Author: Lillian Eden

A genome-wide screen in live hosts reveals new secrets of parasite infection

Researchers in the Lourido Lab performed the first genome-wide screen of Toxoplasma gondii in live hosts, revealing genes that are important for infection but previously undetected in cell culture experiments. 

Greta Friar | Whitehead Institute
July 8, 2024

Apicomplexan parasites are a common cause of disease, infecting hundreds of millions of people each year. They are responsible for spreading malaria; cryptosporidiosis – a severe childhood diarrheal disease; and toxoplasmosis – a disease that endangers immune compromised people and fetuses, and is the reason why pregnant women are told to avoid changing cat litter. Apicomplexan parasites are very good at infecting humans and many other animals, and persisting inside of them. The more that researchers can learn about how apicomplexans infect hosts, the better they will be able to develop effective treatments against the parasites.

To this end, researchers in Whitehead Institute Member Sebastian Lourido’s lab, led by graduate student Christopher Giuliano, have now completed a genome-wide screen of the apicomplexan parasite Toxoplasma gondii (T. gondii), which causes toxoplasmosis, during its infection of mice. This screen shows how important each gene is for the parasite’s ability to infect a host, providing clues to genes’ functions. In the journal Nature Microbiology on July 8, the researchers share their approach for tracing lineages of parasites in a live host, and some specific findings of interest—including a possible anti-parasitic drug target.

From dish to animal

Researchers in Lourido’s lab previously developed a screen to test the function of every T. gondii gene in cells in a dish in 2016. They used CRISPR gene editing technology to make mutant parasites in which each lineage had one gene inactivated. The researchers could then assess the importance of each gene to a parasite’s fitness, or ability to thrive, based on how well the mutants missing that gene did. If a mutant died off, this implied that its inactivated gene is essential for the parasite’s survival.

This screen taught the researchers a lot about T. gondii’s biology but faced a common limitation: the parasites were studied in a dish rather than a live host. Cell culture provides an easier way to study parasites, but the conditions are not the same as what parasites face in an animal host. A host’s body is a more complex and dynamic environment, so it may require parasites to rely on genes that they don’t need in the artificial setting of cell culture.

To overcome this limitation, researchers in Lourido’s lab figured out how to repeat the T. gondii genome-wide screen, which their colleagues in the lab had previously done in cell culture, in live mice. This was a massive undertaking, which required solving various technical challenges and running a large number of parallel experiments. T. gondii has around eight thousand genes, so the researchers performed pooled experiments, with each mouse getting infected by many different mutants—but not so many as to overwhelm the mouse. This meant that the researchers needed a way to more closely monitor the trajectories of mutants in the mouse. They needed to track the lineages of parasites that carried the same mutation over time, as this would allow them to see how different replicate lineages of a particular mutant performed.

“This is an outstanding resource,” says Lourido. “The results of the screen reveal such a broader spectrum of ways in which the parasites are interacting with hosts, and enrich our perception of the parasites’ abilities and vulnerabilities.”
The researchers added barcodes to the CRISPR tools that inactivated a gene of interest in the parasite. When they harvested the parasites’ descendants, the barcode would identify the lineage, distinguishing replicate parasites that had been mutated in the same way. This allowed the researchers to use a population-based analytical approach to rule out false results and decrease experimental noise. Then they could draw conclusions about how well each lineage did. Lineage tracing allowed them to map how different populations of parasites traveled throughout the host’s body, and whether some populations grew better in one organ versus another.

The researchers found 237 genes that contribute to the parasite’s fitness more in a live host than in cell culture. Many of these were not previously known to be important for the parasite’s fitness. The genes identified in the current screen are active in different parts of the parasite, and affect diverse aspects of its interactions with a host. The researchers also found instances in which parasite fitness in a live host increased when a gene was inactivated; these genes may be, for example, related to signals that the host immune system uses to detect the parasites. Next, the researchers followed up on several of the fitness-improving genes that stuck out as of particular interest.

Genes that make the difference in a live host

One gene that stuck out was GTP cyclohydrolase I (GCH), which codes for an enzyme involved in the production of the essential nutrient folate. Apicomplexans rely on folate, and so the researchers wanted to understand GCH’s role in securing it for the parasite. Cell culture media contains high levels of folate, and in this nutrient-rich environment, GCH is not essential. However, in a live mouse, the parasite must both scavenge folate and synthesize it using the metabolic pathway containing GCH. Lourido and Giuliano uncovered new details of how that pathway works.

Although previously GCH’s role was not fully understood, the importance of folate for apicomplexans is a well-known vulnerability that has been used to design anti-parasitic therapies. The anti-folate drug pyrimethamine was commonly used to treat malaria, but many parasites have developed resistance to it.

Some drug-resistant apicomplexans have increased the number of GCH gene copies that they have, suggesting that they may be using GCH-mediated folate synthesis to overcome pyrimethamine. The researchers found that combining a GCH inhibitor with pyrimethamine increased the efficacy of the drug against the parasites. The GCH inhibitor was also effective on its own. Unfortunately, the currently available GCH inhibitor targets mammalian as well as parasitic folate pathways, and so is not safe for use in animals. Giuliano and colleagues are working on developing a GCH inhibitor that is parasite-specific as a possible therapy.

“There was an entire half of the folate metabolism pathway that previously looked like it wasn’t important for parasites, simply because people add so much folate to cell culture media,” Giuliano says. “This is a good example of what can be missed in cell culture experiments, and what’s particularly exciting is that the finding has led us to a new drug candidate.”

Another gene of interest was RASP1. The researchers determined that RASP1 is not involved in initial infection attempts, but is needed if the parasites fail and need to mount a second attempt. They found that RASP1 is needed to reload an organelle of the parasites called a rhoptry that the parasites use to breach and reprogram host cells. Without RASP1, the parasites could only deploy one set of rhoptries, and so could only attempt one invasion.

