David Calkins - US grants

DESCRIPTION (Adapted from applicant's abstract): The question of which ganglion cells send color signals to the brain is unresolved. The M and P construct provides that achromatic information is encoded by parasol cells, and chromatic information by midget cells that project, respectively, to the M and P layers of the LGN. However, K layers in the LGN, ventral to each M and P layer, project to the cytochrome oxidase-rich blue/yellow and red/green color blobs in layers 2 and 3 of VI, and it is known in the case of the blue/yellow pathway, that there are blue/yellow small bistratified ganglion cells (i.e., not midget cells) that project to the LGN K cells. The central hypothesis of the present proposal is that ganglion cells that innervate other K cells in the LGN (i.e., alpha-CamK-positive gamma and epsilon cells) (1) are sufficient to provide additional vision channels, and (2) include ganglion cells that pools spatially co-extensive signals from M and L cones via an opponent bipolar cell to underlie red/green color vision. The specific aims to test this hypothesis in macaque retina are: (1) to measure the spatial distribution of gamma and epsilon cells using alpha-CamK-staining of the retina; (2) to establish their presynaptic circuitry via electron microscopy of serial sections through stained and injected cells; and (3) to determine the localization of glutamate receptors to bipolar cell dendrites via electron microscopy of serial sections through stained and injected cells.

This competitive application seeks support for the continuation of a highly successful Claude Pepper Older Americans Independence Center at Harvard Medical School's Division on Aging and its affiliated institutions. The primary goal of the Center continues to be to develop junior geriatricians into academic leaders with effective research, teaching, and clinical capabilities within a traditionally excellent research environment. This joint effort involving investigators in basic and clinical departments has been actively developing interventional strategies to promote independence in older Americans, a high priority objective of Healthy People 2000. It has also excelled in identifying and training outstanding clinicians for research. Careers in areas central to the mission of the National Institute on Aging. The Intervention Study (S) proposes to address problems associated with medication utilization in the elderly. The Intervention Development Study (IDS) aims to improve treatment efficacy for urinary incontinence. Two research resource cores (RRCs) are proposed for the enhancement of the intervention Study, the Intervention Development Study, support of ongoing research, and the development of new projects, especially those of junior faculty. The research cores are: RRC A: Subject recruitment; and RRC B: Basic Science and Engineering. A Research Development Core (RDC) will provide educational and career development opportunities for junior faculty and research associates. Fifteen experienced, well-funded faculty scientists, many of whom are established geriatrician investigators, will serve as potential mentors for junior faculty in seven areas: (1) geriatric medicine, (2) public health epidemiology, (3) cardiovascular renal, (4) endocrinology/metabolism, (5) neurosciences, (6) genetics cell/molecular biology, and (7) biomedical engineering physics. A Demonstration and Information Dissemination Project (DIDP) will provide information regarding improved treatment measures and disseminate the results of the intervention research to patients, care providers, academic faculty, and the public at large. The aim of the proposed Leadership/Administrative Core is to continue to maintain an environment that nurtures innovative, socially responsive, multidisciplinary research and training that will result in gr eater independence for older Americans. Ultimately we aim to develop more specific, more effective treatment that will improve and lengthen independence while at the same time decrease health care costs for our older Americans. We also aim to continue to develop junior faculty into independent scientific investigators who can apply basic research findings to clinical geriatric problems. This can best be accomplished within an Older Americans Independence Center.

In order to support research on basic biology of aging, Harvard Medical School's Division on Aging and its affiliated institutions propose to establish an NIA-funded Nathan Shock Basic Biology of Aging Center. The goals of this proposed Center are to provide state-of-the-art infrastructure, resources and services for basic scientists to study the molecular basis of the aging process and the development of age-associated diseases. More specifically, the research resource cores proposed are designed to facilitate and further accelerate the aggressive pursuit of hypotheses concerning the genetics of aging, from the molecule to the organism. As examples, the Center will support investigations on DNA instabilities as potential causal factors in cancer and aging, gene expression changes during aging and disease, and the role of aging and longevity genes in determining healthy lifespan. Three Research Resource cores are proposed: (1) Analysis of Gene Mutations; (2) Gene Transcript Recovery and Gene Development; and (3) Transgenetic and Genetic Model Systems. In addition to technical services and resources, these cores will also provide training courses on subjects in molecular gerontology. In addition, a Research Development Core, will provide a complete training program for junior faculty interested in aging research, and encourage senior faculty in other disciplines to consider applying their expertise to questions in aging, and a program Enrichment Core will provide administrative oversight, organize workshops and special seminars, promote utilization of Core facilities, develop an international network of contacts, and seek long-term permanent sources of funding.

