Martha Neuringer - US grants

An unresolved and controversial question in human nutrition is whether infants have a specific nutritional need for docosahexaenoic acid (DHA, 22:6w3), a long-chain omega-3 fatty acid which is a major structural component of membranes in the retina and brain. Current US infant formulas contain linolenic acid, a dietary precursor of DHA, but do not directly provide DHA as does human milk. Our previous studies of rhesus monkeys demonstrated that diets low in omega-3 fatty acids during gestation and infancy led to reduced brain and retina DHA levels, deficits in retinal function, slower visual acuity development and changes in visual attention. These findings led to increases in the amount of linolenic acid in many infant formulas, and prompted studies of premature human infants which found similar effects of omega-3 fatty acids on all of these measures. Our current work is evaluating the influence of different amounts and forms of dietary omega-3 fatty acids on visual, cognitive and behavioral development. We are comparing three groups of rhesus monkeys fed a diet low in linolenic acid (18:3w3), a diet high in linolenic acid, or a diet containing DHA and simulating the fatty acid composition of primate milk. During the last year we completed all functional testing through 18 months of age, including behavioral and evoked potential measures of visual acuity and contrast sensitivity, electroretinographic studies of retinal function, and behavioral tests of visual attention, recognition memory, short-term working memory and response inhibition. Effects of low levels of linolenic acid were found on visual tests and on measures of visual attention, but supplementation with DHA showed no benefit compared with diets high in linolenic acid. In a new series of studies of immune system function, animals fed DHA showed reduced lymphocyte response to mitogen stimulation, suggesting altered immune function.

We identified a spontaneous retinal degeneration in two rhesus females with the same father and different, unrelated mothers. We are characterizing the disease to determine if it provides a nonhuman primate model of retinitis pigmentosa, a hereditary retinal disease leading to visual impairment or blindness in 4 of every 1000 humans worldwide. The electroretinogram (ERG) was used to assess retinal function in the affected females and 14 age-matched, unaffected monkeys reared under the same conditions. Retinal photographs and fluorescein angiograms were obtained several times, and in one affected animal retinal pathology was documented by light and electron microscopy at 20 months of age. In both affected females, rod and cone ERG a-wave and b-wave amplitudes were reduced to 15-30% of normal by 7 months of age and declined further by 2 years. By 2 years, rod B-wave implicit times were prolonged. In the first animal, incipient ophthalmoscopic changes were visible by 3 months; at 20 months, histopathological examination revealed virtually complete absence of rod photoreceptors but some preservation of cones, particularly in the central retina. The second animal did not show obvious ophthalmoscopic changes at 7, 14 or 20 months, but at 14 years demonstrates widespread bilaterally symmetrical peripheral degeneration sparing the posterior pole. This animal is negative for herpes B and cytomegalovirus. We are now screening her DNA for candidate gene mutations known to produce human retininis pigmentosa, and efforts are underway to produce offspring by in vitro fertilization-embryo transfer. This spontaneous retinal degeneration closely resembles human retinitis pigmentosa both electrophysiologically and morphologically, and thus could provide a unique primate model of the disease. Because only higher primates share critical structural features of the human retina, including the presence of a macula which is essential for central vision, such a model could greatly facilitate the testing of therapeutic strategies including nutritional and drug treatments, gene therapy and retinal transplantation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project was designed to test a potential genetic therapy for age-related macular degeneration (AMD), the most prevalent form of blindness in the elderly. In its most severe form AMD results in uncontrolled invasion of the retina by choroidal blood vessels leading to hemorrhage, retinal detachment and destruction of the macular retina causing central blindness. Efforts at therapy development have largely focused on stopping vessel growth with anti-angiogenic agents. The current state-of-the-art treatment involves monthly injection of anti-angiogenic antibodies into the vitreous. Although this treatment is effective in stopping disease progression in a majority of patients, frequent intraocular injections pose risk of infection and can present an unacceptable burden to some patients. We are testing a potential gene therapy that could require only one injection to maintain long-term therapeutic effectiveness. The use of nonhuman primates for these studies is critical because only monkeys and apes have a macula and other features closely resembling the human retina. It is our goal that these experiments will lead to an effective treatment for AMD in humans.

DESCRIPTION (provided by applicant): Degenerative diseases of the retina are cumulatively the most common causes of untreatable blindness. These conditions, which include age-related macular degeneration and retinitis pigmentosa, are characterized by progressive loss of cells in the outer retina. Stem cells hold great promise for treating these diseases by repopulating cells that have been lost. A cell type that will be central to the success of this strategy is the retinal pigment epithelium (RPE). Recent technological breakthroughs make possible for the first time the production of recipient-specific donor cells through reprogramming of pluripotency in adult cells and directed differentiation. However, it is not known how RPE cells produced by these and other methods compare with respect to key biological characteristics, including immunogenicity and mitochondrial senescence, when transplanted to the healthy or diseased retina. These issues are critical to the potential use of such cells for retinal disease therapy. The goal of this proposal is to study the functionality of RPE cells generated from experimentally induced pluripotent stem cells in vitro and after transplantation to the rodent or nonhuman primate retina, including their immunogenicity and their ability to rescue visual loss in a rodent model of retinal degeneration. The project will make innovative use of unique resources, including allograft and autograft stem-cell-derived rhesus monkey RPE cell lines and a naturally-occurring nonhuman primate model of macular disease. The proposal has three specific aims: 1) To generate rhesus macaque RPE cells from three sources of pluripotent stem cells--embryonic stem cells (ESCs), ESCs derived by somatic cell nuclear transfer (SCNT- ESCs), and induced pluripotent stem (iPS) cells--and evaluate their function in arresting visual decline in the RCS retinal degeneration model. 2) To investigate the immunology of these RPE cell types transplanted as allograft or autografts into the retina of adult rhesus monkeys and after retreatment of the same eye or fellow eye. 3) To characterize the differences in immune response between young and senescent rhesus monkeys, including those with age-related maculopathy which parallels intermediate human AMD. This translational research project will provide information key to the success of cell therapy in the retina. It will provide insights as to the most appropriate cell source to repopulate lost retinal cells with the aim of preserving or restoring vision, and will generate novel data on the immune response to such therapeutic intervention and the best approach to avoid graft rejection. The proposal will address these issues in an animal model with an eye and immune system that most closely resembles that of humans and in a manner closely mirroring potential clinical practice.