Identifying the function of RASP1 in infection also demonstrated the importance of studying how parasites interact with different cell types. In cell culture, researchers typically culture parasites in fibroblasts, a connective tissue cell. The researchers found that parasites could invade fibroblasts with or without RASP1, suggesting that this cell type is easy for them to invade. However, when the parasites tried to invade macrophages, an immune cell, those without RASP1 often failed, suggesting that macrophages present the parasites with more of a challenge, requiring multiple attempts. The screen uncovered other probable cell-type specific pathways, which would not have been found using only model cell types in a dish.

The screen also highlighted a previously unnamed gene that the researchers are calling GRA72. Previous studies suggested that this gene plays a role in the vacuole or protective envelope that the parasite forms around itself. The Lourido lab researchers confirmed this, and discovered additional details of how the absence of GRA72 disrupts the parasite vacuole.

A rich resource for the future

Lourido, Giuliano, and colleagues hope that their findings will provide new insights into parasite biology and, especially in the case of GCH, lead to new therapies. They intend to continue pulling from the treasure trove of results—their screen identified many other genes of interest that require follow-up—to learn more about apicomplexan parasites and their interactions with mammalian hosts. Lourido says that other researchers in his lab have already used the results of the screen to guide them towards relevant genes and pathways in their own projects.

“This is an outstanding resource,” says Lourido, who is also an associate professor of biology at MIT. “The results of the screen reveal such a broader spectrum of ways in which the parasites are interacting with hosts, and enrich our perception of the parasites’ abilities and vulnerabilities.”

Brady Weissbourd named Searle Scholar

With an eye on regenerative medicine, Weissbourd's lab will study how jellyfish manage to constantly integrate new neurons into their nervous system.

David Orenstein | The Picower Institute for Learning and Memory
July 8, 2024

Scientists who dream of a future in which regenerative medicine has advanced enough to enable repairs in human nervous systems currently have more questions than answers. As a recently named Searle Scholar, MIT Assistant Professor Brady Weissbourd will seek to learn some of the needed fundamentals by studying a master of neural regeneration: the jellyfish, Clytia hemisphaerica.

Weissbourd, a faculty member in the Department of Biology and The Picower Institute for Learning and Memory, has helped to pioneer use of the seafaring species in neuroscience research for many reasons. It is transparent for easy imaging, reproduces rapidly, and shares many basic nervous system properties with mammals despite diverging evolutionarily 600 million years ago (just after the development of the earliest nervous systems). Meanwhile, with about 10,000 neurons, the jellyfish fills a gap in the field in terms of that degree of complexity.

But what Weissbourd didn’t appreciate until he began experimenting with the jellyfish was that they are also incredibly good at refreshing and rebuilding their nervous systems with new cells. After becoming the first researcher to develop the ability to genetically manipulate the organism, he started teasing out how its highly distributed nervous system (there is no central brain), was organized to enable its many behaviors. When he ablated a subnetwork of cells to test whether it was indeed responsible for a particular feeding behavior, he found that within a week it had completely regrown. Moreover, he has observed that the jellyfish constantly produce and integrate new cells, even in the absence of major injury.

Looking for the logic

The finding raised a proverbial boatload of intriguing questions that his support of $100,000 a year for the next three years from the Searle Scholars Program will help him pursue.

“Where are these newborn neurons coming from in both the normal and regenerative contexts?” Weissbourd asked. “What rules guide them to the correct locations to rebuild these networks, both to integrate these newborn neurons into the network without messing it up and also to recreate it during regeneration? Are the rules the same or different between these contexts?”

Additionally, by using a combination of techniques such as imaging neural activity during behavior, sequencing gene expression cell by cell, and computational modeling, Weissbourd’s lab has discerned that within their web-like mesh of neurons, jellyfish harbor more than a dozen different functional subnetworks that enable its variety of different behaviors. Can all the subnetworks regenerate? If not, why do some forgo the remarkable ability? Among those that do regenerate, do they all do so the same way? If they employ different means, then learning what those are could provide multiple answers to the question of how new neurons can successfully integrate into existing neural networks.

Building on support provided by a Klingenstein-Simons Fellowship Weissbourd earned last year, he’ll be able to pursue experiments designed to understand the “logic” of how jellyfish manage neural regeneration.

“The ability to understand how nervous systems regenerate has significant implications for regenerative medicine,” Weissbourd said.

A complete 3D ‘wiring diagram’

As part of the new award, Weissbourd also plans to create a major new resource for jellyfish neurobiology to advance not only this project, but also the research of any other scientist who wants to study the organism. Working with collaborator Jeff Lichtman, a professor of molecular and cellular biology at Harvard University, Weissbourd will create a complete 3D reconstruction of a jellyfish’s nervous system at the subcellular resolution enabled by electron microscopy. The resource, which Weissbourd plans to provide openly online, will amount to a full “wiring diagram” of a jellyfish where every circuit connection can be mapped.

Being able to see how every neural circuit is constructed in a whole animal will enable Weissbourd to answer questions about how the circuits are built and therefore how new neurons integrate. Having a complete and detailed view of every circuit will improve the computational models his lab is building to predict how anatomy helps give rise to function and behavior. And given that new neurons are being born, migrating and integrating all the time, Weissbourd said, the imaging will also likely yield a snapshot of neural regeneration in action in its many stages.

Weissbourd said he was grateful for the honor of being named a Searle Scholar, which not only provides support for his lab’s work, but also welcomes him into a new community of young scientists.

“I’m honored and super excited,” Weissbourd said. “I’m excited to interact with the other scholars as well.”

 

“Vaults” within germ cells offer more than safekeeping

Ribonucleoprotein (RNP) granules are believed to preserve maternal mRNA within eggs and developing embryos. The Lehman Lab reveals that a specific type of RNP granule also plays an active role in translating the mRNA that is crucial for specifying germ cells.

Shafaq Zia | Whitehead Institute
July 2, 2024

Maternal messenger RNAs (mRNAs), located within the cytoplasm of an immature egg, are crucial for jump starting development. Following fertilization, these mRNAs are passed onto the zygote, the first newly formed cell. Having been read from the maternal DNA genetic code, they serve as the sole templates for protein production essential for early development until the zygote’s own genes become active and take over.

