Author: Julie Pryor

RNA-targeting enzyme expands the CRISPR toolkit

by Jennifer Michalowski |

Researchers at MIT’s McGovern Institute have discovered a bacterial enzyme that they say could expand scientists’ CRISPR toolkit, making it easy to cut and edit RNA with the kind of precision that, until now, has only been available for DNA editing. The enzyme, called Cas7-11, modifies RNA targets without harming cells, suggesting that in addition to being a valuable research tool, it provides a fertile platform for therapeutic applications.

“This new enzyme is like the Cas9 of RNA,” says McGovern Fellow Omar Abudayyeh, referring to the DNA-cutting CRISPR enzyme that has revolutionized modern biology by making DNA editing fast, inexpensive, and exact. “It creates two precise cuts and doesn’t destroy the cell in the process like other enzymes,” he adds.

Up until now, only one other family of RNA-targeting enzymes, Cas13, has extensively been developed for RNA targeting applications. However, when Cas13 recognizes its target, it shreds any RNAs in the cell, destroying the cell along the way. Like Cas9, Cas7-11 is part of a programmable system; it can be directed at specific RNA targets using a CRISPR guide. Abudayyeh, McGovern fellow Jonathan Gootenberg, and their colleagues discovered Cas7-11 through a deep exploration of the CRISPR systems found in the microbial world. Their findings are reported today in the journal Nature.

Exploring natural diversity

DNA tools in the CRISPR toolkit (red) are approaching capacity, but researchers are now beginning to find new tools to edit RNA (blue). Image: Steven Dixon

Like other CRISPR proteins, Cas7-11 is used by bacteria as a defense mechanism against viruses. After encountering a new virus, bacteria that employ the CRISPR system keep a record of the infection in the form of a small snippet of the pathogen’s genetic material. Should that virus reappear, the CRISPR system is activated, guided by a small piece of RNA to destroy the viral genome and eliminate the infection.

These ancient immune systems are widespread and diverse, with different bacteria deploying different proteins to counter their viral invaders.

“Some target DNA, some target RNA. Some are very efficient in cleaving the target but have some toxicity, and others do not. They introduce different types of cuts, they can differ in specificity—and so on,” says Eugene Koonin, an evolutionary biologist at the National Center for Biotechnology Information.

Abudayyeh, Gootenberg, and Koonin have been scouring genome sequences to learn about the natural diversity of CRISPR systems—and to mine them for potential tools. The idea, Abudayyeh says, is to take advantage of the work that evolution has already done in engineering protein machines.

“We don’t know what we’ll find,” Abudayyeh says, “but let’s just explore and see what’s out there.”

As the team was poring through public databases to examine the components of different bacterial defense systems, a protein from a bacterium that had been isolated from Tokyo Bay caught their attention. Its amino acid sequence indicated that it belonged to a class of CRISPR systems that use large, multiprotein machines to find and cleave their targets. But this protein appeared to have everything it needed to carry out the job on its own. Other known single-protein Cas enzymes, including the Cas9 protein that has been widely adopted for DNA editing, belong to a separate class of CRISPR systems—but Cas7-11 blurs the boundaries of the CRISPR classification system, Koonin says.

The enzyme, which the team eventually named Cas7-11, was attractive from an engineering perspective, because single proteins are easier to deliver to cells and make better tools than their complex counterparts. But its composition also signaled an unexpected evolutionary history. The team found evidence that through evolution, the components of a more complex Cas machine had fused together to make the Cas7-11 protein. Gootenberg equates this to discovering a bat when you had previously assumed that birds are the only animals that fly, thereby recognizing that there are multiple evolutionary paths to flight. “It totally changes the landscape of how these systems are thought about, both functionally and evolutionarily,” he says.

Precision editing

McGovern Fellows Jonathan Gootenberg and Omar Abudayyeh in their lab. Photo: Caitlin Cunningham

When Gootenberg and Abudayyeh produced the Cas7-11 protein in their lab and began experimenting with it, they realized this unusual enzyme offered a powerful means to manipulate and study RNA. When they introduced it into cells along with an RNA guide, it made remarkably precise cuts, snipping its targets while leaving other RNA undisturbed. This meant they could use Cas7-11 to change specific letters in the RNA code, correcting errors introduced by genetic mutations. They were also able to program Cas7-11 to either stabilize or destroy particular RNA molecules inside cells, which gave them the ability to adjust the levels of the proteins encoded by those RNAs.

Abudayyeh and Gootenberg also found that Cas7-11’s ability to cut RNA could be dampened by a protein that appeared likely to also be involved in triggering programmed cell death, suggesting a possible link between CRISPR defense and a more extreme response to infection.

The team showed that a gene therapy vector can deliver the complete Cas7-11 editing system to cells and that Cas7-11 does not compromise cells’ health. They hope that with further development, the enzyme might one day be used to edit disease-causing sequences out of a patient’s RNA so their cells can produce healthy proteins, or to dial down the level of a protein that is doing harm due to genetic disease.

“We think that the unique way that Cas7-11 cuts enables many interesting and diverse applications,” Gootenberg says, noting that no other CRISPR tool cuts RNA so precisely. “It’s yet another great example of how these basic-biology driven explorations can yield new tools for therapeutics and diagnostics,” he adds. “And we’re certainly still just scratching the surface of what’s out there in natural diversity.”

