RECENT RESEARCH GRANTS
September 2006
Project Dates: January 1, 2007 - December 31, 2007
In vivo investigation of optic nerve formation and connectivity within the mouse brain
Nadean L. Brown, PhD
Children’s Hospital Research Foundation, Cincinnati, OH
The goal of this project is to understand how retinal neurons grow out of the mammalian eye, assemble into the functional nerves and establish the correct connections with the brain. Each of these steps is essential for the images an eye sees to be properly interpreted by the brain. To accomplish this we created a transgenic mouse model in which the developing optic nerve is labeled in living mouse embryos. We propose to place the growing retina from these embryos in culture, by itself, or with the appropriate brain tissues and study optic nerve formation. Using this system we will test the ability of the factor oncomodulin, which stimulates adult optic nerve regeneration, to direct embryonic optic nerve formation. We will also test the ability of oncomodulin to restore mutant optic neuron outgrowth in the brain. These studies will provide crucial information about the requirements for initially creating the optic nerve versus regenerating it. Such increased understanding will contribute to the development of more effective therapies to prevent or halt vision loss in patients with glaucoma.
Developmental determinants of retinal ganglion cell regenerative ability- year 2
Jeffrey, L. Goldberg, MD, PhD
Bascom Palmer Eye Institute,
In glaucoma, axons of mature retinal ganglion cells (RGCs) do not regenerate into the optic nerve. The vast majority of regeneration research has focused on identifying extrinsic glial-associated inhibitors of regeneration. This has been fruitful, yet overcoming the inhibitory environment leads to only a small fraction of regenerative response. On the other hand, RGCs have lost their intrinsic ability to rapidly regenerate their axons during development, but the molecular mechanism is entirely unknown. This affords us an opportunity to approach this problem from a wholly new perspective, to screen developmentally-regulated RGC genes for an ability to improve axon growth, using retinal ganglion cells in culture and, later, in vivo. This approach has the opportunity to open a conceptual breakthrough into the failure of RGC regeneration, to lead to entirely new molecular insights and thus to new strategies to “revert” mature RGCs to their greater embryonic axon growth ability. Thus we hope to bring novel approaches to the study and ultimately the treatment of glaucoma.
Three Dimensional Reconstruction of the Lamina Cribrosa using Second Harmonic Imaging Microscopy
Don S. Minckler, MD, MS
University of California, Irvine
Glaucoma causes progressive loss of vision accompanied by changes in the structure of the optic nerve head typically as increased cupping. Advancing age and increasing eye pressure are risk factors for progression of glaucoma. Experimental and pathology studies have demonstrated that the initial injury in glaucoma is precisely in the lamina cribrosa or scleral portion of the optic nerve head where nerve cells from the retina form the connection to the orbital nerve and ascend toward the rain. The lamina cribrosa can be likened to a porous Swiss cheese-like material that hardens with age that is stretched and bent by elevated eye pressure. Though numerous studies have examined the lamina cribrosa, detailed knowledge as to the effects of pressure on its organization and structure are very limited. We propose to use a new technology to visualize the three dimensional structure of the lamina cribrosa at very high resolution using non-invasive second harmonic imaging microscopy (SHIM). This technique allows for direct measurement of the structural changes in the lamina caused by pressure that avoid many of the problems and artifacts of past methods. These data should provide critically important insights as to how pressure causes vision damage.
The role of glial NF-kappaB in retinal ganglion cell loss in glaucoma
Valery Shestapolov, PhD
Bascom Palmer Eye Institute,
This project aims to investigate the effect of the cellular environment, specifically the neural glia, on the survival of retinal ganglion cells (RGCs), neurons that are critical for communicating visual information to the brain. The death of these neurons, which communicate visual information to the brain, causes blindness in major ocular disorders like glaucoma. The results of the preliminary experiments allowed me to generate the hypothesis that the nuclear fator-kappaB (NF-kB) played the key role in converting the normally supportive neuronal environment into a noxious, reactive one. We will use our expertise and a unique research tool, a transgenic mouse strain possessing genetically inactivated NF-kB complex, to test this hypothesis. This inactivation occurs specifically in astrocytes thus allowing a direct evaluation of impact of activated glia on RGC death in a mouse model of glaucoma. This mouse strain will allow us to address the main objectives of this proposal and examine whether the genetic inactivation of this complex will protect these neurons. By comparing neuronal death rates in normal and transgenic mice, we will determine the effect of NF-kB activation directly in live animal retinas. This knowledge may provide novel targets for both prevention and molecular therapy of glaucoma.
