RECENT RESEARCH GRANTS

Protein aggregation in glial cells of the optic nerve: Role in glaucoma
Principal Investigator:  Irina G. Surgucheva, PhD
VA Medical Center, Kansas City, Missouri
The optic nerve acts like an electric cable with over a million wires and is responsible for carrying images from the eye to the brain.  This electric cable is composed of the endings of cells called retinal ganglion cells and another type of cell called astrocytes, which support normal functions of neuron endings in the optic nerve.  In glaucoma, patients' astrocytes die in the optic nerve because of excessive accumulation of abnormal proteins.  This project will investigate why these abnormal proteins accumulate in cells.  The project also hopes to unveil the mechanisms of death of astrocytes due to the accumulation of these abnormal proteins and to find substances which might prevent their death.

Using immune-based strategy to awaken dormant retinal stem cells: A therapeutic approach to glaucoma - Year 2
Principal Investigator:  Michal Schwartz, PhD
Weizmann Institute of Science, Rehovot, Israel
While there are treatments to lower pressure in the eye, thereby preventing continued damage, there is currently no cure for glaucoma nor any therapy capable of inducing cell renewal in the damaged tissue.  It has been suggested that stem cells, which can differentiate to form numerous cell types, could be used to replace nerve cells in the retina damage by the disease.  Stem cells exist in the mammalian eye but are dormant.  The first year of this grant was devoted to exploring the reasons that the ocular stem cells are unable to divide and form new nerve cells.  In this second year, an immunological approach will be used as a basis for awakening the quiescent stem cell population.  The goal is that activation of these dormant stem cells can be developed into a promising therapy for glaucoma.

Spring 2009
Project Dates: July 1, 2009 - June 30, 2010
A minimally invasive drug delivery approach to modify the ECM and promote neural regeneration in a model of glaucoma
Principal Investigator:  Erin B. Lavik, SB, SM, ScD
Case Western Reserve University, Cleveland, Ohio
Neural degeneration in glaucoma arises from the loss of retinal ganglion cells (RGCs) and is accompanied by extensive remodeling of the extracellular matrix (the external environment of the optic nerve cells) that inhibits repair.  This project proposes to make the environment permissive for repair by delivering a drug, AG1478, that has been shown to alter this environment and promote optic nerve regeneration.  The drug will be delivered from injectable microspheres.  The project hypothesizes that the combination of neural progenitor cells to replace lost RGCs cells with sustained delivery of AG1478 will promote regeneration.

DNA methylation changes associated with ganglion cell injury
Principal Investigator:  Shannath Merbs, MD, PhD
Johns Hopkins University, Baltimore, Maryland
Alterations in the expression of genes can lead to disease.  One way to alter gene expression without changing the DNA sequence of a gene, is to chemically modify the DNA by methylation.  Abnormal DNA methylation leads to some cancers, and it is possible that methylation changes could also contribute to the development of non-neoplastic diseases like glaucoma.  This project will look for DNA methylation changes associated with RGC death.  Drugs to manipulate DNA methylation have been used in the treatment of cancer and might someday prove useful for the treatment of glaucoma.

Math5 target genes in retinal ganglion cell formation
Principal Investigator:  Xiuqian Mu, MD, PhD
State University at Buffalo, New York
This proposal focuses on the function of a transcription factor named Math5 in RGC formation.  Transcription factors are proteins that regulate the activities of other genes.  Genetic studies have shown that Math5 is required for RGC formation, but how it functions is not known, in that we do not know what genes are turned on and off by Math5.  The objective of this proposal is to identify these genes.  The project will utilize a recently developed technology, ChiP-seq, which uses specific antibody and ultrahigh-throughput DNA sequencing to identify the genes Math5 controls.  This information will help future efforts to generate RGCs in cell culture for cell replacement therapy for glaucoma.

Micro & nanotechnology-based bioplatforms for high-throughput analysis of axon-glial interactions in glaucomatous neuropathy - Year 2
Principal Investigator:  David W. Sretavan, MD, PhD
University of California San Francisco
Improved management of glaucoma requires better understanding of disease mechanisms that damage the axons of RGC cells at the optic nerve head.  This work is aimed at developing a new type of highly versatile microplatform that incorporates advances in micro and nanotechnology to provide researchers with unprecedented control over key experimental parameters.  This renewal grant proposes to use this bioplatform to conduct high-throughput experimentation on RGC axons to investigate whether axon biology and survival may be affected by the presence of Eph/ephrin signaling molecules.  This cellular signaling system was recently demonstrated to be closely linked to onset of RGC axon damage in disease. 

