Keep An Eye On It

Art Courtesy of Luna Aguilar.

What if you suddenly had blurry vision, couldn’t recognize familiar faces, or had difficulty adapting to dimly lit places? This is the reality for people with age-related macular degeneration, also known as AMD, one of the most prevalent causes of vision loss that affects around 200 million people in the world.

In AMD, damage occurs in the macula, an oval-shaped area at the center of the retina. The retina consists of a layer of cells known as photoreceptors, which are crucial for converting light entering the eye into signals sent to the brain. The macula is specifically responsible for sharp and central vision. Thus, someone with AMD usually has difficulty deciphering fine details. There are limited effective therapies for the disease—current treatments such as vitamins and minerals only slow disease progression, but do not stop or reverse it.

Yale scientists are among those who have joined the cause to find out more about AMD disease pathology. From discovering possible therapeutic targets for AMD and other neurodegenerative diseases to uncovering a quantum chemistry reaction in the retina, their findings could not only inform potential AMD treatments, but also offer applications far beyond the eye.

A Window Into Neurodegeneration

In a recent study published in Nature Communications, Yale Assistant Professor Brian Hafler and a team of Yale researchers found that AMD, which is itself a neurodegenerative disease of the retina, could serve as a system for understanding other neurodegenerative diseases such as Alzheimer’s disease and multiple sclerosis. To arrive at this finding, they developed a novel approach to understanding AMD and its cellular pathology.

Hafler and his team utilized single-cell data and machine learning techniques to pinpoint the populations of cells in the retina that play a prominent role in the disease progression of AMD. This study built upon previous research in the retina which highlighted the overall role of inflammation in the pathology of macular degeneration, Hafler explained. The team isolated 70,973 individual retinal cells from seventeen different human retinas with different stages of disease and healthy controls. “This allowed us to build a unique road map into the genetic networks driving inflammation in macular degeneration and hopefully to develop new therapeutic targets,” Hafler said.

To analyze these cells, the team designed a novel collection of machine learning tools which they termed “Cellular Analysis with Topology and Condensation Homology,” or CATCH. At the core of CATCH is a method known as diffusion condensation, which identifies similar groups of cells based on how they are pulled toward the weighted average of neighboring cells in space. This method enabled the team to pinpoint two populations of activated glial cells (cells whose primary role is to support neurons): astrocytes and microglia. Astrocytes provide neuroprotective, structural, and metabolic nourishment to nerve cells, while microglia are the immune cells of the brain and mount responses to pathogens. Both were found to be activated in the early phase of AMD. 

Surprisingly, similar activation profiles were found to dominate the early phases of other neurodegenerative diseases, such as Alzheimer’s disease and multiple sclerosis. This association led the researchers to believe that early stages of neurodegenerative disease progression generally utilize a common mechanism involving the activation of glial cells. It also suggests that the retina can potentially be a unique system for developing new therapeutic strategies to treat neurodegenerative diseases.

Then, using single-cell data from Alzheimer’s and multiple sclerosis studies, Hafler and his team were able to characterize specific cellular interactions that induce inflammation, which may be a common characteristic of neurodegenerative disease progression. They first identified interleukin-1β, a protein that signals immune cells to mount and induce a response, that was derived from the microglial cells activated in AMD. Using a computational technique, they found that interleukin-1β signals for astrocyte activation are pro-angiogenic, meaning that they enhance blood vessel formation. This observation lined up with the typical symptoms observed in wet AMD, an advanced stage of AMD. In late stages of AMD, blood vessels can abnormally form, grow, and leak beneath the macula. This bleeding can distort the retina and impair one’s central vision. 

Hafler’s study suggests that targeting astrocytes and microglia should be further considered when attempting to treat neurodegenerative diseases. Anti-angiogenic medications are currently the primary treatment, but they are only effective in advanced stages of the disease. To fill in the gap, interleukin-1β may be an effective target. With Hafler’s deep understanding of AMD both in a clinical and research setting, his results show promise towards moving forward in the fight against AMD. “My clinical practice is what drives my benchwork in the lab,” Hafler said. “When medical research is applied to patient care, we can uniquely translate novel therapeutic approaches for diseases like AMD.”

How Does Melanin Protect The Retina?

