An international research collaboration has uncovered some clues to macular degeneration’s onset, namely, the discovery of a microscopic mineral “scaffolding” around which drusen deposits begin to form. Drusen are tiny protein and fat deposits in the retina that, when detected through a dilated eye exam, are a hallmark sign of the early stages of age-related macular degeneration (AMD).
BrightFocus grantee Richard B. Thompson, PhD, and co-investigator and senior author Imre Lengyel, PhD, published February 3 in the print edition of the Proceedings of the National Academy of Sciences (PNAS) detailing their evidence about how tiny spheres of mineralized calcium phosphate, called hydroxyapatite (HAP), may precede drusen formation and signal the onset of AMD.
The presence of drusen, or tiny protein and fat deposits in the retina, is taken as a clinical sign of AMD, and used for diagnosis during a dilated eye exam.
Before now, nobody understood how drusen formed and grew. Once they reach a clinically relevant size, it’s believed they contribute to AMD by preventing essential nutrients from reaching the eye’s photoreceptors, or light-sensing cells.
Photoreceptors, like all cells, have a life cycle, and when they die, they are typically discarded into the retinal pigmented epithelium (RPE), a thin layer of “housekeeping” cells that nourish and maintain the retina. From there, they’re broken down and transported out of the eye as a waste product.
It’s believed that drusen interfere with this “recycling” process by attracting various fat and protein particles that are mixed up in this cellular debris—a process that’s accelerated once more photoreceptors become undernourished and die. Thompson, Lengyel, et al, believe that the mineralized spheres they’ve discovered attract these proteins and fats to their surface to form drusen. It starts when an insoluble shell made of HAP forms around naturally occurring lipid droplets; then protein and fat molecules form around that. Over years, the globules build.
HAP is common in the body—it comprises the hard part of bones and teeth—but it had never been identified in that part of the eye before.
The discovery was widely reported on science news sites, and Thompson was quoted in a UMD new release. “We had no idea that HAP might be involved,” he said. “That’s what makes this work so exciting. It opens up a lot of new research opportunities.”
An Imaging Success Story
Thompson is an associate professor of biochemistry and molecular biology at the University of Maryland. Last year, he and Lengel received a BrightFocus grant to investigate the role of zinc and HAP in inducing drusen formation. Lengyel is a senior research fellow in the Institute of Ophthalmology at University College, London, and a member of its faculty in Brain Sciences.
For years, the two researchers have been collaborating on ways to detect and quantify microscopic levels of zinc and other heavy metals in the eye and brain. Their research has focused on determining whether these metallic catalysts—themselves unbound from cellular structures—may in turn contribute to biochemical changes leading up to disease.
The challenge, in this investigation, was to find a way to produce images of the tiny spheres of mineralized calcium, just a few microns—or thousandths of a millimeter—across. They did this through post-mortem examination of 30 eyes from donors between 43 and 96 years old, using fluorescent dyes to stain the particles.
Resulting images, like the one above, show their detective work has paid off. “We found these miniscule hollow spheres inside all of the eyes and all the deposits that we examined, from donors with and without AMD,” explains Lengyel in a UCL release about the discovery. “Eyes with more of these spheres contained more drusen. The spheres appear long before drusen become visible on clinical examination.” Whether they are a cause or symptom of AMD is still unclear, but either way, they have diagnostic value.
“The fluorescent labelling technique that we used can identify the early signs of drusen build-up long before they become visible with current methods. The dyes that we used should be compatible with existing diagnostic machines. If we could develop a safe way of getting these dyes into the eye, we could advance AMD diagnoses by a decade or more and could follow early progression more precisely,” Lengyel said. It’s possible that further strategies to prevent build-up could potentially stop AMD from developing altogether.
“If we could get to the spheres before the fat and protein build-up, we could prevent further growth,” Lengyel said. “This can already be done in the lab, but much more work is needed before this could be translated into patients.”
In an interesting side note, some of the mineral spheres identified in the eye samples were coated with amyloid beta, which is linked to Alzheimer’s disease. This has led the researchers to speculate whether the same technique developed to identify spheres for AMD diagnosis might also aid in the early diagnosis of Alzheimer’s.
In addition to his and Lengyel’s decades-long collaboration, credit for the discovery is shared by dozens of researchers worldwide, some of them serving as coauthors of the report.
“Our discovery opens up an exciting new avenue of scientific research into potential new diagnostics and treatments, but this is only the beginning of a long road,” added Thompson.
In a statement, UMD Vice President for Medical Affairs E. Albert Reece, MD, PhD, MBA, who also is dean of its medical school, said: “Dr. Thompson and his colleagues have provided new insight into the deep mechanisms of this terrible disease [AMD], and in doing so, they have created new avenues of research that have the potential to help millions of people.”
Heavy Metal Experts: Know Your Researchers!
The collaboration between BrightFocus grantee Richard Thompson, PhD, and co-principal investigator Imre Lengyel, began over a decade ago, when they met at a zinc conference.
As it happened, they’re both into “heavy metals”—not music, but looking at how elemental metals become un-bonded at the molecular level and re-aggregate in the body to influence health and disease.
By “looking,” they mean measuring, which sets theirs apart from other investigations.
“We’ve measured metal ions in the ocean. We’ve measured them inside cells. We’ve taken pictures of them. We’re all about that,” Thompson said.
“There’s some skill to doing these measurements,” he adds. “Most biochemists aren’t trained this way. We’ve been taught by guys like marine chemists – people who care about copper in the ocean. They’ve developed the techniques that we’ve adapted.”
Using a technique called fluorescent spectroscopy, they were able to isolate zinc deposits in a part of the eye underlying the retinal pigmented epithelium (RPE). They published the finding in 2007.
‘There’s some skill to doing these measurements. We’re all about that.’
—Principal Investigator Richard Thompson, PhD
“We used one of the fluorescent dyes Richard is working with, and we stained the back of the eye. And although most of that zinc is bound to certain proteins, a large proportion still is captured by the fluorescent sensor, and leaves a nice big contrast,” Lengyel said.
“The color, or the intensity of the fluorescence matches what we’re trying to measure,” Thompson explained.
“The best pictures are taken in Imre’s lab [in London], because he’s got a beautiful microscope,” he added. “These nice three-dimensional images—they’re otherworldly!”
Watch a video about the discovery
The researchers also measured other metals, like copper and iron, in the eye, but “the most highly concentrated metals are zinc and calcium,” Lengyel said. So their next step was to isolate drusen deposits, measure them with high resolution, and quantify their elemental content. That’s how they discovered that calcium levels are exceedingly high—“so high that it’s unreal unless the calcium is in a crystalline form,” Lengyel said. Crystal structures affect the signaling differently.
In every sub-RPE drusen deposit they analyzed, they found “a lot of calcium, but it must be in a crystalline form,” Lengyel concluded. “So that becomes the hydroxyapatite story.”
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