The Autism Tissue Program, or ATP, is pleased to announce 8 new research projects which have been reviewed and approved by the Tissue Advisory Board. These projects involve a wide range of disciplines to better understand the neurobiology, neuropathology, and neurochemistry of autism, using state of the art techniques which will help better identify genetic and environmental influences of autism. These projects would not be possible without the resources provided by Autism Speaks and the Autism Tissue Program, and represent both new and collaborative ideas which were closely reviewed by the ATP Tissue Advisory Board.

How Might Environmental Factors Contribute to Autism?

Richard Deth, Ph.D.
Department of Pharmaceutical Science
Northeastern University

Alternative Splicing of Brain Methionine Synthase in Autism

The incidence of autism has increased dramatically in recent years, suggesting that environmental factors may play a role in causing the condition. Previous studies suggest that toxins such as mercury, lead, arsenic and other substances affect the development of neurons through oxidative stress and inflammation. One mechanism by which these toxins act is to lower the concentration of the antioxidant glutathione in the brain. An important regulator of the production of glutathione is the enzyme methionine synthase. Methionine synthase is also important in the function of the neurotransmitter dopamine which helps to regulate brain activity. Certain vitamins, such as folate and vitamin B-12, which have proven effective in treating autism, act to increase the activity of methionine synthase.

While the expression of glutathione and methionine synthase has been previously studied in serum from individuals affected with autism, this study will examine how this enzyme is expressed in brain tissue. The function of the brain may not be well characterized by what is found in blood. Dr. Deth will be collaborating with Dr. Antonio Persico who has isolated DNA and mRNA using brain tissue, thereby expanding the use of an already existing resource.

Significance: If the research finds differences in the methionine synthase associated with autism, it will provide researchers with more information about potential genetic susceptibilities which may interact with environmental factors.

Exploring Genetic Influences in Autism Spectrum Disorders

Arthur Beaudet, M.D.
Department of Molecular and Human Genetics
Baylor College of Medicine

Testing A Mixed Epigenetic/Genetic and De Novo (MEGDI) Model for Autism

The genetics of autism spectrum disorder are very complex and while many studies have reported specific changes in changes in DNA sequence or genotype in individuals affected with autism, some changes in gene expression are not detected by traditional techniques. Changes in the DNA code may be observed through mutations in the DNA code, or through chemical modifications of the DNA which produces changes in gene expression. This is called an epigenetic or “de novo” mutation and may be induced spontaneously or through environmental exposure. This area of study, called “epigenetics”, refers to changes in DNA which do not result from a change in the sequence of the molecules in the DNA, but from the addition of a methyl group (a simple carbon atom with three hydrogen atoms attached) to a section of the DNA known as a “CpG” island. Mutation of the gene which regulates coding of a protein responsible for methylation of DNA is responsible for Rett's Syndrome, a condition considered to be on the autism spectrum. This abnormal methylation of DNA which can adversely affect the functionality of the brain cell may contribute to other types of autism as well.

Utilizing the most advanced technologies available to study methylation patterns, this project will compare and contrast the methylation patterns in brain tissue from donors and the pattern identified from blood samples in individuals affected with autism. As part of an expanded project, blood from family members will also be analyzed to determine how these epigenetic factors are inherited.

Significance: This research will help scientists better understand the role of genetic factors in autism spectrum disorder that may not be revealed through changes in the DNA sequence itself.

How Does the Brain Develop Differently in Autism?

Gene Blatt, Ph.D.
Department of Anatomy and Neurobiology
School of Medicine
Boston University

Studies of the Fusiform Face Area, Broca's Area, and Wernicke's Area in Autism

Autistic individuals typically exhibit weak social skills due to deficits in language, attention, communication, and the ability to interpret the emotions of others. When autistic individuals interact, they spend less time looking at the faces of others with whom they are communicating, and when they do, they tend to focus on one particular facial feature. They miss important facial expression cues that are essential to normal communication.

