Research Area: Cellular & Molecular Neuroscience

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

Controlling RNA in living cells

Anne Trafton |

MIT researchers have devised a new set of proteins that can be customized to bind arbitrary RNA sequences, making it possible to image RNA inside living cells, monitor what a particular RNA strand is doing, and even control RNA activity.

The new strategy is based on human RNA-binding proteins that normally help guide embryonic development. The research team adapted the proteins so that they can be easily targeted to desired RNA sequences.

“You could use these proteins to do measurements of RNA generation, for example, or of the translation of RNA to proteins,” says Edward Boyden, an associate professor of biological engineering and brain and cognitive sciences at the MIT Media Lab. “This could have broad utility throughout biology and bioengineering.”

Unlike previous efforts to control RNA with proteins, the new MIT system consists of modular components, which the researchers believe will make it easier to perform a wide variety of RNA manipulations.

“Modularity is one of the core design principles of engineering. If you can make things out of repeatable parts, you don’t have to agonize over the design. You simply build things out of predictable, linkable units,” says Boyden, who is also a member of MIT’s McGovern Institute for Brain Research.

Boyden is the senior author of a paper describing the new system in the Proceedings of the National Academy of Sciences. The paper’s lead authors are postdoc Katarzyna Adamala and grad student Daniel Martin-Alarcon.

Modular code

Living cells contain many types of RNA that perform different roles. One of the best known varieties is messenger RNA (mRNA), which is copied from DNA and carries protein-coding information to cell structures called ribosomes, where mRNA directs protein assembly in a process called translation. Monitoring mRNA could tell scientists a great deal about which genes are being expressed in a cell, and tweaking the translation of mRNA would allow them to alter gene expression without having to modify the cell’s DNA.

To achieve this, the MIT team set out to adapt naturally occurring proteins called Pumilio homology domains. These RNA-binding proteins include sequences of amino acids that bind to one of the ribonucleotide bases or “letters” that make up RNA sequences — adenine (A), thymine (T), uracil (U), and guanine (G).

In recent years, scientists have been working on developing these proteins for experimental use, but until now it was more of a trial-and-error process to create proteins that would bind to a particular RNA sequence.

“It was not a truly modular code,” Boyden says, referring to the protein’s amino acid sequences. “You still had to tweak it on a case-by-case basis. Whereas now, given an RNA sequence, you can specify on paper a protein to target it.”

To create their code, the researchers tested out many amino acid combinations and found a particular set of amino acids that will bind each of the four bases at any position in the target sequence. Using this system, which they call Pumby (for Pumilio-based assembly), the researchers effectively targeted RNA sequences varying in length from six to 18 bases.

“I think it’s a breakthrough technology that they’ve developed here,” says Robert Singer, a professor of anatomy and structural biology, cell biology, and neuroscience at Albert Einstein College of Medicine, who was not involved in the research. “Everything that’s been done to target RNA so far requires modifying the RNA you want to target by attaching a sequence that binds to a specific protein. With this technique you just design the protein alone, so there’s no need to modify the RNA, which means you could target any RNA in any cell.”

RNA manipulation

In experiments in human cells grown in a lab dish, the researchers showed that they could accurately label mRNA molecules and determine how frequently they are being translated. First, they designed two Pumby proteins that would bind to adjacent RNA sequences. Each protein is also attached to half of a green fluorescent protein (GFP) molecule. When both proteins find their target sequence, the GFP molecules join and become fluorescent — a signal to the researchers that the target RNA is present.

Furthermore, the team discovered that each time an mRNA molecule is translated, the GFP gets knocked off, and when translation is finished, another GFP binds to it, enhancing the overall fluorescent signal. This allows the researchers to calculate how often the mRNA is being read.

This system can also be used to stimulate translation of a target mRNA. To achieve that, the researchers attached a protein called a translation initiator to the Pumby protein. This allowed them to dramatically increase translation of an mRNA molecule that normally wouldn’t be read frequently.

“We can turn up the translation of arbitrary genes in the cell without having to modify the genome at all,” Martin-Alarcon says.

The researchers are now working toward using this system to label different mRNA molecules inside neurons, allowing them to test the idea that mRNAs for different genes are stored in different parts of the neuron, helping the cell to remain poised to perform functions such as storing new memories. “Until now it’s been very difficult to watch what’s happening with those mRNAs, or to control them,” Boyden says.

These RNA-binding proteins could also be used to build molecular assembly lines that would bring together enzymes needed to perform a series of reactions that produce a drug or another molecule of interest.

Study reveals a basis for attention deficits

Anne Trafton |

More than 3 million Americans suffer from attention deficit hyperactivity disorder (ADHD), a condition that usually emerges in childhood and can lead to difficulties at school or work.

A new study from MIT and New York University links ADHD and other attention difficulties to the brain’s thalamic reticular nucleus (TRN), which is responsible for blocking out distracting sensory input. In a study of mice, the researchers discovered that a gene mutation found in some patients with ADHD produces a defect in the TRN that leads to attention impairments.

The findings suggest that drugs boosting TRN activity could improve ADHD symptoms and possibly help treat other disorders that affect attention, including autism.

“Understanding these circuits may help explain the converging mechanisms across these disorders. For autism, schizophrenia, and other neurodevelopmental disorders, it seems like TRN dysfunction may be involved in some patients,” says Guoping Feng, the James W. and Patricia Poitras Professor of Neuroscience and a member of MIT’s McGovern Institute for Brain Research and the Stanley Center for Psychiatric Research at the Broad Institute.

Feng and Michael Halassa, an assistant professor of psychiatry, neuroscience, and physiology at New York University, are the senior authors of the study, which appears in the March 23 online edition of Nature. The paper’s lead authors are MIT graduate student Michael Wells and NYU postdoc Ralf Wimmer.

Paying attention

Feng, Halassa, and their colleagues set out to study a gene called Ptchd1, whose loss can produce attention deficits, hyperactivity, intellectual disability, aggression, and autism spectrum disorders. Because the gene is carried on the X chromosome, most individuals with these Ptchd1-related effects are male.

