Pathway analysis: A receptor that regulates signaling at synapses, the junctions between neurons, has abnormal protein interactions in mice that model fragile X syndrome.

Researchers have identified a new mechanism that may underlie a runaway cell-signaling pathway in fragile X syndrome. The results were published 22 January in Nature Neuroscience¹.

Fragile X syndrome is caused by a mutation that inactivates the fragile X mental retardation protein, or FMRP, which regulates gene expression by stalling the machinery that translates genetic messages into proteins. Because FMRP silences many genes that are activated by metabotropic glutamate receptors, or mGluRs, researchers are developing fragile X syndrome treatments that block mGluRs.

However, studies also suggest that mGluR activity may be enhanced in individuals with fragile X syndrome through a separate mechanism involving FMRP that is unrelated to its role in regulating gene expression.

In the new study, researchers show that in male mice lacking FMR1, the fragile X gene, mGluR binds preferentially to an altered version of a scaffolding protein, called homer1a (H1A). H1A is a short version of the homer1 protein that permanently activates mGluR signaling. 

Homer proteins connect mGluRs with other cell-signaling molecules at the synapse, or junction between neurons, most notably the SHANK3 protein. A SHANK3 mutant disrupts the protein’s ability to bind to homer proteins, leading to autism symptoms in mice.

Deleting the H1A gene in fragile X mice restores mGluR binding to the longer homer proteins and normalizes many of the consequences of overactive mGluR5 signaling, the study found. These include excess protein synthesis in the brain.

Mice that lack both H1A and FMR1 also have fewer seizures and exhibit more typical anxiety levels than FMR1 mutants alone, which tend to be less anxious than controls, the study found.

Blocking the interaction between the H1A protein and mGluR also restores mGluR signaling in fragile X mice to more typical levels.

However, other features of fragile X syndrome are not recovered when H1A activity is inhibited, the study found. These include enhanced long-term depression, which is a dampening of signals at the junctions between neurons after they fire.

This suggests that H1A binding to mGluRs only partially accounts for the role the receptor plays in fragile X syndrome.

However, compounds that modulate the interaction between mGluR and H1A may still be potential therapeutics for fragile X syndrome, the researchers say.

References:

1: Ronesi J.A. et al. Nat. Neurosci. Epub ahead of print (2012) PubMed

Time span: Researchers used 108 postmortem brains to map how a form of gene regulation changes from before birth to old age.

Researchers have charted patterns of DNA methylation — a chemical alteration to DNA that modifies gene expression — in the planning center of the brain from before birth to old age. The results were published 10 February in The American Journal of Human Genetics¹ and are also available in a searchable online database.

Results show that several genes switch their methylation status as the brain matures. These include the autism-associated gene neurexin-1, which functions at synapses, the junctions between neurons, and a number of genes that have been linked to schizophrenia. Altered methylation during crucial developmental stages could contribute to neurological disorders, the researchers say.

DNA methylation is one form of epigenetic regulation, which influences gene expression without changing the DNA sequence. Epigenetic modifications can be inherited, but also vary over an individual’s lifespan, often in response to experience.

In a study published last year, researchers show that several autism-associated genes are regulated through DNA methylation.

In the new study, researchers examined DNA methylation patterns in 108 postmortem brains ranging in age from fetal week 14 to more than 80 years. They analyzed more than 27,000 sites that are prone to methylation in the regulatory regions of about 14,500 genes in the prefrontal cortex, a brain region important for thinking, planning and social behavior.

The brains of fetuses, young children and individuals older than 10 years of age each have a distinct pattern of methylation, the study found. Fetal brains also show the greatest change over time, with the methylation marks varying at a rate of 80 percent per year in fetal tissue. However, these changes take place at fewer sites than those seen in the brains of young children and individuals older than 10: 865 sites compared with 5,506 and 10,578, respectively.

A number of these sites switch methylation states as brains mature from the fetal stage into childhood, with the majority gaining a new methyl mark, the study found.

Changes in methylation are not always followed by changes in gene expression, the study also found. And, in contrast to previous reports suggesting that methylation serves as a repressor, methylation can result in either increases or decreases in gene expression.

Using the online database, researchers can track the epigenetic modifications that underlie brain development, which may be awry in developmental disorders such as autism.

References:

1: Numata S. et al. Am. J. Hum. Genet. 90, 260-272 (2012) PubMed

A friend of mine who has a 3-year-old son with autism says that he could sing dozens of songs before he ever spoke a word of  command. That sharp contrast between language and music ability is not unusual in children with the disorder.

Though these children often have language difficulties, a significant number appear to have exceptional musical talent, such as perfect pitch.

Given that the brain regions that process speech and song overlap, what explains this disparity in autism? A new brain imaging study seeks to explore its cause and, in so doing, shed light on some of the roots of the disorder.

Researchers used functional magnetic resonance imaging (fMRI) to measure brain activity in 12 low-functioning children with autism with a mean age of 12, and 21 similarly aged controls as they listened to a parent speaking or their favorite song.

Activity in part of the brain called the left inferior frontal gyrus, which is involved in processing both music and speech, is weaker in participants with autism than in controls when they listen to speech, but stronger than in controls when they listen to music, the study found. 

Researchers also analyzed brain structure in these participants and in an additional 24 low-functioning people with autism, by using fMRI to explore the functional connections among brain regions, and a type of structural MRI known as diffusion tensor imaging.

One theory for the language deficits in autism is a problem with the long-range connections in the brain. In the new study, the researchers found some structural differences between the two groups in various parts of the brain, but conclude that problems with the brain’s long-range connections are unlikely to account for language deficits in the people with autism.

They suggest instead that changes in local connectivity or lower-level auditory processing may account for the differences in the way the brains of people with autism respond to speech and song. For example, speech requires discrimination of quickly changing sounds, whereas the fluctuations in music are slower and therefore may be easier to process.

