Joe G. Hollyfield - US grants

The research proposed in this application is directed at understanding the mechanism through which specific degenerative diseases act to alter retinal metabolism. We will exploit a novel culture preparation which allows both morphological and biochemical analysis to be performed on small retinal buttons from human eyes. We will elevate the levels of cyclic nucleotides or ornithine concentrations in the culture medium to stimulate metabolite imbalance in the disease state. By utilizing the appropriate labeled precursor we will follow the changes in specific molecular components with biochemical techniques and identify the cell type in which these changes take place with autoradiography. Renewal pattern of RNA, protein and lipids will be evaluated with this systematic approach. We will determine how renewal processes in the retina are altered when imbalances in specific metabolites occur and provide fundamental information regarding the mechanism through which degenerative changes are manifest in the human retina.

This project is directed at characterizing the time of appearance of specific neurotransmitter properties during development of the vertebrate retina. It will utilize biochemical, autoradiographic, morphological and physiological techniques to follow the time of appearance of the high affinity uptake for specific neurotransmitters, the time when neurotransmitters are first synthesized during development, time when release of neurotransmitters can first be evoked and the time of post-synaptic receptor appearance. Using dissociated neuroepithelial cells derived from retinal rudiments from the embryo maintained in tissue culture, we will determine the requirements for cell-to-cell contacts during retinal differentiation for the appearance of specific neurotransmitter properties. The project will also involve an analysis of the factors which regulate membrane assembly in photoreceptors. It will systematically evaluate the involvement of cyclic nucleotide metabolism, phosphorylation reactions, and calcium fluxes in establishing the burst of membrane assembly at the base of rod outer segments which follows the onset of the light cycle. The project will also evaulate the involvement of environmental lighting in setting the rates of metabolism of RNA and protein synthesis within the retina. A basic understanding of the regulation of retinal development, renewal processes and rates of retinal metabolism will provide important new information to understanding the control of these processes in retinal health and disease.

A source of human tissues has been secured which provides over 50 pairs of eyes per year with postmortem times under one hour. The retina and pigment epithelium from these tissues will be used for cell biological studies. The overall goal of this program is to identify the unique cell biological features of the human retina, both spatially and temporally which may underlie some of the basic processes which lead to lesions in the retina in response to inherited retinal diseases such as retinitis pigmentosa or macular degeneration. Specific aims of this study are to evaluate foveal versus peripheral retina in the synthesis of IRBP. Comparisons will be made of peripheral versus foveal cones in the synthesis of a glycoprotein which labels with galactose and is transported to the cone pedicle. An evaluation will be made of the ability of the fovea and peripherial retina to synthesize melatonin as well as to establish the regional distribution of HIOMT immunoreactivity. In addition, comparisons will be made of rods versus cones in the peripheral retina and foveal cones versus peripheral cones with respect to the endocytotic capabilities of the inner segments of these photoreceptor types using horseradish peroxidase, dextran and 125I-IRBP as tracers. The pigment epithelium from these donor eyes will be utilized to evaluate differences in the synthetic capabilities of this epithelium in submacular versus peripheral regions. Specific emphasis will be placed on components of the interphotoreceptor matrix which are synthesized by the pigment epithelial cells and secreted from their apical surface. Retinal tissues will also be utilized for continuation of studies on neurotransmitters in the inner retina with emphasis on GABA and acetylcholine. Techniques used in this project will include organ culture, radioactive tracers, polyacrylamide gel electrophoresis, fluorography, chromatography, immunocytochemistry, autoradiography and electron microscopy.

This project is directed at characterizing the time of appearance of specific neurotransmitter properties during development of the vertebrate retina. It will utilize biochemical, autoradiographic, morphological and physiological techniques to follow the time of appearance of the high affinity uptake for specific neurotransmitters, the time when neurotransmitters are first synthesized during development, time when release of neurotransmitters can first be evoked and the time of post-synaptic receptor appearance. Using dissociated neuroepithelial cells derived from retinal rudiments from the embryo maintained in tissue culture, we will determine the requirements for cell-to-cell contacts during retinal differentiation for the appearance of specific neurotransmitter properties. The project will also involve an analysis of the factors which regulate membrane assembly in photoreceptors. It will systematically evaluate the involvement of cyclic nucleotide metabolism, phosphorylation reactions, and calcium fluxes in establishing the burst of membrane assembly at the base of rod outer segments which follows the onset of the light cycle. The project will also evaulate the involvement of environmental lighting in setting the rates of metabolism of RNA and protein synthesis within the retina. A basic understanding of the regulation of retinal development, renewal processes and rates of retinal metabolism will provide important new information to understanding the control of these processes in retinal health and disease.

