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A Jolt to the System

For CALS geneticist Barry Ganetzky, insight into the genetic underpinnings of traumatic brain injury began by knocking out fruit flies.

As a linebacker for the UW–Madison Badgers, Chris Borland made a name for himself as a hard-hitting tackler. His senior year, he was selected as a first-team All-American as well as the top linebacker and defensive player in the Big Ten Conference.

A third-round draft pick, Borland seemed destined for a headline career in the National Football League. But during a full-contact practice at the San Francisco 49ers summer training camp in August 2014, Borland got his “bell rung” by a 290-pound fullback during a routine exercise. Though Borland felt dazed, he played through—as he’d done dozens of times before.

Like many football players, Borland had endured his share of hard hits, including two diagnosed concussions. This particular hit, however, got him thinking seriously about the future, and about the negative effects that repeated collisions could have on his long-term physical and cognitive health. Even so, he went on to play a dynamite rookie year.

Then, after the season was over, Borland quit.

The announcement shocked the sports world. Borland was 24 years old and healthy, yet chose to walk away from a $2.3 million, four-year contract.

“I just honestly want to do what’s best for my health,” Borland explained on ESPN’s Outside the Lines. “From what I’ve researched and what I’ve experienced, I don’t think it’s worth the risk.”

With their repeated hits, football players—along with boxers—are at increased risk of developing chronic traumatic encephalopathy (CTE), a degenerative brain disease marked by memory loss, depression, suicidal thoughts, aggression and dementia. Of 91 brains donated to science by former NFL players, 87 have tested positive for CTE. It’s seen as a likely contributing factor to nine suicides by current and retired football players over the past decade.

Borland didn’t want to share that fate.

“To me, Chris Borland is a hero. He walked away before he made the big bucks and he was very explicit about why he quit—that it was not worth it to him,” says CALS genetics professor Barry Ganetzky, whose findings about the central nervous system in fruit flies are shedding light on what hard hits do to humans.

Ganetzky isn’t a sports guy, but he started paying attention to football-related brain injuries after the 2012 suicide of New England Patriots linebacker Junior Seau, intrigued by the biological processes driving this tragic phenomenon.

“I started wondering, what’s the link between a blow to the head and neurodegeneration 10 or 20 years down the line? When I started digging into the scientific literature, it became clear that we know very little,” says Ganetzky, who held the Steenbock Chair for Biological Sciences for 20 years. “And my usual response is, well, if we don’t understand something about the brain, then we should be studying it in flies.”

Fruit flies, officially known as Drosophila melanogaster, are a widely studied model organism, with a vast arsenal of genetic and molecular tools available to support that work. Flies reproduce rapidly and are easy to work with, enabling swift research progress. They are well suited for brain research because they have nerve cells, neural circuitry and a hard skull-like cuticle remarkably similar to our own, allowing scientists to conduct probing experiments that would be difficult in rodent models—and impossible in human subjects.

Fly models already exist to study Alzheimer’s, Parkinson’s and a number of other neurological diseases. Why not concussion? But there wasn’t a model available.

Then Ganetzky remembered work he’d done decades earlier.

“It occurred to me that I knew how to make flies have a concussion, and I had done it 40 years ago as a post-doc,” says Ganetzky. “I thought, ‘That’s it!’”

It was a simple thing: As a post-doctoral researcher at the California Institute of Technology, Ganetzky decided to see if any of his flies happened to be bang-sensitive mutants, flies that display seizures and paralysis after given a high-powered swirl on a vortex machine. But he didn’t have a vortex nearby, so he decided to just bang the vials against his hand.

“After a couple of sharp whacks, some of the flies were hanging out at the bottom of the vial, stunned. Others were on their backs, obviously knocked out. And after a few minutes, they all got up and started walking around again,” recalls Ganetzky.

He immediately knew the flies weren’t bang-sensitive—it’s an extremely rare mutation—but Seau’s death helped Ganetzky realize they had displayed symptoms “very similar in many respects to the empirical definition of a concussion.”

After developing and validating the new fly model, Ganetzky and UW genetics professor David Wassarman have been able to charge forward with brain injury research. The model has already been used to reveal key genes involved in the body’s response to brain injury. It’s also poised to help unlock medical applications, including a genetic test for high-risk individuals and an assortment of promising drugs and treatments.

In addition to helping athletes in contact sports, these advances will benefit the millions of Americans each year who experience traumatic brain injury due to falls, car accidents and violent assaults.

“At the most fundamental level, we just want to understand how traumatic brain injury works,” explains Ganetzky. “However, this is a major medical problem for which there are not many good—or any good—treatments or therapies or preventives, and so that is part of our motivation. If we can learn the genes and the molecules and the pathways, can we come up with interventions?”

Ganetzky was raised in a working-class neighborhood in Chicago by a candy salesman father and a homemaker mother. Growing up, he had an abundance of natural curiosity and asked a lot of tough questions—and often questioned the answers he received. While this trait caused him some problems as a youth, it came to serve him well in science.

