Research Area: Cellular & Molecular Neuroscience

​synapses, vesicles, electrophysiology, protein complexes, signaling, small molecules, disease-linked genes, dendrites, cell death, brain disorders, neurodevelopment

The craving state

by Jennifer Michalowski |

This story originally appeared in the Winter 2022 issue of BrainScan.

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For people struggling with substance use disorders — and there are about 35 million of them worldwide — treatment options are limited. Even among those who seek help, relapse is common. In the United States, an epidemic of opioid addiction has been declared a public health emergency.

A 2019 survey found that 1.6 million people nationwide had an opioid use disorder, and the crisis has surged since the start of the COVID-19 pandemic. The Centers for Disease Control and Prevention estimates that more than 100,000 people died of drug overdose between April 2020 and April 2021 — nearly 30 percent more overdose deaths than occurred during the same period the previous year.

In the United States, an epidemic of opioid addiction has been declared a public health emergency.

A deeper understanding of what addiction does to the brain and body is urgently needed to pave the way to interventions that reliably release affected individuals from its grip. At the McGovern Institute, researchers are turning their attention to addiction’s driving force: the deep, recurring craving that makes people prioritize drug use over all other wants and needs.

McGovern Institute co-founder, Lore Harp McGovern.

“When you are in that state, then it seems nothing else matters,” says McGovern Investigator Fan Wang. “At that moment, you can discard everything: your relationship, your house, your job, everything. You only want the drug.”

With a new addiction initiative catalyzed by generous gifts from Institute co-founder Lore Harp McGovern and others, McGovern scientists with diverse expertise have come together to begin clarifying the neurobiology that underlies the craving state. They plan to dissect the neural transformations associated with craving at every level — from the drug-induced chemical changes that alter neuronal connections and activity to how these modifications impact signaling brain-wide. Ultimately, the McGovern team hopes not just to understand the craving state, but to find a way to relieve it — for good.

“If we can understand the craving state and correct it, or at least relieve a little bit of the pressure,” explains Wang, who will help lead the addiction initiative, “then maybe we can at least give people a chance to use their top-down control to not take the drug.”

The craving cycle

For individuals suffering from substance use disorders, craving fuels a cyclical pattern of escalating drug use. Following the euphoria induced by a drug like heroin or cocaine, depression sets in, accompanied by a drug craving motivated by the desire to relieve that suffering. And as addiction progresses, the peaks and valleys of this cycle dip lower: the pleasant feelings evoked by the drug become weaker, while the negative effects a person experiences in its absence worsen. The craving remains, and increasing use of the drug are required to relieve it.

By the time addiction sets in, the brain has been altered in ways that go beyond a drug’s immediate effects on neural signaling.

These insidious changes leave individuals susceptible to craving — and the vulnerable state endures. Long after the physical effects of withdrawal have subsided, people with substance use disorders can find their craving returns, triggered by exposure to a small amount of the drug, physical or social cues associated with previous drug use, or stress. So researchers will need to determine not only how different parts of the brain interact with one another during craving and how individual cells and the molecules within them are affected by the craving state — but also how things change as addiction develops and progresses.

Circuits, chemistry and connectivity

One clear starting point is the circuitry the brain uses to control motivation. Thanks in part to decades of research in the lab of McGovern Investigator Ann Graybiel, neuroscientists know a great deal about how these circuits learn which actions lead to pleasure and which lead to pain, and how they use that information to establish habits and evaluate the costs and benefits of complex decisions.

Graybiel’s work has shown that drugs of abuse strongly activate dopamine-responsive neurons in a part of the brain called the striatum, whose signals promote habit formation. By increasing the amount of dopamine that neurons release, these drugs motivate users to prioritize repeated drug use over other kinds of rewards, and to choose the drug in spite of pain or other negative effects. Her group continues to investigate the naturally occurring molecules that control these circuits, as well as how they are hijacked by drugs of abuse.

Distribution of opioid receptors targeted by morphine (shown in blue) in two regions in the dorsal striatum and nucleus accumbens of the mouse brain. Image: Ann Graybiel

In Fan Wang’s lab, work investigating the neural circuits that mediate the perception of physical pain has led her team to question the role of emotional pain in craving. As they investigated the source of pain sensations in the brain, they identified neurons in an emotion-regulating center called the central amygdala that appear to suppress physical pain in animals. Now, Wang wants to know whether it might be possible to modulate neurons involved in emotional pain to ameliorate the negative state that provokes drug craving.

These animal studies will be key to identifying the cellular and molecular changes that set the brain up for recurring cravings. And as McGovern scientists begin to investigate what happens in the brains of rodents that have been trained to self-administer addictive drugs like fentanyl or cocaine, they expect to encounter tremendous complexity.

McGovern Associate Investigator Polina Anikeeva, whose lab has pioneered new technologies that will help the team investigate the full spectrum of changes that underlie craving, says it will be important to consider impacts on the brain’s chemistry, firing patterns, and connectivity. To that end, multifunctional research probes developed in her lab will be critical to monitoring and manipulating neural circuits in animal models.

