Maureen Neitz - US grants

DESCRIPTION (provided by applicant): Most of our daily activities are performed under light levels where our vision is based on cone photoreceptors. Our long term goal is to contribute to an understanding of the basic mechanisms underlying cone-based vision, how these mechanisms are disrupted in vision disorders and how vision loss might be prevented or reversed. For this application a multidisciplinary approach using psychophysics, adaptive optics imaging, electrophysiology, and molecular biology will be used to address three specific aims: Specific Aim 1. Identify variations linked to the X-chromosome cone photopigment gene array that are associated with shifts in the relative numbers of human L and M cones. There is astounding individual variation in the L:M cone ratio among humans with normal color vision. To test hypotheses about the mechanism responsible for determining whether a cone is M or L, the region of the X-chromosome containing the cone photopigment genes will be examined in a large sample of males with normal color vision who have known differences in the ratio of L:M cones. Specific Aim 2. Determine the consequences for the human cone mosaic of identified genetic differences that are proposed to affect the photoreceptor topography. Adaptive optics imaging coupled with retinal densitometry will be used to visualize structural and functional changes in the cone photoreceptors and their topographical arrangement in the retina as the result of genetic mutations. Specific Aim 3. Explore an amazing plastic neural mechanism that is hypothesized to allow information from the environment to instructively reorganize neural connections throughout life. We will characterize a recently discovered plasticity of the adult visual system in which the long term effects of altered chromatic experience have been found to change the color vision of adults. This will allow hypotheses about the role of neural plasticity in establishing and maintaining proper function of the visual system to be tested.

The long term goal of this research is to understand the molecular biologic basis of vision. The immediate goal of this proposal is to determine the fundamental properties of X-linked genes that encode cone pigments. To that end the specific aims are: 1. To determine (i) the number of visual pigment genes on the X-chromosome, (ii) the number of genes that encode long-wavelength sensitive pigments, and (iii) the spacing and arrangement of genes encoding middle and long wavelength sensitive pigments. 2. To characterize the cone pigments that underlie protanomalous color vision, to determine the structure of the genes that produce protanomalous pigments, and to investigate the genetic mechanisms that give rise to anomalous pigments. A model to explain the molecular genetic basis of red-green human vision formulated by Nathans et al. (Science 232:198-202, 1986) has gained wide acceptance. Key features of this model include that (i) the average number of visual pigment genes on the X-chromosome is approximately three and (ii) all individuals with normal color vision have a single long wavelength sensitive cone pigment gene. However, the available data do no support these and other basic aspects of the Nathans et al. theory. The significance of the research proposed here is that an understanding of the molecular biology of vision must be built from a base of facts about the most fundamental properties of the genes that encode the cone pigments. Theories conceived to explain the individual differences in normal color vision, common color defects, and rarer more debilitating visual defects, as well as the experiments designed to test those theories, will depend critically on knowing the possibilities allowed by the number of pigment genes, the variety of genes producing different pigments, the relative frequencies of those genes and their arrangements. Knowledge of these basic facts can guide the search for understanding of riot only the properties of the cones--their spectral sensitivities and their ratios--that underlie the diversity of human vision, but also of how the circuits for processing visual information arise. The basic features of the X-linked cone pigment genes from males with normal and color defective vision will be investigated by Southern hybridization analysis and genomic DNA will be used in the polymerase chain reaction to amplify segments of the X-linked visual pigment genes for nucleotide sequence analysis. The color vision and visual sensitivity of the subjects will be examined in detail using psychophysical methods and the electroretinogram.

