FUNDING PERIOD: APRIL 1, 2008 - MARCH 31, 2010

Ron Ofri, Ph.D.
Hebrew University of Jerusalem
Jerusalem, Israel
Expression of ion channels during ganglion cell apoptosis. Implications for the pathogenesis of glaucomatous optic neuropathy & relevance for neuroprotective treatment
$100,000

Loss of vision in glaucoma patients is caused by death of ganglion cells in the retina. Therefore, preventing the death of these cells may preserve vision in glaucoma patients. Obviously, development of a drug that will prevent cell death depends on understanding the underlying mechanisms of the cell death process. We believe that changes in electrical currents may be the 'trigger' which leads to cell death in glaucoma, and propose that if these changes can be prevented, cell death and loss of vision can be prevented. Lamotrigine is such an inhibitor, which is safely used to treat epilepsy patients. Therefore, we propose to test lamotrigine in a rat glaucoma model. We will test the drug's effect on the function and structure of the retina, and on molecular and genetic events in the ganglion cells, of these rats. Positive results may lead to the testing of the drug in human glaucoma patients.

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Beatrice Yue, Ph.D.
University of Illinois at Chicago
Chicago, IL
Functional Roles of Optineurin in RGC-5 cells
$100,000

Glaucoma is a major blinding disease.  In this application, we propose two specific aims to study the roles of optineurin, a glaucoma gene, in two cellular functions in neuronal cells.  One of the functions is related to how proteins are transported inside the cells and the other governs whether and how neurites extend or grow in the cells.  Our goal is to provide new information regarding the involvement or roles of optineurin in these important functions to help elucidate why and how abnormal levels or mutations of optineurin may lead to glaucoma.   The study may enhance our understanding of mechanisms leading to neuronal cell loss in glaucoma and facilitate development of new treatment modalities for patients with glaucoma.

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Adriana Di Polo, Ph.D.
University of Montreal
Montreal, Canada
Novel drug-based neuroprotective therapies for glaucoma
$100,000

At present, there is no cure for glaucoma and the only treatment available is to lower intraocular pressure using drugs or surgery. A significant proportion of patients continue to have disease progression and vision loss despite successful reduction of eye pressure. Thus, current therapeutic strategies for glaucoma are insufficient and new approaches to slow disease progression are urgently needed. The overall aim of his proposal is to characterize the clinical potential of galantamine, a member of the acetylcholinesterase family, for the treatment of glaucoma. Galantamine as neuroprotective therapy in glaucoma has several competitive advantages including: efficacy to delay retinal ganglion cell degeneration in experimental glaucoma, clinical history for Alzheimer's disease that could lead to rapid clinical start-up, good safety profile, and excellent drug pharmacokinetics. In specific aim 1, we will determine whether galantamine can protect vision after sustained periods of ocular hypertension damage. In specific aim 2, we will elucidate the mechanisms by which galantamine confers RGC neuroprotection in glaucoma. The outcome of this research may lead to more effective drug-based therapies for the treatment of glaucoma. Understanding the molecular pathways underlying galantamine-induced neuroprotection may provide key insight for the design of small molecule neuroprotective compounds with high specificity and few side effects.

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Abigail S. Hackam, Ph.D.
Bascom Palmer Eye Institute
Miami, FL
Neuroprotection of retinal ganglion cells by Muller glia: Investigation of the role of the Wnt signaling pathway.
$100,000

The retina is a thin tissue at the back of the eye that is essential for our view of the world. In glaucoma, a certain type of cell in the retina called the retinal ganglion cell begins to lose its ability to function and eventually dies, leading to visual difficulties and eventual loss of sight. Our research addresses several important questions: What causes retinal ganglion cells to sicken and die in glaucoma? Which genes and proteins are implicated in retinal ganglion cell demise? Can we develop new methods to save retinal ganglion cells and preserve vision?

We recently discovered that molecules called 'Wnts' promote the survival of various types of cells, suggesting that they may be used to protect retinal ganglion cells. We believe that protective Wnts are normally increased during glaucoma but are not at high enough levels to save retinal ganglion cells. To test this idea, we will determine whether delivering extra Wnt molecules protects retinal ganglion cells grown in a dish and we will identify the genes and proteins involved. We will also test whether another cell type in the retina called Muller glia, which are known to secrete high levels of Wnt molecules, can help save retinal ganglion cells from dying. Finally, we will look at donated enucleated glaucomatous eyes to determine whether Wnts are elevated during glaucoma.
           
These experiments will determine whether Wnts can protect retinal ganglion cells from dying, will identify how Wnts promote survival and will elucidate the role of Muller glia in glaucoma. The results of this study will provide new insights into glaucoma and may reveal novel directions towards developing preventive therapies.

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XiangRun Huang, Ph.D.
Bascom Palmer Eye Institute
Miami, FL
Glaucomatous Damage Mechanisms: Axonal Cytoskeleton and Relation to Birefringence of Retina Nerve Fiber Layer
$100,000

Glaucoma, a leading cause of blindness worldwide, damages a tissue in the eye called the retinal nerve fiber layer, or RNFL. To aid in diagnosis of glaucoma, doctors use optical instruments to image the RNFL, hoping to detect abnormalities before significant loss of vision.

Our laboratory has studied the optical properties of normal RNFL for many years and has made a number of important discoveries. We now feel that we understand most of the optical properties of normal RNFL and we have some ideas about how these properties relate to the cellular structures inside nerve fibers. For example, we have shown that one clinically important property, called birefringence, is due to long, very thin filaments, called microtubules, that exist inside all nerve fibers. Microtubules are one component of the complex internal structure of nerve fibers.
           