Imaging technology developed by investigator Ed Boyden will also enable nanoscale protein visualization brain-wide. An important goal will be to identify a neural signature of the craving state. With such a signal, researchers can begin to explore how to shut off that craving — possibly by directly modulating neural signaling.

Targeted treatments

“One of the reasons to study craving is because it’s a natural treatment point,” says McGovern Associate Investigator Alan Jasanoff. “And the dominant kind of approaches that people in our team think about are approaches that relate to neural circuits — to the specific connections between brain regions and how those could be changed.” The hope, he explains, is that it might be possible to identify a brain region whose activity is disrupted during the craving state, then use clinical brain stimulation methods to restore normal signaling — within that region, as well as in other connected parts of the brain.

To identify the right targets for such a treatment, it will be crucial to understand how the biology uncovered in laboratory animals reflects what’s happens in people with substance use disorders. Functional imaging in John Gabrieli’s lab can help bridge the gap between clinical and animal research by revealing patterns of brain activity associated with the craving state in both humans and rodents. A new technique developed in Jasanoff’s lab makes it possible to focus on the activity between specific regions of an animal’s brain. “By doing that, we hope to build up integrated models of how information passes around the brain in craving states, and of course also in control states where we’re not experiencing craving,” he explains.

In delving into the biology of the craving state, McGovern scientists are embarking on largely unexplored territory — and they do so with both optimism and urgency. “It’s hard to not appreciate just the size of the problem, and just how devastating addiction is,” says Anikeeva. “At this point, it just seems almost irresponsible to not work on it, especially when we do have the tools and we are interested in the general brain regions that are important for that problem. I would say that there’s almost a civic duty.”

Study finds a striking difference between neurons of humans and other mammals

Anne Trafton |

McGovern Institute Investigator Mark Harnett. Photo: Justin Knight

Neurons communicate with each other via electrical impulses, which are produced by ion channels that control the flow of ions such as potassium and sodium. In a surprising new finding, MIT neuroscientists have shown that human neurons have a much smaller number of these channels than expected, compared to the neurons of other mammals.

The researchers hypothesize that this reduction in channel density may have helped the human brain evolve to operate more efficiently, allowing it to divert resources to other energy-intensive processes that are required to perform complex cognitive tasks.

“If the brain can save energy by reducing the density of ion channels, it can spend that energy on other neuronal or circuit processes,” says Mark Harnett, an associate professor of brain and cognitive sciences, a member of MIT’s McGovern Institute for Brain Research, and the senior author of the study.

Harnett and his colleagues analyzed neurons from 10 different mammals, the most extensive electrophysiological study of its kind, and identified a “building plan” that holds true for every species they looked at — except for humans. They found that as the size of neurons increases, the density of channels found in the neurons also increases.

However, human neurons proved to be a striking exception to this rule.

“Previous comparative studies established that the human brain is built like other mammalian brains, so we were surprised to find strong evidence that human neurons are special,” says former MIT graduate student Lou Beaulieu-Laroche.

Beaulieu-Laroche is the lead author of the study, which appears today in Nature.

A building plan

Neurons in the mammalian brain can receive electrical signals from thousands of other cells, and that input determines whether or not they will fire an electrical impulse called an action potential. In 2018, Harnett and Beaulieu-Laroche discovered that human and rat neurons differ in some of their electrical properties, primarily in parts of the neuron called dendrites — tree-like antennas that receive and process input from other cells.

One of the findings from that study was that human neurons had a lower density of ion channels than neurons in the rat brain. The researchers were surprised by this observation, as ion channel density was generally assumed to be constant across species. In their new study, Harnett and Beaulieu-Laroche decided to compare neurons from several different mammalian species to see if they could find any patterns that governed the expression of ion channels. They studied two types of voltage-gated potassium channels and the HCN channel, which conducts both potassium and sodium, in layer 5 pyramidal neurons, a type of excitatory neurons found in the brain’s cortex.

 

Former McGovern Institute graduate student Lou Beaulieu-Laroche is the lead author of the 2021 Nature paper.

They were able to obtain brain tissue from 10 mammalian species: Etruscan shrews (one of the smallest known mammals), gerbils, mice, rats, Guinea pigs, ferrets, rabbits, marmosets, and macaques, as well as human tissue removed from patients with epilepsy during brain surgery. This variety allowed the researchers to cover a range of cortical thicknesses and neuron sizes across the mammalian kingdom.

The researchers found that in nearly every mammalian species they looked at, the density of ion channels increased as the size of the neurons went up. The one exception to this pattern was in human neurons, which had a much lower density of ion channels than expected.

The increase in channel density across species was surprising, Harnett says, because the more channels there are, the more energy is required to pump ions in and out of the cell. However, it started to make sense once the researchers began thinking about the number of channels in the overall volume of the cortex, he says.

In the tiny brain of the Etruscan shrew, which is packed with very small neurons, there are more neurons in a given volume of tissue than in the same volume of tissue from the rabbit brain, which has much larger neurons. But because the rabbit neurons have a higher density of ion channels, the density of channels in a given volume of tissue is the same in both species, or any of the nonhuman species the researchers analyzed.

