Research Area: Cellular & Molecular Neuroscience

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

Divide and conquer

Anne Trafton |

Cell populations are remarkably diverse—even within the same tissue or cell type. Each cell, no matter how similar it appears to its neighbor, behaves and responds to its environment in its own way depending on which of its genes are expressed and to what degree. How genes are expressed in each cell—how RNA is “read” and turned into proteins—determines what jobs the cell performs in the body.

Traditionally, researchers have taken an en masse approach to studying gene expression, extracting an averaged measurement derived from an entire cell population. But over the past few years, single cell sequencing has emerged as a transformative tool, enabling scientists to look at gene expression within cells at an unprecedented resolution. With single-cell technologies, researchers have been able to examine the heterogeneity within cell populations; identify rare cells; observe interactions between diverse cell types; and better understand how these interactions influence health and disease.

This week in Science, researchers from the labs of Broad core institute members Aviv Regev and Feng Zhang, of MIT and MIT’s McGovern Institute respectively, report on their newest contribution to this field: Div-Seq, a method that enables the study of previously intractable and rare cell types in the brain. The study’s first authors, Naomi Habib, a postdoctoral fellow in the Regev and Zhang labs, and Yinqing Li, also a postdoc in the Zhang lab, sat down to answer questions about this groundbreaking approach.

Why is it so important to study neurons at the single cell level?

Li: Neuropsychiatric diseases are often too complex to find an effective treatment, partly because the neurons, that underly the disease are heterogeneous. Only when we have a full atlas of every neuron type at single-cell resolution—and figure out which ones are the cause of the pathology—can we develop a targeted and effective therapy. With this goal in mind, we developed sNuc-Seq and Div-Seq to make it technologically possible to profile neurons from the adult brain at significantly improved resolution, fidelity, and sensitivity.

Scientifically, what was the need that you were trying to address when you started this study?

Habib: Going into this study we were specifically interested in studying so-called “newborn” neurons, which are rare and hard to find. We think of our brain as being non-regenerative, but in fact there are rare, neuronal stem cells in specific areas of the brain that divide and create new neurons throughout our lives. We wanted to understand how gene expression changed as these cells developed. Typically when people studied gene expression in the brain they just mashed up tissue and took average measurements from that mixture. Such “bulk” measurements are hard to interpret and we lose the gene expression signals that come from individual cell types.

When I joined the Zhang and Regev labs, some of the first single cell papers were coming out, and it seemed like the perfect approach for advancing the way we do neuroscience research; we could measure RNA at the single cell level and really understand what different cell types were there, including rare cells, and what they contribute to different brain functions. But there was a problem. Neurons do not look like regular cells: they are intricately connected. In the process of separating them, the cells do not stay intact and their RNA gets damaged, and this problem increases with age.

So what was your solution?

Habib: Isolating single neurons is problematic, but the nucleus is nice and round and relatively easy to isolate. That led us to ask, “Why not try single nucleus RNA sequencing instead of single cell sequencing?” We called it “sNuc-Seq.”

It worked well. We get a lot of information from the RNA in the nucleus; we can learn what cell type we’re looking at, what state of development it’s in, and what kind of processes are going on in the cell—all of the key information we would want to get from RNA sequencing.

Then, to make it possible to find the rare newborn neurons, we developed Div-Seq. It’s based on sNuc-Seq, but we introduce a compound that incorporates into DNA and labels the DNA while it’s replicating, so it’s specific for newly divided cells. Because we already isolated the nuclei, it’s fairly simple from there to fluorescently tag the labeled cells, sort them, and get RNA for sequencing.

You tested this method while preparing your paper. What did you find?

Habib: We studied “newborn” neurons from the brain across multiple time-points. We could see the changes in gene expression that occur throughout adult neurogenesis; the cells transition from state-to-state—from stem cells to mature neurons—and during these transitions, we found a coordinated change in the expression of hundreds of genes. It was beautiful to see these signatures, and they enabled us to pinpoint regulatory genes expressed during specific points of the cell differentiation process.

