This research was supported by BrightFocus
The week of July 20-24 brought the International Society for Eye Research (ISER) biennial meeting to U.S. shores. In San Francisco, ISER 2014 drew 800 scientists from 37 countries to hear about the latest research into the world's leading vision diseases.
Most of the eye experts who attend ISER are directly involved in research activities. They were there to learn, to cross-pollinate their own research, and to present findings.
It's an elite group and, impressively, a third of current National Glaucoma Research (NGR) grantees (12 out of the 35 researchers) were presenters at ISER, displaying their work alongside former grantees and members of the BrightFocus Scientific Review Committee who also shared their latest work.
It can’t all be described in this space, but here’s a partial recap of ISER 2014, focusing on early-stage discoveries aimed at treating and curing glaucoma.
All Eyes on Trabecular Meshwork
Gaze deeply into someone’s eyes, and you won’t see their trabecular meshwork (TM). Nonetheless, this built-in drainage system tends to the eye’s health and comfort. The TM is nature’s way of disposing of aqueous humor, the viscous fluid that continually moistens delicate eye tissue and helps maintain intraocular pressure (IOP).
After circulating through the eye, aqueous humor reaches the TM by sliding through a microscopically thin passageway beneath the cornea. Next, it drains through the TM’s spongy membranes and collects in a connecting passageway known as Schlemm’s canal (SC), where is absorbed into blood vessels and eliminated through the bloodstream.
When people speak of the “outflow pathway” in glaucoma, the TM is what they mean. The TM handles the bulk of fluid volume, about 80 percent, and also regulates flow; it’s here where the greatest resistance to aqueous flow is has been measured. To an increasing degree, glaucoma researchers are focusing on the TM and SC, as places where things go wrong and lead to IOP elevations associated with glaucoma.
Counting the Pores in Schlemm's Canal
A 2013-15 BrightFocus grantee, Darryl Overby, PhD, of Imperial College, London, and co-investigator Ernst Tamm, MD, of University of Regensburg, Germany, have investigated factors that control pore formation in the eye’s drainage pathway. In particular, their work focuses on a single layer of cells that forms the outer layer of Schlemm’s canal, known as SC endothelium. Normally, the aqueous humor collecting in Schlemm’s canal passes through this thin, porous lining and is dispersed to blood vessels. If the SC endothelium has too few pores, that could impede fluid drainage from the eye, and raise intraocular pressure (IOP)
Overby reported on experiments involving SC cells cultured from eyes donated by glaucoma patients and normal controls. SC cells in glaucomatous eyes had reduced pore-forming ability and increased stiffness, compared to controls. In separate experiments, stiffness was associated with additional variables believed to contribute to endothelial dysfunction, including tissue changes known as stress fibers and focal adhesions. All these factors may be involved in IOP elevation. These researchers identified at least 10 genes associated with these changes, and also reported on the elevated expression of several proteins that may be involved.
Understanding the TM Homeostasis
Janice Vranka, PhD, of Oregon Health Sciences University, is investigating the hemodynamic properties of the TM. Vranka is a 2014-16 BrightFocus grantee.
She described the TM as a versatile structure that maintains IOP by altering resistance to aqueous humor outflow. It does so primarily by exerting changes in the extracellular matrix (ECM), or molecules lying outside of cells that provide structural and biochemical support to surrounding cells. When pressure is elevated, TM cells sense that and respond with an attempt to restore IOP to acceptable levels--a dynamic adjustment process called IOP homeostasis.
Complicating this picture, the TM has the ability to respond differentially to elevated IOP. The eye drains in a complex manner, and regions of the TM differ in terms of their drainage rate and response to high versus low pressure.
Using human TM cells grown in culture, Vranka seeks to define the molecular differences between high and low-flow regions of the TM. She hypothesized that when IOP goes up, the ECM response will trigger gene differences in high versus low pressure areas. What she found is that while many ECM genes in both high and low-flow regions are downregulated in response to elevated IOP, “there’s quite a laundry list of genes with different dynamics.” Some genes downregulate in low-flow regions and upregulate in high-flow regions, and others do the opposite. Understanding all of these events from a molecular standpoint may provide insight into how to regulate IOP by more completely opening the TM as a therapeutic strategy in glaucoma.