The interphotoreceptor matrix (IPM), referred to clinically as the subretinal space, is the site of interactions between the photoreceptors and pigment epithelium (RPE) which are of fundamental importance to vision. This is the site of a variety of activities which include: retinal attachment; cell-cell recognition before phagocytosis; visual pigment chromophore exchange; establishment of specialized photoreceptor micro-environments; and photoreceptor alignment. The degree of involvement of the components present in the IPM in these activities is not well understood. Defects in the maintenance of the IPM could underlie a variety of clinical disorders which occur at this interface, such as retinal detachment, some forms of macular degeneration, as well as some forms of retinitis pigmentosa. Failure to restore IPM structures could prevent successful reattachment of detached retinas. The long term goal of this research is to elucidate the fundamental processes that establish and maintain the IPM in order to understand better the requirements for photoreceptor survival and optimal function. To achieve this goal, we propose to examine the organization, cellular origin, function, biosynthesis, and development of the multiple constituents of the IPM. With radioactive tracer studies and chromatography in conjunction with cell and organ culture, we will determine the cellular origin of the major IPM components such as chondroitin sulfate proteoglycan (CSPG) in cone matrix sheaths and the wheat germ agglutinin (WGA) binding proteins in rod matrix domains. We will examine the turnover of IPM components in vivo in mice by following changes in specific activity of IPM components after systemic tracer administration. We will determine whether the cone matrix domains function as a barrier to macromolecular movement within the IPM. Finally, we will establish using immunocytochemistry and electron microscopy whether the IPM proteoglycans are present in a higher order of organization as proteoglycan aggregates and establish the various cells which contribute the three components of the aggregate. Forthcoming from these efforts will be a better understanding of the fundamental processes that establish and maintain the IPM and the requirements of these IPM components for photoreceptor survival and optimal function.

The broad, long-term objective of this proposal is to elucidate the structure-function relationships of specific interphotoreceptor matrix (IPM) macromolecules, especially as they relate to supporting the maintenance, health and survival of photoreceptor cells of the retina and retinal pigment epithelium (RPE). During the upcoming grant period, the focus will be on GP147, a prominent glycoprotein in the insoluble IPM, and hyaluronan (HA), recently identified as a component of the insoluble IPM. The Specific Aims are: Specific Aim 1: To test the hypothesis that GP147, identified during the previous grant period as a major glycoprotein in the insoluble IPM of human retina, is differentially synthesized and distributed around rods and cones. Emphasis is on determining the distribution of GP147 in macula and peripheral retina, identifying the cells responsible for its synthesis, the structure of the glycoconjugates and isolation of the GP147 gene. Specific Aim 2: To test the hypothesis that the mouse IPM contains a homologue to GP147. Emphasis is on the expression and synthesis of GP147 during retinal development and in the rate of turnover of GP147 in the adult. Specific Aim 3: To test the hypothesis that GP147 and hyaluronan (HA), also identified during the previous gant period as a component of the insoluble IPM, form associative interactions. Emphasis is on determining whether GP147 interacts with the IPM through associations with HA. Specific Aim 4: To test the hypothesis that photoreceptors and/or the RPE are involved in the synthesis of hyaluronan (HA) present in the insoluble IPM. Emphasis is on the cellular origin and development of IPM HA. Because of the strategic location of the IPM, components of this matrix may be targets for genetic disorders that affect this interface which may include retinal detachment, central serous retinopathy, some forms of macular degeneration and retinitis pigmentosa.