We were also able to look at where regeneration occurs. We decided to look in the spinal cord because there is a lot of interest in understanding the potential of regeneration to help with spinal cord injury. Div-Seq enabled us to scan millions of neurons and isolate the small percentage that were dividing and characterize each by its RNA signature. We found that within the spinal cord there is ongoing regeneration of a specific type of neuron—GABAergic neurons. That was an exciting finding that also showed the utility of our method.

Are the data you get from this method compatible with data from previous single-cell techniques?

Li: Because this method is specifically designed to address the particular challenges of profiling neurons, the data from this method is distinct from that obtained from previous single-cell techniques. Since the data was new to this approach, a novel computational tool was developed in this project in order to fully reveal the rich information, which is now available to the scientific community.

Are there other benefits of using this method?

Habib: Single nucleus RNA-seq enables the study of the adult and aging brain at the single cell level, which is now being applied to study cellular diversity across the brain during health and disease. Our approach also makes it easier to explore any complex tissue where single cells are hard to obtain for technical reasons. One important aspect is that it works on frozen and fixed tissue, which opens up opportunities to study human samples, such as biopsies, that may be collected overseas or frozen for days or even years.

Additionally, Div-Seq opens new ways to look at the rare process of adult neurogenesis and other regenerative processes that might have been challenging before. Because Div- Seq specifically labels dividing cells, it is a great tool to use to see what cells are dividing in a given tissue and to track gene expression changes over time.

What is the endgame of studying these processes? Can you put this work in context of human health and disease?

Li: We hope that the methods in this study will provide a starting point and method for future work on neuropsychiatric diseases. As we expand our understanding of cell types and their signatures, we can start to ask questions like: Which cells express disease associated genes? Where are these cells located in the brain? What other genes are expressed in these cells, and which might serve as potential drug targets? This approach could help bridge human genetic association studies and molecular neurobiology and open new windows into disease pathology and potential treatments.

Habib: These two methods together enable many applications, which were either very hard or impossible to do before. For example, we characterized the cellular diversity of a region of the brain important for learning and memory—the first region affected in Alzheimer’s disease. Having that understanding—knowing what the normal state of cells is at the molecular level and what went wrong in each individual cell type—can advance our understanding of the disease and perhaps aid in the search for a treatment. We are also excited by the prospect of finding naturally-occurring regeneration in the brain and spine, which could have implications for the field of regenerative medicine in treating, for example, neuronal degeneration or spinal injury.

Paper cited:

Habib N, Li Y, et al. Div-Seq: Single nucleus RNA-Seq reveals dynamics of rare adult newborn neurons. Science. Online July 28, 2016.

Seeing RNA at the nanoscale

Anne Trafton |

Cells contain thousands of messenger RNA molecules, which carry copies of DNA’s genetic instructions to the rest of the cell. MIT engineers have now developed a way to visualize these molecules in higher resolution than previously possible in intact tissues, allowing researchers to precisely map the location of RNA throughout cells.

Key to the new technique is expanding the tissue before imaging it. By making the sample physically larger, it can be imaged with very high resolution using ordinary microscopes commonly found in research labs.

“Now we can image RNA with great spatial precision, thanks to the expansion process, and we also can do it more easily in large intact tissues,” says Ed Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT, a member of MIT’s Media Lab and McGovern Institute for Brain Research, and the senior author of a paper describing the technique in the July 4 issue of Nature Methods.

Studying the distribution of RNA inside cells could help scientists learn more about how cells control their gene expression and could also allow them to investigate diseases thought to be caused by failure of RNA to move to the correct location.

Boyden and colleagues first described the underlying technique, known as expansion microscopy (ExM), last year, when they used it to image proteins inside large samples of brain tissue. In a paper appearing in Nature Biotechnology on July 4, the MIT team has now presented a new version of the technology that employs off-the-shelf chemicals, making it easier for researchers to use.

