Jeffrey L. Goldberg - US grants

[unreadable] DESCRIPTION (provided by applicant): We propose to investigate why mature retinal ganglion cells (RGCs) fail to regenerate their axons after injury or in degenerative disease. Mature RGCs fail to regrow their axons once severed, yet axons in the embryonic CNS can regenerate after injury. The loss of this embryonic regenerative ability correlates with the developmental loss of RGCs' intrinsic ability to rapidly extend axons in vitro and in vivo. Interestingly, neonatal RGCs are signaled to decrease their intrinsic axon growth ability by amacrine cells, and at the same time, RGCs increase in their intrinsic ability to elongate dendrites. This switch in intrinsic regenerative ability is dependent on new gene expression, but the molecular basis is currently unknown. This is the first concrete demonstration that an intrinsic mechanism may limit regeneration. In this proposal we will use new, high-throughput technology to investigate the molecular mechanism for this loss of intrinsic axon growth ability. In the first aim we will use microarrays and methods to purify and culture RGCs and amacrine cells to broadly identify genes that RGCs change through development from embryo to early adult, and identify the subset of genes that RGCs change in response to contact with amacrine cells. In the second aim we will use powerful transfection and RNAi techniques as well as a new high-throughput imaging/analysis technology to screen the candidate genes identified in the first aim, to investigate the molecular basis of RGCs' decreased axon and increased dendrite growth abilities. This will be the first use of micro arrays to globally assay a purified neuron's gene expression through development, but with the added significance of using the candidate genes to screen for specific axon and dendrite growth phenotypes. Our goal is to revert mature, postnatal RGCs to their embryonic axon growth ability, and thereby to develop new treatments to promote RGC regeneration after injury in ocular diseases including glaucoma, retinal ischemia, optic neuritis and optic neuropathies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose to investigate why mature, central nervous system (CMS) neurons fail to regenerate after injury, and how this failure depends on the developmental control of gene expression. Mature retinal ganglion cells (RGCs) fail to regenerate after their axons are severed, yet axons in the embryonic CMS can regenerate after injury. The loss of this embryonic regenerative ability correlates with the loss of RGCs1 intrinsic ability to rapidly extend axons. Interestingly, neonatal RGCs do not decrease their axon growth ability by an intrinsic aging mechanism, but rather they are signaled to do so by amacrine cells. In this proposal we will investigate the cellular and molecular mechanism for this loss of intrinsic axon growth ability. We demonstrate that the amacrine-signaled decrease in RGC axon growth ability correlates with an increased ability to elongate dendrites, and is dependent on new gene expression in RGCs. In the first aim we will use an in vivo model in which amacrine cells largely fail to develop to ask whether amacrine cells are required for the developmental loss of intrinsic axon growth ability by RGCs. In the second aim we will characterize the amacrine cell membrane-associated cue that is sufficient to signal embyronic RGCs to decrease their axon growth ability, and use microarrays to develop a list of candidates genes. In the third aim we will use powerful transfection and RNAi techniques to investigate the molecular basis of RGCs1 decreased axon and increased dendrite growth abilities. Our goal is to revert mature, postnatal RGCs to their embryonic axon growth ability, and to enhance RGC regeneration after optic nerve injury in vivo. Our ultimate goal is to develop new treatments to promote RGC regeneration after injury in ocular diseases including glaucoma, retinal ischemia, optic neuritis and optic neuropathies, and to extend our understanding to more broadly promote CMS regeneration, for example after spinal cord injury or in neurodegenerative disease.