This revised competing renewal proposal seeks support to continue a highly successful, NIA-funded Mentored Clinical Scientist Development Program Award. The program represents a major, multidisciplinary and interdepartmental collaboration among the medical school, school of public health, as well as the affiliated institutions in the Longwood Medical Area and around Boston. We have increased our efforts at recruiting candidates widely, including those who are members of under-represented minorities in aging research, and those who are from other medical schools including schools of osteopathy. We have provided information on eight superb prospective trainee candidates of which five are women, three are African American, one is Hispanic, and one is a doctor of osteopathy working with underserved elderly (including Native Americans) in rural Maine. Many of these candidates are interested in pursuing research in public and social medicine, especially the care of minority elderly in the underserved, inner-city and underserved rural communities of America. The proposed Program is designed to support and enhance research experience in several interrelated gerontologic disciplines. Six specific areas will be emphasized. These are (1) cardiovascular disease, (2) cell proliferation disorders, (3) neurodegenerative disease and dementia, (4) endocrine/renal dysfunction, (5) geriatric syndromes, and (6) public health. More than 50 experienced, well-funded faculty scientists (20 of whom are women), many of whom are established gerontologic investigators, will serve as potential primary or secondary mentors for the clinician scientist trainees. A dual mentoring system has been implemented to ensure ongoing exposure to gerontologic/geriatric expertise and orientation. Ultimately, we plan to develop the trainees into clinician investigators who can facilitate the translation of important research findings into improved care for all older Americans.

