CHOP protein is known to be over-expressed in ADRP photoreceptors 
and is a major factor, the ablation of which leads to attenuation of pathogenic hallmarks of different degenerative disorders 
. Therefore, it was logical for us to test the hypothesis that creating a deficiency in CHOP protein could be used as a therapy for ADRP photoreceptors. Thus, in this study, we not only demonstrated that CHOP protein could not be considered as a potential therapeutic target for treating ADRP photoreceptors but also revealed the cause of accelerated retinal degeneration in ADRP retinas deficient in CHOP.
Despite the fact that the T17M RHO
retina is known to rapidly degenerate 
, the T17M RHO
CHOP−/− retinas demonstrated a much more severe loss of the a-wave amplitudes of the scotopic electroretinogram. For example, in 1-month-old T17M RHO
CHOP−/− retinas, we observed a 76% loss of a-wave amplitude compared to T17M RHO
mice and no significant difference in the b-wave amplitude, suggesting first, that the main physiological changes occur in the T17M RHO
CHOP−/− photoreceptors and second, that the ADRP photoreceptor cells experiencing the activation of the UPR due to expression of the human T17M RHO
transgene respond to CHOP ablation more rapidly than bipolar cells. Despite the fact that the reduction of ERG b-wave amplitudes shows just one trend in the T17M RHO
CHOP−/− retina, the loss of photoreceptors is evidently responsible for the lessening of b-wave amplitude and the fact that the CHOP−/− retinas do not demonstrate loss of either a- or b-wave amplitudes and do exhibit normal ERG favors this hypothesis. In 2- and 3-month-old T17M RHO
and T17M RHO
CHOP−/− mice, both the a- and b-wave amplitudes were almost identical because the T17M RHO
retinas fully degenerate by this time point. In 1 month-old mice, the measurement of implicit time of the a- wave ERG amplitudes also reveals that it is was significantly longer in T17M RHO
CHOP−/− mice compared to all other groups and indicates a delay in the response of photoreceptors to the light flash (P
<0.001). The implicit time for the b-wave ERG amplitude is changed slowly in T17M RHO
CHOP−/− mice and only by 3 month of age. The response of bipolar cells to the light stimulation of photoreceptors is postponed. This fact indicates that despite no differences in the b-wave ERG amplitudes between T17M RHO
and T17M RHO
CHOP−/− mice is observed, the bipolar cells respond slower suggesting that functional changes in these mice could be obvious at later time points.
The functional loss of photoreceptors in T17M RHO CHOP−/− retinas, which is already accelerated by 1 month, could be a result of photoreceptor cell death, which we confirmed by SD-OCT, histological and IHC analyses. In the T17M RHO CHOP−/− retinas, the average thickness of the ONL in the superior and inferior regions were reduced by 24% and 22%, respectively. The number of nuclei in the ONL was dramatically reduced by 33% suggesting that in these animals, the photoreceptor cells were undergoing morphological changes. In addition, the ablation of CHOP protein in T17M RHO retina at least partially prevents the RHO protein from its trafficking to the OS and promotes its aggregation increasing the burden of misfolded protein in photoreceptors. At this point we do not know a precise localization of mistrafficking rhodopsin. To answer the question of whether rhodopsin retains within the Endoplasmic Reticulum or forms agglomerates in the cytosol of PR, additional experiments such as electron microscopy should be conducted.
Therefore, the photoreceptor cell death could be hastened in T17M RHO CHOP−/− mice by modified cellular signaling, thus leading to advanced retinal degeneration by 1 month of age. Therefore, we next performed RNA and protein analyses.
Behram et al. 
noted the relationship between the RHO
and CHOP genes at the level of post-transcriptional regulation of RHO
mRNA during activation of the UPR. That study revealed that this control occurs through transcriptional regulation of miR-708 by CHOP protein and assigned the CHOP protein a cytoprotective function. Despite the significant contribution of this study to progressing our understanding of mammalian gene expression networks, the precise role of CHOP protein ablation in ADRP retinas experiencing activation of the UPR has not been studied. Therefore, we performed experiment in which we analyzed the level of miR-708 in ADRP retina. We found no difference between all four groups of animals when analyzed by one-way Anova suggesting that in ADRP retina: 1) the CHOP ablation does not promote modulation of the miR-708 expression (T17M RHO
CHOP+/+ vs T17M RHO
CHOP−/−) as it has been proposed for the MEFs treated with thapsigardin 
and 2) the ER stress (T17M RHO
) down-regulates the level of miR-708 in the CHOP deficient retina (CHOP−/− vs T17M RHO
CHOP−/−). Overall, the conclusion we made supports the general finding presented by Behram et al. 
that the combination of ER stress and CHOP ablation promotes reduction of the miR-708 (wild-type vs T17M RHO CHOP−/−, P
While looking for the cellular mechanism involved in the rapid retinal degeneration observed in T17M RHO CHOP−/− mice, we tested the RHO mRNA expression and found that the levels of mouse and human RHO mRNA were significantly reduced in T17M RHO CHOP−/− compared to T17M RHO retinas, suggesting that either photoreceptor cell death or transcriptional inhibition is responsible for this down-regulation. It also points the fact that the additional regulatory pathways besides the transcriptional regulation of the RHO by miR-708 could be responsible for accelerated retinal degeneration of T17M RHO CHOP−/− mice.