Many maternal mRNAs are stored in ribonucleoprotein (RNP) granules, which are a type of membrane-less compartments, or condensates, within eggs and developing embryos. These granules are believed to preserve the mRNA in a “paused” state until the encoded proteins are needed for specific developmental processes upon fertilization of the egg cell. Then, certain developmental signals kick in to instruct the RNP granules to release the stored mRNA so the instructions can be translated into a functional protein.

One type of RNP granules called germ granules is found in embryo germplasm, a cytoplasmic region that gives rise to germ cells, which become the eggs or sperms of adult flies. Whitehead Institute Director Ruth Lehmann studies how germ cells form and transmit their genetic information across generations. Her lab is particularly interested in understanding how germ granules in embryos localize and regulate maternal mRNAs.

Now, Lehmann, along with graduate student Ruoyu Chen and colleagues, has uncovered that the role of germ granules in fruit flies (Drosophila melanogaster) extends beyond safeguarding maternal mRNAs. Their findings, published in the journal Nature Cell Biology on July 4, demonstrate that germ granules also play an active role in translating, or making into protein, a specific maternal mRNA, called nanos, crucial for specifying germ cells and the abdomen of the organism.

“Traditionally, scientists have thought of RNP granules as a dead zone for translation,” says Chen. “But through high-resolution imaging, we’ve challenged this notion and shown that the surface of these granules is actually a platform for translation of nanos mRNA.”

RNP granules act as vaults

Within a developing embryo, various fate-determining proteins dictate whether a cell will become a muscle, nerve, or skin cell in a fully-formed body. Nanos, a gene with conserved function in Drosophila and humans, guides the production of Nanos protein which instructs cells to develop into germline. Mutations in the nanos gene cause sterility in animals.

During early embryonic development, Nanos protein also helps establish the body plan of the fruit fly embryo — it specifies the posterior end or abdominal region, and guides the ordered development of tissues along the length of the body, from head to tail. In embryos with impaired Nanos function, the consequences are fatal.

“When Nanos protein isn’t functioning properly, the fruit fly embryos are really short,” says Chen. “This is because the embryo has no abdomen, which is basically half of its body. Nanos also has a second function that is conserved from flies to humans. This function is very local and instructs the cells with lots of Nanos to become germ cells. ”

Given Nanos’ vital role, embryos must safeguard instructions for its production until the embryo reaches a specific stage of development, when it is time to define the posterior region. Previous work has indicated that germ granules in the germplasm and germ cells can act like vaults, shielding the nanos mRNA from degradation or premature translation.

However, while the mRNA instructions for building the protein are distributed throughout the embryo, Nanos protein is found only in regions where germ granules reside. The mRNA does not get translated elsewhere in the embryo because of a regulatory protein called Smaug, named after the golden dragon depicted in J. R. R. Tolkien’s 1937 novel The Hobbit. Smaug binds to a non-protein coding segment of the mRNA known as the 3’ untranslated region (3’ UTR), extending beyond the protein-coding sequence, effectively suppressing the translation process.

For Lehmann, Chen, and their colleagues, this hinted at an intriguing relationship between nanos mRNA and germ granules. Are the granules essential for translating nanos mRNA into a functional protein? And if they are, is their role primarily to serve as a safekeeping place to evade repression by Smaug or do they actively facilitate the translation of nanos mRNA too?

To answer these questions, the researchers combined high-resolution imaging with a technique called the SunTag system to directly visualize the translation of nanos mRNA within Drosophila germ granules at the single-molecule level.

Unlike green fluorescent protein tagging, where a single fluorescent molecule is used, the SunTag system allows scientists to recruit multiple GFP copies for an amplified signal. First, a small protein tag, known as the SunTag, is fused with the protein-producing region of the nanos mRNA. As the mRNA instructions undergo translation, GFP molecules stick to the newly synthesized SunTag-Nanos protein, resulting in a bright fluorescent signal. Overlying this translation signal with fluorescent probes specifically labeling the mRNA then allows researchers to precisely visualize and track when and where the translation process is taking place.

“Using this system, we’ve discovered that when nanos mRNA is translated, it protrudes slightly from the surface of the granules like snakes peeking out of a box,” says Chen. “But they can’t fully emerge; a part of their sequence, specifically their “back” end, the 3’ UTR, remains tucked inside the granules. When the RNA is not translated, like during oogenesis, the tip coils back and is hidden inside the granule.”

With their high-resolution SunTag imaging technique, Lehmann, Chen and their colleagues have directly added to the work of other researchers with similar observations: mRNAs in the process of translation are in an extended configuration, while the 5’UTR curls back to the 3’UTR when the mRNAs are repressed.

Flipping on nanos translation

Then, the researchers went on to take a closer look at how these granules help initiate translation, while Smaug is able to inhibit the same nanos mRNA molecules from being translated in other areas of the embryo. They hypothesized that the untranslated region (UTR) of nanos mRNA, which remains concealed within the granules, might be playing a pivotal role in the translation process by localizing the mRNA instructions within germ cell granules. This localization, they speculated, protects the mRNA from Smaug’s inhibitory actions and facilitates Nanos protein production, so the posterior region can develop properly.

However, counter-intuitive to a simple protection model, they found that rather than being depleted, Smaug is enriched within germ granules, indicating that additional mechanisms within the RNP granule must counteract Smaug’s inhibitory effects. To explore this, the researchers turned to another regulatory protein called Oskar, which is known to interact with Smaug.

Discovered by Lehmann in a 1986 study, and named after a character in the German novel The Tin Drum, the oskar gene in Drosophila is known to help with the development of the posterior region. Later research has revealed that, during the development of oocytes, Oskar acts as a scaffold protein by initiating the formation of germ granules in germ cells and directing mRNA molecules, including nanos, towards the granules.

To gain a deeper understanding of Oskar’s full role in translational regulation in germ granules and its interaction with Smaug, the researchers engineered a modified version of Oskar protein. This altered Oskar protein retained its ability to initiate the formation of germ granules and localize nanos mRNA within them. However, Smaug no longer localized to the germ granules assembled by this altered Oskar.