In spite of our progress with normal RNFL, we still know little about the changes in optical properties caused by glaucoma, nor do we understand the changes in cellular structure that enable us to detect abnormal RNFL in patients. In this project, therefore, we are tackling these important questions. Because change in RNFL structure precedes RNFL loss, detecting early structural change can open a window during which treatment might prevent or even reverse glaucomatous damage.
           
We are pursuing three specific aims in this project. First, we will use special stains to label different internal components of the nerve fibers in the RNFL to learn how they are distributed at different stages of glaucoma. Second, we will compare these components, trying to learn which is most sensitive to damage. Third, we will look specifically at the relation between structural damage, particularly damage to microtubules, and changes in RNFL birefringence. A unique feature of our research is that the studied structures relate directly to clinically-detectable properties of the RNFL. Thus, we expect the results to lead directly to improved sensitivity for RNFL damage in clinical testing of patients.

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C. Ross Ethier, Ph.D.
Imperial College London
London, United Kingdom
Suitability of Hydrostatic Pressure Model for Studying Glaucoma
$100,000

The pressure in the eye is elevated in most forms of glaucoma. We know this leads to loss of retinal ganglion cell function, and hence vision loss, but we are not sure how this happens. Recently, investigators have exposed retinal ganglion cells and another supporting cell type (optic nerve head astrocytes) to elevated pressure, and studied their behaviour. They showed that pressure had important effects, but the way the pressure was applied to the cells may not be suitable for studying what occurs in glaucoma. In our research we will study whether this way of applying pressure to cells is useful for understanding the response of cells in glaucoma. To do this we will repeat previous experiments, but in such a way that possible confounding effects are removed from the experiments. We will also make direct measurements of oxygen levels and pH near the cells in a novel way that will help determine if the cells are being inadvertently exposed to a toxic environment in these experiments.

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Raghu R. Krishnamoorthy, Ph.D.
University of North Texas Health Science Center
Fort Worth, TX
Regulation of endothelin B receptor expression in glaucomatous optic neuropathy
$100,000

Glaucoma is a neurodegenerative disease in which a select group of nerve cells in the eye undergo cell death. Increase eye pressure due to poor drainage of a fluid inside the eye is a major cause of glaucoma. Hence, most medications for glaucoma are aimed at reducing the pressure within the eye by either better drainage or reducing the rate at which fluid is formed inside the eye. This has proved to be an effective way to treat glaucoma. However, despite reducing the pressure, sometimes the nerve cells in the eye continue to die slowly. It would be very beneficial if we have additional approaches called neuroprotection (meaning: protection of nerve cells) to more effectively treat glaucoma. Scientific studies have shown that there is an increase in a protein called endothelin receptors in glaucoma patients. The proposed study will determine if endothelin receptors produce some of the nerve damage seen in glaucoma. Understanding how endothelin receptors are damaging to nerve cells, will provide valuable information to block these receptors and thereby provide protection to nerve cells in the eye from further damage. This could lead to development of neuroprotection drugs as additional treatments to effectively treat glaucoma.

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Mortimer M. Civan, M.D.
University of Pennsylvania
Philadelphia, PA
Pannexin regulation of aqueous humor inflow and outflow
$100,000

The only approach proven to delay the onset and slow the progression of glaucomatous blindness is to lower the pressure built up within the eye [the intraocular pressure (IOP)] by the formation of the aqueous humor by the ciliary epithelium. The purine substances adenosine triphosphate (ATP) and adenosine (formed from ATP) are normally produced by cells in the eye, and participate in the regulation of IOP. Adenosine acts on one surface (the aqueous humor side) of the ciliary epithelium to increase the rate of formation of fluid and increase IOP, while ATP acts on the body side of the ciliary epithelium to reduce the rate of formation of aqueous humor. The delivery of adenosine and ATP to these surfaces ultimately depends on the release of ATP. The aim of this research is to identify how ATP is released at the two surfaces of the ciliary epithelium and also by cells of the aqueous humor outflow pathway, and to use this information to develop a new strategy for lowering IOP.

Despite the likely importance of ATP and adenosine in regulating IOP, the mechanisms of cellular ATP release triggering this regulation have been unclear. Our preliminary results are consistent with a role for a newly discovered family of channels in releasing ATP into the aqueous humor, in addition to two other possible channels previously considered. We plan to determine whether ATP is released by different mechanisms at the aqueous humor surface and body surface of the ciliary epithelium and in cells derived from the outflow pathway of aqueous humor. If ATP is indeed delivered to these sites by different mechanisms, the stage would be set for applying ATP faster at one surface and slower at another. Reducing the rate of ATP release at the aqueous surface of the ciliary epithelium and/or increasing the rate of ATP release at the body surface could provide the rational basis for a future novel method of treating glaucoma.

First, we wish to use blockers of the different pathways to test which ATP-release channel is most important for cells from the two surfaces of the ciliary epithelium and from the aqueous outflow pathway.

Second, we shall test our conclusions based on drug studies by measuring ATP release after reducing expression of the key newly-discovered channel, using tools of molecular biology. Third, we shall use electrical and fluorescence techniques to see if the newly-discovered channels also help link cells within the ciliary epithelium, thereby facilitating aqueous humor formation. Fourth, we shall test the conclusions drawn from our studies of isolated cells by measuring the effects of drugs upon the rate of aqueous humor formation measured directly in isolated, arterially perfused bovine eyes.

