This lab focuses on developing and analyzing animal models of retinal degeneration and testing potential treatments for human disease. We are focused on 5 areas: (1) Disease mechanisms and gene therapy for retinoschisis, a leading cause of juvenile macular degeneration in males, using a knockout mouse we developed. (2) Screening for defects in retinal functional and morphology in other murine genetic models to explore the possible role of these genes play in normal and diseased retina. (3) Mechanisms of disease and treatment in other animal models of retinal degeneration, including possible adverse effects of treatment. (4) Mechanisms producing functional, electroretinographic (ERG), changes in animal models and human retinal degeneration. (5) Human clinical trials for retinal disease. Techniques of analysis include light and electron microscopy, immunohistochemistry, biochemistry, molecular biology and measuring retinal functional responses using the electroretinogram (ERG). X-linked juvenile retinoschisis (XLRS): We previously established retinal expression profiles of the retinoschisin (RS) gene with age and localization of the protein in the mouse retina with immunohistochemistry (IHC) and showed that gene therapy could partially restore normal structure and function in the adult RS knockout (RSKO) mice. As we move towards human gene therapy, our focus is on understanding the molecular interactions, biochemistry and sub-cellular localization of the RS protein. To understand and be able to control RS expression we are isolating and characterizing the mouse RS promoter region. Gene expression arrays comparing wild type and RSKO mice have been carried out to help understand the way RS influences the expression of other genes. We cloned and sequenced the human RS gene, coupled it to the GFP reporter gene and generated vectors containing either the RS-GFP or the RS and GFP genes alone. These vectors will be used to track RS secretion and movement in the normal mouse retina which will aid in the development of gene therapy. We are continuing to explore the efficacy of viral gene transfer in restoring normal structure and function in the RSKO mouse at difference ages and with different vectors and and routes of administration. We are investigating the structural and functional consequences of aging and gene therapy in this model. A major goal is to understand the function of RS protein in the retina. Various biochemical techniques are being used to describe its molecular targets and interactions. Using immunohistochemistry at the light and electron microscopic levels we are localizing the retinal structures with which retinoschisn is associated. Screening of murine models: Mutations in a gene coding for an enzyme involved in the ELOngation of Very Long chain fatty acids that maps to 6q14 (ELOVL4) causes STGD3 disease in humans. This is an autosomal dominant atrophic macular degeneration characterized by deterioration in central vision resulting from atrophy of the retinal pigment epithelium (RPE) and accumulation of lipofuscin in the RPE, with loss of photoreceptors in the central retina. In order to learn more about the function of the ELOVL4 protein in vivo, an ELOVL4 heterozygous knockout mouse was generated and we investigated its retinal phenotype. Total knockout of ELOVL4 is fetal lethal. We observed subtle changes in the retina consistent but with no decrement in retinal function. Since ELOVL4 protein in these mice was approximately one-half that in wild type mice and we did not see major changes in retinal structure or function, these results indicate that the phenotype of STGD3 probably does not result from haploinsufficiency, that is, a loss of function due to reduced levels of the protein. This conclusion is important in guiding further work on the causes of this and other forms of macular degeneration. We have done functional screening on several additional murine models with gene mutations: Rab38 heterzygous and homozygous cht; homozygous and heterozygous Pcdh15av-5J and Pcdh15av-Jfb ; RanBP2 heterozygous knockout; myocilin ; NRL knockout and transgenics; RPE65 knockout and transgenics; MATH5 knockout; recoverin knockout. Each of these mutations is in retinal proteins which may play a role in retinal disease. We?ve particularly focused on mutations in Pcdh15 which cause Usher?s syndrome type 1F in humans resulting in deafness and progressive retinal degeneration. Mice with the above mutations in this gene are deaf, but have not been tested for retinal structure and function to see if they are suitable models for Usher?s syndrome. We measured ERG responses and retinal cell number and size at several ages in mice with 2 different mutations in this gene. Mechanisms of retinal disease and treatment: We investigated the role of the Kir4.1 potassium channel in generation of the photopic negative (PhNR) ERG response which is highly preserved, relative to other ERG potentials, in some animal models of photoreceptor degeneration but not others. Measurements of density of these channels by immunohistochemistry and western blot indicate different degrees of change with disease type and age and blocking agents strongly suggest these channels produce the PhNR. This work will lead to some important information on the homeostatic mechanisms and maintenance of retinal circuits in retinal disease. In the area of disease mechanisms we explored the role of immune system cytokines in the retina. Recent work has shown that various components of the immune system may be involved in the etiology of age related macular degeneration (AMD) including macrophage recruitment and complement factor H. Light induced retinal degeneration in rodents, in some respects, models the disease processes and phenotype of AMD. We are investigating the role of immune system cytokines in this model using gene knockout mice, retinal histology, biochemistry and gene expression array technology. We investigated the protective effects of AAV-mediated delivery of lens epithelial derived growth factor (LEDGF) in RCS rats, a model of retinitis pigmentosa, and the possible role of small heat shock proteins HSP25 and alpha-B-crystallin. AAV delivery of LEDGF slows retinal degeneration in RCS rats, but not through an increase in Hsp25 and alpha-B-crystallin expression. Human clinical trials: Human Protocol 03-EI-0033. X-Linked Juvenile Retinoschisis - Clinical and Molecular Studies. (PI: P.A. Sieving). A genotype-phenotype study of XLRS which results in splitting of the retinal layers. A better understanding of XLRS development might lead to improved treatments through gene transfer. Human Protocol 03-EI-0179. Investigation of the Effect of Dietary Docosahexaenoic Acid (DHA) Supplementation on Macular Function in Subjects with Autosomal Dominant Stargardt-Like and Autosomal Recessive Stargardt Macular Dystrophy. (PI: P.A. Sieving). Evaluate DHA supplementation to improve macular function in patients with Stargardt and Stargardt-like macular dystrophies. DHA fatty acid is essential for normal brain and eye development. Human Protocol 03-EI-0234. A Phase I Study of NT-501-10 and NT-501-6A.02, Implants of Encapsulated Human NTC-210 Cells Releasing Ciliary Neurotrophic Factor (CNTF), in Patients with Retinitis Pigmentosa. (PI: P.A. Sieving). Evaluate safety of CNTF implant in the human eye of retinal degeneration subjects. CNTF protects against retinal degeneration in animal models. Study addresses a major treatment challenge of delivery directly into the human eye. Human Protocol 03-EI-0255. Pilot Study on the Effect of Vitamin A Supplementation on Cone Function in Retinitis Pigmentosa (RP). (PI: P.A. Sieving). A protocol to evaluate whether high dose oral vitamin A will improve retinal function acutely in patients with RP.