Cohesive unit: A unique partnership brings together researchers from different European countries, in different phases of research, and from both academia and the pharmaceutical industry.

A $38.7 million project in the European Union — the largest single grant for autism research in the world — aims to bring together academic labs and pharmaceutical companies, including Roche, Eli Lilly and Pfizer, to speed the move from basic to clinical research.

The effort, called European Autism Interventions – A Multicentre Study for Developing New Medications, or EU-AIMS, focuses on improving translational research, including developing stem cells, animal models and a European network for running clinical trials. Launched in April, it is led by Roche and researchers at King's College London.

“If we want to find new medicines, we need to lower the barrier for investment,” says Luca Santarelli, head of neuroscience at Roche. “That means we need to collectively progress the science, translational research and clinical trial technology.”

Half the funding for the five-year project comes from the Innovative Medicines Initiative, a European public-private initiative designed to help develop better and safer medicines, and half from the European Federation of Pharmaceutical Industry Associations. Autism Speaks, a U.S.-based research and advocacy organization that contributed $1 million to the project, is another collaborator.

“I think it’s a great first effort and will dramatically contribute to the understanding, treatment and de-stigmatization of autism in Europe,” says Randall Carpenter, president of Seaside Therapeutics, a Massachusetts-based biotech company testing drugs for autism and fragile X syndrome, and head of the grant review committee. “They have been visionary in their thought process and tried to integrate and learn from everyone else who has tried these projects.”

For example, the researchers plan to make the resources that emerge from the project, such as databases and a collection of biological samples, able to link with existing autism repositories. 

The products of the project are also considered ‘pre-competitive,’ meaning the information can be shared freely. “Pre-competitive applies to areas where the field is young and needs to progress to a critical point where companies can invest in proprietary research,” says Santarelli.

Trial training:

A central thrust of the EU-AIMS project is to create a network of centers of expertise for the various components of autism research.

“We want to develop better animal models, better understanding of the biology of disease, and better clinical scales and endpoints,” says Santarelli. “If you do that in isolation, you have less power than doing it as a team.”

One group aims to generate induced pluripotent stem (iPS) cells from people with autism to try to identify cellular abnormalities, and to develop new technology for drug discovery assays.

Researchers specializing in animal models plan to generate new strains that robustly show the core symptoms of autism: social deficits and repetitive and restricted behaviors. They also aim to develop reproducible behavioral assays and anatomical and functional biomarkers, such as differences in brain structure or behavior, that can ultimately be used to test drugs. 

The animals will be housed in a central animal repository, and breeding pairs can be shared with other researchers.

Part of the translational effort will be to develop new molecular imaging techniques that can be used across different species.

“We want to put everything together and try to come up with a holistic view,” says Declan Murphy, professor of neurodevelopmental sciences at King’s College London, and the academic lead on the effort.

For example, he says, the researchers don’t just want to measure glutamate and GABA, two chemical messengers in the brain, in live animals. They instead hope to understand how these markers relate to autism-linked mutations and how they are modulated by different drugs.

On the clinical side, researchers plan to work closely with families, for example by studying infants who have siblings with autism and are therefore at higher-than-average risk of developing the disorder. They plan to follow approximately 300 of these so-called ‘baby sibs’, as well as 500 children with autism, in search of early biomarkers that can predict autism, and to assess whether those biomarkers change over time. 

“What they are doing is very comprehensive and commendable in terms of trying to integrate investigators and bringing in pharma to the table,” says Antonio Hardan, assistant professor of psychiatry and behavioral science at Stanford University in California, who is not involved in the project. Forty million dollars, he points out, “is a great catalyzer.”

One of the most important aspects of the project is the development of six clinical centers — with more planned — across Europe. “If we identify new treatments, we can roll them out to clinical populations immediately,” says Murphy.

The researchers also plan to work with Europe’s drug regulatory body, the European Medicines Agency, to determine the best outcome measures.

For a disorder as diverse as autism, it’s especially important to have access to large numbers of people for clinical trials. Most clinical trials in autism have been comparatively small, with 50 or fewer participants. That makes it difficult to identify subgroups that might respond to the treatment, says Hardan.

Given that the effort spans at least seven different countries and intends to include individuals who speak as many languages, the researchers face the unique challenge of developing and validating tools that work across the various languages and cultures. 

The U.S. has its own autism clinical trials network, known as the Research Units on Pediatric Psychopharmacology Autism Network, but it has few sites and does not incorporate a basic science component.

“No other effort [in the autism field] is bringing basic science and clinical trials under one umbrella,” says Hardan. “These countries have done it before politically, so they can probably [work] together here also.”

Preliminary results from a placebo-controlled trial of the antibiotic minocycline in children with fragile X syndrome suggest the drug alleviates some aspects of the disorder, according to research presented Friday at the International Meeting for Autism Research in Toronto.

In the study, researchers analyzed data from 48 children and teens, ages 3 to 16, with fragile X syndrome, one of the most common forms of inherited intellectual disability. Participants took either the drug or the placebo for three months, and then switched to the other arm of the study, a design known as a double-blind cross-over clinical trial.

Minocycline significantly improved scores on the Clinical Global Impression rating scale, a seven-point measure used to assess symptom severity and response to treatment, lead investigator Randi Hagerman, medical director of the MIND Institute at the University of California, Davis, reported at the conference.

The researchers also saw improvement on the anxiety and mood subscales of the Visual Analog Scale, which is designed to measure conditions such as pain. Because it is a subjective test, it is typically used to measure changes in an individual.

In the trial, the researchers did not see significant differences between placebo and drug treatments in the language or aggression scales of this test, nor in the Aberrant Behavior Checklist, another test used to assess treatment effects. The drug seems to be safe and well tolerated, Hagerman says.

Previous research in animal models of fragile X has shown that minocycline improves some symptoms of the disorder, including anxiety. It also helps to mature neural connections between hippocampal cells grown in culture, says Hagerman. Mouse models of fragile X have an abundance of immature neural connections.

The drug may act through a variety of mechanisms. It is an anti-inflammatory and also slows protein synthesis, which is thought to be overactive in fragile X.

