Joseph J. Carroll - US grants

DESCRIPTION (provided by applicant): This proposal addresses 2 of the program objectives of the NEI Retinal Diseases Panel: - Explore the topographical and regional differences of the retina and the relationship of this topography to disease progression. - Continue to develop and apply noninvasive technologies such as functional magnetic resonance imaging (fMRI), ocular coherence tomography, adaptive optics, and confocal imaging to better understand retinal function and changes in disease states. The overall purpose of this proposal is to characterize the topography of the cone mosaic in normal and diseased retinae, to examine the factors that might govern this topography, and to assess photoreceptor function in these mosaics. I have discovered novel cone degeneration mechanisms linked to mutations in the cone photopigments. The experiments in this proposal will help clarify the deleterious effects of disruptions in cone pigment expression, both on the appearance of the cone mosaic and on visual performance. This proposal is the first step in an effort to build a research program that will contribute to the understanding of fundamental biological processes underlying cone vision and vision disorders. By examining how topographical disruptions in the cone mosaic affect visual function (contrast sensitivity, acuity, sensitivity), I will gain novel insight into the structure-function relationship on a cellular level. Moreover, the methodological approach developed in this proposal will be translatable to other retinal diseases that are more complex in nature. Through a unique collaborative effort, I propose to combine psychophysical, electrophysiological, and genetic techniques with in vivo imaging techniques (such as optical coherence tomography & adaptive-optics ophthalmoscopy) to address the following aims: Specific Aim 1 - Characterize the S-cone submosaic in normal and tritan subjects and its relationship to the overall topography of the retina. Specific Aim 2 - Examine the effects of mutations in the L/M-photopigment gene array on the viability of the cones and the organization of the photoreceptor mosaic. Specific Aim 3 - Determine the consequences of cone-opsin mutations and disorganization of the cone mosaic for cone and visual system function. PUBLIC HEALTH RELEVANCE: The majority of our visual activity relies on the cone photoreceptors in the retina. This proposal employs a multidisciplinary approach (using high-resolution retinal imaging, genetic analysis, and electrophysiological tests) to investigate how mutations in the cone pigments affect cone-photoreceptor structure & function. The work in this proposal will serve as the foundation for translation of this same approach to the future study of other retinal degenerations, which will accelerate progress for the effective implementation of novel therapies.

The Engineering & Translational Imaging (E&TI) Module is the newly redesigned Image Analysis Module that also encompasses functions of our former Animal Resources Module. The re-development and melding of Module functions was recommended by our Core Advisory Committee to reflect the changing emphases in Module use, to take better advantage of the exceptional skills of our existing personnel, and to accommodate the research interests of new members added to our group. We consider the new Module highly innovative. We also believe it will spur innovative research because of its emphasis on technology development. This Module is expected to be unusually important for fostering collaboration between those who develop new imaging technologies and those who use them - including the clinical faculty in ophthalmology - to everyone's benefit. Our former Animal Resources Module provided shared space located within the school's animal care area that is outfitted with equipment dedicated to performing ophthalmic examinations and procedures on research animals. This essential function is retained, but moved into the E&TI Module for two compelling reasons: (1) the animal area is increasingly used for retinal imaging and functional analyses of animal eyes (rather than for procedures), which is precisely parallel to the human subjects imaging supported by the former Image Analysis Module (now renamed the E&TI), and (2) the former Animal Resource Module had no support staff. Our newly designed E&TI Module will have an expanded role for the Module assistant who aids with studies of human subjects to include studies of research animals. The Module will also have an engineer to maintain existing instruments, modify them to suit investigators' needs, and - importantly - to develop new instruments and new image processing and data analysis software. This Module staffing will bring significantly added value to those who do animal research. Further, the translational aspect of our Core investigators' research will be strongly supported by melding support for animal and human eye imaging under the same Module umbrella. The highly innovative and well integrated functions of the newly designed E&TI Module therefore are: (1) to provide engineering support for maintaining, modifying and developing novel imaging technologies, (2) to assist with generating and analyzing images of animal and human eyes, both for testing new devices and for studying ocular phenotypes, and (3) to facilitate studies involving imaging of human subjects in collaboration with clinician scientists through the services of a Module assistant who functions as clinical research coordinator. This Module is expected to significantly enhance productivity and provide important cost benefits by making available equipment and services that could not be supported on individual investigator's grants including specialized imaging instruments, an engineer skilled in working on optical devices, and a clinical research coordinator to facilitate human subjects research