In mice, the researchers found that the part of the brain most affected by the loss of Ptchd1 is the TRN, which is a group of inhibitory nerve cells in the thalamus. It essentially acts as a gatekeeper, preventing unnecessary information from being relayed to the brain’s cortex, where higher cognitive functions such as thought and planning occur.

“We receive all kinds of information from different sensory regions, and it all goes into the thalamus,” Feng says. “All this information has to be filtered. Not everything we sense goes through.”

If this gatekeeper is not functioning properly, too much information gets through, allowing the person to become easily distracted or overwhelmed. This can lead to problems with attention and difficulty in learning.

The researchers found that when the Ptchd1 gene was knocked out in mice, the animals showed many of the same behavioral defects seen in human patients, including aggression, hyperactivity, attention deficit, and motor impairments. When the Ptchd1 gene was knocked out only in the TRN, the mice showed only hyperactivity and attention deficits.

Toward new treatments

At the cellular level, the researchers found that the Ptchd1 mutation disrupts channels that carry potassium ions, which prevents TRN neurons from being able to sufficiently inhibit thalamic output to the cortex. The researchers were also able restore the neurons’ normal function with a compound that boosts activity of the potassium channel. This intervention reversed the TRN-related symptoms but not any of the symptoms that appear to be caused by deficits of some other circuit.

“The authors convincingly demonstrate that specific behavioral consequences of the Ptchd1 mutation — attention and sleep — arise from an alteration of a specific protein in a specific brain region, the thalamic reticular nucleus. These findings provide a clear and straightforward pathway from gene to behavior and suggest a pathway toward novel treatments for neurodevelopmental disorders such as autism,” says Joshua Gordon, an associate professor of psychiatry at Columbia University, who was not involved in the research.

Most people with ADHD are now treated with psychostimulants such as Ritalin, which are effective in about 70 percent of patients. Feng and Halassa are now working on identifying genes that are specifically expressed in the TRN in hopes of developing drug targets that would modulate TRN activity. Such drugs may also help patients who don’t have the Ptchd1 mutation, because their symptoms are also likely caused by TRN impairments, Feng says.

The researchers are also investigating when Ptchd1-related problems in the TRN arise and at what point they can be reversed. And, they hope to discover how and where in the brain Ptchd1 mutations produce other abnormalities, such as aggression.

The research was funded by the Simons Foundation Autism Research Initiative, the National Institutes of Health, the Poitras Center for Affective Disorders Research, and the Stanley Center for Psychiatric Research at the Broad Institute.

Neuroscientists discover a gene that controls worms’ behavioral state

Anne Trafton |

In a study of worms, MIT neuroscientists have discovered a gene that plays a critical role in controlling the switch between alternative behavioral states, which for humans include hunger and fullness, or sleep and wakefulness.

This gene, which the researchers dubbed vps-50, helps to regulate neuropeptides — tiny proteins that carry messages between neurons or from neurons to other cells. This kind of signaling is important for controlling physiology and behavior in animals, including humans. Deletions of the human counterpart of the vps-50 gene have been found in some people with autism.

“Given what is reported in this paper about how the gene works, coupled with findings by others concerning the genetics of autism, we suggest that the disruption of the function of this gene could promote autism,” says H. Robert Horvitz, the David H. Koch Professor of Biology and a member of MIT’s McGovern Institute for Brain Research.

Horvitz and Martha Constantine-Paton, an MIT professor of brain and cognitive sciences and member of the McGovern Institute, are the senior authors of the study, which appears in the March 3 issue of the journal Current Biology. The paper’s lead authors are former MIT postdocs Nicolas Paquin and Yasunobu Muruta.

Influencing behavior

Neuropeptides, which are involved in brain functions such as reward, metabolism, and learning and memory, are released from cellular structures called dense-core vesicles.

In the new study, the researchers found that the vps-50 gene encodes a protein that is important in the generation of such vesicles and in the release of neuropeptides from them.

They discovered the protein in the worm Caenorhabditis elegans, where it is found primarily in nerve cells. In those cells, vps-50 associates with both synaptic vesicles and dense-core vesicles, which release neurotransmitters such as dopamine and serotonin. The researchers showed that vps-50 is required for maturation of the dense-core vesicles and also regulates activity of a proton pump that acidifies the vesicles. Without the proper acidity level, the vesicles’ ability to produce neuropeptides is impaired.

The researchers also found distinctive behavioral effects in C. elegans worms, which normally change their speed depending on food availability and whether they have recently eaten.

“Worms are the fastest when food (bacteria) is absent, presumably because they are looking for food,” Paquin says. “When they reach food, they slow down, but when you make them hungry for 30 minutes before putting them on food, they slow down even more.”

Worms lacking vps-50 behaved as if they were hungry — moving slowly through a food-rich area even when they were well fed, the researchers found. This suggests that the worms without vps-50 are unable to signal that they are full and continue to behave as if they are hungry. The researchers also found an equivalent gene in mice and showed that it can compensate for loss of the worm version of vps-50, showing that the two genes have the same function.

Human link

One important question raised by the study is how the mouse and human versions of vps-50 affect behavior in those animals, Horvitz says. Although this study focused on switching between hunger and fullness, neuropeptide signaling has been previously shown to control other alternative behaviors such as sleep and wakefulness and also to control social behaviors, such as anxiety.

The researchers suggest that studies of vps-50 might shed light on aspects of autism, because the human version of the gene is missing in some people with autism. Furthermore, a protein known as UNC-31, which is also located in dense-core vesicles has also been linked with autism in humans and mice. When mutated in worms, UNC-31 produces behavioral effects similar to those caused by vps-50 mutations.

“For these reasons, we hope that our studies of vps-50 will provide insights into human neuropsychiatric disorders,” Horvitz says.

The research was funded by the National Institutes of Health and the Simons Center for the Social Brain at MIT.

McGovern neuroscientists reverse autism symptoms

Anne Trafton |

Autism has diverse genetic causes, most of which are still unknown. About 1 percent of people with autism are missing a gene called Shank3, which is critical for brain development. Without this gene, individuals develop typical autism symptoms including repetitive behavior and avoidance of social interactions.

