The hT17M Rho transgenic mouse model is a very useful tool for understanding the activation of ER stress in adRP. T17M mutant rhodopsin has been described previously in cell lines and X. laevis, and to our knowledge this is the first time it has been studied in a mammalian model. We confirm that the mutant hT17M Rho induces ER stress in the T17M+/− ERAI+/− mice at P15 due to its retention within the ER or mislocalization to the cytoplasm (). Further study using specialized microscopic techniques will be required to determine whether the mutant protein forms agglomerates in the cytoplasm of hT17M Rho cells.
Consistent with our immunohistological analysis of T17M+/− ERAI+/− retinas demonstrating the splicing of the Xbp1 mRNA and activation of the IRE pathway, we found that several genes involved in UPR are over-expressed at P15 in hT17M Rho transgenic retinas. Results of the protein analysis also confirm the activation of the UPR in transgenic retina. For example, the PERK signaling is activated in P15 hT17M Rho retina. However, the ATF6 pathway is not modified.
It is important to note here that the hT17M Rho
mouse model has an extra copy of Rho
. To confirm that this induction of UPR genes was not due to Rho
overdose but rather the mutant human transgene, we analyzed the hT17M Rho
mice carrying one copy of mouse Rho
), which were not available for us at the time of the original experiment. Interestingly, these mice demonstrated the same level of modulation of the UPR-associated gene expression compared to hT17M Rho
(Supplementary Fig. S2, Supplementary Table S3. http://www.iovs.org/content/53/7/3792/suppl/DC1
). In T17M+/−
mice, we observed up-regulation of the UPR genes along with elevated Bcl2 family and apoptotic genes expression as early as P15 (Supplementary Fig. S2, http://www.iovs.org/content/53/7/3792/suppl/DC1
Elevation of UPR-associated gene expression also was observed in wild-type retinas injected subretinally with AAV5-hT17M Rho-GFP when compared to AAV5-hRho-GFP injections (Supplementary Fig. S1, Supplementary Table S4, http://www.iovs.org/content/53/7/3792/suppl/DC1
). To support our model further, we checked expression of hRho
(transgene) and mRho
) in T17M Rho
and C57BL/6 retinas at P12, P21, and P30 (). Surprisingly, we observed that when the mRho
expression declines, the hRho
expression increases suggesting a compensatory effect. The total Rho protein in hT17M Rho
, however, was lower compared to wild-type at P15 () indicating that the hT17M Rho
photoreceptors do not over-produce Rho protein.
In hT17M Rho, increases in the expression of the molecular chaperones Bip, Cnx, and Hsp901B, suggesting that the hT17M Rho ER is overloaded with misfolded proteins, and this overload leads to the activation of ER stress responses (). Markers of the PERK and IRE1 pathways are up-regulated in hT17M Rho retinas. For example, expression of the eIf2α and Atf4 genes increases starting at P15. However, the downstream marker, Chop gene, is up-regulated significantly by P12, earlier then the expression of its upstream regulators. The basal level of peIf2α protein appears to be sufficient to stimulate the transcription of the nuclear factor ATF4. Even a slight up-regulation of ATF4 at P12 is sufficient to induce the expression of the Chop gene. The IRE1 pathway also is up-regulated in hT17M Rho retinas by P12. However, it is induced only significantly at P15, which correlates with our immunohistochemical data on splicing of the Xbp1 mRNA (). The ATF6 pathway does not show any specific trend and does not appear to have a significant role in hT17M Rho degeneration (). Therefore, our immunohistochemical, gene expression, and Western blot analyses suggest that ER stress is activated in hT17M Rho retinas.
An analysis of ERAD gene expression shows no significant changes compared to the control (Supplementary Table S1, http://www.iovs.org/content/53/7/3792/suppl/DC1
). However, the autophagy degradation pathway is up-regulated in hT17M Rho
retinas starting at P15 (Supplementary Fig. S3, http://www.iovs.org/content/53/7/3792/suppl/DC1
). This up-regulation appears to be connected to the over-expression of the UPR and Atf4
genes that has been shown to induce autophagy.16
At this point, it is not clear whether autophagy exerts a pro-survival effect in hT17M Rho
retinas by degrading mutant agglomerates or promotes a pro-death, non-apoptotic form of programmed cell death involving the bulk degradation of the cytoplasmic contents. Additional experiments to analyze hT17M Rho
retinas lacking the Atg5
genes will be necessary to answer this question.