It also lowers levels of matrix metalloproteinase 9 or MMP9, an enzyme that has several functions, including breaking down the extracellular matrix MMP9 levels are elevated in many people with fragile X and some people with autism, Hagerman says.

The researchers hope to do a longer-term clinical trial, as well as to test the drug in children with autism, she adds. They have applied for funding to study levels of MMP9 as a biomarker in children with fragile X and in those with autism, as well as how these children respond to minocycline.

For more reports from the 2012 International Meeting for Autism Research, please click here.

Autism researchers need to build a culture of data-sharing. That’s the message that Thomas Insel, director of the National Institute of Mental Health (NIMH), wanted to convey Friday at the International Meeting for Autism Research (IMFAR) in Toronto.

Grants from the National Institutes of Health (NIH) already require that researchers add their data to the National Database of Autism Research (NDAR) within a year of publication. That’s important so that data can be reused in new ways, for example to create larger datasets with greater statistical power, said Susan Daniels, acting director of the NIMH’s Office of Autism Research Coordination, who filled in for Insel as a speaker at the conference.

Given shrinking research budgets, data-sharing is likely to become even more important. In Insel’s words, presented by Daniels at the conference, “Share data to protect the future of your funding and funding for autism research as a whole."

After growing steadily for most of the past decade, funding for autism research has plateaued, a trend Insel pointed to at IMFAR last year.

However, autism funding is growing faster than that for other neurological disorders, such as multiple sclerosis, as well as for neuroscience in general.

In 2010, a combination of 18 public (82 percent) and private (18 percent) organizations gave out a total of $408 million. The Simons Foundation, SFARI.org’s parent organization, is the second largest funder after the NIH.

Research on lifespan issues, including functioning of adults with autism and treatments tailored to this group, get the smallest chunk of funding, less than two percent. This issue used to be lumped together with research into autism services, but it is now its own category because it is considered so important, said Daniels. Services garner 16 percent of overall funding.

For more reports from the 2012 International Meeting for Autism Research, please click here.

Children and teens with autism — or the related disorders schizophrenia and attention deficit hyperactivity disorder, among others — are often prescribed antipsychotic drugs from an early age.

But little is known about the long-term effects these drugs may have on the brain.

Research on schizophrenia suggests that many of these drugs affect brain volume: Antipsychotics appear to shrink the brain and the mood stabilizer lithium seems to enlarge it. What has been unclear so far is whether these changes result from the treatment or are a normal part of the disease course.

A new study confirms that the observed changes in brain size are the result of drug treatment. Researchers treated rats with either the antipsychotic haloperidol or with lithium every day for eight weeks, roughly equivalent to five years for humans.

According to brain scans taken before and after treatment, haloperidol shrank the volume of gray matter, which contains neuronal cell bodies, by six percent, and lithium increased gray matter volume by three percent. The findings were published 8 May in Biological Psychiatry.

The study leaves many questions unanswered. It’s not clear, for example, whether these changes reflect part of the benefits of the drugs, or whether they are a negative side effect. Also, the researchers used normal rats rather than those engineered to mimic the symptoms of schizophrenia or autism, so they can't say whether the drugs would have different structural effects in those animals.

Still, the findings could be relevant for children with autism. About one in three people with autism are treated with antipsychotics to relieve irritability, aggression and sleeplessness. In fact, the only two drugs approved by the Food and Drug Administration to treat autism, risperidone and aripiprazole, are both antipsychotics. (Other drugs, including antidepressants and stimulants, are commonly prescribed as well.)

Although neither risperidone nor aripiprazole was tested in the current study, they share some molecular mechanisms with haloperidol. Risperidone binds to receptors for brain-derived neurotrophic factor, a protein that stimulates the growth of new neurons and neural connections.

Lithium has been shown to reverse some of the signs of fragile X syndrome, an autism-related disorder, in animal models. Preliminary evidence suggests it can improve irritability in people with the syndrome. 

Given that autism is thought to be a disorder of brain connectivity, and that changes in brain size are associated with the disorder, it seems crucial to understand the long-term effects of drugs used to treat it. 

Methyl marks: A study of pregnant women could help clarify the gene-environment interactions that raise autism risk.

By studying pregnant women who already have a child with autism, researchers hope to understand how epigenetic changes — those that affect gene expression but don’t directly alter DNA — during pregnancy influence risk of the disorder.

One group is using a method called CHARM, or comprehensive high-throughput arrays for relative methylation, to assess methylation, the addition of a methyl group to DNA that typically turns off gene expression. The researchers are studying how methylation changes during pregnancy and in young children over time, as well as in response to specific environmental exposures.

A second group is measuring ‘methylation capacity,’ an indirect measure of how well a cell can methylate genes, in pregnant women to determine whether abnormal levels can predict a later autism diagnosis for the child.

Both projects, presented yesterday at the International Meeting for Autism Research in Toronto, are part of the Early Autism Risk Longitudinal Investigation (EARLI), which aims to sort out the genetic and environmental factors that contribute to autism.

EARLI results:

Launched in 2009, EARLI is focused on pregnant women who already have a child with autism and have a higher-than-normal risk of having another. About 10 to 25 percent of siblings of a child with autism will go on to develop the disorder, compared with an average risk of about 1 percent. (One of EARLI’s goals is to get a better handle on that number.)

Researchers at a number of sites across the U.S. are collecting blood and other biological samples from these women and their children as well as data on their diet, medication use, medical conditions, pesticide exposure, vaccine use, occupational history and additional factors. The children get behavioral assessments beginning at 6 months of age and through 36 months, when they can be tested for autism. EARLI ultimately aims to enroll 1,200 mothers.

Because the study is prospective, meaning that data are collected over time, the researchers say they hope it will help clarify how genetic and environmental factors interact to raise autism risk. Epigenetics, which can be influenced by the environment, is one way to examine this interaction. (Retrospective studies rely on participants to recall information about the past and so are more subject to bias.)

One of the two EARLI projects presented yesterday aims to link methylation data from women during and after pregnancy with environmental exposure. Preliminary findings show that methylation appears to be stable throughout pregnancy.