Mapping the cellular circuits behind spitting

by Raleigh McElvery | MIT Biology |

For over a decade, researchers have known that the roundworm Caenorhabditis elegans can detect and avoid short-wavelength light, despite lacking eyes and the light-absorbing molecules required for sight. As a graduate student in the Horvitz lab, Nikhil Bhatla proposed an explanation for this ability. He observed that light exposure not only made the worms wriggle away, but it also prompted them to stop eating. This clue led him to a series of studies that suggested that his squirming subjects weren’t seeing the light at all — they were detecting the noxious chemicals it produced, such as hydrogen peroxide. Soon after, the Horvitz lab realized that worms not only taste the nasty chemicals light generates, they also spit them out.

Now, in a study recently published in eLife, a team led by former graduate student Steve Sando reports the mechanism that underlies spitting in C. elegans. Individual muscle cells are generally regarded as the smallest units that neurons can independently control, but the researchers’ findings question this assumption. In the case of spitting, they determined that neurons can direct specialized subregions of a single muscle cell to generate multiple motions — expanding our understanding of how neurons control muscle cells to shape behavior.

“Steve made the remarkable discovery that the contraction of a small region of a particular muscle cell can be uncoupled from the contraction of the rest of the same cell,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, Howard Hughes Medical Institute Investigator, and senior author of the study. “Furthermore, Steve found that such subcellular muscle compartments can be controlled by neurons to dramatically alter behavior.”

Roundworms are like vacuum cleaners that wiggle around hoovering up bacteria. The worm’s mouth, also known as the pharynx, is a muscular tube that traps the food, chews it, and then transfers it to the intestines through a series of “pumping” contractions.

Researchers have known for over a decade that worms flee from UV, violet, or blue light. But Bhatla discovered that this light also interrupts the constant pumping of the pharynx, because the taste produced by the light is so nasty that the worms pause feeding. As he looked closer, Bhatla noticed the worms’ response was actually quite nuanced. After an initial pause, the pharynx briefly starts pumping again in short bursts before fully stopping — almost like the worm was chewing for a bit even after tasting the unsavory light. Sometimes, a bubble would escape from the mouth, like a burp.

After he joined the project, Sando discovered that the worms were neither burping nor continuing to munch. Instead, the “burst pumps” were driving material in the opposite direction, out of the mouth into the local environment, rather than further back into the pharynx and intestine. In other words, the bad-tasting light caused worms to spit. Sando then spent years chasing his subjects around the microscope with a bright light and recording their actions in slow motion, in order to pinpoint the neural circuitry and muscle motions required for this behavior.

“The discovery that the worms were spitting was quite surprising to us, because the mouth seemed to be moving just like it does when it’s chewing,” Sando says. “It turns out that you really needed to zoom in and slow things down to see what’s going on, because the animals are so small and the behavior is happening so quickly.”

To analyze what’s happening in the pharynx to produce this spitting motion, the researchers used a tiny laser beam to surgically remove individual nerve and muscle cells from the mouth and discern how that affected the worm’s behavior. They also monitored the activity of the cells in the mouth by tagging them with specially-engineered fluorescent “reporter” proteins.

They saw that while the worm is eating, three muscle cells towards the front of the pharynx called pm3s contract and relax together in synchronous pulses. But as soon as the worm tastes light, the subregions of these individual cells closest to the front of the mouth become locked in a state of contraction, opening the front of the mouth and allowing material to be propelled out. This reverses the direction of the flow of the ingested material and converts feeding into spitting.

The team determined that this “uncoupling” phenomenon is controlled by a single neuron at the back of the worm’s mouth. Called M1, this nerve cell spurs a localized influx of calcium at the front end of the pm3 muscle likely responsible for triggering the sub-cellular contractions.

M1 relays important information like a switchboard. It receives incoming signals from many different neurons, and transmits that information to the muscles involved in spitting. Sando and his team suspect that the strength of the incoming signal can tune the worm’s behavior in response to tasting light. For instance, their findings suggest that a revolting taste elicits a vigorous rinsing of the mouth, while a mildly unpleasant sensation causes the worm spit more gently, just enough to eject the contents.

In the future, Sando thinks the worm could be used as a model to study how neurons trigger subregions of muscle cells to constrict and shape behavior — a phenomenon they suspect occurs in other animals, possibly including humans.

“We’ve essentially found a new way for a neuron to move a muscle,” Sando says. “Neurons orchestrate the motions of muscles, and this could be a new tool that allows them to exert a sophisticated kind of control. That’s pretty exciting.”

Having more conversations to boost brain development

by Jennifer Michalowski |

Engaging children in more conversation may be all it takes to strengthen language processing networks in their brains, according to a new study by MIT scientists.

Childhood experiences, including language exposure, have a profound impact on the brain’s development. Now, scientists led by McGovern Institute investigator John Gabrieli have shown that when families change their communication style to incorporate more back-and-forth exchanges between child and adult, key brain regions grow and children’s language abilities advance. Other parts of the brain may be impacted, as well.

In a study of preschool and kindergarten-aged children and their families, Gabrieli, Harvard postdoctoral researcher Rachel Romeo, and colleagues found that increasing conversation had a measurable impact on children’s brain structure and cognition within just a few months. “In just nine weeks, fluctuations in how often parents spoke with their kids appear to make a difference in brain development, language development, and executive function development,” Gabrieli says. The team’s findings are reported in the June issue of the journal Developmental Cognitive Neuroscience.

“We’re excited because this adds a little more evidence to the idea that [the brain] is malleable,” adds Romeo, who is now an assistant professor at the University of Maryland College Park.

