The new tool, called Antibody Positioned RNA Amplification (APRA), was created in the lab of James Eberwine, a professor in the department of pharmacology at the University of Pennsylvania School of Medicine. It allows for a detailed analysis in intact cells -- in this case, neurons -- of mRNA (messenger ribonucleic acid), which is essential for the transportation of genetic information from DNA to protein-producing areas.
APRA was created to study mRNA molecules involved with the Fragile X Mental Retardation Protein (FMRP), which when missing leads to Fragile X syndrome, the most common form of inherited mental retardation, especially in males. APRA and the mRNA-protein interactions it unveiled are detailed in the Feb. 6 issue of the journal Neuron.
Intense interest has focused on FMRP since Illinois researchers William T. Greenough and I.J. Weiler of the Beckman Institute for Advanced Science and Technology, co-authors on the Neuron paper, first reported in 1997 that the Fragile X protein is synthesized at synapses, the connections through which nerve cells communicate.
Using an approach in which FMRP was produced in bacteria, Greenough's postdoctoral associate Andrea Beckel-Mitchener, also a co-author, confirmed the binding of FMRP mRNA with the numerous molecules identified with APRA at Penn. Among them, she found the glucocorticoid receptor, a protein necessary for the regulation of circulating levels of adrenal corticosteroids following exposure to stress.
Weiler found lowered levels of the receptor protein in synaptic preparations. Beckel-Mitchener then carried the confirmation a step further. Looking in the hippocampus in normal mouse brains, she found glucocorticoid receptors in cell bodies and their associated dendrites. In experimental mice modified to not produce FMRP, the receptors were found in cell bodies but were virtually absent in the dendrites.
Previous research at Stanford University Medical Center and the University of Minnesota had found a deficiency in corticocosteroid regulation after exposure to mild stress in children with Fragile X syndrome. A reduced level of the glucocorticoid receptor, in the absence of the FMRP protein, Weiler said, would plausibly affect corticorsteroid feedback mechanisms, leading to prolonged levels of anxiety in Fragile X patients.
"We have shown that a particular messenger RNA -- the product of a particular gene which encodes the glucocorticoid receptor -- is bound to the Fragile X mental retardation protein, and it is differentially regulated in its expression in neurons of animals than can make FMRP versus animals that cannot," said Greenough, who holds a Swanlund Endowed Chair at Illinois. "In essence, we've shown that a major symptom of Fragile X syndrome is accounted for, in principle, by the binding of FMRP to the gene product, the messenger RNA that encodes this receptor."
The technique and protein-mRNA discoveries offer a new view of Fragile X syndrome and a potential approach to develop new treatments, said Weiler, an adjunct professor of psychology. "Fragile X up to now has been considered to involve a single missing protein, but it turns out that the protein involved is probably governing the production and localization of a group of sub proteins, making this a multigenic disease," she said.
"We are very pleased with this new methodology that Dr. Eberwine has developed," Weiler said, "because for the first time FMRP and its associated mRNAs for this subset of proteins have been localized in situ -- in an intact neuron -- rather than in homogenate brain tissue. So we feel that he has been able to localize the culprits in the act, so to speak."
Now, Weiler said, the entire group of proteins identified by APRA needs to be studied. "Now we can go on to define what it is that Fragile X patients are most basically needing -- in what part of the brain and at what ages," she said. "There is some hope for narrowing down the paths of possible remedial steps that could be taken."
APRA is described in more detail in a news release issued by the Penn School of Medicine (http://health.upenn.edu/News/News_Releases/feb03/FragileX.html).
The research was funded through grants from the FRAXA Research Foundation, National Institute of Child Health and Human Development, National Institute on Aging and the National Institute of Mental Health.
Illinois researchers were Greenough, Weiler, Beckel-Mitchener and doctoral student T. Patrick Purk, all of the Beckman Institute, and Lei Liu of the W.M. Keck Center for Comparative and Functional Genomics. Liu also is director of bioinformatics in the Illinois Biotechnology Center.
Other researchers were Eberwine and Kevin Miyashiro at Penn, Salvatore Carbonetto of McGill University, and Kevin G. Becker and Tanya Barret of the National Institute of Aging.
Journal
Neuron