DESCRIPTION (provided by applicant): Neurons in the central nervous system (CNS), which includes the eye, brain and spinal cord, fail to regenerate their axons after injury and in neurodegenerative diseases. The failure of retinal ganglion cells'(RGCs1) axons to traverse an injured optic nerve leads to permanent loss of vision. In the spinal cord, a similar failure leads to permanent loss of motor and sensory function. Thus the failure of CNS regeneration underlies a major clinical need. Over the prior 25 years, research has attributed this failure to the presence of negative, inhibitory, glial-associated substrates, as well as to a lack of sufficient positive, growth-promoting neurotrophic signals. Blocking inhibitory signals and providing neurotrophic factors can slightly enhance functional recovery, although typically only a few axons regenerate. It is known that trophic factors must be presented to the growth cone to stimulate axon elongation. Interestingly, direct mechanical tension will coax axonal growth cones to elongate independent of trophic factors, suggesting that trophic factors signal growth cones to create tension on the axon. After binding to their receptors, neurotrophic factors are rapidly endocytosed into 100-200 nm diameter "signaling endosomes" and retrogradely transported intact down the axon to the cell body. If signaling endosomes were instead artificially held at the growth cone, could they more effectively stimulate axon growth? These questions have been difficult to address because of endosomes'nanometer scale. >We have identified two approaches by which magnetic nanoparticles could be used to manipulate axons to overcome inhibitory substrates and to enhance the trophic signaling of axon growth. Here we propose to design and validate the use of magnetic nanoparticles to enhance regenerative axon growth by RGCs by (1) directly manipulating mechanical tension on the growth cone after surface attachment of magnetic nanoparticles, and (2) directly manipulating localization of neurotrophic signaling in signaling endosomes inside RGC axons after endocytosis of magnetic nanoparticles. In our first two aims we will test magnetic nanoparticle size arid coatings to optimize surface binding (Aim 1) and endocytosis into signaling endosomes (Aim 2). In our third aim we will use these optimized magnetic nanoparticles to stimulate axon regeneration in cultured RGCs by application of external magnetic fields. Our long term goal at a basic science level is to better understand the interplay between trophic signaling and mechanical tension in axon growth, and at a translational level to develop nanotechnologies into therapeutic approaches for treating CNS-related diseases.

DESCRIPTION (provided by applicant): There is no 3-dimensional model for studying neural tissues, outside of explanting whole tissues from animals. For example, in the retina, degenerations of retinal ganglion cells (RGCs) in diseases like glaucoma or of photoreceptors and retinal pigment epithelium in diseases like age-related macular degeneration have generated considerable interest understanding the integration of stem cells or stem cell-derived neurons into neural tissues. But study of cell replacement therapies for such diseases outside of the whole animal has focused on a limited group of experimental approaches, either examining the explanted whole retina in culture, or studying the individual cell types in 2-dimensional cultures. Missing is any opportunity to learn about cellular development or integration in the 3-dimensional (3D) environment these cells normally experience. In addition, there are no 3-dimensional organ replacement therapeutic approaches for neural tissue. Here we will reverse this paradigm and create a new model system that combines in vitro advantages of experimental control and screening capability with in vivo advantages of studying neurons in their 3D environment, building towards organ replacement therapies for the nervous system. We will re-create the retina from its basic cell types in culture using 3D biodegradable scaffolds to provide a critical new model system to study how the retina develops, functions, and operates in response to disease or injury, and to study how stem cell-derived neurons synaptically integrate with their neighbors. Specifically, we will create 3D tissue models of the neural retina with retinal ganglion cells and amacrine cells, characterize and optimize their synaptic connectivity, and examine the ability of retinal progenitor cells added to these 3D tissue models to proliferate, differentiate, and integrate synaptically. Our goal is to develop 3D neural tissues for studying neural development, and ultimately for tissue replacement therapies. PUBLIC HEALTH RELEVANCE: Here we will create for the first time a three-dimensional neural tissue, the retina, ex vivo. This will allow for a new way to study stem cell integration into neural tissues and, as importantly, will build towards tissue replacement therapies for the nervous system.

