By Cindy Landrum
Photography by Josh Wilson

Study lays groundwork for developing drugs to treat or prevent addiction in humans

To most people, fruit flies are aggravating little pests that show up in their kitchen when they leave ripening bananas or other fruit on the counter too long.

But geneticist Trudy Mackay, who has directed the Clemson University Center for Human Genetics since it opened in 2018, has a much more endearing moniker for the flying insects.

“They are wonderful little beasties,” she says.

And she gets them drunk, plies them with cocaine, exposes them to heavy metals and manipulates their DNA. It’s all a part of her quest to discover the genetic roots of complex traits — traits that are influenced by multiple genes — that are important to human health.

The Center for Human Genetics is a state-of-the-art research and education facility in Self Regional Hall on the campus of the Greenwood Genetic Center in Greenwood, South Carolina. Mackay, widely recognized as a leading authority on the genetics of complex traits, is showing a visitor around the facility when she stops in the hallway at the door to what appears to be a closet.

It’s not.

Inside, there is what looks, at first glance, to be a cooler. But the air is a balmy 77 degrees. “Here’s where we keep the flies,” Mackay says. Trays of test tubes fill the shelves. The vials hold hundreds of thousands of the common fruit fly, Drosophila melanogaster.

You may wonder how flying insects smaller than a grain of rice could answer questions that have puzzled geneticists for decades — how they may reveal why some people who use drugs or drink alcohol become addicted while others do not. Or unlock the secret to longevity. Or explain why a genetic disorder severely affects some members of a family but only causes minor symptoms in others.

Despite the stark differences in physical appearances, Drosophila melanogaster has much more in common with humans than one would think at first glance.

“The remarkable thing is that 75 percent of human disease genes actually have a counterpart in the little fruit fly,” says Mackay, a U.S. National Academy of Sciences member who started working with the fruit fly in graduate school. “Our findings can be translated into humans.”

“They are wonderful little beasties.”

Nearly Impossible

It’s difficult to study some traits in humans.

Take substance abuse. The National Survey on Drug Use and Health estimates that approximately 20 million people in the U.S. age 12 and over have a substance abuse problem. About 5.5 million adults reporting in that same survey used cocaine in 2019, and about 1 million people abused methamphetamine. But researchers know many more people use drugs or drink alcohol and don’t become addicted. What they don’t know is why.

“We can’t give people cocaine or not. We can’t give people alcohol or not,” Mackay explains. “Scientists can do association analysis of people who abuse drugs and those who have taken them but don’t become abusers. But those studies are difficult to do, and there’s always going to be the complication of not being able to control the environment. People self-medicate. They typically don’t do it with just one drug. Often, there’s smoking and nicotine involved, and there may be more than one drug of abuse. It’s a thorny problem that is almost impossible to solve in humans.”

Full Control

But controlling both genetics and environment in fruit flies is easy, thanks partly to Mackay’s decision nearly 20 years ago when she was on the faculty of North Carolina State University.

Her lab needed some fruit flies to use in its research, so Mackay asked Richard Lyman, one of the lab’s research scientists, to go to the State Farmers Market in Raleigh during the peach season to collect some. Lyman captured over 1,000 pregnant female fruit flies. After he returned to the lab, he inbred the flies for 20 generations. The inbreeding virtually eliminated genetic variation in each of the lines.

Those flies were the start of the Drosophila Genetic Reference Panel, a valuable resource Mackay’s lab developed for researchers from all over the world to use. The original panel of 200 lines now comprises 1,200 fly lines with fully sequenced genomes. Lyman also moved to the Clemson Center for Human Genetics to direct the Drosophila Research Core.

Mackay’s latest collaborative research involved giving male and female flies a fixed amount of sucrose or cocaine-laced sucrose over two hours.

“We just dissolve the drugs in a sugar-water solution and give it to them,” says Brandon Baker, who recently earned his Ph.D. in genetics at Clemson and worked in Mackay’s lab both at the Center for Human Genetics and at North Carolina State University. “When we do that, we actually can see some effects that we see in humans in the flies.”

The flies exposed to cocaine in this experiment showed impaired locomotor activity and increased seizures.

It’s what Mackay and her collaborators, including her husband and fellow scientist Robert Anholt, did next — and how they did it — that broke new ground.