MIT graduate students Fei Chen and Asmamaw Wassie are the lead authors of the Nature Methods paper, and Chen and graduate student Paul Tillberg are the lead authors of the Nature Biotechnology paper.

A simpler process

The original expansion microscopy technique is based on embedding tissue samples in a polymer that swells when water is added. This tissue enlargement allows researchers to obtain images with a resolution of around 70 nanometers, which was previously possible only with very specialized and expensive microscopes.

However, that method posed some challenges because it requires generating a complicated chemical tag consisting of an antibody that targets a specific protein, linked to both a fluorescent dye and a chemical anchor that attaches the whole complex to a highly absorbent polymer known as polyacrylate. Once the targets are labeled, the researchers break down the proteins that hold the tissue sample together, allowing it to expand uniformly as the polyacrylate gel swells.

In their new studies, to eliminate the need for custom-designed labels, the researchers used a different molecule to anchor the targets to the gel before digestion. This molecule, which the researchers dubbed AcX, is commercially available and therefore makes the process much simpler.

AcX can be modified to anchor either proteins or RNA to the gel. In the Nature Biotechnology study, the researchers used it to anchor proteins, and they also showed that the technique works on tissue that has been previously labeled with either fluorescent antibodies or proteins such as green fluorescent protein (GFP).

“This lets you use completely off-the-shelf parts, which means that it can integrate very easily into existing workflows,” Tillberg says. “We think that it’s going to lower the barrier significantly for people to use the technique compared to the original ExM.”

Using this approach, it takes about an hour to scan a piece of tissue 500 by 500 by 200 microns, using a light sheet fluorescence microscope. The researchers showed that this technique works for many types of tissues, including brain, pancreas, lung, and spleen.

Imaging RNA

In the Nature Methods paper, the researchers used the same kind of anchoring molecule but modified it to target RNA instead. All of the RNAs in the sample are anchored to the gel, so they stay in their original locations throughout the digestion and expansion process.

After the tissue is expanded, the researchers label specific RNA molecules using a process known as fluorescence in situ hybridization (FISH), which was originally developed in the early 1980s and is widely used. This allows researchers to visualize the location of specific RNA molecules at high resolution, in three dimensions, in large tissue samples.

This enhanced spatial precision could allow scientists to explore many questions about how RNA contributes to cellular function. For example, a longstanding question in neuroscience is how neurons rapidly change the strength of their connections to store new memories or skills. One hypothesis is that RNA molecules encoding proteins necessary for plasticity are stored in cell compartments close to the synapses, poised to be translated into proteins when needed.

With the new system, it should be possible to determine exactly which RNA molecules are located near the synapses, waiting to be translated.
“People have found hundreds of these locally translated RNAs, but it’s hard to know where exactly they are and what they’re doing,” Chen says. “This technique would be useful to study that.”

Boyden’s lab is also interested in using this technology to trace the connections between neurons and to classify different subtypes of neurons based on which genes they are expressing.

The research was funded by the Open Philanthropy Project, the New York Stem Cell Foundation Robertson Award, the National Institutes of Health, the National Science Foundation, and Jeremy and Joyce Wertheimer.

From cancer to brain research: learning from worms

by Elizabeth Dougherty |

In Bob Horvitz’s lab, students watch tiny worms as they wriggle under the microscope. Their tracks twist and turn in every direction, and to a casual observer the movements appear random. There is a pattern, however, and the animals’ movements change depending on their environment and recent experiences.

“A hungry worm is different from a well-fed worm,” says Horvitz, David H. Koch Professor of Biology and a McGovern Investigator. “If you consider worm psychology, it seems that the thing in life worms care most about is food.”

Horvitz’s work with the nematode worm Caenorhabditis elegans extends back to the mid-1970s. He was among the first to recognize the value of this microscopic organism as a model species for asking fundamental questions about biology and human disease.