DESCRIPTION (provided by applicant): It is currently unknown why mature, central nervous system (CNS) neurons fail to regenerate after injury, and how this failure depends on developmental changes in gene expression. Mature RGCs fail to regenerate after their axons are severed, yet axons in the embryonic CNS can regenerate after injury. The loss of this embryonic regenerative ability correlates with the loss of RGCs' intrinsic ability to rapidly extend axons. Here we propose to investigate the regulation of RGC axon growth and regenerative capacity by the Kruppel-Like family of transcription factors. Specifically, we have identified a family of developmentally regulated transcription factors in the Kruppel-Like Factor (KLF) family that regulate axon growth of RGCs in vitro and in vivo. In the first aim we determine how KLF family members differ in their recruitment of co-activators and co-repressors, and how such co- factors regulate RGC axon growth with the KLFs. In the second aim we will characterize the gene targets of KLF family members in RGCs in vitro and in vivo. In the third aim we will ask whether manipulating expression of multiple KLFs in vitro and in vivo alters RGC axon growth during development or after optic nerve injury. Our goal is to revert mature, postnatal RGCs to their embryonic axon growth ability, and to enhance RGC regeneration after optic nerve injury in vivo. Our ultimate goal is to develop new treatments to promote RGC regeneration after injury in ocular diseases including glaucoma, retinal ischemia, optic neuritis and optic neuropathies, and to extend our understanding to more broadly promote CNS regeneration, for example after spinal cord injury or in neurodegenerative disease. PUBLIC HEALTH RELEVANCE: Retinal ganglion cells (RGCs) lose their ability to regenerate, but it is not known why. Developmentally regulated transcription factors in the Kruppel-Like Factor family may control RGCs' intrinsic capacity for rapid axon growth and regeneration, but the cellular and molecular details of this process remain to be discovered. Our hope in investigating this process is to understand why RGCs fail to regenerate after injury or in degenerative diseases such as glaucoma and ischemic optic neuropathy.

DESCRIPTION (provided by applicant): Non-arteritic anterior ischemic optic neuropathy (NAION) is the most common cause of optic nerve-related acute loss of vision in the US;there is no effective treatment. NAION causes injury to optic nerve axons, leading to dysfunction and death of retinal ganglion cells (RGCs). Interventions to enhance RGC regeneration could be applied before RGC death, to reverse dysfunction by allowing RGCs to reconnect with their targets in the brain. Enhancement of optic nerve regeneration is a major goal for patients with NAION and other neuropathies. The lack of regeneration-promoting therapies in NAION and other diseases reflects barriers to regeneration in the injured central nervous system (CNS), including growth-inhibitory proteins associated with myelin and the glial scar. Strategies to promote regeneration by overcoming these barriers have shown efficacy in animal models, but novel strategies and translation to the clinic are needed. We have performed a phenotypic screen using a library of novel drug-like triazine compounds on primary mammalian neurons, and have identified 4 compounds capable of increasing neurite growth on a substrate of inhibitory CNS myelin. These compounds a) act on different neuronal types, including RGCs, b) are potent, c) overcome inhibition in several assays relevant to CNS injury, and d) may act by novel mechanisms. We have now shown that one compound, AA4F05, promotes regeneration in an animal model of retinal injury, as well as in a model of spinal cord injury. AA4F05 and its relatives are exciting candidates to lead to novel drugs for promoting regeneration of RGCs and other CNS neurons. Although AA4F05 has favorable chemical properties and is active both in vitro and in vivo, there has been no attempt to optimize its activity or pharmacokinetics. The present proposal will use AA4F05 as a starting point for the development of new compounds with the potential to substantially improve regeneration of damaged axons from RGCs. Derivatives will be tested in primary neurons in vitro (primary and secondary screens), and the best candidates will be tested in 2 models of optic nerve injury. PUBLIC HEALTH RELEVANCE: Diseases of the optic nerve (optic neuropathies) are a leading cause of impaired vision in the US. This proposal is designed to develop and test novel therapeutic chemicals that can later be developed into drugs for treatment of optic neuropathies.