“This building plan is consistent across nine different mammalian species,” Harnett says. “What it looks like the cortex is trying to do is keep the numbers of ion channels per unit volume the same across all the species. This means that for a given volume of cortex, the energetic cost is the same, at least for ion channels.”

Energy efficiency

The human brain represents a striking deviation from this building plan, however. Instead of increased density of ion channels, the researchers found a dramatic decrease in the expected density of ion channels for a given volume of brain tissue.

The researchers believe this lower density may have evolved as a way to expend less energy on pumping ions, which allows the brain to use that energy for something else, like creating more complicated synaptic connections between neurons or firing action potentials at a higher rate.

“We think that humans have evolved out of this building plan that was previously restricting the size of cortex, and they figured out a way to become more energetically efficient, so you spend less ATP per volume compared to other species,” Harnett says.

He now hopes to study where that extra energy might be going, and whether there are specific gene mutations that help neurons of the human cortex achieve this high efficiency. The researchers are also interested in exploring whether primate species that are more closely related to humans show similar decreases in ion channel density.

The research was funded by the Natural Sciences and Engineering Research Council of Canada, a Friends of the McGovern Institute Fellowship, the National Institute of General Medical Sciences, the Paul and Daisy Soros Fellows Program, the Dana Foundation David Mahoney Neuroimaging Grant Program, the National Institutes of Health, the Harvard-MIT Joint Research Grants Program in Basic Neuroscience, and Susan Haar.

Other authors of the paper include Norma Brown, an MIT technical associate; Marissa Hansen, a former post-baccalaureate scholar; Enrique Toloza, a graduate student at MIT and Harvard Medical School; Jitendra Sharma, an MIT research scientist; Ziv Williams, an associate professor of neurosurgery at Harvard Medical School; Matthew Frosch, an associate professor of pathology and health sciences and technology at Harvard Medical School; Garth Rees Cosgrove, director of epilepsy and functional neurosurgery at Brigham and Women’s Hospital; and Sydney Cash, an assistant professor of neurology at Harvard Medical School and Massachusetts General Hospital.

Dealing with uncertainty

by Jennifer Michalowski |

As we interact with the world, we are constantly presented with information that is unreliable or incomplete – from jumbled voices in a crowded room to solicitous strangers with unknown motivations. Fortunately, our brains are well equipped to evaluate the quality of the evidence we use to make decisions, usually allowing us to act deliberately, without jumping to conclusions.

Now, neuroscientists at MIT’s McGovern Institute have homed in on key brain circuits that help guide decision-making under conditions of uncertainty. By studying how mice interpret ambiguous sensory cues, they’ve found neurons that stop the brain from using unreliable information.

“One area cares about the content of the message—that’s the prefrontal cortex—and the thalamus seems to care about how certain the input is.” – Michael Halassa

The findings, published October 6, 2021, in the journal Nature, could help researchers develop treatments for schizophrenia and related conditions, whose symptoms may be at least partly due to affected individuals’ inability to effectively gauge uncertainty.

Decoding ambiguity

“A lot of cognition is really about handling different types of uncertainty,” says McGovern Associate Investigator Michael Halassa, explaining that we all must use ambiguous information to make inferences about what’s happening in the world. Part of dealing with this ambiguity involves recognizing how confident we can be in our conclusions. And when this process fails, it can dramatically skew our interpretation of the world around us.

“In my mind, schizophrenia spectrum disorders are really disorders of appropriately inferring the causes of events in the world and what other people think,” says Halassa, who is a practicing psychiatrist. Patients with these disorders often develop strong beliefs based on events or signals most people would dismiss as meaningless or irrelevant, he says. They may assume hidden messages are embedded in a garbled audio recording, or worry that laughing strangers are plotting against them. Such things are not impossible—but delusions arise when patients fail to recognize that they are highly unlikely.

Halassa and postdoctoral researcher Arghya Mukherjee wanted to know how healthy brains handle uncertainty, and recent research from other labs provided some clues. Functional brain imaging had shown that when people are asked to study a scene but they aren’t sure what to pay attention to, a part of the brain called the mediodorsal thalamus becomes active. The less guidance people are given for this task, the harder the mediodorsal thalamus works.

The thalamus is a sort of crossroads within the brain, made up of cells that connect distant brain regions to one another. Its mediodorsal region sends signals to the prefrontal cortex, where sensory information is integrated with our goals, desires, and knowledge to guide behavior. Previous work in the Halassa lab showed that the mediodorsal thalamus helps the prefrontal cortex tune in to the right signals during decision-making, adjusting signaling as needed when circumstances change. Intriguingly, this brain region has been found to be less active in people with schizophrenia than it is in others.

group photo of study authors
Study authors (from left to right) Michael Halassa, Arghya Mukherjee, Norman Lam and Ralf Wimmer.