In a study of mice, MIT researchers have now shown that they can reverse some of those behavioral symptoms by turning the gene back on later in life, allowing the brain to properly rewire itself.

“This suggests that even in the adult brain we have profound plasticity to some degree,” says Guoping Feng, an MIT professor of brain and cognitive sciences. “There is more and more evidence showing that some of the defects are indeed reversible, giving hope that we can develop treatment for autistic patients in the future.”

Feng, who is the James W. and Patricia Poitras Professor of Neuroscience and a member of MIT’s McGovern Institute for Brain Research and the Stanley Center for Psychiatric Research at the Broad Institute, is the senior author of the study, which appears in the Feb. 17 issue of Nature. The paper’s lead authors are former MIT graduate student Yuan Mei and former Broad Institute visiting graduate student Patricia Monteiro, now at the University of Coimbra in Portugal.

Boosting communication

The Shank3 protein is found in synapses — the connections that allow neurons to communicate with each other. As a scaffold protein, Shank3 helps to organize the hundreds of other proteins that are necessary to coordinate a neuron’s response to incoming signals.

Studying rare cases of defective Shank3 can help scientists gain insight into the neurobiological mechanisms of autism. Missing or defective Shank3 leads to synaptic disruptions that can produce autism-like symptoms in mice, including compulsive behavior, avoidance of social interaction, and anxiety, Feng has previously found. He has also shown that some synapses in these mice, especially in a part of the brain called the striatum, have a greatly reduced density of dendritic spines — small buds on neurons’ surfaces that help with the transmission of synaptic signals.

In the new study, Feng and colleagues genetically engineered mice so that their Shank3 gene was turned off during embryonic development but could be turned back on by adding tamoxifen to the mice’s diet.
When the researchers turned on Shank3 in young adult mice (two to four and a half months after birth), they were able to eliminate the mice’s repetitive behavior and their tendency to avoid social interaction. At the cellular level, the team found that the density of dendritic spines dramatically increased in the striatum of treated mice, demonstrating the structural plasticity in the adult brain.

However, the mice’s anxiety and some motor coordination symptoms did not disappear. Feng suspects that these behaviors probably rely on circuits that were irreversibly formed during early development.
When the researchers turned on Shank3 earlier in life, only 20 days after birth, the mice’s anxiety and motor coordination did improve. The researchers are now working on defining the critical periods for the formation of these circuits, which could help them determine the best time to try to intervene.

“Some circuits are more plastic than others,” Feng says. “Once we understand which circuits control each behavior and understand what exactly changed at the structural level, we can study what leads to these permanent defects, and how we can prevent them from happening.”

Gordon Fishell, a professor of neuroscience at New York University School of Medicine, praises the study’s “elegant approach” and says it represents a major advance in understanding the circuitry and cellular physiology that underlie autism. “The combination of behavior, circuits, physiology, and genetics is state-of-the art,” says Fishell, who was not involved in the research. “Moreover, Dr. Feng’s demonstration that restoration of Shank3 function reverses autism symptoms in adult mice suggests that gene therapy may ultimately prove an effective therapy for this disease.”

Early intervention

For the small population of people with Shank3 mutations, the findings suggest that new genome-editing techniques could in theory be used to repair the defective Shank3 gene and improve these individuals’ symptoms, even later in life. These techniques are not yet ready for use in humans, however.

Feng believes that scientists may also be able to develop more general approaches that would apply to a larger population. For example, if the researchers can identify defective circuits that are specific for certain behavioral abnormalities in some autism patients, and figure out how to modulate those circuits’ activity, that could also help other people who may have defects in the same circuits even though the problem arose from a different genetic mutation.

“That’s why it’s important in the future to identify what subtype of neurons are defective and what genes are expressed in these neurons, so we can use them as a target without affecting the whole brain,” Feng says.

How maternal inflammation might lead to autism-like behavior

Anne Trafton |

In 2010, a large study in Denmark found that women who suffered an infection severe enough to require hospitalization while pregnant were much more likely to have a child with autism (even though the overall risk of delivering a child with autism remained low).

Now research from MIT, the University of Massachusetts Medical School, the University of Colorado, and New York University Langone Medical Center reveals a possible mechanism for how this occurs. In a study of mice, the researchers found that immune cells activated in the mother during severe inflammation produce an immune effector molecule called IL-17 that appears to interfere with brain development.

The researchers also found that blocking this signal could restore normal behavior and brain structure.

“In the mice, we could treat the mother with antibodies that block IL-17 after inflammation had set in, and that could ameliorate some of the behavioral symptoms that were observed in the offspring. However, we don’t know yet how much of that could be translated into humans,” says Gloria Choi, an assistant professor of brain and cognitive sciences, a member of MIT’s McGovern Institute for Brain Research, and the lead author of the study, which appears in the Jan. 28 online edition of Science.

Finding the link

In the 2010 study, which included all children born in Denmark between 1980 and 2005, severe infections (requiring hospitalization) that correlated with autism risk included influenza, viral gastroenteritis, and urinary tract infections. Severe viral infections during the first trimester translated to a threefold risk for autism, and serious bacterial infections during the second trimester were linked with a 1.5-fold increase in risk.

Choi and her husband, Jun Huh, were graduate students at Caltech when they first heard about this study during a lecture by Caltech professor emeritus Paul Patterson, who had discovered that an immune signaling molecule called IL-6 plays a role in the link between infection and autism-like behaviors in rodents.

Huh, now an assistant professor at the University of Massachusetts Medical School and one of the paper’s senior authors, was studying immune cells called Th17 cells, which are well known for contributing to autoimmune disorders such as multiple sclerosis, inflammatory bowel diseases, and rheumatoid arthritis. He knew that Th17 cells are activated by IL-6, so he wondered if these cells might also be involved in cases of animal models of autism associated with maternal infection.

“We wanted to find the link,” Choi says. “How do you go all the way from the immune system in the mother to the child’s brain?”

