Ruben Adler - US grants

The pathogenesis of photoreceptor degenerative diseases remains obscure. An example in human beings is retinitis pigmentosa, characterized by proressive photoreceptor loss, which frequently occurs on a genetic basis. The working hypothesis to be tested here is that photoreceptor survival and differentiation are regulated by molecular signals from their microenvironment, and that defects in these trophic factors might be involved in the pathogenesis of retinal degenerations of genetic origin. This hypothesis will be tested using retinal cells from normal and "Rd" mutant mice. Homozygous Rd mice undergo photoreceptor degenerations early in postnatal life. Extensive use will be made of in vitro systems available in our laboratory. Reaggregation cultures will be used to study the role of cell-cell interactions in photoreceptor development. Interactions of normal and Rd retinal cells with each other and with pigment epithelial cells will be studied in combined aggregates. Monolaver cultures will be used to test the responsiveness of normal and Rd retinal cells to putative sources of trophic agents, such as i) retinal extracts from normal and Rd mice; ii) pigment epithelium conditioned medium; iii) interphotoreceptor maxtrix, and iv) purified extracellullar matrix molecules. Survival and differentiation of photoreceptors and neurons in monolayers and reaggregates will be analyzed with light and electronmicroscopy, autoradiography, lectin cytochemistry and immunocyochemistry using rhodopsin antibodies. It is our expectation that this work will identify molecular agents capable of regulating photoreceptor survival and differentiation, and of altering the natural evolution of retinal degenerations of genetic origin.

Long term goals of this project are to improve our understanding of cellular and molecular mechanisms controlling the survival and differentiation of visual neurons, and to test the hypothesis that these phenomena are regulated by the interactions of visual neurons with A) their targets of innervation; b) the neurons from which they receive afferent connections; and c) the glial cells with which they are associated. It is hoped that further understanding of these developmental mechanisms will increase our chances to intervene upon injured visual neurons to promote their regeneration. Purified monolayer cultures from chick embryo neural retina or optic lobe neurons will be the basic experimental system. Neuronal differentiation will be evaluated by i) the presence of cellular machineries necessary for neurotransmitter-related activities; ii) morphological specialization of the neurons; iii) neuritic development; and iv) development of synapsis. Putative sources of regulatory influences will be retinotectal interactions (studied using co-cultures, tissue extracts of conditioned media), and glia-neuron interactions (using purified monolayers of optic lobe or neural retina non-neuronal cells). The role of substratum-attached materials in the elongation and directionality of neurites will also be studied. Molecules responsible for activities that might be detected in tissue extracts or conditioned media will be characterized and fractionated. The involvement of taurine in the regulation of survival and differentiation of cultured retina cells will be investigated. Finally, some of the properties of the cultured nonneuronal cells will be studied, particularly with respect to the high affinity uptake of neurotransmitters.

Photoreceptor death causes blindness in retinal degenerations such as retinitis pigmentosa and age-related macular degeneration. Although genetic mechanisms trigger several forms of these diseases, cell-cell interactions and microenvironmental factors are also likely to be involved in their pathogenesis. The search for trophic agents supporting the survival and differentiated function of photoreceptor cells is therefore important for the development of strategies for their prevention and/or treatment. Embryonic cell transplantation -- an approach actively explored in many laboratories as a possible treatment for retinal degenerations -- could also benefit from new information on photoreceptor survival- and differentiation-promoting factors. Retinoids of the vitamin A family have been known for many years to be necessary for photoreceptor health and survival, but their mechanisms of action remain poorly understood. New avenues for their investigation have been opened in recent years by the almost explosive growth in information about retinoic acid and its receptors. As part of our long term interest in the study of trophic factors for photoreceptor cells, we have recently observed regulatory effects of retinoic acid and other retinoids upon isolated mouse photoreceptor cells in vitro. These culture systems thus appear as suitable experimental systems for detailed analysis of these issues. The studies proposed here will involve embryonic and neonatal mouse retinas in situ and in explant cultures, as well as purified cultures of retinal neurons and photoreceptors. In situ hybridization, the polymerase chain reaction, and immunocytochemical and autoradiographical methods will be used in a multidisciplinary approach to investigate the expression and distribution of retinoic acid receptors, their developmental regulation, and the responses of retinal neurons and photoreceptors to retinoic acid and other retinoids. It is anticipated that these studies will generate new information regarding the role of retinoids in photoreceptor differentiation and survival, thus contributing to the design of strategies for the prevention and potential treatment of blinding retinal degenerations.

