In this study, we report the first demonstration of small molecule repression of rod gene expression for the potential treatment of dominant retinitis pigmentosa. The expression of Crx, Otx2, Nrl, and Nr2e3 in mature rods is consistent with their role in the maintenance of proper photoreceptor gene expression and homeostasis.
32 We screened compounds that were previously identified as Nr2e3 interacting compounds (using high-throughput FRET and cell-based Nr2e3:Ncor interaction screens) for their ability to modulate rod gene expression in developing retinal cells in vitro. We found that a subset of these compounds inhibited the expression of rhodopsin in the screen and we further characterized one of these, PR1, for its ability to inhibit expression of rod genes in developing and mature rod photoreceptors. The activity of PR1 on rod photoreceptors is similar to loss-of-function mutations in
Nr2e3, and significantly reduces expression from the rhodopsin promoter in HEK293T cells transfected with
Nr2e3,
Crx, and
Nrl, and affects the binding of Nr2e3 to Crx and Nrl. Together the data support the conclusion that PR1 interacts with Nr2e3 and acts to antagonize its activity, most likely through its binding with transcriptional cofactors.
The results from the previous screens indicate that PR1 can interact with Nr2e3, most likely through the ligand binding domain, and inhibit the interactions with the corepressor Ncor. Our data also demonstrate that PR1 can inhibit expression from the rhodopsin promoter, driven by Nr2e3-Crx-Nrl cotransfection in HEK cells. Somewhat paradoxically, immunoprecipitation shows that PR1 causes an increase in the binding of Nr2e3 with Crx and Nrl. This may be due to changes in the ligand binding domain of Nr2e3 upon PR1 binding. The protein Crx is thought to interact with Nr2e3 through the DNA binding domain (DBD),
19 and so it is unlikely that PR1 would affect this interaction directly. Several Nr2e3 variants associated with enhanced S-cone syndrome reduce Nr2e3 binding with Nrl (p.L336P, p.L353V, and p.R385P); however, two of these impair Nrl/Crx-mediated transactivation of the
Rhodopsin promoter (p.L336P,p.L353V ), while another potentiates transactivation.
33 Thus, there is a complex relationship between the binding affinity of Nrl and Nr2e3 and the ability of this complex to activate rod gene transcription, and this may relate to interactions with corepressors and coactivators, which are not completely understood for this system. Additionally, Nr2e3 homodimerization is affected by many disease-causing mutations in the DBD of Nr2e3, and these mutations can both potentiate and repress transactivation of the
Rhodopsin promoter.
34 It is also possible that the effect of PR1 on decreasing transcription factor-mediated activation of the
Rho promoter could be in part due to changes in Nr2e3 homodimerization, and we cannot detect changes in homodimer formation from the co-IP results.
The primary effect we observe after treatment of the retina, either in vitro or in vivo, is a reduction in the expression of rod photoreceptor expressed genes, like
Rho and
Gnat1. However, not all rod genes are reduced to the same extent; we see no changes in
Gnb1 expression, for example. Moreover, both
Nrl and
Nr2e3 are significantly reduced by PR1 treatment at either P0 or P12, and some of the effects we observe on rod gene expression may be due to the reduction in these transcription factors. Similar results are seen after conditional knockout of
Nrl in adult mice, in that rod genes are more affected than cone genes.
13 Additionally, fewer cone genes are upregulated following knockout in the adult compared to germline knockout of
Nrl, possibly due to developmental changes in the methylation status of cone gene promoters.
13 Nonetheless, this partial “reprogramming” via conditional knockout of
Nrl is sufficient to prevent photoreceptor degeneration in the
Rho knockout model of RP,
13 similar to our findings with the
RhoP23H and
Pde6brd1 models in vitro. Together, these studies demonstrate that downregulation of rod gene program in degenerative diseases that primarily affect rods may be an effective strategy for treatment in humans. Suppression of rhodopsin or other commonly mutated rod genes with siRNAs may also be similarly effective.
35,36
Interestingly, our effects on rod gene expression are much more pronounced than the effects reported from
Nr2e3 loss-of-function models, such as the
rd7 mouse and the targeted knockout of
Nr2e3.
19,21,23,24,32 In these mice, rod genes like
Rho and
Gnat1 are only modestly reduced, while we observed large changes with PR1 treatment. This difference may be due to our finding that PR1 also decreases the expression of
Nrl, while these genetic mutations do not, and Nrl may be sufficient to drive rod gene expression in the absence of Nr2e3. It also remains possible that PR1 inhibits
Nrl expression through an Nr2e3-independent mechanism. In addition to changes in the expression of rod genes,
rd7 mice show a derepression of some cone genes. Similarly, mutations in
Nr2e3 can cause enhanced S-cone syndrome and patients present increased sensitivity to blue light.
37 We did not observe very large increases in S-opsin expression that we expected from antagonism of Nr2e3, particularly in the mature retina. It is possible that PR1 selectively affects the ability of Nr2e3 to act as a transcriptional activator (inhibiting its ability to form a complex with Crx and Nrl), but has less of an effect on its repressor functions.
38 However, it is also possible that acute loss of Nr2e3 has different effects than developmental deletions. Future studies will be needed to distinguish between these alternatives.
In addition to Nr2e3, there are several other nuclear receptors that regulate photoreceptor development and maintenance (see Ref. 39 for review), and might constitute targets for the treatment of retinal disease. Retinoid X receptors, thyroid hormone receptor-beta 2, and Coup-TF are important in cone photoreceptor development, regulating the expression of S- and M-opsin. In addition to Nr2e3, Nr1d1 (RevErbα), and Nr3b2 (ERRβ) are important in rod photoreceptor development and maintenance.
40,41 Retinoic acid receptor–related orphan receptor beta (RORβ) coordinates the development of rods and cones, by activating expression of
Nrl.
42 Although our evidence suggests that Nr2e3 is a potential target of PR1, it is also possible that one of these other nuclear receptors binds PR1 as well. For example, if PR1 also inhibits the activity of RORβ, this might explain the changes we observe in Nrl expression.
In conclusion, we demonstrate that a small molecule can regulate Rho expression in developing and mature retina, and could provide a novel approach to the treatment of dominant forms of RP. The ability to modulate rod and cone gene expression may also have utility in recessive forms of the disease, since conditional deletion of Nrl in mature mice provided rod protection in the Rho–/– mouse. The possibility for small molecule reprogramming of a cell affected with a disease causing mutation to a related cell that does not express the mutant allele might also have application in other genetic disorders.