[unreadable] DESCRIPTION (provided by applicant): Our goal for this exploratory proposal is to understand how neuronal aging depends upon morphological phenotype in the human retina. We will generate a comprehensive genetic profile of aging photoreceptors by combining immunocytochemical identification of rods and different spectral types of cone with serial analysis of gene expression (SAGE). This information will allow us to compare cell-type specific patterns that could either contribute to or impede age-related photoreceptor degeneration. Age-related deficits in vision are a costly, debilitating and psychologically onerous consequence of overall senescence of the brain. These deficits are associated with many physiological changes in the neural retina, including most prominently the bss of rods and cones. Photoreceptors are also primarily targeted in the leading age-related retinal disease, macular degeneration. The susceptibility of photoreceptors to aging and disease depends upon both neuronal phenotype and individual variation. For example, rods are far more vulnerable than cones, while psychophysical studies indicate that blue-light sensitive "S" cones may decline more rapidly than green- and red-sensitive M and L cones. These losses are not uniform across different individuals. While extrinsic factors in the eye must contribute to photoreceptor decline, we hypothesize that the differential susceptibility of distinct photoreceptor phenotypes corresponds to an intrinsic pattern of cell-type specific gene expression. We have developed a unique and highly sensitive technology to construct, probe and compare complete gene libraries (cDNA) from the RNA harvested from select, labeled neurons in aldehyde-preserved retina. This "micro-harvesting" allows us to compare with great precision expressed sequences across different neuronal cell types. We will construct cDNA libraries from rods, S and MIL cones in young and aged human central retina. From these cDNAs, we propose to use a SAGE protocol modified for small amounts of RNA to (1) determine how gene expression within each photoreceptor cell type varies between human donors of the same age, (2) test whether aging correlates with patterned changes in gene expression for photoreceptors and whether expression correlates with photoreceptor survival, and (3) determine how different photoreceptor cell types respond genetically to aging. The information generated will provide a basis for more extensive investigations of the mechanisms that mediate neuronal aging in the retina and brain. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our long-term objective is to understand the early molecular events leading to the death of retinal ganglion cells (RGCs) and their axons in glaucoma. The defining feature of glaucoma is sensitivity to intraocular pressure (IOP), and elevated IOP represents a significant risk factor for the disease. Lowering IOP pharmacologically is the standard treatment to slow the disease, but there is no cure because the neurobiological mechanisms linking RGC degeneration to pressure remain unresolved. The death of RGCs in glaucoma demonstrates key aspects of neuronal death in other degenerative diseases, most prominently somatic (cell body) loss via apoptosis and axonal degeneration. In other diseases, somatic and axonal degenerative are often linked to elevated intracellular Ca2+, and Ca2+-dependent cascades are also likely to contribute to RGC degeneration in glaucoma. Our studies demonstrate that for RGCs exposed to elevated pressure in culture, rapidly increased intracellular Ca2+ predicates both somatic and axonal loss. These observations raise the questions of whether pressure-induced RGC death is dependent on increased intracellular Ca2+ and, if so, what is the mechanism of this dependence. We have hypothesized that pressure-induced RGC degeneration involves the activation of a mechanosensitive channel that directly gates an increase in intracellular Ca2+. In support of this hypothesis, we recently identified in RGCs the capsaicin-sensitive, vanilloid-1 transient receptor potential (TRPV1) channel. TRPV1 is characterized by a robust Ca2+ conductance that contributes to pressure sensitivity and Ca2+-dependent cell death in other systems. Here we will probe the relationships between pressure-induced changes in intracellular Ca2+, RGC death and TRPV1 activation using an in vitro preparation of purified RGCs optimized for studying somatic degeneration and a retinal explant preparation optimized for studying axonal degeneration ex vivo. By applying pharmacological tools to these systems we will (1) test the Ca2+-dependence of pressure-induced RGC degeneration and the contribution of TRPV1 to pressure-induced increases in RGC intracellular Ca2+ and (2) determine the dependence of pressure-induced RGC degeneration on TRPV1 activation. Finally, by applying genetic tools for gene inhibition and over-expression developed in our laboratory and a TRPV1 knock-out mouse we will (3) test the relationship between TRPV1 expression and RGC susceptibility to pressure-induced degeneration. PUBLIC HEALTH RELEVANCE:. With the aging of the population, glaucoma will afflict nearly 80 million people worldwide by 2020, making the disease the leading cause of irreversible blindness. Glaucoma remains incurable, largely because our understanding of how pressure sensitivity translates to RGC degeneration is incomplete. The work proposed here will explore a viable molecular mechanism for contributing to RGC susceptibility to pressure-related injury and test its relevance as a novel therapeutic target.

DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanisms of neurodegeneration in glaucoma and find new ways to abate it. Vision loss in glaucoma involves selective loss of retinal ganglion cell (RGC) neurons through two broad degenerative programs: in the optic projection, affecting RGC axons, and in the retina, affecting RGC dendrites, synapses and cell bodies. Degeneration arises from sensitivity to intraocular pressure (IOP), but IOP-lowering regimens do not always slow progression. Thus, to intervene at the neuronal level requires a better understanding of how the RGC pathway responds to IOP-related stressors and whether this response includes mechanisms to counter loss of function. Our objective in this project is focused on characterizing one such mechanism involving the TRPV1 (transient receptor potential vanilloid-1) receptor. Our central hypothesis is that TRPV1 counters RGC degeneration by enhancing excitatory activity in response to IOP- related stress. In other systems, increased TRPV1 at the neuronal membrane maintains cytoskeletal integrity and augments synaptic excitation by enhancing Ca2+ activity in response to stress. We propose a similar role for TRPV1 in RGCs, having established TRPV1 as a robust Ca2+ channel in RGCs that, when activated, increases excitation and influences their survival. We will test our hypothesis using both acute (microbead occlusion) and chronic (DBA2J) mouse models for which we have mapped key RGC degenerative outcome measures. For Aim 1, we will apply the acute model to a TRPV1 knock-out mouse to identify TRPV1- dependent axonal and retinal outcomes and their progression in RGC degeneration. For Aim 2, we will compare in the acute and chronic models IOP-dependent changes in TRPV1 expression and localization and link these changes to RGC subcellular compartments to identify structural correlates of TRPV1's action. For Aim 3 we will measure in both models how changes in IOP influence TRPV1's contribution to RGC excitation and determine if modulating TRPV1 sensitivity promotes survival. These new studies will capitalize on our completed studies of TRPV1 and a unique toolbox already in place to illuminate a novel cascade that could counter and slow stress-induced loss of function associated with glaucoma.

