2022 Shaffer Research Grants

We identified a potential therapeutic target for treating RGC loss and axon degeneration in glaucoma and other optic neuropathies. Our results suggest that Tppp3 is an RGC marker that promotes RGC survival and axon regeneration in an optic nerve trauma model, which can be further combined with other strategies for glaucoma and other optic neuropathies.

Ocular hypertension (OH) is the major risk factor for glaucoma, the leading cause of irreversible blindness. Lowering intraocular pressure (IOP) is the only proven treatment. All forms of OH are caused by increased outflow resistance via the conventional outflow pathway comprising the trabecular meshwork (TBM). In open angle OH, pathology is intrinsic to the TBM. This research project focuses on a secondary form of open angle OH caused by treatment with pharmaceutical glucocorticoids (GCs) in the eye. We hypothesize that a protein called ELAVL1is responsible for stabilizing fibronectin mRNA in response to GCs. We will investigate this idea in cultured TBM cells by perturbing ELAVL1 gene expression and assessing the effects on fibronectin stability in the presence and absence of dexamethasone. Study results will be of high impact if our hypothesis is correct, providing the preliminary data for a full study to validate the mechanism in vivo and determine the mechanisms regulating ELAVL1. The ultimate goal is to learn whether GCs directly stimulate expression of a gene, via its GRE, that is responsible for ELAVL1 activation.

Glaucoma is a neurodegenerative eye disease that is a leading cause of irreversible vision loss around the world. Higher than normal eye pressure is the most common risk factor for glaucoma, but the mechanisms linking high eye pressure and eventual blindness are not well understood. We tested the hypothesis that pressure-induced changes to the physical structure of glial cells in the retina early in disease progression contributes importantly to the eventual degeneration of retinal neurons. Using a mouse model of glaucoma, we found that glial cells shrink in size soon after the onset of elevated eye pressure. This glial shrinkage may contribute importantly to the eventual death of retinal neurons and vision loss.

Schlemm’s canal (SC) is a circular channel located in the eye’s anterior chamber responsible for regulating the outflow of aqueous humor. Elevated intraocular pressure (IOP) induces fluid flow-induced shear stress on SC cells, triggering nitric oxide (NO) production by SC to regulate IOP homeostasis. Our project aimed to elucidate the role of primary cilia (PC) in regulating NO production in SC cells. We have made significant progress in this direction, demonstrating that PC regulate endothelial nitric oxide synthase in SC cells and developing an optimized monitoring system that can measure NO production and dynamics in SC cells under shear stress conditions. Additionally, our findings indicate that the phosphoinositide 3-kinase pathway is critical for eNOS activation in response to shear stress in SC cells. We are currently working on generating mice with disrupted PC in SC cells to investigate the effects of this disruption on IOP regulation. We expect that our results will be essential for a comprehensive understanding of the regulatory mechanisms that govern IOP homeostasis and facilitate the development of novel drugs for more efficient and safe treatment of ocular hypertension and glaucoma.

Glaucoma is characterized by the damage and death of retinal ganglion cells (RGC) and their retinal nerve fiber layer axons, leading to vision loss. Clinicians rely on diagnostic tests, such as visual field studies and optical coherence tomography (OCT), to diagnose and monitor glaucoma. However, these tests are currently insensitive to detecting glaucoma early and monitoring minute changes in its progression. This proposal will address this need by designing and testing a new OCT imaging device, called visible-light OCT (vis-OCT). Vis-OCT uses visible wavelengths (560 nm +/- 50 nm) to provide ultra-high resolution, three-dimensional imaging (1 to 2 microns) of the retinal layer anatomy. The ultra-high-resolution of vis-OCT enables sub-laminae to be distinguished within the inner plexiform layer (IPL), a site where RGC’s synapse to other retinal neurons. The ability to discern these connections opens the possibility of sensitively detecting RGC damage by measuring the thickness of the sub-laminae. Vis-OCT imaging can be technically challenging due to artifacts from patient eye motion. To solve this issue, we will construct a vis-OCT device with fundus tracking, enabling the acquisition of high-quality images even in the presence of eye motion. Using this novel device, we will image IPL sub-laminae and make measurements of the thicknesses in varying glaucoma stages. This study will provide insight into whether we can detect RGC damage at ultra-high resolution in the IPL. Ultimately, vis-OCT could offer a new imaging biomarker for detecting glaucoma early and monitoring its progression.

Vision loss in glaucoma results from damage to the retinal ganglion cells (RGCs), which are the neurons that connect the eye to the brain. RGC axons from throughout the retina converge at the optic nerve head to exit the eye. A critical insult to RGC axons occurs at the optic nerve head, where they interact extensively with another resident cell type, astrocytes. Astrocytes are thought to play both neuroprotective and neurotoxic roles in complex neurodegenerative diseases, such as glaucoma and Alzheimer’s. Our studies will provide important molecular information and tools for manipulating optic nerve head astrocytes to understand and treat glaucoma. We will use cutting edge single-nuclear RNA sequencing technology to dissect out the molecular pathways that define neurotoxic versus neuroprotective astrocytes in optic nerve heads of glaucoma and normal mouse eyes. We will also develop new viral tools for selectively manipulating genes in optic nerve head astrocytes that can be used for gene therapy approaches by the glaucoma research community. These studies will pave the path for the development of a new avenue of glaucoma therapies directed towards optic nerve head astrocytes.