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David J. Calkins, Ph.D.
Vanderbilt University Medical Center
Nasville, TN
TRPV1: A Novel Neuroprotective Target in Glaucoma
$100,000

Glaucoma is the leading cause of irreversible blindness worldwide and is estimated to afflict some 80 million people by 2020. Most people think of glaucoma as a disease about pressure in the eye, because high pressure is associated with the disease and lowering pressure often is helpful for slowing vision loss in glaucoma. In fact, glaucoma is irreversible because it damages the fibers of the optic nerve, which is part of the brain and therefore limited in its ability to heal. Our research is important because it is the first attempt to understand how these fibers respond to pressure in the eye and whether blunting this response could prevent vision loss in glaucoma. Our team is comprised of neurobiologists with expertise in how nerve fibers respond to pressure. Thus, our research will help identify new drugs that will reduce the loss of the optic nerve in glaucoma by making its fibers insensitive to pressure in the eye.

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Jamie E. Craig, Ph.D.
Flinders University of South Australia
Adelaide,
Genome Wide Association studies in Glaucoma using equimolar DNA pooling
$100,000

This research will identify common genetic markers which lead to glaucoma.  We will use well-characterized groups of people with and without glaucoma, and carefully compare common variation in genes in these populations. Powerful technique of  high density gene screening to identify multiple genetic risk factors for glaucoma will be employed.

A novel genomic methodology, the construction of DNA pools, will be utilized in order to prioritize genomic regions important in glaucoma and complete a very large-scale project with excellent value for the requested budget.

The significance to people with glaucoma is that the level of risk of an unaffected individual developing glaucoma is currently not able to be determined. A definitive diagnosis of glaucoma may be difficult to make in teh early stages of disease. Patients also still are often diagnosed after major irreversible damage has already occurred. Glaucoma treatment currently balances the benefits of slowing disease progression against the risks of treatment. It would therefore be highly useful to understand genetic associations of glaucoma so earlier and better treatment could be directed to those individuals at high risk, while freeing other lower risk individuals of the need for treatment.

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John G. Flanagan, Ph.D.
Toronto Western Hospital
Toronto, Ontario
Canada
Protein expression of human optic nerve astrocytes and lamina cribrosa cells following exposure to different modes of biomechanical strain and hypoxia
$100,000

The 'big picture' question is what happens at the very earliest stages of glaucoma that results in a healthy optic nerve becoming glaucomatous? We attempt to reproduce the stresses and strains both mechanical, i.e. high pressure in the eye, and vascular, i.e. poor blood supply to the optic nerve, that are likely present in the very earliest stages of glaucoma.  We then harvest cells from optic nerves donated for research and expose them to conditions similar to those found in early glaucoma.  We measure the responses by analysing the different type of proteins the cells produce when stressed.  Our research is unique in that it involves collaborations between engineers who understand the biomechanics of pressure induced stress, scientists who can grow cells and measure their reactions to stress, and clinicians who can interpret the results in a way that may ultimately benefit their patients with glaucoma.

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Raquel L. Lieberman, Ph.D.
Georgia Institute of Technology
Atlanta, GA
Development of pharmacological chaperone therapy for inherited primary and juvenile open angle glaucoma
$100,000

Our aim is to develop a new therapy for inherited glaucoma, many cases of which are caused by mutations in a protein called myocilin. Myocilin forms part of the trabecular extracellular matrix, or TEM, which is important in regulating eye pressure. When the matrix doesn't function correctly, eye pressure increases, leading to retinal degeneration and vision loss. Human trabecular meshwork (HTM) cells, which produce the matrix, recognize mutations in myocilin and prevent mutant myocilin from being secreted to the TEM. Instead, mutant myocilin remains in the interior of HTM cells, causing the HTM cells to die.  This results in a disrupted TEM, increased eye pressure, and eventually glaucoma. We hope to discover a drug molecule that interacts with mutant myocilin inside the HTM cells and restores secretion of myocilin to the TEM.  The ability to secrete mutant myocilin will have two desired effects: (1) the mutant protein will not accumulate in the HTM cells and thereby help keep these cells alive and (2) the TEM will be restored and better control intraocular pressure. Taken together, this approach will retard retinal degeneration associated with glaucoma. We will accomplish our long-term goal by first unveiling the molecular structure of myocilin, and then identifying and testing drug candidates that bind to specific grooves on the surface of myocilin.

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Teri L. Belecky-Adams, Ph.D.
Trustees of Indiana University
Indianapolis, IN
Bmp7 and glaucoma
$100,000

Glaucoma is a group of diseases that often have a build up of fluid within the eye that creates pressure on the optic nerve, killing retinal ganglion cells, the cells that communicate with the rest of the brain.  There are also disruptive changes to the glial cells in the optic nerve, cells that normally play a supportive role to the ganglion cells.  Our  proposal focuses on understanding the role of a growth factor, Bmp7, in both the increased survival of ganglion cells and the disruptive changes the glial cells undergo during glaucoma.  It is our hope that novel therapies may one day be included in treatment of glaucoma which will help save the ganglion cells and decrease disruptive changes to the glial cells.