The researchers then studied whether the mutant protein had any effect on nanos mRNA translation. In the germ cells with this mutant version of Oskar, the researchers saw a significant reduction in the translation of nanos mRNA. These findings, combined, suggested that Oskar regulates nanos translation in fruit fly embryos by recruiting Smaug to the granules and then counteracting its repression of translation.

“Condensates composed of RNAs and proteins are found in the cytoplasm of pretty much every cell and are thought to mediate mRNA storage or transport,” says Lehmann, who is also a professor of biology at the Massachusetts Institute of Technology. “But our results provide new insights into condensate biology by suggesting that condensates can be also used to specifically translate stored mRNAs.”

Indeed, in the oocyte, the germ granules are silent and only become activated when the egg is fertilized.

“This suggests that there might also be other ‘on and off switches’ governing translation within condensates during early development,” adds Lehmann. “How this is achieved and whether we could engineer this to happen at will in these and other granules is a question for the future.”

Boston Globe: Mary-Lou Pardue, MIT professor whose anti-bias efforts lifted women in science, dies at 90

Her research formed the foundation for understanding the structure of chromosomes.

Bryan Marquard | Boston Globe
July 7, 2024

Amid the clatter of lunchtime dishes, Mary-Lou Pardue sat across from Nancy Hopkins one day in 1994 in a café not far from the Massachusetts Institute of Technology, reading a letter Hopkins had drafted.

Both were MIT professors and scientists, and Hopkins, the younger of the two, had gathered data showing women on the faculty were routinely discriminated against in numerous ways. Hopkins wanted to send her findings to the school’s president, but sought a blessing of sorts from Dr. Pardue, the first woman in MIT’s School of Science to be inducted into the National Academy of Sciences.

“I chose Mary-Lou as the person whose judgment would mean the most to me. I had this huge respect for her as a scientist before I even met her,” Hopkins recalled in an interview.

Dr. Pardue read the letter “very slowly and put it down on the table and said, ‘I agree with this letter, every word. I want to sign it and think you should send it to the president,’ ” Hopkins said. “And that changed my life, and ultimately it changed MIT. That was, to me, the defining moment for women at MIT.”

A highly regarded cellular and molecular biologist whose work formed the foundation for key advancements and discoveries in understanding the structure of chromosomes, Dr. Pardue died June 1 in Youville Assisted Living in Cambridge.

She was 90, had been diagnosed with Parkinson’s disease, and her health had been failing.

The first Boris Magasanik professor of biology at MIT, Dr. Pardue had also been an American Academy of Arts and Sciences fellow, and was a past president of the Genetics Society of America and the American Society for Cell Biology.

Her efforts at MIT 30 years ago with Hopkins and other female professors, however, are still having a ripple effect through academia across the country and around the world.

When Dr. Pardue told Hopkins she wanted to sign the letter about bias against women and send it to MIT’s president, “I knew the world had shifted,” said Hopkins, whose efforts with Dr. Pardue and others were documented in “The Exceptions,” a 2023 book by New York Times reporter Kate Zernike, who initially broke the story as a Boston Globe reporter.

“I could sense the power of it: Two women, saying the same thing, one of them a member of the National Academy of Sciences,” Hopkins said. “She looked at me and felt the same thing, that two women together had power.”

They reached out to other tenured female professors at MIT, and almost all co-signed the letter, which they presented to the president. In 1995, MIT created the Committee on the Status of Women Faculty, whose 1999 report documented the systematic bias that women in the School of Science were facing.

That report, and MIT’s subsequent efforts to address its failings, led to similar efforts at universities across the country.

“It was life-changing, but that it could change the world? This is not something that occurred to me then,” Hopkins said, laughing at the memory.

As a young scientist, Dr. Pardue and Joseph Gall, who had been her doctoral adviser at Yale University, developed an “in situ hybridization” technique that “led to many discoveries, including critical advancements in developmental biology, our understanding of embryonic development, and the structure of chromosomes,” MIT said in its tribute to Dr. Pardue.

“In situ hybridization was a crucial step toward genomics. In some ways you could call it the first genomic technique,” said Allan Spradling, an investigator at the Howard Hughes Medical Institution.

“Her research is underappreciated,” said Spradling, who also is a former director of the embryology department at the Carnegie Institution for Science. “It’s all tied into so many momentous events in the history of genomics.”

Kerry Kelley, who formerly managed Dr. Pardue’s lab, and is now manager of the Yilmaz Lab at the Koch Institute for Integrative Cancer Research, said that “Mary-Lou was a giant of her time.”

Continuing to work in her lab after the onset of Parkinson’s, Dr. Pardue was “gracious, kind, smart as a whip, and just full of great stories,” Kelley said.

The techniques that Dr. Pardue and Gall developed are now used in thousands of labs around the world, said Thomas Cech, a former post-doctoral student of Dr. Pardue’s who shared the 1989 Nobel Prize in Chemistry.

“It was one of those discoveries which seemed important at the time and certainly attracted many of us to her laboratory,” he said, “but in retrospect, we had no idea how powerful this would become.”

Born on Sept. 15, 1933, Mary-Lou Pardue grew up in and around Lexington, Ky.

Her father, Louis Pardue, was a dean at Virginia Tech. Her mother, Mary Allie Marshall Pardue, had been a teacher before marrying. Her younger brother, William, who died in 2016, was a scientist in the nuclear industry.

Dr. Pardue graduated in 1955 from the College of William and Mary with a bachelor’s degree in biology.

After working in research, she received a master’s in radiation biology in 1959 from the University of Tennessee and a doctorate in biology in 1970 from Yale University.

She also did postdoctoral work at the University of Edinburgh before seeking a faculty position in the United States. MIT turned her down with a letter at first, and then recruited her for an associate professor position in 1972 after hearing about her work and lectures, “which I thought was as sincere an apology as you can get,” she said with a laugh in a video forum that MIT posted online.

Dr. Pardue, whose marriage in her graduate student years ended in divorce, was an avid hiker in New Hampshire’s White Mountains who also took on distant challenging terrain. She agreed to the Genetics Society presidency because on the way home from an international meeting in India she could go trekking in Nepal’s Annapurna range.