Decoding Myocilin Structure-Function Relationship
The protein myocilin is strongly linked with inherited forms of primary open angle glaucoma (POAG). In mutant forms, myocilan aggregates are found in the endoplasmic reticulum, or sac-like membranes of the TM cells, where they contribute to IOP misregulation and cell death. Over90% of these mutations occur in the olfactomedin domain of myocilin.
Rebecca Donegan, PhD, and Raquel Lieberman, PhD, both of the Georgia Institute of Technology, presented their work characterizing the crystal structure of myocilin OLF, which was supported by a 2008-11 NGR grant to Lieberman. This accomplishment will lend insight into disease-associated mutations, amyloidogenic regions, and the currently unknown functions of both myocilin OLF and native myocilin. In addition, “it will provide a starting point for structure-based drug design for the treatment of myocilin glaucoma,” Donegal stated.
Caveolae: The Sentries of IOP?
Michael Elliott, PhD, of the University of Oklahoma, continues his inquiry into the CAV-1 gene’s influence on the tissue of the TM and nearby Shlemms’ canal. He’s a 2013-15 BrightFocus grantee and 2013 recipient of NGR’s Thomas R. Lee Award.
The CAV-1 gene is expressed in several places in the eye (and also outside of the eye) and is known to form caveolae, flask-shaped particles with their neck opening to the plasma membrane. Caveolae have “spring-like” properties, Elliott says. It’s hypothesized they serve as mechanical sensors that regulate fluid drainage, and that mutations in the CAV-1 gene may render this sensor defective.
“In cells where they’re abundant, caveolae act like springs; they can sense pressure in the cell,” he said, whereas in the knock-out mouse model he’s developed, the membrane seems to lose its ability to respond to pressure.
At one time Elliott, a lipid and protein biochemist, focused exclusively on the retina. That changed when a scientist pal sent him an email that drew him into the CAV-1 story. Months later, he connected with his collaborators, Ernest Tamm, PhD, an outflow anatomy expert at the University of Regensburg (Germany), who’s imaging and characterizing the response to pressure in the caveola tissue; and Dan Stamer, PhD, of Duke University, a member of BrightFocus Scientific Review Committee (SRC) and an innovator on outflow measurements, who’s corroborating Elliott's results.
Now the team is preparing to publish. At ISER, Elliott showed numerous pictures of caveolae from Tamm and others. Ironically, some of the images were from studies unrelated to glaucoma—illustrating how answers sometimes lurk in unexpected places.
In fact, it was at ISER 2012, in Berlin, where Elliott and Tamm first began discussing CAV-1, and shortly thereafter, Elliott applied for the BrightFocus grant, which permitted him to study this glaucoma pathway—also a whole new pathway for him. Chalk up another successful research collaboration to ISER!
Connecting the Dots Between Genes, SNPs, and Disease
Ours is an age when the causes of disease—and the ways to arrest them—are studied at the molecular level. Genome-wide association studies (GWAS) are part of that, an outgrowth of having the entire human genome sequenced and accessible in a computer data base. GWAS use rapid gene scanning technologies to locate DNA fragments that reflect variations in sequencing, known as SNPs (single-nucleotide polymorphisms) and may be associated with a disease. SNPs from individuals with the disease are then cross-checked with similar sequences in disease-free controls, to see if the same pattern is seen in a large number of individuals with a disease, which points to a possible pathway.
Importantly, GWAS help locate the general “locus,” or region of DNA sequencing which may be associated with a disease, but they cannot, on their own, specify whether genes “cause” a disease. SNPs vary from alleles, or mutations in that they are only a gene fragment, not the entire gene, and may exert only a partial influence. Many diseases, including primary open-angle glaucoma (POAG), the most prevalent form, are “multifactorial” diseases – meaning they’re caused by multiple factors, including age, stress, and genetic susceptibility—rather than a single mutation. GWAS are ideally suited for studying complex disease like this as a way of approximating and locating the genetic contribution.
End of A GWAS Era, Already?
David Mackey, MD, of Lions Eye Institute in Nedlands, Australia, co-moderated a session on glaucoma genetics. A 2014-16 BrightFocus grantee, Mackey is using advanced imaging techniques to link genetic variations with structural anomalies in the optic nerve head.
He’s part of a major worldwide collaborative research effort, the International Glaucoma Genetics Consortium, which is biobanking genetic information from hundreds of individuals with glaucoma, and using it to tease apart the genetic contributions. Data from that group are forthcoming on intraocular pressure, cup-to-disk ratio, disc size, and other markers of risk associated with POAG.
