma and especially Noxa (3-fold increase) are
dramatically increased. Following the binding to anti-apoptotic
proteins and the activation of Bax/Bak, PUMA-induced apoptosis
proceeds through a typical mitochondrial pathway . There-
fore, we assume that on P12, the over-expression of Puma
ER Stress and S334ter Rhodopsin
PLoS ONE | www.plosone.org 10March 2012 | Volume 7 | Issue 3 | e33266
associates with the mitochondria membrane permeabilization
transition pore (MPTP), which eventually leads to the cleavage
and release of the AIFf1 protein and to the activation of caspase
(see below). In addition, the increase in Noxa expression correlates
with the upregulation of the Hif1a gene, which controls the
expression of Noxa, on P10 . Both Bid and PUMA trigger the
mitochondrial apoptotic pathway leading to cytochrome C and
AIF1 release from the mitochondria as demonstrated in our study
The expression of the BH3-only Bim protein was elevated from
P10 to P15 in S334ter-4 Rho retinas. The BH3-only BIM protein
is an important initiator and regulator of the intrinsic pathway
because BIM interacts with anti-apoptotic Bcl-2 proteins and the
multidomain pro-apoptotic effector proteins BAX and BAK .
Because Bcl-2 expression was not modified, the increase in Bim
expression may be associated with the upregulation of the Jnk
pathway or the downregulation of the pro-survival Erk2 pathway
in transgenic retinas. Recently, links between Bim and cJnk and
between Bim and Erk signaling have been established .
Therefore, a decline in the expression of pro-survival Erk2 in P10
to P21 could regulate the Bim gene expression in S334ter-4 Rho
retinas. An additional study has revealed that the level of Bim
mRNA is positively regulated by C/EBPa and CHOP following
ER stress , and this finding is in agreement with our results
demonstrating an increase in CHOP expression in S334ter-4 Rho
Proteasomal degradation and autophagy are the two main
mechanisms that control protein clearance in the cell. Unlike
proteasomal degradation, autophagy degrades soluble and aggre-
gated proteins. The molecular mechanisms responsible for the
regulation of autophagy have not been completely elucidated;
however, a recent study has demonstrated that severe hypoxia may
lead to ER stress and may induce ATF4-dependent autophagy
through LC3 as a survival mechanism . In a study by Wang
et al., the over-expression of KDEL (ER resident) receptors also
activated autophagy . It is apparent that the upregulation of
the UPR genes increases the expression of KDEL receptors on the
ER and this could promote autophagy in S334ter-4 Rho
photoreceptors. The expression of lysosomal-associated membrane
protein 2 or Lamp2 was induced significantly on P10. Our results
are in agreement with the study of hypoxia-induced Lamp2
activation  in which the authors have proposed that hypoxia
induces a high turnover of autophagic generation and degradation
The activation of calpains in transgenic retinas has been
demonstrated . Kaur et al. have shown that in S334ter Rho
line 3 (a more rapidly degenerating line), the activation of calpain
3, which was measured using an in situ enzymatic assay on unfixed
cryosections reaches a peak on P12. In our study of the S334ter
Rho line 4 (a slower degenerating line), we discovered that on P15
the activation of calpains (1 and 2) is already pronounced (2-fold
increase) and progresses along with retinal degeneration until P30.