PROJECT SUMMARY Age-related macular degeneration (AMD) is the most common cause of legal blindness in the elderly in developed countries and a leading cause of blindness worldwide. A growing body of evidence supports protective roles for nutritional factors, including the carotenoids lutein (L) and zeaxanthin (Z), which concentrate in the fovea to form the macular pigment, and omega-3 (?-3) fatty acids. However, the typical American diet is low in these nutrients and therefore, there is an urgent need to better understand the role that these nutrients play in retinal aging and disease and their potential to reduce the risk of AMD. Only nonhuman primates with a macula and fovea provide an accurate translational model to address this issue. We have available three exceptional resources: groups of aging macaque monkeys with life-long controlled diets devoid of L and Z and providing either adequate or deficient ?-3 fatty acid content, and showing signs of early AMD; aging monkeys fed diets high in fat and sugar and mimicking the typical American diet; and a large colony of aging macaques fed a healthy diet to address the effects of normal aging. Comparison of these groups of animals provide a truly unique opportunity to understand the roles of dietary factors in retinal aging and disease. This proposal will use these unique resources to address the following aims: 1) Define the relationships in vivo among macular pigment and metrics of macular health in primate eyes across the lifespan and across a wide range of dietary histories including including life-long absence of L/Z. 2) Define the relationships ex vivo among L/Z, the molecular components of lipofucsin, retinal morphology and diet-related retinal pathology, and correlate these findings to the corresponding measurements obtained in vivo. Collectively, these aims will quantify longitudinal changes in macular pigment and the concentration and distribution of components of lipofuscin in the retina and RPE, will characterize changes in retinal structure with age and retinal disease, and will correlate these measurements between in vivo and ex vivo assessment modalities to enhance the interpretation of in vivo imaging. We expect the outcome of the proposed work to have a significant impact on the understanding of the role of macular pigment and dietary fat in aging and retinal diseases including AMD. We also expect to provide data critical to the current controversy on the role of lipofuscin components in AMD. Our studies also will provide exceptional opportunities to examine the evolution of retinal aging and disease, as well as to obtain high quality clinicopathological correlations in multiple metrics of retinal health. Because of the highly translational nature of these nonhuman primate studies, they are expected to have a significantly high impact on understanding the role of nutrition in maintaining retinal health and prevention or treatment of retinal disease.

Project Summary Blindness can be caused by many genetic mutations that lead to degeneration of the retina, but most of these diseases have no treatments. The development of safe and effective treatments critically depends on the ability to test them in appropriate animal models before using them in human patients. Because nonhuman primates are the only animals with retinal structure like humans, including the macula that underlies central vision, they have the potential to provide the most accurate and informative models of blinding diseases. Indeed, the lack of such models has been identified as a major impediment to the rapid translation of promising therapies to clinical use for preventing and treating blindness. We have spent many years screening the large macaque colony at the Oregon National Primate Research Center for naturally-occurring retinal diseases. In the last year we found a family of rhesus monkeys with Bardet-Biedl syndrome, a disorder resulting in severe retinal degeneration combined with kidney disease. We identified the cause as a mutation in the BBS7 gene, genotyped a pedigree including at least 50 carriers, and examined the nature of the retinal degeneration by histopathology. We now propose to propagate this model and define the time course of the degeneration so that it can be used for future efforts to develop treatments. The specific aims of this proposal are: 1. To generate and cryopreserve rhesus embryos confirmed to have the BBS7 mutation. We will use eggs and sperm collected from BBS7 carriers to produce embryos by in vitro fertilization, and use genetic sequencing of embryo biopsies to select embryos with two copies of the disease-causing mutation. 2. To produce live affected infants by transfer of selected blastocysts to surrogate mothers, using methods optimized for decades by the ONPRC Assisted Reproductive Technologies Core. 3. To characterize the time course of the disease in affected infants from birth by comprehensive retinal imaging and functional assessments like those used in human patients. This spontaneously-occurring monkey disorder closely mirrors Bardet-Biedl syndrome as seen in human patients. In addition to providing a model of this specific genetic disease, it also provides a model for the large family of similar retinal degenerations called retinitis pigmentosa that together are a major inherited cause of blindness. This discovery provides us with a unique opportunity to characterize a primate model of an inherited retinal degeneration so that, in the future, we can use these animals to test cell replacement approaches to therapy for this entire class of blinding disorders. Our goal is to preserve vision in these animals and in human patients with this and similar blinding diseases.