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Judith A. West-Mays, Ph.D.
McMaster University
Hamilton, Ontario
Canada
The Roles of MMPs in TGFbeta induced Glaucoma
$100,000

The goal of our research is to determine what differentiates an eye displaying glaucoma symptoms from a normal eye. The glaucoma eye is different in the sense that it expresses different concentrations and combinations of molecules that are typically found in a normal eye. Some of these molecules are involved in formation of scar tissue (TGFβ) or breakdown of scar tissue (MMPs). We wish to determine how manipulation of MMPs can influence the development of scarring in the eye, which can lead to glaucoma. Also, we wish to establish the time at which these molecules begin to differ in quantity, which may explain the worsening of symptoms characteristic of glaucoma over time.

Previously, no one has attempted to determine the role of MMPs in glaucoma induced by TGFβ and research has shown that halting the activity of MMPs may be beneficial in reducing the severity of ocular disorders. Characterizing MMP activity in an animal model where TGFβ is artificially in abundance is important because glaucoma is often characterized by increased levels of TGFβ. It is crucial that the roles of MMPs are characterized in a glaucoma-like environment. If our predictions hold true for the role of MMPs in glaucoma, MMP inhibition may be an excellent treatment option for some glaucoma patients. Additionally, if a timeline for disease progression in the TGFβ model is successfully documented, it will greatly facilitate the generation of new methods of treatment, as various therapies could be implemented at a time known to be prior to symptom expression and the effects of the treatment monitored.

First, we wish to determine a time prior to, during and post glaucoma symptom expression that will allow for these various timepoints to be studied and their importance to symptom progression. Secondly, we wish to determine how and where MMPs are involved in this model of glaucoma. Finally, we wish to determine if inhibition of MMPs will prevent the animals from developing glaucoma.

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Brad Fortune, Ph.D.
Legacy Health System
Portland, OR
Imaging Retinal Nerve Fiber Layer Pathology in Experimental Glaucoma
$100,000

Structural abnormalities within the retinal nerve fiber layer (RNFL) may be one of the earliest signs of damage in glaucoma. The majority of eyes with elevated intraocular pressure already have clinically detectable RNFL defects before reproducible abnormalities first appear in the visual field. Thus RNFL evaluation has become part of the clinical standard of care for all glaucoma patients and suspects. Previous studies suggest that alteration of the structural elements within retinal axons precedes complete, irreversible loss of those axons and that this intermediate stage can be detected using clinical imaging technology. This study is designed to test the hypothesis that degradation of axonal internal elements known as neurofilaments and/or microtubules is a clinically detectable process or disease stage, and that it precedes complete degeneration and loss of axons from the RNFL and optic nerve.

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FUNDING PERIOD: APRIL 1, 2007 – MARCH 31, 2009

Michael G. Anderson, Ph.D.
The University of Iowa
Iowa City, IA
Project:Genetics of Central Corneal Thickness in Mice
90,000

The cornea is the outermost covering of the eye.  Given the importance of the cornea to vision, it is somewhat surprising that the cornea exhibits vast person-to-person variability, especially regarding its overall thickness (referred to as central corneal thickness, CCT). Recently, it has become clear that differences in CCT are more than a mere anatomical curiosity. CCT strongly associates with several ocular diseases, particularly glaucoma.  The Ocular Hypertension Treatment Study (OHTS, a large, multicenter, prospective clinical glaucoma study) recently found that a thinner CCT predicts the development of primary open angle glaucoma. The pathophysiological reason for this association remains unknown. Our experiments offer an innovative approach that will allow CCT to be studied at the molecular level using experiments with mice. Knowing the genes that influence CCT, even in mice, will immediately suggest new mechanistic hypotheses that can be carried back into human studies.

Our work also addresses an important health disparity issue that exists among patients with glaucoma.  Advancing age and elevated intraocular pressure are two well known risk factors for developing glaucoma. Race is another. Both the incidence of glaucoma and the incidence of thin corneas are much more common among African Americans than other ethnicities. For unknown reasons, African Americans with glaucoma have on average more severe disease that is more resistant to treatment in comparison to patients of other ethnicities. The reason for these associations may in part reflect the biological connections that link CCT and glaucoma. Our studies in mice offer an innovative approach for identifying molecules influencing CCT and experimentally testing their relevance to glaucoma.

Ultimately, results of these studies will contribute to an improved understanding of the risk factors that contribute to glaucoma, with the long term goal being the development of improved therapies and outcomes for all people affected by glaucoma.

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Dong Feng Chen, M.D., Ph.D.
Schepens Eye Research Institute
Boston, MA
Project: Astroglial Contributions to Glaucoma
$90,000

Glaucoma is a leading cause of blindness in the world and affects an estimate 3 million Americans. In most cases, glaucoma is caused by elevated intraocular pressure that damages the optic nerve and eventually leads to death of retinal ganglion cells (cells that extend nerve fibers to form the optic nerve). Current therapy for glaucoma is directed at lowering intraocular pressures, but very often, it fails to prevent the progressive neuron and vision loss associated with the disease. Therefore, finding ways that protect the optic nerve and c from injury are crucial to the development of more efficacious treatment for glaucoma. Retinal astroglial cells are supporting cells of the retina. Accumulating evidence suggests that when these supporting cells respond to nerve injury, they induce detrimental effect on the nerve and retinal ganglion cells, thus, may contribute critically to the development and progression of the disease. In the present application, we hypothesize that responses of these supporting cells are directly responsible for glaucoma-induced optic nerve damage and retinal ganglion cell death by producing neurotoxic agents, triggering inflammation, and generating an inhospitable environment. The objective of this research plan will define the functional roles of these supporting cells in optic nerve and retinal ganglion cell damage in glaucoma and eventually, design drugs that prevent neuronal damage and vision loss by targeting to the function of these cells. We have proposed three Specific Aims:

• Aim I will investigate the functional roles of these supporting cells in optic nerve and retinal ganglion cell degeneration in a glaucoma mouse model as well as test the effect of a potential chemical that can eliminate the detrimental effects of the supporting cells.
  