DESCRIPTION (provided by applicant): The broad, long-term objective of this application is to define in molecular terms the linkage between the accumulation of soft drusen below the retinal pigment epithelium (RPE) in the macula and the increased risk of developing age-related macular degeneration (AMD). The presence of soft drusen in the macula is the hallmark risk factor for developing AMD. Surprisingly little is known of the composition or origin of drusen. To this end a novel method for drusen isolation has been developed that allows the collection of microgram quantities of drusen from donor eye tissue. At the time of isolation, different drusen sub-types can be identified and separated for use in studies that will characterize their molecular composition. The diagnostic utility of drusen in AMD can be likened to that of blood levels of cholesterol in atherosclerosis. The presence and abundance of drusen, like the level of cholesterol in the blood, indicates the degree to which a patient is at risk for developing the disease. Because of the relationship of drusen and AMD, understanding the composition of different drusen sub-types will provide important information on possible pathways that are causally involved in drusen development. Novel proteins or common modifications of proteins present in drusen, should provide insight as to potential drug targets of therapeutic agents to treat AMD. The current application is focused on exploiting this drusen isolation procedure to define the molecular composition, distribution and cellular origin of drusen sub-types in normal and AMD tissues. The three specific aims are: (1) To test the hypothesis that different sub-populations of drusen can be isolated from donor eye tissue. (hard vs. soft, foveal vs. peripheral, old vs. older, spherical vs. flat, amber vs. clear, opaque vs. granular, etc.). (2) To test the hypothesis that different structural features of drusen sub-populations reflect different molecular composition (light microscopy, histochemistry, electron microscopy, SDS/PAGE, Western blotting, mass spectrometry). (3) To test the hypothesis that some of the molecules present in drusen are novel and are not found in Bruch's membrane.

[unreadable] DESCRIPTION (provided by applicant): The vision research program of the Cleveland Clinic Foundation's (CCF) Cole Eye Institute proposes a program of research infrastructure development that will facilitate the study of the structure, development, and function of the eye and visual system in health and disease, with the goal of preventing or curing disorders of the eye that lead to blindness. The investigators benefiting from this support use the most sophisticated research techniques available, including those of genomics, proteomics, electrophysiology, and cell and molecular biology. The two resource modules that are proposed will help achieve this goal: [unreadable] [unreadable] I. Proteomics/Protein Chemistry Resource Module (Assistance in protein identification using amino acid analysis, 2D gel electrophoresis, mass spectroscopy for custom research needs.) [unreadable] [unreadable] II. Microscopy/Histology Resource Module (Histology support for tissue analysis using confocal microscopy, electron microscopy, brightfield, darkfield, phase contrast or fluorescence microscopy, immunocytochemistry, and computer-aided image analysis.) [unreadable] [unreadable] This is a new application submitted by the Principal Investigator and 15 other funded vision scientists. The scientists include six with eight eligible NEI grants. The nine other basic and clinical investigators are funded by other sources. Of the latter, four have submitted NEI R01 applications, and two others plan to do so in the future. The CCF has committed to support the Vision Research Core Center by constructing new laboratory space; purchasing a wide range of new equipment for the resource modules; providing salary support for an administrator to manage the grant; and providing partial support for investigator salaries. This Vision Research Infrastructure Development Grant will bring important Core Resource services needed by these investigators. Their research activities will be enhanced by these services and their collaborative interactions will be strengthened and extended through this program. [unreadable] [unreadable]

The broad, long-term objective of this proposal is to define in molecular terms the linkage between the oxidative modifications of proteins in the outer retina, their recognition by the immune system and the vulnerability of the outer retina to attack, leading to the disease processes underlying age-related macular degeneration (AMD). Work completed during the last grant period revealed that AMD eye tissues contain high levels of proteins modified by the adduction of oxidation fragments of the long chain polyunsaturated fatty acid, docosahexaenoic acid (DHA). Many of the proteins modified by this and other adducts are found in drusen and Bruch's membrane. Furthermore, autoantibodies against these unique carboxyethylpyrrole adducts (CEP) are more abundant in the circulation (plasma) of individuals with AMD than are found in agematched individuals without AMD. From these results the following hypothesis has emerged regarding an initiating stimulus for AMD: (a). Because of the high concentration of DHA in the photoreceptors-RPE complex coupled with the vulnerability of DHA to oxidative damage, CEP-adducts are slowly generated over time in the outer retina, (b). These CEP-adducts represent new epitopes foreign to the immune system resulting in the generation of autoantibodies against CEP. (c). Anti-CEP-antibodies in turn are involved in activation of the complement attack pathway at the Bruch's membrane-RPE interface in response to the continued generation of CEP epitopes in tissues of the outer retina. To test this hypothesis we immunized normal mice with CEP-adducted mouse serum albumin. Our prediction was that systemic immunization with CEP would sensitize mice to endogenous CEP-adducted proteins generated in the outer retina during the normal course of aging. In turn the immune system would respond by attacking the cells where CEP epitopes are most readily generated. Analysis of these mice demonstrated focal lesions in the RPE and photoreceptors that mimic geographic atrophy, the blinding end-stage atrophy associated with dry AMD. This new mouse model for AMD will be further characterized in normal mice and the CEP-immunizations will be extended to mice with genetic defects in complement pathway molecules and their regulators. A new model for AMD in the mouse will be an important a new resource for use in the preclinical testing of therapeutics designed to prevent or limit the progression of AMD.