The long term goal of this research is to understand the molecular genetics of vision based on cone photopigments. To achieve this goal, we must first establish a relationship between color vision phenotype and genotype. This requires a clear, accurate understanding of the genotype. Recent evidence indicates that the structure of the arrays underlying normal color vision are radically different than previously appreciated. Thus, the immediate goal of the work proposed here is to gain understanding of the structure of the X-linked pigment gene array. What are the numbers and rations of pigment genes in individual arrays, how do the numbers vary, and what role do "extra" pigment genes in play in normal vision and in vision abnormalities? What recombination mechanisms play a role in altering the structure of the array; which alterations are associated with color vision defects and more serious vision disorders? How are individual differences in the amino acids of the opsins related to pigment function; what changes are associated with color vision defects, or other vision disorders? Each of the specific aims of this proposal builds on a different new discovery from our laboratories. Each discovery has far reaching implications for understanding vision based on these genes. 1) We have evidence that some individuals do not have intact middle-wave genes, but instead have incomplete genes. 2) We have evidence that individual differences in the numbers and ratios of genes that underlie normal color vision are far greater than had been imagined. 3) We identified a mutation in the genes underlying protanomalous color vision that does not change the absorption peak of the pigment, yet it alters pigment function to produce the difference between anomalous trichromacy and dichromacy. Toward exploring the implications of these findings the specific aims of our research are: 1) To investigate gene rearrangements among the X- linked visual pigment genes with regard to the frequency of occurrence of incomplete middle- or long-wave genes in normal vision and vision defects, and the genetic mechanisms that produce the deletions. 2) To characterize the basic structure of the X-linked visual pigment gene array with regard tot he number and ration of long- and middle-wave genes in the arrays of observers with normal color vision and those with color vision defects. 3) To characterize athe pigments underlying the color vision defects, protanopia and protanomaly, with regard to spectral sensitivity, optical density, and bleaching and regeneration kinetics, and to explore the relationship between gene sequence differences and these functional differences in encoded pigments. Visual capacities of males with normal and defective color vision will be examined in detail using psychophysical methods and the electroretinogram. The X-linked pigment genes will be investigated using Southern analysis, the polymerase chain reaction,a nd DNA sequence analysis.

How plastic is the adult nervous system? Can an animal's behavior be changed by delivering a gene encoding a light sensitive protein to the appropriate neuron in the retina. If the answer to this question is yes, it means that the nervous systems is sufficiently plastic to allow remodeling of the adult neural circuitry to the extent that it would bring about a change in behavior. If the answer to this question is yes, it will have an enormous impact on our understanding of neural plasticity, it will open new vistas for gene therapy for human disease, and i will allow us to create a model system to directly probe the mechanisms by which neural circuits are established and remodeled. Most New World primates, including squirrel monkeys, have a single visual pigment gene on the X-chromosome. However, three alleles occur at this locus and each allele encodes a spectrally distinct pigment. The three alleles correspond to the L and M pigment genes that are the basis for red-green color vision in humans, and the monkeys have a single X- chromosome, and thus are a model for a form of inherited red-green color vision deficiency common among human males. This form of color vision loss in humans is caused by deletion of all but one of the visual pigment genes on the X chromosome. To have normal, trichromatic color vision, male humans must have at least one L and ne M pigment gene. Female squirrel monkeys who are heterozygous at the X-linked pigment gene locus have trichromatic color vision similar to that of humans. Thus, the neural circuitry necessary to establish trichromatic color vision is present in this species, and is clearly utilized when a third visual pigment gene is present during development. We propose to add, by subretinal injection of recombinant adeno-associated virus carrying a human L opsin gene, a third cone type to the adult male squirrel monkey retina. The three cone types will be the endogenous S and M cones of the male squirrel monkey retina, plus M clones transduced with the rAAV virus and expressing a human L pigment. We will monitor color vision behavior both before and after subretinal injection to determine whether the animal's color vision changes from dichromatic to trichromatic.

DESCRIPTION (provided by applicant): The long-term goals of the work proposed in this grant application are to study neural plasticity of the adult mammalian visual system by adding new sensory input. Experiments that enhance rather than ablate sensory input offer a new avenue of research for answering fundamental questions about the plasticity of the adult visual system and have important implications for the rational design of gene therapy and retinal prostheses. We have chosen red-green color vision as a model system in which gain of function can be monitored quantitatively at the level of the retina using the electroretinogram and at the level of the visual cortex with behavioral tests of color vision. As a first step toward achieving our long term goals, we propose two specific aims. Specific Aim 1. To determine whether adding a third cone type via viral mediated delivery of a photopigment gene to the retina in a dichromatic adult squirrel monkey can expand his sensory capacity and change color vision behavior from dichromatic to trichromatic. Specific Aim 2. To determine whether expressing a long-wavelength sensitive pigment gene in a subset of UV cones via viral mediated gene delivery to the gerbil retina can expand the gerbil's color vision capacity to include making color discriminations in the red-green region of the spectrum.

