Marie E. Burns - US grants

DESCRIPTION (provided by applicant): The absorption of photons in rods and cones of the retina activates a cascade of biochemical reactions (phototransduction cascade) that generates the electrical response to light. The activation and deactivation of the cascade ultimately limits the amplitude and kinetics of the transduced signal, and thus the sensitivity and temporal resolution of vision. The overall goal of this study is to understand the mechanisms that turn off the light response in intact mouse photoreceptors. Gene targeting techniques will be used to manipulate the function of a subset of proteins that have been suggested to play key roles in deactivation of the cascade, and the resulting changes in the photoresponses of single rod cells will be determined by electrical recording. Using this approach, we will address 3 important questions: (1) How rapidly does rhodopsin become phosphorylated, and what determines this time course? (2) What are the functional consequences of arrestin translocation on the photoresponse? (3) Are the photoreceptor-specific splice variants of the RGS9 complex uniquely suited for deactivating transducin/PDE, and how? and 4) What are the mechanisms that speed transducin/PDE deactivation during light adaptation? This research addresses 1 of the objectives recommended by the Retinal Diseases Panel (http://www.nei.nih.gov/strategicplanning/np_retinal.asp#obj), which is to "Analyze the mechanisms underlying light adaptation and recovery following phototransduction and understand the changes in neural coding in light/dark adaptation." This research will help clarify the initial steps in the normal visual process, as well as the pathogenesis of diseases that arise from failures of deactivation, such as in some forms of retinitis pigmentosa and nightblindness. In a broader context, these experiments will provide insights into the mechanisms of deactivation of G protein cascades, which all eucaryotic cells use to transduce extracellular signals into intracellular responses.

? DESCRIPTION (provided by applicant): Retinal degeneration is the leading cause of blindness and costs society billions of dollars annually in disability and lost productivity, a burden that is predicted to worsen as baby-boomers age. All current treatments for retinal degeneration, including experimental therapeutics like stem cell or gene replacement therapy, are thought to be most effective when degeneration is caught in its earliest stages. Unfortunately, the earliest detectable symptom of degeneration is often visual impairment, which is only detectable after a large number of retinal cells have died and disappeared. The ability to discern the first signs of cell stress, prior to apoptosis and degeneration, could significantly improve the likelihood of delaying or preventing vision loss. One common early indication of pending degeneration is activation of retinal microglia, the resident retinal immune cells, which can proliferate, migrate to and phagocytose injured neurons. The proposed work will define the earliest changes in microglia during photoreceptor degeneration. Using high-resolution, adaptive optics imaging, we will determine the 3-dimensional morphology and dynamics of individual retinal microglia in vivo, both in healthy tissue and during the earliest stages of photoreceptor stress and degeneration (Aim 1). We will also determine the signals that trigger microglial migration to the outer retina and specify photoreceptor engulfment (Aim 2). Finally, we will determine the time course by which circulating macrophages infiltrate the outer retina, and evaluate the degree to which both of these cell types promote or undermine photoreceptor survival (Aim 3). This work will contribute to the long-term goal to monitor and manipulate microglial dynamics in vivo so as to provide earlier detection of and assessment of treatments for retinal degenerative disease.

PROJECT SUMMARY This application seeks funds for junior investigators and trainees to attend the biennial Federation of American Societies of Experimental Biology (FASEB) Science Research Conferences on the ?The Biology and Chemistry of Vision?. Since its inception in 1985, this has been one of the most successful and influential meetings focused on photoreceptor biology. The major goal of the meeting is to bring together established and young scientists to discuss the most recent advances in the broad and diverse fields of photoreceptor biology and pathobiology. This focused meeting is held in an intimate setting (small conference center with shared meals) for a relatively small number of researchers (~150) in order to catalyze scientific interchange. The program combines high-quality, formal presentations of novel experimental results with informal interactions in a friendly, collegial atmosphere. The meeting also has tremendous educational, career- development and networking value for junior investigators. Accordingly, the funding requested in this application will be used for covering the travel expenses and conference fees for the young scientists, including pre- and post-doctoral trainees and junior investigators at the level of assistant professor. The program emphasizes the most recent, and often most controversial, research in the field, address timely questions and identifies emerging trends, all of which catalyzes collaborations for future work. In 2017, the program will include 9 platform sessions, 4 poster sessions, two data-blitz presentations, two keynote speakers, and a career development workshop.