DESCRIPTION (provided by applicant): Why do neurons in the mature central nervous system (CNS) die after injury? White matter ischemia (stroke) leads to axon injury and then in most cases to death of CNS neurons. Thus, for example, ischemic optic neuropathy leads to retinal ganglion cell (RGC, a type of CNS neuron) dysfunction and death, and permanent loss of vision. Although many of the downstream molecular pathways of cell death and apoptosis are under intensive study, the upstream signals that regulate survival and axon regeneration after axon injury are not known. Recent evidence suggests that RGCs die after axon injury for two reasons: they are cut off from target-derived trophic signals, and they become less responsive to such signals. Trophic responsiveness, survival and regeneration can be enhanced by elevating cyclic AMP (cAMP), but the signal transduction pathways remain largely unstudied. Here we will use the rodent retina and optic nerve as a model system for CNS neurons and their axonal, white matter pathways, respectively, and test the hypothesis that compartmentalized signaling on a family of scaffold proteins called AKAPs regulate survival and regeneration signaling in a novel model of white matter stroke in the optic nerve. In three Specific Aims, we will (1) identify the specific adenylyl cyclases responsible for cAMP signaling in primary neurons~ (2) identify the specific AKAP-related signaling pathways potentiated by cAMP and contributing to neuronal survival and regeneration~ and (3) determine whether manipulating AKAP-mediated signalosomes in vivo regulates neuronal survival and regeneration in vivo after ischemic axon injury. We hope through these experiments to determine the molecular basis for the failure of RGC survival after ischemic axon injury, and ultimately to develop new treatments to maintain CNS neuronal survival after white matter ischemia.

DESCRIPTION (provided by applicant): Like all neurons of the central nervous system (CNS), retinal ganglion cells fail to regenerate, and often die, after damage to the optic nerve such as occurs in optic nerve stroke or other optic neuropathies. Loss of axonal connectivity and neuronal death that occurs after damage to the optic nerve results in vision loss. While therapeutics targeting secondary damage after neuronal insult have shown benefit in reducing functional deficits after neuronal damage, there are currently no approved agents capable of addressing axon regenerative failure, the primary cause of visual dysfunction after optic nerve damage. As such, novel approaches capable of improving axonal growth (neuroregeneration) have potential to restore visual capacity after damage to the optic nerve. LRP1 was recently identified as a novel receptor of myelin-associated inhibitors (MAIs), the components of degraded myelin responsible for the extrinsic component of regenerative failure. We have shown in vivo that infusion of the LRP1 antagonist RAP into the CNS after injury results in attenuation of RhoA activity, the critical signal involved in extrinsic causes of regenerative failure. Direct inhibition of RhoA enhances neuronal regeneration in rodent models and a pan-RhoA inhibitor has shown evidence of efficacy in humans in exploratory clinical trials. However, current therapeutic candidates have several critical limitations such as lack of neuronal specificity and poor bioavailability limiting drug delivery. In contrast, RAP is readily available o the CNS from the peripheral circulation. Because RAP is both readily soluble and can be delivered to the CNS via multiple doses, it possesses desirable therapeutic advantages over current pan-RhoA inhibitors. As beneficial results have already been observed using direct infusion to the injury site, we first wish to assess whether peripheral administration of RAP has comparable beneficial effects on the signaling events associated with regenerative failure after optic nerve insult. To accomplish this, an intravenous administration protocol capable of resulting in sufficient levels of RAP in the CNS must first be established. We will then perform long term studies (8-week injury course) to assess histological regeneration of damaged neurons with RAP treatment. As LRP1 has been shown to be a critical facilitator of myelin- mediated neuroregenerative failure, we hypothesize that therapeutic application of RAP will result in significant neuronal regeneration of retinal ganglion cells in the optic nerve. Additionally, the unique biological characteristics of RAP such as CNS bioavailability and specific RhoA inhibition in neurons could make it a superior therapeutic approach to the current pan-RhoA inhibitors. As such, RAP is an important candidate to bring through pre-clinical proof-of-concept testing as a high-value potential therapeutic for restoring axonal growth after damage to the optic nerve.