The Vanderbilt Vision Research Center (WRC) includes faculty in the College of Arts & Science, Peabody College of Education, School of Engineering and School of Medicine. The high level of performance of WRC researchers derived from Core grant support also synergizes with campus-wide investments in biomedical research and training at Vanderbilt. We request continued support for five modules. (1) The Animal Care Module provides specialized surgical support and veterinary care of nonhuman primates and other large animals and murine electroretinogram phenotyping. (2) The Computer Module provides hardware installation and maintenance, software development for visual displays and real-time data acquisition and analysis, and webpage maintenance and data acquisition. (3) The Imaging Module facilitates acquisition and analysis of functional brain imaging, optical imaging, confocal and standard microscopy and other imaging data. (4) The Gene & Protein Analysis Module provides timely and economical access to genomic, proteomic and histology services. (5) The Shop Module designs, fabricates and repairs specialized optical, mechanical and electronic instruments. Administrative support ensures continued smooth and stable operation of the WRC research and training missions. Modules are directed by investigators with history of NEI funding, have talented and experienced staff and provide services that are otherwise not available or would be prohibitively expensive or slow. During the last grant period more than three NEI-funded investigators used each module at least moderately. WRC investigators produced five hundred publications that made fundamental contributions to basic and clinical visual science. This Core grant has increased collaborations within and between basic and clinical vision researchers across the Vanderbilt campus and with other institutions. This Core grant has enhanced recruitment of world-class eye and vision researchers resulting in continued extensive NEI-sponsored research at Vanderbilt.

? DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate how aging and intraocular pressure (IOP) influence retinal ganglion cell (RGC) degeneration in glaucoma and to leverage this knowledge to identify novel therapies based on neuronal protection, repair, and regeneration. This is an important goal, since all of vision is encoded by action potentials propagated along RGC axons in the optic projection. These axons degenerate steadily from adulthood to death and are susceptible early in glaucoma. Axonal signals are determined primarily by excitatory, glutamatergic synapses summed and integrated in the RGC dendritic arbor. The objective here is focused on understanding how RGC axon degeneration in the optic projection in aging and glaucoma relates to synapse degradation in the retina. To gain this understanding, experiments will test a novel central hypothesis: that early axonal stress due to aging and IOP induces self-repair and adaptive remodeling of the RGC synaptic complex to prolong signaling, similar to homeostatic plasticity of excitatory synapses in other systems. This dynamic relationship stands in stark contrast to the most prevalent current hypothesis in which early and irrevocable synaptic and dendritic pruning drives RGC axon loss. A series of rigorous, quantitative and functional assays will test this remodeling hypothesis by leveraging both chronic (DBA2J) and inducible (microbead occlusion) models of glaucoma. Experiments in Aim 1 will vary IOP and map changes in synaptic and cytoskeletal components to dendritic complexity and axonal function for different RGC types. Aim 2 will assess how synaptic and dendritic changes for RGC types characterized by axon function depend on age and whether aging influences the response to elevated IOP. Finally, Aim 3 will use established transgenic tools to modulate axonal and somatic degeneration and determine for key ages and IOPs whether dendrites and synapses in individual RGC types are conserved or undergo remodeling independently. These innovative studies combining neurochemical, morphological, and physiological measures will enrich the understanding of how synaptic and axonal activity interrelate at the molecular level and lay the foundation for novel therapeutics based on neuronal self-repair.