The second looked at metabolic biomarkers in pregnant women. The researchers had previously found that women who have children with autism have an abnormal methylation capacity, defined as the ratio of S-adenosyl methionine (SAM) to S-adenosylhomocysteine (SAH) SAM is a critical ingredient of methylation. The methylation process, in turn, produces SAH.

EARLI gave the team the opportunity to determine whether this pattern is present in pregnancy and, if so, whether it predicts the child’s risk of autism.

The researchers found that about 15 percent of the 60 women have low methylation capacity compared with 100 controls. However, they won't know for about three years, when the children can reliably be diagnosed with autism, whether this is predictive of the disorder.

Still, the 15 percent figure is interesting because it aligns with the risk for siblings of children with autism, notes lead investigator Jill James, director of the Metabolic Genomics Laboratory at Arkansas Children’s Hospital Research Institute.

For more reports from the 2012 International Meeting for Autism Research, please click here.

Simon says: Children who have autism struggle with joint attention, the ability to engage others’ attention. 

Toddlers with autism who receive behavioral interventions that improve joint attention — engaging and following others’ focus — have better language ability five years later than do controls, according to a study published in May in the Journal of the American Academy of Child and Adolescent Psychiatry¹.

Studies have shown that early behavioral interventions may be among the best available treatments for autism, but they are not effective for all children. Few studies have documented the long-term outcomes of this type of intervention.

In a 2006 study, researchers enrolled 58 children with autism in behavioral interventions for 30 hours a week, for five to six weeks.

The researchers divided the children into three groups². In one group, the intervention focused on joint attention, for example by repeating back to the child whatever he or she said. In the second, researchers encouraged the children to participate in symbolic play, such as pretending that a doll can drive a car. The third group of children represented controls, and received standard treatment with no particular emphasis on joint attention or play skills.

After the intervention, the children who learned joint attention skills were more likely to engage the attention of their mothers, and those who learned play skills were more likely to engage in unprompted symbolic play.

In the new study, researchers followed up on 40 of these 58 children. Overall, regardless of the treatment group, the children who spontaneously engaged in symbolic play at 3 years of age have better language ability five years later, the study found.

Toddlers who participated in one of the two interventions also developed a better vocabulary compared with controls. Those who were closer to 3 years of age when they began the intervention improved more than those who were closer to 4, the study found.

References:

1: Kasari C. et al. J. Am. Acad. Child Adolesc. Psychiatry 51, 487-495 (2012) PubMed

2: Kasari C. et al. J. Child Psychol. Psychiatry 47, 611-620 (2006) PubMed

Love hormone: After a single dose of oxytocin, fathers were less willing to stop gazing at their babies.

Two new studies on the so-called ‘trust hormone’ oxytocin, presented today at the International Meeting for Autism Research in Toronto, suggest new avenues for using the drug to treat autism.

One study found that treating fathers with the hormone boosts levels in their infants. The second showed that when children with autism play with a parent, their oxytocin levels rise to those of controls.

Oxytocin, a hormone first discovered for its role in childbirth, governs social bonding and is released both in the brain and peripherally in the body.

Mutations in genes that regulate oxytocin release are thought to raise the risk of autism or autism-like traits, although some findings are contradictory. Some studies suggest that when people with autism sniff oxytocin, their performance on social tasks improves.

Ruth Feldman, professor of psychology at Bar-Ilan University in Israel, studies the development of the parent-child bond, and the role that oxytocin plays.

Feldman’s previous research has shown that mothers and fathers both have an increase in oxytocin levels in saliva when holding and playing with their babies. Her team is studying how treatment with oxytocin in parents affects hormone levels in their children, as well as how the drug might be used to treat autism and other disorders.

In one study, Feldman and her team gave 35 fathers a single dose of oxytocin by nasal puff before playing with their babies. (They didn't study mothers because they had not yet been given approval to give the hormone to breastfeeding women.)

These fathers showed a significant increase in saliva oxytocin levels after playing with their infants, from an average concentration of 1,000 picomoles at baseline to 6,000 after play. The fathers were less willing to stop gazing at the infants, and the infants held the gaze longer, says Feldman. Fathers given a placebo didn’t show a significant rise in oxytocin or change in behavior.

Strikingly, infants showed a similar increase in hormone levels, even though they hadn’t been given any drugs, notes Feldman. That may prove relevant for treating children at risk of developing autism.

“Giving oxytocin to a parent has a parallel effect on the child, which may open a new direction for intervention for young high-risk infants,” says Feldman.

In the second study, her team analyzed oxytocin levels in parents, and in preschool-aged children with high-functioning autism, before and after a play session.

Mothers of children with autism showed no difference in oxytocin levels compared with controls, before or after play, the study found.

As might be expected, the children with autism spent less time gazing at parents and engaging in joint attention — when the parent gestures for the child to pay attention to something — compared with controls.

The children with autism had lower baseline levels of oxytocin compared with controls, a finding in line with previous research showing that adults with the disorder have low levels of the hormone.

After playing with a parent for 20 minutes, oxytocin levels in the children with autism rose to levels similar to those of controls, and remained elevated for about 40 minutes before falling back down.

“This is the first result we know of in peripheral oxytocin of young children with autism,” says Feldman. “These findings, if replicated and further understood, can serve as a baseline of intervention, maybe pharmacological or behavioral, to maintain a high level of oxytocin.”

For more reports from the 2012 International Meeting for Autism Research, please click here.

Cell explosion: Zebrafish embryos with low levels of KCTD13 protein (left) show a surge of new brain cells compared with controls (middle) or those with too much KCTD13 (right).

By creating genetically engineered fish, two independent groups have identified genes in an autism hotspot on chromosome 16 that influence head size and brain development. One of the studies appears today in Nature¹.

A 29-gene stretch of chromosome 16 known as 16p11.2, or 16p, is deleted or duplicated in roughly one percent of individuals with autism and duplicated in some individuals with schizophrenia. Researchers have struggled to sort out which genes in the region contribute to features of these disorders.