The specific conclusions of this kind of structural study are up for debate, especially given recent research pointing to a potential flaw in brain imaging studies of children with developmental disabilities. But the findings provide support for incorporating music into behavioral therapy.

Previous research has shown that making music a part of therapy may help children with autism learn social skills and language, but more rigorous research is needed on the topic. It has also shown that children with the disorder process emotional cues from music, despite having difficulty understanding facial emotions.

Perhaps song will offer a way for lower-functioning children with autism to better learn to communicate.

Self destruction: Antibodies carried in the blood of mothers of children with autism (top), but not those of mothers of typically developing children (bottom) attack proteins in mouse brains.

The possibility that autoimmune mechanisms are a contributing factor in autism spectrum disorders has been entertained for decades, ever since early studies suggested that individuals with autism have a family history of autoimmune disease^1,2.

Much of the early data were acquired from a fairly small number of individuals with autism. However, a 2009 Danish study examined autoimmune disorders in more than 600,000 children born between 1993 and 2004, and found an association between autism and both rheumatoid arthritis and celiac disease¹. In fact, the study concluded that the risk of autism more than doubles for children who have a mother with one of these disorders.

The presence of autoantibodies, which are immune proteins that mistakenly attack the body’s own cells, in both diseases raises the possibility of a relationship between maternal autoantibodies and autism. In this model, maternal autoantibodies cross the placenta and enter the fetal brain, leading to alterations in its development. A variety of data published in the past few years provide evidence that such a model is biologically plausible.

The passage of maternal antibodies across the placenta is a well-known mechanism for fetal immune protection. Maternal antibodies reach all fetal tissues, even the brain. In adults, the entry of circulating soluble molecules and cells into brain tissue is limited by the blood-brain barrier, but in recent years it has become increasingly clear that the brain is less of an immune-privileged organ than it was previously considered to be. 

In the fetus, the blood-brain barrier is not fully formed, making the developing brain vulnerable to blood-borne substances. In fact, acquired changes or genetic impairments in cognition and behavior have been shown to be a consequence of circulating brain-specific antibodies that can alter function if they gain access to brain tissue³.

Case studies:

The children of patients with systemic lupus erythematosus (SLE) provide compelling data supporting this hypothesis. Congenital heart block, a type of arrhythmia, and skin rash are clearly transmissible to offspring by autoantibodies that are commonly present in mothers with SLE.

The children of mothers with SLE also have a high frequency of learning disorders⁴. This effect has been linked to autoantibodies in mice, but not in humans, however. Intriguingly, many anti-DNA antibodies that are characteristic of SLE cross-react with the N-methyl-D-aspartate receptor (NMDAR), which is involved in learning and memory⁵.

We have shown that pregnant female mice harboring DNA- and NMDAR-specific antibodies have pups with abnormal fetal brain development. When the pups are born, their reflexes don’t develop as quickly as those of controls and, as adults, they have selected impairments in cognitive tasks⁶.

Several investigators have identified the presence of antibodies that bind to human fetal brain tissue in a subset of women who have children with autism^7,8. When researchers gave these antibodies to pregnant mice and monkeys, they caused abnormal behavior in their offspring⁹.

In a 2003 study, researchers gave serum with anti-brain antibodies from mothers of children with autism to pregnant mice. The offspring had deficits in social behavior and motor skills, as well as cerebellar abnormalities¹⁰.

In a subsequent study, pregnant mice were given immunoglobulin antibodies isolated from the blood of mothers of children with autism. In this case, the offspring were more anxious during adolescence, had alterations in sociability and were more sensitive to noise than controls were¹¹.

Researchers have also administered similar antibodies to pregnant rhesus monkeys. The offspring had more social deficits, increased motor activity and increased repetitive behaviors compared with offspring born to mothers given immunoglobulin from mothers of typically developing children¹².

The results from these studies suggest that maternal antibodies targeting the brain can affect brain development in their offspring, resulting in altered cognition, behavior and motor skills.

Risk rate:

By studying families in the Simons Simplex Collection (SSC), we have confirmed that mothers of children with autism are five times more likely to have anti-brain antibodies than are members of a control group consisting of healthy women of childbearing age. The SSC is a database of genetic and clinical information from families that have one child with autism, and unaffected parents and siblings, funded by SFARI.org’s parent organization.

Anti-nuclear antibodies, which are directed against the cell’s nucleus, are characteristic of many autoimmune diseases. We found that they are elevated in the blood of mothers of children with autism who also carry anti-brain antibodies compared with those who do not have anti-brain antibodies. This is consistent with the theory that autoimmunity predisposes mothers to having brain-reactive antibodies and giving birth to children with autism.

Interestingly, mothers with rheumatoid arthritis are as likely to have anti-brain antibodies as are mothers of children with autism. We are investigating whether genetic variants that have been linked to rheumatic arthritis and celiac disease are present in mothers who have both anti-brain antibodies and a child with autism.

Confirming an immune mechanism for some proportion of autism cases may help identify at-risk pregnancies, by allowing pregnant women or women planning to become pregnant to be screened for harmful anti-brain antibodies. It could eventually lead to the development of drugs that block these antibodies, thereby preventing autism from developing in vulnerable offspring.

These observations suggest that genetic studies in autism should be integrated with investigations into environmental exposures, including the maternal immune repertoire, in order to fully understand the genetic susceptibility of autism. Studying the targets of harmful anti-brain antibodies may also provide insights into disease mechanisms and pathways — which is a top priority for our future studies.

Betty Diamond is head of the Center for Autoimmune and Musculoskeletal Disorders at The Feinstein Institute for Medical Research in Long Island, New York. Lior Brimberg is a postdoctoral fellow in her laboratory. Peter Gregersen is head of the Robert S. Boas Center for Genomics and Human Genetics at the Feinstein Institute.