PROJECT SUMMARY Glaucoma is a leading source of irreversible blindness worldwide. The disease causes degeneration of retinal ganglion cells (RGCs) and their axons through sensitivity to intraocular pressure (IOP). Many patients continue to lose vision despite efforts to manage IOP. Thus, an unmet clinical need is a treatment that addresses RGC degeneration directly. Our long-term goal is to address this need by identifying new therapeutic targets based on neuronal repair, protection and restoration. Consistent with this goal, in the current cycle we discovered a powerful form of intrinsic RGC protection involving the TRPV1 (transient receptor potential vanilloid-1) cation channel. We found that RGC spontaneous excitation and axon signaling is enhanced in response to elevated IOP through up-regulation, translocation and increased activation of TRPV1. Silencing TRPV1 by knock-out (Trpv1-/-) or pharmacological antagonism eradicates this enhancement, raises the threshold for RGC axon signaling, and accelerates by two-fold axon degeneration in our inducible model. Our objective now is to investigate how TRPV1 exerts this compensatory influence in RGCs and whether TRPV1-mediated enhancement could be harnessed therapeutically in glaucoma by increasing RGC resistance to stress. To do so, we will test the central hypothesis that in glaucoma, increased TRPV1 in RGC dendrites stabilizes cytoskeletal and synaptic structures by potentiating glutamatergic signaling, resulting in enhanced excitation. This hypothesis is supported by published results from the current cycle that form the premise for this continuation. While TRPV1 expression is normally low, we found that elevated IOP induces a transient increase in RGC TRPV1 mRNA and protein. This up-regulation involves translocation of TRPV1 to RGC dendrites proximal to glutamatergic synapses, where it supports focal increases in Ca2+ and amplified depolarizing currents. These changes parallel the IOP-induced enhancement of RGC spontaneous axon activity that is absent in Trpv1-/- mice. To link these observations mechanistically and test their therapeutic value, we will combine physiological, cell-imaging and molecular tools to probe TRPV1 function in RGCs using inducible (microbead occlusion) and chronic (DBA2J mouse) models of glaucoma. Experiments in Aim 1 will test how TRPV1 influences RGC dendrite and synapse survival during progression and whether this influence differs between RGC types identified physiologically and morphologically. Aim 2 will measure TRPV1's influence on glutamatergic signaling in RGC types, its interactions with synaptic and dendritic proteins, and whether its actions in glaucoma depend upon activation of the mechanosensitive TRPV4 subunit. Finally, Aim 3 will probe whether manipulating TRPV1 activation and expression boosts excitatory enhancement, prevents degeneration, and improves visual performance. These innovative studies offer a new understanding of progression in glaucoma at an unprecedented level of detail with the goal for new therapies based on manipulating neuronal physiology and signaling.

This study will test the central hypothesis that our patented, minimally erythropoietic, form of erythropoietin (EPO), EPO-R76E, protects retinal ganglion cells (RGCs) from glaucoma pathogenesis by directly activating the Nrf2 pathway in these cells. Oxidative stress is known to contribute significantly to glaucoma pathogenesis. EPO can decrease oxidative stress through activation of Nrf2 signaling to result in increased expression of antioxidant proteins from the antioxidant response element (ARE). We will test our hypothesis through the following Specific Aims: 1) Determine how EPO-R76E influences the Nrf2 signaling pathway; 2) Compare EPO-R76E-induced Nrf2 pathway activation in RGCs, astrocytes, and Müller cells; and 3) Measure EPO-R76E induced signaling and the efficacy of EPO-R76E microparticles in a non-human primate model of glaucoma. We will use the microbead occlusion model of glaucoma in both species. We will utilize genetic and pharmacological approaches to determine the pathways activated and the relative contributions of astrocytes, Müller cells, and RGCs in EPO-R76E induced Nrf2/ARE activation. We will use cell-type specific recombinant adeno-associated viruses and promoters, pathway inhibitors, flow cytometry, and microscopy. Finally, we will test the efficacy of inherently-antioxidant microparticle loaded with EPO-R76E in a clinically relevant species, the squirrel monkey. We expect to gain greater insight into glaucoma pathogenesis leading to the identification of druggable targets. Further, we expect to demonstrate that EPO- R76E microparticles are a safe and effective IOP-independent treatment for glaucoma.!