Using zebrafish allows for an unbiased screen of individual genes, says Nicholas Katsanis, professor of cell biology at Duke University in Durham, North Carolina, and lead investigator of the Nature study. “We tested all of them with exactly same protocol, with no prior expectation of what we were going to find,” he says.

His study shows that suppressing a little-known gene called KCTD13 in zebrafish leads to a 20 percent increase in head size. Conversely, expressing too much of the gene leads to a 20 percent decrease in head size.

This seems to mirror what happens in people. Individuals lacking one copy of 16p often have abnormally large heads, dubbed macrocephaly, whereas those with an extra copy tend to have abnormally small heads, or microcephaly.

Another report, published 1 May in Disease Models and Mechanisms², finds that the vast majority of genes in the 16p region affect brain development. In most cases, gene expression must be lowered by at least 75 percent to see an effect. But for two of the genes — ALDOA and KIF22 — cutting the expression in half, which mirrors the loss of a single copy in people, leads to abnormal brain and body shapes.

Studying zebrafish “looks like a good way of prioritizing the genes responsible for a specific part of the syndrome,” especially for clear-cut anatomical features, notes Wendy Chung, assistant professor of pediatrics and medicine at Columbia University in New York, who was not involved in either study.

“The limitation is, you can’t really see autism in a zebrafish,” says Chung, principal investigator of the Simons Variation in Individuals Project, funded by SFARI.org's parent organization, which aims to study people with deletions or duplications of 16p. One obvious next experiment, she says, would be to search for KCTD13 mutations in people who have autism and big heads.

Heads together:

At a grant review meeting in Paris last March, Katsanis happened to sit next to Jacques Beckmann, a medical geneticist at the University of Lausanne in Switzerland. Beckmann told Katsanis about his 2010 results, showing that individuals who are missing a copy of 16p tend to be overweight and have large heads, whereas those with a duplication of the region show the opposite features.

Katsanis says he immediately thought of zebrafish. Since 2008, he and his colleagues have created a library of more than 200 types of fish carrying disease-linked genetic mutations. “It was like a light bulb,” Katsanis recalls. “I thought, ‘Well, I can’t measure obesity and I can’t do behavior, but I can measure head size.’”

Within two months, Katsanis and his team were manipulating 16p genes in the fish. To mimic the effect of a genetic duplication, they injected human RNA of each of the 29 genes in the 16p region into zebrafish embryos and measured their heads four days later. (It’s not possible to measure head size much later than that, Katsanis says, because RNA degrades quickly and any effect it may have will be lost.)

To recapitulate a 16p deletion, the researchers exposed the fish embryos to morpholinos, synthetic molecules that curb the expression of specific genes. In this experiment, the morpholinos reduced expression of each target gene by about 70 percent.

Only 1 of the 29 genes influenced head size: KCTD13. “Essentially nothing” is known about its function, according to Katsanis. But in zebrafish and mouse cell lines, the researchers showed that knocking down the gene leads to an overgrowth of new neurons in the developing brain.

Another piece of evidence came last July, when Katsanis presented preliminary data from this work at a conference in Newport, Rhode Island. He learned that James Gusella, professor of neurogenetics at Harvard Medical School, had found an individual with autism who carries an unusual 16p rearrangement that includes a mutation in KCTD13. What’s more, a study published last year reported a family in which six members, four of whom have autism, are missing a small, five-gene section of 16p. One of those genes is KCTD13.

“This study does not exclude the possibility that there are other genes in the human region of 16p11.2 that also contribute to head size,” Katsanis says. “But it suggests that this one gene is the major driver.”

Active zone:

Because genetically engineered zebrafish are quick and inexpensive to make, at least compared with transgenic mice, they may also be useful for screening hundreds of drugs as potential therapies and for insight on relevant biological pathways.

Others say that approach may be premature for KCTD13. “There’s a long way from a chemical screen to a therapeutic, but especially when it involves the proliferation of brain cells,” notes Hazel Sive, a member of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts.

Sive led the second new zebrafish study on 16p genes. Her team found that when ALDOA gene expression is cut by half, the fish show an abnormal response to touch and develop a narrow forebrain. When KIF22 levels are reduced by half, the fish develop smaller brains and a bent tail.

ALDOA encodes an enzyme involved in metabolism. Two case studies have linked the gene to mental retardation³ and language delay⁴, and postmortem studies have found high levels of expression in the brains of people with schizophrenia and depression compared with controls⁵.

ALDOA interacts with SHANK3, a protein needed for neuronal connections that has been strongly linked to autism. KIF22 is important for chromosome alignment, but little is known about its role in the brain.

Perhaps most intriguingly, Sive says, 20 of 22 genes she tested in the 16p region affect brain development. “We think this is a very active set of genes, much more active than your usual handful of genes,” she says.

Taken together, the studies suggest that the interaction of several genes drives the diverse symptoms of individuals carrying 16p mutations. Zeroing in on specific genes might help tease apart those relationships, Sive says. “We need all the hints we can get if we’re going to understand the genetics of autism.”

References:

1: Golzio C. et al. Nature Epub ahead of print (2012) [Abstract](TK)

2: Blaker-Lee A. et al. Dis. Model Mech. Epub ahead of print (2012) Abstract

3: Beutler E. et al. Trans. Assoc. Am. Physicians 86, 154-166 (1973) PubMed

4: Kreuder J. et al. N. Engl. J. Med. 334, 1100-1104 (1996) PubMed

5: Beasley C.L. et al. Proteomics 6, 3414-3425 (2006) PubMed

Brain teaser: Although commonly used, functional magnetic resonance imaging offers only an indirect measure of neuronal activity. 

Combining functional magnetic resonance imaging (fMRI) of rat brains with a technique that uses light to detect neuronal activity can help researchers hone in on the source of the activity, according to a study published 6 May in Nature Methods¹. 

To identify the brain regions involved in a particular task, researchers often rely on fMRI, a non-invasive imaging method that detects changes in blood flow and oxygen levels in the brain. But this is an indirect measure of neuronal activity, and cannot determine whether neurons in the region are sending or receiving a signal. It also cannot discriminate between the activity of neurons and other brain cells, such as glia, which are support cells in the brain.