“She was a fun person to be around,” said Susan A. Gerbi, the George Eggleston professor of biochemistry emerita at Brown University, and a graduate school contemporary of Dr. Pardue’s at Yale.

“She had a twinkle in her eye, which you can see even if you look at the seminar she gave on YouTube,” Gerbi said. “And she was very smart and had good insights.”

Over the years, Dr. Pardue was close to her brother’s family, spending time with them during the winter holidays and going along on skiing and camping trips.

“A lot of times you run into scientists who are quite intelligent and can’t relate to people on a personal level,” said her nephew, Todd Pardue of Fairfax Station, Va. “She would take the time to talk to you. She was a very special person.”

He and his sister, Sara Pardue Gibson of Columbus, Ohio, are their aunt’s closest survivors. Plans for a celebration of Dr. Pardue’s life and work are pending.

While fielding questions during her MIT talk that was recorded for a video, Dr. Pardue smiled and said in a voice still rich with the Kentucky accent of her youth that as a researcher, “the greatest joy is when an experiment you didn’t think would work, works.”

Such clear, concise lessons were among those she imparted to generations of young scientists who worked in her labs, including at MIT, where she was a professor for more than 30 years.

“She was a great mentor who was as proud of her scientific children and grandchildren as she was of her own accomplishments. That’s not the way all scientists look at things,” said Ky Lowenhaupt, manager of the Biophysical Instrumentation Facility at MIT.

Lowenhaupt said Dr. Pardue “was a role model of what women in science can be at a time when there weren’t a lot of those, and a trailblazer as a woman — but also a trailblazer as a scientist who didn’t do things along the path that other people took.”

She’s fighting to stop the brain disease that killed her mother before it gets her

Jonathan Weissman is the senior author on a recent study on silencing a prion protein's expression. Prions cause devastating neurodegenerative disorders such as dementia, Huntington's, Parkinson's, and Lou Gehrig's disease. Silencing genes represents a step towards a therapeutic model for treating these diseases in humans.

Karen Weintraub | USA TODAY
June 27, 2024

CAMBRIDGE, Mass. ‒ Sonia Vallabh watched helplessly as her 51-year-old mother rapidly descended into dementia and died. It didn’t take long for Vallabh to realize she was destined for the same rare genetic fate.

Vallabh and her husband did what anyone would want to do in their situation: They decided to fight.

Armed with little more than incredible intellect and determination they set out to conquer her destiny.

A dozen years later, they’ve taken a major step in that direction, finding a way to shut off enough genetic signals to hold off the disease.

And in the process of trying to rescue Vallabh, they may save many, many others as well.

In a paper published Thursday in the prestigious journal Science, Vallabh and her husband, Eric Minikel, and their co-authors offer a way to disrupt brain diseases like the one that killed her mother.

The same approach should also work against diseases such as Huntington’s, Parkinson’s, Lou Gehrig’s disease and even Alzheimer’s, which result from the accumulation of toxic proteins. If it works as well as they think, it could also be useful against a vast array of other diseases that can be treated by shutting off genes.

“It doesn’t have to be the brain. It could be the muscles. It could be the kidneys. It could be really anywhere in the body where we have not easily been able to do these things before,” said Dr. Kiran Musunuru, a cardiologist and geneticist at the University of Pennsylvania’s Perelman School of Medicine, who wasn’t involved in the research but wrote a perspective accompanying the paper.

So far, they’ve proven it only in mice.

“The data are good as far as they go,” Vallabh said this week from her office at the Broad Institute of Harvard and the Massachusetts Institute of Technology, where she has worked since getting a Ph.D. at Harvard. She had already gotten a law degree from the university, but she and Minikel, then a transportation planner, both pursued biology degrees after her mother’s death. Now, they work together at the Broad.

“We’re far from this being a drug,” Vallabh said. “There’s always, always reason for caution. Sadly, everything is always more likely to fail than succeed.

“But there is justifiable reason for optimism.”

A terrible disease

The disease that killed Vallabh’s mother was one of a group of conditions called prion diseases. These include mad cow disease, which affects mostly cattle, scrapie, which affects sheep, and Creutzfeldt-Jakob disease, which kills about 350 Americans a year ‒ most within months of their first symptom.

These diseases are triggered when the prion protein found in all normal brains starts misfolding for some reason, as yet unknown.

“Prion disease can strike anybody,” Vallabh said, noting the 1 in 6,000 risk to the general population.

Though prion diseases are, in some cases, contagious, a federal study earlier this year concluded that chronic wasting disease, found in deer, elk and moose, is very unlikely to pass to people who eat the meat of sick animals.

In Vallabh’s case, the cause is genetic. Vallabh discovered after her mother’s death that she carries the same variant of the same gene that caused her mother’s disease, meaning she will certainly develop it.

The only question is when.

“The age of onset is extremely unpredictable,” Vallabh said. “Your parent’s age of onset doesn’t actually predict anything.”

How the gene-editing tool works

Vallabh and Minikel approached colleagues at the Whitehead Institute a biomedical research institute next to the Broad. They asked to collaborate on a new gene-editing approach to turn off Vallabh’s disease gene. The technique developed by Whitehead scientists is called CHARM (for Coupled Histone tail Autoinhibition Release of Methyltransferase).

While previous gene-editing tools have been described as scissors or erasers, Musunuru described CHARM as volume control, allowing scientists to tune a gene up or down. It has three advantages over previous strategies, he said.

The device is tiny, so it fits easily inside the virus needed to deliver it. Other gene-editing tools, like CRISPR, are bigger, which means they need to be broken into pieces and much more of the virus is needed to deliver those pieces to the brain, risking a dangerous immune reaction.

CHARM, Musunuru said, is “easier to deliver to hard-to-deliver spaces like the brain.”

At least in the mouse, it also seems to have reached throughout the brain, making the desired genetic change without other, unwanted ones, Musunuru said.

And finally, the research team figured out a way to turn the gene editor off after its work was done. “If it’s sticking around, there’s the potential for genetic mischief,” Musunuru said.