PROJECT SUMMARY Glaucoma is a leading causes of blindness and along with other optic neuropathies is characterized by the loss of retinal ganglion cells (RGCs). Increased intraocular pressure (IOP) management is the current standard of care for glaucoma patients, but fails to stop the irreversible loss of RGCs and progressive visual dysfunction. Vision restoration through RGC replacement therapy, one of the NEI?s Audacious Goals program, could be a potential solution, and considerable progress has been made in understanding the molecular signals that regulate RGC specification from human stem cells, as well as in RGC transplant and integration in rodents. However, when considering translation of laboratory advances to human testing, rodent models are limited by critical differences in retinal physiology, and proof-of-concept in non-human primates would greatly increase confidence and aid in therapeutic development before moving to human testing. Thus there is a considerable need for a tractable non-human primate model. Here we will establish a squirrel monkey-induced glaucoma model and the parameters to study human stem cell-derived RGC integration and potential vision restoration in a retina and visual system closer to those of human. Through this 5-year proposal we will achieve critical milestones, including validating the monkey glaucoma model, studying key structural and functional measures using innovative new modalities that should be portable between monkey and humans, and demonstrating the model?s ability to move across institutions. All of this will be accomplished in the setting of studying RGC transplant: differentiation, migration, local integration and synapse formation, growth down the optic nerve, and targeting to distal brain nuclei, with the goal of vision restoration.

PROJECT SUMMARY: ADMINISTRATIVE MODULE The Vanderbilt Vision Research Center (VVRC) was founded in 1989 as a cross-institutional, interdisciplinary collaboration between Vanderbilt University and Vanderbilt University Medical Center. The VVRC has a history innovative vision research, spanning the eye and its diseases to cognitive processing and integration of visual information. Faculty from the School of Medicine, College of Arts & Science, School of Engineering and the Peabody College of Education and Human Development combine through strong institutional support and strategic faculty appointments to sustain excellence in vision science. The VVRC's long-term mission is to leverage novel technologies, strategies and partnerships to (1) understand the biological substrates of vision and mechanisms of diseases affecting the visual system and (2) leverage this knowledge to develop and test new therapeutic strategies for vision-threatening conditions. To this end, we support eight well-coordinated service modules. Animal Services, Histology, Instrumentation and Computation represent cores intrinsic to VVRC facilities, while Genomics, Cell Imaging, In Vivo Imaging, and Proteomics utilize an internal scholarship system to subsidize use of the world-class institutional cores for which Vanderbilt is known. The purpose of the VVRC Administrative Module is to facilitate the integration and communication of activities within and use of the separate service modules. VVRC Director Calkins will remain primary investigator of the overall core. The administrative module is rounded out by Program Manager Jill Brott. Together with the Directorship Committee, which includes the directors of each service module, the administrative module oversees all daily activities within the VVRC and the use of all core funds. The Specific Aims of this module are to (1) facilitate flow of service requests to appropriate module director and relevant staff, (2) maintain electronic records of service module usage for distribution to module directors, (3) reconcile all financial ledgers against expenditures and personnel encumbrances, (4) facilitate purchase of supplies and equipment necessary for VVRC service modules and oversee their equitable usage, (5) mediate dispersion of information related to all vision research activities, and (6) interact with administrative services for other centers and institutes in support of core function. In these ways, the administrative module will promote innovation in vision research at Vanderbilt by serving as the hub of financial, organizational, and educational activities related to the VVRC mission.

PROJECT SUMMARY: HISTOLOGY SERVICE MODULE The Vanderbilt Vision Research Center (VVRC) includes faculty investigators with a strong interest in discerning structure-function relationships in the visual pathways. These include inferences based on whole tissue analysis, single cell labeling, and localization of molecular components of biochemical cascades in involved in intra- and extracellular signaling. Such inferences require access to expert histological processing and labeling. The purpose of the VVRC Histology Module is to provide a comprehensive service for all tissue preparation, sectioning and staining/labeling for investigator laboratories needing supplemental provision in these areas not covered by staff members supported by their individual grants. In the current funding period the histology service trained 15 staff members and 10 students/fellows and contributed material for 17 investigators, 10 of whom authored 41 publications using the service. Availability of this module during the current period saved VVRC investigators $262,184 in histology services. A survey of researcher plans indicates that the use of this service will increase, with moderate to extensive use by 21 of 36 VVRC investigators. The histology module, housed in the research space of the Department of Ophthalmology & Visual Sciences/Vanderbilt Eye Institute, is directed by VVRC Director and P30 Primary Investigator David Calkins, PhD. Using this space and personnel supported in part by this Core mechanism, the VVRC Histology Module will: (1) assist or supervise preparation of visual system tissues suitable for sectioning; provide a broad range of (2) tissue embedding capabilities and (3) sectioning of visual system structures; (4) support a diverse array of histological and immuno-labeling stains; (5) provide access to automated conventional microscopy and image processing software; and (6) train members of the vision research community on basic histological techniques. These services and resources will enhance the scope of experimentation NEI-funded VVRC investigators conduct, expand the training of students and fellows involved in vision science, and promote collaboration by providing histological support to those who otherwise would not have such capabilities, including early-career vision scientists and clinician-scientists competing for extramural funding for their laboratories.