Abstract Although mature retinal ganglion cells (RGCs) are normally unable to regenerate axons following optic nerve damage, studies from several labs, including those participating in this collaboration, have identified cellular, molecular, and physiological manipulations that enable some RGCs to regenerate injured axons from the eye to the brain. In spite of these efforts, however, the number of RGCs that survive after optic nerve injury and successfully regenerate axons into the brain remains small, thereby limiting meaningful visual recovery. The proposed research will combine a strong pro-regenerative therapy with novel transcriptomic and proteomic approaches and cutting-edge bioinformatic methods to identify new transcripts and proteins associated with the initiation and execution of a successful regenerative program. We will investigate the temporal sequence of changes in gene expression, protein translation, and protein transport down the regenerating optic nerve as mature RGCs undergo a transition from a normal intact state into a robust growth state, identify transcripts and proteins selectively expressed in the subset of RGCs that successfully extends axons into the nerve, and characterize the RGC subtypes with the highest potential to regenerate axons. 100-150 of the top candidate genes identified in the discovery phase will be tested for their ability to promote axon outgrowth in immunopurified RGCs in culture, and lead candidates from the intermediate screen will be tested for their ability to substantially augment levels of optic nerve regeneration in vivo, either in isolation or in combination with established pro-regenerative therapies. These latter studies will investigate the targeting of axons to appropriate central visual nuclei and tests of visual recovery. The integrated approach proposed here directly addresses the goal of identifying novel molecular targets to re- establish visual circuitry after injury to the visual system.

? DESCRIPTION (provided by applicant): Ischemic optic neuropathy is a type of central nervous system (CNS) white matter stroke that causes permanent blindness due to retinal ganglion cell (RGC) axon damage and cell death. Although such white matter ischemia comprises an important source of morbidity in humans, the molecular mechanisms contributing to neuronal dysfunction and death remain poorly understood, and there exist few effective therapies to prevent or restore loss of vision. A major goal for this research is to define the signaling pathways defective in neurons following ischemic injury, so that novel therapeutic regimens that confer neuroprotection and neuroregeneration may be rationally designed. The myocyte enhancer factor 2 (MEF2) transcription factor family is an important mediator of pro-survival signaling in neurons that is activated by both retrograde neurotrophin stimulation and neuronal depolarization. Transcriptional activity of MEF2A and MEF2D, the primary MEF2 isoforms expressed in RGCs, is regulated by post-translational modification of distinct amino acid residues by extracellular signal-regulated kinase 5 (ERK5) catalyzed phosphorylation and calcineurin (CaN) phosphatase catalyzed dephosphorylation. MEF2A/D, ERK5, and CaN are all binding partners for the scaffold protein muscle A-kinase anchoring protein ? (mAKAP?/AKAP6), a scaffold protein expressed within the retina primarily in RGCs. In this application we will test the novel hypothesis that mAKAP? signalosomes serve as critical nodes in the neuronal signal transduction network by orchestrating MEF2 activation in neuroprotection and axon growth. In Aim 1 using primary cultures, we will determine the regulation and function of MEF2A/D post- translational modifications, including phosphorylation and sumoylation, by mAKAP? signalosomes in response to brain-derived neurotrophic factor (BDNF) and depolarizing stimuli. In Aim 2 using conditional MEF2 knock-out mice and a photochemically-induced ischemic optic neuropathy (PCI-ION) model, we will test whether MEF2A and MEF2D transcriptional activity are required for RGC survival in stroke and for the pro-survival and regenerative effects of neurotrophic factors such as BDNF after intravitreal injection. In Aim 3, we propose to enhance neuroprotection and regeneration following PCI-ION by increasing ERK5 signaling and MEF2 activity using intravitreal injection of adeno-associated virus (AAV) gene therapy vectors to express constitutively active MEK5 and MEF2 proteins in RGCs in vivo. Data from these experiments will advance our basic understanding of how MEF2 is regulated in neurons and how compartmentalized signaling regulates neuronal survival and regeneration, ultimately contributing to new therapeutic strategies for stroke and optic neuropathy.