“It suggests that in a relatively short period of time, the brain can change in positive ways,” says Romeo.

30 million word gap

In the 1990s, researchers determined that there are dramatic discrepancies in the language that children are exposed to early in life. They found that children from high-income families heard about 30 million more words during their first three years than children from lower-income families—and those exposed to more language tended to do better on tests of language development, vocabulary, and reading comprehension.

In 2018, Gabrieli and Romeo found that it was not the volume of language that made a difference, however, but instead the extent to which children were engaged in conversation. They measured this by counting the number of “conversational turns” that children experienced over a few days—that is, the frequency with which dialogue switched between child and adult. When they compared the brains of children who experienced significantly different levels of these conversational turns, they found structural and functional differences in regions known to be involved in language and speech.

After observing these differences, the researchers wanted to know whether altering a child’s language environment would impact their brain’s future development. To find out, they enrolled the families of fifty-two children between the ages of four and seven in a study, and randomly assigned half of the families to participate in a nine-week parent training program. While the program did not focus exclusively on language, there was an emphasis on improving communication, and parents were encouraged to engage in meaningful dialogues with their children.

Romeo and colleagues sent families home with audio recording devices to capture all of the language children were exposed to over two full days, first at the outset of the program and again after the nine-week training was complete. When they analyzed the recordings, they found that in many families, conversation between children and their parents had increased—and children who experienced the greatest increase in conversational turns showed the greatest improvements in language skills as well as in executive functions—a set of skills that includes memory, attention, and self-control.

 

graph depicting cortical changes
Clusters where changes in cortical thickness are significantly correlated with changes in children’s experienced conversational turns. Scatterplots represent the average change in cortical thickness as a function of the pre-to-post changes in conversational turns.

MRI scans showed that over the nine-week study, these children also experienced the most growth in two key brain areas: a sound processing center called the supramarginal gyrus and a region involved in language processing and speech production called Broca’s area. Intriguingly, these areas are very close to parts of the brain involved in executive function and social cognition.

“The brain networks for executive functioning, language, and social cognition are deeply intertwined and going through these really important periods of development during this preschool and transition-to-school period,” Romeo says. “Conversational turns seem to be going beyond just linguistic information. They seem to be about human communication and cognition at a deeper level. I think the brain results are suggestive of that, because there are so many language regions that could pop out, but these happen to be language regions that also are associated with other cognitive functions.”

Talk more

Gabrieli and Romeo say they are interested in exploring simple ways—such a web or smartphone-based tools—to support parents in communicating with their children in ways that foster brain development. It’s particularly exciting, Gabrieli notes, that introducing more conversation can impact brain development when at the age when children are preparing to begin school.

“Kids who arrive to school school-ready in language skills do better in school for years to come,” Gabrieli says. “So I think it’s really exciting to be able to see that the school readiness is so flexible and dynamic in nine weeks of experience.”

“We know this is not a trivial ask of people,” he says. “There’s a lot of factors that go into people’s lives— their own prior experiences, the pressure of their circumstances. But it’s a doable thing. You don’t have to have an expensive tutor or some deluxe pre-K environment. You can just talk more with your kid.”

International Dyslexia Association recognizes John Gabrieli with highest honor

by Julie Pryor |

Cognitive neuroscientist John Gabrieli has been named the 2021 winner of the Samuel Torrey Orton Award, the International Dyslexia Association’s highest honor. The award recognizes achievements of leading researchers and practitioners in the dyslexia field, as well as those of individuals with dyslexia who exhibit leadership and serve as role models in their communities.

“I am grateful to the International Dyslexia Association for this recognition,” said Gabrieli, who is the Grover Hermann Professor of Health Sciences and Technology, a professor of brain and cognitive sciences, and a member of MIT’s McGovern Institute for Brain Research. “The association has been such an advocate for individuals and their families who struggle with dyslexia, and has also been such a champion for the relevant science. I am humbled to join the company of previous recipients of this award who have done so much to help us understand dyslexia and how individuals with dyslexia can be supported to flourish in their growth and development.”

Gabrieli, who is also the director of MIT’s Athinoula A. Martinos Imaging Center, uses neuroimaging and behavioral tests to understand how the human brain powers learning, thinking, and feeling.  For the last two decades, Gabrieli has sought to unravel the neuroscience behind learning and reading disabilities and, ultimately, convert that understanding into new and better education interventions—a sort of translational medicine for the classroom.

“We want to get every kid to be an adequate reader by the end of the third grade,” Gabrieli says. “That’s the ultimate goal: to help all children become learners.”

In March of 2018, Gabrieli and the MIT Integrated Learning Initiative—MITili, which he also directs—announced a $30 million-dollar grant from the Chan Zuckerberg Initiative for a collaboration between MIT, the Harvard Graduate School of Education, and Florida State University. This partnership, called “Reach Every Reader” aims to make significant progress on the crisis in early literacy – including tools to identify children at risk for dyslexia and other learning disabilities before they even learn to read.

“John is especially deserving of this award,” says Hugh Catts, Gabrieli’s colleague at Reach Every Reader. Catts is a professor and director of the School of Communications Science and Disorders at Florida State University. “His work has been seminal to our understanding of the neural basis of learning and learning difficulties such as dyslexia. He has been a strong advocate for individuals with dyslexia and a mentor to leading experts in the field,” says Catts, who is also received the Orton Award in 2008.