Choi and Huh launched the study as postdocs at Columbia University and New York University School of Medicine, respectively. Working with Dan Littman, a professor of molecular immunology at NYU and one of the paper’s senior authors, they began by injecting pregnant mice with a synthetic analog of double-stranded RNA, which activates the immune system in a similar way to viruses.

Confirming the results of previous studies in mice, the researchers found behavioral abnormalities in the offspring of the infected mothers, including deficits in sociability, repetitive behaviors, and abnormal communication. They then disabled Th17 cells in the mothers before inducing inflammation and found that the offspring mice did not show those behavioral abnormalities. The abnormalities also disappeared when the researchers gave the infected mothers an antibody that blocks IL-17, which is produced by Th17 cells.

The researchers next asked how IL-17 might affect the developing fetus. They found that brain cells in the fetuses of mothers experiencing inflammation express receptors for IL-17, and they believe that exposure to the chemical provokes cells to produce even more receptors for IL-17, amplifying its effects.

In the developing mice, the researchers found irregularities in the normally well-defined layers of cells in the brain’s cortex, where most cognition and sensory processing take place. These patches of irregular structure appeared in approximately the same cortical regions in all of the affected offspring, but they did not occur when the mothers’ Th17 cells were blocked.

Disorganized cortical layers have also been found in studies of human patients with autism.

Preventing autism

The researchers are now investigating whether and how these cortical patches produce the behavioral abnormalities seen in the offspring.

“We’ve shown correlation between these cortical patches and behavioral abnormalities, but we don’t know whether the cortical patches actually are responsible for the behavioral abnormalities,” Choi says. “And if it is responsible, what is being dysregulated within this patch to produce this behavior?”

The researchers hope their work may lead to a way to reduce the chances of autism developing in the children of women who experience severe infections during pregnancy. They also plan to investigate whether genetic makeup influences mice’s susceptibility to maternal inflammation, because autism is known to have a very strong genetic component.

Charles Hoeffer, a professor of integrative physiology at the University of Colorado, is a senior author of the paper, and other authors include MIT postdoc Yeong Yim, NYU graduate student Helen Wong, UMass Medical School visiting scholars Sangdoo Kim and Hyunju Kim, and NYU postdoc Sangwon Kim.

How a single gene contributes to autism and schizophrenia

Anne Trafton |

Although it is known that psychiatric disorders have a strong genetic component, untangling the web of genes contributing to each disease is a daunting task. Scientists have found hundreds of genes that are mutated in patients with disorders such as autism, but each patient usually has only a handful of these variations.

To further complicate matters, some of these genes contribute to more than one disorder. One such gene, known as Shank3, has been linked to both autism and schizophrenia.

MIT neuroscientists have now shed some light on how a single gene can play a role in more than one disease. In a study appearing in the Dec. 10 online edition of Neuron, they revealed that two different mutations of the Shank3 gene produce some distinct molecular and behavioral effects in mice.

“This study gives a glimpse into the mechanism by which different mutations within the same gene can cause distinct defects in the brain, and may help to explain how they may contribute to different disorders,” says Guoping Feng, the James W. and Patricia Poitras Professor of Neuroscience at MIT, a member of MIT’s McGovern Institute for Brain Research, a member of the Stanley Center for Psychiatric Research at the Broad Institute, and the senior author of the study.

The findings also suggest that identifying the brain circuits affected by mutated genes linked to psychiatric disease could help scientists develop more personalized treatments for patients in the future, Feng says.

The paper’s lead authors are McGovern Institute research scientist Yang Zhou, graduate students Tobias Kaiser and Xiangyu Zhang, and research affiliate Patricia Monteiro.

Disrupted communication

The protein encoded by Shank3 is found in synapses — the junctions between neurons that allow them to communicate with each other. Shank3 is a scaffold protein, meaning it helps to organize hundreds of other proteins clustered on the postsynaptic cell membrane, which are required to coordinate the cell’s response to signals from the presynaptic cell.

In 2011, Feng and colleagues showed that by deleting Shank3 in mice they could induce two of the most common traits of autism — avoidance of social interaction, and compulsive, repetitive behavior. A year earlier, researchers at the University of Montreal identified a Shank3 mutation in patients suffering from schizophrenia, which is characterized by hallucinations, cognitive impairment, and abnormal social behavior.

Feng wanted to find out how these two different mutations in the Shank3 gene could play a role in such different disorders. To do that, he and his colleagues engineered mice with each of the two mutations: The schizophrenia-related mutation results in a truncated version of the Shank3 protein, while the autism-linked mutation leads to a total loss of the Shank3 protein.

Behaviorally, the mice shared many defects, including strong anxiety. However, the mice with the autism mutation had very strong compulsive behavior, manifested by excessive grooming, which was rarely seen in mice with the schizophrenia mutation.

In the mice with the schizophrenia mutation, the researchers saw a type of behavior known as social dominance. These mice trimmed the whiskers and facial hair of the genetically normal mice sharing their cages, to an extreme extent. This is a typical way for mice to display their social dominance, Feng says.

By activating the mutations in different parts of the brain and at different stages of development, the researchers found that the two mutations affected brain circuits in different ways. The autism mutation exerted its effects early in development, primarily in a part of the brain known as the striatum, which is involved in coordinating motor planning, motivation, and habitual behavior. Feng believes that disruption of synapses in the striatum contributes to the compulsive behavior seen in those mice.

In mice carrying the schizophrenia-associated mutation, early development was normal, suggesting that truncated Shank3 can adequately fill in for the normal version during this stage. However, later in life, the truncated version of Shank3 interfered with synaptic functions and connections in the brain’s cortex, where executive functions such as thought and planning occur. This suggests that different segments of the protein — including the stretch that is missing in the schizophrenia-linked mutation — may be crucial for different roles, Feng says.

The new paper represents an important first step in understanding how different mutations in the same gene can lead to different diseases, says Joshua Gordon, an associate professor of psychiatry at Columbia University.