PROJECT SUMMARY There is an important unmet need in glaucoma to develop next-generation functional imaging modalities. The advent of high resolution retinal imaging technologies, specifically, optical coherence tomography and adaptive optics scanning laser ophthalmoscopy, has enabled evaluation of retinal anatomical features at a cellular level in living human eyes. However, assessment of anatomy only provides limited information about the health and function of the retinal tissue. There remains a pressing need for molecular and metabolic endpoints in evaluating glaucoma, where structural changes may take many years to appear, often associated with significant vision loss. Currently available techniques for assessment of retinal blood flow, oxygen saturation, and mitochondrial function in humans have limited depth discrimination and require separate instruments, hampering accurate and comprehensive quantification of retinal ganglion cell physiology. Thus, we propose to develop and validate an innovative depth-resolved retinal metabolic imaging system, scanning protocols and analysis algorithms to non-invasively, quantitatively and simultaneously assess retinal oxygen and mitochondrial metabolism in glaucoma, and in response to candidate neuroprotective or regenerative therapies, both in animal models and in human subjects. We will perform pilot studies to demonstrate and validate the technology's capability to provide metabolic measures relevant to vision restorative therapies in animals and humans. The availability of novel retinal metabolic measures will for the first time introduce a physiologically-based outcome measure for disease characterization and also for candidate therapies relevant to regenerative ophthalmology and vision restoration. In addition, the proposed research will have a significant impact on advancing: 1) knowledge of retinal metabolic dysfunction in animal models of human glaucoma and other optic neuropathies, 2) translation of candidate regenerative therapies that improve retinal metabolic function in pre-clinical studies, and 3) clinical assessment and evaluation of available and emerging therapies for restoring vision in glaucoma, as well as in other degenerative retina/optic nerve diseases.

Stanford Vision Research Core: Overall Component PROJECT SUMMARY The Stanford vision research community is comprised of an impressive array of faculty bridging all levels of vision research, from molecular to cellular to circuits to systems, from development to adult to disease. The Stanford Vision Research Core grant will bring 4 modules to this community: (1) Advanced Computing/Computational Core, (2) Device Design and Development, (3) Neurogenetics of Vision, and (4) Imaging Structure and Function. These cores will be positioned to amplify the considerable resources Stanford University is devoting to the growth of vision research, including new faculty recruiting in the Department of Ophthalmology and the Stanford Neurosciences Institute and new space allocation to wet- and dry-lab vision research, as well as commitments from the Department of Ophthalmology for additional administrative capacities. Bringing these 4 cores to this community will help us achieve a number of specific outcomes. 1) We will extend the reach of vision research among the NEI-funded investigators at Stanford: by providing core resources and services to investigators, this grant will centralize specialty capacity, allowing faculty to benefit from the ready availability of such expertise. 2) We will accelerate discoveries in these laboratories: the availability of new resources that specifically target areas of need across the vision research community at Stanford will allow research to move more quickly into new, cutting edge areas of innovation. 3) We will promote inter-disciplinary collaboration that bridges molecular through systems level vision research: The selection of these 4 cores also carries a specific intention to bring vision research at Stanford into a ?next-generation? position bridging across disciplines. Offering these tools, with cell- and species-compatible vectors, device development, advanced imaging, and the computational power to extract relevant data from these, will facilitate this bridging. Finally, 4) We will attract new faculty at junior through senior levels into vision research: by providing tools specific to vision research, and making these tools available broadly to the Stanford research community, we will facilitate entry into vision research by both seasoned investigators in other fields, and newly recruited junior investigators poised to become the next generation of leaders in vision research.