Working with postdoctoral researcher Norman Lam and research scientist Ralf Wimmer, Halassa and Mukherjee designed a set of animal experiments to examine the mediodorsal thalamus’s role in handling uncertainty. Mice were trained to respond to sensory signals according to audio cues that alerted them whether to focus on either light or sound. When the animals were given conflicting cues, it was up to them animal to figure out which one was represented most prominently and act accordingly. The experimenters varied the uncertainty of this task by manipulating the numbers and ratio of the cues.

Division of labor

By manipulating and recording activity in the animals’ brains, the researchers found that the prefrontal cortex got involved every time mice completed this task, but the mediodorsal thalamus was only needed when the animals were given signals that left them uncertain how to behave. There was a simple division of labor within the brain, Halassa says. “One area cares about the content of the message—that’s the prefrontal cortex—and the thalamus seems to care about how certain the input is.”

Within the mediodorsal thalamus, Halassa and Mukherjee found a subset of cells that were especially active when the animals were presented with conflicting sound cues. These neurons, which connect directly to the prefrontal cortex, are inhibitory neurons, capable of dampening downstream signaling. So when they fire, Halassa says, they effectively stop the brain from acting on unreliable information. Cells of a different type were focused on the uncertainty that arises when signaling is sparse. “There’s a dedicated circuitry to integrate evidence across time to extract meaning out of this kind of assessment,” Mukherjee explains.

As Halassa and Mukherjee investigate these circuits more deeply, a priority will be determining whether they are disrupted in people with schizophrenia. To that end, they are now exploring the circuitry in animal models of the disorder. The hope, Mukherjee says, is to eventually target dysfunctional circuits in patients, using noninvasive, focused drug delivery methods currently under development. “We have the genetic identity of these circuits. We know they express specific types of receptors, so we can find drugs that target these receptors,” he says. “Then you can specifically release these drugs in the mediodorsal thalamus to modulate the circuits as a potential therapeutic strategy.”

This work was funded by grants from the National Institute of Mental Health (R01MH107680-05 and R01MH120118-02).

Single gene linked to repetitive behaviors, drug addiction

by Jill Crittenden |

Making and breaking habits is a prime function of the striatum, a large forebrain region that underlies the cerebral cortex. McGovern researchers have identified a particular gene that controls striatal function as well as repetitive behaviors that are linked to drug addiction vulnerability.

To identify genes involved specifically in striatal functions, MIT Institute Professor Ann Graybiel previously identified genes that are preferentially expressed in striatal neurons. One identified gene encodes CalDAG-GEFI (CDGI), a signaling molecule that effects changes inside of cells in response to extracellular signals that are received by receptors on the cell surface. In a paper to be published in the October issue of Neurobiology of Disease and now available online, Graybiel, along with former Research Scientist Jill Crittenden and collaborators James Surmeier and Shenyu Zhai at the Feinman School of Medicine at Northwestern University, show that CDGI is key for controlling behavioral responses to drugs of abuse and underlying neuronal plasticity (cellular changes induced by experience) in the striatum.

“This paper represents years of intensive research, which paid off in the end by identifying a specific cellular signaling cascade for controlling repetitive behaviors and neuronal plasticity,” says Graybiel, who is also an investigator at the McGovern Institute and a professor of brain and cognitive sciences at MIT.

McGovern Investigator Ann Graybiel (right) with former Research Scientist Jill Crittenden. Photo: Justin Knight

Surprise discovery

To understand the essential roles of CDGI, Crittenden first engineered “knockout” mice that lack the gene encoding CDGI. Then the Graybiel team began looking for abnormalities in the CDGI knockout mice that could be tied to the loss of CDGI’s function.

Initially, they noticed that the rodent ear-tag IDs often fell off in the knockout mice, an observation that ultimately led to the surprise discovery by the Graybiel team and others that CDGI is expressed in blood platelets and is responsible for a bleeding disorder in humans, dogs, and other animals. The CDGI knockout mice were otherwise healthy and seemed just like their “wildtype” brothers and sisters, which did not carry the gene mutation. To figure out the role of CDGI in the brain, the Graybiel team would have to scrutinize the mice more closely.

Challenging the striatum

Both the CDGI knockout and wildtype mice were given an extensive set of behavioral and neurological tests and the CDGI mice showed deficits in two tests designed to challenge the striatum.

In one test, mice must find their way through a maze by relying on egocentric (i.e. self-referential) cues, such as their turning right or turning left, and not competing allocentric (i.e. external) cues, such as going toward a bright poster on the wall. Egocentric cues are thought to be processed by the striatum whereas allocentric cues are thought to rely on the hippocampus.

In a second test of striatal function, mice learned various gait patterns to match different patterns of rungs on their running wheel, a task designed to test the mouse’s ability to learn and remember a motor sequence.

The CDGI mice learned both of these striatal tasks more slowly than their wildtype siblings, suggesting that the CDGI mice might perform normally in general tests of behavior because they are able to compensate for striatal deficits by using other brain regions such as the hippocampus to solve standard tasks.