A second study, published in PNAS, found a quantum chemistry reaction that could explain how melanin protects the retina from age-related macular degeneration. Yale scientist Douglas Brash, a physicist by training and co-author of the study, did not expect to investigate AMD. But one day, he performed an experiment on melanocytes, which are special melanin-producing cells. Melanin is a natural pigment that shows up across the body, from the eyes to the skin. In the skin, melanin accumulates with UV-light exposure. In the retina, melanin exists in tiny granules at the photoreceptor layer; however, its function is almost completely unknown. Brash wanted to see what would happen when melanocytes were UV-irradiated. Cells that are UV-irradiated develop a specific type of DNA damage called cyclobutane dimers. 

Brash eventually showed that, when exposed to UV radiation, melanin was oxidized by free radicals—meaning that its chemical structure lost electrons—to produce dioxetane, a chemical compound on melanin that then splits to give a molecule with a similar high-energy state to ultraviolet light in sunlight. The radicals and dioxetanes continued long after the UV light was turned off. Dioxetane’s high-energy state was a specific kind called a triplet state, which is capable of initiating reactions that ordinary chemistry cannot. He also knew that melanin was found in many places in the body, such as the eye and the ear, and the two radicals behind its oxidation, superoxide and nitric oxide, were found in many conditions such as inflammation. 

“These [are] events that can’t not happen. Why aren’t we dead?” Brash recalled thinking. Could the high-energy reaction cause deafness and blindness? A surprising clue to the exact opposite conclusion came from Ulrich Schraermeyer, an ophthalmologist at the University of Tubingen in Germany, who had heard about Brash’s work with melanin chemistry. Schraermeyer had an idea that completely opposed the norm ten years ago. He suggested that perhaps melanin actually had a protective role in the retina. 

For years, he had been working on studies to show that when melanin was associated with another molecule called lipofuscin, the retina was less susceptible to macular degeneration. Lipofuscin, a pigment that accumulates in the retina with age, is associated with neurodegeneration in AMD, but its exact composition is unclear. While Schraermeyer was convinced of the critical involvement of melanin in AMD prevention, he could not figure out the chemistry. And while Brash was intrigued by melanin having a protective role, the mechanism would need to be proven.

In Schraermeyer’s initial experiments, he proved many drugs could actually slow or prevent macular degeneration in mice and monkeys. Brash noticed that these drugs were all chemicals that could create triplet states, the unique high-energy chemical state that Brash had previously created in melanin after it was treated with radicals. This led to their theory that the dioxetane in melanin that led to the triplet state was the step responsible for melanin’s protective role in the retina.

In his initial experiments, Schraermeyer showed that under electron microscopy, a type of imaging technique used to visualize subcellular structures, melanin was often seen together with lipofuscin in the retina in what is called melanin-lipofuscin (MLF) granules. He observed that MLF granules accumulated in the eyes of humans above the age of sixty. Building on this observation, the group showed that the toxic lipofuscin component of MLF granules could be degraded by treating mice with a non-melanin molecule that was in a triplet state. The degradation was blocked if mice also received a molecule that siphons the triplet energy away.  Thus, it seemed like melanin chemiexcitation, using chemicals to create a high-energy state, and melanin-lipofuscin association could be studied as a pathway for lipofuscin degradation. 

Schraermeyer believes that upregulating melanin in the retina could be a therapeutic target. Having already shown that people lose melanin in the retina with age, he theorizes that the melanin is being used up in its protective role throughout one’s life. Brash, on the other hand, is convinced about the importance of dioxetane chemistry, but not so much about melanin itself. “I’m willing to bet that as you get older, the melanin may well contribute to AMD, so it’s like a double-edged sword,” said Brash. Brash’s therapeutic goal is to get triplet-state precursors into the eye so that dioxetane chemistry can be harnessed for AMD prevention. 

Seeing Eye-to-Eye

While Hafler and Brash took two very different approaches to characterizing some of the underlying mechanisms of AMD, their findings both pave a new way forward for the development of potential treatments. With scores of scientists studying AMD from various specialties and backgrounds, the pursuit of an effective treatment that accounts for multiple mechanisms grows increasingly hopeful—while potentially also addressing diseases beyond the retina as well.