Researchers have used animal studies to address some of these issues. In one study, they scanned monkey brains while using an electrical probe to record neuronal activity. However, the metal probe can interfere with the magnetic signals detected during fMRI, making it difficult to interpret the results².

In the new study, the researchers combined fMRI with a technique that records the activity of neurons using light rather than electrical activity. This allowed them to use a small optical probe inserted into a rat’s skull, which they found does not interfere with fMRI signals.

The researchers gave rats sitting in an fMRI scanner a slight shock in their front paws, and then recorded the response in the somatosensory cortex, a brain region that responds to touch. They also detected neuronal activity by injecting rat brains with an indicator dye that fluoresces in response to calcium, which is released when neurons fire.

Neuronal activity is so closely tied to the fMRI signal that in some cases one can predict the other, the study found. However, by using a dye that discriminates between neurons and glia, the researchers found that in some cases glial activity, rather than neuronal activity, contributes to the fMRI response.

The results suggest that interpreting these fMRI signals is not as simple as previously thought, the researchers say.

The researchers also plan to use techniques such as optogenetics to detect signaling from certain subpopulations of neurons. For example, researchers could express a different indicator in neurons that inhibit signals compared with those that activate signals and parse out the relative contribution of each to an fMRI response.

References:

1: Schulz K. et al. Nat. Methods Epub ahead of print (2012) PubMed

2: Oeltermann A. et al. Magn. Reson. Imaging 25, 760-774 (2007) PubMed

Rowdy rodents: Mice lacking SHANK2, an autism-linked protein that functions at neuronal junctions, are hyperactive.

Mice lacking the autism-associated gene SHANK2 show autism-like behaviors similar to those seen in mice lacking SHANK3, another member of the same gene family. But SHANK2 and SHANK3 mice have distinct alterations at neuronal junctions. The results, which were presented at the 2011 Society for Neuroscience annual meeting, were published 29 April in Nature.

SHANK2 and SHANK3 proteins sit at the signal-receiving ends of neurons and help to organize other proteins at synapses, the junctions between neurons. A number of mutations in each SHANK protein have been found in individuals with autism.

Researchers have also engineered several mouse models lacking different regions of the SHANK3 gene. All of these mice exhibit varying degrees of social deficits, repetitive behaviors, such as obsessive self-grooming, and anxiety.

In the new study, researchers created mice lacking either one or both copies of the SHANK2 gene. As with SHANK3 mutant mice, the SHANK2 mice show less interest than controls in other mice and engage in long bouts of repetitive self-grooming. They also have significantly heightened anxiety and hyperactivity, fearing brightly lit corridors and covering twice as much ground as controls do when exploring an open space.

The synapses of neurons from the SHANK2-deficient mice have more NMDA and AMPA receptors, which transmit activating signals to neurons, and more SHANK3 protein compared with controls. By contrast, the synapses from SHANK3 mutant mice have fewer copies than controls do of most versions of these receptors.

Neurons from SHANK2-deficient mice also have fewer dendritic spines, the branches of neurons that receive activating signals, and have weaker excitatory signaling compared with controls.

The results suggest that mutations in different SHANK proteins lead to distinct synaptic alterations that would respond differently to therapies, even though they have a similar effect on behavior, the researchers say.

Alysson Muotri, assistant professor of pediatrics at the University of California, San Diego, has given a whole new meaning to the tradition of the tooth fairy.

A couple of years ago, Muotri and his collaborators developed a way to use stem cells from dental pulp, collected from the innards of lost teeth, to create induced pluripotent stem (iPS) cells — adult cells that have been reprogrammed to a more flexible state.

Muotri is planning to build a bank of such cells from children with autism. By differentiating the stem cells into neurons, he says he hopes to understand the molecular and cellular changes that underlie the disorder, particularly for so-called idiopathic autism — with no known cause — which makes up the bulk of cases.

The tooth fairy approach avoids a trip to the doctor and a skin biopsy, both of which can be especially distressing for children with autism. (iPS cells are typically derived from skin samples.) And teeth can be sent from anywhere in the world.

Ultimately, neurons derived from an individual with autism could be used to select the best therapy for that person. Muotri is already trying a simplified version of this approach, which he presented last month at the Translational Neuroscience Symposium in Switzerland.

Starting with samples from a child in Brazil, researchers discovered the child has a translocation, or DNA swap, affecting two genes: VPRVP, which is involved in the cell cycle but not expressed in the nervous system, and TRPC6, a calcium channel expressed in parts of synapses, the connections between neurons, throughout development.

Gene expression studies revealed that iPS cells derived from the child produce only half the normal amount of TRPC6 protein. After differentiating iPS cells in a dish into cortical neurons, researchers found his cells have fewer dendritic spines — small bumps on neurons that receive inputs from other cells — than those derived from controls. 

Analyzing a second child who has similar outward symptoms to the child with the translocation, researchers found some molecular similarities. Although this child does not have a deletion or translocation of TRPC6, he also has fewer spines on iPS cell-derived neurons. 

What’s more, both have fewer glutamate synapses, neuronal connections that mediate excitatory signals. Muotri says that is similar to neurons from people with Rett syndrome, a rare inherited disorder that shares some symptoms of autism.

He speculates that MeCP2, the gene that is disrupted in Rett syndrome, regulates TRPC6. When they examined iPS cell-derived neurons from someone with Rett, they found low expression of the TRPC6 gene, he says. These results have not yet been published.

How significant these differences are is a matter of some debate. Some scientists argue that the variability inherent in making iPS cells makes it difficult to determine whether the disparities can be attributed to the disorder itself or merely to small differences in the cells’ origins.

Muotri says he hopes that the National Institutes of Health or other organizations will spearhead an iPS cell consortium to try to answer some of these questions. Academic labs such as his own don’t have the resources to create and bank large numbers of cell lines, he says. In fact, the tooth fairy project is on hold, because the lab doesn’t have enough people to process the cells.

