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.

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 Unpublished data implicate other mutations in this gene in some cases of autosomal recessive retinitis pigmentosa (RP).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.

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.

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.