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. 2014 Jan 31;9(1):e87942.
doi: 10.1371/journal.pone.0087942. eCollection 2014.

Modifier genes as therapeutics: the nuclear hormone receptor Rev Erb alpha (Nr1d1) rescues Nr2e3 associated retinal disease

Nelly M Cruz  1 , Yang Yuan  2 , Barrett D Leehy  3 , Rinku Baid  4 , Uday Kompella  5 , Margaret M DeAngelis  6 , Pascal Escher  7 , Neena B Haider  1
Affiliations

Modifier genes as therapeutics: the nuclear hormone receptor Rev Erb alpha (Nr1d1) rescues Nr2e3 associated retinal disease

Nelly M Cruz et al. PLoS One. .

Abstract

Nuclear hormone receptors play a major role in many important biological processes. Most nuclear hormone receptors are ubiquitously expressed and regulate processes such as metabolism, circadian function, and development. They function in these processes to maintain homeostasis through modulation of transcriptional gene networks. In this study we evaluate the effectiveness of a nuclear hormone receptor gene to modulate retinal degeneration and restore the integrity of the retina. Currently, there are no effective treatment options for retinal degenerative diseases leading to progressive and irreversible blindness. In this study we demonstrate that the nuclear hormone receptor gene Nr1d1 (Rev-Erbα) rescues Nr2e3-associated retinal degeneration in the rd7 mouse, which lacks a functional Nr2e3 gene. Mutations in human NR2E3 are associated with several retinal degenerations including enhanced S cone syndrome and retinitis pigmentosa. The rd7 mouse, lacking Nr2e3, exhibits an increase in S cones and slow, progressive retinal degeneration. A traditional genetic mapping approach previously identified candidate modifier loci. Here, we demonstrate that in vivo delivery of the candidate modifier gene, Nr1d1 rescues Nr2e3 associated retinal degeneration. We observed clinical, histological, functional, and molecular restoration of the rd7 retina. Furthermore, we demonstrate that the mechanism of rescue at the molecular and functional level is through the re-regulation of key genes within the Nr2e3-directed transcriptional network. Together, these findings reveal the potency of nuclear receptors as modulators of disease and specifically of NR1D1 as a novel therapeutic for retinal degenerations.

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Conflict of interest statement

Competing Interests: Co-authors Neena B, Haider and Margaret D. Angelis are PLOS ONE Editorial Board Members. This does not alter the authors′ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. rd7 phenotypes are suppressed in N6 B6.Cg-Mor7 AKR:Nr2e3rd7/rd7 mice.
(A, B) Hematoxylin and eosin staining of retinal sections from affected (A) and suppressed (B) F2 B6.Cg-Mor7 AKR:Nr2e3rd7/rd7 P30 animals. Retinal dysplasia was absent in the suppressed rd7 homozygote animals. (C, D) Labeling of retinal sections with anti-OPN1SW shows that the S-cone population is restored to a normal level in suppressed F2 Cg.AKR/J-Nr2e3rd7/rd7 animals (D), compared to affected animals (C). (E) Chart showing distribution of the 95 retinal genes that map to the Mor7 interval. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer, OS: outer segments, RPE: retinal pigment epithelium.
Figure 2
Figure 2. Strain specific alleles and differential expression of Nr1d1.
(A) C57BL/6J and AKR/J chromatograms of polymorphisms identified in the ligand-binding domain of Nr1d1. (B) ClustalW2 sequence alignment of amino acid sequences from C57BL/6J, AKR/J, rat, chimpanzee and human. Stars indicate identity in all sequences, while dots indicate conserved amino acids. (C) C57BL/6J and AKR/J chromatograms of polymorphisms identified in the Nr1d1 5′UTR region. (D) ClustalW2 sequence alignment across species reveals the consensus is in accordance with AKR/J sequence. Stars indicate nucleotide conservation in all species. (E) Nr1d1 relative expression in P30.5 AKR/J and C57BL/6J retinas (mean ± SD of mean, n = 3, p = 0.0024). (F) Nr1d1 relative expression in P30.5 C57BL/6J, CAST/EiJ and NOD.NOH-H2nb1 retinas (p<0.05).
Figure 3
Figure 3. GFP expression in P30 rd7 retina electroporated with GFP.Nr1d1AKR/J at P0.
(A) GFP expression (green) is apparent in the outer nuclear layer (ONL) and inner nuclear layer (INL) of the retina and co-localizes with the nuclear marker DAPI (blue) (B). INL: inner nuclear layer, ONL: outer nuclear layer, RPE: retinal pigment epithelium. Scale bar = 50 µm.
Figure 4
Figure 4. Gene delivery of Nr1d1 suppresses pan-retinal spotting, retinal dysplasia and function in Nr2e3 rd7/rd7 mice.
(A–F) Fundus photographs of control and rd7 injected retinas: (A) B6 (uninjected), (B) rd7 (uninjected), (C) GFP injected, (D) GFP.Nr2e3B6 injected, (E) GFP.Nr1d1AKR/J injected, (F) GFP.Nr1d1 B6 injected. (G–J) DAPI staining (blue) shows rescue of defects in retinal morphology 30 days after electroporation into rd7 neonatal retinas. (G) GFP control, (H) Nr2e3B6 injected, (I) GFP control, (J) Nr1d1 AKR/J injected. L: left, R: right, GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. Scale bar = 50 µm. (K, L) Representative scotopic (K) and photopic (L) electroretinograms from animals 4 month after injection with GFP (blue) or GFP.Nr1d1AKR/J (red).
Figure 5
Figure 5. Expression of phototransduction genes Opn1sw and Gnat2 is rescued in rd7 retinas upon Nr1d1 delivery.
Quantitative real time PCR shows that Nr1d1 delivery results in down-regulation of the phototransduction genes Opn1sw and Gnat2 in rd7 retinas (mean ± SD of mean, n = 3, p<0.05), to near normal levels.
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