One shot on goal

While researchers, including Vallabh, continue to work to perfect an approach, the clock for Vallabh and others is ticking.

Right now there’s no viable treatment and if it takes too long to develop one, Vallabh will miss her window. Once the disease process starts, like a runaway train, it’ll be much harder to stop than it would be to just shut the gene off in the first place.

The more prion protein in the brain, the more likely it is to misfold. And the more likely it is for the disease to spread, a process that co-opts the natural form of the protein and converts it to the toxic form.

That’s why getting rid of as much of it as possible makes sense, said Jonathan Weissman, the senior author of the study, who leads a Whitehead lab.

“The biology is really clear. The need (for a cure) is so compelling,” Weissman said.

Every cell in the brain has the gene for making the prion protein. By silencing even 50% of those genes, Weissman figures he can prevent the disease. In mice, CHARM silenced up to 80% to 90%.

“We’ve figured out what to deliver. Now we have to figure out how to deliver it,” he said.

Another of the paper’s co-authors, the Broad’s Ben Deverman, published a study late last year showing he could deliver a gene-therapy-carrying virus throughout the brain. Others are developing other viral delivery systems.

Vallabh and Minikel have hedged their bets, helping to develop a so-called antisense oligonucleotide, or ASO, which uses another path for stopping the gene from making the prion protein.

The ASO, which is in early trials in people by a company called Ionis Pharmaceuticals, requires regular treatment rather than the one-and-done of gene therapy. Recruitment for that trial had to be paused in April because the number of would-be volunteers outstripped the available slots.

Vallabh isn’t ready yet to start any treatment yet herself.

“She has one shot on goal,” Musunuru said. “At some point, she’ll have to decide what’s the best strategy.”

In the meantime, the clock Vallabh can’t see continues to tick toward the onset.

She and Minikel stay exceedingly busy with their research along with their daughter, almost 7, and 4-year-old son ‒ both born via IVF and preimplantation genetic testing to ensure they wouldn’t inherit her genetic curse. (They were super lucky, Vallabh notes, to be living in Massachusetts where IVF is at least “approachable” financially.)

“There is a mountain ahead of us,” Vallabh said of the path to a cure. “There’s still a lot of hurdles, there’s still a lot to figure out.”

A day in the life — graduate student and genomics researcher Neha Bokil

Neha Bokil is studying mechanisms that regulate expression of genes located on the X and Y chromosomes in order to better understand sex-biased conditions that predominantly affect one sex.

Shafaq Zia | Whitehead Institute
June 25, 2024

Graduate student Neha Bokil moves around the Page lab with urgency. Today, she’s running an experiment using white blood cells from patients with varying numbers of X and Y chromosomes.

The lab of Whitehead Institute Member David Page investigates the role of the X and Y chromosomes beyond determining sex. While most females have two X chromosomes (XX) and most males have one X and one Y chromosome (XY), there are individuals whose sex chromosome constitution varies from this, having instead, for example, XXY, XXX, or XXXXY. With the goal of understanding why certain conditions are more prevalent in one sex versus than the other, Bokil is using this experiment to explore if and how cellular processes, such as gene regulation, vary among individuals with these atypical combinations of sex chromosomes.

Partially hidden in the cell culture hood, Bokil finally locates what she’s been searching for: a pipette for dispensing 99 microliters of the cell suspension she’s meticulously prepared this afternoon, a type of culture where cells float in nutrient-rich liquid, free to function and grow.

Bokil carefully extracts this volume and transfers it to a flat plate — also called a 96-well plate — with tiny holes for growing small cell samples. Now, it’s a waiting game until she can find out how these cells are growing, and whether their proliferation rate depends on the number of sex chromosomes in a cell.

Bokil dives into the intricacies of human genetics every day, hoping her work will eventually help reshape how sex differences are understood in medicine and improve treatment outcomes. The dynamic research Bokil is conducting at Whitehead Institute is her calling, but she has other passions as well. Here’s what a typical day in her life as a graduate student looks like, both in and outside the lab.

An inherited love of numbers

When she isn’t rushing out the door, Bokil loves brewing and savoring the perfect cup of morning chai, a traditional South Asian loose-leaf tea with milk. Every family has their own recipe, and Bokil makes hers with ginger, a touch of cardamom, and some sugar.

“Chai is comforting at any time, but I’ve noticed my mood vastly improves when I’m able to have a cup in the morning,” she says.

On her walk to the Whitehead Institute, she often listens to Bollywood songs. But these predilections — chai and Indian cinema — are more than just rituals for her. They symbolize tradition and cherished connections with family and friends.

In fact, family bonds have greatly influenced Bokil’s career path. As a child, she loved mathematics. It wasn’t a trait passed on genetically, but one that flourished through moments of connection with her grandmother, a math teacher in India. During summer visits to Bokil’s family in the U.S., she’d enthusiastically impart her passion for numbers onto her granddaughter. By the time Bokil went to high school and later college, she had become fluent in the language of logic and patterns.

“My time with her made me realize just how beautiful and fun math is, and I could see its practical applications in everyday life, all around me,” Bokil says.

For her PhD, she sought to combine her undergraduate training in mathematics and molecular biology to tackle a real-world problem. With genetics at the crossroads of these disciplines, and the Page Lab leading the way in transforming scientific understanding of X and Y chromosomes beyond reproduction, Bokil knew she had to get involved.

This morning, as she sits at her desk, poring over a research paper before an afternoon lab meeting, she ponders how insights from the study could enhance her manuscript writing process. Bokil’s graduate project uses a collection of cell lines derived from patients with atypical numbers of X and Y chromosomes to investigate mechanisms that regulate — or dial up and down the expression of — genes located on one of the X chromosomes in females called the “inactive” X chromosome.

Although the X and Y sex chromosomes in mammals began as a pair with similar structures, over time, the Y chromosome underwent degeneration, leading to the loss of numerous active genes. In contrast, the X chromosome preserved its original genes and even gained new ones. To maintain balance in gene expression across the two sexes — XX and XY — an evolutionary mechanism called X chromosome inactivation emerged.

