Many scientists spend their lives conducting tests on a single gene but, in their quest to develop a drug that will repair injured spinal cords, researchers at The Miami Project to Cure Paralysis are testing thousands of genes in hundreds of thousands of nerve cells in any given week.
In fact, in a recent study, they tested the effect that 440 million different chemicals have on primary neurons—a number that astonishes even Vance Lemmon, one of the co-authors of the study.
“That’s mind boggling,” said Lemmon, professor of neurological surgery and the Walter G. Ross Distinguished Chair in Developmental Neuroscience at the University of Miami Miller School of Medicine. “I think we’ve tested more ‘things’ on primary neurons than the rest of the world combined. And that’s given us lots of insight into the kinds of gene families that are really powerful at turning axon growth on or off.”
Over the past 16 years, Lemmon and his longtime collaborator, Vice Provost for Research John Bixby, professor of pharmacology and neurological surgery, have pioneered the use of a high-tech, automated process known as high-content screening to study neurons. Along with computational biochemist Hassan Al-Ali, they use what Lemmon calls this “amazing microscope” to search for the genes and compounds that promote or prevent the regeneration of axons, the threadlike part of the nerve cells that conduct electrical impulses to other cells.
Although axons regenerate in other parts of the body, they typically remain disconnected in the spinal cord and brain after injury, leaving people with permanent deficits, including paralysis.
“If your axons are broken you can’t send signals down to move your muscles and you can’t get signals from your body telling you where your knee is, or that you bumped into something,” Lemmon said. “It works both ways—you lose your sensory information and you lose your voluntary and involuntary control of bodily functions.”
Which is why the LemBix Lab, as the Miami Project’s Laboratory for Axon Growth and Guidance is better known, became one of the first medical school labs in the world to purchase an automated high-powered microscope that can take pictures of cells and, combined with sophisticated image analysis, document how they respond to thousands of different treatments.
“Our original idea in 2003 was to use automation to reduce the bias that comes from studying your favorite proteins,” Bixby said. “We had no idea how rich a source of information we’d be able to mine.”
Sixteen years and two microscopes later, Lemmon said he can’t even begin to count how many neurons they’ve incubated, stained, and placed into the 24, 96, or 348 individual wells that each microscope plate contains.
“I’d say we’ve conducted millions of tests on cells with different treatments to look for those that alter the activity of genes,” Lemmon said. “In one set of experiments we identified three related proteins that bind to DNA and turn on and off many other genes, promoting nerve growth in vitro, or in the dish. When we tested them in vivo, or in an animal model, each of them made axons grow. That was a very exciting result for us.”
Published in the journal ACS Medicinal Chemistry last year, their study of the effect of 440 million different chemicals on primary neurons also produced an encouraging result. They found nine chemicals that made axons grow in vitro at extremely low doses, making them good candidates for drug investigations. Interestingly, when compared to earlier results focusing on a family of enzymes known as kinases, they found that some inhibitors of these kinases made axons grow farther and faster than the nine new compounds. They think the kinase inhibitors are so effective because they turn several kinases off simultaneously.
“By turning off many different ‘brakes’ on axon growth we can have a huge effect so it’s really hyper-charging axon growth,” Lemmon said.
But nothing has hyper-charged the LemBix lab’s research like technology. Without the automated microscope, Lemmon said, they would never have been able to test 440 million different chemicals, which took only a few months to complete.
“Did I mention that what we do is mind-boggling?” Lemmon said. “I can’t even begin to fathom those numbers.”
Source: University of Miami