“It’s a richly deserved honor,”says Sanjay Sarma, the Fred Fort Flowers (1941) and Daniel Fort Flowers (1941) Professor of Mechanical Engineering at MIT. “John’s research is a cornerstone of MIT’s efforts to make education more equitable and accessible for all. His contributions to learning science inform so much of what we do, and his advocacy continues to raise public awareness of dyslexia and helps us better reach the dyslexic community through literacy initiatives such as Reach Every Reader. We’re so pleased that his work has been recognized with the Samuel Torrey Orton Award,” says Sarma, who is also Vice President for Open Learning at MIT.

Gabrieli will deliver the Samuel Torrey Orton and Joan Lyday Orton Memorial Lecture this fall in North Carolina as part of the 2021 International Dyslexia Association’s Annual Reading, Literacy and Learning Conference.

 

 

MIT Technology Review names McGovern Fellows top innovators under 35

by Julie Pryor |

McGovern Institute Fellows Omar Abudayyeh and Jonathan Gootenberg have both been named to MIT Technology Review’s annual list of exceptional innovators under the age of 35. The annual list recognizes “exceptionally talented technologists whose work has great potential to transform the world.”

Abudayyeh was named to the 2020 list for developing a CRISPR-based test for COVID-19; a diagnostic technology that now has potential to rapidly and economically detect a wide variety of diseases.

This year, Gootenberg is being recognized for his work with CRISPR gene editing technologies to develop a cellular engineering “toolkit” that will help scientists better understand — and treat — diseases that affect millions worldwide.

“I’m honored that our lab’s work on molecular tools for cellular engineering is being recognized for its potential impact on diagnostics and therapeutics for patients.” — Jonathan Gootenberg

During their time in the Zhang lab, Abudayyeh and Gootenberg engineered new genome editing tools based on enzymes that they and others discovered from scanning bacterial CRISPR systems. In 2018, Gootenberg and Abudayyeh became the first members of the McGovern Institute Fellows program, which supports the transition to independent research for exceptional recent PhD graduates.

“It’s exciting that alternative uses of CRISPR beyond gene editing are being recognized, including for sensing and diagnosing diverse disease states and that certain CRISPR-based COVID-19 diagnostic assays already authorized for patient use,” says Abudayyeh.

CRISPR-based COVID-19 test using paper strips. Photo: Broad Institute

“Omar and Jonathan’s combination of basic discovery and synthetic biology continues to deliver ever more powerful tools for probing and controlling cell activity,” says McGovern Institute Director Robert Desimone. “Such tools are key to the immense challenge of understanding brain function, and treating dysfunction, the goal of the McGovern Institute.”

Now Abudayyeh and Gootenberg is expanding the boundaries of cellular engineering tools, to encompass not only genome editing but also transcriptome control and cell-state sensing — powerful technologies that can change or correct how cells behave without permanently changing their genome. Just as CRISPR has helped decode the role of genes in disease and provided a method for changing gene sequences, the pair’s cellular engineering tools reveal how cells in the body transform in response to disease and provide new means of curing disease. It is the potential of these tools to usher in a new era of cellular discoveries and treatments that caught the attention of the editors at MIT Technology Review.

“We get more than 500 nominations for the list every year, and getting that list down to 35—a task not only for the editors at MIT Technology Review but also for our 30+ judges—is one of the hardest things we do each year,” says Tim Maher, Managing Editor of MIT Technology Review. “We love the way the final list always shows what a wide variety of people there are, all around the world, working on creative solutions to some of humanity’s hardest problems.”

Gootenberg and Abudayyeh continue to work together to build a comprehensive toolkit to both understand and engineer human cells. Gootenberg and his fellow honorees will be featured at the upcoming EmTech MIT conference, MIT Technology Review’s annual flagship event that offers a perspective on the most significant developments of the year, with a focus on understanding their potential business and societal impact. EmTech MIT will be held online September 28-30, 2021.

Squishy, stealthy neural probes

by Jennifer Michalowski |

Slender probes equipped with electrodes, optical channels, and other tools are widely used by neuroscientists to monitor and manipulate brain activity in animal studies. Now, scientists at MIT have devised a way to make these usually rigid devices become as soft and pliable as their surroundings when they are implanted in the brain. Their new multifunctional devices are less intrusive than traditional neuroscience probes and remain functional for months after implantation, enabling long-term studies of neural circuits in animal models.

Researchers led by McGovern Institute scientist Polina Anikeeva built the new devices by embedding their functional components in a water-absorbing hydrogel. Each device begins as stiff probe able to penetrate brain tissue. But once it is in place, the hydrogel absorbs water and the device transforms.

“When it’s dry, it’s completely rigid. Its mechanics are dominated by mechanics of the polymers and metals that went into it,” explains Anikeeva, who is also an associate professor in the Departments of Materials Science and Engineering and Brain and Cognitive Sciences. “When it’s fully hydrated, it has the [mechanical] properties of the brain.”

Anikeeva and colleagues reported on the new devices in the June 8 issue of Nature Communications.

Stealthy probes

Neural probes made out of metal or hard plastics have been invaluable in neuroscience research, allowing scientists to sense electrical activity within the brain, supply drugs to specific locations, or deliver neuron-activating pulses of light.

In 2015, Anikeeva and her group developed multifunctional probes, which are equipped with the tools to do all of these things. Although these polymer based devices were more biocompatible than metals and semiconductors, which can cut like tiny knives through the soft, jiggly tissue of the brain, their mechanics were still orders of magnitude away from those of neural tissue. Most neural probes can be used for a few weeks, until scar tissue forms around them and interferes with their function.

