Yvonne Ou, MD - US grants

DESCRIPTION (provided by applicant): This K08 application for Yvonne Ou, MD describes a five year strategy designed to enhance her research and professional skills with the career goal of becoming an independent clinical scientist advancing our understanding of the neurodegenerative mechanisms underlying glaucoma and improving its treatment. Dr. Ou completed her MD at Harvard Medical School, ophthalmology residency at the University of California, Los Angeles, and a two-year glaucoma fellowship at Duke University. The K08 award will provide Dr. Ou, now an assistant professor at the University of California, San Francisco (UCSF), with the support necessary to accomplish the following goals: 1) to conduct laboratory studies of neuronal loss in an animal model of glaucoma, with emphasis on developing new skills in cellular neurobiology, mouse genetics, and in vivo retina imaging; 2) to become expert in retinal ganglion cell (RGC) synapse and dendrite biology both through formal didactics as well as publishing and presenting in the field; 3) to develop an independent research career. Although the exact mechanisms of glaucomatous optic neuropathy are unknown, the final common pathway is RGC death. This research examines the compartmentalized neurodegeneration of RGCs in response to elevated intraocular pressure, one of the main risk factors of glaucoma. Preliminary evidence suggests that synaptic and dendritic changes may be early events in the neurodegenerative process prior to cell death. Using a laser-induced ocular hypertension mouse model, the first aim will determine RGC synapse density in both the retina and dorsal lateral geniculate nucleus (dLGN). Initial characterization demonstrates RGC post-synaptic loss in the retina and RGC pre-synaptic loss in the dLGN. Aim 2 will examine dendrite morphology longitudinally using confocal laser scanning ophthalmoscopy to image the retinas of live CB2-GFP mice, in which a subset of relatively large transient OFF-aRGCs are genetically labeled. The third aim will evaluate RGC axonal territory and synaptic density in the dLGN of axons projecting from the non- glaucomatous eye using a novel genetic tool. Key components of the career development plan include: 1) Laboratory investigations of neuronal loss in a glaucoma animal model; 2) Mentorship from a multidisciplinary group of scientists and clinical scientists with expertise in RGC biology, rodent disease models, and in vivo retina imaging; 3) Formal didactics to expand neurobiology knowledge; 4) Presentation of data in the format of supervised manuscript and grant submissions, and presentation of work at meetings; 5) Planned transition to independence including professional development. A scientific advisory and mentorship committee has been assembled to ensure progression towards independence. The career development plan will be carried out at UCSF, which provides an outstanding environment to conduct this research, including state-of-the-art facilities and a rich and supportive community of clinical scientists critical to the success of this young investigator. PUBLIC HEALTH RELEVANCE: Glaucoma, the leading cause of irreversible blindness worldwide, is a disease in which the retinal ganglion cells of the optic nerve die, leading to gradual visual field loss and eventual blindness. A major deficit in our treatment of glaucoma is that it is often initiated after there is already evidence of retinal ganglion cell death and visua field loss. Improved understanding of which parts of the retinal ganglion cell are vulnerable in glaucoma will potentially uncover novel diagnostic and therapeutic targets for glaucoma patients.

Precise connections between synaptic partners are shaped during development to ensure proper neural circuit function, but how these connections are disassembled and rearranged after injury is less well understood. Glaucoma provides an excellent model to explore circuit plasticity when a postsynaptic neuron is injured. Furthermore, the ability to diagnose and treat glaucoma at an optimal stage before irreversible retinal ganglion cell (RGC) loss occurs requires a comprehensive understanding of inner retina circuit disassembly and plasticity. However, major gaps exist in knowledge about how RGCs are disconnected from and potentially rewired with their excitatory presynaptic partners, bipolar cells (BCs), how this remodeling affects RGC function, and the potential mechanistic role of microglia in circuit disassembly. Such knowledge is required to successfully develop optic nerve regeneration strategies that depend on functional circuit rewiring. The overall objective of this application is to determine the connectivity, function, and potential mechanisms of circuit disassembly and remodeling following intraocular pressure (IOP) elevation. The central hypothesis is that specific microcircuits in the injured adult retina may exhibit plasticity in terms of connectivity and function, with microglia playing an important role in synapse pruning. The hypothesis will be tested in the following specific aims: 1) Determine the specificity and timing of anatomic circuit rewiring in diseased adult retina; 2) Determine if diseased adult retina has the capacity for functional plasticity; 3) Identify the contributions of microglia in the mechanism of circuit disassembly. The approach includes biolistic transfection of individual RGCs, sub-micron imaging, electrophysiological measurements, and novel genetic tools to study bipolar-ganglion cell connectivity, RGC function, and microglia in experimental rodent glaucoma. The proposal is innovative because it examines the potential for cell-type specific rewiring, circuit-specific synapse pruning, and functional plasticity, concepts that shift the paradigm in understanding RGC degeneration in glaucoma. The proposed research is significant, because the resulting identification of both vulnerable and resilient retinal microcircuits to target will open new research horizons, particularly in novel psychophysics testing paradigms, drug development, and RGC regeneration or neuroprotection strategies. Finally, these experiments will fundamentally expand knowledge of how adult neural circuits react and rearrange in the face of injury.

