Yang Hu - US grants

? DESCRIPTION (provided by applicant): Injuries of mature central nervous system (CNS) axons result in loss of vital functions due to the failure of CNS axons regeneration. Neutralizing extracellular inhibitory molecules yields only limited regeneration or functional recovery in vivo, suggesting a critical role for neuron-intrinsic factors. As it has become apparent that the PTEN/mTOR pathway is critical for CNS axon regeneration, understanding the regrowth control of this pathway represents the first step toward developing novel therapeutic approaches to neural injury. Unfortunately, mTOR over-activity can result in tumor formation, metabolic diseases, and neurological disorders. It is therefore critically important to identify the specific downstream effectors by which PTEN/mTOR promotes axon regeneration, and to isolate them from other targets that mediate mTOR's deleterious effects. Using the anatomical and technical advantages of retinal ganglion cell and optic nerve as a CNS injury model, we have identified crucial regulators of axon growth, and are now ideally positioned to elucidate the downstream mechanisms by which PTEN/mTOR stimulates regeneration in mature CNS axons and identify translational targets of PTEN/mTOR govern adult CNS axon regeneration. These effectors are ideal therapeutic targets to promote regeneration in CNS injury and diseases, which can be selectively activated without activating other, potentially harmful pathways, thus to assist in safely translating our findings into novel neural repair treatments to preserve vital functions in patients with CNS injuries.

PROJECT SUMMARY Optic neuritis is one of the most common clinical manifestations of Multiple Sclerosis (MS). It causes severe visual loss due to inflammatory demyelination of the optic nerve (ON) and subsequent degeneration of ON and retinal ganglion cells (RGCs). The significant unmet clinical need for neuroprotectants is due to the lack of understanding of the key upstream signals that trigger the neurodegenerative cascade. Our previous studies demonstrated that both acute and chronic ON injury induce endoplasmic reticulum (ER) stress in RGCs. We were able to protect the injured RGC soma and axons if we blocked the detrimental effects of ER stress by manipulating two key downstream molecules of the unfolded protein response (UPR) in opposite ways: a) deletion of CCAAT/enhancer binding protein homologous protein (CHOP), and/or b) activation of X-box binding protein 1 (XBP-1). Thus axon injury-induced ER stress may be a common mechanism of neuronal damage and targeting neuronal ER stress may have considerable therapeutic neuroprotective potential in diseases associated with axonopathy. The rodent experimental autoimmune encephalomyelitis (EAE) model induced by immunization with myelin proteins replicates many clinical symptoms and pathological signs of MS, including optic neuritis and significant RGC soma and axon loss. ER stress has been detected in white and grey matter of MS patients' brains and in EAE mice. We confirmed the role of neuronal ER stress in autoimmune-induced neurodegeneration in EAE. Furthermore, exciting recent studies of axonal Wallerian degeneration have shown that several key molecules involved in axonal NAD+ metabolism are critical for axonal degeneration. SARM1 (Sterile Alpha and TIR Motif 1), for example, is negatively regulated by axonal NAD+ synthetic enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) to induce axon degeneration; deletion of SARM1 or activation of axonal NMNATs results in axon protection. Here we propose to test the hypothesis that modulating both intrinsic neuronal ER stress and NAD+ metabolism will synergistically prevent both RGC soma and axon (ON) degeneration and preserve vision in EAE/optic neuritis. We anticipate that this study will unambiguously identify novel therapeutic targets and that our findings will ultimately be translated safely into innovative neuroprotective treatments for patients with MS and optic neuritis.