“Each SNP mutation may only provide a partial explanation, but it may identify the candidate gene for a treatment, or a pathway in which to direct a therapeutic approach,” Mackey said. “The pathway is the main thing that allows us to move into therapeutics.”
Similar international collaborations already have located SNPs associated with risk factors in two other multifactorial vision diseases, age-related macular degeneration and myopia.
As useful as GWAS are, Mackey predicted that soon they will be superseded by faster and cheaper ways to sequence the entire exome, or set of protein-coding genes known as “exons” in the human genome. Exons comprise about one percent of the human genome, but mutations in exons are more likely to have severe consequences than those in the remaining 99 percent of genetic material. The goal is to be able to identify genetic variations responsible for rare “Mendelian” diseases as well as common disorders, without the high costs associated with GWAS and whole-genome sequencing.
“The genetics of glaucoma may be thought of as a jigsaw puzzle; we have some of the pieces in place, but many more to find and fit,” Mackey said. “Our project will add to the existing knowledge of what causes glaucoma and contribute more pieces to the puzzle … helping to prevent vision loss at earlier stages and identify new glaucoma genes that may hold potential as treatment targets.
TBK1 and Normal Tension Glaucoma
BrightFocus Grantee John Fingert, MD, PhD, of the University of Iowa, described his BrightFocus-supported research into the TANK binding kinase (TBK1) gene. Duplications of TBK1 have been associated with normal tension glaucoma (NTG), a mystifying late-onset form of glaucoma where optic nerve damage and vision loss occur despite normal IOP. Somehow, there’s a loss of retinal ganglion cells (RGC), the nerve cells carrying signals from the eye to the brain. NTG exerts a strong family inheritance, but the mechanism behind it remains unclear, Fingert said. There's evidence pointing to disregulation of autophagy, the process by which cells isolate and digest proteins, organelles, and other materials.
For their research into NTG, Fingert and colleagues developed a transgenic mouse model with the human TBK1 gene incorporated into its genome; and also cultured RGC-like neurons from an NTG patient known to have TBK1 gene duplication, using pleuripotent techniques. Using these tools, they were able to link TBK1 gene duplication to key markers of abnormal autophagy.
“For the first time in my career, I’m able to report anecdotally on three different genes interacting with autophagy [in NTG],” Fingert proudly remarked. His co-investigator on the BrightFocus grant is Budd Tucker, PhD.
Genome Studies Involving POAG
Janey Wiggs, MD, PhD, a Harvard ophthalmologist and medical geneticist, had some pithy things to say about GWAS and genetic research into glaucoma. She is a co-principal investigator on a 2014-16 BrightFocus grant to Baojian Fan, MD, PhD, at the Massachusetts Eye and Ear Infirmary in Boston.
Whereas gene variations associated with early-onset forms of glaucoma, especially those with familial inheritance, tend to exert a large biologic effect, SNPs associated with late-onset disease (such as POAG) tend to exert smaller, complex effects, and are harder to study, Wiggs said.
For GWAS to have impact in these areas, “we need more genes, and we need to learn more about the genes we’ve identified,” Wiggs said.
Also, at the end of the day, “for complex genes, there may not be causative mutations,” Wiggs acknowledged. “Glaucoma is not just apples or oranges—it’s really the whole fruit basket. It’s the common endpoint of multiple pathways.”
So far, GWAS have been successful at identifying five genomic regions significantly associated with POAG: CDKN2BAS, SIX1/SIX6, CAV1/CAV2, TMCO1, and 8q22.
Wiggs herself was part of the study reporting a significant association between POAG and the SIX6 gene locus, which plays a role in ocular development and has been associated with the morphology of the optic nerve (Carnes et al, 2014). That work, which was not affiliated with BrightFocus, utilized data from two large genetic studies, NEIGHBOR and GLAUGEN, for its 256 cases and 256 controls.
She credited collaborative research and gene consortiums, like the IGC, with making large gene sampling possible.
However, in addition to sample size, for a GWAS to be successful, it’s important to define and adhere to shared definitions of “cases” versus “controls,” she said.
And that’s not easy. According to Wiggs: “If you really want to have a good day, put 30 glaucoma experts in a room and ask them to define what glaucoma is….
“That will take the whole day!”
This content was last updated on: Thursday, July 2, 2015
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