Dr. Simon John published his latest research study “FYN regulates aqueous humor outflow and IOP through the phosphorylation of VE-CADHERIN”

Simon John, PhD, the Robert L. Burch III Professor of Ophthalmic Sciences, is a leading researcher in the ophthalmology field, with a focus on glaucoma. Dr. John’s interest in biology has always been broad and varied, starting out his scientific career in studying insects and their dual functions as pollinators and pests, and then moving on to the symbiotic relationship between the bacteria rhizobium and legumes. He then decided to pursue his interest in human genetics and complex diseases, eventually leading to his groundbreaking work in developing the glaucoma mice model, which is now widely used by researchers in studying homologous genes and development of glaucoma in humans.

Glaucoma, one of the most common neurodegenerative diseases, is estimated to affect around 80 million people worldwide. Dr. John and his research lab focus on studying the causes of glaucoma with the purpose of developing new treatments. The lab is working to understand how intraocular pressure (IOP) is normally regulated, and how its elevation causes glaucoma, damaging retinal ganglion cells which connect the eye to the brain and damaging the optic nerve. As part of this, they study the anatomy and development of ocular drainage tissues, such as Schlemm’s canal and trabecular meshwork, that determine IOP.

In their latest research, Simon John and his team of collaborators have published their paper titled “FYN regulates aqueous humor outflow and IOP through the phosphorylation of VE-CADHERIN" in Nature Communications. As senior author of the paper, Dr. John shared insight with us about the team’s journey to publication.

How does your paper “FYN regulates aqueous humor outflow and IOP through the phosphorylation of VE-CADHERIN” tie into the larger picture of your lab’s mission?

This paper fits into our studies of intraocular pressure and how Schlemm’s canal (SC) regulates and mediates aqueous humor drainage and IOP. The rate of drainage or outflow of aqueous humor from the eye is a critical determinant of IOP. Aqueous enters the lumen of Schlemm’s canal from where it rapidly drains into veins that are connected to Schlemm’s canal. The inner wall of Schlemm’s canal is the final barrier that aqueous humor has to pass to enter the canal’s lumen and drain from the eye. Despite its central role in controlling IOP, traditional treatments have not targeted the inner wall of SC. Our studies characterizing SC aim to change this. This paper addressed the path by which aqueous humor enters the lumen of SC and how eye pressure modulates the rate of aqueous humor entry into SC.

Tell us about main findings of the paper.

The molecular mechanisms by which the inner wall cells of SC sense and respond to pressure changes to regulate aqueous humor drainage and IOP are largely undefined. We tested the role of mechano-responsive signaling molecules. We show that increasing IOP result in mechano-responsive phosphorylation of the cell adhesion molecule VE-CADHERIN (CDH5) in cells of SC’s inner wall. We show that the enzyme FYN and possibly other related enzymes mediate this phosphorylation in response to increasing IOP. VE-CADHERIN holds adherens junctions between SC cells together and its increased phosphorylation at higher IOP destabilizes these intercellular junctions. This in turn acts to lower IOP by allowing more aqueous humor to flow around the borders of SC cells and enter the lumen of SC. These findings demonstrate that adherens junctions regulate aqueous humor drainage. They identify an important role of mechanotransducive signaling within SC cells in maintaining IOP homeostasis and implicate FYN as a potential target for developing IOP-lowering treatments.

What inspired you to collaborate with your co-authors to write this paper? How important was collaboration to the success of this project?

Collaboration is vital for success bringing additional perspectives, skill sets and strengths. Outside of my laboratory, we collaborate with Krish Kizhatil and Revathi Balasubramanian in this area. Both are former trainees of mine. I am proud that all of my former trainees who applied for faculty positions have been successful. There is nothing more rewarding than watching former trainees grow independently and flourish as key, next-generation figures making a difference in the ophthalmic sciences. I am pleased to have good relationships with former trainees. We know each other well, allowing us to exploit our individual strengths and so we make effective teams.

When in my laboratory, Krish was vital in developing methods to study SC at high resolution around the entire ocular circumference and in being the first to characterize the detailed development of SC including demonstration that it has molecular characteristics that are between those of blood vessels and lymphatic vessels. He brought this experience and knowledge as well as his deep expertise in cell biology and microscopy to this project. Revathi, is skilled in single cell genomic approaches. While in my lab, she molecularly characterized SC using these and additional approaches, both in adults and during development, providing a new molecular foundation of understanding to drive the field. Her work is also revolutionizing understanding of SC with a focus on development and childhood glaucoma. She brought these skills in genomics to this project.

What were some of the unique challenges of this project?

The delicate scleral tissue being studied has a morphology that is very easily disturbed during analyses. Imaging, sampling and analyzing SC at high resolution around the entire ocular circumference required extensive and time-consuming microcopy. Additionally, testing and developing antibody labeling methods presented further challenges. Also, the development of rapid fixation protocols was critical to fixing the studied eyes at the set eye pressures, without disturbing that pressure.

What are the next steps after this project?

We are continuing to characterize molecular processes by which SC and trabecular meshwork coordinately regulate aqueous humor drainage/IOP and malfunction in glaucoma. This includes developing new tools to target the inner wall of SC, experimentally and for treatments, including viral vectors that can enable gene therapies.

Our lab’s studies have also demonstrated that metabolic disturbances occur very early in glaucoma and that supporting metabolism and mitochondrial health with natural metabolites is potently protective in mouse models, with initial success subsequently shown in clinical trials. The role of metabolism and the development of treatments to induce metabolic and cellular resilience against glaucoma, as well as to improve regenerative strategies, is an important ongoing focus. We are particularly interested in the use of natural molecules that are expected to be relatively safe and accessible.

And finally, what advice would you give younger scientists looking to advance their careers?

At the time I started my lab, there was a huge resistance against using mice to study glaucoma, as the mythology at the time was that mice eye anatomy and development was not analogous to humans. I took a risk of starting my lab in attacking those problems- we worked out the anatomy and development of the mouse eye, which showed that mice were a valid model for humans, and developed the now widely-used glaucoma mouse model. It was a niche, because others weren’t taking it seriously, and it was high risk, but it made us different from other labs at the time.

Younger scientists have got to keep up that passion and motivation and think deeply about the science- but they’ve also got to be bold. They’ve got to be well-informed about the particular risks they need to take, and they’ve got to push it. Obviously, they need to develop grant writing skills, but otherwise I think it’s about being persistent and educated. Very good work, very stringent and high-quality work, and not just filling in details if they can, especially in this difficult research climate. They need to be trying to figure out what they can do that’ll really make a difference.