References:

1: Atladóttir H.O. et al. Pediatrics 124, 687-694 (2009) PubMed

2: Keil A. et al. Epidemiology 21, 805-808 (2010) PubMed

3: Diamond B. et al. Nat. Rev. Immunol. 9, 449-456 (2009) PubMed

4: Lahita R.G. Psychoneuroendocrinology 13, 385-396 (1988) PubMed

5: DeGiorgio L.A. et al. Nat. Med. 7, 1189-1193 (2001) PubMed

6: Lee J.Y. et al. Nat. Med. 15, 91-96 (2009) PubMed

7: Croen L.A. et al.. Biol. Psychiatry 64, 583-588 (2008) PubMed

8: Singer H.S. et al. J. Neuroimmunol. 194, 165-172 (2008) PubMed

9: Enstrom A.M. et al. Curr. Opin. Investig. Drugs 10, 463-473 (2009) PubMed

10: Dalton P. et al. Ann. Neurol. 53, 533-537 (2003) PubMed

11: Singer H.S. et al. J. Neuroimmunol. 211, 39-48 (2009) PubMed

12: Martin L.A. et al. Brain Behav. Immun. 22, 806-816 (2008) PubMed

Climbing cases: An epidemiology study shows that the rates of autism rise linearly for children born between 1992 and 2003 based on their year of birth.

The likelihood of being diagnosed with autism has increased for children born each year since 1992, especially for individuals at the higher-functioning end of the autism spectrum, reports a study published 7 December in The International Journal of Epidemiology¹.

The results provide a lens through which to investigate theories about the rising prevalence of the disorder. Specifically, they suggest that greater social awareness of the disorder and changes to diagnostic standards are responsible for the increase, the researchers say.

Autism diagnoses have risen significantly in the past few years: For example, studies report a 634 percent increase in autism cases in California between 1987 and 2003. The question is: Why?

Epidemiological studies suggest that increased awareness of autism and changes to diagnostic criteria account for much of this trend. Others have proposed that environmental factors may be fueling the steep rise.

In the new study, researchers looked at more than six million individuals born in California between 1992 and 2003 to investigate the relative contribution of three factors to the likelihood of an autism diagnosis: the year they were born, the year they were diagnosed and the age at which they received a diagnosis.

The study found that individuals are most likely to be diagnosed with autism around age 3. Specifically, 3-year-olds in California are 37 times more likely to receive a diagnosis than 2-year-olds. This is consistent with the age at which symptoms of the disorder become obvious.

The study also found that the later an individual was born, the higher the likelihood that they would be diagnosed with autism. The yearly increase in autism rates is linear, with each subsequent birth year carrying with it a higher risk of autism than the previous one. For example, individuals born in 1999 are about four times more likely to have an autism diagnosis than those born in 1992, and people born in 2003 have almost 17 times the odds, the study found.

This trend is more pronounced for high-functioning individuals with autism than for those with greater social and language impairments. For example, high-functioning individuals with autism born in 2002 are almost 15 times more likely to have an autism diagnosis than those born in 1992. Lower-functioning individuals born the same year have only four times the odds.

The overall number of individuals with autism diagnosed in a given year does not rise as significantly, the study found. For example, there were only four times the number of autism diagnoses in 2003 compared with 1994. This finding suggests that the year in which someone was born has a bigger effect on the likelihood of receiving a diagnosis than the year in which they were diagnosed with the disorder.  

For a theory about the rise in autism prevalence to fit the new data, it has to explain the gradual increase in rates of diagnosis over time and the skew toward diagnosing high-functioning people with autism. Greater social awareness fits these data, the researchers say. However, because the rise in diagnosis is tied to an individual’s year of birth, and not the year of diagnosis, this theory only fits if parents become aware of the disorder when their child is a certain age.

Broadening of diagnostic criteria also matches the pronounced trend toward diagnosing more children at the high-functioning end of the spectrum, the researchers say.

On the other hand, environmental factors fit poorly with the data. For environmental factors to explain the change in prevalence, they would have to increase incrementally over that time period, which would increase the number of diagnoses across all ages.

References:

1: Keyes K.M. et al. Int. J. Epidemiol. (2011) Epub ahead of print PubMed

Building bridges: In 6-month-old babies who later develop autism, white matter bundles have more structural integrity than do those in infants who do not develop the disorder. 

The development of white matter tracts, the nerve bundles that join one brain region to another, is different in babies who go on to develop autism compared with those who do not, according to a new study.

Researchers scanned the brains of infant siblings of children with autism — who have an increased risk of developing the disorder themselves — several times during their first two years of life. The so-called ‘baby sibs’ who go on to receive a diagnosis of autism at 24 months of age have distinct brain patterns at 6 months and abnormal neural development from 6 to 24 months, according to the study. The results were published 17 February in the American Journal of Psychiatry¹.

“The story is that autism is an unfolding process, not something that happens in the third trimester and then is done,” says lead investigator Joseph Piven, professor of psychiatry at the University of North Carolina-Chapel Hill. “We see the brain changing over time in a dynamic way.”

It’s too early to speculate how these specific brain changes might ultimately lead to autism, the researchers say.

But the findings add to other published and unpublished studies identifying neural signatures of autism in 6-month-olds.

The new study “was music to my ears, because it superimposes on other infant-sib studies finding all kinds of brain signatures by 6 months,” says Charles Nelson, professor of pediatrics and neuroscience at Harvard Medical School, who was not involved in the new study. Nelson is using a different imaging technology to measure baby sibs’ brain waves. “It’s making us all wish that we’d started [scanning] even younger.”

These kinds of biomarkers, especially if paired with information from genetic and behavioral screens, could potentially identify children with autism long before symptoms appear, Piven says. Early detection would ideally lead to interventions that improve behaviors.

“It gives you this sense that you can jump in during this window when the brain is unfolding and maybe make a difference,” he says.

Abnormal structure:

Since 2007, Piven’s group and three other centers in the Infant Brain Imaging Study have been collecting longitudinal data on hundreds of baby sibs, including several types of brain scans, blood samples for future genetic studies and, beginning at 2 years old, standardized tests for autism.