The team then decided to give the mice a completely different type of test that relies on the striatum. Because the striatum is strongly activated by drugs of abuse, which elevate dopamine and drive motor habits, Crittenden and collaborator Morgane Thomsen (now at the University of Copenhagen) looked to see whether the CDGI knockout mice respond normally to amphetamine and cocaine.

Psychomotor stimulants like cocaine and amphetamine normally induce a mixture of hyperactive behaviors such as pacing and focused repetitive behaviors like skin-picking (also called stereotypy or punding in humans). The researchers found however, that the drug-induced behaviors in the CDGI knockout mice were less varied than the normal mice and consisted of abnormally prolonged stereotypy, as though the mice were unable to switch between behaviors. The researchers were able to map the abnormal behavior to CDGI function in the striatum by showing that the same vulnerability to drug-induced stereotypy was observed in mice that were engineered to delete CDGI in the striatum after birth (“conditional knockouts”), but to otherwise have normal CDGI throughout the body.

Controlling cravings

In addition to exhibiting prolonged, repetitive behaviors, the CDGI knockout mice had a vulnerability to self-administer drugs. Although previous research had shown that treatments that activate the M1 acetylcholine receptor can block cocaine self-administration, the team found that this therapy was ineffective in CDGI knockout mice. Knockouts continued to self-administer cocaine (suggesting increased craving for the drug) at the same rate before and after M1 receptor activation treatment, even though the treatment succeeded with their sibling control mice. The researchers concluded that CDGI is critically important for controlling repetitive behaviors and the ability to stop self-administration of addictive stimulants.

mouse brain images
Brain sections from control mice (left) and mice engineered for deletion of the CDGI gene after birth. The expression of CDGI in the striatum (arrows) grows stronger as mice grow from pups to adulthood in control mice, but is gradually lost in the CDGI engineered mice (“conditional knockouts”). Image courtesy of the researchers

To better understand how CDGI is linked to the M1 receptor at the cellular level, the team turned to slice physiologists, scientists who record the electrical activity of neurons in brain slices. Their recordings showed that striatal neurons from CDGI knockouts fail to undergo the normal, expected electrophysiological changes after receiving treatments that target the M1 receptor. In particular, the neurons of the striatum that function broadly to stop ongoing behaviors, did not integrate cellular signals properly and failed to undergo “long-term potentiation,” a type of neuronal plasticity thought to underlie learning.

The new findings suggest that excessive repetitive movements are controlled by M1 receptor signaling through CDGI in indirect pathway neurons of the striatum, a neuronal subtype that degenerates in Huntington’s disease and is affected by dopamine loss and l-DOPA replacement therapy in Parkinson’s disease.

“The M1 acetylcholine receptor is a target for therapeutic drug development in treating cognitive and behavioral problems in multiple disorders, but progress has been severely hampered by off-target side-effects related to the wide-spread expression of the M1 receptor,” Graybiel explains. “Our findings suggest that CDGI offers the possibility for forebrain-specific targeting of M1 receptor signaling cascades that are of interest for blocking pathologically repetitive and unwanted behaviors that are common to numerous brain disorders including Huntington’s disease, drug addiction, autism, and schizophrenia as well as drug-induced dyskinesias. We hope that this work can help therapeutic development for these major health problems.”

This work was funded by the James W. (1963) and Patricia T. Poitras Fund, the William N. & Bernice E. Bumpus Foundation, the Saks Kavanaugh Foundation, the Simons Foundation, and the National Institute of Health.

New programmable gene editing proteins found outside of CRISPR systems

by Jennifer Michalowski |

Within the last decade, scientists have adapted CRISPR systems from microbes into gene editing technology, a precise and programmable system for modifying DNA. Now, scientists at MIT’s McGovern Institute and the Broad Institute of MIT and Harvard have discovered a new class of programmable DNA modifying systems called OMEGAs (Obligate Mobile Element Guided Activity), which may naturally be involved in shuffling small bits of DNA throughout bacterial genomes.

These ancient DNA-cutting enzymes are guided to their targets by small pieces of RNA. While they originated in bacteria, they have now  been engineered to work in human cells, suggesting they could be useful in the development of gene editing therapies, particularly as they are small (~30% the size of Cas9), making them easier to deliver to cells than bulkier enzymes. The discovery, reported September 9, 2021, in the journal Science, provides evidence that natural RNA-guided enzymes are among the most abundant proteins on earth, pointing toward a vast new area of biology that is poised to drive the next revolution in genome editing technology.

The research was led by McGovern Investigator Feng Zhang, who is the James and Patricia Poitras Professor of Neuroscience at MIT, a Howard Hughes Medical Institute investigator, and a Core Institute Member of the Broad Institute. Zhang’s team has been exploring natural diversity in search of new molecular systems that can be rationally programmed.

“We are super excited about the discovery of these widespread programmable enzymes, which have been hiding under our noses all along,” says Zhang. “These results suggest the tantalizing possibility that there are many more programmable systems that await discovery and development as useful technologies.”