ADMINISTRATIVE CORE SUMMARY The goal of the Administrative Core for the Stanford Vision Research Core grant is to provide overall coordination, scientific and budgetary oversight to the P30 projects. The Administrative Core will foster and coordinate activities between the four Service/Resource Cores: (1) Advanced Computing/Computational Core, (2) Device Design and Development, (3) Neurogenetics of Vision, and (4) Imaging Structure and Function. The Administrative Core Director will liaison directly with the 4 Core Directors, an internal Steering Committee, and an External Advisory Board to enact decisions made across these groups. The Administrative Core Director will ensure the functioning of the Administrative Core with the following functions: 1) encourage entry into vision research by investigators not yet funded by the NEI, through advertisement, collaborative meetings and quarterly presentations to the vision research community as part of a new vision research minisymposium; 2) promote new collaborative activity by ensuring dissemination of new approaches and technologies, transfer of reagents and exchange of ideas across the campus; 3) create and maintain scheduling for Core resources and an evaluative process for Core usage; 4) Establish and coordinate meetings of the Steering Committee to oversee Core usage and overall strategy, budgetary decision making, and personnel management, and establish and coordinate meetings of the External Advisory Board to provide feedback on scientific feedback and strategy to the P30 PI and Steering Committee; and 5) assure fiscal responsibility of the cores to maximize breadth and utility in serving Aims 1 and 2 above.

PROJECT SUMMARY Here we propose a Stanford University NEI T32 Vision Science Training Grant. Based on excellence in postdoctoral training among our vision science faculty, our goal in this application is to provide a vision- specific research training program with integrated clinical experience to the talented trainees at Stanford. Specifically, we seek training support for post-doctoral fellows. The 20 primary vision research faculty in the Stanford Vision Training Program includes 14 PhDs and 6 MD/PhDs of all academic ranks. The vision faculty has strengths in diverse areas, including molecular and cellular vision biology, vision encoding and circuitry, development and genetics, in vivo imaging, higher order visual behavior and perception, mechanisms of diseases, and different approaches to the treatment of diseases. There are multiple institutional grants and programs that support the faculty, along with many individual faculty grants. Together, the primary vision faculty is funded by 98 grants totaling over 20 million dollars, of which 28 grants are from the NEI, 50 grants are from the NIH (including NEI), and the rest are from the Department of Defense, National Science Foundation, and various foundations. Three faculty mentors are Howard Hughes Medical Institute investigators. Exciting developments for the Stanford Vision Training Program include the recruitment of a significant new cohort of vision research faculty to Stanford, including the new Chair of the Department of Ophthalmology, and unparalleled institutional resources committed by the department and by the Stanford University School of Medicine. Formal classroom, clinical and laboratory training under the auspices of carefully crafted training plans, new quarterly and annual vision research symposia, and oversight by both an Executive Committee and External Reviewer(s) will allow this Stanford Vision Training Program to produce future leaders in vision research who are able to tackle the most interesting and important questions and open new horizons at Stanford and beyond.

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.

This project will use a combination of structural and functional measurements to test the hypothesis that early- stage damage in human glaucoma occurs first in the inner plexiform layer (IPL) of the retina ? especially its OFF sub-lamina ? as suggested by murine glaucoma models. In the first Aim, we will use a novel visible-light optical coherence tomograph (VIS OCT) to study structural changes in the retina of glaucoma patients. The newly developed VIS OCT has sufficient image contrast and resolution to segment the IPL boundaries and to define sub-lamination in volumetric OCT data, something not currently possible with existing near-infrared OCT instruments. We will make comparative measurements within the IPL and between the IPL, the ganglion cell layer (GCL) and the retinal nerve fiber layer (RNFL). Because data from mouse models of glaucoma suggests that early damage occurs preferentially within the OFF sub-lamina of the IPL, we will make separate VIS OCT measurements biased for the OFF- and ON-sublaminae of the IPL and use machine learning approaches to determine whether a similar damage process can be demonstrated in human. To test whether OFF-pathway function is preferentially lost in glaucoma, we will use a novel Steady-State Visual Evoked Potential (SSVEP) paradigm that employs sawtooth increments and decrements to bias the measurement to ON vs OFF pathways, respectively, a paradigm our data suggests discriminates glaucoma from control patients. The second Aim will optimize this SSVEP measurement for testing localized areas of the visual field. The third Aim will make comparative measurements of visual-field, VIS OCT and SSVEP loss patterns in a large sample of glaucoma patients and in age- and sex-matched controls. Thickness and interface reflectivity amplitude maps derived from VIS OCT imaging of the RNFL, GCL and IPL including sublaminae will be correlated topographically with visual field defects to assess the relative sensitivity of our structural biomarkers at and near visual field locations with demonstrable losses on conventional (Humphrey) perimetry. Similarly, SSVEP responses from different locations in the visual field will be correlated topographically with visual field loss patterns and to VIS OCT losses, with special emphasis on correlating structural damage in OFF vs ON sub-laminae of the IPL with the functional correlates derived from regional decremental and incremental SSVEPs. Separately and in combination, our structural and functional measurements are designed to provide strong tests of the biological hypothesis that the OFF pathway is preferentially damaged in human glaucoma, and to reveal new biomarkers for the disease.