The new study analyzed diffusion tensor imaging (DTI) scans from 92 of these infants, 28 of whom received an autism diagnosis at 24 months.

DTI measures the integrity of brain connections by tracking the movement of water molecules. In white matter, water tends to move along nerve fibers in one direction, whereas in gray matter, the tissue in which neuronal cell bodies reside, “water moves all over the place,” says Jason Wolff, a postdoctoral fellow in Piven’s lab.

This study examined 15 white matter tracts, each chosen because it had been previously linked to autism. In scans done at 6 months, 12 tracks were significantly different in the babies that would go on to develop autism than in those who would not.

More specifically, babies who would later develop autism had higher ‘fractional anisotropy,’ or FA, a measure of how strongly water moves in one direction. “A higher fractional anisotropy means the bundles are more developed and water moves more efficiently along them than across them,” Wolff says.

FA increases with age, and is typically thought to mark brain maturity². “You’d expect it to be lower in autism,” notes Kevin Pelphrey, director of the Yale Child Neuroscience Lab, who was not involved in the study.

Still, he points out that it’s hard to interpret what a high FA means at 6 months because white matter undergoes so many changes during early development. “Just showing that there’s a difference is important,” Pelphrey says. “What more FA or less FA means at this point, I’m not sure.”

For instance, a high FA in the autism group could mean that the millions of nerve fibers that make up the white matter bundles are not getting pruned appropriately at 6 months.

One major caveat in the new study is the lack of a control group of babies whose older siblings do not have autism. Pelphrey’s studies suggest that baby sibs have unusual brain signatures even if they don’t go on to develop autism.

Another important factor is time: The brain changes rapidly in the first few years of life. For example, the study found that at 24 months, toddlers with autism have lower FA values than those who do not. Piven plans to continue to scan the same group of children until age 3, because other baby-sib studies have shown that some children lose their autism diagnoses between age 2 and 3.

These abnormal trajectories of brain development only come to light when studies follow children over many years, Pelphrey notes. “That seems to be the story in autism, over and over again,” he says. “At this point, I think all imaging studies should be longitudinal or we shouldn’t be publishing them.”

References:

1: Wolff J.J. et al. Am. J. Psychiatry Epub ahead of print (2012) Abstract

2: Gao W. et al. AJNR Am. J. Neuroradiol. 30, 290-296 (2009) PubMed

University of Hertfordshire

Just over a year ago, I wrote about a handful of research groups creating robots that can engage children who have autism using speech, facial expressions or body movements. These 'social' bots ranged from a miniature dinosaur and a dancing yellow snowman to several sophisticated, full-size humanoids.

The eclectic collection now includes a boy that can sense when it's touched, a floating female head that expresses a wide range of emotions and a low-cost fuzzy penguin that can track a child's eye movements. Descriptions of all three appear in the latest conference proceedings of the IEEE Engineering in Medicine and Biology Society.

For many years, engineers at the University of Hertfordshire in the U.K. have been developing KASPAR (above), a child-size robot that can make simple facial expressions. For KASPAR's most recent iteration, the team has covered its body with sensors, dubbed 'RoboSKIN', that record when and where it's touched.

Because exploring the environment through touch is a key part of early motor and social development, KASPAR's new skin could add an extra dimension to play sessions with children who have autism. In the new report, the researchers observed preschoolers with autism interacting with KASPAR. They showed that the robot was well received, and that it usually responds appropriately to various kinds of physical interactions that involve touch — for example, by saying, “This is nice,” if its feet are tickled, or, “Ouch,” if its nose is pinched.

The second new bot, created by an Italian team, may help children with autism develop the facial recognition skills they often lack. FACE, or Facial Automation for Conveying Emotions, is an extremely realistic female face that can effortlessly turn from annoyed to alert or tense to contented.

In a typical therapy session with FACE, a child with autism sits in a room full of cameras and wears a specialized T-shirt to measure respiration rate, a finger band to record skin temperature and a baseball cap for eye tracking. A therapist can program the robot to role-play a particular social scenario, such as the bot and the child paying attention to the same object. Later, the therapist can review the collected data and monitor the child's progress.

G. Fischer

In contrast with KASPAR and FACE, which are used in schools or clinics and shared by many children, the third new bot aims to be cheap and portable, so a child could use it at home. The Penguin for Autism Behavioral Interventions, or PABI, can move its eyes, beak, head and wings and record a child’s body and head movements.

In the future, its creators at the Worcester Polytechnic Institute in Massachusetts hope that it will be able to respond to a child — by imitating speech or gestures, for instance — without the involvement of a parent or therapist. In another mode, a therapist, even one who is miles away, could theoretically control PABI’s movements with a joystick while it plays with the child.

Considering the wide range of benefits that robots have been shown to bring children with autism, I say bring on the singularity.

White noise: Children with autism who are hypersensitive to sound may have more severe behavioral problems than controls do. 

Brain imaging can detect acute sensitivity to sound in people with autism, according to a study published 25 January in Neurophysiology¹.

The results suggest that sound sensitivity originates in the auditory cortex, a brain region responsible for sound perception. 

In addition to the core features of autism — deficits in social skills, language impairments, and repetitive and restrictive behaviors — many people with the disorder have acute sensitivity to sensory stimuli, such as loud noises or bright lights. Because a fear of crowded, noisy spaces can lead to social isolation and calming stereotyped behaviors, these sensitivities might even underlie the core deficits of autism, some researchers say.

Despite its potential importance for tailoring interventions for children with the disorder, however, sensory sensitivity is understudied in autism.

In the new study, researchers used magnetoencephalography (MEG) — a non-invasive brain-imaging technique that can capture large groups of neurons working together — to investigate sound sensitivity in 18 boys with autism and 12 age-matched controls. MEG detects a neural response to sound both 50 and 100 milliseconds after a stimulus, called the M50 and the M100, primarily in the auditory cortex, an indicator of auditory perception.

Of the 18 participants with autism, 9 have acute sensitivity to sound as measured by the Sensory Profile, a parental questionnaire that gauges sensory sensitivity.