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is a scourge of the elderly, with some stage of this potentially blinding disease present in approximately a third of individuals over 75 years of age in the US. There is substantial evidence that AMD is an inflammatory disease involving dysregulation of the complement pathway. We found that the hapten, CEP (carboxyethylpyrrole), generated by oxidative damage to docosahexaenoic acid (DHA), is present as an adduct on drusen proteins in AMD donor eyes. In addition, autoantibodies against CEP were more abundant in the circulation (plasma) of individuals with AMD than in age-matched individuals without AMD. Because DHA is abundant in the outer retina where light and high oxygen levels provide a permissive environment for oxidative damage, this tissue is a likely source of CEP-adducts. Since CEP-adducts is antigenic, we speculated that as the outer retina generates these adducts, the immune system becomes responsive to these new epitopes and over time begins to attack the cells that are the source of this fragment. To test this hypothesis we immunized normal mice with CEP-adducted mouse serum albumin (CEP-MSA). Our prediction was that systemic immunization with CEP would sensitize mice to endogenous CEP-adducts generated in the outer retina during the normal course of aging. In turn the immune system would respond by attacking the cells where CEP epitopes are most readily generated. We found that immunized mice develop antibodies to CEP, fix complement component-3 in Bruch's membrane, accumulate drusen below the RPE during aging, and develop lesions in the RPE mimicking geographic atrophy, the end-stage condition characteristic of dry AMD. The research proposed here uses this mouse model for mechanistic studies on the role of complement activation pathways in producing the pathology, the role of antibody in causing the lesions observed, and the possibility of prevention of the lesions through manipulation of complement activation or suppression of the pathology with oral tolerance. Outcomes from these studies have the potential to lead to the identification of new therapeutic pathways to prevent AMD. PUBLIC HEALTH RELEVANCE: The loss of central vision from age-related macular degeneration is the bane of the elderly in industrialized nations. Until recently so little was known about the mechanism of this disease that there was no potential for developing animal models for this disorder. This has now changed with the findings that oxidative damage to retinal lipids is a source of an inflammatory signal that may target this tissue for immune system mediated pathology. This inflammatory signal has been identified, synthesized and used to produce AMD-like lesions in mice. Studies proposed in this grant use this mouse model to understand several aspects of the disease process and explore possible routes of therapeutic intervention.

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is a scourge of the elderly, with some stage of this potentially blinding disease present in approximately a third of individuals over 75 years of age in the US. There is substantial evidence that AMD is an inflammatory disease involving dysregulation of the complement pathway. We found that the hapten, CEP (carboxyethylpyrrole), generated by oxidative damage to docosahexaenoic acid (DHA), is present as an adduct on drusen proteins in AMD donor eyes. In addition, autoantibodies against CEP were more abundant in the circulation (plasma) of individuals with AMD than in age-matched individuals without AMD. Because DHA is abundant in the outer retina where light and high oxygen levels provide a permissive environment for oxidative damage, this tissue is a likely source of CEP-adducts. Since CEP-adducts is antigenic, we speculated that as the outer retina generates these adducts, the immune system becomes responsive to these new epitopes and over time begins to attack the cells that are the source of this fragment. To test this hypothesis we immunized normal mice with CEP-adducted mouse serum albumin (CEP-MSA). Our prediction was that systemic immunization with CEP would sensitize mice to endogenous CEP-adducts generated in the outer retina during the normal course of aging. In turn the immune system would respond by attacking the cells where CEP epitopes are most readily generated. We found that immunized mice develop antibodies to CEP, fix complement component-3 in Bruch's membrane, accumulate drusen below the RPE during aging, and develop lesions in the RPE mimicking geographic atrophy, the end-stage condition characteristic of dry AMD. The research proposed here uses this mouse model for mechanistic studies on the role of complement activation pathways in producing the pathology, the role of antibody in causing the lesions observed, and the possibility of prevention of the lesions through manipulation of complement activation or suppression of the pathology with oral tolerance. Outcomes from these studies have the potential to lead to the identification of new therapeutic pathways to prevent AMD.