Loss of retinal ganglion cells (RGCs) in glaucoma and traumatic and other optic neuropathies results in permanent partial or complete blindness. Molecular mechanisms that may oppose this RGC death remain an area of active investigation and potential high impact, as bridging RGC survival in chronic optic neuropathies has high potential to preserve or restore vision. Multiple signal transduction pathways have been implicated in RGC neuroprotection, including cAMP and neurotrophic factor-induced mitogen-activated protein kinase (MAPK) signaling pathways. How these pathways synergistically promote RGC survival and elicit their downstream effects remains unknown. Recent data from our labs support a model in which signalosomes organized by the perinuclear scaffold protein muscle A-Kinase Anchoring Protein ? (mAKAP?/AKAP6?) mediate cAMP-dependent signaling and potentiate neuroprotective MAPK signaling, resulting in Ets Like-1 protein (Elk-1) transcription factor activation and RGC survival. The identification of this intracellular cAMP signaling compartment specifically relevant to neuroprotection provides a mechanism for spatially distinct cAMP action and should inform the design of strategies providing therapeutic specificity greater than global cAMP elevation with adenylyl cyclase activators or cAMP analogs. In this application, we propose three Specific Aims to test this model and to elucidate the mechanism conferring the synergy between cAMP and neurotrophic factor signaling in neuroprotection. Specific Aim 1: Defining Neuroprotective Gene Expression. Using single- cell RNA transcriptome sequencing (scRNA-seq), we will study to what degree similar gene transcription programs are induced by different neuroprotective interventions, including generalized versus compartmentalized cAMP elevation, determine whether individual RGC subtypes are preferentially regulated by cAMP and neurotrophic factor signaling, and identify gene candidates whose altered expression may be critical for neuroprotection in response to therapeutic intervention. Specific Aim 2: Role of Perinuclear Compartmented cAMP Signaling in RGC Neuroprotection. Using new tools to promote or inhibit cAMP and Ca2+ in special intracellular compartments, we will test whether Ca2+-cAMP signaling at RGC mAKAP? signalosomes is uniquely sufficient and/or necessary for RGC neuroprotection after optic nerve crush. Specific Aim 3: Crosstalk Between cAMP- and Neurotrophic Factor-Dependent RGC Neuroprotection. To test whether cAMP and neurotrophic factors promote neuroprotection through co-regulation of ERK1/2-dependent Elk-1 activation, mice with gain- and loss-of-function for Elk-1 in RGCs will be subjected to optic nerve crush and compared for their response to additional treatment with exogenous neurotrophic factors and AAV-mediated mAKAP? signaling compartment enhancement. Together, these Specific Aims will provide molecular insights into the signaling pathways and the altered gene expression that can confer RGC neuroprotection in vivo, while providing proof-of-concept for new strategies to prevent loss of vision in RGC disease.