“The key is to identify how the different mutations alter brain function in different ways, as done here,” says Gordon, who was not involved in the research. “Autism strikes early in childhood, while schizophrenia typically arises in adolescence or early adulthood. The finding that the autism-associated mutation has effects at a younger age than the schizophrenia-associated mutation is particularly intriguing in this context.”

Modeling disease

Although only a small percentage of autism patients have mutations in Shank3, many other variant synaptic proteins have been associated with the disorder. Future studies should help to reveal more about the role of the many genes and mutations that contribute to autism and other disorders, Feng says. Shank3 alone has at least 40 identified mutations, he says.

“We cannot consider them all to be the same,” he says. “To really model these diseases, precisely mimicking each human mutation is critical.”

Understanding exactly how these mutations influence brain circuits should help researchers develop drugs that target those circuits and match them with the patients who would benefit most, Feng says, adding that a tremendous amount of work needs to be done to get to that point.

His lab is now investigating what happens in the earliest stages of the development of mice with the autism-related Shank3 mutation, and whether any of those effects can be reversed either during development or later in life.

The research was funded by the Simons Center for the Social Brain at MIT, the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard, the Poitras Center for Affective Disorders Research at MIT, and National Institute of Mental Health.

Tasting light

Anne Trafton |

Human taste receptors are specialized to distinguish several distinct compounds: sugars taste sweet, salts taste salty, and acidic compounds taste sour. Now a new study from MIT finds that the worm Caenorhabditis elegans has taken its powers of detection a step further: The worm can taste hydrogen peroxide, triggering it to stop eating the potentially dangerous substance.

Being able to taste hydrogen peroxide allows the worm to detect light, which generates hydrogen peroxide and other harmful reactive oxygen compounds both within the worm and in its environment.

“This is potentially a brand-new mechanism of sensing light,” says Nikhil Bhatla, the lead author of the paper and a postdoc in MIT’s Department of Biology. “All of the mechanisms of light detection we know about involve a chromophore — a small molecule that absorbs a photon and changes shape or transfers electrons. This seems to be the first example of behavioral light-sensing that requires the generation of a chemical in the process of detecting the light.”

Bhatla and Robert Horvitz, the David H. Koch Professor of Biology, describe the new hydrogen peroxide taste receptors in the Jan. 29 online issue of the journal Neuron.

Though it is not yet known whether there is a human equivalent of this system, the researchers say their discovery lends support to the idea that there may be human taste receptors dedicated to flavors other than the five canonical ones — sweet, salty, bitter, sour, and savory. It also opens the possibility that humans might be able to sense light in ways that are fundamentally different from those known to act in vision.

“I think we have underestimated our biological abilities,” Bhatla says. “Aside from those five, there are other flavors, such as burnt. How do we taste something as burnt? Or what about spicy, or metallic, or smoky? There’s this whole new area that hasn’t really been explored.”

Beyond bitter and sweet

One of the major functions of the sense of taste is to determine whether something is safe, or advantageous, to eat. For humans and other animals, bitterness often serves as a warning of poison, while sweetness can help to identify foods that are rich in energy.

For worms, hydrogen peroxide can be harmful because it can cause extensive cellular trauma, including damaging proteins, DNA, and other molecules in the body. In fact, certain strains of bacteria produce hydrogen peroxide that can kill C. elegans after being eaten. Worms might also ingest hydrogen peroxide from the soil where they live.

Bhatla and Horvitz found that worms stop eating both when they taste hydrogen peroxide and when light shines on them — especially high-energy light, such as violet or ultraviolet. The authors found the exact same feeding response when worms were exposed to either hydrogen peroxide or light, which suggested to them that the same mechanism might be controlling responses to both stimuli.

Worms are known to be averse to light: Previous research by others has shown that they flee when light shines on them. Bhatla and Horvitz have now found that this escape response, like the feeding response to light, is likely caused by light’s generation of chemicals such as hydrogen peroxide.

The C. elegans worm has a very simple and thoroughly mapped nervous system consisting of 302 neurons, 20 of which are located in the pharynx, the feeding organ that ingests and grinds food. Bhatla found that one pair of pharyngeal neurons, known as the I2 neurons, controls the animal’s response to both light and hydrogen peroxide. A particular molecular receptor in that neuron, gustatory receptor 3 (GUR-3), and a molecularly similar receptor found in other neurons (LITE-1) are critical to the response. However, each receptor appears to function in a slightly different way.

GUR-3 detects hydrogen peroxide, whether it is found naturally in the environment or generated by light. There are many GUR-3 receptors in the I2 neuron, and through a mechanism that remains unknown, hydrogen peroxide stimulation of GUR-3 causes the pharynx to stop grinding. Another molecule called peroxiredoxin, an antioxidant, appears to help GUR-3 detect hydrogen peroxide.

While the GUR-3 receptor responds much more strongly to hydrogen peroxide than to light, the LITE-1 receptor is much more sensitive to light than to hydrogen peroxide. LITE-1 has previously been implicated in detecting light, but until now, it has been a mystery how a taste receptor could respond to light. The new study suggests that like GUR-3, LITE-1 indirectly senses light by detecting reactive oxygen compounds generated by light — including, but not limited to, hydrogen peroxide.

Kenneth Miller of the Oklahoma Medical Research Foundation published a paper in 2008 describing LITE-1 and hypothesizing that it might work by detecting a chemical product of light interaction. “This paper goes one step beyond that and identifies molecules that LITE-1 could be sensing to identify the presence of light,” says Miller, who was not part of the new study. “I thought it was a fascinating look at the complex gustatory sensory mechanism for molecules like hydrogen peroxide.”

Not found in humans

The molecular family of receptors that includes GUR-3 and LITE-1 is specific to invertebrates, and is not found in humans. However, peroxiredoxin is found in humans, particularly in the eye, so the researchers suspect that peroxiredoxin might play a role in detecting reactive oxygen species generated by light in the eye.

The researchers are now trying to figure out the exact mechanism of hydrogen peroxide detection: For example, how exactly do these gustatory receptors detect reactive oxygen compounds? The researchers are also working to identify the neural circuit diagram that defines how the I2 neurons interact with other neurons to control the worms’ feeding behavior. Such neural circuit diagrams should provide insight into how the brains of worms, and people, generate behavior.