A strain of mice lacking the TRPC6 gene is available but has been little studied. Neurons from adult mice, however, have fewer glutamate synapses, similar to those seen in human TRPC6-deficient cells made from the boys with autism, says Muotri.

Muotri and his collaborators are trying to figure out whether these findings can be used for personalized therapy. Hyperforin, a component of the herbal remedy St. John’s wort, has been shown to activate TRPC6 channels. In neurons, hyperforin increases activation of TRPC6, says Muotri.

The outcome of the story is somewhat muddy, as expected in a trial of one. One of the children was given St. John’s wort, which is available over the counter, by his parents. After several months of treatment, the father and therapists reported a behavioral change, and the school reported improved attention, but the mother says she did not see any changes.

If there is behavioral change, “We don’t know if it’s due to the drug or not,” says Muotri. “But it’s very exciting that we found a kid with a similar phenotype to the child with the translocation and saw similar molecular defects in iPS cell-derived neurons.”

Rising rates: Eric Fombonne says the new CDC report does not necessarily mean that autism prevalence is increasing.

In March, the Centers for Disease Control and Prevention (CDC) released what on the surface seemed to be an alarming report. It found that the rate of autism has increased 23 percent — from 1 in 110 children to 1 in 88 — in the past two years, and a whopping 78 percent between 2002 and 2008¹.

The findings immediately raised questions about how much of the rise is because of heightened awareness of autism or changes in the way it is diagnosed, and how much can be attributed to a true increase in its incidence.

The CDC data were collected in 2008 at 14 sites across the U.S. that are part of the Autism and Developmental Disabilities Monitoring Network. Researchers looked at medical and education records for 337,093 8-year-olds and determined whether they met diagnostic criteria for autism spectrum disorders.

Eric Fombonne, Canada Research Chair in child psychiatry at McGill University in Montreal, has spent the past 20 years analyzing the prevalence of autism. He digs into the CDC data with SFARI.org, explaining that big differences among different states and among children of different ethnicities, driven by the challenge of identifying all children with autism, are likely to be major contributors to the apparent increase. 

SFARI.org: Do the numbers in the CDC report represent a real increase in the prevalence of autism?

Eric Fombonne: This interpretation is only one of many. My approach has been to look at these data and systematically examine how alternative explanations could explain these trends. The idea that the incidence is increasing should only be accepted if alternative explanations can confidently be ruled out.

SFARI.org: What are some of these other factors?

EF: Differences in ascertainment [the ability to identify cases in surveys] and in diagnostic concepts and criteria are still significant issues.

Variability in prevalence among different sites provides evidence of ascertainment difficulties. First, it is important to realize that the current CDC rate of 1.1 percent is only an average that is obtained by pooling together different figures and is not more ‘true’ than each estimate that contributes to it.

Click to enlarge image

CDC

Vast variability: There is a more than four-fold difference between the prevalence of autism in Alabama, which has the lowest rate, and Utah, which has the highest. The U.S. average rate is highlighted in red. States that used only health records (green) tend to have lower rates than those that used both health and educational records (blue).

The variability in ingredients of this average is also important to consider. In the CDC study, there is a roughly four-fold difference in the estimates between Alabama, the state with the lowest prevalence (4.8 per 1,000 children), and Utah, the state with the highest prevalence (21.2 per 1,000). (See graph, right.) Nobody interprets these site differences as differences in incidence. It’s a difference in ascertainment.

This variability within the same study can be compared to that between surveys conducted six years apart: 6.6 per 1,000 children according to a 2002 survey and 11.3 per 1,000 children according to the most recent survey, from 2008.

If the four-fold difference in the new survey does not raise concerns about different incidence rates across states, why should a less than two-fold rate difference in surveys conducted six years apart raise concerns about an ‘epidemic’?  

SFARI.org: Were you surprised by the numbers in the new report?

EF: No. I predicted three years ago that we would see such an increase². Some states are improving their identification of children with autism due to better services, heightened public awareness and the development of local expertise. This will continue in the future, especially in states or population subgroups where identification of children with autism is currently lagging behind. I therefore predict that we will see additional increases in national U.S. estimates.

SFARI.org: Why is there so much variability?

EF: [Researchers] try their best to apply uniform methods to identify children with autism in all areas, but they cannot do it as efficiently across states. The prevalence is probably severely underestimated in certain areas. Alabama, Florida and Wisconsin, for example, have rates that are much too low compared with the averages in the U.S. or other parts of the world, which are more or less at one percent. 

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CDC

Margin of error: The states with the highest prevalence, Utah and New Jersey, also have the widest error bars because of their small sample sizes. States that used only medical records to assess prevalence (open circles) tend to have lower rates than those that used both medical and education records (filled circles).

Some states use only medical records, others use both medical and educational records. If next time all states include educational sources, the rates will go up, not because of an increased incidence but because the method will be more efficient at identifying cases. (See figure, left.)

SFARI.org: The changes in prevalence seem to vary among different ethnic backgrounds. From 2006 to 2008, rates among non-Hispanic whites have increased 16 percent, compared with 42 percent among blacks and 29 percent among Hispanics. Is that significant?

EF: Yes. Traditionally, there has been systematic under-ascertainment of these groups, especially Hispanics, in the U.S.

If you look backward to 2006, rates were much lower in children from these ethnic backgrounds, and they were therefore expected to rise relatively more than in more mainstream groups. Indeed, the rates have now disproportionately increased in these ethnic minorities. That is good; it means more children from ethnic minorities are being identified, and hopefully accessing support services. Note, however, that the overall prevalence among Hispanic and black children is still much lower than that among white children by factors ranging from 17 to 33 percent. As a consequence, in future CDC surveys, the rates will ‘catch up’ in these groups, leading to further increases in mean rates in the U.S.

SFARI.org: Do the states with the highest rates reflect the true prevalence of autism?

EF: That is more difficult to evaluate. However, I noted some methodological  peculiarities in the survey methods employed in the two states with the highest rates.