? DESCRIPTION (provided by applicant): The Cole Eye Institute Vision Science Training Program (CEIVSTP) is specifically designed to train pre-doctoral Ph.D. students, medical students and post-doctoral fellows to become future leaders in ophthalmology research. One of the major long-term goals of CEIVSTP is to limit and eventually merge the boundaries that now separate clinical and basic research in ophthalmology. This training program is uniquely positioned to achieve this goal for the following reasons: First, the trainers in this program have been selected for their ability to perform outstanding ophthalmology research with a translational focus on eye diseases. Second, the clinical mentors in the program are highly successful clinicians with proven track records in research and a commitment to the goals of the training program. Third, both a basic and clinician scientist will mentor each trainee. Fourth, the housing of this program under one roof in the Cole Eye Institute establishes a cohesive and comprehensive environment particularly suited to this purpose. Fifth, the close interactions between medical students and Ph.D. students during the research training, will promote communication between the clinical and basic areas and foster scientific networks and collaborations that can continue into the future. The strong didactic, research and clinical component of this program together with the novel curriculum of the Molecular Medicine Ph.D. program will provide a solid foundation in basic scientific principles in the context of human ocular biology and disease. This proposal seeks funding for outstanding pre-doctoral graduate students, medical students working toward a masters level thesis and post-doctoral fellows committed to ophthalmic research.

Abstract Administrative Module The Administrative Module will oversee the efficient use of resources and the equitable utilization of the services provided in the resource modules supported by this P30 Core grant for vision research. It will provide strong leadership to oversee the efficient utilization of support from this NEI P30 Vision Core Grant. It will monitor the efficient utilization of funds distributed from this award to the individual modules. An Executive Committee will be established to review and monitor the progress and efficiency of each resource module supported by this grant. This module will establish a clear line of authority to resolve any conflicts or problems that may arise that would alter the efficient use of the module services by participating investigators. An external advisory board will meet each year to review the activities supported by this NEI P30 Core Grant and offer suggestions for improvement. The leadership of this module will see that primary access to the core module will be for NEI R01 funded investigators; that newly recruited investigators will be able to obtain data for new NEI R01 grant submissions with help from the resource modules; that the resource modules will help foster collaborative interactions between RO1 funded investigators and newly recruited investigators; that the resource modules will be helpful in attracting investigators from other departments in the institution to research on the visual system; and that the resource modules will be highly visible as an attractive departmental resource as we continue to expand our research faculty.

The overall aim of this NEI Core Grant for Vision Research is to facilitate the application of important technologies in the analysis of the structure, function and disease mechanisms that affect the visual system. The long-term goal of the investigators served by this grant is to preventing blindness and restoring vision. To this end we will develop a series of core modules that will provide state-of-the-art instrumentation and technologies required for vision research. The three core modules are: (1) Ocular Imaging Module; (2) Functional Vision Module; and (3) Molecular Informatics Module. These modules contain equipment that will be maintained and operated by highly skilled technicians that are supervised by established R01 funded investigators. Consolidating these technologies into these modules allow us to bring these techniques and skills to all potential users in our program, which we anticipate will increase productivity in the user laboratories and avoid duplication of equipment and effort. Successful operation of these three modules will bring the best of these technologies to each of the NEI RO1 supported laboratories in this institution. We also envision that these core modules will enhance and sustain the collaborative scientific environment that is a strong feature of the research group supported by these modules. The modules will also enhance productivity and increase collaboration among the participating investigators. These modules will also allow junior investigators to have access to services and research strategies that will help generate preliminary data to support new RO1 application.