They identified specific cell clusters in the fruit fly brain affected by acute cocaine exposure, a discovery that potentially lays the groundwork for the development of drugs to treat or prevent addiction in humans.

To assess the effects of cocaine consumption on brain gene expression, the researchers dissected the fly brains and dissociated them into single cells. Using next-generation sequencing technology, they made libraries of the expressed genes for individual cells. The study looked at nearly 89,000 cells, each of which has thousands of transcripts. Through sophisticated statistical analysis, the researchers could group them into 36 distinct cell clusters.

“We identified the regions of the brain which are important. Now, we can see what genes are expressed when exposed to cocaine and whether there are Federal Drug Administration-approved drugs that could be tested, perhaps first in the fly model. We’ve already spotted several of these genes,” Mackay says.

Researchers can now leverage this work to understand potential therapies, explains Mackay, the Self Family Endowed Chair in Human Genetics.

First Time

The study marks the first time scientists have scanned the entire brain of the fly in one comprehensive experiment to reveal new brain regions involved in the response to cocaine, says Susan Harbison, who has worked with Mackay and is now an investigator with the National Institutes of Health’s National Heart, Lung and Blood Institute in Bethesda, Maryland.

Harbison says that before this work, the only way to identify which brain regions responded to cocaine was very laborious with no guarantee of success.

Wen Huang, an assistant professor in Michigan State University’s Department of Animal Sciences who did postdoctoral research in Mackay’s lab when she was at North Carolina State, calls the work “a huge advance.”

“Without getting to this kind of single-cell cellular detail, what you have is a very blurred picture,” he says.

Huang compares it to somebody attending a large party and taking a picture of everybody who was there. “The pixels are pretty big. You can’t really look at all the details,” he says. “With this type of technology, you can look at every single cell and identify different cell types based on their gene expression. It’s just like when we look at people. You can recognize them as a whole, but they have subtle details that tell them apart.”

Additional research by Baker identified nearly 1,000 genes that were involved in variation in voluntary consumption of cocaine and methamphetamine.

Against the Grain

Baker says when he first began researching cocaine and methamphetamine, the drug abuse research field did not readily accept fruit flies.

“Trudy taught me it’s OK to go against the grain, that it’s OK not to do exactly what’s popular if you have a good belief that it’s going to work,” he says. “We’ve been able to show through our hard work that flies are an excellent system to find genes that are important.”

Going against the grain is nothing new to Mackay.

“When I worked with her,” explains Harbison, “I was fascinated by her idea that one could understand complex traits studying naturally occurring variants across the genome and that by applying quantitative genetics to the data, one could examine the genetic architecture of a trait holistically. Her approach differed completely from classic reductionist approaches.

“She’s been at the forefront of the move from single-gene analysis to the elucidation of entire genetic networks influencing complex traits,” Harbison continues. “The payoff has been tremendous in terms of increasing our understanding of complex trait biology.”

They identified specific cell clusters in the fruit fly brain affected by acute cocaine exposure, a discovery that potentially lays the groundwork for the development of drugs to treat or prevent addiction in humans.

Drawn to Genetics

Mackay’s interest in the emerging field of genetics started when she took an advanced biology class in 10th grade that included genetics, along with laboratory experiments working with Drosophila.

“It just fascinated me,” she says. “I’ve always enjoyed quantitative biology, the math part of it, and anybody who likes animals gets fascinated by how they’re all the same breed, but they all look different. Unlike other fields, people first studying genetics in school are fairly easily divided into two groups and two groups only: ‘I don’t get it’ and ‘I get it.’ It’s this great divide with no middle ground. I was in the ‘I get it’ group. It just clicked for me.”

Her first project as an undergraduate at Dalhousie University in Canada didn’t involve fruit flies. Instead, she studied Sprirorbis borealis, an aquatic worm prevalent off the coast of Nova Scotia that decided between two kinds of seaweed as its lifetime substrate.

“The big question was why did they choose? Was there any genetic variation to it? Indeed, there was, but it was the 1970s and there was no way to figure out anything other than they were genetically different because we had no genetics in non-model organisms,” she says.

Mackay decided the more purely genetic questions drew her in, so she picked the fruit fly as her research vehicle.

