A major factor limiting the use of mice to study glaucoma is the scarcity of high-IOP mutants. To correct this, we are collecting a panel of new mouse mutants with altered IOP and glaucoma. We are currently developing more than 30 new mutants. By characterizing these mutants, we will provide valuable insights into the mechanisms determining IOP and leading to glaucoma.
As examples, we determined that mutations in one gene (Tdrd7) impede the formation of intracellular structures known as stress granules. Our findings suggest that the mutated gene causes increased susceptibility to oxidative stress, resulting in IOP elevation with age. In humans, mutations in the same gene caused cataracts and glaucoma in some patients. Current data also suggest that mutations in this gene alter susceptibility to glaucoma following pediatric cataract extraction. As another example, we determined that mutations in the gene encoding a novel protease (Prss56) cause angle closure glaucoma (ACG) in mice, and ACG with reduced ocular size in human patients. ACG is a subset of glaucoma affecting 16 million people and blinds more people worldwide than any other form of glaucoma. We continue to investigate the normal functions of this gene and its role in disease.
Abnormal resistance to aqueous humor drainage through the ocular drainage structures underlies higher IOP in glaucoma, but is poorly understood. We have developed new methods and resources to study drainage structure development, microanatomy and function. We focus on the trabecular meshwork and Schlemm’s canal (SC), with a major focus on SC. Current effort is focused on understanding the source of resistance and regulation of aqueous humor drainage. We have characterized the detailed development of Schlemm’s canal and discovered that it is a novel class of vessel with features of both lymphatic and blood vessels.
We have characterized roles for various genes that cause developmental glaucoma, including genes for transcription factors (Foxc2, Foxc2, and Lmx1b), bone morphogenetic protein (Bmp4), collagen type IV α1 (Col4a1), cytochrome p450 enzyme (Cyp1b1) and the TEK tyrosine kinase receptor (angiopoietin receptor). Mutations in all of these genes cause human glaucoma associated with developmental abnormalities (developmental glaucoma).
Despite the developmental abnormalities in human patients, and for unknown reasons, IOP usually becomes elevated during young adulthood. We plan detailed characterization of drainage structure development and function in mice with mutations in selected developmental glaucoma genes. These studies will greatly advance our molecular understanding of the mechanisms responsible for elevated IOP. We also characterize modifier genes that modulate developmental glaucoma phenotypes. Importantly, we identified the tyrosinase gene as a modifier whose deficiency exacerbates defects in both Cyp1b1 and Foxc1 mutant mice. Tyrosinase provides L-DOPA (L-3,4-dihydroxyphenylalanine), that protects against the developmental abnormalities.
In pigmentary glaucoma (PG), abnormally dispersed iris pigment and cell debris enter the ocular drainage structures, leading to increased IOP. DBA/2J mice develop a form of PG. The glaucoma results from mutations in related genes encoding melanosomal proteins (Gpnmb and Tyrp1). The mutant genes alter melanosomes, allowing toxic intermediates of pigment production to leak from these structures, causing iris disease and subsequent PG. To induce glaucoma, the Gpnmb mutation must also be present in immune cells. Replacing the bone marrow (which gives rise to immune cells) of DBA/2J mice with normal donor bone marrow restored important aspects of ocular immunity and prevented the disease components associated with the Gpnmb mutation. These findings are the first evidence that an abnormal immune response may contribute to development of PG. Not all mouse strains with pigment dispersion are equally susceptible to developing PG. We are characterizing the strain differences that determine whether pigment dispersion leads to high IOP and PG. The genetics of the progression from pigment dispersion to high IOP and PG is complex, involving at least seven loci, and we are working to identify the most important genes.
Our understanding of IOP and its roles in glaucoma will greatly benefit from improved methods of data collection and far denser data sets. Thus, we work with engineers to develop new technologies to allow continuous IOP assessments using wirelessly powered and remotely interrogated implantable technology. To allow treatment we have also collaborated to develop new methods of controlling IOP. We are characterizing the innervation of the ocular drainage structures and have characterized parameters that allow the use of electrical stimulation to decrease IOP. We are also collaborating to develop a new human glaucoma implant that would increase ocular fluid drainage but have a controllable resistance and self-cleaning properties to remove biofouling. Although further development is required, systems combining such devices may allow the future control of IOP through closed-loop biofeedback.
The neurobiology of pressure-induced cell death in glaucoma is poorly understood. DBA/2J mice provide a tractable model for dissecting pathways of cell death in glaucoma and for investigating neuroprotective strategies. Using DBA/2J mice, we determined that distinct degeneration pathways kill the RGC soma and axons. The proapoptotic molecule BAX is required for RGC death but not for axon degeneration. Resolving a long-standing issue, we experimentally demonstrated that the RGC axon is locally insulted in the optic nerve in glaucoma. The insult occurs within a small and specialized region of the optic nerve head that is rich in astrocytes. This suggests that abnormal neuroglial interactions may be critical in glaucoma.
To better understand the molecular pathophysiology of early glaucoma, we developed an approach that exploits gene expression data to characterize very early stages of this complex disease. We succeeded in characterizing a temporally ordered sequence of glaucoma stages, including stages that precede conventionally detectable damage. Our studies are providing key new insights into glaucoma and identifying potential therapeutic targets. For example, we demonstrated that the complement cascade and endothelin systems are up-regulated very early in glaucoma and may contribute to axon injury. Abrogating the function of both complement component C1qa and endothelin receptors is highly protective, highlighting the power of targeting early pathologic events.
Additionally, we discovered that a single radiation treatment profoundly protects DBA/2J mice from developing glaucoma (96 percent of eyes completely protected in the initial study). We are not aware of any other neuroprotective effects this substantial. Localized x-ray irradiation of a single eye is sufficient to protect that eye from glaucoma, but does not protect an untreated fellow eye. Thus, the protective effect is local and not mediated by a circulating factor. We discovered that a subclass of immune cells known as monocytes enters eyes at a very early stage of glaucoma that precedes neural damage. These cells may be necessary to initiate or propagate neural damage in glaucoma, and the radiation treatment modifies eyes so that these cells do not enter, but further studies are required to fully understand their importance.
Recently, we discovered that levels of nicotinamide adenine dinucleotide (NAD) decrease in the aging retina. NAD is a central molecule in energy metabolism, in mitochondrial health and in other critical cellular processes. We hypothesized that decreased NAD renders retinal ganglion cells susceptible to damage when IOP becomes elevated, thus permitting glaucoma development and progression. This is consistent with our discovery that mitochondrial abnormalities are among the very first detrimental changes to occur within retinal ganglion in a mouse model of glaucoma. Importantly and supporting our hypothesis, we continued to show that treating DBA/2J mice with nicotinamide, the amide form of vitamin B3, increases NAD levels and profoundly protects from glaucoma. This has prompted a significant focus on the role of metabolism in mouse and human glaucoma.