“For some experiments, this may not matter,” Anikeeva says. “But for other experiments, it does. If, for example, you’re interested in how a neuron evolves over the course of long-term behavior, or aging, or development, it’s important to keep track of the same tissue or the same cells. And that was challenging [with rigid probes].”

To enable longer experiments, Anikeeva’s team began to think about making multifunctional probes out of a material that is more compatible with the brain. “We wanted to create a device that would be stealthy, so the brain wouldn’t know that it’s there,” she says. To be useful, the device would still need some amount of hard material. But electrodes, microfluidic chambers, and optical channels can be tiny—just a fraction of the width of a human hair. “Even if they’re made out of polymer or soft metal, if you make them that small, they become sufficiently soft that they will be able to move with the brain and not cause damage,” Anikeeva says. It is the polymer matrix that surrounds these functional components that gives neural probes their shape and rigidity, which despite causing problems once inside the brain, is essential for implantation.

 

Seongjun Park, a graduate student in Anikeeva’s group, and Hyunwoo Yuk, another MIT graduate student who had been working with hydrogels in Xuanhe Zhao’s mechanical engineering lab, discussed the problem and proposed a probe that took advantage of that material. Because of hydrogels’ tunable nature, they could be used to build a device that was both stealthily squishy and piercingly rigid. By fine-tuning the chemistry, the team could ensure that after the device was implanted, its hydrogel would absorb just enough water to closely match the mechanics of the brain.

Hydrogel glue

Other researchers had previously developed neural probes wrapped in a hydrogel covering, but Anikeeva’s team wanted the hydrogel to be the bulk of the device. They would use the swellable material to bundle together the functional elements and fill the space between them.

To do so, they assembled the fibers that would give their device its desired function—an electrode array fiber for sensing neural activity, an optical fiber for delivering light to manipulate signaling, and a fluidic fiber for delivering drugs and genes—and chemically treated them so that they would adhere directly to the components of a hydrogel.

 

They then dipped the treated fibers into a solution of a hydrogel-forming compound called alginate. By exposing the solution to light, they triggered the alginate to polymerize, ultimately creating a thin strand of the hydrogel with the functional fibers embedded within it.

When it is first pulled out of the solution, Anikeeva says, the hydrogel-based device is like a wet noodle, with its components moving freely within it like the bendable bristles of a wet paintbrush. As the hydrogel dries, the fibers become firmly affixed to one another and the entire device stiffens—much like a drying paintbrush.

Long-term tracking

To test the devices, Anikeeva’s team implanted them into mice, targeting anxiety circuits deep within the brain. They behaved exactly as they had hoped—easily penetrating into the tissue, then returning to their “wet noodle” state and remaining in place without triggering a foreign body response in the brain. After more than six months of recording neural activity, the probes remained fully functional.

Anikeeva says her team’s squishy new probes are the first multifunctional neural devices to remain effective in living animals for this prolonged period. The improved longevity of the devices compared to their predecessors means researchers will be able to use them to track and manipulate neuronal behavior during long-term processes such as learning, disease progression, and aging.

The team is already working on the next-generation of hydrogel probes, which will further take advantage of the material’s unique properties to control the release of drugs or other compounds within the brain and improve the devices’ biocompatibility. And with a simplified fabrication process in development, Anikeeva says it may soon be possible for neuroscientists to manufacture the stealthy probes in their own labs.

Exploring the unknown

by Jennifer Michalowski |

View the interactive version of this story in our Summer 2021 issue of BrainScan.

 

McGovern Investigator Ed Boyden.

McGovern Investigator Ed Boyden says his lab’s vision is clear.

“We want to understand how our brains take our sensory inputs, generate emotions and memories and decisions, and ultimately result in motor outputs. We want to be able to see the building blocks of life, and how they go into disarray in brain diseases. We want to be able to control the signals of the brain, so we can repair it,” Boyden says.

To get there, he and his team are exploring the brain’s complexity at every scale, from the function and architecture of its neural networks to the molecules that work together to process information.

And when they don’t have the tools to take them where they want to go, they create them, opening new frontiers for neuroscientists everywhere.

Open to discovery

Boyden’s team is highly interdisciplinary and collaborative. Its specialty, Boyden says, is problem solving. Creativity, adaptability, and deep curiosity are essential, because while many of neuroscience’s challenges are clear, the best way to address them is not. In its search for answers, Boyden’s lab is betting that an important path to discovery begins with finding new ways to explore.

They’ve made that possible with an innovative imaging approach called expansion microscopy (ExM). ExM physically enlarges biological samples so that minute details become visible under a standard laboratory microscope, enabling researchers everywhere to peer into spaces that once went unseen (see video below).

To use the technique, researchers permeate a biological sample with an absorbent gel, then add water, causing the components of the gel to spread apart and the tissue to expand.

This year, postdoctoral researcher Ruixuan Gao and graduate student Chih-Chieh (Jay) Yu made the method more precise, with a new material that anchors a sample’s molecules within a crystal-like lattice, better preserving structure during expansion than the irregular mesh-like composition of the original gel. The advance is an important step toward being able to image expanded samples with single-molecule precision, Gao says.

A revealing look

By opening space within the brain, ExM has let Boyden’s team venture into those spaces in new ways.