March 2006
Project Dates: July 1, 2006 – June 30, 2007
Functional Magnetic Resonance Imaging (fMRI) of Function-Specific Vision Loss in
Glaucoma is a family of disorders known to affect the vision of almost 3 million Americans. If left untreated, glaucoma eventually results in the death of cells in the eye that relay visual information to the brain. Animal studies have shown that the loss of these cells, in turn, has detrimental consequences for cells in the brain. There are three primary pathways that relay different aspects of the visual scene from the eye to the brain: the magnocellular, the parvocellular, and the koniocellular pathway. The aim of this proposal is to determine if any of the three primary visual pathways is affected differentially by glaucoma. Functional magnetic resonance imaging (fMRI) will be used to compare cortical responses to visual stimuli that differentially stimulate one of the three primary visual pathways. These experiments should demonstrate which, if any, of these functionally distinct neural pathways is most affected by human glaucoma. Understanding how the visual pathway from the optic nerve to the brain is affected by glaucoma will provide insights into the pathology of the disease, which may guide future research for neuroprotective, genetic, and molecular therapies.
CD44-osteopontin interaction in axonal outgrowth of retinal ganglion neurons
Barbara Grimpe, PhD
The Miami Project to Cure Paralysis, Miami, FL
Progressive irreversibly blinding diseases, collectively called glaucoma, that in most cases produce increased pressure within in the eye, cause damage to the optic nerve. To design strategies to rescue the injured nerve, it is necessary to understand the underlying processes in nerve growth. Therefore, it is essential to identify proteins that are involved in axonal outgrowth of retinal ganglion cells (RGCs) during development as well as in the mature central nervous system (CNS). To identify these proteins, my laboratory uses a completely new approach that involves the design of a computer program suite collecting protein names from literature relevant to “nerve regeneration”. We were able to identify two proteins that interact with each other that have never been investigated regarding their importance in axonal outgrowth of RGCs. Using a method that can eliminate a specific protein, we were able to demonstrate the role of these two proteins in neurite growth of embryonic RGCs. Further experiments will investigate the expression pattern of these two proteins in embryonic as well as mature brains. We will also use genetically modified mice, so-called knock out mice, to perform additional studies on the biological function of these proteins in the visual system.
Intranasal Application of Neuroprotective Agents in Rats with Glaucoma
Linda K. McLoon, PhD
Glaucoma is a major eye disease whose cause is still unknown. There is evidence to suggest that disruption of the blood flow to the retina and optic nerve in patients with glaucoma may in party explain the loss of the cells of the retina in these patients. We have characterized a model of retinal and optic nerve injury that is caused by hypoxia to these tissues, which is loss of oxygen due to temporary disruption of the blood supply. We will test a novel method of drug administration, intranasal application, to determine whether this method of treatment can rescue the retinal cells and optic nerve axons that had been exposed to a short-lived disruption of blood flow resulting in ischemia. We will examine the potential efficacy of insulin growth factor-1, a hormone with known neuroprotective effects in stroke, retinal and spinal cord injury, but whose systemic side effects from high doses are not acceptable for patient use. In addition, we will test erythropoietin, another neuroprotective candidate molecule. Intranasal drug application results in higher effective doses to the tissues of the nervous system than systemic applications. Previous studies have shown intranasal application of drugs can be effective in protection of neurons of the brain and spinal cord after injury.