PROJECT SUMMARY Glaucoma is the most common cause of irreversible blindness due to the death of retinal ganglion cells (RGCs) and degeneration of optic nerve (ON). Neuroprotectants that promote RGC survival, stem cell-derived RGCs to replace lost RGCs after transplantation and regeneration therapies to stimulate RGC soma and axon regeneration are promising neural repair strategies for vision restoration in glaucoma patients. A relevant translation-enabling animal glaucoma model is critical, but the current available ocular hypertension models, such as laser photocoagulation caused trabecular meshwork (TM) impairment and microbead occultation of TM, are technically challenging and often involved multiple treatments with unstable IOP elevation and irreversible ocular tissue damage. It is crucially important and in urgent need to develop a simple, reliable, and more importantly, reversible experimental ocular hypertension/glaucoma model in the animal species that closely resembles human glaucoma, such as nonhuman primate (NHP). To take on this challenge, we recently developed and characterized a simple procedure of intracameral injection of silicone oil (SO) in mouse eyes that blocks the pupil, causes accumulation of aqueous humor in the posterior chamber and induces ocular hypertension, which can be removed to lower IOP to normal and faithfully replicates post-operative secondary glaucoma in human patients. NHP's visual system closely resembles human anatomy, especially has macula and lamina cribrosa that do not present in mouse. NHP glaucoma model is most likely to predict human responses to ocular hypertension and therapies. We propose to extend our successful mouse SO-induced reversible ocular hypertension model into a novel NHP glaucoma model, success of which will enable us for the first time to test neuroprotective and regenerative strategies together with IOP lowering situation that faithfully mimic clinical scenarios in the closest animal species to human.

PROJECT SUMMARY Glaucoma is the most common cause of irreversible blindness and will affect more than 100 million people between 40 to 80 years old by 2040. It causes severe visual loss due to degeneration of optic nerve (ON) and retinal ganglion cells (RGCs). There is a significant unmet clinical need for neuroprotectants. Our previous studies of ON traumatic injury and glaucoma demonstrated that both acute and chronic ON injury induce endoplasmic reticulum (ER) stress in RGCs. We were able to protect the injured RGC soma and axons if we blocked the detrimental effects of ER stress by manipulating two key downstream molecules of the unfolded protein response (UPR) in opposite ways: a) deletion of CCAAT/enhancer binding protein homologous protein (CHOP), and/or b) activation of X-box binding protein 1 (XBP-1). Thus axon injury-induced ER stress may be a common mechanism of neuronal damage and targeting neuronal ER stress may have considerable therapeutic neuroprotective potential in diseases associated with axonopathy. As the first step, we propose to identify novel ER stress modulators by screening chemical libraries with cell-based high throughput screen (HTS) assays; and then to validate whether these agents promote RGC and ON survival and preserve visual function in mouse glaucoma models. Recently, exciting recent studies of axonal Wallerian degeneration have shown that several key molecules involved in axonal NAD+ metabolism are critical for axonal degeneration. SARM1 (Sterile Alpha and TIR Motif 1), for example, is negatively regulated by axonal NAD+ synthetic enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) to induce axon degeneration; deletion of SARM1 or activation of axonal NMNATs results in axon protection. Thus, we will test the hypothesis that modulating both intrinsic neuronal ER stress and NAD+ metabolism will synergistically prevent both RGC soma and axon (ON) degeneration and preserve vision in glaucoma. This study may generate novel combinatory therapeutic strategies that lead to more efficient neuroprotection in patients. And finally, we will develop novel in vivo imaging tools for RGC morphology and function studies and acquire much needed insights into the mechanism of RGC ER stress initiation. We expect the results through these studies will provide essential information for clinical application of ER stress modulation, and establish translatable techniques and biomarkers that will greatly facilitate clinical management of glaucoma patients.

NEUROGENETICS OF VISION PROJECT SUMMARY Genetics and in particular, genetic-based approaches such as transgenes and viruses that enable researchers to express proteins of interest in desired sets of neurons in the retina and brain, are pivotal to make significant progress in modern basic visual neuroscience and toward the treatment of visual disorders. The last decade has brought forth a wide and powerful arsenal of genetic tools for identifying the neurons that comprise the visual system of flies and rodents. Because they enable delivery of a wide range of gene cargo, such tools also allow for selective manipulation of cells of interest. Indeed, in comparison to just a decade ago, nowadays it is straightforward to label a given cell type in vivo, and thereby visualize its unique morphology, and then compare it to other cells of different types, selectivity target them for electrophysiology, calcium- or voltage-dye imaging, and then reversibly silence or activate them. Last but not least, genetic tools can be leveraged to explore the signature pattern of RNA expression present in different cell types in order to probe their homology across species and/or relevance to mutations associated with human diseases.