Our finding correlates with the study by Kaur et al. proposing that
the proteolytic activity of calpains persists at times when the
nuclear DNA has already disintegrated . In agreement with
these data, we found that the caspase-12 protein was cleaved in
P15 S334ter-4 Rho retina as a result of activated calpains. Later,
however, its activity measured in P21 and P30 S334ter-4 retinas
was diminished. Evidently, transient activation of caspase-12 in
P15 retina is sufficient to trigger the ER stress-associated apoptosis
to contribute to a self-destructive program in S334ter-4 Rho
photoreceptors. In addition, it has been proposed that caspase-12
is not required for caspase-dependant ER stress-induced apoptosis
Therefore, we proposed that active calpains, together with the
BH3-only proteins, Noxa, Puma, Bik, and Bid, compromised the
MPTP in S334ter-4 Rho retinas and control a mitochondria-
induced apoptosis. In support of this hypothesis, we detected the
translocation of cleaved AIF1 from the mitochondria to the cytosol
in S334ter-4 Rho retinas on P15. This data suggests that the
S334ter-4 Rho mitochondria experience MPTP events that
provoke caspase-independent apoptosis. To our knowledge, this
is the first demonstration of AIF1 release from S334ter-4 Rho
mitochondria. Meanwhile, in contrast to the study by Kaur et al.
, the activation of caspase-dependant apoptosis through
cytochrome C release from the mitochondria was not detected in
our experiments. We did not observe difference in cytochrome C
release between SD and S334ter-4 Rho mitochondria. However,
this discrepancy between our study and the study by Kaur et al.
can be explained by differences in the experimental approaches.
Kaur et al. performed the analysis using fixed cryostat retinal
sections, whereas we analyzed protein cytoplasmic fractions in
which we had confirmed the absence of mitochondrial protein
Although we did not observe the cytosolic release of cytochrome
C from mitochondria, an increase in the Apaf1 gene expression
was detected suggesting the caspase-dependent activation of
apoptosis. It is possible that the induction of Apaf1 expression in
S334ter-4 Rho retinas is related to the upregulation of the p53
gene that controls APAf1 , Bik, Noxa and Puma. Therefore,
p53 gene expression and the translocation of p53 to the
mitochondria during the progression of ADRP should be
examined in S334ter-4 Rho retinas. Despite studies demonstrating
that retinal degeneration in rd1 mice occurs independent of p53
, others have demonstrated that the p53 gene plays a role in
the regulation of photoreceptor apoptosis in inherited retinal
The expression of photoreceptor-specific transcription factors
Nrl and Crx declined steadily in S334ter-4 Rho retinas between
P10 and P21 and was reduced significantly in P21 retinas. These
results suggest that in addition to the progressive collapse of
photoreceptors in S334ter-4 Rho retinas, the transcriptional
inhibition of Nrl and Crx may also take place. For example, it
has been proposed that the over-expression of leukemia inhibitory
factor (LIF), which is highly induced in developing ADRP mice
retinas that express a mutant rhodopsin protein , reduces Crx
and Nrl-dependent transcription . Another explanation of the
transcriptional inhibition of the Nrl and Crx transcription factors
is linked to the inhibition of histone deacetylases (HDAC) that are
diminished during retinal degeneration  and affect the RNA
levels of these genes . Apparently, the level of Hdac expression
is modified in S334ter-4 Rho retina. In support of this hypothesis
we observed the elevation in Apaf1 gene expression (Figure 5) that
has been proposed to depend on the Hdac gene expression .
The future study of HDAC expression would also shed light on the
upregulation of the Apaf1 gene in S334ter-4 Rho photoreceptors.
Our results describe mechanisms by which ER stress may be
involved in the retinal pathology of S334ter-4 Rho rats, and how
ER stress may be connected to mitochondrial dysfunction (Fig.S1).
During hypoxia, the ER homeostasis in S334ter-4 Rho photore-
ceptors is compromised, which causes the activation of the UPR.
The persistence of the UPR in S334ter-4 Rho photoreceptors
leads to the upregulation of caspase-12 and BH3-only pro-
apoptotic proteins, that together with calpains, induce MTPT.