New mechanism of MMP-9 regulation and its role in glaucoma
Andrei Surguchov, PhD
Kansas University Medical Center, Kansas City, MO
Metalloproteinase-9 (MMP-9) is an enzyme that is implicated in retinal damage and alterations in the optic nerve in glaucoma. Despite the important function of MMP-9 in glaucoma, its role in pathology is not completely understood. Recent data suggest that defects in MMP-9 production leading to its excessive accumulation may be a key step in glaucoma and probably other eye diseases. In our previous studies we have found new potent activators of MMP-9 production that may play a significant role in ocular diseases. This upregulation is induced by synucleins- proteins that are expressed in the retina and optic nerve. Synculeins are implicated in neurodegenerative diseases, including glaucoma, but their exact role in these illnesses is not known. In this application we propose to investigate the mechanism of increase MMP-9 production caused by synucleins and elucidate the implication of this mechanism in eye pathologies. Studies proposed in this application will explore entirely new mechanism of MMP-9 regulation. Since defects in MMP-9 control play an important role in glaucoma and some retinal diseases, the results received will provide important insight into understanding the mechanisms and developing treatments for these ocular pathologies.
Chemical Genetic Screen for Compounds that Enhance Secretion of Mutant Myocilin
Douglas Vollrath, MD, PhD
Stanford University School of Medicine, Stanford, CA
We are studying an inherited form of glaucoma that affects thousands of Americans. The advantage of studying this form of glaucoma is that, unlike more common forms of glaucoma, the mutant gene that causes this genetic disorder is known. We are trying to understand how the mutant protein encoded by the gene causes the disease. We hope that by solving this tractable problem, we will gain insight into some of the causes of more common forms of glaucoma. Our Current results show that, unlike the normal version of the protein, the mutant protein has an abnormal shape and is not properly released from cells. We have also found that when cells derived from the front of the eye make the mutant protein, they become sick and die. These particular cells are known to be important in draining fluid from the eye, so their loss could well explain how the mutant gene/protein causes glaucoma. Interestingly, we found that when these cells are grown at temperatures a little below body temperature, the mutant protein is released from the cells and the cells no longer die. We propose to find drugs that stimulate release of the mutant protein from cells at body temperature. We hope that identification of such compounds will lead to development of new forms of therapy for this type of glaucoma and encourage similar investigations into other forms of glaucoma.
September 2005
Project Dates: January 1, 2006 – December 31, 2006
Aquaporins 4 & 9 expression in glaucoma progression
Adnan Dibas, PhD
University of North Texas Health Science Center, Fort Worth, TX
Very little is known about how and why the optic nerve is progressively damaged in glaucoma, a leading disease for blindness worldwide. Conditions known to cause glaucoma such as elevated intraocular pressure, hypoxia, and ischemia were found to be associated with changes in the expression of aquaporin water channels in non-ocular tissues (e.g. brain, neurons, & muscle). Therefore, it is of great importance to follow the expression of levels of aquaporins in the retina and optic nerve head using a rat model of glaucoma with elevated pressure and chemically induced model of optic nerve degeneration by the injection of endothelin-1. Currently glaucoma medication involves only pressure lowering medication, however, vision loss continues. Therefore, the identification of additional mechanisms that continue to promote vision loss will assist in the development of combination therapy of lowering pressure and preventing vision loss in glaucoma.
Study of Optineurin Mutations on its Binding Activity to Huntingtin, mGluR1α, RNF11 and MYO6 Proteins
Tayebeh Rezaie, PhD
University of Connecticut Health Center, Famington, CT
Glaucoma characterized by a specific pattern of retinal ganglion cell death with painless failure of peripheral vision. The disorder affects more than 70 million people worldwide with profound visual impairment and blindness. Adult-onset Primary Open-Angle Glaucoma (POAG) is the most common form of this condition. We identified mutations in a new gene that we named Optineurin (OPTN) in a group of our familial cases. It is predicted that insufficiency or absence of the normal function of the optineurin protein results in the retinal ganglion cell death as commonly seen in patients with glaucoma. The proposed study aims to investigate the potential downstream consequences of OPTN mutations on the binding ability of this protein to foud of its well-known protein partners. This proposal anticipates establishing further knowledge on biological and biochemical pathways that play a major role in the pathophysiology of the protein encoded by this glaucoma gene. This study will help to raise our basic understanding of how the predicted protection of the normal optineurin protein is impaired in glaucoma patients with defective mutations in this gene.
