Neena B. Haider - US grants

The rd/rd7 mutant mice display retinal degeneration and have white, evenly spaced spots over their entire retina at one month of age and subsequently develop mottled retinal pigmentation and a 50% photoreceptor cell degeneration by 16 months of age. The rd7 mouse carries a mutation in the photoreceptor cell-specific nuclear receptor (NR2E3) gene. Mutations in the human NR2E3 gene are associated with a unique retinal dystrophy, Enhanced S Cone syndrome (ESCS). Most inherited human retinal diseases affect mature photoreceptor distribution by reducing the numbers of receptors in the mosaic through apoptotic mechanisms. A common finding is that disease-causing photoreceptor- specific genes alter key structures or functions within these cells that lead to cell death. ESCS is unique in that it manifests as greater numbers of a subtype of photoreceptors, showing a major increase in the least populous cone subtype, the S-cones; with varying degrees of retinal degeneration. The goal of this study is to functional characterize mNR2E3 and gain insight into mechanisms involved in the correct development and function of the retina. This goal will be accomplished by investigating the following aims: 1. determining the temporal and spatial expression pattern of NR2E3, 2. identifying factors that interact with NR2E3, and 3. identifying downstream effector genes.

The rd/rd7 mutant mice display retinal degeneration and have white, evenly spaced spots over their entire retina at one month of age and subsequently develop mottled retinal pigmentation and a 50% photoreceptor cell degeneration by 16 months of age. The rd7 mouse carries a mutation in the photoreceptor cell-specific nuclear receptor (NR2E3) gene. Mutations in the human NR2E3 gene are associated with a unique retinal dystrophy, Enhanced S Cone syndrome (ESCS). Most inherited human retinal diseases affect mature photoreceptor distribution by reducing the numbers of receptors in the mosaic through apoptotic mechanisms. A common finding is that disease-causing photoreceptor- specific genes alter key structures or functions within these cells that lead to cell death. ESCS is unique in that it manifests as greater numbers of a subtype of photoreceptors, showing a major increase in the least populous cone subtype, the S-cones; with varying degrees of retinal degeneration. The goal of this study is to functional characterize mNR2E3 and gain insight into mechanisms involved in the correct development and function of the retina. This goal will be accomplished by investigating the following aims: 1. determining the temporal and spatial expression pattern of NR2E3, 2. identifying factors that interact with NR2E3, and 3. identifying downstream effector genes.

DESCRIPTION (provided by applicant): Retinal degenerations are a group of genetically heterogeneous disorders that can often be classified according to the type of pathology observed in rod and cone photoreceptors. These diseases affect one in every 4000 individuals and it is clear the severity of disease is strongly affected by genetic factors. This large group of disorders includes retinitis pigmentosa, macular degeneration, Bardet-Biedl syndrome, Usher syndrome, and enhanced S-cone syndrome (ESCS), each of which has photoreceptor degeneration as a major component of the disease phenotype. Our long-term goal is to understand the transcriptional networks regulating photoreceptor generation and maintenance, which will enable us to identify novel targets that may be amenable for improved treatment strategies for retinal disease. Our studies and those of others demonstrate that the nuclear receptor Nr2e3 functions in multiple transcriptional networks to regulate the development and maintenance of photoreceptor cells. The PI was the first to report that mutations in human Nr2e3 cause the recessive ESCS, and mutations in mouse Nr2e3 cause excess production of blue opsin expressing cone cells with progressive retinal degeneration. Recent findings also demonstrate that mutations in human Nr2e3 can have significant variability in phenotypic manifestation causing a milder ESCS phenotype, Goldman Favre syndrome, or dominant retinitis pigmentosa. This underscores the importance of Nr2e3-directed transcriptional pathways in retinal disease and suggests the existence of human modifier genes influencing these diseases. The objective of this proposal is to identify genetic modifiers of retinal degeneration in the mouse model Nr2e3rd7/rd7. We utilize two approaches: a genetic mapping strategy, and a candidate gene approach to perform our studies. Aim 1 is to identify the genetic modifier of Nr2e3rd7/rd7, referred to as Mor7, on the AKR/J strain background using a positional cloning approach. We have mapped this modifier gene and developed a congenic line (N9) that ameliorates Nr2e3rd7/rd7 associated retinal degeneration. Aim 2 is to determine whether the nuclear receptor Nr1d1, a cofactor of Nr2e3, can modify Nr2e3rd7/rd7 associated retinal degeneration. We will test our hypothesis by over-expressing Nr1d1 in newborn Nr2e3rd7/rd7 mice to determine if Nr1d1 can rescue retinal degeneration and retinal explant experiments to determine the effects of altered Nr2e3 or Nr1d1 expression on rod or cone photoreceptor cell fate. Our studies will greatly enhance understanding of genetic factors that influence severity of retinal disease, and, provide potentially powerful targets for improved therapies to treat or prevent multiple forms of retinal disease involving photoreceptor degeneration. PUBLIC HEALTH RELEVANCE: Retinal diseases are debilitating disorders that affect millions of individuals worldwide and can often lead to complete blindness. We propose to use mouse models to identify genes that can correct retinal disease. These genes will be potentially powerful targets that can be used to treat not one but many forms of retinal disease.

PROJECT SUMMARY/ABSTRACT: Microglia are resident myeloid-lineage cells in both the CNS and in the eye and function in the maintenance of normal tissue. Retinal microglia can become activated and/or dysregulated during disease, and thus affect disease progression in retinitis pigmentosa. Understanding the biology of microglia is a challenge due to absence of markers and molecular microglia signatures. Recently, we identified a homeostatic molecular microglia signature which provides new tools for investigating retinal microglial biology and the possibility of targeting retinal microglia for the treatment of retinitis pigmentosa. Using our new microglial markers, we investigated microglia in the rd1 murine models of RP. We found increased numbers of resident microglia but no infiltration of monocytes in the retinal. Most importantly, we found that intravitreal transfer of microglia from animals with RP into normal animals, resulted in photoreceptor loss. Consistent with this, intravitreal transfer of retinal microglia from normal animals into animals with RP reduced photoreceptors loss. We hypothesize that in RP, microglia proliferate and acquire a cytotoxic phenotype mediated by intrinsic activation of the TREM2-APOE pathway which suppresses microglia homeostatic molecular properties and leads to uncontrolled chronic inflammation and photoreceptors damage. Treatments aimed to target microglia by suppressing the TREM2-APOE pathway is associated with activation of both the TGF? pathway and MERTK which abrogates the inflammatory microglial phenotype and restores retinal microglial homeostatic properties. This provides a new direction for studying RP and development of novel therapies that target microglia. We believe that an innovative feature of our approach is that it is a mutation- independent approach that applies to RP independent of the genetic mutation. In addition, there is a translational aspect to the proposed work as we will investigate human eyes with our recently described microglial antibodies. We will address the following specific aims: Aim 1. Identify molecular pathways affected in retinal microglia in mouse models and human RP. Aim 2. Target the TREM2-APOE-SPP1 pathway to inhibit MGnD-cytotoxic microglia in rd mice. Aim 3. Restore M0-homeostatic microglia via TGF?1-MERTK signaling in rd mice.