2020 Shaffer Research Grants

This study examined the composition of the insoluble material that accumulates in the eyes of patients with pseudoexfoliation syndrome and pseudoexfoliation glaucoma. We found that the material consists largely of three proteins, fibrillin-1, LOXL1, and LEFTY2. We also examined the composition of aqueous humor collected from the eyes of control and affected individuals, and found that the concentration of LEFTY2 was significantly higher in those patients with pseudoexfoliation glaucoma. Future experiments will evaluate the use of LEFTY2 as a biomarker for the condition and examine its role in disease progression.

The use of IOP-lowering drugs, the current mainstay treatment for glaucoma, is often insufficient to prevent progressive visual loss in patients. Therefore, recent work on potential glaucoma treatments have shifted to assessment of neuroprotective strategies to promote neuronal survivability and thereby preserve visual function. Like other neurodegenerative diseases in the brain, glaucoma in humans and animal models often follows a course of initial cell death due to the primary insult followed by a progressive secondary loss, the latter which can ultimately be responsible for most cell loss and the resulting visual dysfunction. Our strategy is therefore to determine the mechanism(s) responsible for secondary cell loss in glaucoma to create novel preventive treatments. Our recent work has shown directly that gap junctions (GJs), portals that connect neighboring retinal neurons, play a crucial role in the propagation of secondary cell loss under a number of insult conditions, including glaucoma, in mouse models. Importantly, blockade or genetic ablation of GJs in glaucomatous mouse eyes models largely prevents secondary cell loss and thereby preserves visual function. Toward our ultimate goal of creating novel neuroprotective treatments for glaucoma patients, it is important to determine whether our results in a rodent model of glaucoma can be recapitulated in non-human primates. We therefore created a new glaucoma model for the New World monkey marmoset to determine whether blockade of GJs can protect retinal neurons and optic nerve axons to thereby prevent visual impairment associated with the disease. Our preliminary results using the new non-human primate model supports our hypothesis that retinal GJs form targets for innovative treatments to preserve cell health and visual function in glaucoma.

The subconjunctival space of the eye is often used in the treatment of eye diseases. Drugs can be injected there. However, the drugs disappear or exit the subconjunctival space so that the drug-effects wear off. Glaucoma surgeries open the subconjunctival space to lower eye pressure by allowing fluid inside of the eye to exit. However, these surgeries can fail where the intraocular fluid becomes trapped and can no longer exit the subconjunctival space. Therefore, it is important to understand fluid outflow from the subconjunctival space. If this process can be slowed down, better drug delivery can be developed. If this process can be enhanced, glaucoma surgeries may work better. In this proposal, we proposed to understand the nature of subconjunctival outflow. Using donated eyes from multiple species (including humans), we identified that lymphatics are the biological structures which drain the subconjunctival space. We then confirmed this in live human subjects. Then, we used mouse models to demonstrate that subconjunctival lymphatics can be positively and negatively manipulated using various drugs. Therefore, in the future, we hope to be able to use subconjunctival lymphatic manipulation as a way to improve the treatment of eye diseases through better drug delivery and glaucoma surgery.

Retinal ganglion cells communicate information from the retina to the brain via the optic nerve. Ganglion cells degenerate and eventually die in glaucoma by mechanisms that are not fully understood. The mainstay of glaucoma therapy is lowering the intraocular pressure (IOP), but this is not effective in all cases. It would therefore be important to identify therapies that protect retinal ganglion cells by a different mechanism. Ganglion cells and their axons in the optic nerve are surrounded by a meshwork of supporting cells – astrocytes – that respond to nerve injury by initiating a response that attempts to aid and protect the neurons in their vicinity. The main purpose of our work is to stimulate protective responses of astrocytes and use them therapeutically. We identified the transcription factor Runx1 as one of the regulatory molecules that is expressed in astrocytes and mediates some of the functions of reactive astrocytes. Directly stimulating Runx1 itself would have an influence on many genes and pathways and might lead to side effects such as the growth of aberrant blood vessels. It would therefore be preferable to find downstream targets of Runx1 that have a more specific neuroprotective effect. In our work, we have identified several candidate molecules and we have shown neuroprotective activity for at least one of them.

Glaucoma is defined as a type of optic nerve damage which cannot be reversed at the present time. Many factors can lead to susceptibility of optic nerve damage. We hypothesize the stability and integrity of the tissues surrounding the optic nerve is one of the key factors. Supported by GRF, we used cutting edge technology, Atomic Force Microscopy to test this hypothesis using our mouse model with known systemic tissue abnormalities caused by genetic defect. We discovered the tissue surrounding the optic nerve is stiffer which is commonly seen in human patients suffering from glaucoma and is also an aging process. We plan to determine the causality of tissue stiffness in glaucoma using our established mouse model and techniques. This may provide an opportunity to new treatment.

Glaucoma is one of the most common causes of blindness. Although researchers have been studying its underlying biological basis for decades, blindness still occurs far too often. New scientific breakthroughs have provided unique opportunities to study the disorder from a fresh perspective, with the ultimate goal of discovering novel therapies. One breakthrough is the development of induced pluripotent stem cell (iPSC) technology, which allows investigators to turn white blood cells or skin cells into virtually any other cell type in the body – including eye tissue. Another breakthrough is the ability to analyze the expression pattern of every gene in a cell, which provides a window into the function of that cell, and how that function goes awry in disease. In this proposal, we plan on generating eye tissue from iPSCs made from individuals with Lowe Syndrome, a rare genetic disorder that leads to congenital cataracts and glaucoma. We will study the gene expression pattern in eye tissue derived from the iPSCs to find novel pathways involved in the development of glaucoma.

Loss of vision in patients with glaucoma is the result of disconnection between the eye and brain. The death of the cells connecting the eye to the brain causes this blindness. Mammalian models of glaucoma result in similar cell death to that seen in humans while zebrafish cells are resistant to cell death under similar conditions. We tested whether the key cell death gene BAX behaves similarly in human and zebrafish cells. Surprisingly, we found the behavior of these genes as they pertain to localization to the cytoplasm (survival) and mitochondria (death) were similar in both species. This suggests that the protection from cell death seen in zebrafish might be downstream from BAX. Future identification of this protective mechanism might suggest ways to prevent cell death in glaucoma patients.

A major impediment to the development of new treatments to preserve and/or restore vision in glaucoma is the large number of potential therapeutic targets to be evaluated. This frequently involves significant expense and time for each candidate target. We have devised a strategy to allow for the assessment of multiple candidates at once in terms of their potential to protect the retina and optic nerve from further degeneration and their potential to aid in restorative therapies. We have optimized several key components of that strategy in essential proof-of-principle experiments.