How a single gene contributes to autism and schizophrenia

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.

How we make emotional decisions

Some decisions arouse far more anxiety than others. Among the most anxiety-provoking are those that involve options with both positive and negative elements, such choosing to take a higher-paying job in a city far from family and friends, versus choosing to stay put with less pay.

MIT researchers have now identified a neural circuit that appears to underlie decision-making in this type of situation, which is known as approach-avoidance conflict. The findings could help researchers to discover new ways to treat psychiatric disorders that feature impaired decision-making, such as depression, schizophrenia, and borderline personality disorder.

“In order to create a treatment for these types of disorders, we need to understand how the decision-making process is working,” says Alexander Friedman, a research scientist at MIT’s McGovern Institute for Brain Research and the lead author of a paper describing the findings in the May 28 issue of Cell.

Friedman and colleagues also demonstrated the first step toward developing possible therapies for these disorders: By manipulating this circuit in rodents, they were able to transform a preference for lower-risk, lower-payoff choices to a preference for bigger payoffs despite their bigger costs.

The paper’s senior author is Ann Graybiel, an MIT Institute Professor and member of the McGovern Institute. Other authors are postdoc Daigo Homma, research scientists Leif Gibb and Ken-ichi Amemori, undergraduates Samuel Rubin and Adam Hood, and technical assistant Michael Riad.

Making hard choices

The new study grew out of an effort to figure out the role of striosomes — clusters of cells distributed through the the striatum, a large brain region involved in coordinating movement and emotion and implicated in some human disorders. Graybiel discovered striosomes many years ago, but their function had remained mysterious, in part because they are so small and deep within the brain that it is difficult to image them with functional magnetic resonance imaging (fMRI).

Previous studies from Graybiel’s lab identified regions of the brain’s prefrontal cortex that project to striosomes. These regions have been implicated in processing emotions, so the researchers suspected that this circuit might also be related to emotion.

To test this idea, the researchers studied mice as they performed five different types of behavioral tasks, including an approach-avoidance scenario. In that situation, rats running a maze had to choose between one option that included strong chocolate, which they like, and bright light, which they don’t, and an option with dimmer light but weaker chocolate.

When humans are forced to make these kinds of cost-benefit decisions, they usually experience anxiety, which influences the choices they make. “This type of task is potentially very relevant to anxiety disorders,” Gibb says. “If we could learn more about this circuitry, maybe we could help people with those disorders.”

The researchers also tested rats in four other scenarios in which the choices were easier and less fraught with anxiety.

“By comparing performance in these five tasks, we could look at cost-benefit decision-making versus other types of decision-making, allowing us to reach the conclusion that cost-benefit decision-making is unique,” Friedman says.

Using optogenetics, which allowed them to turn cortical input to the striosomes on or off by shining light on the cortical cells, the researchers found that the circuit connecting the cortex to the striosomes plays a causal role in influencing decisions in the approach-avoidance task, but none at all in other types of decision-making.

When the researchers shut off input to the striosomes from the cortex, they found that the rats began choosing the high-risk, high-reward option as much as 20 percent more often than they had previously chosen it. If the researchers stimulated input to the striosomes, the rats began choosing the high-cost, high-reward option less often.

Paul Glimcher, a professor of physiology and neuroscience at New York University, describes the study as a “masterpiece” and says he is particularly impressed by the use of a new technology, optogenetics, to solve a longstanding mystery. The study also opens up the possibility of studying striosome function in other types of decision-making, he adds.

“This cracks the 20-year puzzle that [Graybiel] wrote — what do the striosomes do?” says Glimcher, who was not part of the research team. “In 10 years we will have a much more complete picture, of which this paper is the foundational stone. She has demonstrated that we can answer this question, and answered it in one area. A lot of labs will now take this up and resolve it in other areas.”

Emotional gatekeeper

The findings suggest that the striatum, and the striosomes in particular, may act as a gatekeeper that absorbs sensory and emotional information coming from the cortex and integrates it to produce a decision on how to react, the researchers say.

That gatekeeper circuit also appears to include a part of the midbrain called the substantia nigra, which has dopamine-containing cells that play an important role in motivation and movement. The researchers believe that when activated by input from the striosomes, these substantia nigra cells produce a long-term effect on an animal or human patient’s decision-making attitudes.