  
• Aim II will further prove the concept by defining the roles of these supporting cells using a genetically engineered mouse models.
  
  
• Aim III will take advantage of the new technology of cDNA microarray to determine the molecular basis underlying the detrimental effects of the supporting cells after nerve injury.  Results derived from this study will provide important information for developing new therapeutic strategies to treat glaucoma and other brain and optic nerve damage.

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Dharamainder Choudhary, Ph.D.
University of Connecticut Health Center
Farmington, CT
Project: Mechanistic Effects of CYP1B1 Variations in POAG
$90,000

Our group first (1997) linked the presence of CYP1B1 mutations with Primary Congenital Glaucoma (PCG) phenotype in patients. Recently, CYP1B1 mutations have also been shown in other eye diseases with anterior chamber abnormalities and in juvenile and adult onset Primary Open Angle Glaucoma (POAG). This involvement in a wide spectrum of eye diseases suggests a crucial role of CYP1B1 in maintaining the physiology of the eye during embryonic to adult stages of life. We hypothesize that CYP1B1 may act as a key factor, which modulates the effect of other known POAG causative genes e.g., myocilin, optineurin and WDR36. We have a number of genomic DNA samples of glaucoma subjects who have previously been shown to be positive for mutations in one of the above-mentioned genes. We will screen for the presence of CYP1B1 mutations in these glaucoma families. The anticipated CYP1B1 mutations observed will be functionally tested by our previously established biochemical and molecular protocols. The positive co-existence of mutations in CYP1B1 and other known glaucoma genes will aid in establishing a common protocol for examining CYP1B1 mutations in individuals with family background of possessing mutations in either myocilin, optineurin or WDR36. This will help in early identification of the individuals who are at higher risk of developing glaucoma phenotype at early stages of adult life.

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Haiyan Gong, M.D., Ph.D.
Boston University School of Medicine
Boston, MA
Project: Outflow Area and Its Role in Glaucoma Pathogenesis
$90,000

Glaucoma is typically associated with elevated intraocular pressure (IOP) caused by increased aqueous outflow resistance. In previous studies of bovine eyes, we found that the area available for outflow increases when IOP and outflow resistance decreases, suggesting a relationship between available area for outflow and outflow resistance. The morphological correlation of reduction in the area available to outflow, with increasing IOP, seems to be related to a progressive collapse of the Schlemm’s canal and a previously unrecognized, progressive herniation of the inner wall and the JCT into the collector channel ostia. A similar herniation of the inner wall into the collector channel ostia was also seen when the eyes were fixed even at 0 mmHg raising the prospect that initially reversible herniations can become irreversible. We hypothesize that the herniations we have documented will be found to be reversible in eyes without glaucoma but can become irreversible, leading to increased outflow resistance and IOP. The results of this study will help us to better understand glaucoma pathophysiology and will also better direct us to the anatomical sites where the pathological resistance resides.

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Xuri Li, Ph.D.
NIH/NEI
Bethesda, MD
Project: Neuroprotection of Retinal Ganglion Cells by VEGF-B
$88,920
 
Glaucoma is the most prevalent form of adult optic neuropathy characterized by the degeneration and death of the retinal ganglion cells (RGCs). Molecules with neuroprotective effect on the endangered RGCs are therefore much desired to preserve and rescue the RGCs and thus the vision of glaucoma patients. This research proposal is therefore designed to test in vivo the neuroprotective effect of one such candidate molecule, the vascular endothelium growth factor B (VEGF-B), and to further characterize the molecular and cellular mechanisms underlying. VEGF-B has been shown to be a critical neuroprotective factor in the brain. Our current work has shown that VEGF-B may play important roles in the retina. Based on our preliminary studies, we hypothesize that VEGF-B may have a neuroprotective effect on the retinal ganglion cells. To test this hypothesis, we will use multiple approaches and methods, including both normal and VEGF-B transgenic mice, both protein and gene transfer, both gain and loss of function analysis, to investigate the neuroprotective effect of VEGF-B on retinal ganglion cells in vivo. The outcome of this study may lead to possibilities of novel therapy for glaucoma patients and more insight into the course of glaucoma.

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Brian A. Link, Ph.D.
Medical College of Wisconsin
Milwaukee, WI
Project: Identifying Glaucoma-Promoting Genes Using Zebrafish
$90,000
(This project is additionally supported by the Glaucoma Research Foundation, San Francisco, California)

Glaucoma is a leading cause of blindness world-wide and yet our understanding of the genetic basis of this neurodegenerative disease remains largely unknown.  One reason for this is that glaucoma is really a collection of diseases and each form is thought to be caused by mutations in multiple genes.  The main goal of this study is to identify genes that cause ganglion cell degeneration when intraocular pressure (IOP) is elevated, a key feature of many forms of glaucoma.  Using zebrafish that show elevated IOP with out associated ganglion cell degeneration, we will screen for ‘interacting’ mutations that lead to injury response pathways in retinal ganglion cells and eventually cause blinding degeneration.   Zebrafish are ideal for these studies because they share similar ocular anatomy, physiology and genetics as humans.  In addition, zebrafish can be raised in very large numbers at modest cost, facilitating complex forward genetic endeavors.   Once the key genes for glaucoma are identified, genetic tests can be developed and ultimately, research for targeted therapies can begin.