Natural adaptation

Programmable enzymes, particularly those that use an RNA guide, can be rapidly adapted for different uses. For example, CRISPR enzymes naturally use an RNA guide to target viral invaders, but biologists can direct Cas9 to any target by generating their own RNA guide. “It’s so easy to just change a guide sequence and set a new target,” says graduate student and co-first author of the paper, Soumya Kannan. “So one of the broad questions that we’re interested in is trying to see if other natural systems use that same kind of mechanism.”

Zhang lab graduate student Han Altae-Tran, co-author of the Science paper with Soumya Kannan. Photo: Zhang lab

The first hints that OMEGA proteins might be directed by RNA came from the genes for proteins called IscBs. The IscBs are not involved in CRISPR immunity and were not known to associate with RNA, but they looked like small, DNA-cutting enzymes. The team discovered that each IscB had a small RNA encoded nearby and it directed IscB enzymes to cut specific DNA sequences. They named these RNAs “ωRNAs.”

The team’s experiments showed that two other classes of small proteins known as IsrBs and TnpBs, one of the most abundant genes in bacteria, also use ωRNAs that act as guides to direct the cleavage of DNA.

IscB, IsrB, and TnpB are found in mobile genetic elements called transposons. Graduate student Han Altae-Tran, co-first author on the paper, explains that each time these transposons move, they create a new guide RNA, allowing the enzyme they encode to cut somewhere else.

It’s not clear how bacteria benefit from this genomic shuffling—or whether they do at all.  Transposons are often thought of as selfish bits of DNA, concerned only with their own mobility and preservation, Kannan says. But if hosts can “co-opt” these systems and repurpose them, hosts may gain new abilities, as with CRISPR systems which confer adaptive immunity.

“A lot of the things that we have been thinking about may already exist naturally in some capacity,” says Altae-Tran.

IscBs and TnpBs appear to be predecessors of Cas9 and Cas12 CRISPR systems. The team suspects they, along with IsrB, likely gave rise to other RNA-guided enzymes, too—and they are eager to find them. They are curious about the range of functions that might be carried out in nature by RNA-guided enzymes, Kannan says, and suspect evolution likely already took advantage of OMEGA enzymes like IscBs and TnpBs to solve problems that biologists are keen to tackle.

Comparison of Ω (OMEGA) systems with other known RNA-guided systems. In contrast to CRISPR systems, which capture spacer sequences and store them in the locus within the CRISPR array, Ω systems may transpose their loci (or trans-acting loci) into target sequences, converting targets into ωRNA guides. Image courtesy of the researchers.

“A lot of the things that we have been thinking about may already exist naturally in some capacity,” says Altae-Tran. “Natural versions of these types of systems might be a good starting point to adapt for that particular task.”

The team is also interested in tracing the evolution of RNA-guided systems further into the past. “Finding all these new systems sheds light on how RNA-guided systems have evolved, but we don’t know where RNA-guided activity itself comes from,” Altae-Tran says. Understanding those origins, he says, could pave the way to developing even more classes of programmable tools.

This work was made possible with support from the Simons Center for the Social Brain at MIT; National Institutes of Health Intramural Research Program; National Institutes of Health grants 1R01-HG009761 and 1DP1-HL141201; Howard Hughes Medical Institute; Open Philanthropy; G. Harold and Leila Y. Mathers Charitable Foundation; Edward Mallinckrodt, Jr. Foundation; Poitras Center for Psychiatric Disorders Research at MIT; Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT; Yang-Tan Center for Molecular Therapeutics at MIT; Lisa Yang; Phillips family; R. Metcalfe; and J. and P. Poitras.

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.”

Some brain disorders exhibit similar circuit malfunctions

Anne Trafton |

Many neurodevelopmental disorders share similar symptoms, such as learning disabilities or attention deficits. A new study from MIT has uncovered a common neural mechanism for a type of cognitive impairment seen in some people with autism and schizophrenia, even though the genetic variations that produce the impairments are different for each condition.

In a study of mice, the researchers found that certain genes that are mutated or missing in some people with those disorders cause similar dysfunctions in a neural circuit in the thalamus. If scientists could develop drugs that target this circuit, they could be used to treat people who have different disorders with common behavioral symptoms, the researchers say.

“This study reveals a new circuit mechanism for cognitive impairment and points to a future direction for developing new therapeutics, by dividing patients into specific groups not by their behavioral profile, but by the underlying neurobiological mechanisms,” says Guoping Feng, the James W. and Patricia T. Poitras Professor in Brain and Cognitive Sciences at MIT, a member of the Broad Institute of Harvard and MIT, the associate director of the McGovern Institute for Brain Research at MIT, and the senior author of the new study.

Dheeraj Roy, a Warren Alpert Distinguished Scholar and a McGovern Fellow at the Broad Institute, and Ying Zhang, a postdoc at the McGovern Institute, are the lead authors of the paper, which appears today in Neuron.

Thalamic connections

The thalamus plays a key role in cognitive tasks such as memory formation and learning. Previous studies have shown that many of the gene variants linked to brain disorders such as autism and schizophrenia are highly expressed in the thalamus, suggesting that it may play a role in those disorders.