DESCRIPTION (provided by applicant): Inherited retinal degenerations affect approximately 1 in 1,500 individuals in the US, and the prospect of treating these devastating diseases is a daunting task. While exciting progress has been made in the development of therapeutic strategies for these conditions in animal models, we currently lack sensitive tools to directly and immediately assess how various interventions might affect retinal health in human patients - clinical tools for assessing retinal structure and function are relatively insensitive and macroscopic. As such, there is a critical need to develop sensitive, noninvasive, cellular-resolution techniques for assessing photoreceptor structure and function. Adaptive optics imaging systems correct for the eye's optical aberrations, allowing imaging of individual rod and cone photoreceptor cells in the living human retina. Moreover, the same adaptive optics tools used to image the photoreceptor mosaic offer the potential to probe retinal function on a cellular scale, completely noninvasively. From a clinical perspective, these tools are in relative infancy, and we propose to help accelerate their translation through the following specific aims: 1) Define the therapeutic potential in human patients with achromatopsia, 2) Determine how L and M opsin mutations affect the integrity of the photoreceptor mosaic, and 3) Define the retinal phenotype in patients with retinitis pigmentosa and Usher syndrome, and determine how changes in the foveal cone mosaic affect visual function and sensitivity. The specific retinal degenerations were chosen because they represent a wide range of rod and cone involvement, are current or emerging targets for treatment efforts, and are a current strength of our collaborative team. This work is expected to have a significant positive impact, with the high-resolution genotype-phenotype relationships identified here providing a better understanding of the therapeutic potential in patients with inherited retinal degenerations as well as producing validated tools for assessing photoreceptor structure and function with cellular resolution. This proposal addresses 3 emerging needs identified in the NEI's Publication, Vision Research: Needs, Gaps, and Opportunities: Characterize the macula and perifoveal regions of the retina to better understand the predilection of the macula for disease, Translate high- resolution retinal imaging technologies, like adaptive optics, into cost-effective and easy-to-use platforms for routine clinical use, and Develop novel, noninvasive imaging techniques for monitoring electrical or metabolic activity of retinal neurons in vivo, ideally at the spatial resolution of photoreceptors or better for early detection of disease and monitoring of therapeutic intervention.

DESCRIPTION (provided by applicant): The eye and vision research group at the Medical College of Wisconsin requests continuing support for its Core program. Thirteen Core investigators work on many tissues of the eye and on the visual system, with areas of research ranging from molecular mediators of oxidative stress to imaging of the normal and diseased human retina. Over the nearly 35 years of Core support, the Core Modules have been continuously updated so that they efficiently and equitably serve the needs of the group as its composition and research interests have evolved. In addition to helping support individual research, the Core program also brings together investigators with diverse skills stimulating collaboration on research questions of common interest. With rapid changes in technology and an increasing expectation for interdisciplinary research, shared resources and a mechanism to share new skills have become essential for continued research success. The goal of the Core program is therefore to enhance the independent and collaborative investigations of Core participants by providing both economical infrastructural support for services that cannot be readily supported by individuals, and access to current techniques. The latter is accomplished by knowledgeable. Module directors, experienced staff, continuously upgraded instrumentation, and Core Modules that are designed to work synergistically with one another and with institutional research resources. Support is requested for four updated Modules: Biochemistry-Molecular Biology Module, Cell Culture Module, Morphology & Microscopic Imaging Module, and a significantly redesigned Engineering & Translational Imaging Module that stresses development of novel imaging technologies. Past success of the research group can be partly attributed to the availability of Core-supported Modules. Continued Core support is critical to enhancing the quality of research and to maintaining the cohesiveness and productivity of Medical College eye and vision researchers.

? DESCRIPTION (provided by applicant): Strategies for treating degenerative retinal diseases are evolving at a rapid pace; however there exist major gaps impeding progress towards the ultimate audacious goal of regenerating neurons in the eye to restore sight. Technologies for monitoring the presence and health of individual photoreceptors and ganglion cells in living animal and human retinae are desperately needed. These tools would provide critical insight into the pathogenesis of a number of retinal and neuro-degenerative diseases; such insight is a requisite first step to developing the appropriate therapeutic approaches for a given patient/disease. Furthermore, improved visualization of cellular structure and function in patients with retinal degenerative diseases will permit scientists and clinicians to more precisely target and monitor the outcome of their therapeutic interventions. We have assembled a multidisciplinary research team uniquely equipped to address this major technological need. Drawing on our extensive experience in developing adaptive optics and retinal imaging tools, we propose to develop and disseminate four complementary platform/enabling technologies. We will leverage our existing collaborative relationships among all five participating sites, synergisic expertise, and access to extensive animal models along with an unrivaled patient population for testing these technologies. The specific technologies we propose to develop are: 1) Real-time retinal motion compensation, allowing retinal cellular-resolution imaging even in cases of extreme involuntary eye motion, like nystagmus; 2) Adaptive longitudinal chromatic aberration correction, allowing multi-wavelength, cellular-resolution retinal imaging; 3) Super- resolution line scanning ophthalmoscopy, to non-invasively image previously inaccessible cells and provide the largest image resolution improvement (> 50%) since the original demonstration of ophthalmic adaptive optics; and 4) High-throughput, opto-physiological method for assessing photoreceptor function with cellular resolution, providing a sensitive biomarker for assessing the function of regenerated/restored cells. A major strength of this application is that through our collaborative network we will validate the utility of these new technologies using regenerative therapies in both pre-clinical and clinical settings. This work will have a significant positive impact by enabling diagnosis of retinal disease and monitoring of retinal structure and function with unprecedented sensitivity and resolution. Finally, the focus of the proposed technologies will be photoreceptor and retinal ganglion cell imaging to explicitly advance the audacious goal, but they will not be limited to assessing any one therapeutic approach or cell type. Rather they will be generalizable and broadly applicable to all retinal cell types, retinal diseases, and therapeutic strategies.