The results of the questionnaire also showed that individuals with autism who did not score above a hypersensitivity cutoff are still more sensitive to sound than are controls.

The researchers also observed differences in their MEGs. The children with autism and hypersensitivity have delayed M50 and M100 responses after hearing a noise transmitted directly into their ear canal compared with children with autism but no hypersensitivity. The latter group also has a delayed response compared with controls, however. The results suggest that delays in the M50 and M100 responses correlate with the severity of an individual’s sensitivity to sound, the researchers say.

Children with heightened sound sensitivity and autism also have greater ‘dipole moments’ on the M50 response — an indicator of the magnitude of neuron activity — than do controls, the study found. 

The children with autism and sound sensitivity do not have a more severe form of autism than those who are not hypersensitive to sound, based on the Autism Diagnostic Observation Schedule, or ADOS, and the Autism Screening Questionnaire, the study found. However, the greater their sensitivity to sound, the more severe their behavioral problems are, as measured by the Child Behavior Checklist, a parental questionnaire focused on behavioral and emotional problems.

MEG could serve as an objective method to diagnose sound sensitivity, which in turn could lead to tailored behavioral therapies for children with autism, the researchers say.

References:

1: Matsuzaki J. et al. Neuroreport 23, 113-118 (2012) PubMed

Growing up: Using the right combination of cues, immature cells (top) derived from fetal brain tissue can be coaxed to develop into mature neurons at about four weeks in culture (bottom).

Simulating neuronal development in culture with cells derived from human brain tissue offers a new way to study the function of autism-linked genes, according to research published in the February issue of Molecular Psychiatry¹.

The study, conducted by Daniel Geschwind's team at the University of California, Los Angeles, represents a growing movement toward creating human-based models of early brain development. The researchers used human neural progenitor cells derived from fetal brain tissue to generate neurons. They then studied the expression of genes linked to autism as the cells developed into mature neurons over an eight-week period.

By grouping the genes into networks, or modules, based on when they are co-expressed in culture, the researchers show that autism-related genes act very early in brain development and tend to be closely connected to one another.

The researchers say the cells provide a way around some of the shortcomings of animal models. For example, they can be used to study the effects of simultaneous mutations in multiple genes, something that is challenging to do in animals.

Previous research suggests that some cases of autism may result from mutations in two or more genes, so having a method to study these interactions is especially important.

"I think as a strategy, it's terrific," says David Amaral, director of research at the University of California, Davis MIND Institute, who was not involved in the study. "It's a human model system that allows you to probe how particular genes that have been implicated in neuropsychiatric disorders are regulated."

Next up, neurexin:

Geschwind, professor of neurology and psychiatry, and his collaborators were especially interested in using their new model to investigate the role of neurexin-1 (NRXN1), a gene that is involved in synapse formation and has been linked to both autism and schizophrenia.

They found that expression levels of NRXN1 increased after four weeks in culture, the point at which neurons began to form, but levels of neurexin-3, a related gene, decreased. This is the first finding to suggest that these highly homologous genes may have opposite effects in the developing brain.

The researchers were able to confirm this expression pattern in fetal brain slices and in mouse brain data from the Allen Brain Atlas, which suggests that the expression pattern could be studied further using mouse models of human diseases, including autism.

"It was really nice to get in vivo confirmation of the data we were getting from the cells," says Genevieve Konopka, assistant professor of neuroscience at the University of Texas Southwestern Medical Center in Dallas, and lead investigator of the study.

NRXN1 was also one of the more highly connected genes in the modules the authors created, suggesting that it acts in concert with other autism-related genes and may influence multiple molecular pathways that may contribute to the disease. Other highly connected autism candidate genes included SCN1A and SLC9A6.

Follow-up studies could help elucidate the precise functions of these gene networks. The researchers also plan to use the cells to track the expression patterns of new autism-related genes as they're identified, says Konopka.

New culture:

As cell culture techniques improve, the notion of recreating complex human disorders "in a dish" is fast becoming a reality².

Many researchers are using induced pluripotent stem (iPS) cells, usually obtained from skin samples, to generate human neurons in culture. This approach has some advantages: The cells are easy to obtain and can be linked to specific patients, which makes it easier for researchers to relate results back to disease phenotypes.

But others are concerned that the multiple reprogramming steps necessary to create iPS cells and differentiate them into mature neurons may ultimately create neurons that are genetically different from those in the brain.

In contrast to iPS cells, neural progenitor cells are obtained from human brain tissue and don't have to be reprogrammed, so they allow researchers to more closely model the development process as it occurs in nature.

The progenitor cells used in this study are easy to work with, says Konopka, and are more developed than stem cells, so they differentiate into neurons in weeks instead of months. But they are more difficult to obtain than iPS cells. They are isolated from aborted fetuses, which is technically challenging and may be ethically controversial for some researchers.

There is also no way to specify what brain areas the cells come from. Geschwind and Konopka found that the cells differentiated into numerous neuronal subtypes, however, including those of the frontal cortex, which is of particular interest to autism researchers.

Their study focused on autism-related genes, but the approach could be readily adapted to study gene variants associated with other neurodevelopmental disorders, such as epilepsy and schizophrenia.

One can imagine using this more general approach to complement animal models and iPS cell studies of autism, which allow researchers to study specific behavioral and clinical phenotypes. For example, researchers could use the neural progenitor model to determine where and when a gene of interest is normally expressed during development, then look for abnormalities in that gene in neurons derived from iPS cells taken from individuals with autism.

"As far as I'm concerned, the more models we have, the better," says James Ellis, senior scientist at the University of Toronto's Hospital for Sick Children, who has used iPS cells to model Rett syndrome in culture³.  

Konopka is also interested in using the neural progenitor model system to investigate possible pharmacotherapies for autism. "It's a really nice system for manipulating genes," she says. The model system enables researchers to manipulate multiple genes at once, which could be used to create different in vitro autism phenotypes and to screen large numbers of molecules or drugs against these phenotypes as possible treatments.