Fall 2008
Project Dates: January 1, 2009 - December 31, 2009
Analysis of mitochondrion involvement in the pathogensis of Primary Open Angle Glaucoma
Principal Investigator: Alberto Izzotti, MD, PhD
Primary Investigator: Sergio Claudio Sacca, MD
University of Genoa, Italy
Glaucoma is a degenerative multifactorial disease recognizing oxidative stress as a main pathogenic determinant. Because glaucoma does not recognize major environmental risk factors, we deem of interest to focus our attention on the main endogenous sources of oxidative damage that is mitochondrion. The aim of this study is to analyze mitochondrion status in the trabecular meshwork TM, i.e. the tissues involved in the regulation of aqueous humor outflow from the anterior chamber, as collected from glaucomatous patients as compared to unaffected controls. The expression of normal and mutant myocilin gene will be related to the path-physiology of glaucoma as analyzed in TM. We will evaluate by real-time PCR genetic polymorphisms, expression of mitochoncrion-related genes, apoptosis-related genes (BCL-2, BAX, BAK), myocilin and Cytochrome c oxidase. Results are expected to highlight the alterations occurring in mitochondria, the principal endogenous source of oxygen free radicals, during glaucoma. Obtained results will be matched with available clinical data thus being useful to identify diagnostic and prognostic molecular markers for glaucoma. Accordingly, this study will identify biomarkers bearing putative prognostic attitude in clinical trials.

Single-Cell Imaging of Optic Nerve Astrocytes in Glaucoma
Tatjana Jakobs, MD
Assistant Cell Biologist, Neurosurgery Research
Massachusetts General Hospital, Boston, MA
Ganglion cells are the only neurons in the retina that send axons to the brain via the optic nerve. Glaucoma leads to a progressive and irreversible loss of these cells and thereby severs the connection of an otherwise functional retina with the brain. Recent experimental evidence suggests that a non-neuronal cell type (astrocytes) in the optic nerve might play an active role in the disease. Our goal is to visualize individual astrocytes in more detail than has been possible before. We will use a transgenic mouse strain in which optic nerve astrocytes are labeled with a fluorescent protein. We will experimentally increase the intraocular pressure in these animals as a short-term model of glaucoma and examine the optic nerves for pathological changes. Furthermore, we will cross this mouse line with one that develops a form of hereditary glaucoma. The resulting mouse line will be useful to follow damage in the optic nerve head, especially during early stages of the disease, and to evaluate whether various treatment methods of glaucoma affect these cells.

In Vivo Imaging of Retinal Ganglion Cells – A New Model to Study Neuroprotection in Glaucoma
Christopher Kai Shun Leung, MD, MB ChB, BMedSc, MSc
Assistant Professor, Ophthalmology and Visual Sciences
University Eye Center, The Chinese University of Hong Kong
The goal of this project is to investigate the use of a novel in vivo imaging technique to monitor the longitudinal profile of RGCs damage in glaucoma and to study the longitudinal treatment response of RGCs to a neuroprotectant – brain-derived neurotrophic factor (BDNF). An experimental model of glaucoma is induced by laser photocoagulation at the limbus in a strain of transgenic mice (Thy-1 CFP) that express cyan fluorescent protein (CFP) under the control of a Thy-1 promoter. By using a modified confocal scanning laser ophthalmoscope, fluorescent spots representing the expression of Thy-1 CFP in RGCs can be visualized and counted. RGCs damage is detected as loss of fluorescent signals. BDNF is considered to be neuroportective if it could either prevent the decrease of Thy-1 CFP expression or increase the expression of Thy-1 CFP in fading RGCs. This imaging model offers a unique opportunity to monitor RGCs longitudinally and non-invasively, and will provide a new paradigm to study neuroprotection in glaucoma.

