2021 Shaffer Research Grants

Our study has two major findings. First, when we examined the parents, siblings and children of glaucoma patients of Haitian origin, we found that nearly one-third had suspected or confirmed glaucoma. Moreover, many of these suspected or confirmed glaucoma patients had not previously had eye examinations, so the glaucoma would have been missed if we had not performed this type of “cascade screening.” Second, we realized that the overall knowledge level of a community is the main driver of the community members’ willingness to undergo screening, and have devised a way to evaluate an entire community’s glaucoma knowledge level by performing en masse surveys during church services. Together, by utilizing the church as a way of public announcement/education and by screening close relatives of glaucoma patients, we can prevent blindness by diagnosing glaucoma in high-risk individuals early.

Previously, we showed that intraocular pressure elevation triggered macrophage/microglia activation and reactive astrocyte formation in the retina (PMID 33147455). Further, we showed that the Glucagon-like peptide 1 receptor (GLP-1R) agonist NLY01 suppressed macrophage/microglia reactivity and astrocyte transformation to rescue RGCs in a mouse model of hypertensive glaucoma. GLP-1R agonists is a popular class of medication in use for over 15 years to treat type II diabetes, a disease that increases the risk for glaucoma. Findings of our database study, by showing reduced glaucoma risk in diabetic patients who used GLP-1R agonists as opposed to other diabetic medications, lend compelling support to evaluate GLP-1R agonists as novel therapy for glaucoma, and provide a preliminary impetus for clinicians to preferentially consider GLP-1R agonists in the medical management of diabetes.

My research has the potential long-term impact to better inform which RGC types and retinal microcircuits need to be probed in patients in order to diagnose glaucoma before significant cell loss occurs. We have explored excitatory and inhibitory synapses following the induction of experimental glaucoma in mice to understand which specific circuits in the retina are most affected by intraocular pressure elevation. Our results show that loss of synapses follows different pattern in space and time for excitatory vs inhibitory circuits. This novel knowledge has the potential to identify specific circuits to focus future research in order to develop a more sensitive screening tool for glaucoma and/or monitor the most resilient functional circuits at late stages of the disease, when conventional clinical tests can no longer follow the progression of glaucoma.

The goal of this project was to determine whether neural stem cells could be used to regenerate the connections between the eye and the brain by acting as a relay for the connections damaged in glaucoma. We found that neural stem cells transplanted into the visual system made connections with the eye and that genetic manipulations could increase the number of these connections. Additionally, the transplanted neural stem cells extended connections into the appropriate location within the brain. These findings build the foundation for vision restorative treatments such as whole eye transplants.

Glaucoma is one of the three most heritable human diseases, indicating that genes are very important in its pathogenesis. We previously discovered that mutations in two genes, myocilin (MYOC) or TANK binding kinase 1 (TBK1), cause human glaucoma and we engineered models with analogous mutations in these genes and showed they also develop glaucoma. Mutations in the TBK1 gene are one of the most common, known-molecular causes of human glaucoma, however, the mechanism by which a mutation in this gene causes nerve damage and blindness is not yet known. One way to investigate the biological processes that lead to glaucoma is to examine which genes are activated as the disease develops and progresses. In this application we propose to identify the pattern of gene activation that occurs eyes that develop glaucoma due to a TBK1 gene mutation. We will use a powerful new method, single cell RNA sequencing, to analyze retinal tissue. Our experiments will show which genes are activated in key cells/tissues of the eye at various times during the development of glaucoma. This data will provide a description of what goes wrong in the eyes of patients that have glaucoma at the finest molecular level. We will use these data as roadmap to describe the checkpoints of glaucoma development and to identify opportunities to prevent or halt progression of the damage to the eye that causes vision loss in glaucoma.

Retinal ganglion cells (RGCs) connect the eye and the brain, allowing for visual perception, and these cells are known to be damaged in various blinding disorders such as glaucoma. The degeneration of RGCs is known to occur in a compartmentalized fashion, with the axonal compartment degenerating via different mechanisms than the somatodendritic compartment. While previous studies have demonstrated the ability to study certain aspects of RGC neurodegeneration with human pluripotent stem cells (hPSCs), they have lacked a focus upon the compartmentalized nature of RGC neurodegeneration. Thus, the goal of this study was to examine how RGC neurodegeneration occurs within different cellular compartments. To accomplish this, RGCs were differentiated from human induced pluripotent stem cells (iPSCs) and grown within microfluidics chips to allow for the recruitment of RGC axons away from the somatodendritic compartments, which allowed for an analysis of cellular changes within discrete compartments of RGCs. Additionally, we tested the effects of astrocytes specifically upon the axonal compartment, characteristic of glaucoma, in both quiescent and reactive states. In these studies, we found that RGCs grown in the presence of reactive astrocytes along the axonal compartment exhibited a reduction in neurite outgrowth compared to healthy astrocytes, mimicking some aspects of the phenotypes observed during the progression of glaucomatous neurodegeneration. Taken together, the results of this study demonstrate the ability to study neurodegenerative phenotypes in RGCs differentiated from human stem cells, with the development of this model in a manner that more faithfully captures the precise cellular interactions and orientations in a human cellular model. The results of this study will expand our understanding of the cellular changes that occur in human RGCs, and will support future studies including disease modeling and drug screening.