References:

1: Konopka G. et al. Mol. Psychiatry 17, 202-214 (2012) Abstract

2: Tiscornia G. et al. Nat. Med. 17, 1570-1576 (2011) Abstract

3: Hotta A. et al. Nat. Methods 6, 370-376 (2009) Abstract

Mini model: Fruit flies have fewer genes than people do, but carry versions of 75 percent of human disease-causing genes. 

A new study reveals the three-dimensional structure of fruit fly chromosomes, which groups together active and inactive genes. The results were published 3 February in Cell¹.

Not long ago, gene expression was believed to be essentially linear, with regulatory regions controlling the expression of adjacent genes. DNA is now known to wrap around a structure called the nucleosome, which fine-tunes gene expression by altering how tightly the molecule is wound. The new work provides a glimpse at yet another level of gene regulation: the organization of chromosomes in three-dimensional space.

The fruit fly genome, although much smaller than that of humans, contains versions of about 75 percent of genes involved in human disease. For example, researchers have learned about both fragile X syndrome and autism by studying mutant flies that model these disorders. 

In the new study, the researchers constructed a genome-wide map of chromosomal structure in fruit flies.

They treated the chromosomes with a chemical that preserves their three-dimensional shape, and then used an enzyme to chop up the DNA molecules at a specific sequence of nucleotides, the building blocks of DNA. Finally, they fused these fragments together, allowing the DNA to bond not just to where it was cut, but also to nearby clusters, creating chimeric, or hybrid, sequences.

By sequencing more than 362 million of these chimeras, the researchers were able to create a picture of the proximity of different genetic regions when DNA is tightly packaged into chromosomes.

Chromosomes are folded into distinct domains that are separated by insulator proteins. These proteins form a physical barrier between regions harboring genes with different levels of expression, the study found. Each of these domains is characterized by a specific pattern of histone modifications, which are chemical alterations to nucleosomes that change the way they interact with DNA.

For example, genes with the H3K4 trimethyl mark, which is known to activate gene expression, cluster together in domains that primarily contain active genes. Other regions group together genes with histone marks that turn off expression, such as H3K27 trimethyl.

Modifying gene expression in this way, without changing the underlying DNA sequence, is called epigenetics. Researchers are beginning to understand the function of epigenetics in disease, and the role it is likely to play in regulating the complex gene-environment interactions that underlie autism.

Understanding how chromosomes are organized in fruit flies provides a basis on which to begin to understand this level of epigenetic regulation in other organisms and in disease, the researchers say.

References:

1: Sexton T. et al. Cell 148, 458-472 (2012) PubMed

Genome Research Limited

Risk regions: Tourette syndrome is associated with duplications or deletions of chunks of DNA that span genes that regulate brain signaling.

Genes implicated in Tourette syndrome overlap with those involved in autism, according to an analysis of rare DNA duplications and deletions in people with the disorder, published in the March issue of Biological Psychiatry¹.

Several studies in the past few years have shown that rare duplications and deletions of large pieces of chromosomes, called copy number variants (CNVs), are more common in individuals with autism and schizophrenia than in controls. These studies have also revealed significant overlap in the genetic profiles of various neurodevelopmental disorders, including autism, schizophrenia, bipolar disorder, intellectual disability and attention deficit hyperactivity disorder.

In the new study, researchers investigated the role of rare CNVs in individuals with Tourette syndrome, a neurological disorder characterized by involuntary physical and verbal tics. They looked at 460 individuals with the syndrome and 1,131 controls.

Unlike in autism and schizophrenia, people with Tourette syndrome do not have more rare CNVs than do controls, the study found. However, they are more likely to have three large CNVs greater than one megabase in length. The largest of these is a duplication of almost 52 megabases that spans 447 genes.

The researchers also looked at the function of 2,646 genes that are disrupted by rare CNVs in individuals with Tourette syndrome. Their analysis suggests that the histamine signaling pathway plays an important role in the disorder. This pathway regulates the immune system as well as important chemical messengers in the brain and gut.

It also implicates the gamma-aminobutyric acid (GABA) pathway, which dampens signals in the brain and has been linked to autism. One large CNV that overlaps with several GABA-related genes is responsible for much of this association.

The study found significant overlap between the CNVs and genes that have been associated with autism. The researchers looked at 36 genes and 10 genomic regions strongly implicated in autism, called ‘ASD implicated,’ and another 103 genes for which studies show some association with autism, called ‘ASD-implicated,’ and another 103 genes for which studies show some association with autism, called ‘autism candidates’².

About 2 percent of individuals with Tourette syndrome have CNVs that disrupt ‘ASD-implicated’ genes compared with only 0.4 percent of controls, the study found. And almost twice as many individuals with the syndrome have CNVs that disrupt either ‘ASD-implicated’ or ‘autism-candidate’ genes than do controls: 10.2 compared with 5.7 percent.

The genes implicated in both Tourette syndrome and autism include CNTNAP2, DISC1, AUTS2, and the 16p11.2 and 22q11.21 genomic regions.

The study did not find significant overlap between Tourette syndrome CNVs and genes implicated in intellectual disability and schizophrenia.

References:

1: Fernandez T.V. et al. Biol. Psychiatry 71, 392-402 (2012) PubMed

2: Pinto D. et al. Nature 466, 368-372 (2010) PubMed

Using game mechanics to engage users and solve real-world problems is one of the hottest online trends, encompassing healthcare, finance and even science.

And there’s plenty of proof it works. Players of the protein-folding game Foldit, for example, helped solve the three-dimensional structure of an enzyme involved in replication of the HIV virus.

Now, Sebastian Seung, professor of computational neuroscience at the Massachusetts Institute of Technology, and his students aim to do the same for neuroscience. They have created a website called Eyewire, where users can help map out the complex neural wiring of the retina, a part of the central nervous system.