Does Tau Dysfunction Play a Role in Glaucoma?
Keith R.G. Martin, MA, DM, MRCP, FRCOphth
University Lecturer and Consultant in Ophthalmology, Center for Brain Repair
Cambridge Centre for Brain Repair, United Kingdom 
Loss of sight in glaucoma is due to the death of neurons in the retina. Exactly how and why neurons die in glaucoma not yet fully understood. Previous work suggests that blockage of the transport of survival factors from the brain to retinal neurons contributes to cell death in glaucoma. Similar transport problems also occur in other neurodegenerative conditions such as Alzheimer’s disease and multiple sclerosis. In these diseases, dysfunction of a protein called tau contributes to disrupted cellular transport. Tau is a small protein that stabilizes the tracks along which motor proteins transport their cargo (e.g. neuronal survival factors), much like the sleepers that keep railroad tracks firmly in place. Dysfunction of tau causes the tracks to become disorganized and transport fails. We have strong preliminary evidence that tau dysfunction occurs in experimental glaucoma. This is exciting because drugs that modulate tau are available, including lithium and also newer agents with more favorable side-effect profiles. We will test whether these drugs reduce neuron death in glaucoma and help to preserve sight.

Development of Functional Assay for WDR36 - Year 2
Michael Walter, PhD
Professor, Medical Genetics
University of Alberta, Edmonton, Canada
Finding the genes that cause glaucoma is the first step in improving early diagnosis and treatment of patients suffering from glaucoma. WDR36 has been proposed as a new primary open angle glaucoma gene. However the role of WDR36 in glaucoma is controversial. While a number of nucleotide changes of WDR36 have been found in elevated frequency in glaucoma patients versus non-affected controls, proof that these alterations are disease-causing mutations awaits demonstration that these alterations result in actual defects in WDR36 function. We have developed an assay to test the consequences of these WDR36 DNA sequence changes. We have found that WDR36 mutations alter cellular processes, but only when a second gene is also mutated. We will test if mutations of this second gene also cause glaucoma, and will investigate the cellular processes in which both genes are involved to determine the role of such processes in glaucoma. 

Spring 2008
Project Dates: July 1, 2008 - June 30, 2009
ATOH7 (Math5) Mutations in Optic Nerve Aplasia
Tom Glaser, MD, PhD
Associate Professor, Internal Medicine
University of Michigan Medical School, Ann Arbor
Retinal ganglion cell (RGC) neurons and their axons in the optic nerve are the targets of glaucoma disease pathology. This project studies ATOH7, a major gene discovered by the project team that controls the first step in the formation of RGCs from embryonic retinal stem cells. The project explores how mutations, identified within or near ATOH7, cause congenital absence of the optic nerve in two families. In one, they will compare the molecular properties of normal and mutant ATOH7 protein products. In the other, they will find the exact DNA change that causes this disease by high-resolution genomic analysis. Complementary studies will test whether halving the ATOH7 gene dosage affects the number of optic nerve axons. The results should help to guide future studies on RGC regeneration and optic nerve disease.


Characterization of Modifiers for Open-Angle Glaucoma by Candidate Gene Screening and Genome Wide Linkage Study
Vincent Raymond, MD, PhD
Professor, Departments of Ophthalmology and Anatomy-Physiology 
Université Laval Hospital Research Center, Quebec City, Canada
Genetic factors play a major role in the etiology of glaucoma. Fourteen chromosomal regions encode genes for primary open-angle glaucoma (POAG), the most common form of glaucoma, but only three of these genes have been identified: myocilin, optineurin and WDR36. The surprising occurrence of older individuals with healthy vision, despite the fact that they are carriers of myocilin mutations, raises the possibility that “good” genes, named protective modifier genes, maintain healthy vision by counteracting the effects of “bad” genes. The investigators recently found evidence for at least one of these modifier genes in the world’s largest known glaucoma family. The goal of this study is to discover these modifier genes. Their identification should offer novel and powerful approaches for discovering drugs to treat and perhaps prevent glaucoma.


Searching for a Molecular Mechanism to Awaken Dormant Retinal Stem Cells: A Therapeutic Approach to Glaucoma
Michal Schwartz, PhD
Professor of Neuroimmunology, Department of Neurobiology
Weizmann Institute of Science, Rehovot, Israel
While treatments are available to lower pressure in the eye, and thereby prevent continued damage from glaucoma, there is currently no cure for glaucoma nor any therapy capable of inducing cell renewal in the damaged tissue. Stem cells, which can differentiate to form numerous cell types, might be used to replace nerve cells in the retina that have been lost to glaucoma. Stem cells exist in the human eye but are dormant. Dr. Schwartz will explore the reasons why ocular stem cells are unable to divide and form new nerve cells, and to use this information as a basis for therapy aimed at awakening these stem cells in order to circumvent the need for donor stem cells.