PROJECT SUMMARY/ABSTRACT We propose the establishment of a UCSF-Proctor Clinician Vision Scholars K12 program for the mentoring and training of talented and diverse faculty Scholars in high quality, reproducible scientific research and the development of successful academic careers in ophthalmology and vision sciences. UCSF is one of the world?s leading health sciences institutions and this program will build on existing strengths in clinical and translational sciences, bioengineering, and career development. The objectives of the program are to 1) mentor and train exceptional and diverse junior faculty in developing methodological expertise and impactful research programs; and 2) transition the K12 Scholars to become independent investigators and successful academic careers. Two clinician-scientists with complementary scientific expertise and leadership skills will co-direct the program. An accomplished group of Mentors with broad expertise and strong records of mentorship and collaboration will provide the Scholars with structured mentorship across three Tracks. Track 1 is focused on epidemiology/randomized clinical trials/global health, Track 2 on basic and translational discovery science, specifically around the themes of ocular genetics & therapeutics, visual system injury, plasticity, & regeneration, and neurodegeneration, and Track 3 on bioengineering and innovation investigations. Key activities in the training plan include: 1) high-quality mentored research; 2) a customized didactic curriculum tailored to the Track; 3) career development and academic success skills; 4) detailed plan for transition to independence; and 5) training in the responsible conduct of research. The career development and transition to independence activities emphasize oral and written communication, grant writing, leadership, networking, responsible conduct of research, and scientific management skills. An Advisory Committee will work with the program directors to select K12 Scholars, monitor their progress, and identify opportunities for program quality improvement. An External Advisory Board will also provide input on program evaluation and strategic planning. The planned duration of appointments is three years and the projected number of junior faculty Scholars is two training at any given time. The intended outcomes for Scholars are engagement in rigorous, reproducible research, the development of funded independent ophthalmology and vision science research programs with a lasting impact on the field and a focus on improving human health and alleviating blindness, and the advancement of successful academic careers as leaders and mentors in the field.

Project Summary / Abstract Retinal ganglion cells (RGCs) are the output neurons of the retina responsible for transmitting information about the visual world from the eye to the brain. Thus, RGC damage and loss, a characteristic of many disorders of the visual system, has the direct consequence of vision impairment, or blindness when RGC loss is more severe. Our translation-enabling approach builds on a very well-established, thoroughly characterized and validated experimental glaucoma (EG) model. This affords our study the distinct advantage of conducting each of the proposed hypothesis-driven experiments within the framework of a reliable model of RGC degeneration that closely recapitulates the anatomical changes and pathophysiological processes observed in human glaucoma. Moreover, our preliminary results establish the feasibility of our approach, demonstrating that we have already achieved successful transplantation of human induced pluripotent stem cell (iPSC)-derived RGCs into the EG retina, while also characterizing major barriers that require targeted solutions. Hence, we propose to employ a series of manipulations to both donor RGCs and the recipient EG retina in order to overcome the existing barriers to RGC replacement and thus make a giant leap forward toward realization of the audacious goal to restore vision in persons blinded by glaucoma or other optic neuropathies. Each of our Aims is soundly based on existing knowledge of the relevant biology and will lead to meaningful enhancement of donor RGC survival, integration, and function in the glaucomatous EG retina. We will utilize rigorous quantitative electrophysiological and anatomical assessments for testing the hypothesis at the core of each Aim. Aim 1 will target neuroinflammation to improve the long-term survival of transplanted RGCs. We will create hypoimmunogenic iPSCs and manipulate the host retinal environment using systemic immunosuppressive agents or inhibition of microglial activation. Aim 2 will augment donor cell survival and integration through modulation of cellular age, with host retinal glia experimentally induced to an immature state through cellular rejuvenation. Aim 3 will enhance the connectivity and axon outgrowth of donor RGCs in the retina. Donor RGCs will be edited to express hM3Dq DREADD receptors for chemogenetic stimulation and mTOR activators. Thrombospondin will be overexpressed in host retinal astrocytes and donor RGCs, leveraging astrocyte-derived factors that promote axonal outgrowth and synaptogenesis. Together, these Aims will generate a wealth of knowledge and resources for the scientific community and bring us significantly closer to the reality of vision restoration through RGC replacement.