Jeremy Nathans - US grants

The proposed research aims to define the functional properties of visual pigments at a molecular level. Specifically, the work focuses on: (1) the mechanisms by which each pigment tunes the absorption spectrum of the chromophobe (ll-cis retinal) to the correct wavelengths, (2) the conformational changes that occur in the visual pigments upon photoexcitation, and (3) the sites of contact between visual pigments and the peripheral membrane proteins with which they interact, transducin/G-protein, rhodopsin kinase, and arrestin/48-K protein. Two complementary approaches will be used. In the first, human and bovine visual pigments will be produced in large quantities in tissue culture cells by expression of their cloned DNAs. Specific mutations will be constructed in these DNAs so as to produce visual pigments with defined alterations. These mutant pigments will be characterized with respect to their spectroscopic and enzymatic properties. ln the second approach, naturally occurring visual pigment variants will be studied in individuals with inherited variations in color vision. These variants are quite common (8% of caucasian males have an inherited anomaly in their color vision) and represent a rich source of material for study. By testing the color vision of human subjects, analyzing their visual pigment genes, and producing the altered pigment we can correlate psychophysical, molecular genetic, spectroscopic and enzymatic data. A rare and clinically significant color vision defect, blue cone monochromasy, will be investigated by molecular genetic methods. These studies should reveal its molecular basis and provide diagnostic DNA probes for genetic counseling. The visual pigments resemble in structure and function many other cellular receptors, including alpha and beta adrenergic, Ml and M2 muscarinic, and substance K receptors. The molecular mechanisms of visual pigment action defined by the proposed studies will therefore be of general interest to pharmacologists and physiologists.

DESCRIPTION: (Adapted from applicant's abstract) This grant has the following objectives: (1) identify and characterize novel transcription factors in the human retina by molecular cloning and immunolocalization, and by biochemical studies of the recombinant proteins; (2) characterize regulatory DNA sequences that determine cone cell-specific expression of the human red-, green- and blue-sensitive visual pigment genes by determining the ability of these sequences to direct expression of linked reporter genes in transgenic mice; (3) construct transgenic mice in which defined retinal cell types have been ablated by selective expression of diphtheria toxin, both to study the physiological and developmental effects of this cell loss and as an aid to identifying cDNA clones specific for the target cells by differential screening. These three lines of research are expected to converge to elucidate the regulatory network of transcription factors and their targets. An additional result from this work will be a number of reagents--antibodies, cDNA and genomic clones, characterized regulatory sequences, and transgenic lines--that will be of general use to the retinal research community. While the proposed work is not targeted directly at human disease, past experience shows that elucidating normal mechanisms is a prerequisite for elucidating pathogenic mechanisms.

The goal of this research program is to develop an in vitro assay for the functional characterization of the retina-specific ABC transporter, ABCR/RIM. Mutations in the gene encoding ABCR/RIM are responsible for Stargardt disease (STGD), an autosomal recessive early onset form of macular degeneration, and have also been reported in a subset of patients with age related macular degeneration (AMD). Unpublished data implicate other mutations in this gene in some cases of autosomal recessive retinitis pigmentosa (RP). We aim to (1) identify the transported substrate and (2) develop methods for producing recombinant human ABCR/RIM that can be used to test the effect of mutations on the structure, function, and stability of this protein. Identifying the transported molecule(s) may reveal new compounds or new roles for known compounds. Determining whether the transported molecule is transported in conjunction with specific binding proteins, and studying its synthesis, degradation, and processing may lead both to the identification of additional candidate genes for retinal diseases, and to the identification of additional targets for drug development. Production of a large number of different ABCR/RIM proteins, each carrying a mutation found in humans with STGD, AMD, or RP will allow us to determine the biochemical defect in each case, and will allow a correlation to be drawn between the clinical characteristics of the affected subjects and the biochemical defects resulting from the mutations they carry.

[unreadable] DESCRIPTION (provided by applicant): The retinal pigment epithelium (RPE) plays a critical role in maintaining the health and viability of photoreceptor cells. Current evidence implicates RPE dysfunction in the pathogenesis of a variety of retinal degenerative disorders, including age-related macular degeneration (AMD), the most common cause of severe visual loss in the elderly population. The current proposal is aimed at investigating signal transduction within the RPE relevant to photoreceptor-RPE interactions and retinal disease. One line of research is focused on bestrophin, the protein product of the vitelliform macular dystrophy (VMD; Best disease) gene, which we have recently shown to be a chloride channel. VMD is one of several early onset inherited macular degenerations which serve as models for AMD. Investigating the structure, function, and regulation of bestrophin should provide insight into: (1) the signal transduction cascade(s) that mediate light-dependent regulation of RPE function in general and chloride flux in particular, and (2) the pathogenic mechanism of VMD. The second line of research is focused on peropsin, a G-protein-coupled receptor that we discovered several years ago, and that is found only within the apical microvilli of the RPE. We are aiming to: (1) identify the intracellular signaling cascade activated by peropsin, and (2) identify peropsin's ligand.