One such gene is called Ptchd1, which Feng has studied extensively. In boys, loss of this gene, which is carried on the X chromosome, can lead to attention deficits, hyperactivity, aggression, intellectual disability, and autism spectrum disorders.

In a study published in 2016, Feng and his colleagues showed that Ptchd1 exerts many of its effects in a part of the thalamus called the thalamic reticular nucleus (TRN). When the gene is knocked out in the TRN of mice, the mice show attention deficits and hyperactivity. However, that study did not find any role for the TRN in the learning disabilities also seen in people with mutations in Ptchd1.

In the new study, the researchers decided to look elsewhere in the thalamus to try to figure out how Ptchd1 loss might affect learning and memory. Another area they identified that highly expresses Ptchd1 is called the anterodorsal (AD) thalamus, a tiny region that is involved in spatial learning and communicates closely with the hippocampus.

Using novel techniques that allowed them to trace the connections between the AD thalamus and another brain region called the retrosplenial cortex (RSC), the researchers determined a key function of this circuit. They found that in mice, the AD-to-RSC circuit is essential for encoding fearful memories of a chamber in which they received a mild foot shock. It is also necessary for working memory, such as creating mental maps of physical spaces to help in decision-making.

The researchers found that a nearby part of the thalamus called the anteroventral (AV) thalamus also plays a role in this memory formation process: AV-to-RSC communication regulates the specificity of the encoded memory, which helps us distinguish this memory from others of similar nature.

“These experiments showed that two neighboring subdivisions in the thalamus contribute differentially to memory formation, which is not what we expected,” Roy says.

Circuit malfunction

Once the researchers discovered the roles of the AV and AD thalamic regions in memory formation, they began to investigate how this circuit is affected by loss of Ptchd1. When they knocked down expression of Ptchd1 in neurons of the AD thalamus, they found a striking deficit in memory encoding, for both fearful memories and working memory.

The researchers then did the same experiments with a series of four other genes — one that is linked with autism and three linked with schizophrenia. In all of these mice, they found that knocking down gene expression produced the same memory impairments. They also found that each of these knockdowns produced hyperexcitability in neurons of the AD thalamus.

These results are consistent with existing theories that learning occurs through the strengthening of synapses that occurs as a memory is formed, the researchers say.

“The dominant theory in the field is that when an animal is learning, these neurons have to fire more, and that increase correlates with how well you learn,” Zhang says. “Our simple idea was if a neuron fires too high at baseline, you may lack a learning-induced increase.”

The researchers demonstrated that each of the genes they studied affects different ion channels that influence neurons’ firing rates. The overall effect of each mutation is an increase in neuron excitability, which leads to the same circuit-level dysfunction and behavioral symptoms.

The researchers also showed that they could restore normal cognitive function in mice with these genetic mutations by artificially turning down hyperactivity in neurons of the AD thalamus. The approach they used, chemogenetics, is not yet approved for use in humans. However, it may be possible to target this circuit in other ways, the researchers say.

The findings lend support to the idea that grouping diseases by the circuit malfunctions that underlie them may help to identify potential drug targets that could help many patients, Feng says.

“There are so many genetic factors and environmental factors that can contribute to a particular disease, but in the end, it has to cause some type of neuronal change that affects a circuit or a few circuits involved in this behavior,” he says. “From a therapeutic point of view, in such cases you may not want to go after individual molecules because they may be unique to a very small percentage of patients, but at a higher level, at the cellular or circuit level, patients may have more commonalities.”

The research was funded by the Stanley Center at the Broad Institute, the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, the James and Patricia Poitras Center for Psychiatric Disorders Research at MIT, and the National Institutes of Health BRAIN Initiative.

Queen of hearts

Anne Trafton |

Amphibians and humans differ in many ways, but Laurie Boyer, a professor of biology and biological engineering at MIT, is particularly interested in one of those differences. Certain types of amphibians and fish can regenerate and heal their hearts after an injury. In contrast, human adults who have experienced trauma to the heart, such as in the case of a heart attack or exposure to certain medications, are unable to repair the damage. Often, the injured heart ends up with scar tissue that can lead to heart failure.

Recent research in this area now indicates that mice, and even humans, have some capacity for cardiac repair for a short period after birth. But after even just a few days of age, that ability starts to shut off. “The heart has very limited ability to repair itself in response to injury, disease, or aging,” Boyer says.

Alexander Auld, a postdoc in the Boyer Lab, studies the key cellular mechanisms that lead heart cells to mature and lose regenerative potential. Specifically, he’s interested in understanding how cardiomyocytes, the heart cells responsible for pumping blood, develop an ability to contract and relax repeatedly. Auld tests the function of proteins that serve as signals to assemble the cardiac muscle structure after birth. The assembly of these structures coincides with the loss of regenerative ability.

“I’m trying to piece together: What are the different mechanisms that push cardiomyocytes to assemble their contractile apparatus and to stop dividing?” Auld says. “Solving this puzzle may have potential to stimulate regeneration in the adult heart muscle.”