“We would so like to find a way to use these findings to relieve anxiety disorder, and other disorders in which mood and emotion are affected,” Graybiel says. “That kind of work has a real priority to it.”

In addition to pursuing possible treatments for anxiety disorders, the researchers are now trying to better understand the role of the dopamine-containing substantia nigra cells in this circuit, which plays a critical role in Parkinson’s disease and may also be involved in related disorders.

The research was funded by the National Institute of Mental Health, the CHDI Foundation, the Defense Advanced Research Projects Agency, the U.S. Army Research Office, the Bachmann-Strauss Dystonia and Parkinson Foundation, and the William N. and Bernice E. Bumpus Foundation.

From genes to brains

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.”

Genome Editing with CRISPR – Cas9

This animation depicts the CRISPR-Cas9 method for genome editing – a powerful new technology with many applications in biomedical research, including the potential to treat human genetic disease. Feng Zhang, a leader in the development of this technology, is a faculty member at MIT, an investigator at the McGovern Institute for Brain Research, and a core member of the Broad Institute.


Calcium reveals connections between neurons

A team led by MIT neuroscientists has developed a way to monitor how brain cells coordinate with each other to control specific behaviors, such as initiating movement or detecting an odor.

The researchers’ new imaging technique, based on the detection of calcium ions in neurons, could help them map the brain circuits that perform such functions. It could also provide new insights into the origins of autism, obsessive-compulsive disorder and other psychiatric diseases, says Guoping Feng, senior author of a paper appearing in the Oct. 18 issue of the journal Neuron.

“To understand psychiatric disorders we need to study animal models, and to find out what’s happening in the brain when the animal is behaving abnormally,” says Feng, the James W. and Patricia Poitras Professor of Neuroscience and a member of the McGovern Institute for Brain Research at MIT. “This is a very powerful tool that will really help us understand animal models of these diseases and study how the brain functions normally and in a diseased state.”

The lead author of the Neuron paper is McGovern Institute postdoc Qian Chen.

Performing any kind of brain function requires many neurons in different parts of the brain to communicate with each other. They achieve this communication by sending electrical signals, triggering an influx of calcium ions into active cells. Using dyes that bind to calcium, researchers have imaged neural activity in neurons. However, the brain contains thousands of cell types, each with distinct functions, and the dye is taken up nonselectively by all cells, making it impossible to pinpoint calcium in specific cell types with this approach.

To overcome this, the MIT-led team created a calcium-imaging system that can be targeted to specific cell types, using a type of green fluorescent protein (GFP). Junichi Nakai of Saitama University in Japan first developed a GFP that is activated when it binds to calcium, and one of the Neuron paper authors, Loren Looger of the Howard Hughes Medical Institute, modified the protein so its signal is strong enough to use in living animals.

The MIT researchers then genetically engineered mice to express this protein in a type of neuron known as pyramidal cells, by pairing the gene with a regulatory DNA sequence that is only active in those cells. Using two-photon microscopy to image the cells at high speed and high resolution, the researchers can identify pyramidal cells that are active when the brain is performing a specific task or responding to a certain stimulus.

In this study, the team was able to pinpoint cells in the somatosensory cortex that are activated when a mouse’s whiskers are touched, and olfactory cells that respond to certain aromas.

This system could be used to study brain activity during many types of behavior, including long-term phenomena such as learning, says Matt Wachowiak, an associate professor of physiology at the University of Utah. “These mouse lines should be really useful to many different research groups who want to measure activity in different parts of the brain,” says Wachowiak, who was not involved in this research.

The researchers are now developing mice that express the calcium-sensitive proteins and also exhibit symptoms of autistic behavior and obsessive-compulsive disorder. Using these mice, the researchers plan to look for neuron firing patterns that differ from those of normal mice. This could help identify exactly what goes wrong at the cellular level, offering mechanistic insights into those diseases.

“Right now, we only know that defects in neuron-neuron communications play a key role in psychiatric disorders. We do not know the exact nature of the defects and the specific cell types involved,” Feng says. “If we knew what cell types are abnormal, we could find ways to correct abnormal firing patterns.”

The researchers also plan to combine their imaging technology with optogenetics, which enables them to use light to turn specific classes of neurons on or off. By activating specific cells and then observing the response in target cells, they will be able to precisely map brain circuits.

The research was funded by the Poitras Center for Affective Disorders Research, the National Institutes of Health and the McNair Foundation