This process is known to randomly silence one X chromosome in each XX pair, ensuring that both sexes have an equal dosage of genes from the X chromosome. However, in recent years, the Page lab has discovered that there are powerful distinctions within females’ pair of X chromosomes, and the so-called “inactive” X chromosome is far from passive. Instead, it plays a crucial role in regulating gene expression on the active X chromosome.

“That’s not all,” adds Bokil. “There are still genes expressed from that “inactive” X chromosome. Cracking how these genes are regulated could answer longstanding questions about sex differences in health.”

Bokil is unraveling this genetic mystery with the help of chemical tags called histone marks. These tags cling to a family of proteins that function like spools, allowing long strands of DNA to coil around them — like thread around a bobbin — so genetic information remains neatly packaged within the cell’s nucleus.

This complex of DNA, RNA, and proteins is called chromatin, the genetic material that eventually forms chromosomes. Chromatin also lays the groundwork for gene regulation by keeping some genes tightly wound around the histones, rendering them inaccessible, and unwinding others for active use.

Certain histone marks are associated with open chromatin structure and active gene expression, while others indicate closed chromatin structure and gene silencing. By examining the specific histone marks on proteins near genes on the “inactive” X chromosome, Bokil aims to decipher if and how these genes are turned on and off.

She’s particularly interested in a group of genes that have counterparts on the Y chromosome. These genes, known as homologous X-Y gene pairs, are typically dosage-sensitive and play a crucial role in regulating essential processes throughout the body like the transcription of DNA into RNA and the translation of RNA into proteins.

Celebrating small triumphs

Graduate school can feel like a marathon — progress is slow but every small step counts towards a breakthrough. For Bokil, stumbling upon a captivating scientific puzzle has been a stroke of luck she deeply appreciates. In fact, the mystery of how genes are controlled on the “inactive” X chromosome has not only shaped her scientific pursuits but also her artwork — on one quiet evening at home, she found herself inspired to capture an experiment, called CUT&RUN, in her painting.

During the early days of her PhD, Bokil spent hundreds of hours using this technique to identify the precise locations of histone protein and DNA interactions. Right as she was prepared to expand these experiments across multiple cell lines, the COVID-19 hit, throwing her plans — and progress — off course.

During these challenging times, Bokil found solace in her cultural roots and the warmth of community. She began teaching virtual BollyX classes — a dance similar to Zumba, but on Bollywood tunes — every Tuesday evening as a means to stay connected, a commitment she’s upheld ever since throughout her time in graduate school.

Beyond nurturing a sense of togetherness through dance, Bokil is committed to mentoring in science and celebrating improbable victories along a tedious research journey.

“I had a former lab mate who used to do what she called a data dance every time she had a graph she felt happy with,” Bokil recalls. “I think that should catch on a little bit more because it’s always a really good feeling to see how these experiments that have taken up so much of your time and effort are leading somewhere.”

Sara Prescott named Pew Scholar in the Biomedical Sciences

Assistant Professor Sara Prescott and her lab plan to test whether and how neurons have a role in airway remodeling, which goes awry in many diseases.

David Orenstein | The Picower Institute for Learning and Memory
June 17, 2024
Whitehead Institute Member Siniša Hrvatin named a 2024 McKnight Scholar

The McKnight Endowment Fund for Neuroscience has selected Whitehead Institute Member Siniša Hrvatin as one of ten early career scientists to receive a 2024 McKnight Scholar Award, supporting his research on mechanisms underlying certain animals’ capacity to enter states of torpor and hibernation.

Merrill Meadow | Whitehead Institute
June 20, 2024
In Memoriam: Mary-Lou Pardue, 1933-2024

Mary-Lou Pardue, Professor Emeritus of Biology, dies at 90

Lillian Eden | Department of Biology
June 17, 2024

Known for her rigorous approach to science and pioneering research, Pardue paved the way for women scientists at MIT and beyond

Mary-Lou Pardue, professor emerita in the Department of Biology, died on June 1, 2024. She was 90.

Early in her career, Pardue developed a technique called in situ hybridization with her PhD advisor Joseph Gall, which allows researchers to localize genes on chromosomes. This led to many discoveries, including critical advancements in developmental biology, our understanding of embryonic development, and the structure of chromosomes. She also studied the remarkably complex way organisms respond to stress, such as heat shock, and discovered how telomeres, the ends of chromosomes, in fruit flies differ from those of other eukaryotic organisms during cell division.

“The reason she was a professor at MIT and why she was doing research was first and foremost because she wanted to answer questions and make discoveries,” says longtime colleague and Professor Emerita Terry Orr-Weaver. “She had her feet cemented in a love of biology.”

In 1983, Pardue was the first woman in the School of Science at MIT to be inducted into the National Academy of Sciences. She served as Chairman for the Section of Genetics from 1991 to 1994 and as a Council Member from 1995 to 1998. Among other honors, she was named a Fellow of the American Academy of Arts and Sciences, where she served as a Council Member, and a Fellow of the American Association for the Advancement of Science.  She also served on numerous editorial boards and review panels, and as the vice president, president, and chair of the Genetics Society of America and president of the American Society for Cell Biology.

Her graduate students and postdoctoral scholars included Alan Spradling, Matthew Scott, Tom Cech, Paul Lasko, and Joan Ruderman.

In the minority

Pardue was born on Sept. 15, 1933, in Lexington, Kentucky. She received a BS in Biology from the College of William and Mary in 1955, and she was awarded an MS in Radiation Biology from the University of Tennessee in 1959. In 1970, she was awarded a PhD in Biology for her work with Gall at Yale University.

As one of the senior women faculty who co-signed a letter to the Dean of Science at MIT about the bias against women scientists at the institute, Pardue’s career was inextricably linked to the slowly rising number of women with advanced degrees in science. During her early years as a graduate student at Yale, there were a few women with PhDs — but none held faculty positions. Indeed, Pardue assumed she would spend her career as a senior scientist working in someone else’s lab, rather than running her own.

