Modelling Autism in a Dish

Human iPSCs derived from patients with Rett syndrome
Monday, 15 November 2010

A collaborative effort between researchers at the Salk Institute for Biological Studies and the University of California, San Diego, successfully used human induced pluripotent stem (iPS) cells derived from patients with Rett syndrome to replicate autism in the lab and study the molecular pathogenesis of the disease.

Their findings, published in the Nov. 12, 2010, issue of Cell, revealed disease-specific cellular defects, such as fewer functional connections between Rett neurons, and demonstrated that these symptoms are reversible, raising the hope that, one day, autism maybe turn into a treatable condition.


This is Alysson R. Muotri, Ph.D., of

the University of California,

San Diego. Credit: UCSD.

"Mental disease and particularly autism still carry the stigma of bad parenting," says lead author Alysson Muotri, Ph.D., an assistant professor in the Department of Molecular and Cellular Medicine at the University of California, San Diego School of Medicine.

"We show very clearly that autism is a biological disease that is caused by a developmental defect directly affecting brain cells."

"This work is important because it puts us in a translational mode," said Muotri.

"It helps expand and deepen our understanding of autism, from behavioural disorder to developmental brain disorder. We can now look for and test drugs and therapies and see what happens at a cellular and molecular level. That's something we've never been able to do with human autistic neurons before."

Rett syndrome is a neurological disorder and the most physically disabling of the autism spectrum disorders. Primarily affecting girls, the symptoms of Rett syndrome often become apparent just after they have learned to walk and say a few words. Affected newborns display normal development until six months to 1½ years of age, "after which behavioural symptoms begin to emerge," Muotri said.

"Progressively, motor functions become impaired. There may be hypotonia or low muscle tone, seizures, diminished social skills and other autistic behaviours."

Then, the seemingly normal development slows down and eventually the infants regress, loosing speech and motor skills, developing stereotypical movements and autistic characteristics.

Almost all cases of the disease are caused by a single mutation in the MeCP2 gene, which is involved in the regulation of global gene expression, leading to a host of symptoms that can vary widely in their severity.

"Rett syndrome is sometimes considered a 'Rosetta Stone' that can help us to understand other developmental neurological disorders since it shares genetic links with other conditions such as autism and schizophrenia," says first author Carol Marchetto, Ph.D., a postdoctoral researcher in the Laboratory of Genetics at the Salk Institute.

Human induced pluripotent stem (iPS) cells

derived from patients with Rett syndrome

allow researchers to replicate autism in the lab

and study the molecular pathogenesis of the

disease. Credit: Illustration: Courtesy of Jamie

Simon, Salk Institute for Biological Studies.

In the past, scientists had been limited to study the brains of people with autistic spectrum disorders via imaging technologies or post mortem brain tissues. The new research goes further. Muotri and colleagues at the Salk Institute for Biological Studies and Pennsylvania State University developed a culture system using induced pluripotent stem cells (iPSCs) derived from RTT patient's skin fibroblasts – cells that typically give rise to connective tissues. Instead, the human RTT-iPSCs were reprogrammed to generate functional neurons that, compared to normal control cells, featured fewer synapses, reduced spine density, smaller soma size, altered calcium signalling and electrophysiological defects – all indications that the deleterious alterations to human RTT neurons begin early in development.

"It is quite amazing that we can recapitulate a psychiatric disease in a Petri Dish," says lead author Fred Gage, Ph.D., a professor in the Salk's Laboratory of Genetics and holder of the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Diseases.

"Being able to study Rett neurons in a dish allows us to identify subtle alterations in the functionality of the neuronal circuitry that we never had access to before."

Marchetto started with skin biopsies taken from four patients carrying four different mutations in the MeCP2 gene and a healthy control. By exposing the skin cells to four reprogramming factors, she turned back the clock, triggering the cells to look and act like embryonic stem cells. Known at this point as induced pluripotent stem cells, the Rett-derived cells were indistinguishable from their normal counterparts.


Neurons generated from Rett-iPS cells

form fewer synapses, the specialized

signal transmission points between

brain cells. Synapses are shown in

red and dendrites, which function as

signal receivers, are shown in green.

Credit: Image: Courtesy of Dr. Carol

Marchetto, Salk Institute for

Biological Studies.

It was only after she had patiently coaxed the iPS cells to develop into fully functioning neurons — a process that can take up to several months — that she was able to discern differences between the two. Neurons carrying the MeCP2 mutations had smaller cell bodies, a reduced number of synapses and dendritic spines, specialized structures that enable cell-cell communication, as well as electrophysical defects, indicating that things start to go wrong early in development.

Since insulin-like growth factor 1 (IGF-1) — a hormone which, among other things, has a role in regulating cell growth and neuronal development — was able to reverse some of the symptoms of Rett syndrome in a mouse model of disease, the Salk researchers tested whether IGF-1 could restore proper function to human Rett neurons grown in culture.

"IGF-1 treatment increased the number of synapses and spines reverting the neuronal phenotype closer to normal," says Gage.

"This suggests that the autistic phenotype is not permanent and could be, at least partially, reversible."

Muotri said IGF1 appeared to rescue some RTT-iPSCs, reverting some neuronal defects, though exactly how IGF1 works remains unknown and requires further investigation.

"This suggests, however, that synaptic deficiencies in Rett syndrome, and likely other autism spectrum disorders, may not be permanent," Muotri said.

Muotri is particularly excited about the prospect of finding a drug treatment for Rett syndrome and other forms of autism:

"We now know that we can use disease-specific iPS cells to recreate mental disorders and start looking for new drugs based on measurable molecular defects."

About autism:
Autism spectrum disorder (ASD) is a range of complex, varying neurodevelopment disorders characterized by social impairments, communication difficulties and restricted, repetitive and stereotyped patterns of behavior. It is not known what causes ASD. Scientists have identified a number of genes associated with the disorder, but environmental factors likely play a role too.
Autistic disorder (sometimes called autism or classical ASD) is the most severe form. Milder conditions include Asperger syndrome, Rett syndrome, Childhood Disintegrative Disorder and Pervasive Developmental Disorder Not Otherwise Specified (usually referred to as PDD-NOS).
The Centers for Disease Control and Prevention reports the prevalence of autism to be approximately 1 in every 110 births in the United States. An estimated 1.5 million Americans live with the effects of ASD, which occurs in all ethnic and socioeconomic groups and affects every age group, though males are four times more likely to have ASD than females.


Source: Salk Institute and University of California - San Diego
Contact: Gina Kirchweger and Contact: Scott Lafee
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ZenMaster

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