BH3-only proteins are regarded as the initial responders to incoming stress signals.17
In our experiments, we observed the overexpression of Bax
, and Noxa
during ER stress, which suggests that pro-apoptotic stimuli are induced in hT17M Rho
photoreceptors. Over-expression of BH3-only genes appears to be related to p53 activation in hT17M Rho
retinas. A link between ER stress and the activation of p53 has been proposed recently.18
x and Bik
also have been shown to be regulated translationally by p53,19,20
and the release of Bid proteins from Bcl-xL sequestration can occur in a p53-dependent manner.21
These data indicate the need for further study of p53 activation in hT17M Rho
The mechanisms by which pro-apoptotic activation occurs include posttranslational alterations, such as phosphorylation (Bad and Bik/Nbk) or proteolytic cleavage (the truncation of Bid by caspase-8).17
Therefore, we next asked the question: “Is there a direct link between overproduction of these proteins and their activation”? Based on our results, there does appear to be a connection. Our analysis of the retinal mitochondrial fraction shows a 55% increase in the release of AIF1 from the hT17M Rho
mitochondria, indicating that the mitochondria permeability transition pore (MPTP) already exists by P21. This result implies that the over-expression of BH3-only proteins correlates with an increase in their post-translational modification.
Pro-survival markers, such as Akt signaling and Bcl2 over-expression, are present in hT17M Rho
retinas. Akt signaling controls the dissociation of Bad protein from the Bcl-2/Bcl-X complex and the loss of its pro-apoptotic function.22
In our experiments, Bad
gene expression was found not to be elevated in hT17M Rho
retinas, and it is possible that the Akt-controlled posttranscriptional phosphorylation of Bad affects its transcriptional level. Another signaling molecule, the anti-apoptotic gene Bcl-2
, is up-regulated in hT17M Rho
retinas after P12, at which point the expression of the Chop
gene already is increased. The Bcl-2
gene is known to be a transcriptional control target of the CHOP transcriptional factor.23
Therefore, after P15, when Chop
expression decreases, Bcl2
gene expression increases. Both pro-survival pathways are up-regulated in hT17M Rho
retinas in an attempt to balance the expression of pro-apoptotic Bcl2 proteins and restore homeostasis in hT17M Rho
photoreceptors. Simultaneous activation of pro- and anti-apoptotic pathways in our experiment, however, could be accounted for by different dynamics of degeneration in different retinal cell types. For example, while rod photoreceptors already are undergoing suicide by apoptosis, neighboring cells, such as cone photoreceptors and retinal ganglia cells, still are struggling to prevent apoptosis.
Finally, we found that ER stress and mitochondria-associated apoptosis lead to the final demise of photoreceptors (, A). Both caspase-3 and caspase-12 are up-regulated. However, caspase-3 is up-regulated earlier than caspase-12, suggesting that other cellular pathways lead to the activation of the executioner caspase. These findings are consistent with the rate of adRP progression in transgenic retinas measured by OCT (C). At 1 month, transgenic mice already have lost 50% of their photoreceptors, resulting in the downregulation of the transcriptional factors Nrl and Crx starting as early as P15 and P18. These changes apparently are responsible for decrease in the level of Rho protein in hT17M Rho retina.
To our knowledge, for the first time, we demonstrated that the ER stress response is involved in retinal degeneration in hT17M Rho retinas, and correlates strongly with the up-regulation of autophagy markers and the activation of mitochondrial signaling via the up-regulation of pro-apoptotic Bcl2 genes. The results of our experiments allow us to identify the following genes that could be potential therapeutic targets for adRP treatment: Atg5, Atg7, Bax, Bid, Bik, and Noxa.