SiRNA Strategy to Ameliorate Transforming Growth Factor-β-Induced Adverse Effects
Beatrice Yue, PhD
Lions Of Illinois Eye Research Institute,
Researchers have identified a gene, transforming growth factor-β (TGFβ), as an important factor involved in the pathogenesis of glaucoma, a major blinding disease. Elevated amounts of TGFβ are found in the aqueous humor of patients with primary open angle glaucoma, the most common form of glaucoma. TGFβ has also been shown to elevate intraocular pressure (IOP) and promote extracellular matrix accumulation in the trabecular meshwork in both cell culture and perfusion organ cultures. In the current application, we will determine whether blocking TGFβ activity would halt of minimize its adverse consequences. Specifically, we plan to employ a new technology called RNA interference (RNAi) to “silence” or knock down expression of the receptor for TGFβ to specifically block the TGFβ action. We have already used this strategy to successfully suppress ocular inflammation and fibrosis in a mouse model and believe that there is a high probability of positive outcome for the proposed project. Information obtained will lead to development of novel therapeutic modalities for glaucoma.
Ganglion Cell Regeneration – Year 2
Deborah L. Stenkamp, PhD
The ganglion cells of the vertebrate retina are responsible for relaying visual information to the brain. Damage to these cells and to their axons, the optic nerve, results in irreversible visual loss in most vertebrates. Glaucoma, a disease that results in damage to the optic nerve ad the progressive death of ganglion cells, is a major cause of blindness in humans, because the ganglion cells cannot be revived or restored. In fish, however, retinal damage typically results in regeneration of retinal neurons, including retinal ganglion cells, and the outgrowth of their axons toward the brain. Our goal is to determine whether regenerated ganglion cells develop in a manner consistent with the restoration of visual function. In the proposed renewal funding period, we will pursue this goal by determing whether a template of healthy ganglion cells facilitates regeneration, and by determining the extent to which regeneration is possible in a zebrafish model for glaucoma. This study may serve as a basis for designing ganglion cell regeneration strategies that result in optimal reestablishment of visual function in humans with vision loss due to glaucoma.
March 2005
Project Dates: July 1, 2005 – June 30, 2006
Developmental Determinants of Retinal Ganglion Cell Regenerative Ability
Jeffrey L. Goldberg, MD, PhD
Bascom Palmer Eye Institute,
In glaucoma, axons of mature retinal ganglion cells (RGCs) do not regenerate into the optic nerve. The vast majority of regeneration research has focused on identifying extrinsic glial-associated inhibitors of regeneration. This has been fruitful, yet overcoming the inhibitory environment leads to only a small fraction of regenerative response. On the other hand, RGCs have lost their intrinsic ability to rapidly regenerate their axons during development, but the molecular mechanism is entirely unknown. This affords us an opportunity to approach this problem from a wholly new perspective, to screen developmentally-regulated RGC genes for ability to improve axon growth. High-throughput technology, using a >$300,000 automated imaging/analysis workstation to collect and quantify axon growth is a critical feature of these experiments, allowing the broad screening of hundreds of candidate genes in the time it would normall take to screen only a few selected candidates. This unbiased, high-throughput screening approach has the opportunity to open a conceptual breakthrough into the failure of RGC regeneration, to lead to entirely new molecular insights and thus to new strategies to “revert” mature RGCs to their greater embryonic axon growth ability. Thus we hope to bring novel approaches to the study and ultimately the treatment of glaucoma.
Quantitative Analysis of Glaucoma Progression in DBA/2J Mice
Robert W. Nickells, PhD
University of Wisconsin, Madison, WI
The discovery of a line of mice that develops chronic glaucoma has generated a great deal of enthusiasm to use this model to study the molecular pathology of this disease, and to test drugs that may have a therapeutic benefit. Preliminary studies on the natural history of the disease in these mice, however, have suggested that the disease is variable, much in the same way it is in humans. In order to establish this model for meaningful future studies, a detailed examination of the rates of ganglion cell loss and optic nerve degeneration are reguired. This proposal is aimed at studying and quantifying the loss of ganglion cells in mice that acquire glaucoma as they age. The study makes use of novel molecular marker that allows us to stain living ganglion cells blue, so that we can monitor the rate and pattern of cell loss in isolated retinas of mice at different ages.