Micro & Nanotechnology-Based Bioplatforms for High-Throughput Analysis of Axon-Glial Interactions in Glaucomatous Neuropathy
David W. Sretavan, MD, PhD.
Professor of Ophthalmology, University of California San Francisco
Better understanding of the causes of damage to the axons of retinal ganglion cells should lead to improved treatment of glaucoma. This project will develop a new type of highly versatile microplatform for glaucoma research that incorporates advances in micro and nanotechnology to provide researchers with unprecedented control over key experimental parameters. With this bioplatform, researchers will be able to conduct high-throughput experimentation simultaneously on a hundred axons, providing the amount of data that currently might require several dozen rounds of experimentation. This project will fabricate and test this new generation of micro/nano research bioplatforms with the ultimate aim of using these devices to analyze cellular communication between retinal axons and glial cells.


Characterizing Microglial Activation in a Mouse Model of Glaucoma
Xianjun Zhu, PhD
Research Scientist, The Jackson Laboratory, Bar Harbor, ME
Mice provide valuable models for molecular and mechanistic studies of glaucoma pathogenesis and for the rational development of neuroprotective therapy. DBA/2J mice provide an inherited glaucoma model that accurately reproduces many hallmarks of human glaucoma. Microglia are cells that appear to play an important role in glaucoma. However, their role is not clearly defined. This project aims at determining how the expression of various microglial genes change during DBA/2J glaucoma and to assess the relationship of these changes to glaucomatous damage. The researchers will also assess the role of a microglial enzyme in DBA/2J glaucoma. This will be one of the first experiments to functionally test the role of a specific microglial molecule in glaucoma.

Fall 2007
Project Dates: January 1, 2008 - December 31, 2008
Genetic Characterization of a Novel Canine Model of Heritable Angle Closure Glaucoma
Markus H. Kuehn, PhD
Assistant Professor, Ophthalmology and Visual Sciences
The University of Iowa, Iowa City
In primary angle closure glaucoma (PACG), the iris blocks the drainage of fluid from the eye through the trabecular meshwork. In the U.S., PACG accounts for about 10 percent of glaucoma, but in other countries, particularly in Asia, it represents the majority of cases. To date, genes associated with PACG have not been identified. The researchers recently identified a pedigree of Basset hounds afflicted with hereditary PACG, with features similar to those observed in humans. Preliminary genetic studies point to small regions of their genome which most likely contain the disease - causing mutation. The proposed project seeks to identify this mutation. Discovery of the responsible gene will enhance understanding of how this disease develops and may aid in early detection of at-risk persons and improve the ability to evaluate the effectiveness of treatment regimens.

The Role of Extracellular Matrix Interactions in Retinal Ganglion Cell Survival and Growth Factor Neuroprotection
Paulo D. Koeberle, PhD
Assistant Professor, Division of Anatomy, Dept. Surgery
University of Toronto, Ontario, Canada
Glaucoma is a progressive disease that results in the programmed cell death of retinal ganglion cells (RGCs). A number of naturally occurring proteins known as neurotrophic factors have been shown to promote RGC survival and regeneration. The therapeutic use of neurotrophic factors has been limited due to a number of factors, including the loss of effectiveness when they are delivered for prolonged periods. Dr. Koeberle’s research suggests that one factor contributing to the loss of effectiveness is the activation of enzymes that degrade the extracellular matrix surrounding nerve cells. This study will identify those critical matrix components and the signaling cascades that help promote cell survival in concert with signaling pathways that are activated by neurotrophic factors. This will lead to the development of new avenues for using neurotropic factors as effective therapeutics for glaucoma.

Genome-Wide Association Study of Normal-Tension Primary Open Angle Glaucoma
Mansoor Sarfarazi, PhD
Professor of Human Molecular Genetics
University of Connecticut Health Center
While elevated intraocular pressure (IOP) is the most important known risk factor for glaucoma, approximately 30 percent of primary open-angle glaucoma in the United Stataes can be accounted for by non-IOP dependent risk factors, most commonly referred to as normal tension glaucoma (NTG). Dr. Sarfarazi’s group previously identified a defective gene that is primarily involved with the inherited forms of NTG. But for the majority of cases no specific gene is known. This study will use a subgroup of NTG cases and a similar number of matched control subjects and scan the genome with over 1.8 million land marked DNA markers. It is anticipated that a specific DNA marker will be identified that is highly associated with the NTG phenotype. Identification of such a DNA marker will lead the researchers to a specific gene or a known biological pathway, providing an early method of detection for NTG and promoting subsequent development of an effective medical therapy.

