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Telltale Protein Defects Mark Fragile X Pathways

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A team of scientists led by National Institute of Mental Health Health (NIMH) grantees has identified a trove of proteins involved in synaptic plasticity and neuronal growth—some of them likely implicated in mental retardation and perhaps other neurodevelopmental disorders like autism. A new technique they developed revealed, in living neurons, a swath of secondary damage caused by the primary protein defect in Fragile X syndrome, the most common inherited form of mental retardation. Gene knockout mice modeling this defect showed abnormalities in the distribution and quantities of some of the affected secondary proteins and the genetic material that makes them.

A melding of genomics and proteomics, the new method, called Antibody Positioned RNA Amplification (APRA), can be applied in similar studies of other systems and cells, say James Eberwine, Ph.D., University of Pennsylvania, and colleagues, who report their findings in the February 6, 2003, issue of Neuron.

Fragile X symptoms in children can range from mild learning disabilities to severe mental retardation, often accompanied by physical and behavioral abnormalities. While it's known that a glitch in the gene that makes Fragile X mental retardation protein (FMRP) causes the syndrome, scientists have had only a murky picture of the downstream consequences of this defect on other genes, proteins, and a neuron's function. Defective FMRP can have such devastating effects because as an "RNA binding protein" it influences many other proteins in critical brain centers, like the hippocampus, a memory hub. FMRP regulates the synthesis and transport of a bevy of here-to-fore unknown associated proteins. Like a dispatcher in a truck depot, FMRP manages the shuttling of these "cargo proteins" from the cell's nucleus to supply the needs of its working parts, or cytoplasm.

"Much of the cargo turns out to be the genetic material (RNA) that makes proteins vital to synaptic maturation and communication between neurons—which breaks down if the 'dispatcher' can't do its job," explained Eberwine.

To discover FMRP's cargo proteins in cultured mouse hippocampal neurons, the researchers devised an intricate methodology (APRA) that takes advantage of the specific affinity that antibodies and short strands of genetic material have for particular genes and proteins. They joined an antibody that binds to FMRP with genetic material that, in turn, binds to genes associated with FMRP. The antibody positions the molecular probe close to the FMRP cargo so that it can detected. The researchers then copied enough of this molecularly-engineered bait to fish for FMRP-associated genes in the pool of genes expressed in the human brain. About 60 percent of the catch proved to be genes directly associated with FMRP, again, many involved in synaptic plasticity and neuronal maturation.

When the researchers examined expression of genes that make some of these proteins in a genetically modified strain of knockout mice lacking the FMRP gene, they found that some differed in quantity and/or distribution within cells from normal mice. For example, one of FMRP's cargo proteins turns out to be the receptor in cells' cytoplasm that binds to glucocorticoids, hormones secreted in response to stress. Unlike normal mice, the knockout mice lacked glucocorticoid receptors in the branch-like dendrites where synapses form. They also showed less receptors in synapses, suggesting a breakdown in transport or local synthesis of the cargo protein. Children with Fragile X syndrome show stress hormone abnormalities when mildly stressed, as well as heightened anxiety and related learning deficits. The absence of FMRP may disrupt intricate feedback mechanisms in which stress hormones play a pivotal role, suggest the researchers.

Since 15-40 percent of people with Fragile X syndrome show autistic behavior, the researchers suspected that some FMRP cargo proteins might also be associated with autism. Among the 81 proteins, 15 mapped to the same chromosomal locations as candidate autism genes. Mutations in some of the genes that code for these proteins may contribute to autism and other disorders characterized by autistic-like social impairment and stereotyped behavior, they suggest.

Also participating in the study were: Drs. William Greenough, Ivan Jeanne Weiler, Andrea Beckel-Mitchener, Lei Liu, and T. Patrick Purk, University of Illinois; Dr. Kevin Miyashiro, University of Pennsylvania; Drs. Kevin Becker, Tanya Barret, National Institute on Aging (NIA); Dr. Slavatore Carbonetto, McGill University.

In addition to NIMH and NIA, the research was also funded by the National Institute of Child Health and Human Development (NICHD), and the FRAXA Research Foundation.

 

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NIMH, NIA, and NICHD are part of the National Institutes of Health (NIH), the Federal Government's primary agency for biomedical and behavioral research. NIH is a component of the U.S. Department of Health and Human Services.

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About the National Institute of Mental Health (NIMH): The mission of the NIMH is to transform the understanding and treatment of mental illnesses through basic and clinical research, paving the way for prevention, recovery and cure. For more information, visit the NIMH website.

About the National Institutes of Health (NIH): NIH, the nation's medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit the NIH website .