Our study and several other studies, have demonstrated that ER
stress- and mitochondria-induced apoptosis culminate in the
activation of caspase-3 in S334ter-4 Rho retinas. We believe that
the activation of both ER stress- and mitochondria-originated
ER Stress and S334ter Rhodopsin
PLoS ONE | www.plosone.org 11March 2012 | Volume 7 | Issue 3 | e33266
apoptotic signals occur at approximately the same time (P12–P15)
during retinal development in S334ter-4 Rho rats. In favor of this
hypothesis, the expression of pro-apoptotic Bcl2 genes was
significantly elevated in P12. Future experiments have to be
conducted to establish a direct link between activation of the UPR
and MPTP in S334ter-4 Rho rats. We also demonstrate that the
relative expression of the UPR, pro-apoptotic, and oxidative-
related genes in S334ter-4 Rho retinas have a temporal
progression between P10 and P18. It is apparent that once
triggered, cell death is executed rapidly and even the temporal
expression of some genes in the P10–P15 retinas leads to apoptotic
cell death. It is important to emphasize that in addition to caspase-
dependent apoptosis occurring in S334ter-4 Rho photoreceptors, a
caspase-independent pathway is induced by the release of AIF1
from the mitochondria. A study by Hong et al. has demonstrated a
direct link between the release of the AIF1 factor from the
mitochondria and the over-activation of PARP-1  suggesting
that our observation of AIF1 release could be considered as
additional proof of a caspase-independent pathway that occurs
simultaneously in photoreceptor cells. However, additional studies
are needed to determine if AIF1 release contributes to the
proposed non-apoptotic cell death in S334ter Rho photoreceptors
Our findings indicate a number of genes that are potential
therapeutic targets for ADRP gene therapy in S334ter Rho
photoreceptors. This list includes but is not limited to Bik, Bim,
Noxa, Puma and Bid proteins, calpains and caspase-12 proteins.
Clearly, further studies are required to shed more light on the
mechanisms involved in the induction of apoptosis, such as
knocking down the expression of these genes in S334ter Rho
Materials and Methods
The animal protocol was carried out with approval from the
Review Board for Animal Studies at the University of North Texas
Health Science Center (Approval Number # 2009/10-46-AO5)
and in accordance with the guidelines of the Association for
Research in Vision and Ophthalmology Statement for the Use of
Animals in Ophthalmic and Vision Research. All efforts were
made to minimize the number and the suffering of the animals
Homozygous S334ter rhodopsin transgenic rats (line 4) were
maintained in the UNTHSC housing facility and were bred with
WT Sprague-Dawley (SD) rats to generate heterozygous S334ter-4
Rhodopsin rats. Therefore, the SD rats were used as WT controls
in our experiments. The animals were sacrificed on P10, P12, P15,
P18 and P21 for RNA and protein analyses. All rats were
maintained in specific pathogen-free (SPF) conditions with a 12-
hour light and 12-hour dark daily cycle.
RNA preparation and Real-Time PCR Analysis
Retinas from SD and S334ter-4 rats at P10, P12, P15, P18 and
P21 of development were isolated. Total RNA was isolated from
the individual retinas from each strain using an RNeasy Mini kit
(Qiagen, Valencia, CA) (P10, N=4; P 12, N=6; P15, N=5, P18
N=6; P21, N=6). Using individual retinal extracts of SD and
S334ter-4 Rho retinas and a high capacity cDNA Reverse
transcription Kit (Applied Biosystems); two cDNAs were prepared
from each RNA sample. Each cDNA (10 ng) was subjected to
qRT-PCR using Applied Biosystems TaqMan assays on 96-well
plates (validated for each selected gene) on a One Step Plus
instrument (Applied Biosystems, Foster City, CA) to compare the
number of cycles (Ct) needed to reach the midpoint of the linear
phase. All observations were normalized to the GAPDH
housekeeping gene. The replicated RQs (Relative Quantity) values
for each biological sample was average. Biological samples from
each strain were used for the qPCR data analysis.
A semi-quantitative RT-PCR analysis of spliced Xbp1 was
performed as described . Quantification of the spliced portion
of the XbP1 cDNA was performed by obtaining ratio of spliced
Xbp1 to normalized unspliced Xbp1.