At the University of Illinois in Chicago, he figured he’d become a chemist for the good career prospects. He ended up switching to the biological sciences, however, after a 10-week honors biology research experience in a Drosophila lab that expanded into a two-year project. From that point forward he stuck with flies, earning his doctoral degree at the University of Washington and then doing his post-doc work at Caltech.

In 1979, Ganetzky joined the University of Wisconsin–Madison, where he chose to focus his research program on exploring temperature-sensitive paralytic mutants, flies that behave normally at room temperature, but then start to tremble and twitch—or pass out—when things heat up. For each mutant he identified, he sought to uncover the faulty gene involved, and thus better understand how brain cells work.

Over the decades, this approach enabled Ganetzky’s team to discover a number of critical genes and molecular pathways involved in brain cell signaling, including those required for the release of neurotransmitters. That body of work established Ganetzky as one of the foremost leaders in neurogenetics. Some of his findings shed light on human genetic diseases and led to a test that’s now routinely used to assess the safety of new pharmaceutical drugs. For his contributions, Ganetzky was elected in 2006 to the National Academy of Sciences, the nation’s preeminent scientific society.

After Ganetzky’s “eureka moment” about fly concussions in spring 2012, he immediately reached out to colleague David Wassarman, a genetics professor in the UW–Madison School of Medicine and Public Health. Wassarman, who studies human neuronal disorders using fruit flies, had already been attending Ganetzky’s lab meetings for a few years after some of their research findings linking the innate immune response and neurodegeneration dovetailed.

“I did a demonstration of fruit fly concussion for David, and I remember his response very well,” says Ganetzky. “His jaw kind of dropped, and he said, ‘If you’re not going to study that, then I want to.’”

It was exactly the response that Ganetzky had been hoping for. With retirement looming on the horizon, Ganetzky needed a trusted and enthusiastic collaborator to help pursue the work—someone who would be willing to take on more and more as time went on. Wassarman was game.

“I wanted to put both feet in,” says Wassarman. “I said, ‘If we’re going to do it, let’s do it.’”

As a first order of business, Wassarman developed a tool capable of delivering a consistent “dose” of brain injury to flies. The result, known as the High-Impact Trauma (HIT) device, utilizes a metal spring to slam a vial of flies against a firm foam surface. In this setup, it’s important to note, the brain injury the flies experience is caused by the rapid acceleration and deacceleration of their bodies; it’s not necessarily about a direct hit to the head.

“Quite often, as with football players, it can happen because they are running fast and then meet an immovable object. The concussion is caused by a kind of whiplash, where the brain is ricocheting off the inside the skull, and that’s what’s causing the damage,” says Ganetzky. “That’s what we’re doing here with the flies.”

Ganetzky and Wassarman found that flies injured using the HIT device exhibit many of the classic symptoms of traumatic brain injury (TBI) seen in humans. As they reported in the Proceedings of the National Academies of Science in 2013, flies show temporary incapacitation and loss of coordination immediately after injury. Those that survive severe injury go on to develop long-term symptoms: activation of the innate immune response, neurodegeneration and early death.

These TBI flies have the potential to reveal much-needed insights—and medical interventions—for the millions of Americans who experience traumatic brain injury each year. According to the U.S. Centers for Disease Control and Prevention, TBIs cause around 2.5 million emergency room visits, 283,600 hospitalizations and 52,800 deaths each year. Top causes are falls, motor vehicle accidents, and blows or jolts to the head or body, including sports-related concussions. Bomb blasts can cause brain trauma in soldiers in combat zones. Across the country, as many as 6.5 million people are believed to be struggling with the consequences of TBI, and the total economic cost of this health issue is estimated to be $76 billion per year.

In a demonstration of the power of the TBI model, Rebeccah Katzenberger, a senior research specialist in Wassarman’s lab, subjected 179 genetically unique strains of flies to four strikes of the HIT device—meant to simulate a series of severe brain injuries—and then monitored them for death at 24 hours post-injury, a data point that serves as an easy-to-measure proxy for the various negative events unfolding inside the body.

The results revealed a huge diversity of responses, underscoring the fact that genotypes matter when it comes to TBI response. Some strains were particularly susceptible to death, losing as many as 57 percent of the flies in those first 24 hours, while others were much more resilient, losing just 7 percent. The team then identified the genes that possibly made a difference, publishing their findings in eLife in March 2015.

“Now we have these 100 genes, and scientists can start looking at them in more detail,” says Wassarman. “A lot of them are genes that had never really been implicated in traumatic brain injury before. I think this is going to be one of our big contributions.”

These findings, the researchers note, may help explain why people respond so differently to similar brain injury events, and may help lead to a genetic test to identify high-risk individuals.
“Once we understand those genetic links, we’ll be able to test people and tell them, ‘Look, you probably shouldn’t play football. You should play non-contact sports,’” explains Ganetzky.