If you look at the figure (above, left), Utah has the highest rate but also the biggest error bar. That is because it had an extremely low sample size, a surveyed population of about 2,100 children, compared with more than 36,000 in Alabama and even over 50,000 in Georgia. Also, New Jersey surveyed a population of 7,000, under the 10,000 initial CDC requirement to be included in the study. Utah and New Jersey were added to the study at a later stage and relied on different sampling strategies (using school districts as compared to entire counties). The consequence is that the denominators used to calculate prevalence in these states had to be estimated with different methods, leaving open the question of their validity.

It is also possible that, because New Jersey historically has been good about financing special education and other services, it attracted more families that have children with autism. Or those families are more likely to stay there.

When I reviewed the data, I also found that children with autism from the Utah sample are unusual in several ways. They have the lowest rate of mental retardation, 13 percent compared with the U.S. average of 38 percent. They also have the lowest male-to-female ratio, 2.7 to 1, compared with the typical average of 4.6 to 1. Taken together, these characteristics are really puzzling, because typically you expect a greater sex ratio at the higher-functioning end of the spectrum. The opposite pattern reported in Utah points to a very atypical sample.

SFARI.org: Do all these factors explain the increase?

EF: We do not know. We cannot demonstrate that all this adds up to the entire increase. Equally, we also cannot infer that the incidence is increasing, which the CDC researchers rightly point out in their abstract.

Irrespective of whether the incidence is increasing, the rate is important to know because it has implications for services.

SFARI.org: How can we better determine whether there is a true increase?

EF: Given its means and funding power, the CDC is the best machine to do this presently in the U.S. Their methods are quite consistent across surveys and over time. It is worth noting, however, that the CDC surveys do not typically screen for autism in children in mainstream schools or home-schooled children. Children are also not directly evaluated for diagnostic confirmation. The CDC has provided convincing data on small subsamples that its indirect approach to diagnosis is valid, but we do not know if this holds true across sites or over time.

One option is to follow large birth cohorts prospectively, as is being done in Norway. If you follow them over time, you can capture most kids with autism at a later age. But the costs would be prohibitive.

I have other ideas, but I’m not sure I would put my money or energy into it. The whole point of demonstrating changes in incidence is to identify causes of autism, such as environmental events, that could potentially be controlled to prevent disease occurrence. But so far, the exposures we know of, such as prenatal exposure to various medications and possibly some neurotoxins or infectious agents, have only weak associations (if any) with autism and can at most explain only a tiny fraction of the increase. We need more persuasive evidence. 

References:

1: Autism and Developmental Disabilities Monitoring Network Surveillance Year 2008 Principal Investigators MMWR Surveill. Summ. 61, 1-19 (2012) PubMed

2: Fombonne E. Pediatr. Res. 65, 591-598 (2009) PubMed

Neural links: Diffusion tensor imaging, which detects the flow of water in the brain, maps connections between brain regions.

The bundles of nerve fibers that connect two regions important for language are abnormal in the brains of children with autism, according to a study published 5 April in the American Journal of Neuroradiology¹.

This difference is more significant in individuals who have both autism and language impairment than in those who have autism alone. It is also more pronounced in the left hemisphere than in the right, the study found.

Scientists have found abnormalities in the structure of white matter, which contains the connecting processes of neurons, and in the connections between brain regions in individuals with autism. In the new study, researchers looked specifically at the superior longitudinal fasciculus, or SLF, a bundle of nerve fibers that connects two language regions called Broca’s area and Wernicke’s area.  

They used a method called diffusion tensor imaging, or DTI, which measures the rate of diffusion of water molecules along long swaths of white matter. Overall, the 35 children with autism in the study have greater diffusion of water in the SLF compared with 25 controls, the researchers found.

This may be because the neurons in these fibers bundle together less tightly, allowing the water to diffuse more freely and lessening the connectivity between brain regions, the researchers say.

These alterations are more pronounced in the 17 participants who have both autism and language impairment than in the 18 children who have autism alone. Overall, all children, including controls, who have lower scores on the Clinical Evaluation for Language Fundamentals test show more diffusion than those with higher scores. This is particularly true in the left hemisphere of the brain, according to the study.

These findings suggest that white-matter abnormalities in the SLF may underlie the language deficits seen in autism, the researchers say.

References:

1: Nagae L.M. et al. Am. J. Neuroradiol. Epub ahead of print (2012) PubMed

Muted markers: Clinical trials for autism rely on behavioral assessments of children, like those used for diagnosing the disorder, rather than on quantitative biomarkers.

The past few years have seen an unprecedented number of clinical trials for experimental drugs to treat autism-related disorders, most notably fragile X syndrome. But as the trials progress, scientists are calling for better methods to measure the drugs’ effectiveness.

“There’s been a big focus on basic science research, but not on quality clinical research,” says Randall Carpenter, president of Seaside Therapeutics, a company in Cambridge, Massachusetts, that is running several clinical trials. “That’s an impediment now because we have all these targets but don’t have the tools to properly do clinical testing.”

The ideal solution, of course, would be a biomarker such as a pattern of brain activity that can be used to quantitate response to treatment. Although the hunt is on for such a biomarker, it’s unlikely to be available in the near future. 

“In the short term, will have to work with tools that are already in use,” says Carpenter. Given that the autism field has seen few placebo-controlled, blinded and randomized drug trials, “there is not much known about how to measure efficacy in clinical trials of people with autism,” he says.

The best-studied tools for assessing autism were developed primarily for diagnosing the disorder, not measuring response to treatment.

“Existing tools were perfected to capture “trait variables” — relatively constant features of an individual over time — but they are not well-designed to capture shifts with respect to autism symptoms,” says John Constantino, professor of psychiatry and pediatrics at Washington University in St. Louis. They are also often cumbersome and time-consuming, he says, not ideal qualities for use in large-scale clinical trials.

The ABCs:

Most ongoing clinical trials for fragile X use parts of the Aberrant Behavior Checklist (ABC), a 58-item questionnaire with five different subscales — irritability, hyperactivity, lethargy/withdrawal, stereotypy and inappropriate speech — that was originally developed for use in people with intellectual disability. These trials rely on the ABC largely because it was used in clinical trials of risperidone and aripiprazole, antipsychotics that are approved to treat irritability in autistic spectrum disorders. Those trials used the irritability subscale of the ABC.