Areas of research and brain disorders page
Graduate student Oz Wassie examines expanded brain tissue. Photo: Justin Knight

In work led by Deblina Sarkar (who is now an assistant professor at MIT’s Media Lab), Jinyoung Kang, and Asmamaw (Oz) Wassie, they showed that they can pull apart proteins in densely packed regions like synapses so that it is easier to introduce fluorescent labels, illuminating proteins that were once too crowded to see. The process, called expansion revealing, has made it possible to visualize in intact brain tissue important structures such as ion channels that help transmit signals and fine-scale amyloid clusters in Alzheimer’s model mice.

Another reaction the lab has adapted to the expanded-brain context is RNA sequencing—an important tool for understanding cellular diversity. “Typically, the first thing you do in a sequencing project is you grind up the tissue, and you lose the spatial dimension,” explains Daniel Goodwin, a graduate student in Boyden’s lab. But when sequencing reactions are performed inside cells instead, new information is revealed.

Confocal image showing targeted ExSeq of a 34-panel gene set across a slice of mouse hippocampus. Green indicates YFP, magenta indicates reads identified with ExSeq, and white indicates reads localized within YFP-expressing cells. Image courtesy of the researchers.

Goodwin and fellow Boyden lab members Shahar Alon, Anubhav Sinha, Oz Wassie, and Fei Chen developed expansion sequencing (ExSeq), which copies RNA molecules, nucleotide by nucleotide, directly inside expanded tissue, using fluorescent labels that spell out the molecules’ codes just as they would in a sequencer.

The approach shows researchers which genes are turned on in which cells, as well as where those RNA molecules are—revealing, for example, which genes are active in the neuronal projections that carry out the brain’s communications. A next step, Sinha says, is to integrate expansion sequencing with other technologies to obtain even deeper insights.

That might include combining information revealed with ExSeq with a topographical map of the same cells’ genomes, using a method Boyden’s lab and collaborators Chen (who is now a core member of the Broad Institute) and Jason Buenrostro at Harvard have developed for DNA sequencing. That information is important because the shape of the genome varies across cells and circumstances, and that has consequences for how the genetic code is used.

Using similar techniques to those that make ExSeq possible, graduate students Andrew Payne, Zachary Chiang, and Paul Reginato figured out how to recreate the steps of commercial DNA sequencing within the genome’s natural environment.

By pinpointing the location of specific DNA sequences inside cells, the new method, called in situ genome sequencing (IGS) allows researchers to watch a genome reorganize itself in a developing embryo.

They haven’t yet performed this analysis inside expanded tissue, but Payne says integrating in situ genome sequencing (IGS) with ExM should open up new opportunities to study genomes’ structure.

Signaling clusters

Alongside these efforts, Boyden’s team is working to give researchers better tools to explore how molecules move, change, and interact, including a modular system that lets users assemble sets of sensors into clusters to simultaneously monitor multiple cellular activities.

Molecular sensors use fluorescence to report on certain changes inside cells, such as the calcium that surges into a neuron after it fires. But they come in a limited palette, so in most experiments only one or two things can be seen at once.

Graduate student Shannon Johnson and postdoctoral fellow Changyang Linghu solved this problem by putting different sensors at different points throughout a cell so they can report on different signals. Their technique, called spatial multiplexing, links sensors to molecular scaffolds designed to cling to their own kind. Sensors built on the same scaffold form islands inside cells, so when they light up their signals are distinct from those produced by other sensor islands.

Eventually, as new sensors and scaffolds become available, Johnson says the technique might be used to simultaneously follow dozens of molecular signals in living cells. The more precise information they can help people uncover, the better, Boyden says.

“The brain is so full of surprises, we don’t know where the next big discovery will come from,” he says. With new support from the recently established K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience, the Boyden lab is positioned to make these big discoveries.

“My dream would be to image the signaling dynamics of the brain, and then perturb the dynamics, and then use expansion methods to make a map of the brain. If we can get those three data sets—the dynamics, the causality, and the molecular organization—I think stitching those together could potentially yield deep insights into how the brain works, and how we can repair it in disease states.”

Abnormal brain connectivity may precede schizophrenia onset

by Jill Crittenden |

The cerebellum is named “little brain” for its distinctive structure. Although the cerebellum was long considered only for its role in maintaining the balance and timing of movements, it has become evident that it is also important for balanced thoughts and emotions, belying the diversity of functions that “little brain” implies.

In a new study published in Schizophrenia Bulletin, McGovern Research Affiliate and Northeastern University Professor of Psychiatry Susan Whitfield-Gabrieli shows for the first time that cerebellar dysfunction actually precedes the onset of psychosis in schizophrenia, a brain disorder characterized by severe thought and emotional imbalances.

“This study exemplifies the concept of “neuroprediction,” the discovery of brain-based biomarkers that allow early detection and therefore early intervention for mental disorders,” says Whitfield-Gabrieli.

Cerebellar connectivity and schizophrenia

Early evidence that the cerebellum is involved in more than movement came from numerous reports that people with brain damage originating in the cerebellum can have severely disordered thought processes. Now cerebellar abnormalities have been identified in numerous neurodevelopmental and neuropsychiatric conditions including autism, attention-deficit hyperactivity disorder (ADHD), Alzheimer’s disease, and schizophrenia.