The leap from worm to human might seem great and perilous, but in fact they share many fundamental biological mechanisms, one of which is programmed cell death, also known as apoptosis. Horvitz shared the Nobel Prize in Physiology or Medicine in 2002 for his studies of cell death, which is central to a wide variety of human diseases, including cancer and neurodegenerative disorders. He has continued to study the worm ever since, contributing to many areas of biology but with a particular emphasis on the nervous system and the control of behavior.

In a recently published study, the Horvitz lab has found another fundamental mechanism that likely is shared with mice and humans. The discovery began with an observation by former graduate student Beth Sawin as she watched worms searching for food. When a hungry worm detects a food source, it slows almost to a standstill, allowing it to remain close to the food.
Postdoctoral scientist Nick Paquin analyzed how a mutation in a gene called vps-50, causes worms to slow similarly even when they are well fed. It seemed that these mutant worms were failing to transition normally between the hungry and the well-fed state.

Paquin decided to study the gene further, in worms and also in mouse neurons, the latter in collaboration with Yasunobu Murata, a former research scientist in Martha Constantine-Paton’s lab at the McGovern Institute. The team, later joined by postdoctoral fellow Fernando Bustos in the Constantine-Paton lab, found that the VPS-50 protein controls the activity of synapses, the junctions between nerve cells. VPS-50 is involved in a process that acidifies synaptic vesicles, microscopic bubbles filled with neurotransmitters that are released from nerve terminals, sending signals to other nearby neurons.

If VPS-50 is missing, the vesicles do not mature properly and the signaling from neurons is abnormal. VPS-50 has remained relatively unchanged during evolution, and the mouse version can
substitute for the missing worm gene, indicating the worm and mouse proteins are similar not only in sequence but also in function. This might seem surprising given the wide gap between the tiny nervous system of the worm and the complex brains of mammals. But it is not surprising to Horvitz, who has committed about half of his lab resources to studying the worm’s nervous system and behavior.

“Our finding underscores something that I think is crucially important,” he says. “A lot of biology is conserved among organisms that appear superficially very different, which means that the
understanding and treatment of human diseases can be advanced by studies of simple organisms like worms.”

Human connections

In addition to its significance for normal synaptic function, the vps-50 gene might be important in autism spectrum disorder. Several autism patients have been described with deletions that include vps-50, and other lines of evidence also suggest a link to autism. “We think this is going to be a very important molecule in mammals,” says Constantine-Paton. “We’re now in a position to look into the function of vps-50 more deeply.”

Horvitz and Constantine-Paton are married, and they had chatted about vps-50 long before her lab began to study it. When it became clear that the mutation was affecting worm neurons in a novel way, it was a natural decision to collaborate and study the gene in mice. They are currently working to understand the role of VPS-50 in mammalian brain function, and to explore further the possible link to autism.

The day the worm turned

A latecomer to biology, Horvitz studied mathematics and economics as an undergraduate at MIT in the mid-1960s. During his last year, he took a few biology classes and then went on to earn
a doctoral degree in the field at Harvard University, working in the lab of James Watson (of double helix fame) and Walter Gilbert. In 1974, Horvitz moved to Cambridge, England, where he worked with Sydney Brenner and began his studies of the worm.

“Remarkably, all of my advisors, even my undergraduate advisor in economics here at MIT, Bob Solow, now have Nobel Prizes,” he notes.

The comment is matter-of-fact, and Horvitz is anything but pretentious. He thinks about both big questions and small experimental details and is always on the lookout for links between the
worm and human health.

“When someone in the lab finds something new, Bob is quick to ask if it relates to human disease,” says former graduate student Nikhil Bhatla. “We’re not thinking about that. We’re deep in
the nitty-gritty, but he’s directing us to potential collaborators who might help us make that link.”

This kind of mentoring, says Horvitz, has been his primary role since he joined the MIT faculty in 1978. He has trained many of the current leaders in the worm field, including Gary Ruvkun
and Victor Ambros, who shared the 2008 Lasker Award, Michael Hengartner, now President of the University of Zurich, and Cori Bargmann, who recently won the McGovern’s 2016 Scolnick Prize in Neuroscience.

