Shaffer Research Grants

Glaucoma is a leading cause of blindness in the United States and affects nearly 80 million people worldwide. Glaucoma results from increased pressure in the eye. Drainage structures in the eye regulate this pressure. In several cases of glaucoma and especially congenital glaucoma, drainage structures are affected. To address this, we need to understand the genetics of drainage structure development. We have identified an important signaling system in drainage structure development and will determine its mechanistic role in development using genetic mouse models. A mechanistic understanding will lead to the development of new treatment targets for congenital glaucoma.

Ocular structures, including the ciliary body, trabecular meshwork, and retina are insulin-sensitive tissues that express aquaporin water channels. Recent epidemiological studies suggest that people with insulin resistance (i.e., diminished cellular sensitivity to insulin) often have higher eye pressure than people with normal insulin signaling. However, how insulin resistance leads to elevated eye pressure and its implications on the retinal ganglion cell death in glaucoma remain largely undetermined. This project will investigate the underlying processes of insulin resistance-induced glaucomatous changes in the eye and the visual pathway by examining the aquaporin-4 water channel and cell damage in the ciliary bodies, trabecular meshwork, inner retina, and optic nerve axons upon direct exposure to a specific insulin receptor signaling blocker. We will develop cell and animal models to test the hypothesis that insulin resistance facilitates glaucomatous damage of the eyes and the visual pathway via the aquaporin-4 water channel, leading to increased aqueous inflow, decreased aqueous outflow, and disrupted cerebrospinal fluid dynamics that can increase eye pressure, decrease metabolic brain waste clearance, and induce retinal ganglion cell damage and vision loss. The outcomes of this study will be of impact in determining if insulin resistance and aquaporin-4 are cardinal to glaucoma, and whether targeting insulin receptor signaling and water channel function in the eye and the visual pathway can be a therapeutic regimen for glaucoma.

Glaucoma is an optic neuropathy where there are progressive changes to the optic nerve head; however, all current treatments are directed towards eye pressure lowering. Though there are many exciting drug, gene, and cell therapies that may treat the optic neuropathy underlying glaucoma, there is no FDA-approved or research method to achieve targeted optic nerve head delivery. We have pioneered optic nerve head delivery systems in rabbits and are seeking to extend this to animals with more similar optic nerve heads to humans. The goal of this research is to develop a targeted optic nerve head delivery system in pigs, which share several key features with human optic nerve heads. The Shaffer Grant will be instrumental in performing the necessary studies to eventually translate targeted drug delivery systems for the optic nerve to humans. Successful completion of this project will enable future development of paradigm-shifting therapies for glaucoma treatment.

Glaucoma is a leading cause of vision loss and blindness, characterized by the progressive degeneration of retinal ganglion cells (RGCs), the projection neurons of the retina that convey visual information to the brain. Aging is a risk factor for glaucoma development and progression likely due to the increased susceptibility of aged RGCs to age-related insults. Importantly, the aging process is also characterized by the increased presence of reactive and dysfunctional astrocytes, which are known to play a prominent role in neurodegeneration and neuroinflammation in several contexts. Hence, we hypothesize that aged RGCs exhibit increased susceptibility to the neurotoxic effect promoted by reactive astrocytes. Animal models are powerful systems to study aging phenotypes, however, they often insufficiently mimic the degenerative state found within patients. Human pluripotent stem cells (hPSCs) constitute a physiologically relevant in vitro human system that overcomes differences between rodent models and human cells. Thus, hPSC-derived RGCs and astrocytes will be used to test our hypothesis in this study. hPSC-derived RGCs will be incubated with a small molecule able to induce age-related features in RGCs. Furthermore, to better model the highly compartmentalized nature of RGCs and the specific location of reactive astrocytes in the axonal compartment, microfluidic platforms will be employed to examine how reactive astrocytes affect age-related RGCs, in a human model relevant to glaucoma and aging. This project will allow the establishment of an in vitro model of age-related human RGCs and to understand the role of reactive astrocytes in RGC degeneration throughout aging. This study will also open the avenue for the identification of potential pharmacological targets and therapeutic strategies to protect RGCs and prevent or delay the progression of glaucoma.

Elevated pressure within the eye (IOP) is a major risk factor for the development of glaucoma and lowering IOP remains the only treatment for this disease. However, continued disease progression in patients with reduced IOP indicates that IOP-independent mechanisms contribute to glaucoma. In the healthy eye, inflammation is tightly regulated to protect the delicate tissues necessary for vision, but in glaucoma inflammation in the eye becomes dysregulated and in animal models of glaucoma, microglia activation and inflammation persist even after the IOP has been successfully lowered. Microglia are considered the resident immune cells of the retina and are responsible for normal maintenance of the retina, as well as the local response to injury. Chronic activation of microglia is thought to contribute to the death of retinal ganglion cells (RGCs) in glaucoma by promoting harmful inflammation and in animal models of glaucoma, the extent of microglia activation correlates with the extent of RGC death and optic nerve degeneration. We previously identified a protein, soluble Fas ligand (sFasL), that is essential in preventing inflammation in the healthy eye. Microglia activation and the development of glaucoma coincides with the loss of this protein. Therefore, we hypothesized that sFasL is essential in preventing retinal microglia activation and with the loss of sFasL in glaucoma, the microglia become chronically activated, promoting neurodestructive inflammation that continues even after the IOP is lowered. The question we ask in this project is whether treatment with sFasL after glaucoma injury could “restore” the homeostatic phenotype of activated retinal microglia and prevent the continued progression of disease.