Whitfield-Gabrieli has focused on how symptoms in these disorders correlate with how well the cerebellum is connected to other brain regions, including regions of the cerebral cortex, the characteristically-folded, outer part of the brain. Active connections in the brain of people who are resting or who are engaged in a mental task can be found by functional magnetic resonance imaging (fMRI), a brain scanning technique that detects when and where oxygen is being used by cells. If oxygen usage in two brain regions consistently peaks at the same time while someone is in the scanner, they are considered to be functionally connected.

Connectivity differences prior to psychosis

In her new study, Whitfield-Gabrieli explored whether brain scans could reveal cerebellar abnormalities in people at-risk for schizophrenia.

To do this, she and her colleagues compared cerebellar connectivity among at-risk adolescents and young adults who went on to develop psychosis within the following year versus those that remained stable or improved. The at-risk participants were identified in an international collaboration called the Shanghai At Risk for Psychosis (SHARP) program that recruited people who were seeking help at China’s largest outpatient mental health center. Of the 144 adolescents and young adults at-risk for schizophrenia at the outset of the study, 23 went on to develop the disorder. Notably, this group showed fMRI patterns of cerebellar dysfunction at the outset of the study, before they developed psychosis.

Abnormal brain architecture

All of the brain scans were evaluated to determine the degree to which three specific cerebellar regions were connected to the cerebral cortex, a brain region that does not finish development until young adulthood. The cerebellar regions of interest to Whitfield-Gabrieli are part of the “dentate nuclei,” so named because they look like a set of jagged teeth. Neurons in the dentate nuclei serve to integrate inputs from the rest of the cerebellum and send the compiled information out to the rest of the brain. Whitfield-Gabrieli and colleagues divided the dentate nuclei into three zones according to what parts of the cerebral cortex they are functionally connected to while people are relaxing, doing visual tasks, or engaging in a motor task or receiving some sort of stimulation.

The team found abnormal connectivity for all three zones of the dentate nuclei in the individuals who later went on to develop schizophrenia. Since the connectivity patterns varied across regions within the three zones, with some regions over-connected and others under-connected to the cerebral cortex in the group that developed psychosis, separated high-resolution analyses of the different connections was key.

Previous work established that cerebellar abnormalities are associated with schizophrenia but this study is the first to show that functional connections between the deep cerebellar nuclei and the cerebral cortex might precede disease onset.  “Treatments for mental disorders are inherently reactive to suffering and incapacity. A proactive approach by which abnormal brain architecture is identified prior to clinical diagnosis has the potential to prevent suffering by helping people before they become ill, one of my ultimate goals” said Whitfield-Gabrieli.

This study was supported by the Poitras Center for Psychiatric Disorders Research at MIT), US National Institute of Mental Health (R21 MH 093294, R01 MH 101052, R01 MH 111448, and R01 MH 64023), Ministry of Science and Technology of China (2016 YFC 1306803), European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 749201 and by a VA Merit Award.

New technique corrects disease-causing mutations

by Jill Crittenden |

Gene editing, or purposefully changing a gene’s DNA sequence, is a powerful tool for studying how mutations cause disease, and for making changes in an individual’s DNA for therapeutic purposes. A novel method of gene editing that can be used for both purposes has now been developed by a team led by Guoping Feng, the James W. (1963) and Patricia T. Poitras Professor in Brain and Cognitive Sciences at MIT.

“This technical advance can accelerate the production of disease models in animals and, critically, opens up a brand-new methodology for correcting disease-causing mutations,” says Feng, who is also a member of the Broad Institute of Harvard and MIT and the associate director of the McGovern Institute for Brain Research at MIT. The new findings publish online May 26 and in print June 10 in the journal Cell.

Genetic models of disease

A major goal of the Feng lab is to precisely define what goes wrong in neurodevelopmental and neuropsychiatric disorders by engineering animal models that carry the gene mutations that cause these disorders in humans. New models can be generated by injecting embryos with gene editing tools, along with a piece of DNA carrying the desired mutation.

In one such method, the gene editing tool CRISPR is programmed to cut a targeted gene, thereby activating natural DNA mechanisms that “repair” the broken gene with the injected template DNA. The engineered cells are then used to generate offspring capable of passing the genetic change on to further generations, creating a stable genetic line in which the disease, and therapies, are tested.

Although CRISPR has accelerated the process of generating such disease models, the process can still take months or years. Reasons for the inefficiency are that many treated cells do not undergo the desired DNA sequence change at all, and the change only occurs on one of the two gene copies (for most genes, each cell contains two versions, one from the father and one from the mother).

In an effort to increase the efficiency of the gene editing process, the Feng lab team initially hypothesized that adding a DNA repair protein called RAD51 to a standard mixture of CRISPR gene editing tools would increase the chances that a cell (in this case a fertilized mouse egg, or one-cell embryo) would undergo the desired genetic change.

As a test case, they measured the rate at which they were able to insert (“knock-in”) a mutation in the gene Chd2 that is associated with autism.  The overall proportion of embryos that were correctly edited remained unchanged, but to their surprise, a significantly higher percentage carried the desired gene edit on both chromosomes. Tests with a different gene yielded the same unexpected outcome.

“Editing of both chromosomes simultaneously is normally very uncommon,” explains postdoctoral fellow Jonathan Wilde.  “The high rate of editing seen with RAD51 was really striking and what started as a simple attempt to make mutant Chd2 mice quickly turned into a much bigger project focused on RAD51 and its applications in genome editing,” said Wilde, who co-authored the Cell paper with research scientist Tomomi Aida.