The functions of this Module have evolved over the years in response to changing technology and therefore the changing needs of our group. The Module was originally established as a Biochemistry Module. Three cycles ago when molecular methods came into widespread use, a molecular biology component was added making it the Biochemistry-Molecular Biology Module. Now the order of "Molecular Biology" and "Biochemistry" in the Module name has been reversed to reflect that the use of molecular methods is currently the major activity of the Module. The Module does, however, continue to provide the biochemistry resources that are needed by the participating faculty. This is one of our most heavily used Modules. Its major functions are to provide shared-use instruments for molecular and biochemical analyses that are well maintained and accessible, and to provide skilled technical support to perform a variety of commonly used protocols. There is also an important instructional component to the Module: the Module directors help Core participants develop molecular approaches for answering their research questions, and together the Module directors and technician instruct investigators and their staff in the practical aspects of the experimental procedures and instrument use. In response to the expanding use of the Module for genetic analysis of human samples, we plan to add a part time clinical research coordinator to the Module staff. Access to help with human subjects research was identified by the Core Advisory Committee as the most pressing new staffing need in our Core Modules. With the addition of a clinical research coordinator, this Module will therefore be able to assist investigators with drafting human subjects protocols and with all aspects of recruiting subjects, obtaining samples, and managing patient records.

DESCRIPTION (provided by applicant): Investigators who have achieved independent National Eye Institute (NEI) funding will be provided with additional shared support to enhance their own and University of Washington's capability for conducting vision research. Collaborative studies will be facilitated, and scientists will be attracted to research on the visual system by the presence of this shared support. A modular organizational structure will be maintained, with each module devoted to a specific activity that would be impractical or less efficient to support on an individual research grant. Each module will support a service or resource that enhances or facilitates the research efforts of a CORE group of investigators, each having independent funding. Some sharing of resources and services with non-NEI-funded collaborators and with investigators new to vision research will occur. Junior researchers will also have access to these facilities to improve their ability to attain independent NEI funding. Proposed modules include: Biochemistry/Immunology (B/l), Genotyping/Phenotyping (G/P), Morphological Imaging (M/l), and Psychophysics/Physiology (PIP). Areas of investigation include retinal and choroidal diseases, corneal wound healing, corneal diseases, lens and cataract, glaucoma, strabismus, amblyopia, visual processing, and ocular development. Specific disciplines that will be brought to bear on these problems include: behavioral studies, biochemistry, biostatistics, molecular biology, cell biology, proteomics, immunology, microscopy, microbiology, morphometry, neurophysiology, and pathology. This project will elucidate basic mechanisms that underlie the function of the eye and the visual system and apply this knowledge and other information to the solution of problems in vision and ophthalmology. Collaboration among investigators from the University of Washington and elsewhere will be promoted. This proposal will improve the effectiveness of funding available on individual research project grants.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to provide core support services for 23 investigators at the University of Washington (UW), who together hold 27 NEI ROI grants. The CORE support services are designed (1) to enhance both quality and quantity of research conducted by UW vision scientists, (2) to facilitate collaborations between investigators with different areas of expertise, (3) to allow established investigators to conduct pilot projects and explore new areas, (4) to facilitate innovation within the UW vision science community. In addition to investigators with NEI R01 supported research programs, the Core Modules are anticipated to facilitate vision research in labs of 14 other investigators whose funding is from non-NEI sources with the long-term goal of helping these investigators obtain NEI ROI grant funding. Among these 14 investigators, the short term goals of the core grant are to assist junior investigators establish their labs and apply for NEI ROI support, and to recruit new investigators to vision research. Collectively, UW vision scientists span a very broad range of areas within vision research ranging from investigations of the roles of molecules in vision to assessment of visual performance in awake behaving human and non-human primates. There is a strong emphasis on translational research within the UW vision scientist group with investigation in all areas being targeted at understanding disease mechanisms and development of treatments and preventions for blinding conditions. The Core Grant will support three modules, each of which is staffed by highly skilled research scientists who are also experienced in vision research. The Cellular Biology Services and Shared Instrumentation Module offers microscopy, image analysis, and tissue preparation and sectioning. The Molecular Biology Services and Shared Instrumentation Module offers routine DNA isolation and genotyping, monoclonal antibody production, and immunocytochemistry. The Systems Biology Services and Shared Instrumentation Module offers technical assist with computer programming, magnetic resonance imaging, electroretinograms, and behavioral tests of visual function. PUBLIC HEALTH RELEVANCE: The Core Grant facilitates and enhances the research programs of NEI funded investigators at the University of Washington. These scientists study the visual system with a strong emphasis on translational research. The research supported by this Core Grant contributes to public health by helping to reduce vision loss.