Glaucoma and optic neuropathies causing optic nerve degeneration and retinal ganglion cell (RGC) death are the leading causes of blindness worldwide. However, there are no clinical therapies that halt optic nerve degeneration, RGC death and/or promote axon regeneration. Toward establishing the clinical regenerative therapy, we previously identified an FDA-approved drug, statin as a reagent promoting retinal axon regeneration after optic nerve injury in mice. However, the regenerative and neuroprotective outcomes were still limited. To overcome these issues, experiments in this proposal will focus on the novel combination therapy with statin and matrix bound nanovesicles (MBV), a distinct class of extracellular nanovesicle localized to the extracellular matrix of healthy tissues. We will clarify the molecular mechanisms of how the combined treatment-induced immunomodulation regulates regenerative and neuroprotective outcomes after optic nerve injury in mice. The results of this study will lead to a significant advance in the identification of the novel regenerative therapeutic intervention.

Glaucoma is an eye disease that causes degeneration of the optic nerve and the progressive death of retinal ganglion cells. In most cases, damage to retinal cells begins with increased intraocular pressure. However, treatments that relieve this pressure are not enough to stop the progression of retinal degeneration. Current studies suggest that glaucoma is exacerbated by dysfunctional and over-worked mitochondria, cellular organelles that produce energy and help to maintain cellular homeostasis. The numbers of mitochondria in retinal ganglion cells are extremely high compared to most cells of the central nervous system, and are especially dense in the thin, unprotected portions of axons just at the origin of the optic nerve. In conditions of elevated intraocular pressure, these axons can be damaged, which directly reduces the supply of oxygen and nutrients to the retinal ganglion cells and elevates oxidative stress, which further impairs mitochondrial function. We hypothesize that by preserving the mitochondrial membrane potential, cellular metabolism will be preserved, and retinal ganglion cells and their axons will be protected from damage. Our study uses novel, mitochondria-targeted, high-density aromatic peptide analogs that we hypothesize will protect retinal ganglion cells and the optic nerve in the DBA/2J model of glaucoma. These compounds have potential to promote mitochondrial function in vulnerable neurons and prevent neurodegeneration and blindness in glaucoma patients.

Glaucoma, a leading cause of irreversible blindness, exhibits varying progression rates which are thought to be influenced by both genetic and racial factors. Previous research indicates that individuals of African descent may experience a more accelerated progression of the disease compared to those of European ancestry. Using a score based on many genes (Polygenic Risk Score or PRS), researchers have tried to predict how quickly glaucoma might worsen in a patient. However, these studies have focused on people of European descent. Our Proposal aims to assess whether these gene-based scores are equally effective at predicting glaucoma progression in people of different racial backgrounds. We also aim to investigate whether a combination of PRS with other clinical measurements (like eye pressure) results in more accurate prediction models for glaucoma progression. If successful, this proposal will help better predict which patients are at higher risk of faster glaucoma progression. This could lead to personalized treatment plans and more frequent monitoring for high-risk individuals, ultimately aiming to preserve vision for as long as possible.

Irreversible blindness from glaucoma in the young is a human tragedy especially in resource constrained settings of Sub-Saharan Africa. It takes a tremendous toll on individuals in the prime of their life, their families, and the societies in which they live. No other population group in the world suffers from such high prevalence of an aggressive form of early-onset glaucoma. Our study hopes to identify both known and unknown genes that cause early-onset glaucoma in Africa and identify the frequency of occurrence of these genes. We will describe the phenotypic presentation of early-onset glaucoma in both related and unrelated cohorts; and determine the effect of these mutations on the phenotypic presentation of this type of glaucoma. A detailed understanding of the genetic basis of early onset glaucoma among Africans will provide better ways to identify at-risk individuals early and optimize their clinical treatment to prevent blindness from glaucoma.

Glaucoma is a common condition and a leading cause of blindness. Currently, therapeutic interventions for glaucoma are designed to lower intraocular pressure (IOP), since elevated IOP is a major risk factor of glaucoma and successful/sustained IOP lowering slows disease progression. The drainage system at the anterior angle of the eye helps to carry away fluid produced to maintain the eye shape and function. If the drainage system is blocked, it leads to accumulation of fluid and IOP elevation inside the eye. Based on prior work in our lab, over-expressing the specific small RNAs (microRNAs) could suppress a gene (TSP1) expression. Concomitantly, we found that TSP1 inhibition was shown to facilitate the mice drainage system (outflow of aqueous humour) more than 50%. The goal of this proposal is to provide a preclinical proof-of-concept that increased specific microRNAs improves IOP regulation. If the fundamental procedures to be tested show promising outcomes in the proposed experiments, this novel approach may have broader implications for treatment of blindness caused by glaucoma in clinical settings.