Retinal protein extract for Western blot analysis
Retinal protein extracts were obtained from dissected retinas by
sonication in a buffer containing 25 mM sucrose, 100 mM Tris-
HCl, pH=7.8, and a mixture of protease inhibitors (PMSF,
TLCK, aprotinin, leupeptin, and pepstatin). The total protein
concentration in right and left retinas from individual rat pups was
measured using a Biorad protein assay, and 40 mg of total protein
was used to detect individual proteins. The detection of proteins
was performed using an infrared secondary antibody and an
Odyssey infrared imager (Li-Cor, Inc.). Antibodies that detect the
stress-induced phosphorylated proteins pPERK, pEIF2a, were
from Cell Signaling (1:1000). Antibody detected pATF6 (full land
cleaved form) was from Imgenex (1:1000). Anti-Grp78 and anti-
CHOP (1:1,000) were from Santa-Cruz Biotechnology; the anti-
AIf1 and anti-caspase-12 antibodies (1:1,000) were from Abcam
and cytochrome C was from Santa-Cruz. b-actin was used as an
internal control and was detected by the application of anti-b-actin
Isolation of cytoplasm and mitochondria from S334ter-4
The isolation of the cytosolic fraction from the individual retinas
of five SD and five transgenic rats was performed using the
Mitochondria Isolation Kit for Tissues (Thermo Scientific). The
mitochondria were separated from the cytoplasm using the
Dounce stroke method as recommended by the Termo Scientific
manufacturer. The protein concentration of each fraction was
determined using a Biorad protein assay. To confirm the absence
of mitochondrial contamination in the cytoplasmic fractions, a
western blot was probed with CoxIV antibody (Abcam).
Calpain activity assay
The detection of calpain activity was performed using the
Calpain Activity Assay kit from BioVision in accordance with the
manufacturer’s recommendations and compared the activation of
calpains in S334ter-4 Rho and SD retinal tissues. The detection of
the cleavage substrate Ac-LLY-AFC was performed in a
fluorometer that was equipped with a 400-nm excitation filter
and 505-emission filter.
All data were evaluated and plotted using GraphPad Prism5
software. We analyzed the results using Student’s t-test for
unpaired samples or a two-way ANOVA analysis of variance.
All data are represented as the mean 6 SEM. The P values that
indicated statistical significance in experiments are ‘‘*’’ (P,0.05),
‘‘**’’(P,0.01), and ‘‘***’’ (P,0.001).
in S334ter-4 Rho rats. The ER stress caused by mis-trafficking
The role of ER stress in retinal degeneration
ER Stress and S334ter Rhodopsin
PLoS ONE | www.plosone.org12March 2012 | Volume 7 | Issue 3 | e33266
of truncated rhodopsin protein contributes to the retinal
degeneration in S334ter-4 Rho rats by inducing hypoxic
conditions and compromising the ER homeostasis resulting in
the activation of the UPR. The UPR in ADRP retinas is associated
with the increased expression of the UPR markers, such as the
eiF2 and Atf4 genes (the PERK pathway), the Atf6 gene (the
ATF6 pathway) and the Xbp1 gene (the IRE1 pathway) and with
elevated expression of BH3-only proteins. The BH3-only proteins,
together with activated calpain, directly or indirectly control the
integrity of the mitochondria through the translocation of active
BAX/BAK, which causes an imbalance leading to the release of
AIF1 from the mitochondria. Therefore, during ADRP progres-
sion, the ER stress signal communicates with the mitochondria to
initiate the collapse of the S334ter-4 Rho photoreceptors.
We like to thank Mansi Kunte for helping with RNA preparation and qRT
Conceived and designed the experiments: MG VS OS. Performed the
experiments: VS OS. Analyzed the data: MG JL ML. Contributed
reagents/materials/analysis tools: MG ML. Wrote the paper: MG.
Provided comments: ML JL.
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PLoS ONE | www.plosone.org14 March 2012 | Volume 7 | Issue 3 | e33266