From a previous study by our group 
, we knew that compared to wild-type mice, T17M RHO
mice express the photoreceptor transcriptional factors Nrl and Crx at lower levels and exhibit a major collapse of photoreceptors at P25. Therefore, we then studied Nrl
It is known that while Crx activity regulates the differentiation of both rods and cones, the Nrl transcriptional factor is preferentially expressed in rod photoreceptors, where it is thought to act synergistically with Crx to regulate rhodopsin expression. Mutations in human Nrl
have been associated with ADRP, which is characterized by rod photoreceptor degeneration, and deletion of Nrl
in mice results in complete loss of rod function 
. In our experiment, we found that down-regulation of these transcriptional factors in T17M RHO
CHOP−/− mice is probably responsible for the reduction of RHO
expression in these mice. The observed down-regulation of Nrl
by 95% and 60%, respectively, indicates that the ablation of the CHOP protein accelerates the degeneration of rod photoreceptors. It also points out the fact that other relevant phototransduction cascade genes could be down-regulated in T17M RHO
CHOP−/− leading to significant vision loss.
The results of the ERG and OCT experiments, as well as the results of histological analysis and analysis of RHO
expression, show that under normal conditions, the CHOP−/− retina is not significantly different from that of wild-type mice. Therefore, we further investigated only two groups of animals: T17M RHO
and T17M RHO
CHOP−/− mice. Since PERK and the IRE1 signaling pathways are activated in P15 T17M RHO
, we examined these pathways in 1-month-old T17M RHO
CHOP−/− retinas. We detected significant up-regulation of peIF2α in the P30 T17M RHO
CHOP−/− retina, which suggests that these animals experience long-lasting transcriptional inhibition of gene expression in their photoreceptors. The fact that CHOP ablation leads to the over-production of peIF2α has been demonstrated previously 
. For example, in cardiomyocytes experiencing activation of the UPR, the ablation of CHOP protein leads to an increased peIF2α level, which is considered to be cytoprotective. Under normal conditions, CHOP protein controls the expression of GADD34 that binds the protein phosphatase 1 and, thus, negatively regulates the phosphorylation of eIF2α, leading to reduced protein translation and the inhibition of protein synthesis. Therefore, under CHOP-deficient conditions, decreased expression of GADD34 enhances the phosphorylation of eIF2 α, and this lack of GADD34 is likely to contribute to the prevention of the suppression of protein synthesis. We believe that the inhibition of protein synthesis is long lasting in the T17M RHO
CHOP−/− retina. In T17M RHO
retinas, peIF2α is found to be up-regulated by P15. In P30 T17M RHO
CHOP−/− retinas, this up-regulation most likely leads to transcriptional protein repression and retinal degeneration. Transcriptional inhibition of gene expression from P15 until P30, which is assumed to be the period required to successfully resolve the compromised ER homeostasis, is extended and presumably affects general biosynthesis.
Spliced XBP1 regulates genes that are implicated in protein folding, trafficking, and secretion and thus contributes to the restoration of ER homeostasis and favors cell survival under ER stress 
. In earlier work, we demonstrated splicing of Xbp1 and consequent activation of the IRE1 pathway in T17M RHO
. In the current study, we found that in T17M RHO
CHOP−/− retinas, the level of spliced Xbp1 was decreased compared to controls, suggesting that the function of the pro-survival arm is compromised in these animals. Therefore, protein analysis of T17M RHO
CHOP−/− retinas demonstrates an elevated level of pro-apoptotic PERK signaling and diminished level of the pro-survival Xbp1 UPR arm. Our results were consistent with the findings in a previous study in which ER stress was induced in hippocampus of the Wild-type and CHOP−/− mice by intracerebroventricular injection with tunicamycin. According to that study, CHOP−/− mice showed enhanced hippocampal cell apoptosis and decreased sXBP-1 expression 
To examine the link between the down-regulation of Nrl
gene expression and CHOP ablation in the T17M RHO
CHOP−/− retina, we examined the histone acetyltransferase P300, which is known to be recruited by the CHOP transcription factor 
and to regulate opsin expression along with Crx, Nrl and PolII 
. Additionally, dynamic histone acetylation is controlled by HDACs, which exert an opposite function and are often associated with reduced accessibility of the transcriptional machinery to genes 
The 1-month-old T17M RHO
CHOP−/− retina exhibited a 2-fold higher Hdac1 protein content, suggesting that deacetylation processes prevailed in these mice. Increased histone deacetylation could repress general transcription and accelerate retinal degeneration in these mice. For example, it is known that the hypoacetylation is a major factor associated with severe retinal degeneration in rd1 mice 
. The peak of Hdac activity occurs in rd1 mice at P11, while the peak of TUNEL staining takes place at P13 in the rd1 retina, implying that the observed photoreceptor cell death results from elevated Hdac activity. In addition, it has been shown that HdacI/II inhibitors protect rd1 photoreceptors and strongly reduce photoreceptor cell death by decreasing poly-ADP-ribose-polymerase (PARP).
In addition to an increase in Hdac1 protein expression, we found that the protein level of P300 was decreased in T17M RHO
CHOP−/− retinas, implying that there was negative regulation between CHOP and P300 gene expression. Additionally, the reduction of spliced Xbp1 expression could also contribute to the reduction of P300 expression, as a direct dose-dependent link has been proposed between Xbp1 and P300 
Despite the multiple studies focusing on the silencing of pro-apoptotic CHOP protein for the purpose of developing cellular therapies, we demonstrated that CHOP protein is a survival factor for rod photoreceptors carrying a severe ADRP mutation. The ablation of CHOP protein most likely extends transcriptional inhibition and leads to a reduction of the expression of photoreceptor-specific transcriptional factors and RHO protein, along with an increase in histone deacetylation. These findings suggest that CHOP plays a protective role in rod photoreceptor cells of ADRP affected mouse retinas. However, the role of CHOP protein in other cell types of ADRP mouse retinas needs to be clarified in the future.