Retinal degenerations represent one of the important causes of human blindness. Their prevention and eventual treatment requires understanding the cellular and molecular mechanisms responsible not only for the survival, but also for the differentiation of retinal neurons and photoreceptors. The long term goal of grant EY04859 is to investigate these biological phenomena. The life history of each mature retinal cell includes several rounds of mitotic division, the acquisition of the postmitotic state, migration to one of the developing cell layers, and differentiation into one of the retinal cell types. The cellular and molecular mechanisms that regulate these events remain largely unknown. We are planning to approach these questions by comparing the developmental potential of chick embryo retinal precursor cells in vivo and in vitro. We will use low density, dissociated cultures in which isolated precursor cells grow in the absence of contact-mediated intercellular interactions, as well as other culture techniques that promote that type of interactions. The stage at which each cell undergoes terminal mitosis will be determined by thymidine autoradiography and/or bromodeoxyuridine immunocytochemistry. Photoreceptor differentiation will be characterized using a multidisciplinary approach both at the single cell level (by immunocytochemistry and in situ hybridization with visual pigment-specific probes), and in cell culture extracts (by immunochemistry, Northern blot analysis and the polymerase chain reaction). Tbe experiments will test predictions from several hypotheses, in order to establish whether retinal precursor cells remain uncommitted (plastic) for some time after terminal mitosis, whether their differentiated potential changes as a function of the developmental stage at which they undergo terminal mitosis, whether rods and cones derived from common or from specialized precursors, and whether their differentiation is affected by retinoids and by exposure to cyclic light. Results from these studies should help us to understand not only normal photoreceptor development and function, but also the mechanisms leading to their disease and degeneration.

Retinal degenerations represent one of the important causes of human blindness. Their prevention and eventual treatment requires understanding the cellular and molecular mechanisms responsible not only for the survival, but also for the differentiation of retinal neurons and photoreceptors. The long term goal of grant EY04859 is to investigate these biological phenomena. The life history of each mature retinal cell includes several rounds of mitotic division, the acquisition of the postmitotic state, migration to one of the developing cell layers, and differentiation into one of the retinal cell types. The cellular and molecular mechanisms that regulate these events remain largely unknown. We are planning to approach these questions by comparing the developmental potential of chick embryo retinal precursor cells in vivo and in vitro. We will use low density, dissociated cultures in which isolated precursor cells grow in the absence of contact-mediated intercellular interactions, as well as other culture techniques that promote that type of interactions. The stage at which each cell undergoes terminal mitosis will be determined by thymidine autoradiography and/or bromodeoxyuridine immunocytochemistry. Photoreceptor differentiation will be characterized using a multidisciplinary approach both at the single cell level (by immunocytochemistry and in situ hybridization with visual pigment-specific probes), and in cell culture extracts (by immunochemistry, Northern blot analysis and the polymerase chain reaction). Tbe experiments will test predictions from several hypotheses, in order to establish whether retinal precursor cells remain uncommitted (plastic) for some time after terminal mitosis, whether their differentiated potential changes as a function of the developmental stage at which they undergo terminal mitosis, whether rods and cones derived from common or from specialized precursors, and whether their differentiation is affected by retinoids and by exposure to cyclic light. Results from these studies should help us to understand not only normal photoreceptor development and function, but also the mechanisms leading to their disease and degeneration.

DESCRIPTION (provided by applicant): The long-term goal of application EY04859 is the investigation of the genetic and microenvironmental factors that regulate the survival and differentiation of retinal neurons and photoreceptors. The studies are based on a multidisciplinary approach that involves: i) analysis of genetic influences by gene transfer into retinal cells in vivo and in vitro, ii) experimental manipulation of the cellular microenvironment in ovo and in culture, and iii) characterization of the phenotypic properties expressed by differentiating retinal cells using in situ hybridization, immunocytochemistry, autoradiography, single cell cDNA synthesis, RTPCR, real time PCR, Northern blots and image analysis. Aspects of the dynamics and regulation of photoreceptor cell differentiation that will be studied include: i) the developmental relationships between retinal progenitors that develop as rods or cones, ii) the coordinated expression of visual pigments and other photoreceptor specific-molecules during differentiation, iii) the development of outer segments, and iv) the effects on these phenomena of CNTF and the activin and BMP families of growth factors. The role of the homeobox genes Pax-6 and Chx-1O in the differentiation of retinal neurons and photoreceptors will also be investigated. The information derived from these studies is expected to further our understanding of the mechanisms of retinal development, and may also be relevant for some therapeutic approaches to retinal degenerations.

DESCRIPTION (provided by applicant): Retinal degenerative diseases are a major cause of visual disability and blindness worldwide. Age-related macular degeneration (AMD), for example, is the leading cause of blindness in the elderly in the Western world. Current treatments do little to alter the inexorable loss of vision due to retinal degenerations. Several studies have shown that intraocular injection of factors such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor-2 (FGF2), slows photoreceptor cell death caused by specific mutations or exposure to constant light. However, the clinical usefulness of these findings may be limited, because rescue effects are partial and transient, and some factors appear to have unwanted side effects. Elucidation of the mechanism by which survival factors delay retinal degenerations appears necessary in order to maximize benefits and minimize side effects. Recent studies from our laboratories have suggested that CNTF, BDNF and FGF2 do not act directly on photoreceptors; rather, they appear to act indirectly through other cells, most likely M[unreadable]ller cells. Based on these observations, we propose to investigate the molecular changes triggered by neurotrophic factors in M[unreadable]ller cells. The studies involve the combined use of two complementary and demanding state-of-the-art techniques: the generation of cDNA from individual cells, and their analysis using custom designed retinal cDNA microarrays. We will then establish which of these changes are important for photoreceptor survival. The potential impact of the identification of these molecules is clear, since they could offer new avenues for the treatment of these devastating diseases.