Seung has made it his mission to map the human 'connectome,' the pattern of connections among all the neurons in our brain. (He recently published a book, Connectome, that, in part, explores how such a map could help shed light on developmental disorders thought to be the result of faulty wiring, such as autism and schizophrenia.)

With 100 billion neurons and an estimated 100 trillion connections in the human brain, this is an enormous project.

Seung and his collaborators painstakingly slice sections of tissue and image them using an electron microscope. Individual neurons in the slices are then labeled and computationally stitched together into a cohesive block of connected neurons.

According to Seung, it took postdoctoral candidate Daniel Berger approximately 250 hours of labor to reconstruct a six-micron cube of brain tissue. Scaling that process up to the size of the human brain would take from 100,000 to 1 million years, he estimates, “without coffee breaks.”

Seung’s lab is developing ways of using artificial intelligence to further automate this process, but they aren’t yet good enough to assemble neurons and their connections accurately. In the meantime, he’s turning to crowdsourcing.

Eyewire lets users flip through brain tissue images generated in the lab of collaborator Winfried Denk. Users guide the software in coloring in individual retinal neurons with a few clicks of the mouse, “as if the images were a three-dimensional coloring book,” according to the site. The collective efforts of the players help the computer reconstruct the tree-like neurons in three dimensions.

Seung described the project Wednesday night to a gathering of neuroscience enthusiasts at the Rubin Museum in New York. “We have some addicts already,” he said.

Participation in the project is still limited, but you can sign up to be on the waiting list here. Seung plans to introduce a game to identify synapses in the near future.

Fear of heights: Mice with extra copies of the Rett syndrome gene are less likely than controls to venture onto elevated corridors in a test used to gauge anxiety.

Two genes may be responsible for autism symptoms in mice with extra copies of the Rett syndrome gene, according to a study published 8 January in Nature Genetics¹.

Elevated expression levels of the corticotropin-releasing hormone (CRH) gene and an opioid receptor gene, OPRM1, are associated with anxiety and social deficits, respectively, in the mice, the study shows.

Rett syndrome, a developmental disorder characterized by intellectual disability, autism and motor deficits, is caused by having fewer active copies of the MeCP2 gene, which regulates the expression of thousands of other genes.

A 2004 study showed that mice with extra copies of the MeCP2 gene also show some features of autism, including seizures, motor deficits and repetitive movements². These mice have vision deficits, as a result of their background strain, that make it difficult to assess their anxiety levels and social interest, however.

In the new study, the same team developed mice that express at least two times the typical levels of MeCP2. They were bred from mutant and control mice to minimize deficits caused by inbreeding.

These mice are less likely to enter bright or elevated corridors than control mice are, suggesting that they have heightened anxiety. They also exhibit social deficits: They spend less time investigating other mice than controls do, and prefer to spend time in a chamber with an object rather than in one with another mouse.

The brains of the mutant mice express more than 1,000 genes at different levels compared with controls, the study found. Using the Mouse Genome Informatics database, which aggregates information about existing mouse mutants, the researchers identified 32 of these genes that have been associated with heightened anxiety and social deficits. Of these, nine are expressed at levels that are 50 percent higher or lower than in control mice.

The researchers looked at two of the nine genes to determine the pathways responsible for the autism-related behaviors in these mice: CRH, which codes for a chemical messenger involved in the stress response, and OPRM1, which codes for a signaling receptor located in the brain. Deleting one copy of the CRH in mice with elevated MeCP2 alleviates anxiety, but not social deficits, the study found. By contrast, removing one copy of OPRM1 enhances their sociability, but has no effect on anxiety.

Deleting one copy of a related gene, CRHR1, or treating mice with antalarmin, a drug that targets the stress response pathway, also alleviates anxiety in the mutant mice, the study found.

Honing in on the genes and pathways responsible for autism-related symptoms in mice could help identify relevant targets for new treatments, the researchers say.

References:

1: Samaco R.C. et al. Nat. Genet. 44, 206-211 (2012) PubMed

2: Collins A.L. et al. Hum. Mol. Genet. 13, 2679-2689 (2004) PubMed

Heavy load: Individuals with autism and SHANK2 deletions also carry variations in an autism-linked region of chromosome 15.

By screening the genomes of hundreds of people with autism and analyzing the effects of newly identified mutations in cultured neurons, researchers have clarified the disorder’s complex link to a gene called SHANK2.

Functional mutations in SHANK2 crop up about twice as often in individuals with autism as in typical controls, according to a study published 9 February in PLoS Genetics¹.

The SHANK2 protein buttresses the synapse, or junction between neurons. The new findings add to already robust evidence from genetic studies and animal models that synaptic proteins — notably SHANK3, neurexins and neuroligins — are important in autism, the researchers say.

But a more surprising finding helps to explain why not everyone who has a SHANK2 mutation has autism. The three individuals with autism known to carry SHANK2 deletions also carry rare deletions or duplications — so-called copy number variations, or CNVs — in an autism-linked segment of chromosome 15. This supports the idea that autism arises not from a single genetic glitch, but from multiple hits to the genome.

“I think many people are still thinking about the genome like the old black-and-white movies from the 1950s: The good guy was in white, the bad guy was in black, and everybody knew what was going on,” says lead investigator Thomas Bourgeron, professor of genetics at the University of Paris Diderot.

But studies like this show that a ‘bad’ genetic glitch isn’t necessarily the only bad guy.

“When we find a single mutation in a patient with autism, we can’t say that we’re done,” Bourgeron says. “We still have to work on the whole genome of these patients to understand exactly what’s going on.”

Many hits:

Other research groups have identified spontaneous, or de novo, SHANK2 deletions in two people with autism, as well as in one person with mental retardation and one with speech and developmental delay. Researchers have also found a spattering of SHANK2 single-letter mutations — both de novo and inherited — in people with these conditions.

The new study screened an additional 260 individuals with autism for CNVs and found one individual who carries a de novo SHANK2 deletion. The researchers did not find SHANK2 deletions in more than 5,000 controls.