DESCRIPTION (provided by applicant): Even though the fovea occupies only 0.02% of the total area of the retina, 40% of primary visual cortex is devoted to processing visual signals that arise there. Thus, it is not surprising that diseases affecting the structure or function of the foea are especially devastating to visually guided behaviors. Yet fundamental gaps remain in our concepts of how the fovea develops, how foveal structure is disrupted during aging and disease, and how the fovea interacts with more central visual system structures to determine key features of visual function. The overall goal of this proposal is to address these issues using a novel combination of noninvasive imaging technologies including functional magnetic resonance imaging (fMRI), optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO). Albinism is an inherited disorder characterized by absent or reduced melanin pigment in the eye, and often in the skin and hair, with all types manifesting the visual features of foveal hypoplasia, macular translucency, photosensitivity, refractive errors, nystagmus, impaired stereopsis, altered retinostriate decussation, and reduced visual acuity. These specific retinal and cortical features of albinism make it a perfect experiment of nature to examine the knowledge gaps listed above. We propose to do this through the following specific aims: 1) Define the spectrum of foveal morphology in albinism and assess the relationship between pit morphology and retinal melanin, 2) Identify retinal factors contributing t visual deficits in albinism, and 3) Determine the cortical correlates of visual deficits in albinis. The results of this project will provide a more comprehensive understanding of the interrelationships linking melanin, retinal morphology, and cortical organization. This will offer insight into the basis for vision deficits in albinism, which may alter current phenotype/genotype classifications. This work is expected to have a significant positive impact by providing a new framework for understanding and targeting clinical therapies to alleviate the behavioral manifestations of albinism, and by producing sensitive tools for assessing therapeutic outcomes. This proposal directly addresses emerging needs outlined in the National Eye Institute's August 2012 Publication, Vision Research: Needs, Gaps, & Opportunities, and incorporates specific program objectives of the NEI Retinal Diseases Panel: (1) Characterize the macula and perifoveal regions of the retina to better understand the predilection of the macula for disease. (2) Improve understanding of the roles of neuronal activity and molecular events in the formation of central visual circuits during development. (3) Continue to develop and apply noninvasive technologies such as fMRI, OCT, adaptive optics, and confocal imaging to better understand retinal function and changes in disease states.

PROJECT SUMMARY/ABSTRACT . The NEI?s Audacious Goal Initiative (launched in 2012) put forward the challenge of ?restoring usable vision in humans by regenerating neurons and neural connections in the eye and visual system.? While there is an obvious affinity towards novel therapies, current resource and technology gaps preclude translation of many therapeutic approaches. One such gap pertains to the availability of animal models that share key features of human retinal anatomy, as well as disease models that faithfully emulate the mechanisms and processes seen in patients with retinal degenerations (blinding diseases that might be amenable to regenerative therapies). The absence of readily available cone-dominant mammalian models represents a major technology gap impeding efforts to develop and evaluate regenerative treatment strategies in the retina. We propose to advance two promising model systems that are closer to human visual anatomy and function than the more widely used mouse and rat models. The first is the 13-lined ground squirrel (13-LGS): a diurnal, cone-dominant rodent (~85% cones) with large brain regions dedicated to processing visual information. The second is the tree shrew: a non- rodent, primate-like mammal that is also cone dominant (~95% cones). These models have been used to study visual transduction (13-LGS), outer segment morphogenesis, shedding, and remodeling during hibernation (13- LGS), cone-bipolar cell circuitry (13-LGS), myopia (tree shrew) and central visual processing (tree shrew). However, their use as translation-enabling models for evaluating both survival and integration of regenerated cone photoreceptors has been limited; mainly due to a lack of tools that allow for genetic manipulation of these animals (and thus a dearth of disease models). We propose to advance these species as disease-relevant models through the following Specific Aims: (1) Develop, optimize, and validate imaging methods and functional assays for the 13-LGS and tree shrew; (2) Generate 13-LGS and tree shrew cone photoreceptors from iPSCs in vitro; (3) Create rAAV-mediated retinal degeneration models for the 13-LGS and tree shrew in vivo; (4) Enable germline transgenic 13-LGS models of human disease; (5) Test and optimize integration of transplanted 13- LGS, tree shrew, and human iPSC-derived cones in normal and degenerated 13-LGS and tree shrew retinas. A key feature of this proposal is the validation of these models by comparing their cellular-resolution phenotype with that seen in patients with similar conditions/mutations. Throughout the project, we will share and disseminate our protocols, methods, and data to provide resources for use by the broader vision research community; this will be done using existing and newly-created online tools. A major strength of this application is the multidisciplinary team that has been assembled to take on this challenging project. The team brings the necessary complementary expertise required for model development, stem cell treatment, and evaluation of cell survival, integration, & function. This work will have a significant positive impact by providing not only validated disease models but also generalizable tools with which to create additional models in these and other species.