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 
, in this study, we focus on the relationship between gene expression and the progression of ADRP in transgenic rats expressing truncated S334ter rhodopsin.
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 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 (). 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 Hif1α 
, 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 HIF1α 
. A number of investigators have proposed that retinal hypoxic preconditioning, which leads to HIF1α induction, morphologically and functionally protects retinal cells against light-induced retinal degeneration 
. Others have identified HIF1α 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 Hif1α 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 Hif1α has not been examined previously in S334ter-4 Rho retinas. In this study, we demonstrated that hypoxic conditions leading to oxygen deprivation persisted, at least temporarily, in the developing S334ter-4 Rho retinas. The mechanism by which the Hif1α 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 Hif1α 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 Hif1α 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 H2
, which has been shown to play a protective role in oxygen-deprived dopaminergic neurons in the rat substantia nigra 
. Alternatively, 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, Hif1α 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, eIF2α (eukaryotic translation initiation factor 2α) 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 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 (). 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 (), 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 retrotranslocation 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 peIF2α. In P15 retinas western blot analysis demonstrated the increased production of the peIf2α 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 mitochondrial MPTP after H2
exposure in S334ter-4 Rho photoreceptors 
. 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 ().
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. Therefore, 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 modification 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 transcriptional 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 hypothesis.
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 
. Therefore, we assume that on P12, the over-expression of Puma 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 Hif1α 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 retinas.
Proteasomal degradation and autophagy are the two main mechanisms that control protein clearance in the cell. Unlike proteasomal degradation, autophagy degrades soluble and aggregated 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 in cells.
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 contamination.
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 degeneration 
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 () 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 photoreceptors 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 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 retinas.