Modifier Genes as Therapeutics: The Nuclear Hormone Receptor Rev Erb Alpha (Nr1d1) Rescues Nr2e3 Associated Retinal Disease

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-Erba) 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

Genetic Variations Strongly Influence Phenotypic Outcome in the Mouse Retina

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2-nb1 at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases

Multimodal Regulation Orchestrates Normal and Complex Disease States in the Retina

Regulation of biological processes occurs through complex, synergistic mechanisms. In this study, we discovered the synergistic orchestration of multiple mechanisms regulating the normal and diseased state (age related macular degeneration, AMD) in the retina. We uncovered gene networks with overlapping feedback loops that are modulated by nuclear hormone receptors (NHR), miRNAs, and epigenetic factors. We utilized a comprehensive filtering and pathway analysis strategy comparing miRNA and microarray data between three mouse models and human donor eyes (normal and AMD). The mouse models lack key NHRS (Nr2e3, RORA) or epigenetic (Ezh2) factors. Fifty-four total miRNAs were differentially expressed, potentially targeting over 150 genes in 18 major representative networks including angiogenesis, metabolism, and immunity. We identified sixty-eight genes and 5 miRNAS directly regulated by NR2E3 and/or RORA. After a comprehensive analysis, we discovered multimodal regulation by miRNA, NHRs, and epigenetic factors of three miRNAs (miR-466, miR1187, and miR-710) and two genes (Ell2 and Entpd1) that are also associated with AMD. These studies provide insight into the complex, dynamic modulation of gene networks as well as their impact on human disease, and provide novel data for the development of innovative and more effective therapeutics

Strain specific alleles and differential expression of <i>Nr1d1</i>.

<p>(A) C57BL/6J and AKR/J chromatograms of polymorphisms identified in the ligand-binding domain of <i>Nr1d1.</i> (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 <i>Nr1d1</i> 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) <i>Nr1d1</i> relative expression in P30.5 AKR/J and C57BL/6J retinas (mean ± SD of mean, n = 3, p = 0.0024). (F) <i>Nr1d1</i> relative expression in P30.5 C57BL/6J, CAST/EiJ and NOD.NOH-H2^nb1 retinas (p<0.05).</p

Gene delivery of <i>Nr1d1</i> suppresses pan-retinal spotting, retinal dysplasia and function in <i>Nr2e3</i>^rd7/rd7 mice.

<p>(A–F) Fundus photographs of control and <i>rd7</i> injected retinas: (A) B6 (uninjected), (B) <i>rd7</i> (uninjected), (C) <i>GFP</i> injected, (D) <i>GFP.Nr2e3^B6</i> injected, (E) <i>GFP.Nr1d1^AKR/J</i> injected, (F) <i>GFP</i>.<i>Nr1d1</i>^B6 injected. (G–J) DAPI staining (blue) shows rescue of defects in retinal morphology 30 days after electroporation into <i>rd7</i> neonatal retinas. (G) GFP control, (H) <i>Nr2e3^B6</i> injected, (I) GFP control, (J) <i>Nr1d1</i>^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 <i>GFP</i> (blue) or <i>GFP.Nr1d1^AKR/J</i> (red).</p

Expression of phototransduction genes <i>Opn1sw</i> and <i>Gnat2</i> is rescued in <i>rd7</i> retinas upon <i>Nr1d1</i> delivery.

<p>Quantitative real time PCR shows that <i>Nr1d1</i> delivery results in down-regulation of the phototransduction genes <i>Opn1sw</i> and <i>Gnat2</i> in <i>rd7</i> retinas (mean ± SD of mean, n = 3, p<0.05), to near normal levels.</p