The Morphometry/lmaging module was our most widely and heavily used module in the last funding period. Under its new name. Cellular Biology Services and Shared Instrumentation, it will continue to provide key resources that will be heavily utilized. In 2009, we purchased an Olympus FV1000 confocal microscope in order to meet the growing needs and new directions of NEI-funded investigators. The availability of fluorescent proteins with distinct spectral properties (e.g. cyan, green, yellow, red) has recently increased substantially, enabling investigators to colorize different cell types in the same retina, an approach that facilitates studies of cell-cell interactions. Importantly, fluorescent proteins are more photostable and label live cells. Several NEI-funded investigators have projects that involve labeling and visualization of cells (Wong, Reh, Brockerhoff, Clark, Chao, Neitz). The Olympus FV1000 confocal microscope was acquired because it has the appropriate combination of lasers to specifically excite cyan, green, yellow or red fluorescent protein. Currently, this confocal microscope is central to NEI-funded UW projects investigating (1) retinal function (Deeb, Detwiler, Neitz, Van Gelder), (2) retinal biology in development, disease and regeneration (Brockerhoff, Chao, Clark, Hurley, Reh, Wolf, Wong) and (3) lens organization and development (Clark). Moreover, support from this module enabled investigators to utilize multiphoton microscopy that has been, and continues to be, critical for their in vivo imaging projects in zebrafish (Brockerhoff, Hurley, Clark) and mice (Reh, Wong). Due to the limited resolution of light microscopy, there is increasing need to correlate ultrastructure to observations from light microscopy. This is facilitated by conversion of fluorescent protein labeled cells and tagged proteins into an electron dense material using protocols based on immunolabeling for GFP. Such protocols are, however, not readily transferred to visual structures, including the retina. Mr. Parker, an experienced electron microscopist, is working closely with Dr. Wong to develop protocols that will allow excellent visualization of labeled fluorescent protein and good preservation of the tissue. The benefits of the Cellular Module establishing this approach are far-reaching. For example, the Brockerhoff lab is interested in connectivity of specific types of photoreceptors labeled in live zebrafish, and the Wong lab has several mouse transgenic lines in which neurotransmitter receptors are tagged with YFP, but their localization to synapses need to be ascertained. Moreover, this 'GFP-EM' technique will open new avenues for investigators who need to establish the detailed structure and connectivity of cells identified by light microscopy in normal and diseased conditions.

Administrative Core Component: Project Summary The Administrative Core will oversee the daily running of all resource core components and their staff, and administer the Vision Research Core Grant. The Advisory Group consisting of the PI, the module directors, and the module staff scientists will meet on a quarterly basis to discuss overall core usage and mechanisms to optimize usage and productivity by the core investigators. A Vision Research Core Newsletter is emailed quarterly to all core members. The newsletter contains information describing each core component?s services and shared instrumentation, and includes contact information for component technicians and sign-up information for instruments. The newsletter will also contain information regarding the status of scheduled maintenance or instrument repairs. A Grants Administrator and Financial Analyst will perform accounting and reporting for the Vision Research Core Grant. The PI will receive month accounting statements and will meet with the Financial Analyst to discuss the statements.