The visual pigment rhodopsin is both a G protein-coupled receptor (GPCR) and a critical structural component of the outer segment of photoreceptor cells. Mutations in rhodopsin are the most common cause of autosomal dominant retinitis pigmentosa (RP), a blinding disease that afflicts more than 1.5 million individuals world- wide. Rhodopsin can activate the visual signaling cascade as a monomer, but it self-associates to form dimers and higher order multimers that have been proposed to be important for phototransduction. We discovered that certain rhodopsin mutants that cause RP through unknown mechanisms appear to be function normally as visual pigments but unlike wild type rhodopsin, they fail to dimerize when reconstituted into lipid vesicles. We hypothesize that the lack of dimerization in these mutants prevents normal photoreceptor function, leading eventually to loss of photoreceptor cells, retinal degeneration and RP pathology. The overall goal of this application is to elucidate the biological and pathobiological aspects of photoreceptor functions that rely on rhodopsin dimerization. The RP mutants we propose to analyze provide novel tools and an exciting new opportunity to study the molecular basis of rhodopsin dimerization and the specific role of dimerization in phototransduction and maintaining disc architecture. In our two specific aims, we propose to conduct a comprehensive analysis of rhodopsin dimerization from its molecular basis (and why it fails in RP mutants) to its biological role in living photoreceptors. In the first aim we will use multiscale computational approaches, combined with experimental probing and verification, to test specific hypotheses about the driving forces for rhodopsin dimerization, why dimerization is perturbed in the RP-associated rhodopsin mutants, whether heterodimerization between mutant and wildtype protein occurs and whether we can identify compensatory mutations that restore dimerization. In iterative fashion, predictions from analyses in silico will be tested in direct biochemical assays and the results will serve to refine models and computational investigations. In the second aim we will comprehensively characterize two mouse strains that homozygously express non-dimerizing rhodopsin mutants, one each corresponding to point mutations in TM1 (F45L) and TM5 (F220C) to reflect the different dimer interfaces that are directly implicated. Our goal is to conduct a ?360 degree? analysis of the heterozygous and homozygous knock-in mouse phenotypes by examining photoreceptor morphology, analyzing intracellular targeting of outer segment- resident proteins, biochemical characterization of phototransduction, and electrophysiological assessment of light responses produced by the mutant rods.

DESCRIPTION (provided by applicant): Continued support is requested for interdisciplinary training in the vision sciences at the University of California, Davis. Training is provided by 45 vision scientists (31 preceptors and 14 associate preceptors) across 14 departments that will provide a strong foundation in one or more basic sciences. The goal of the training program is to produce vision scientists who will be capable of establishing independent research programs that will address significant problems in vision science. It will operate under the auspices of existing graduate programs at UC Davis as they offer the broad flexibility needed to achieve our training objectives. Among our 300 pre- and postdoctoral vision science trainees in the past ten years (38 of whom were partially supported by this T32), 90% are active in research through continuing training or in career positions. Among those who have completed all training, 82% are active in research and/or teaching positions at some 71 different colleges, university basic science departments and schools of medicine or veterinary medicine. The training program requests support for 4 predoctoral students (for two years each; 8 slots) and 1 postdoctoral trainee (for one year) to be selected by an Advisory Committee. Internal support mechanisms and extramural grants will be used for the other years of training. The trainees will participate i one or more of five overlapping areas in which our preceptors are clustered: (1) molecular & cellular biology, retinal electrophysiology, and genetics, (2) anterior segment anatomy and physiology, (3) molecular and cellular retinal imaging, (4) systems visual neuroscience, and (5) functional imaging, computational modeling and perception. Each of the 31 preceptors has an active program of vision science research, a strong commitment to training, and extramural funding. Program resources are augmented by a strong institutional commitment, the Center for Visual Sciences and an NEI Core grant. The training program draws on the rigorous research training of the admitting programs, but also requires a one-year course that covers the broader vision sciences and clinical vision science. Graduate trainees will be supported only after their first year of graduate training and will thus be a highly selective group that has completed much of their basic science curriculum. All trainees will participate in an active colloquium series in he vision sciences, Center for Visual Sciences symposia, journal clubs and training in the ethical conduct of research. All trainees will be engaged in vision science research that will be presented at national meetings and submitted to peer-reviewed journals.

SUMMARY ? MOLECULAR CONSTRUCT AND PACKAGING CORE The Molecular Construct and Packaging Core (MCPC) provides 1) molecular construct design and production; 2) packaging of Lentivirus and AAV vectors; and 3) repository, consultation and training in molecular methods to the UC Davis vision science community. The MCPC generates high-quality plasmids and viral vectors with short turn-around times, efficiently and flexibly serving the project goals of our investigators. By being familiar with the needs and goals of individual labs, the MCPC also helps to promote collaboration and cross-fertilization by encouraging the sharing of molecular reagents and knowledge between investigators with similar needs or goals. Because this facility is the only such molecular or viral core facility at UC Davis, this module provides essential support for cell and molecular biology approaches in the vision sciences.