Principal Investigator: William Greenough Ph.D.
Co-Investigator: Ivan Jeanne Weiler, Ph.D.
Beckman Institute, University of Illinois
Project Grants and fellowships funded 1998 through 2003 totalling over $100K per year.
by Andrea Mitchener, 6/2003
Several projects are ongoing in our lab. First, we are identifying and characterizing messenger RNAs that bind to the Fragile X Protein (FMRP). Due to the absence of FMRP in Fragile X Syndrome, normal expression of other proteins is very likely disrupted. We predict that altered expression of these proteins may contribute to the symptoms seen in Fragile X. Using a new technique, Antibody Positioned RNA Amplification (APRA), developed with Dr. Jim Eberwine at the University of Pennsylvania, we have characterized some mRNAs which are bound to FMRP in cultured neurons.
One mRNA target identified by APRA is the glucocorticoid receptor (GR). We have found that GR protein expression is reduced in the hippocampus of FMR1 knockout mice. GR is part of the Hypothalamic-Pituitary-Adrenal (HPA) axis and is necessary for proper functioning of the feedback loop that regulates the physiologic response to stress. Work by Allan Reiss and colleagues suggests that the stress response in Fragile X patients is perturbed: cortisol levels (the glucocorticoid hormone released in response to stress) are higher in patients and show a protracted return to baseline compared to controls. I am examining the response of Fragile X mice to stress. Since the ability to cope with stress can play a critical role in quality of life and affect learning, these studies may suggest a pathway that can be targeted with drug or behavioral interventions.
A second project involves the construction and testing of non-replicating recombinant viral vectors carrying the FMR1 gene. In collaboration with Dr. David Bloom at the University of Florida, we are testing two viral vector systems, Herpes Simplex Virus (HSV) and Adeno-associated Virus (AAV), for their ability to deliver the FMR1 gene into neurons from knockout mice. Our initial tests revealed that the specificity of FMRP expression needed to be improved. We have now redesigned the vector to ensure exclusive neuronal expression and we expect improved results using a new promoter arrangement. These vectors will be useful tools for Fragile X researchers and they will provide valuable information as to the requirements for (and potential pitfalls of) FMR1 gene therapy.
Imjoo Rhyu, Co-investigator, 1/2002
This project will examine detailed shape and structure of dendritic spines (where synapses are found) using high voltage electron microscopy. This technique allows us to examine dendritic spines with very high resolution, and the images obtained can then be used to analyze synapse structure in three dimensions. Using electron microscopy, we will also analyze the distribution of the protein production machinery (polyribosomes) near dendritic spines, which will provide information about whether protein synthesis is altered near synapses in Fragile X Syndrome, as predicted by our other work. A separate project involves whether new neurons are generated to the same degree in Fragile X knockout mice as they are in control mice. Relatively new findings in the field suggest that neurogenesis in specific brain regions is a fundamental process that takes place throughout the life of an animal.We are interested in determining whether the generation of new neurons is altered in Fragile X Syndrome.
by William Greenough, 8/2000
We have determined that FMRP, the protein encoded by the gene that is inoperative in fragile X syndrome, is synthesized at the synapses through which nerve cells communicate in response to neurotransmitters, the chemicals of communication. FMRP is the first protein demonstrated to be regulated in this manner. We have also found, in the FMR1 knockout mouse and in human autopsy samples, neuroanatomical changes that help to understand the disorder. Finally, we have found that the "knockout" mouse model of the syndrome is incapable of the neurotransmitter-activated protein synthesis at synapses. We have also found that the normal brain produces FMRP in response to experience, suggesting that later therapeutic intervention may still be effective, even though the period of early development has passed. Some current research:
A. Basic Research: Understanding the role of the fragile X gene in brain function.
1. Basic Structure of the Fragile X-affected Brain
One area of great importance is understanding the effects of the syndrome on brain development and organization. My laboratory group laid out many of the procedures for quantitative analysis of morphology during our studies of experience- and learning-induced brain plasticity from the 1960's until the 1990's. These procedures are ideal for understanding what is happening in the brain across the lifespan. The most basic questions regard structure and number of synapses, a matter about which there is significant controversy, and one about which most issues can be resolved by quantitative electron microscopy. Different brain regions express FMRP differentially across the lifespan, and these regions can be used to study what happens when FMRP levels rise or fall off. These studies would also look at exuberance in development, the tendency of the brain to make connections that it does not later keep. Our studies of human autopsy samples suggest that pruning of these connections may be impaired in fragile X syndrome and that these extra connections might underlie some of the symptoms of the disorder. Alternatively, the syndrome may involve continual addition of connections.
2. Plasticity of the Fragile X-affected Brain
One fundamental property that could lead to learning deficits is a failure of the brain's normal cellular information storage mechanisms. We have developed excellent behavioral procedures for studying these systems over some 32 years of studying plastic neural mechanisms and can assess their functioning in detail. We are now looking at effects of exposure of fragile X knockout mice to an "enriched" rearing environment to see whether the effects on brain morphology are the same as those reported for normal mice.
B. Studies of the Fragile X Protein and Possible Treatment Directions
In addition to these fundamental studies, because of a number of recent discoveries in our laboratory, we are poised to move quickly now in several promising directions that we believe can have significance for potential treatment.
1. Approaches to understanding the function of FMRP
a) to determine the cellular mechanisms regulating FMRP and synaptically-regulated protein synthesis. We have described a novel "cascade," or signalling pathway, that links synaptic firing to synthesis of FMRP. Working out the details of this pathway could provide clues as to how to bypass the need for FMRP.
b) to determine what FMRP does in the cell. We have determined that the FMR1 knockout mouse is incapable of neurotransmitter-activated protein synthesis at the synapse. This is a significant breakthrough which implies a role for FMRP in synaptic synthesis of other proteins. We are examining how FMRP acts, in hopes of finding ways to substitute for its function.
c) to determine which proteins are dependent upon FMRP for their synthesis. It seems likely that at least some symptoms of fragile X syndrome arise from impaired synthesis of these proteins. Delineating them is a first step towards identifying which proteins may be responsible and ultimately towards bypassing the need for FMRP.
2. Approaches to Treatment Using the Animal Model:
a) to characterize the behavioral syndrome in the knockout mouse, such that the effects of interventions can be evaluated. We are examining an array of behaviors in knockout vs. genetically normal mice.
b) to evaluate molecular interventions as future treatment directions. We are examining effects (on molecular measures) of breeding in a human fragile X gene that has been inserted into an autosome (non X,Y chromosome; collaboration with Robert Bauchwitz, Columbia University) and of restoring the mRNA for the damaged gene to the brain using a viral vector (collaboration with David Bloom, University of Florida).
c) to evaluate a potential drug intervention. We are examining the effects of a drug that affects synaptic communication on the brain structural effects of the knockout mouse syndrome, and the interactions of the drug with developmental experience manipulations. We hope to initiate behavioral studies of the effects of the drug soon. If these studies provide any evidence of efficacy, the drug manufacturer is prepared to support clinical trials.
d) to evaluate the efficacy of behavioral intervention. This requires a reliable knockout behavioral phenotype (a, above). Our goal would be to mimic behavioral therapy in the animal model. We have previously demonstrated brain and behavioral rehabilitation in an animal model of fetal alcohol-induced brain dysfunction, using a motor training intervention.
REFERENCE
Synaptic regulation of protein synthesis and the fragile X protein
William T. Greenough, Anna Y. Klintsova, Scott A. Irwin, Roberto Galvez, Kathy E. Bates, and Ivan Jeanne Weiler
Proc. Natl. Acad. Sci. USA. 2001 June 19; 98 (13): 71017106