The research was funded by the National Science Foundation, the National Institutes of Health, and the Howard Hughes Medical Institute.

New way to turn genes on

Anne Trafton |

Using a gene-editing system originally developed to delete specific genes, MIT researchers have now shown that they can reliably turn on any gene of their choosing in living cells.

This new application for the CRISPR/Cas9 gene-editing system should allow scientists to more easily determine the function of individual genes, according to Feng Zhang, the W.M. Keck Career Development Professor in Biomedical Engineering in MIT’s Departments of Brain and Cognitive Sciences and Biological Engineering, and a member of the Broad Institute and MIT’s McGovern Institute for Brain Research.

This approach also enables rapid functional screens of the entire genome, allowing scientists to identify genes involved in particular diseases. In a study published in the Dec. 10 online edition of Nature, Zhang and colleagues identified several genes that help melanoma cells become resistant to a cancer drug.

Silvana Konermann, a graduate student in Zhang’s lab, and Mark Brigham, a McGovern Institute postdoc, are the paper’s lead authors.

A new function for CRISPR

The CRISPR system relies on cellular machinery that bacteria use to defend themselves from viral infection. Researchers have previously harnessed this cellular system to create gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, telling Cas9 where to make its cut.

In the past two years, scientists have developed Cas9 as a tool for turning genes off or replacing them with a different version. In the new study, Zhang and colleagues engineered the Cas9 system to turn genes on, rather than knock them out. Scientists have tried to do this before using proteins that are individually engineered to target DNA at specific sites. However, these proteins are  difficult to work with. “If you use the older generation of tools, getting the technology to do what you actually want is a project on its own,” Konermann says. “It takes a lot of time and is also quite expensive.”

There have also been attempts to use CRISPR to turn on genes by inactivating the part of the Cas9 enzyme that cuts DNA and linking Cas9 to pieces of proteins called activation domains. These domains recruit the cellular machinery necessary to begin reading copying RNA from DNA, a process known as transcription.

However, these efforts have been unable to consistently turn on gene transcription. Zhang and his colleagues, Osamu Nureki and Hiroshi Nishimasu at the University of Tokyo, decided to overhaul the CRISPR-Cas9 system based on an analysis they published earlier this year of the structure formed when Cas9 binds to the guide RNA and its target DNA. “Based on knowing its 3-D shape, we can think about how to rationally improve the system,” Zhang says.

In previous efforts, scientists had tried to attach the activation domains to either end of the Cas9 protein, with limited success. From their structural studies, the MIT team realized that two small loops of the RNA guide poke out from the Cas9 complex and could be better points of attachment because they allow the activation domains to have more flexibility in recruiting transcription machinery.

Using their revamped system, the researchers activated about a dozen genes that had proven difficult or impossible to turn on using the previous generation of Cas9 activators. Each gene showed at least a twofold boost in transcription, and for many genes, the researchers found multiple orders of magnitude increase in activation.

Genome-scale activation screening

Once the researchers had shown that the system was effective at activating genes, they created a library of 70,290 guide RNAs targeting all of the more than 20,000 genes in the human genome.

They screened this library to identify genes that confer resistance to a melanoma drug called PLX-4720. Drugs of this type work well in patients whose melanoma cells have a mutation in the BRAF gene, but cancer cells that survive the treatment can grow into new tumors, allowing the cancer to recur.

To discover the genes that help cells become resistant, the researchers delivered CRISPR components to a large population of melanoma cells grown in the lab, with each cell receiving a different guide RNA targeting a different gene. After treating the cells with PLX-4720, they identified several genes that helped the cells to survive — some previously known to be involved in drug resistance, as well as several novel targets.
Studies like this could help researchers discover new cancer drugs that prevent tumors from becoming resistant.

“You could start with a drug that targets the mutated BRAF along with combination therapy that targets genes that allow the cell to survive. If you target both of them at the same time, you could likely prevent the cells from developing resistance mechanisms that enable further growth despite drug treatment,” Konermann says.

Scientists have tried to do large-scale screens like this by delivering single genes carried by viruses, but that does not work with all genes.

“This new technique could allow you to sample a larger spectrum of genes that might be playing a role,” says Levi Garraway, an associate professor of medicine at Dana-Farber Cancer Institute who was not involved in the research. “This is really a technology development paper, but the tantalizing results from the drug resistance screen speak to the rich biological possibilities of this approach.”

Zhang’s lab also plans to use this technique to screen for genes that, when activated, could correct the effects of autism or neurodegenerative diseases such as Alzheimer’s. He also plans to make the necessary reagents available to academic labs that want to use them, through the Addgene repository.

The research was funded by the National Institute of Mental Health; the National Institute of Neurological Disorders and Stroke; the Keck, Searle Scholars, Klingenstein, Vallee, and Simons foundations; and Bob Metcalfe.

From genes to brains

by Elizabeth Dougherty |

Many brain disorders are strongly influenced by genetics, and researchers have long hoped that the identification of genetic risk factors will provide clues to the causes and possible treatments of these mysterious conditions. In the early years, progress was slow. Many claims failed to replicate, and it became clear that in order to identify the important risk genes with confidence, researchers would need to examine the genomes of very large numbers of patients.

Until recently that would have been prohibitively expensive, but genome research has been accelerating fast. Just how fast was underlined by an announcement in January from a California-based company, Illumina, that it had achieved a long-awaited milestone: sequencing an entire human genome for under $1000. Seven years ago, this task would have cost $10M and taken weeks of work. The new system does the job in a few hours, and can sequence tens of thousands of genomes per year.

In parallel with these spectacular advances, another technological revolution has been unfolding over the past several years, with the development of a new method for editing the genome of living cells. This method, known as CRISPR, allows researchers to make precise changes to a DNA sequence—an advance that is expected to transform many areas of biomedical research and may ultimately form the basis of new treatments for human genetic disease.