Overall Component: Project Summary The Vision Sciences group at the University of Washington consists of 36 vision researchers who currently hold 24 qualifying NEI R01 grants and two new NEI R01 grants that received excellent priority scores and are likely to be funded. The objective of the Vision Research Core Grant at the University of Washington is to provide critically needed infrastructure to support the research programs of these exceedingly talented investigators and to promote collaborations among members of the group and vision scientists worldwide. The group?s common goals are to elucidate basic mechanism of the visual system and its disorders, and to develop potential treatments and preventions for blinding disorders. To achieve these goals, we have organized facilities, services and shared instrumentation into the following three Components: (1) Cellular Biology Services and Shared Instrumentation (2) Molecular Biology Services and Shared Instrumentation (3) Systems Biology Services and Shared Instrumentation These modules provide resources commonly used by multiple laboratories that are essential to the success of on-going projects as well as to enable new collaborations. The University of Washington (UW), is committed to the support of Vision Science. In 2013, the School of Medicine (UWSOM) established a Vision Sciences Center in a new, state-of-the-art research building on the UW Medicine Research Campus at South Lake Union (SLU3.1). The Vision Sciences Center occupies ~25,000 square feet (sq. ft.), of which ~3000 sq. ft. is dedicated to the Vision Research Core. UWSOM provided $650,000 in funds for the purchase of new equipment for the Vision Sciences Center. The Vision Research Core also maintains equipment on the UW main campus in the Health Sciences Building to accommodate the needs of investigators who chose not to move their labs to SLU3.1. During the last funding cycle, the University provided competitive start-up packages that allowed the recruitment of 7 junior vision research faculty across three departments, and an 8th recruitment is underway. We request support in the form of this Core Grant for Vision Research ? to help maintain shared instrumentation through service contracts ? to provide personnel support for module scientists who assist and train investigators and their staff in the use of the equipment ? to provide select services to avoid duplication of equipment and effort, and enhance the productivity of the UW vision research group ? to help us recruit new vision scientists

The long-term objective of this research is to understand the mechanism by which intermixing of the human long- (L) and middle- (M) wavelength cone photopigment genes give rise to variants that cause aberrant pre- messenger RNA (mRNA) splicing, and lead to vision loss with a diverse set of clinical phenotypes. 95% of our cones are L or M cones, and except at very low light levels when rods are active, all vision is based on cones. The L and M cones play a critical role in the visually guided feedback mechanism responsible for controlling eye growth (emmetropization). Every aspect of seeing, including high acuity and color vision, depends on the L and M cone photopigments and these genes are important risk factors in common eye disorders that plague modern humans. The L and M (LM) cone photopigment genes, designated OPN1LW and OPN1MW, respectively, exhibit high haplotype diversity in exons 2, 3 and 4. The most is known about two variants, designated LIAVA and LVAVA that are associated with photoreceptor dysfunction and severe vision impairment. They are found in patients with a range of clinical diagnoses including high grade myopia, blue cone monochromacy and cone dystrophy. We and others have recently shown that combinations of single nucleotide polymorphisms (SNPs) associated with these variants cause aberrant pre-mRNA splicing. Our preliminary data show that different combinations of the exon 3 polymorphisms shift the ratio of full length to exon 3 skipped mRNA, producing variability in the severity of the splicing defect that will be extremely useful in elucidating the fundamental mechanisms controlling splicing of this exon. Preliminary data obtained using a cell culture-based splicing assay also suggests that haplotypes of exon 3 that yield moderate levels of exon 3 skipping are associated with an average -1.3 diopters of refractive error compared to low/non-skipping variants. The pathophysiology of the different mutations is complex because superimposed on the effects of subnormal amounts of opsin protein produced by the splicing defects, are the effects of some combinations of the amino acids on protein function. To achieve our goal we propose: Aim 1: To investigate the role of combinations of SNPs in exons 2, 3 and 4 of the L and M opsin genes in splicing by 1.1 fully enumerating the transcript isoforms, and measuring their relative abundances. 1.2 investigating and quantitating the effects of exon 3 haplotypes on splicing in cone photoreceptors and particularly those haplotypes that are risk alleles for juvenile onset myopia. Aim 2: To investigate the mechanism of exon 3 skipping using biochemical and molecular biology approaches Aim 3: To evaluate the potential for exon specific U1 snRNAs to rescue the exon 3 skipping phenotype.