DESCRIPTION (provided by applicant): With the increased use of mutant mice and zebrafish in vision research there is a growing need for methods to: (a) visualize individual neuronal morphologies as part of the phenotypic analysis of mutant animals, (b) to mark cells in a noninvasive manner for lineage studies, and (c) to visualize subcellular structures in a sparse subset of living neurons. This proposal has two overall goals. The first goal is to develop, test, and optimize a set of reagents - principally, plasmids and mouse and zebrafish lines - that would be easy to use, provide high quality visualization of cellular morphology and subcellular structures, and be freely available to the research community. In mice, the methodology that we will use is based on a pharmacologically controlled Cre recombinase-estrogen receptor fusion protein that irreversibly activates (by DNA recombination) the production of one or more enzymatic or fluorescent reporter proteins in a sparse subset of cells. In zebrafish, sparse labeling will be achieved by taking advantage of the variegated expression that typically accompanies transgenesis. The second goal is to use these reagents for (a) mapping of retinal ganglion cell morphologies in two lines of transcription factor gene knock-out mice (the POU-homeodomain genes Brn3b and Brn3c), and (b) identifying the spatial distribution of pre- and post-synaptic sites in single identified neurons in both living and fixed retinas in mice and zebrafish. Although the methodology and reagents that we develop will be tested in and applied to the retina, they will be generally useful for the analysis of any part of the nervous system.

DESCRIPTION (provided by applicant): Retinal vascular diseases are major causes of vision loss in the United States and around the world. Age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity are all associated with the growth of new blood vessels (neovascularization) into or on the surface of the retina. They are also associated with the leakage of fluid from the retina blood vessels into the retina (edema) and with bleeding from the new vessels. To better treat these disorders, we need to understand the signaling pathways that control the growth and integrity of retinal blood vessels. We recently discovered a new signaling system that controls the growth of retinal blood vessels. In this pathway, a protein, Norrin, is secreted by Muller glial cells, the most abundant type of glial cell in the retina. Norrin binds with high affinity to a receptor protein, Frizzled4, which, together with two other proteins, Lrp5 and Tspan12, are present on the surface membrane of vascular cells. In humans and in mice, mutations in any of the genes encoding these four proteins lead to insufficient retinal vascular development. The objectives of this proposal are to characterize the response of retinal vascular cells to Norrin/Frizzled4 signaling. We will determine the response to Norrin/Frizzled4 signaling when it is initiated or terminated at different times during development, as well as the role of Norrin/Frizzled4 signaling in neovascularization. Within vascular cells, Norrin/Frizzled4 signaling induces a distinct set of changes in gene expression. We propose to characterize the network of transcription factors that control this response, beginning with one transcription factor, Sox17, which appears to play a central role. We will also explore the role of Norrin/Frizzled4 signaling in the interactions between vascular endothelial cells (the cells that line the inner face of the blood vessels) and pericytes (cells that unsheathe and stabilize the endothelial cells), and between endothelial cells and astrocytes, which form a scaffold along which the endothelial cells migrate as the retinal blood vessels develop. As the retinal vasculature is very similar between mice and humans, and the Norrin/Frizzled4 pathway (as well as other signaling pathways) is highly conserved, our experiments with mice should translate to humans. The experimental methods used include: studying genetically engineered mice in which genes can be activated or inactivated at different times, generating vascular lesions with a laser to study neovasculization, identifying which genes are direct targets of transcription factor binding, and studying the behavior of purified retinal vascular endothelial cells, pericytes, and astrocytes grown outside of the living animal. 1

Project Summary In this renewal application, funding is requesed for continued support of the Johns Hopkins University T32-supported Visual Sciences Training Program (VSTP) (2020-2025). The VSTP is a combined effort of the Wilmer Eye Institute (Department of Ophthalmology) and the Departments of Neuroscience, Molecular Biology and Genetics, Biology, and Institute of Genetic Medicine. The program, administered and directed by PI Donald Zack and co-directors Jeremy Nathans and Amer Riazuddin, provides a multidisciplinary training platform with a diverse faculty covering cover many of the major avenues of modern visual science. The goal of the program is to recruit young, talented scientists into the visual sciences, and to provide them with broad theoretical and methodological research training, which will allow them to contribute to our understanding of the biology of vision and the pathological mechanisms responsible for visual loss in the context of human disease. Johns Hopkins is fortunate to have a large number of investigators exploring different avenues of vision research; their approaches range from the molecular and cellular to the systems levels, and the technologies they employ include cell biology, molecular biology, biochemistry, developmental neurobiology, stem cell biology, electrophysiology, functional imaging, psychophysics, bioinformatics, genomics, and genetics. This diverse vision research community provides a wide variety of research options for VSTP trainees. In this VSTP renewal application, we are proposing to accept two predoctoral students per year and support both of them for two years, and to accept two postdoctoral fellows per year, and to support each of them for one year. As part of our training program, the VSTP, in collaboration with Wilmer Eye Institute and other Hopkins graduate programs, organizes and provides vision-related courses, seminars, and related activities. Additional training in the problems of clinical ophthalmology, with an emphasis on translational problem solving, is available to all VSTP trainees through the medical student ophthalmology program and grand rounds. Through these programs, we hope to continue and expand upon the VSTP?s success in recruiting, inspiring, and training the next generation of vision scientists.