The CRISPR technology, which is based on a natural bacterial defense system against viruses, uses a short strand of RNA as a “search string” to locate a corresponding DNA target sequence. This RNA string can be synthesized in the lab and can be designed to recognize any desired sequence of DNA. The RNA carries with it a protein called Cas9, which cuts the target DNA at the chosen location, allowing a new sequence to be inserted—providing researchers with a fast and flexible “search-and-replace” tool for editing the genome.

One of the pioneers in this field is McGovern Investigator Feng Zhang, who along with George Church of Harvard, was the first to show that CRISPR could be used to edit the human genome in living cells. Zhang is using the technology to study human brain disorders, building on the flood of new genetic discoveries that are emerging from advances in DNA sequencing. The Broad Institute, where Zhang holds a joint appointment, is a world leader in human psychiatric genetics, and will be among the first to acquire the new Illumina sequencing machines when they reach the market later this year.

By sequencing many thousands of individuals, geneticists are identifying the rare genetic variants that contribute to risk of diseases such as autism, schizophrenia and bipolar disorder. CRISPR will allow neuroscientists to study those gene variants in cells and in animal models. The goal, says McGovern Institute director Bob Desimone, is to understand the biological roots of brain disorders. “The biggest obstacle to new treatments has been our ignorance of fundamental mechanisms. But with these new technologies, we have a real opportunity to understand what’s wrong at the level of cells and circuits, and to identify the pressure points at which therapeutic intervention may be possible.”

Culture Club

In other fields, the influence of genetic variations on disease has turned out to be surprisingly difficult to unravel, and for neuropsychiatric disease, the challenge may be even greater. The brain is the most complex organ of the body, and the underlying pathologies that lead to disease are not yet well understood. Moreover, any given disorder may show a wide variation in symptoms from patient to patient, and it may also have many different genetic causes. “There are hundreds of genes that can contribute to autism or schizophrenia,” says McGovern Investigator Guoping Feng, who is also Poitras Professor of Neuroscience.

To study these genes, Feng and collaborators at the Broad Institute’s Stanley Center for Psychiatric Research are planning to screen thousands of cultures of neurons, grown in the tiny wells of cell culture plates. The neurons, which are grown from stem cells, can be engineered using CRISPR to contain the genetic variants that are linked to neuropsychiatric disease. Each culture will contain neurons with a different variant, and these will be examined for abnormalities that might be associated with disease.

Feng and colleagues hope this high-throughput platform will allow them to identify cellular traits, or phenotypes, that may be related to disease and which can then be studied in animal models to see if they cause defects in brain function or in behavior. In the longer term, this high-throughput platform can also be used to screen for new drugs that can reverse these defects.

Animal Kingdom

Cell cultures are necessary for large-scale screens, but ultimately the results must be translated into the context of brain circuits and behavior. “That means we must study animal models too,” says Feng.

Feng has created several mouse models of human brain disease by mutating genes that are linked to these disorders and examining the behavioral and cellular defects in the mutant animals. “We have models of obsessive-compulsive disorder and autism,” he explains. “By studying these mice we want to learn what’s wrong with their brains.”

So far, Feng has focused on single-gene models, but the majority of human psychiatric disorders are triggered by multiple genes acting in combination. One advantage of the new CRISPR method is that it allows researchers to introduce several mutations in parallel, and Zhang’s lab is now working to create autistic mice with more than one gene alteration.

Perhaps the most important advantage of CRISPR is that it can be applied to any species. Currently, almost all genetic modeling of human disease is restricted to mice. But while mouse models are convenient, they are limited, especially for diseases that affect higher brain functions and for which there are no clear parallels in rodents. “We also need to study species that are closer to humans,” says Feng.

Accordingly, he and Zhang are collaborating with colleagues in Oregon and China to use CRISPR to create primate models of neuropsychiatric disorders. Earlier this year, a team in China announced that they had used CRISPR to create transgenic monkeys that will be used to study defects in metabolism and immunity.

Feng and Zhang are planning to use a similar approach to study brain disorders, but in addition to macacques, they will also work with a smaller primate species, the marmoset. These animals, with their fast breeding cycles and complex behavioral repertoires, are ideal for genetic studies of behavior and brain function. And because they are very social with highly structured communication patterns, they represent a promising new model for understanding the neural basis of social cognition and its disruption in conditions such as autism.

Given their close evolutionary relationship to humans, marmoset models could also help accelerate the development of new therapies. Many experimental drugs for brain disorders have been tested successfully in mice, only to prove ineffective in subsequent human trials. These failures, which can be enormously expensive, have led many drug companies to cut back on their neuroscience R&D programs. Better animal models could reverse this trend by allowing companies to predict more accurately which drug candidates are most promising, before investing heavily in human clinical trials.

Feng’s mouse research provides an example of how this approach can work. He previously developed a mouse model of obsessive-compulsive disorder, in which the animals engage in obsessive self-grooming, and he has now shown that this effect can be reversed when the missing gene is reintroduced, even in adulthood. Other researchers have seemed similar results with other brain disorders such as Rett Syndrome, a condition that is often accompanied by autism. “The brain is amazingly plastic,” says Feng. “At least in a mouse, we have shown that the damage can often be repaired. If we can also show this in marmosets or other primate models, that would really give us hope that something similar is possible in humans.”

Human Race

Ultimately, to understand the genetic roots of human behavior, researchers must sequence the genomes of individual subjects in parallel with measurements of those same individuals’ behavior and brain function.

Such studies typically require very large sample sizes, but the plummeting cost of sequencing is now making this feasible. In China, for instance, a project is already underway to sequence the genomes of many thousands of individuals to uncover genetic influences on cognition and intelligence.

The next step will be to link the genetics to brain activity, says McGovern Investigator John Gabrieli, who also directs the Martinos Imaging Center at MIT. “It’s a big step to go from DNA to behavioral variation or clinical diagnosis. But we know those genes must affect brain function, so neuroimaging may help us to bridge that gap.”

But brain scans can be time-consuming, given that volunteers must perform behavioral tasks in the scanner. Studies are typically limited to a few dozen subjects, not enough to detect the often subtle effects of genomic variation.