She moved to Edinburgh, Scotland, to pursue her Ph.D. at the University of Edinburgh. “Edinburgh was, and still is, the best place to go for quantitative genetics,” she says.

There, she worked with Alan Robertson, one of the leading researchers in the field. She put fruit flies in either constant or variable environments and tracked their genetic variation over time.

After spending time on the faculty at Edinburgh, Mackay took the faculty position at North Carolina State in 1987.

Love of Horses

She soon met Anholt. Neither research nor fruit flies brought them together. Instead, it was their mutual love for horses.

A friend asked if she wanted to go on her first fox hunt. “I really wasn’t interested in hurting a poor fox,” she says. After getting assurances they wouldn’t actually catch anything, Mackay agreed to go on the fox hunt at the Red Mountain Hunt Club in Rougemont, North Carolina.

“Trudy was riding a horse. My horse was lame, so I was serving champagne at the start of the hunt,” Anholt recalls.

They later discovered they were both scientists. Eventually, Mackay convinced Anholt, a faculty member at Duke University whose research centered around olfactory behavior, to work with the Drosophila model.

They did their first experiments together at the kitchen table. “I brought odor solutions home. She brought fruit flies home, and we developed an assay to measure behavior to odorants on the kitchen table,” Anholt says.

While at North Carolina State, Mackay’s interest in identifying the genes affecting complex traits continued, and she did the first genotype-phenotype association studies in flies, looking at the relationship between genes and their observable characteristics.

“Those first studies showed that it is much more complex than anyone realized,” Mackay says.

That’s why they developed the Drosophila Genetic Reference Panel.

“In the next phase of our work, we just went to town measuring practically everything we could think of, from sensitivity and resistance to alcohol, lead, cadmium and paraquat, all these different environmental stressors,” says Mackay. “I was interested in lifespan, starvation resistance. Robert was interested in olfactory behavior. We also wanted to figure out the underlying molecular networks, not just one gene at a time but how they interact together. That’s what started us down the RNA sequencing path that culminates today with single-cell RNA sequencing.”

The Center for Human Genetics has already done complete genome sequencing of three patients with rare but undiagnosed genetic disorders and their parents. Two of the three now have diagnoses, and they’re still working on the third.

Advancing Human Genetics

When they moved to Clemson and the Center for Human Genetics, Mackay and Anholt officially combined their labs.

“I tell people Trudy is the brains, and I’m the cheerleader,” Anholt says.

They are continuing their research into complex traits, including substance abuse and lifespan.

“We have lines of flies that live twice as long as the average fly, and we want to know why,” Mackay says.

The National Institutes of Health recently awarded Clemson $10.6 million to establish the Center of Biomedical Research Excellence in Human Genetics in collaboration with the Greenwood Genetic Center. Mackay, Anholt and Richard Steet, the Greenwood Genetic Center’s director of research, will direct the effort, which is the NIH’s first center specifically focused on human genetics.

The center will cover common disorders, such as cardiovascular disease, cancer and neurodegenerative diseases as well as very rare genetic disorders.

The Center for Human Genetics has already done complete genome sequencing of three patients with rare but undiagnosed genetic disorders and their parents. Two of the three now have diagnoses, and they’re still working on the third.

“In one case we solved, the patient had the very first variant ever discovered in that gene,” she says, adding that knowing what the gene is and having a diagnosis could eventually lead to a treatment. “That may be the most exciting thing we’ve done since we’ve been here.”

New discoveries excite Mackay as much now as they did when she started her career:

“We love our jobs because every day is different. We never know what we’re going to find out, what challenges we’re going to meet and what new results we will have. It’s really exciting, like going into the unknown. I think any scientist would say the same thing.”

When Mackay’s students come to her and tell her they got a result they don’t understand, she tells them that’s the beginning of something exciting.

“It’s not science if you know what you’re going to find before you do it. You ask the question, and Mother Nature gives you the answer. You have to go with it because, unless you made a mistake, it’s the answer,” she says. “It may not be what you expect, but you’re going to learn something new.

“That’s the beginning of something exciting. The unexpected can lead to the biggest discoveries.”

Cindy Landrum is a senior science writer in the College of Science.

0 replies

Leave a Reply

Want to join the discussion?
Feel free to contribute!

Leave a Reply

Your email address will not be published. Required fields are marked *