2019 Shaffer Research Grants

The production of energy in retinal ganglion cells is accompanied by metabolic byproducts, many of which are damaging to cellular function. The issue of how these byproducts modulate the excitability of retinal ganglion cells (RGCs) bears heavily on the development, impact, and early detection of optic neuropathies, including glaucoma. Our goals in undertaking this project were to test novel hypotheses about how the metabolic milieu of RGCs affects their electrical signaling of visual information. Our investigations identified the characteristics and biophysical origins of changes to the physiological properties of RGCs due to oxidizing byproducts in the retina. This new knowledge will increase understanding of both normal retinal physiology as well as the pathophysiology of glaucoma. Our novel observations support an emerging model of early stages of glaucoma where increases in oxidizing chemical species during energy production, but not necessarily bioenergetic failure, leads to preferential degeneration of certain subtypes of retinal ganglion cells, resulting in loss of specific aspects of visual capabilities.

Very little is known about how and why the optic nerve is progressively damaged in glaucoma. Conditions known to cause glaucoma such as elevated intraocular pressure (IOP), 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, and muscle). Conditions known to cause glaucoma such as elevated IOP are associated with increased small proteins known as endothelins. Data obtained in the current study suggests that elevation of IOP in rats resulted in the activation of endothelin-producing enzymes known as ECE. Therefore, reduction of ECE activities may be a novel therapeutic in the treatment of glaucoma. 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.

New genetic material can be delivered to cells of the cornea via injection of a viral vector into in to the front chamber of the eye. Whilst this method is largely effective, it substantially increases the risk of patients developing complications, including eye infections, such as endophthalmitis or uveitis. Furthermore, injecting extra fluid into the eye leads to a transient, but dramatic, spike in intraocular pressure that may potentially accelerate vision loss in patients with glaucoma. In this project we explored an alternative gene delivery approach wherein the virus vector was attached to the inside of a contact lens, rather than being injected, and then the contact lens placed on the front of the eye for a brief period (30 minutes). Our main findings were that virus vectors immobilized on to a contact lens remain active, they stay in close contact with the cornea for long periods of time, and are able to penetrate the cornea and successfully deliver new genetic material effectively. These findings represent a positive step towards the development of a non-invasive gene therapy treatment for glaucoma.

Retinal ganglion cell (RGC) death is the hallmark for glaucomatous damage leading to irreversible vision loss and no curative treatments are available. RGC replacement therapy has the advantage of being applied in patients to restore vision. We have identified a set of five chemicals (5C) that can convert fibroblasts into chemically induced RGC-like cells (CiRGCs). These CiRGCs express cardinal genes and function in dish similar to their native counterparts. When transplanted into a rodent glaucoma model, CiRGCs can restore retinal function. CiRGC carries extraordinary translational potential and offer hope for patients with severe, advanced glaucoma who do not have enough residual functional RGCs to be a suitable candidate for traditional gene therapy.

Although glaucoma is frequently treatable with medication or surgery, a large proportion of afflicted individuals are diagnosed too late to prevent the death of key retinal neurons, which are called retinal ganglion cells (RGCs). As RGCs are the only neurons that transmit information from the eye to the brain, their loss results in permanent vision impairment. Although RGCs cannot be naturally regenerated, they can be produced artificially from retinal stem cell cultures. Stem cell cultures potentially offer the promise of recovery for vision loss. However, in order to bring this approach to the clinic, we will need to greatly improve our ability to produce these cells efficiently. We have been developing an approach that will allow RGC production to be enhanced. We have identified several genetic modifications that enhance RGC production, using genes that have been shown to perform this function during the natural development of the eye. Applying these genetic modifications to retinal stem cell models is expected to facilitate the production and transplantation of RGCs for glaucoma therapy.

The GC1 murine model of glaucoma aligns well with the pathophysiology seen in primary open angle glaucoma patients; the disease progresses with age, and mice have moderately elevated IOP, leading to degeneration of the optic nerve. This mouse model is novel, and the work completed in this proposal has already highlighted significant transcriptional changes that may underlie the pathology observed in these mice, and thus may eventually translate to human subjects. In the short term, investigating further the role of cGMP in glaucoma will highlight pertinent pathways in glaucoma pathology that may be targeted with pharmaceuticals to delay progression of the disease. We have already established that an FDA-approved drug, tadalafil, prevents neurodegeneration in two mouse models of glaucoma. We now aim to investigate the mechanisms behind this finding in order to highlight additional downstream drug targets. We are attempting to undertake the first step in this vision; by augmenting inflammatory pathways with drugs in murine chow, we aim to investigate whether cGMP neuroprotection is dependent on inflammatory modulation, and whether this leads to increased degeneration/neuroprotection of retinal ganglion cells. In the long term, this work furthers the exciting finding that modulating cGMP signaling increases retinal ganglion cell survival. The RNA sequencing analysis will provide a wealth of data, which will be published and an open resource for mining, which may highlight other relevant gene targets for future investigation by us and other groups.

Current glaucoma treatment strategies only target IOP, the principal treatable risk factor. Neuroprotective treatments for glaucoma are of great therapeutic need. We have recently discovered a role for nicotinamide (a form of vitamin B3) in protecting the optic nerve in glaucoma by targeting these metabolic processes that change in the retina early during glaucoma. Nicotinamide is well tolerated, inexpensive, and robustly protective and thus may be an ideal therapeutic candidate for human glaucoma patients. To this, recent evidence demonstrate that nicotinamide is low in the sera of primary open angle glaucoma patients and we have been part of a multi-national collaborative group that has demonstrated that oral nicotinamide can increase visual function in existing glaucoma patients. Given nicotinamide’s obvious clinical utility, more research is warranted on understand that exact roles of nicotinamide in retinal ganglion cell and optic nerve health in aging and disease. In this project we have demonstrated that mitochondria rapidly change in morphology during retinal ganglion cell injury at time points prior to gross cell death. In both mouse and rat systems that mimic glaucoma-related insults we report that nicotinamide prevents these early mitochondrial changes and protects from retinal ganglion cell neurodegeneration, extending our knowledge about nicotinamide’s role in glaucoma.

Dr. Williams was awarded the 2021 Shaffer Prize for Innovative Glaucoma Research. The Shaffer Prize, presented annually by Glaucoma Research Foundation, recognizes a researcher whose project best exemplifies the pursuit of innovative ideas in the quest to better understand glaucoma.