One way to enlarge these studies, says Gabrieli, is to image the brain during rest rather than in a state of prompted activity. This procedure is fast and easy to replicate from lab to lab, and patterns of resting state activity have turned out to be surprisingly reproducible; moreover, Gabrieli is finding that differences in resting activity are associated with brain disorders such as autism, and he hopes that in the future it will be possible to relate these differences to the genetic factors that are emerging from genome studies at the Broad Institute and elsewhere.

“I’m optimistic that we’re going to see dramatic advances in our understanding of neuropsychiatric disease over the next few years.” — Bob Desimone

Confirming these associations will require a “big data” approach, in which results from multiple labs are consolidated into large repositories and analyzed for significant associations. Resting state imaging lends itself to this approach, says Gabrieli. “To find the links between brain function and genetics, big data is the direction we need to go to be successful.”

How soon might this happen? “It won’t happen overnight,” cautions Desimone. “There are a lot of dots that need to be connected. But we’ve seen in the case of genome research how fast things can move once the right technologies are in place. I’m optimistic that we’re going to see equally dramatic advances in our understanding of neuropsychiatric disease over the next few years.”

Fifteen MIT scientists receive NIH BRAIN Initiative grants

Anne Trafton |

Today, the National Institutes of Health (NIH) announced their first round of BRAIN Initiative award recipients. Six teams and 15 researchers from the Massachusetts Institute of Technology were recipients.

Mriganka Sur, principal investigator at the Picower Institute for Learning and Memory and the Paul E. Newton Professor of Neuroscience in MIT’s Department of Brain and Cognitive Sciences (BCS) leads a team studying cortical circuits and information flow during memory-guided perceptual decisions. Co-principal investigators include Emery Brown, BCS professor of computational neuroscience and the Edward Hood Taplin Professor of Medical Engineering; Kwanghun Chung, Picower Institute principal investigator and assistant professor in the Department of Chemical Engineering and the Institute for Medical Engineering and Science (IMES); and Ian Wickersham, research scientist at the McGovern Institute for Brain Research and head of MIT’s Genetic Neuroengineering Group.

Elly Nedivi, Picower Institute principal investigator and professor in BCS and the Department of Biology, leads a team studying new methods for high-speed monitoring of sensory-driven synaptic activity across all inputs to single living neurons in the context of the intact cerebral cortex. Her co-principal investigator is Peter So, professor of mechanical and biological engineering, and director of the MIT Laser Biomedical Research Center.

Ian Wickersham will lead a team looking at novel technologies for nontoxic transsynaptic tracing. His co-principal investigators include Robert Desimone, director of the McGovern Institute and the Doris and Don Berkey Professor of Neuroscience in BCS; Li-Huei Tsai, director of the Picower Institute and the Picower Professor of Neuroscience in BCS; and Kay Tye, Picower Institute principal investigator and assistant professor of neuroscience in BCS.

Robert Desimone will lead a team studying vascular interfaces for brain imaging and stimulation. Co-principal investigators include Ed Boyden, associate professor at the MIT Media Lab, McGovern Institute, and departments of BCS and Biological Engineering; head of MIT’s Synthetic Neurobiology Group, and co-director of MIT’s Center for Neurobiological Engineering; and Elazer Edelman, the Thomas D. and Virginia W. Cabot Professor of Health Sciences and Technology in IMES and director of the Harvard-MIT Biomedical Engineering Center. Collaborators on this project include: Rodolfo Llinas (New York University), George Church (Harvard University), Jan Rabaey (University of California at Berkeley), Pablo Blinder (Tel Aviv University), Eric Leuthardt (Washington University/St. Louis), Michel Maharbiz (Berkeley), Jose Carmena (Berkeley), Elad Alon (Berkeley), Colin Derdeyn (Washington University in St. Louis), Lowell Wood (Bill and Melinda Gates Foundation), Xue Han (Boston University), and Adam Marblestone (MIT).

Ed Boyden will be co-principal investigator with Mark Bathe, associate professor of biological engineering, and Peng Yin of Harvard on a project to study ultra-multiplexed nanoscale in situ proteomics for understanding synapse types.

Alan Jasanoff, associate professor of biological engineering and director of the MIT Center for Neurobiological Engineering, will lead a team looking at calcium sensors for molecular fMRI. Stephen Lippard, the Arthur Amos Noyes Professor of Chemistry, is co-principal investigator.

In addition, Sur and Wickersham also received BRAIN Early Concept Grants for Exploratory Research (EAGER) from the National Science Foundation (NSF). Sur will focus on massive-scale multi-area single neuron recordings to reveal circuits underlying short-term memory. Wickersham, in collaboration with Li-Huei Tsai, Kay Tye, and Robert Desimone, will develop cell-type specific optogenetics in wild-type animals. Additional information about NSF support of the BRAIN initiative can be found at NSF.gov/brain.

The BRAIN Initiative, spearheaded by President Obama in April 2013, challenges the nation’s leading scientists to advance our sophisticated understanding of the human mind and discover new ways to treat, prevent, and cure neurological disorders like Alzheimer’s, schizophrenia, autism, and traumatic brain injury. The scientific community is charged with accelerating the invention of cutting-edge technologies that can produce dynamic images of complex neural circuits and illuminate the interaction of lightning-fast brain cells. The new capabilities are expected to provide greater insights into how brain functionality is linked to behavior, learning, memory, and the underlying mechanisms of debilitating disease. BRAIN was launched with approximately $100 million in initial investments from the NIH, the National Science Foundation, and the Defense Advanced Research Projects Agency (DARPA).

BRAIN Initiative scientists are engaged in a challenging and transformative endeavor to explore how our minds instantaneously processes, store, and retrieve vast quantities of information. Their discoveries will unlock many of the remaining mysteries inherent in the brain’s billions of neurons and trillions of connections, leading to a deeper understanding of the underlying causes of many neurological and psychiatric conditions. Their findings will enable scientists and doctors to develop the groundbreaking arsenal of tools and technologies required to more effectively treat those suffering from these devastating disorders.