A molecular copy machine

The Feng lab team next set out to understand the mechanism by which RAD51 enhances gene editing. They hypothesized that RAD51 engages a process called interhomolog repair (IHR), whereby a DNA break on one chromosome is repaired using the second copy of the chromosome (from the other parent) as the template.

To test this, they injected mouse embryos with RAD51 and CRISPR but left out the template DNA. They programmed CRISPR to cut only the gene sequence on one of the chromosomes, and then tested whether it was repaired to match the sequence on the uncut chromosome. For this experiment, they had to use mice in which the sequences on the maternal and paternal chromosomes were different.

They found that control embryos injected with CRISPR alone rarely showed IHR repair. However, addition of RAD51 significantly increased the number of embryos in which the CRISPR-targeted gene was edited to match the uncut chromosome.

“Previous studies of IHR found that it is incredibly inefficient in most cells,” says Wilde. “Our finding that it occurs much more readily in embryonic cells and can be enhanced by RAD51 suggest that a deeper understanding of what makes the embryo permissive to this type of DNA repair could help us design safer and more efficient gene therapies.”

A new way to correct disease-causing mutations          

Standard gene therapy strategies that rely on injecting a corrective piece of DNA to serve as a template for repairing the mutation engage a process called homology-directed repair (HDR).

“HDR-based strategies still suffer from low efficiency and carry the risk of unwanted integration of donor DNA throughout the genome,” explains Feng. “IHR has the potential to overcome these problems because it relies upon natural cellular pathways and the patient’s own normal chromosome for correction of the deleterious mutation.”

Feng’s team went on to identify additional DNA repair-associated proteins that can stimulate IHR, including several that not only promote high levels of IHR, but also repress errors in the DNA repair process. Additional experiments that allowed the team to examine the genomic features of IHR events gave deeper insight into the mechanism of IHR and suggested ways that the technique can be used to make gene therapies safer.

“While there is still a great deal to learn about this new application of IHR, our findings are the foundation for a new gene therapy approach that could help solve some of the big problems with current approaches,” says Aida.

This study was supported by the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, the Poitras Center for Psychiatric Disorders Research at MIT, NIH/NIMH Conte Center Grant (P50 MH094271) and NIH Office of the Director (U24 OD026638).

Michale Fee appointed head of MIT’s Brain and Cognitive Sciences Department

by Tristan Davies | BCS |

McGovern Investigator Michale Fee at work in the lab with postdoc Galen Lynch. Photo: Justin Knight

Michale Fee, the Glen V. and Phyllis F. Dorflinger Professor of Brain and Cognitive Sciences, has been named as the new head of the Department of Brain and Cognitive Sciences (BCS) effective May 1, 2021.

Fee, who is an investigator in the McGovern Institute for Brain Research, succeeds James DiCarlo, the Peter de Florez Professor of Neuroscience, who announced in December that he was stepping down to become director of the MIT Quest for Intelligence.

“I want to thank Jim for his impressive work over the last nine years as head,” says Fee. “I know firsthand from my time as associate department head that BCS is in good shape and on a steady course. Jim has set a standard of transparent and collaborative leadership, which is a solid foundation for making our community stronger on all fronts.” Fee notes that his first mission is to continue the initiatives begun under DiCarlo’s leadership—in academics (especially Course 6-9), mentoring, and diversity, equity, inclusion, and justice—while maintaining the highest standards of excellence in research and education.

“Jim has overseen significant growth in the faculty and its impact, as well as important academic initiatives to strengthen the department’s graduate and undergraduate programs,” says Nergis Mavalvala, dean of the School of Science. “His emphasis on building ties among BCS, the McGovern Institute for Brain Research, and the Picower Institute for Learning and Memory has brought innumerable new collaborations among researchers and helped solidify Building 46 and MIT as world leaders in brain science.”

Fee earned his BE in engineering physics in 1985 at the University of Michigan, and his PhD in applied physics at Stanford University in 1992, under the mentorship of Nobel laureate Stephen Chu. His doctoral work was followed by research in the Biological Computation Department at Bell Laboratories. He joined MIT and BCS as an associate professor in 2003 and was promoted to full professor in 2008.

He has served since 2012 as associate department head for education in BCS, overseeing significant evolution in the department’s academic programs, including a complete reworking of the Course 9 curriculum and the establishment in 2019 of Course 6-9, Computation and Cognition, in partnership with EECS.

In his research, Fee explores the neural mechanisms by which the brain learns complex sequential behaviors, using the learning of song by juvenile zebra finches as a model. He has brought new experimental and computational methods to bear on these questions, identifying a number of circuits used to learn, modify, time, and coordinate the development and utterance of song syllables.

“His work is emblematic of the department in that it crosses technical and disciplinary boundaries in search of the most significant discoveries,” says DiCarlo. “His research background gives Michale a deep appreciation of the importance of every sub-discipline in our community and a broad understanding of the importance of their connections with each other.”

Fee has received numerous honors and awards for his research and teaching, including the MIT Fundamental Science Investigator Award in 2017, the MIT School of Science Teaching Prize for Undergraduate Education in 2016, the BCS Award for Excellence in Undergraduate Teaching in 2015, and the Lawrence Katz Prize for Innovative Research in Neuroscience from Duke University in 2012.

Fee will be the sixth head of the department, after founding chair Hans-Lukas Teuber (1964–77), Richard Held (1977–86), Emilio Bizzi (1986–97), Mriganka Sur (1997–2012), and James DiCarlo (2012–21).