Angle closure glaucoma is a blinding condition that affects 0.5% of the world’s population. It is caused by blockage of the drainage pathways in the eye, leading to acute or chronic elevations in eye pressure and subsequent damage to the optic nerve, which sends visual signals from the eye to the brain. This can lead to swift loss of vision. Disorders of affecting the development or growth of the eye can lead to susceptibility to this subtype of glaucoma. In this proposal we studied a large family with angle closure glaucoma and small eye size, and identified a novel genetic variant in a transcription factor, which regulates the expression of multiple genes in various organ systems. We defined that this transcription factor is expressed in multiple ocular tissues, including the anterior segment, retina, ciliary body, and retinal pigment epithelium suggesting a role in eye development. We found that the variant protein in our family alters the localization of the transcription factor, but does not alter its activity in cellular assays. Through screening a large cohort of individuals with small eyes and angle closure glaucoma, we identified another patient that carries a different mutation in this gene, supporting its role in this condition. Together, our studies help to establish this transcription factor as a risk factor for angle closure glaucoma and small eyes, and pave the path to investigate a new developmental pathway that leads to angle closure glaucoma.

Glaucoma is a leading cause of visual impairment and blindness that leads to progressive dysfunction and ultimately the death of retinal ganglion cells (RGCs). The human retina contains at least 12 different RGC types, each of which is specialized to send information about specific features of the visual scene (e.g. color, contrast, form, motion) to higher visual areas in the brain. Studies in animal models indicate that some RGC types are more vulnerable to injury in glaucoma than others, suggesting that specific visual functions may be lost earlier in disease. Thus, understanding the functions of the different RGC types in the human retina is a necessary first step towards understanding changes that occur in glaucoma. In this study, we demonstrate a novel approach to link genetically defined RGC types with their specific anatomy and function. In doing so, we have identified a previously uncharacterized RGC type in the human retina which is involved in generation of reflexive eye movements that stabilize gaze as we move in the environment. Our results are expected to inform the development of improved objective clinical tests of peripheral visual function in glaucoma.

Here we propose a new strategy for glaucoma, extracellular vesicles (EVs), which possess significant neuroprotective properties, linked to hydrogels for injection into the eye for prolonged EV delivery. Our aim is to develop and optimize EVs to provide neuroprotection for RGCs. The preparations will be tested and optimized using in vitro and in vivo models. This therapy using EVs fits the goals of precision medicine. EVs derived from mesenchymal stem cells (MSCs) have neuroprotective and regenerative properties and are well suited for glaucoma treatment. We will use specific binding peptides that recognize sites on the EVs to link them to alginate hydrogels. These EV/alginate preparations will be tested in vitro. In the second aim, we will test the efficacy of the preparations in a model of glaucoma. Well-developed methods including visual function and histology, and confocal imaging in vivo and in vitro will test efficacy of these EV modifications. The rationale is that medical or surgical intraocular pressure reduction, the only clinically approved glaucoma treatment, neither prevents the main pathophysiological mechanism, retinal ganglion cell (RGC) death, nor axonal loss. Administration route, dosage, and adverse effects limit clinical application of neuroprotective agents. Moreover, the complex pathology of glaucoma necessitates action upon multiple injury mechanisms, including oxidative stress, impaired axonal transport, neuro-inflammation, and excitotoxicity. Stem cells release EVs, nanoparticles that facilitate cell-to-cell communication. MSCs-EVs decrease neuronal cell death after hypox-ia/ischemia in vitro and in vivo, stimulate axonal growth, and attenuate inflammation and oxidative stress. They can be administered cross-species, and their stability, biocompatibility, biological barrier permeability, and low toxicity make them attractive therapeutic delivery vehicles. EVs are taken up by cells; unlike stem cells, which rely upon integration into tissues, or diffusion of secreted contents to the cells, EVs deliver their cargo directly. This study is significant because it will provide the first steps toward a precision therapy for glaucoma by advantageously harnessing properties of EVs and hydrogels, combined together for the first time.