Pardue was an avid hiker and loved to travel and spend time outdoors. She scaled peaks from the White Mountains to the Himalayas and pursued postdoctoral work in Europe at the University of Edinburgh. She was delighted to receive invitations to give faculty search seminars for the opportunity to travel to institutions across the U.S.—including an invitation to visit MIT.

MIT had initially rejected her job application, although the department quickly realized it had erred in missing the opportunity to recruit Pardue. In the end, she spent more than 30 years as a professor in Cambridge.

When Pardue joined, the department had two women faculty members, Lisa Steiner and Annamaria Torriani-Gorini — more women than at any other academic institution Pardue had interviewed. Pardue became an associate professor of Biology in 1972, a professor in 1980, and the Boris Magasanik Professor of Biology in 1995.

The person who made a difference

Pardue was known for her rigorous approach to science as well as her bright smile and support of others.

When Graham Walker, American Cancer Society and HHMI Professor, joined the department in 1976, he recalled an event for meeting graduate students at which he was repeatedly mistaken for a graduate student himself. Pardue parked herself by his side to bear the task of introducing the newest faculty member.

“Mary-Lou had an art for taking care of people,” Walker says. “She was a wonderful colleague and a close friend.”

Troy Littleton, Professor of Biology, Menicon Professor of Neuroscience, and Investigator at the Picower Institute for Learning and Memory — then a young faculty member — had his first experience teaching with Pardue for an undergraduate project lab course.

“Observing how Mary-Lou was able to get the students excited about basic research was instrumental in shaping my teaching skills,” Littleton says. “Her passion for discovery was infectious, and the students loved working on basic research questions under her guidance.”

She was also a mentor for fellow women joining the department, including E.C. Whitehead Professor of Biology and HHMI investigator Tania A. Baker, who joined the department in 1992, and Orr-Weaver, the first female faculty member to join the Whitehead Institute in 1987.

“She was seriously respected as a woman scientist—as a scientist,” recalls Nancy Hopkins, Amgen Professor of Biology Emerita. “For women of our generation, there were no role models ahead of us, and so to see that somebody could do it, and have that kind of respect, was really inspiring.”

Hopkins first encountered Pardue’s work on in situ hybridization as a graduate student. Although it wasn’t Hopkins’ field, she remembers being struck by the implications — a leap in science that today could be compared to the discoveries that are possible because of the applications of gene-editing CRISPR technology.

“The questions were very big, but the technology was small,” Hopkins says. “That you could actually do these kinds of things was kind of a miracle.”

Pardue was the person who called to give Hopkins the news that she had been elected to the National Academy of Sciences. They hadn’t worked together, yet, but Hopkins felt like Pardue had been looking out for her, and was so excited on her behalf.

Later, though, Hopkins was initially hesitant to reach out to Pardue to discuss the discrimination Hopkins had experienced as a faculty member at MIT — Pardue seemed so successful that surely her gender had not held her back. Hopkins found that women, in general, didn’t discuss the ways they had been undervalued; it was humiliating to admit to being treated unfairly.

Hopkins drafted a letter about the systemic and invisible discrimination she had experienced — but Hopkins, ever the scientist, needed a reviewer.

At a table in the corner of Rebecca’s Café, a now-defunct eatery, Pardue read the letter — and declared she’d like to sign it and take it to the Dean of the School of Science.

“I knew the world had changed in that instant,” Hopkins says. “She’s the person who made the difference. She changed my life, and changed, in the end, MIT.”

MIT and the status of women

It was only when some of the tenured women faculty of the School of Science all came together that they discovered their experiences were similar. Hopkins, Pardue, Orr-Weaver, Steiner, Susan Carey, Sylvia Ceyer, Sallie “Penny” Chisholm, Suzanne Corkin, Mildred Dresselhaus, Ann Graybiel, Ruth Lehmann, Marcia McNutt, Molly Potter, Paula Malanotte-Rizzoli, Leigh Royden, and Joanne Stubbe ultimately signed a letter to Robert Birgeneau, then the Dean of Science.

Their efforts led to a Committee on the Status of Women Faculty in 1995, the report for which was made public in 1999. The report captured pervasive bias against women across the School of Science. In response, MIT ultimately worked to improve the working conditions of women scientists across the institute. These efforts reverberated at academic institutions across the country.

Walker notes that creating real change requires a monumental effort of political and societal pressure — but it also requires outstanding individuals whose work surpasses the barriers holding them back.

“When Mary-Lou came to MIT, there weren’t many cracks in the glass ceiling,” he says. “I think she, in many ways, was a leader in helping to change the status of women in science by just being who she was.”

Later years

Kerry Kelley, now a research laboratory operations manager in the Yilmaz Lab at the Koch Institute for Integrative Cancer Research, joined Pardue as a technical lab assistant in 2008, Kelley’s first job at MIT. Pardue, throughout her career, was committed to hands-on work, preparing her own slides whenever possible.

“One of the biggest things I learned from her was mistakes aren’t always mistakes. If you do an experiment, and it doesn’t turn out the way you had hoped, there’s something there that you can learn from,” Kelley says. She recalls a frequent refrain with a smile: “‘It’s research. What do you do? Re-search.’”

Their birthdays were on consecutive days in September; Pardue would mark the occasion for both at Legal Seafoods in Kendall Square with Bluefish, white wine, and lab members and collaborators including Kelley, Karen Traverse, and the late Paul Gregory DeBaryshe.

In the years before her death, Pardue resided at Youville House Assisted Living in Cambridge, where Kelley would often visit.

“I was sad to hear of the passing of Mary-Lou, whose seminal work expanded our understanding of chromosome structure and cellular responses to environmental stresses over more than three decades at MIT. Mary-Lou was an exceptional person who was known as a gracious mentor and a valued teacher and colleague,” says Biology Department Head and Jay A. Stein (1968) Professor of Biology and Professor of Biological Engineering Amy Keating. “She was kind to everyone, and she is missed by our faculty and staff. Women at MIT and beyond, including me, owe a huge debt to Mary-Lou, Nancy Hopkins, and their colleagues who so profoundly advanced opportunities for women in science.”

She is survived by a niece and nephew, Todd Pardue and Sarah Gibson.