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K. Saidas Nair, Ph.D.
The Jackson Laboratory
Bar Harbor, ME
Project: Determining Immune Components of DBA/2J Glaucoma
$90,000

Harmfully high intraocular pressure (IOP) is a major risk factor for glaucoma. Pigment dispersion syndrome (PDS) is a common condition that deposits abnormally liberated iris pigment into the pathway that drains the ocular fluid from the eye.  In some cases, the deposited pigment harms the drain resulting in high IOP and glaucoma. Not all PDS progresses to high IOP and glaucoma suggesting that the pigment itself is not sufficient to induce high IOP. DBA/2J mice have pigment dispersion, mild intraocular inflammation and develop high IOP. We hypothesize that the pigment dispersion and inflammation cooperate to induce the high IOP in these mice. To test this hypothesis, we will use genetically altered DBA/2J mice that are devoid of a subset of immune cells that mediate inflammatory responses. We will determine if the mice develop high IOP and glaucoma in the absence of these cells. Also, we will test if a specific DBA/2J gene predisposes to the inflammation and thereby confers susceptibility to IOP elevation. Our studies may suggest new therapies to prevent progression from PDS to glaucoma by reducing inflammatory responses.

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Kwok-Peng Ng, Ph.D.
Cleveland Clinic Foundation
Cleveland, OH
Project: Glaucoma Biomarker Discovery and Pathogenetics
$90,000

Glaucoma is a group of eye diseases that damages the optic nerve and result in blindness if left untreated. The most common form is primary open-angle glaucoma (POAG). POAG affects over 3 million Americans, a majority of whom do not know they have it. There is currently no cure for glaucoma. We believe that molecular markers exist in the blood of individuals susceptible to developing glaucoma. These molecules are small fragments of proteins that arise from breakdown of proteins during normal cellular functions in the body. Sometimes the levels of certain molecules in the blood change when a person gets sick. The identification of these molecular markers or “biomarkers” will let doctors identify those at risk before clinical evidence of the disease. More importantly, these biomarkers could lead to new therapies for glaucoma. We will use instruments known as mass spectrometers to identify biomarkers in blood samples from individuals with POAG. These biomarkers will be analyzed to ensure they are specific to glaucoma and hence useful for the development of diagnostic tests and new drugs. The long term goals of our research are to develop 1) a blood test for early detection and 2) new treatments for this debilitating disease.

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Robert W. Nickells, Ph.D.
University of Wisconsin-Madison
Madison, WI
Project: Cell Based Neurotrophin Therapy for Glaucoma
$90,000

Glaucoma is a blinding disease characterized by the progressive loss of retinal ganglion cells.  One major line of therapeutic intervention being developed for glaucoma is the introduction of growth factors that can prevent the death of the ganglion cells.  Two major hurdles in this area of investigation is that we have not been able to deliver more than one or two factors at a time and we can not deliver them for long enough periods.  Evidence suggests that the most effective treatment would be the delivery of multiple factors for periods of several months to years.  A possible solution to these problems is the use of progenitor cells.  These cells are similar to stem cells in that they are undifferentiated and can self renew or divide in culture.  These cells can be used as transplants in damaged nerve tissue, where they can infiltrate the regions of damage and make contact with living neurons.  Another advantage of these cells is that we can alter them in the laboratory to express and secrete the complex mixture of growth factors that ganglion cells need to survive.  Once transplanted into the eye, we hypothesize that they will make contact with surviving ganglion cells and provide them with the critical growth factors needed for survival.  Lastly, we expect that these cells will provide the growth factor support for extended periods of time without any additional treatment.  Using progenitor cells for this “cell based therapy” is a relatively new concept for treating glaucoma.  This proposal is designed to address some of the fundamental questions we need to answer to pursue this line of therapy.  Among the questions we will address are:

• Where do the cells go when injected into the vitreous cavity - do they penetrate the retina and make contact with the target ganglion cells?
  
  
• How long do the cells survive in the retina?
  
  
• Can these cells attenuate the rate of ganglion cell loss when engineered to secrete a single growth factor known to promote ganglion cell survival?

To answer these questions, we propose to study their effects on mice that spontaneously develop glaucoma as they age.  If successful, we propose to extend these studies by altering these cells further to secrete a potent mixture of growth factors required for maximal ganglion cell survival.

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Colm O'Brien, M.D.
Mater Misericordiae Hospital
Dublin, Ireland
Project: Modulation of Lamina Cribrosa Matrix Remodeling in POAG
$90,000

This project intends to test the hypothesis of that the use of novel anti-fibrotic therapies will reduce the damage done in glaucoma to the retinal ganglion cells thereby alleviating the resultant loss of vision. At present glaucoma represents the most common form of irreversible blindness in the western world. The exact causes of the different types of glaucoma are not fully understood but in one common form, primary open angle glaucoma it is believed that mechanical strain induced by elevated intra-occular pressure plays a part. This causes changes in the connective tissue of the lamina cribrosa, which is where the optic nerves exit the eye. Previous evidence has shown that soluble growth factors such as the TGF- family can affect the extracellular composition of this tissue. By modulating the expression of the TGF- family we hope to change the connective tissue composition of the lamina cribrosa and thereby reduce the damage to the retinal ganglion cells and reduce loss of visual function.