Previous studies suggest that a cause of this disability may lie in the part of the brain known as the fusiform gyrus, or fusiform face area. Neuroimaging techniques such as magnetic resonance imaging (MRI) show that this part of the brain is less active during face processing. This hyperactivity may “short circuit” the brain's ability to interpret facial cues during social interactions.

This study examines the anatomy of the fusiform gyrus in autistic individuals to determine if there is a difference in neuroanatomy when compared with normal individuals. The study will examine the cellular structure to determine if the circuitry or signal conductivity in the brain is altered. Two other brain regions important to speech, known as the Broca's and the Wernicke's areas, will also be studied in the same manner. The role of GABA, a neurotransmitter found in the central nervous system that has been implicated in autism, 5HT and glutamate transporters and receptors will also be analyzed to determine if they play a role in the circuitry of these brain regions.

Significance: By discovering neurological differences possibly responsible for social perception and speech difficulties, this study may lead to therapies to address potential underlying signal transmission deficits in the brain.

Eric Courchesne, Ph.D.
Department of Neuroscience
University of California at San Diego

Comprehensive Analysis of Regional Microstructural Differences in Autism and Laminar Organization and Gene Expression Profiling in the Dorsolateral Prefrontal Cortex in Autism

Individuals with autism have difficulty with cognitive planning, joint attention and abstract thinking, functions which are often attributed to abnormalities in an area in the frontal lobe of the brain called the dorsolateral prefrontal cortex. Past behavioral, imaging and postmortem research studies provide compelling evidence that the frontal lobe of the brain develops abnormally in children with autism. In fact, the dorsolateral prefrontal cortex is 12% larger in children with autism under five years of age compared to normal children. Furthermore, little is known about defects in the number of neurons found in this area, and their thickness and arrangement when compared with other areas of the brain and when compared with neurotypical brains.

Inadequate information is available to explain the genetic and developmental processes that cause this early brain overgrowth and the related behavioral deficits associated with the dorsolateral prefrontal cortex. Using tissue from ATP, Dr. Courchesne and his colleagues will identify structural abnormalities in the development of the autistic brain. This research will comprehensively analyze the tissue for changes in the number of cells by counting the cells and by looking at the microstructure of the frontal cortex. A comparison of potential defects will then be made by analyzing tissue in other areas of the same brain (the temporal, parietal and occipital lobes). Structural differences between brains of people with autism and those without autism will also be assessed. Abnormal developmental changes over time will also be analyzed by examining brain tissue from various age groups.

One potential way in which the cell size and number in the dorsolateral prefrontal cortex could be altered in autism is because during development, new neurons are not migrating to their proper locations. This hypothesis will be tested by examining expression of “laminar specific markers” in brain tissue which will map the migration patterns in the different layers of the cortex. This part of Dr. Courchesne's study will identify whether or not during brain development, neurons migrate to their proper specific cortical layers, and also describe patterns of gene expression that may be different in individuals with autism. The gene expression patterns identified include the previously mentioned “laminar specific markers” as well as other genes involved in neurogenesis, cell death, and cell to cell signaling.

Significance: Identifying the neural processes that lead to abnormal brain function will provide important information relating the underlying mechanisms of developmental brain abnormalities in autism. Dr. Courchesne will be collaborating with a number of investigators who represent experts in the field of neurobiology, molecular biology, and neuranatomy to better understand the mechanisms by which the dorsolateral prefrontal cortex is impaired in autism.

Understanding How Brain Chemistry Influences Autism

Ricardo Miledi, M.D.
Department of Neurobiology and Behavior
University of California, Irvine

Studies of Neurotransmitter Receptor in the Behavior of Autistic Brain

Autistic individuals often exhibit disabling behaviors such as repetitive movements, self injury and self stimulation. A possible cause of these behaviors may be an imbalance in the brain chemicals known as neurotransmitters in the brains of individuals suffering from autism. Examples of neurotransmitters thought to be important in autism are GABA, dopamine, serotonin and glutamate. These neurotransmitters are one of the means by which signals are relayed between neurons in the brain. The imbalance may be due to alterations in the function of neurotransmitter receptors, that part of neurons which receive the signal from the neurotransmitter.