“The holy grail of regenerative biology would be to stimulate your own heart cells to replenish themselves,” says Boyer, who joined the MIT faculty in 2007. “Before this approach is possible, we need to achieve a deep understanding of the fundamental processes that drive heart development.”

Boyer’s lab studies how many different signals and genes interact to affect heart development. The work will enable a better understanding of how faulty regulation can lead to disease, and may also enable new therapies for people suffering from a variety of heart conditions.

Critical connections

Recently, Boyer’s lab has been studying heart development in people with Trisomy 21, or Down syndrome. Every year, 6,000 babies born in the United States have Down syndrome. Around half have heart defects. The most common heart defect in babies with Down syndrome is a hole in the heart’s center, called an atrioventricular septal defect. It is often repaired with surgery, but the repair can cause scar tissue and cardiovascular complications.

Somatic cells are those that compose an organism’s body; they differ from sex cells, which are used for reproduction. Most people have 46 chromosomes, arranged in 23 pairs, in their body’s somatic cells. In 95 percent of cases, Down syndrome results when a person has three copies of chromosome 21 instead of two –– a total of 47 chromosomes per cell. It’s an example of aneuploidy, when a cell has an abnormal number of chromosomes. Cellular attempts to adapt to the extra chromosome can cause stress on the body’s cells, including those of the heart.

MIT’s Alana Down Syndrome Center (ADSC) brings together biologists, neuroscientists, engineers, and other experts to increase knowledge about Down syndrome. ADSC launched in early 2019, led by Angelika Amon, professor of biology and a member of the Koch Institute for Integrative Cancer Research, along with co-director Li-Huei Tsai, Picower Professor and director of the Picower Institute for Learning and Memory. Amon died at age 53 in 2020 after a battle with ovarian cancer. At MIT, Amon had studied the effects of aneuploidy on cells.

“In my many wonderful scientific and personal discussions with Angelika, who was a beacon of inspiration to me, it became clear that studying Trisomy 21 in the context of heart development could ultimately improve the lives of these individuals,” Boyer says.

Change of heart

To conduct their research, Boyer’s group uses human induced pluripotent cells (hiPSCs), obtained through somatic cell reprogramming. The revolutionary technique was developed by Sir John B. Gurdon and Shinya Yamanaka, who in 2012 won the Nobel Prize in Physiology or Medicine for their work. Reprogramming works by converting specialized, mature somatic cells with one particular function into specialized, mature, cells with a different function.

Boyer’s lab uses hiPSCs from human adults with Down syndrome and converts them into cardiomyocytes through somatic cell reprogramming. Then, they compare those cardiomyocytes with reprogrammed cells from individuals who do not have Down syndrome. This work helps them deduce why the extra chromosome in people with Down syndrome may cause congenital heart defects.

“We can now begin to pinpoint the faulty signals and genes in Trisomy 21 cardiac cells that affect heart development,” Boyer says. “And with that same idea, we can also discover how we might actually be able to ameliorate or fix these defects.”

With this technique, the team can track how aspects of a specific patient’s cell development correlate with their clinical presentation. The ability to analyze patient-specific cells also has implications for personalized medicine, Boyer says. For instance, a patient’s skin or blood cells –– which are more easily obtained –– could be converted into a highly specialized mature cell, like a cardiac muscle cell, and tested for its response to drugs that could possibly cause damage to the heart before they reach the clinic. This process can also be used to screen for new therapies that can improve the outcome for heart failure patients.

Boyer presented the group’s research on Down syndrome at the New England Down Syndrome Symposium, co-organized in November 2020 by MIT, ADSC, Massachusetts Down Syndrome Congress, and LuMind IDSC Foundation.

Heart of the operation

Boyer’s lab employs students at the undergraduate, graduate, and postdoc levels from engineering, life sciences, and computer sciences –– each of whom, Boyer says, brings unique expertise and value to the team.

“It’s important for me to have a lab where everyone feels welcome, and that they feel that they can contribute to these fundamental discoveries,” Boyer says.

The Boyer Lab often works with scholars across disciplines at MIT. “It’s really great,” Auld says. “You can investigate a problem using multiple tools and perspectives.”

One project, in partnership with George Barbastathis, a professor in mechanical engineering, uses image-based machine learning to understand structural differences within cardiomyocytes when the proteins that signal cells to develop have been manipulated. Auld generates high-resolution images that the machine learning algorithms can analyze.

Another project, in collaboration with Ed Boyden, a professor in the Department of Biological Engineering as well as the McGovern Institute for Brain Research, involves the development of new technologies that allow high-throughput imaging of cardiac cells. The cross-pollination across departments and areas of expertise at MIT, Boyer says, often has her feeling like “a kid in a candy shop.”

“That our work could ultimately impact human health is very fulfilling for me, and the ability to use our scientific discoveries to improve medical outcomes is an important direction of my lab,” Boyer says. “Given the enormous talent at MIT and the excitement and willingness of everyone here to work together, we have an unprecedented opportunity to solve important problems that can make a difference in people’s lives.”

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.”

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).