The range of conditions associated with SHANK2 deletions suggested to Bourgeron that other factors are involved in its link to autism.

Other genes, for instance, seem to play a role. Bourgeron’s team found that all three of the individuals with autism who carry de novo SHANK2 deletions also have rare CNVs in 15q11-13, a chromosomal region long associated with autism.

All of the 15q CNVs were inherited, whereas the SHANK2 deletions were spontaneous. “For these patients, it’s like the genome cannot cope with that extra de novo event,” Bourgeron says.

One individual is missing one copy of CYFIP1, a gene located in 15q11-13. It codes for a protein that binds with FMRP, the protein missing in fragile X syndrome. The other two carry an extra copy of CHRNA7, a gene in the same region that encodes a receptor of the acetylcholine neurotransmitter, and has been tied to developmental delay. 

Bourgeron notes that the odds of finding these rare genetic combinations by chance are low. It could be that the additive effect of multiple hits is enough to cause autism, or it could be that some combinations are more powerful than others.

“It may be like nitro and glycerin. Alone they’re OK, but if you mix the two you have to be very careful,” he says.

Other experts point out, however, that many controls also carry 15q CNVs, including those affecting CYFIP1 and CHRNA7. So this particular combination in participants with autism “could just be a coincidence,” says Gudrun Rappold, head of human molecular genetics at the University of Heidelberg, who led the first study tying SHANK2 to people with autism².

Still, Rappold agrees that the multi-hit idea has a lot of evidence behind it. “Autism is a complex disorder, and complex disorders are usually made up of not just one or two or five genes, but a number of genes,” she says.

Synaptic consequences:

Bourgeron’s team also looked for single-letter mutations in SHANK2 by sequencing the exons, or protein-coding parts, of the gene in 455 people with autism and 431 controls. When combined with data from Rappold’s earlier study, the researchers report that in evolutionarily conserved regions of the gene — which are most likely to have functional effects — these mutations show up in 3.4 percent of individuals with autism and 1.5 percent of controls.

Some SHANK2 single-letter mutations appear only in people with autism, some only in controls and some in both groups. The researchers tested what these variants actually do to cells by genetically engineering cultured neurons from rat hippocampus, a brain region important for learning and memory.

They found that mutations that occur in people with autism cause a significant decrease in the density of synapses. In contrast, the mutations carried only by controls have no effect on rat synapses.

These results are similar to those found by Rappold’s group. Last month, she reported that a few SHANK2 mutations cause an array of synaptic defects, including lower synaptic densities and smaller synapses, in cultured mouse neurons.

It's rare to have a study that includes both a large genetic screen and in vitro findings, notes Jacques Michaud, professor of pediatrics at the University of Montreal. "They were very systematic and that's quite impressive." Last year, Michaud's team reported an overrepresentation of mutations in synaptic genes in 95 people with intellectual disability.

Still, it will take an order of magnitude more participants to sort out how these synaptic genes contribute to autism, he says.

"We've realized over the last few years that, for most cases of autism, you're going to have many rare variants that aren't very penetrant," Michaud says. "To find these, and to deal with their interactions, you need to have very large numbers."

Bourgeron intends to further investigate the molecular and behavioral consequences of the gene by studying mutant mice lacking SHANK2. These animals, which his collaborators debuted in November at the Society for Neuroscience annual meeting in Washington, D.C., show social deficits and hyperactivity.

Another approach for the future might be to insert two glitches — in SHANK2 and CYFIP1, for example — into the same animal and observe the effects on brain and behavior, he says.

“There are so many combinations that might be important,” Bourgeron says. “We still have a lot of work to do at the genetic level.”

References:

1: Leblond C.S. et al. PLoS Genetics Epub ahead of print (2012) Full text

2: Berkel S. et al. Nat. Genet. 42, 489-491 (2010) PubMed

Allen Institute for Brain Science

What makes humans so different from our primate cousins? The answer may lie in unique patterns of gene expression soon after birth, primarily in genes required for synapses — the junctions between neurons — to form and function properly.

The findings emerged from an ambitious analysis of postmortem brain tissue collected from humans, chimpanzees and macaques. Researchers measured the expression of 1,200 genes in tissue samples gathered from each species at all different ages, from before birth to the end of life.

The results emphasize the power of time — the ability to examine how gene expression patterns evolve over a lifetime, rather than how they differ at a single moment.

Researchers studying autism are now applying the same approach to brain tissue collected from people with autism, which they hope will shed light on how brain development goes awry early on.

In the primate study, published 2 February in Genome Research, researchers grouped together genes based on the role they play in the cell. They discovered that those that are involved in synapse formation also had the most unique gene expression pattern in humans compared with the other species.

These genes tend to turn off soon after birth in chimps and macaques, but stay on in humans until age 5, the study found. Similarly, the number of synapses in non-human primates peaks soon after birth, but in children continues to bloom until about age 4.

“Humans have much more time to form synaptic connections,” lead researcher Philipp Khaitovich, an evolutionary biologist at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, and the Chinese Academy of Sciences (CAS) in Shanghai, China, told ScienceNow.

Eric Courchesne, director of the Autism Center of Excellence at the University of California, San Diego, is doing a similar type of analysis on brain tissue samples from people with autism.

He and others have found evidence that children with autism have an abnormal pattern of brain development, in which some regions grow more quickly and some more slowly than normal during different stages of development.

The specifics of the pattern are controversial, however, and Courchesne says that postmortem studies of brain tissue may help to sort out these discrepancies.

Carrying out such studies is challenging. A number of efforts to catalogue gene expression during development are underway in postmortem brains from typically developing people, but the availability of tissue samples from people with autism is limited.

“We will not know for quite a while whether we will have sufficient samples at useful ages to see effects,” says Courchesne.

What’s more, the quality of the samples can influence results.

Still, the potential benefits of this type of study for autism research emphasize just how important it is to grow collections of tissue samples.