After identifying the TBI genes, Ganetzky and Wassarman immediately noticed a handful of genes involved in tissue barrier regulation. Tissue barriers—such as the intestinal barrier and the blood-brain barrier—function as biological blockades keeping “bad” things out while allowing “good” things to pass through.

To explore the connection between brain injury and tissue barriers, the duo had Katzenberger conduct a simple, colorful experiment that involves adding bright blue dye to the flies’ food. Under normal conditions, when flies eat the blue-colored food, it stays in the gut, something that is readily observable through the fly exoskeleton. However, after exposure to brain injury—via the HIT device or by having their heads pinched with a forceps—they found that the dye leaks out of the gut and turns the entire body blue, a phenomenon called “smurfing” (after the blue Smurf cartoon characters).

Leaky tissue barriers have previously been observed in rodent models of brain injury as well as in human medical cases. “Somehow this injury to the brain is triggering a series of events that leads to the breakdown of the intestinal barrier,” notes Ganetzky. “So there’s some sort of cross-talk going on between the brain and the intestine, but we don’t fully understand it yet.”

Upon further exploration, Ganetzky and Wassarman were able to confirm that—along with the blue dye—glucose and bacteria were also crossing the intestinal barrier into the fly’s circulatory system, or hemolymph, after brain injury. Homing in on glucose, they found that it plays a causative role in fly death after TBI. “By simply withholding sugar, we were able to keep some of these flies alive, and by a substantial margin,” says Wassarman.

If the findings hold up in rodent models and in human trials, he notes, athletes may one day find themselves advised to avoid certain foods after experiencing concussion.

The bacteria that cross the intestinal barrier appear to be playing more of a long game. Ganetzky and Wassarman believe they are the culprits triggering the innate immune response observed in TBI flies. The innate immune response, also known as the inflammatory response, is the body’s natural reaction to microbial invasion and other stressors. If properly controlled—turned on and off at the right time—it protects the body. If left on, however, it can cause collateral damage throughout the body, including damaging brain cells.

“Here’s what we think is happening: Traumatic brain injury is causing increased intestinal permeability. That causes the bacteria to leak out, which turns on the innate immune response, and that is possibly leading to neurodegeneration down the line,” explains Wassarman.

Ganetzky and Wassarman are intrigued by a concept that is emerging from their work and related studies: that TBI accelerates aging. Some of the key physical outcomes of brain injury—problems with tissue barriers and increased inflammation—are also hallmarks of the natural aging process. More support for this idea came in summer 2015, with the release of a report describing signs of early aging in the brains of war veterans exposed to bomb blasts in Iraq and Afghanistan.

“Somehow a blow to the head is activating all of these pathways related to aging and speeding them all up. Biologically, I think that this is maybe one of the most fascinating things about the whole project,” says Ganetzky, noting that TBI flies are a great model for further exploration.

Even at this early stage, without fully understanding the basic scientific mechanisms involved, the model is already revealing some promising medical applications. As soon as Ganetzky and Wassarman realized that the inflammatory response might lead to neurodegeneration, a treatment suggested itself: Could a simple anti-inflammatory help? They tried giving TBI-injured flies some aspirin mixed in their food. It helped.

“Our studies show that there appears to be a window of time after brain injury when the flies are particularly susceptible to dying. And if we can prevent certain events from happening during this time, then we can prevent death,” says Wassarman. “That’s what we think aspirin is doing—by lowering the innate immune response.”

The next step is to look for drug candidates that work even better than aspirin. Ganetzky and Wassarman are in the process of screening a set of 2,400 compounds, and they’ve already found a handful of very promising ones that can now be tested in rodent models and, ultimately, in human clinical trials.

“It would be wonderful if someday it were possible to offer a simple intervention beyond surgery to help individuals who have suffered a severe traumatic brain injury,” says Wassarman.

There’s a lot left to learn, and Ganetzky and Wassarman are eager to pursue all that the model can tell them. With Ganetzky’s retirement set for early 2016, the work of securing the project’s first federal grant and conducting experiments will largely fall to Wassarman.

But Ganetzky won’t be out of the picture. He continues to keep up on brain injury medical cases and scientific discoveries, and is encouraged by the national conversation about sports and brain injuries that’s starting to gain traction—and by the NFL’s commitment to scientific research in this area.

Some of these advances can be attributed, in part, to Chris Borland, whose post-NFL journey has led him deeper into the world of sports-related brain injury. Borland has submitted to numerous brain scans to support research, and has also become a sought-after speaker, touring the country to raise awareness about the risks of concussion.

It’s that kind of dedication to public service on the part of Borland and many other athletes, along with the excitement of discovery, that’s keeping Ganetzky in the game. Despite his retirement, Ganetzky plans to keep a scaled-back version of his lab running for at least a few more years.