The primary goal of the study is to determine the differences in functional properties of the neurotransmitter receptors of autistic individuals when compared to normal individuals. This will be done by isolating the receptors in brain tissue and studying their genetic makeup, electrophysiological properties and interactions with known medications.

Significance: The results obtained will provide the first functional and structural characterization of neurotransmitter receptors in the autistic brain and their reaction to commonly used medications. The knowledge gained from the effect of drugs on specific brain tissue at the molecular level will improve decision making concerning the use of existing drugs and may lead to improved therapies for autism and specific symptoms of autism.

Patrick Gregory, Ph.D.
Unit of Anatomy
University of Fribourg

Expression of the Calcium-Binding Protein Parvalbumin (PV) and the Autistic Brain

A network of neurons which use GABA as a neurotransmitter, called GABAergic inhibitory neurons, are responsible for signaling across the brain, and control coordinated activities including filtering of signals across sensory modalities. Many investigators have hypothesized that impaired GABAergic signaling may serve as an underlying etiology in autism spectrum disorders. While Dr. Blatt is investigating the expression of GABAergic receptors, Dr. Gregory will be examining the expression of a calcium binding protein, parvalbumin, which is important in the transmission of nerve impulses. In previous research, genetically modified mice known as “knockout mice” have been altered so that they do not express one or several of these calcium-binding proteins. Behavioral studies on these mice showed that they exhibited certain symptoms similar to autistic like behavior (e.g. stereotypic movements and a decrease in some aspects of social interaction). Previous studies by other research groups have shown that these proteins are decreased in particular brain areas of patients with schizophrenia and the absence or decrease in these proteins have also been linked to epilepsy. The goal of this research is to establish if there is a decrease in these proteins in the brains of people with autism by analyzing the brain tissue. In addition to studying expression of parvalbumin in brain tissue, the number of GABAergic inhibitory neurons will be counted.

Significance: If the pathology of autism is better understood this would lead to appropriate treatment interventions. For example, if in fact there is a decrease in calcium binding proteins within the brain, then this could lead to a potential treatment to alleviate the symptoms of autism. The investigations being conducted by both the Gregory and Blatt labs will help determine the role of GABA signaling in autism, both in terms of specific cell type and neurochemical activity.

Kazuhiko Nakamura, MD, PhD
Department of Psychiatry and Neurology
Hamamatsu University School of Medicine

mRNA Expression Analyses Of Serotonin Transporter (5-HTT) and Serotonin Transporter Related Candidate Genes in Postmortem Brains

Many studies suggest that brain chemicals, known as neurotransmitters which relay signals between neurons, play a role in autism. Neurotransmitters act by being released by one neuron and then acting on a receptor on a post-synaptic neuron, relaying a signal in the brain. Neurotransmitters can also be reabsorbed by the parent neuron through transporters which both release and recycle released neurotransmitter actions. Dysfunction in the brain system controlling the neurotransmitter serotonin has been linked to autism. Drugs which slow serotonin reabsorbtion, know as selective serotonin reuptake inhibitors (SSRI's), have a beneficial effect in some autistic individuals. Dr. Nakamura and his colleagues have already examined serotonin expression using PET scans from individuals with autism.

This study seeks to better understand the aspect of the serotonin system in the brain which relates to the reuptake of serotonin by exploring the mRNA (messenger RNA) expression of the serotonin transporter as well as other serotonin genes found in brain tissue. His group will then screen some of the candidate genes (genes that are implicated in causing a disease) using an animal model to determine which are required for normal human development.

Significance: Previous studies using imaging methods such as PET scans suggest that individuals with autism have reduced serotonin transporter expression. This study seeks to confirm and expand these findings by examining brain tissue. Understanding the role of the expression genes, the process in which the gene is switched on at a certain time and then controls serotonin neurotransmission in autism, may lead to new approaches to drug development.