PROJECT SUMMARY: CELL IMAGING MODULE The Vanderbilt Vision Research Center (VVRC) includes faculty investigators with a strong interest in discerning structure-function relationships in the visual pathways. These include inferences based on whole tissue analysis, single cell labeling, and localization of molecular components of biochemical cascades in involved in intra- and extracellular signaling. Such inferences require access to not only expert histological processing and labeling, but also sophisticated high-resolution microscopy imaging platforms. The purpose of the VVRC Cell Imaging Module is to provide a comprehensive resource for light and electron microscopy imaging by supplying support for managerial personnel of the Vanderbilt Cell Imaging Shared Resource (CISR), which includes a new Nikon Center of Excellence housed in the Department of Cell and Developmental Biology (Medical Center North). This support translates on a dollar-for-dollar basis to scholarships issued to VVRC investigators applicable for all CISR services, including the Nikon Center for Excellence. This scholarship system is implemented by the VUMC Office of Research and is utilized instead of a discount or co-pay via the VUMC ILab accounting system. In the current funding cycle, the Cell Imaging service was used by 19 investigators, 15 of whom authored 73 publications using the service, and saved our investigators $207,455 through issuance and utilization of 69 scholarships. In the next cycle, we expect moderate to extensive use by 21 of 36 investigators. The Cell Imaging Module, housed in centralized locations sufficient for 19 independent microscopy platforms, is directed by Associate Professor Rebecca M. Sappington, PhD. Using resources and personnel supported in part by this Core mechanism, the VVRC Cell Imaging Module will provide (1) a broad range of imaging modalities suitable for visual system tissues, (2) state-of-the-art image analysis software and data storage solutions, (3) consistent monitored access to imaging equipment and workstations, (4) imaging consultation on appropriate imaging modalities and sample pre- processing for visual system cell and tissue samples, and (5) training in the full spectrum of imaging modalities available in the CISR. These services and resources will enhance the scope of experimentation NEI-funded VVRC investigators conduct, expand the training of students and fellows involved in vision science, and promote collaboration by providing sophisticated, high-resolution and diverse imaging technology to those who otherwise would not have such capabilities, including early-career vision scientists and clinician-scientists competing for extramural funding for their laboratories.

Project Summary Glaucoma is the world's leading cause of irreversible vision loss. The only modifiable risk factor for glaucoma is intraocular pressure (IOP) and thus all existing medical and surgical approaches target the IOP. Unfortunately, many patients continue to lose vision despite IOP management. Glaucoma involves progressive degeneration of retinal ganglion cells (RGCs), whose axons form the optic projection from retina to brain. Thus, our long-term goal is to develop new treatments to abate this neurodegeneration directly in an IOP- independent manner, based on identification and targeting of critical mechanisms of disease progression. We previously reported that erythropoietin or a mutated version of it, EPO-R76E, are able to preserve the RGCs in models of glaucoma. Our data shows that it modulates neuroinflammation and increases expression of antioxidant proteins. In our parent R01, we propose to use gene therapy strategies in the microbead occlusion model to determine the role of NRF2 and the antioxidant response element in EPO-R76E-mediated neuroprotection in glia and RGCs, separately, in glaucoma. In this supplement application, we propose to expand the scope of investigation to explore the endogenous response of the retina and RGCs to hypertension-induced oxidative stress. The anticipated significant accomplishment of this proposal is identification of new druggable targets early in glaucoma pathogenesis and clarity on the cell types to target, which will increase the safety and efficacy of treatments. It also provides an unique opportunity for Sarah Naguib to be mentored into an independent vision researcher poised for a successful post-doctoral fellowship.