“For anything else — the core symptoms — we have nothing that has been tested before,” says Luca Santarelli, head of neuroscience at the pharmaceutical company Roche, headquartered in Basel, Switzerland.

Companies running clinical trials must define an endpoint before the start of a trial. But most researchers collect additional data along the way, in case the designated endpoint fails but other indicators show an effect. (The new endpoint would then need to be tested in a subsequent trial.)

Roche, which is running clinical trials of an mGluR5 antagonist for fragile X, includes a hypothesis-generating arm in its clinical program to test endpoints that might be sensitive to a given therapeutic approach, says Santarelli. ‘There is no certainty in terms of which will turn out to be successful.”

Based on the emerging data, Roche is building a new instrument tailor-made for use in people with fragile X.

The process for getting new tools approved for clinical trials by the U.S. Food and Drug Administration (FDA), however, can be both time-consuming and expensive. 

“The level of data you have to have to validate these tools for acceptance by the FDA is much higher than [what] you need to publish in an academic journal,” says Carpenter.

Carpenter and his collaborators have instead opted to modify an existing tool, which he discussed at the Translational Neuroscience Symposium in Switzerland in April. They developed a new algorithm, specialized for people with fragile X, for analyzing the results of the ABC.

For example, the lethargy/withdrawal subscale of the ABC includes a number of questions that focus on lethargic behavior. Children with fragile X, however, tend to be hyperactive, so the new algorithm focuses on questions relating to social withdrawal. “We are now validating it in clinical trials to see if it’s sensitive to change,” Carpenter says.

Natural history:

Autism Speaks, a research and advocacy organization, aims to provide some guidance for those embarking on clinical trials. It has convened two working groups to analyze tests that assess social communication deficits, anxiety, and restrictive and repetitive behavior. The results of those efforts have not yet been made public, but are expected to be published soon.

Joseph Horrigan, Autism Speaks’ assistant vice president, says the organization is willing to discuss different clinical outcome measures with researchers.

One of the aspects the organization is evaluating is how well tests developed for other disorders can be used to assess changes in autism symptoms. The Yale-Brown Obsessive Compulsive Scale, for example, is a checklist developed to assess people with obsessive-compulsive disorders. It is sometimes used to measure repetitive behaviors in autism.

“While there are some similarities between the behaviors in those two disorders, there are some significant differences as well,” says Srinivas Rao, chief executive officer of California-based Kyalin Biosciences, a biotech company that is developing an oxytocin-based drug for autism.

Building better tests will, in part, require a better understanding of the natural history of autism.

“Few measures that quantify autism states have been measured in the normal population, so we don’t know how much of a difference is really significant,” says Constantino.

His team is planning to follow both typically developing toddlers and those with autism, using a number of tools to identify changes over time, as well as the most realistic indices to measure those changes.

He and his collaborators are also looking for more efficient and practical ways of evaluating children with autism. They have a paper in press that evaluates a new version of the Childhood Autism Rating Scale, showing that even clinicians with minimal training can reliably score symptom severity based on a 15-minute video of a child¹. 

The potential benefits aren’t limited to drug testing. “For young children, a massive amount of resources are invested in early intervention,” says Constantino, “and there are almost no standardized tools used systematically to track changes over time.”

For more reports from the 2012 Roche Translational Neuroscience Symposium, please click here.

References:

1. Constantino J.N. et al. Neuropsychiatry 2, 1-11 (2012)

Gus Alusi, a British surgeon and cancer researcher, has spent the past couple of years trying out different treatments for his son Kenz, who has fragile X syndrome, one of the most common forms of inherited intellectual disability.

Alusi, his wife Reem, and a team of therapists have tried a number of different drug treatments, such as antidepressants and antipsychotics, hoping to dampen some of the anxiety that goes along with the disorder. 

Alusi videotaped his son’s progress along the way. In early shots, Kenz (pictured at left with his parents) is clearly agitated, whimpering and whining in distress as his mother or therapists try to help him put on clothes and shoes. But in videos taken a year later, he is much calmer. He still has occasional outbursts, but seems better able to pacify himself.

Alusi, who along with his wife has launched a nonprofit advocacy group, Cure4x.org, ascribes the change to the drugs, but also to an intensive regimen of behavioral therapy. The experience has convinced him that medication and behavioral therapy must go hand in hand.

“Behavioral therapy alone is like reading a book in the dark, and medications are like the light,” Alusi said. “You need both to get the full benefit.” (Alusi declined to give the name of the drug that he thinks has most helped his son, saying only that it is approved for pediatric use.)

This approach is not new to psychiatry. Large-scale studies have shown that combining antidepressants with cognitive behavioral therapy is more effective than either treatment alone. People with moderate to severe forms of depression are typically given both.

But this is a tall order for a field that is struggling to develop its first successful drug. No medicines are available to treat the core symptoms of fragile X, though children with the disorder are often treated with drugs approved to alleviate secondary symptoms, such as anxiety, attention deficits and aggression.

Parents, physicians and scientists are anxiously awaiting the results of ongoing clinical trials for a class of drugs, known as mGluR5 antagonists, that have been highly successful in treating animal models of the disorder.

Speaking in April at the Translational Neuroscience Symposium sponsored by pharmaceutical company Roche, Alusi criticized the short length of early-stage clinical trials.

“It’s a waste of time to put my son through clinical trials that last two weeks, which isn’t long enough to see a change in cognition,” he said. “As a parent, I can see you need months of treatment, along with some form of education and stimulation, to produce a tangible benefit beyond simple calming down.”

Randall Carpenter, president of Seaside Therapeutics, which is testing two drugs for fragile X and autism, agrees that this combination is critically important, and something they aim to implement at Seaside. “Even if you normalize brain plasticity and the ability to learn,” he says, “children need training, especially if they are trying make up for lost time.”

For more reports from the 2012 Roche Translational Neuroscience Symposium, please click here.