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Noorjahan Panjwani, Ph.D.
Tufts University School of Medicine
Boston, MA
Project: Identification of TM Cell Markers
$90,000

Glaucoma is the second leading cause of blindness in the world.  Nearly 2.5 million individuals in the US and 70 million worldwide are affected by this blinding disease.  Elevated IOP due to reduction in aqueous outflow facility is a major causal risk factor. In humans, the aqueous outflow pathway is constituted of Schlemm's canal (SC) lined by its endothelial cells, and trabecular meshwork (TM)  which is divided into the corneoscleral (CS) portion with its endothelial-like TM cells and the juxtacanalicular (JCT) portion with its fibroblast-like TM cells.  The functional differences and relative importance of the different cell populations residing in the outflow pathway is a subject of great debate in the field.  While the three cell types of the outflow pathway can be readily distinguished in tissue sections based on distinct morphology, no specific markers have been identified that distinguish JCT and CS cells from one another. Clearly, for the analyses and interrogation of each cell type of the aqueous outflow pathway with respect to its role in the outflow resistance and the pathogenesis of glaucoma, availability of robust markers that would permit preparation of pure cultures of each cell type is a prerequisite. In this application we propose studies to identify differential markers of the three distinct cell types of the aqueous outflow pathway using the most modern, state of the art techniques. It is our hope that characterization of gene expression patterns of distinct cell types of the outflow pathway will lay the foundation for research by novel, more accurate approaches for understanding the pathogenic mechanisms of glaucoma.

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Carla J. Siegfried, M.D.
Washington University
St. Louis, MO
Project: Aqueous Humor Oxidative Stress Markers in Glaucoma
$90,000

Glaucoma, the second leading cause of blindness worldwide, is characterized by damage to the optic nerve which transmits visual information from the eye to the brain.   Most cases are associated with increased eye pressure and decreased outflow through the drainage meshwork in the front of the eye.  Current theories suggest that this age-related disease, as well as other eye conditions like cataract and macular degeneration, may be related to oxidative damage, toxic effects of excess oxygen forming harmful molecules called free radicals.  Our proposed study will examine the aqueous humor, the fluid in the front of the eye, of patients who are undergoing eye surgery. We will measure both a byproduct (hydrogen peroxide) and a protectant (ascorbic acid) of oxidative damage, as well as oxygen levels in different parts of the front of the eye.  By aiding in further understanding of this oxidative mechanism of glaucoma damage, it may lead to innovative therapies for this devastating condition.

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Donald J. Zack, M.D., Ph.D.
Johns Hopkins University
Baltimore, MD
Project: Role of CHOP and ER stress in RGC Degeneration
$90,000

Glaucoma is a major cause of vision loss. It is the second moss common cause of blindness worldwide. In glaucoma, decreased vision is caused by the injury and death of retinal ganglion cells (RGCs), the cells that transmit visual information from the eye to the brain. All current forms of glaucoma treatment (drugs, laser treatment, and surgery) act by lowering the pressure within the eye. Although pressure lowering can be helpful, RGC loss can continue even after pressure reduction. Greater understanding of the molecular mechanisms underlying retinal ganglion cell (RGC) loss will likely make possible the development of more effective diagnostic and treatment strategies. In the work proposed in this application we will explore the molecular mechanisms that actually induce the death of RGCs. In preliminary studies we have shown that a particular pathway, called the stress induced response, is activated in animal models of glaucoma. We propose to study this pathway in more detail, and determine if inhibition of the stress induced response can reduce or prevent the loss of RGCs. If successful, this work could help provide the basis for the development of new strategies for the diagnosis and treatment of glaucoma.

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FUNDING PERIOD: APRIL 1, 2006 – MARCH 31, 2008

Milam Brantley, Jr., M.D., Ph.D.
Washington University
St. Louis, MI
Project: Nmnat1 as a Neuroprotective Agent in Murine Glaucoma
$90,000

Glaucoma is the second leading cause of blindness worldwide and is characterized by degeneration of retinal ganglion cells (RGCs), whose axons make up the optic nerve.  It is generally accepted that degeneration of the RGC axons precedes programmed death of the cell bodies themselves.  Recent tissue culture experiments have shown that increased expression of the Nmnat1 gene can slow the rate of axonal degeneration in the face of axonal injury.  The purpose of this study is to evaluate the ability of overexpression of Nmnat1 to slow axonal degeneration in two mouse models of optic neuropathy.  In the first model, a short period of very high eye pressure will be used to cause RGC damage over 1-2 weeks.  The second model uses application of laser to the eye to moderately and stably raise eye pressure, closely simulating glaucoma.  A virus vector containing the Nmnat1 gene will be injected into eyes from each of these models, and the RGCs will be evaluated both morphologically and functionally to determine if Nmnat1 can protect the RGCs against damage.  This study will efficiently assess the potential of Nmnat1 to serve as an effective anti-glaucoma agent.