“If the science we’ve done has been successful, it’s because I’ve been lucky to have outstanding young researchers as colleagues,” Horvitz says.

Before becoming a mentor, Horvitz had to become a scientist himself. At Harvard, he studied bacterial viruses and learned that even the simplest organisms could provide valuable insights about fundamental biological processes.

The move to Brenner’s lab in Cambridge was a natural step. A pioneer in the field of molecular biology, Brenner was also the driving force behind the adoption of C. elegans as a genetic model organism, which he advocated for its simplicity (adults have fewer than 1000 cells, and only 302 neurons) and short generation time (only three days). Working in Brenner’s lab, Horvitz
and his collaborator John Sulston traced the lineage of every body cell from fertilization to adulthood, showing that the sequence of cell divisions was the same in each individual animal. Their landmark study provided a foundation for the entire field. “They know all the cells in the worm. Every single one,” says Constantine-Paton. “So when they make a mutation and something is weird, they can determine precisely which cell or set of cells are affected. We can only dream of having such an understanding of a mammal.”

It is now known that the worm has about 20,000 genes, many of which are conserved in mammals including humans. In fact, in many cases, a cloned human gene can stand in for a missing
worm gene, as is the case for vps-50. As a result, the worm has been a powerful discovery machine for human biology. In the early years, though, many doubted whether worms would be relevant. Horvitz persisted undeterred, and in 1992 his conviction paid off, with the discovery of ced-9, a worm gene that regulates programmed cell death. A graduate student in Horvitz’ lab cloned ced-9 and saw that it resembled a human cancer gene called Bcl-2. They also showed that human Bcl-2 could substitute for a mutant ced-9 gene in the worm and concluded that the two genes have similar functions: ced-9 in worms protects healthy cells from death, and Bcl-2 in cancer patients protects cancerous cells from death, allowing them to multiply. “This was the moment we knew that the studies we’d been doing with C. elegans were going to be relevant to understanding human biology and disease,” says Horvitz.

Ten years later, in 2002, he was in the French Alps with Constantine-Paton and their daughter Alex attending a wedding, when they heard the news on the radio: He’d won a Nobel Prize, along with Brenner and Sulston. On the return trip, Alex, then 9 years old but never shy, asked for first-class upgrades at the airport; the agent compromised and gave them all upgrades to business class instead.

Discovery machine at work

Since the Nobel Prize, Horvitz has studied the nervous system using the same strategy that had been so successful in deciphering the mechanism of programmed cell death. His approach, he says, begins with traditional genetics. Researchers expose worms to mutagens and observe their behavior. When they see an interesting change, they identify the mutation and try to link the gene to the nervous system to understand how it affects behavior.

“We make no assumptions,” he says. “We let the animal tell us the answer.”

While Horvitz continues to demonstrate that basic research using simple organisms produces invaluable insights about human biology and health, there are other forces at work in his lab. Horvitz maintains a sense of wonder about life and is undaunted by big questions.

For instance, when Bhatla came to him wanting to look for evidence of consciousness in worms, Horvitz blinked but didn’t say no. The science Bhatla proposed was novel, and the question
was intriguing. Bhatla pursued it. But, he says, “It didn’t work.”

So Bhatla went back to the drawing board. During his earlier experiments, he had observed that worms would avoid light, a previously known behavior. But he also noticed that they immediately stopped feeding. The animals had provided a clue. Bhatla went on to discover that worms respond to light by producing hydrogen peroxide, which activates a taste receptor.

In a sense, worms taste light, a wonder of biology no one could have predicted.

Some years ago, the Horvitz lab made t-shirts displaying a quote from the philosopher Friedrich Nietzsche: “You have made your way from worm to man, and much within you is still worm.”
The words have become an informal lab motto, “truer than Nietzsche could everhave imagined,” says Horvitz. “There’s still so much mystery, particularly about the brain, and we are still learning from the worm.”

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.