Spring 2007
Project Dates: July 1, 2007 – June 30, 2008
Assessing Glial Activation in a Mouse Model of Glaucoma
Gareth R. Howell, PhD
Research Scientist
The Jackson Laboratory, Bar Harbor, ME
Glaucoma  is characterized by the degeneration of the optic nerve, which disrupts neurotransmission between the eye and the brain, leading to blindness. Glial cells are thought to play an important role in glaucoma. In a resting state, glial cells are supportive to neurons, but in response to stress, can become activated and damaging. It has been shown that glial cells in the optic nerve become activated in early stages of glaucoma. However, it is not known whether this a primary cause of the disease, or occurs later as the disease progresses. Due to the experimental limitations imposed with human studies, mice are valuable complementary organisms both to study the complex mechanisms of glaucoma and to develop improved therapeutics. Utilizing a mouse model that reproduces important aspects of human glaucomas, we propose to determine the timing and extent of glial activation in relation to glaucomatous damage using a combination of gene and protein expression analyses. This will be one of the most wide-ranging investigations of the role of glial cells in glaucoma to date.

Evaluation of PEX Glaucoma-Associated Autoantigens as Disease Biomarkers and the Role of their Antigenic Targets in Retinal Neurodegeneration
Derek Murphy, PhD
Associate Director, Centre for Human Proteomics
Royal College of Surgeons in Ireland, Dublin
Exploitation of the immune response of glaucoma patients has identified molecules that are of importance for diagnosis, disease development and potentially new therapies for the disease. We have established a unique collaboration between ophthalmologists and molecular biologists to develop protein arrays for the discovery of novel disease markers in glaucoma, and so contribute to the fields of diagnosis and molecular characterization of this disease. To this end, we have profiled the humoral immune responses in pseudoexfoliation syndrome (PEX) glaucoma patients, identifying disease associated autoantibodies in patients' sera. This project can contribute enormously to providing panels of unique markers for the development of a biochip assay to help in the correct diagnosis of this disease.  These markers may also provide novel therapeutic targets for the specific prevention of retinal neural degeneration in glaucoma patients.


Novel Peptides to Understand Herpetic Damage to Human Trabecular Meshwork via Actin Rich Nanotubular Structures
Deepak Shukla, PhD
Assistant Professor, Dept. of Ophthalmology & Visual Sciences
University of Illinois at Chicago 
The infection of human trabecular meshwork (TM) cells with herpes simplex virus leads to elevated intraocular pressure (IOP) and may contribute to the development of glaucoma, which is the second most common cause of permanent blindness in the United States.  HSV-1 infection into TM Is mediated by HVEM receptor in which long actin rich nanotubular structures (LARS) plays a major role during viral spread from one cell to another.  Here, we plan to isolate peptides against HVEM to prevent virus from using HVEM receptors to invade cells and to understand virus interaction with LARS during viral spread.  Our study will allow us to develop novel strategies to reduce the risk of glaucoma and prevent blindness.

Development of a Functional Assay for WDR36
Michael Walter, PhD
Professor and Chair, Department of Medical Genetics
University of Alberta, Canada
The WD40 repeat 36 (WDR36) gene has recently been identified as a new primary open angle glaucoma locus. However, the function of WDR36 and its role in glaucoma pathogenesis are unknown. One of the important challenges presented by an adult-onset disease such as glaucoma is deciding if a DNA change seen in a patient causes the disease or is instead a normal variation that is not associated with the disease. We plan on developing a test that will determine if changes of the WDR36 gene found in glaucoma patients have a functional consequence. This will allow us to determine if WDR36 causes glaucoma. Understanding the actual function of WDR36 could also provide insight into a new cellular pathway to which novel glaucoma therapies can be targeted.

Fall 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 bility- year 2
Jeffrey, L. Goldberg, MD, PhD
Bascom Palmer Eye Institute, Miami, FL
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, CA
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, Miami, FL
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. 

Spring 2006
Project Dates: July 1, 2006 – June 30, 2007
Functional Magnetic Resonance Imaging (fMRI) of Function-Specific Vision Loss in Glaucoma
Robert O. Duncan, PhD
Hamilton Glaucoma Center, San Diego, CA
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
University of Minnesota, Minneapolis, MN
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. 

Fall 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, Chicago, IL
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