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Richard Lee, M.D., Ph.D.
Bascom Palmer Eye Institute
Miami, FL
Project: Proteomic Characterization of Pseudoexfoliation Glaucoma
$90,000

Pseudoexfoliation glaucoma (PEX) is the most common form of open angle glaucoma with an identifiable cause. Pseudoexfoliation glaucoma is defined by the presence of pseudoexfoliation “material” in the anterior segment of the glaucomatous eye, typically layered on the lens capsule in a bull’s eye configuration.  This project will 1) identify the PEX proteins using advanced proteomic approaches and 2) characterize the components of the PEX protein complex using molecular techniques. Preliminary experiments have identified proteins associated with the pathophysiology of PEX glaucoma, but not the causative PEX material proteins themselves. Using differential protein expression analysis, Dr. Lee will identify the PEX material proteins present on PEX patient lens capsules. Antibodies to these PEX proteins will be used to discover the other members of the PEX material complex and understand how PEX material proteins molecularly assemble in the anterior segment of the eye to cause glaucoma.  Identification of PEX-specific proteins and determination of the components of PEX material will provide insight into the pathogenesis of pseudoexfoliation glaucoma. A better understanding of the molecular basis of PEX glaucoma will lead to the development of novel molecularly targeted, non-intraocular pressure dependent therapies that may prevent the formation of PEX material.

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Jeffrey Marchant, Ph.D.
Tufts University School of Medicine
Boston, MA
Project: Matrix Interactions Pertinent to High IOP and Glaucoma
$90,000

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma and results from alteration of the pathway that drains aqueous humor from the anterior eye. While studies have been performed to characterize this pathway in human and non-human primate tissues, the understanding of its organization is still very limited. The outflow pathway is comprised, in part, of a region known as the trabecular meshwork (TM). The TM is a series of cells and connective tissues organized as a filter. Alterations in several TM components have been correlated with elevated IOP. Dr. Marchant has identified a new component in the TM that he hypothesizes will stabilize this region and help to regulate normal IOP. Dr. Marchant will examine the TM in the mouse to evaluate the organization of this and other components thought to regulate IOP. He will then exploit the genetic capabilities of the mouse and examine mutants that have altered expression of key TM regulatory components, as well as evaluate the affect these alterations have on TM function and IOP regulation. Given the similarities between the components in mouse and human TM tissues, these findings will likely be highly relevant to human IOP regulation and thus may lead to intervention strategies for the future.

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Deborah Otteson, B.Sc., Ph.D.
University of Houston
Houston, TX
Project: Regulation of EphA Expression in Retinal Ganglion Cells
$85,000

In the visual system, signaling between Eph/ephrin proteins plays key role in axon path-finding in the optic nerve, determining which retinal ganglion cell (RGC) axons will cross at the optic chaism and regulating the initial positioning of RGC synapses in the brain. Gene expression is largely regulated by factors that bind to regulatory regions called promoters located in the DNA and this project’s hypothesis that the spatial and cellular expression patterns of Eph receptor genes in RGCs are regulated by specific transcriptional regulatory factors. These studies seek to understand the molecular and cellular mechanisms that regulate expression in RGCs by identifying the promoters of the EyhA genes, determining the binding sites for retinal proteins and testing a group of specific transcription factors for their ability to activate the EphA promoters in cultured retinal cells. The long term goals are to identify the network of regulatory mechanisms controlling gene expression in RGCs. This will contribute to our basic knowledge of RGC biology and may identify novel targets for developing new therapeutic agents and will enhance the future development of gene- and cell-based strategies for repair and regeneration of injured RGC axons to restore lost vision in patients with glaucoma.

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Darryl Overby, Ph.D.
Tulane University
New Orleans, LA
Project: How Does Segmental Outflow Affect Outflow Resistance?
$90,000

Glaucoma is a disease typically associated with elevated intraocular pressure (IOP) caused by increased resistance to aqueous humor outflow. Glaucoma is treated clinically by reducing IOP, but the precise factors responsible for controlling IOP and outflow resistance are not well understood. To improve the understanding of outflow resistance, Dr. Overby proposes to study how the hydrodynamic patterns of aqueous humor outflow change in response to stimuli that affect outflow resistance. His hypothesis is because aqueous humor is confined to flow through only a fraction of total area of outflow pathway, outflow resistance is correlated to the area that is actively involved in hydrodynamic filtration of aqueous humor. To test this hypothesis, he will aim to develop a new tracer method that will combine with confocal microscopy to visualize the hydrodynamic patterns of outflow and measure the active area of aqueous humor filtration in human eyes. The second aim is to use this tracer technique to quantify the relationship between outflow resistance and filtration area for changing IOP that causes a change in outflow resistance. The long-term goal of this project is to translate this fundamental understanding of outflow hydrodynamics into a strategy to reduce outflow resistance and IOP in glaucomatous eyes.

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Julia E. Richards , Ph.D.
University of Michigan
Ann Arbor, MI
Project: Genetic Risk Factors and Glaucoma Outcomes
$90,000

Mutations that alter the myocilin (MYOC) gene product are known to cause glaucoma, but it has also been suggested that glaucoma severity might be affected by mutations in the regulatory promoter region that affects level of expression of the gene. Because there are contradictory reports regarding the role of variants in this MYOC regulatory region, it is important to carry out an optimal study aimed at evaluating whether MYOC regulatory variants can affect glaucoma severity or outcomes. Dr. Pawar proposes to use the Collaborative Initial Glaucoma Treatment Study (CIGTS) and Advanced Glaucoma Intervention Study (AGIS) study samples for analyzing this risk factor in the MYOC gene. This sample set offers well-documented clinical information and will help establish whether there is a correlation between the MYOC genotypes and the outcomes of the glaucoma clinical trial samples. In fact, this project may help physicians predict things about severity and outcome for their patients, and assist them in using this information in designing an optimal treatment plan. One of the important features of the proposed work is that it will provide important information whether or not the sequence variant turns out to be predictive of outcome.

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