Role of FMRP in the Regulation of Synaptic Plasticity

The Greenough lab was the first major Neuroscience group to enter the fragile X research field. This group was the first to describe translation of FMRP in response to activation of mGluRs. With more than $1,000,000 from FRAXA Research Foundation over 13 years, Drs. William Greenough and Ivan-Jeanne Weiler at the University of Illinois uncovered the role of FMRP at synapses, leading to much of the subsequent research on fragile X syndrome.

$1,063,000 Grant
William Greenough, PhD
Principal Investigator
Ivan Jeanne Weiler, PhD
Co-Principal Investigator
University of Illinois at Urbana-Champaign
FRAXA Research Grants
$722,000 over 9 Years (2000-2009)
$341,000 over 3 Years (1997-1999)

 

Design and Commercial Production of Mouse Hybridomas to Produce Antibody to FMRP

by Ivan Jeanne Weiler , 1/1/2005

Antibodies to specific proteins, such as FMRP, are currently the most important tools we have to study where the protein goes and what things it interacts with in brain cells. The best antibodies are “monoclonal”, because these cells reproduce indefinitely and will continue to produce this specific antibody. (Antibodies form the basis of the body’s immune system – they recognize and grab onto foreign proteins, viruses, etc. that may pose a threat.)

Most Fragile X researchers still use a monoclonal antibody (1C3) which robustly recognizes FMRP. However, there are problems with it. First, it reacts slightly with another protein, FXR1p, as well as FMRP, so that if we use it to stain tissue, we cannot be sure we are staining only FMRP. This would be important, for example, when determining whether gene therapy had succeeded in helping cells to produce FMRP.

Much of the current research depends on the ability to purify protein clusters which contain FMRP with associated RNA and other proteins, using a technique called immunoprecipitation. For reasons we do not understand, 1C3 fails to precipitate FMRP under normal conditions. Other laboratories have made antibodies to FMRP which will immunoprecipitate, but cannot be used in staining. Our aim is to produce an antibody which will do both.

Because commercial labs have concentrated on developing tricks to elicit monoclonal antibodies with more success than the average university lab, we are contracting with Strategic Biosolutions to produce new monoclonals. We have identified three promising sequences in the FMRP molecule which have not been used before. The company will produce candidate clones based on these sequences and send the clones to us for selection of the best candidates.

If we succeed in obtaining our “dream antibody” we will donate the cells to the Iowa antibody resource which will make the line available to the entire research community.

Studies of the Function of the Fragile X Protein

by Andrea Mitchener, PhD, 6/1/2003

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.

Regulation of FMR1 Gene Expression in Cultured Cells and in Our Mouse Models

by Andrea Mitchener, PhD, 2/1/2000

As outlined in the fellowship application, we are continuing to investigate the rescue of FMR1 knockout mice using Dr. Robert Bauchwitz’ mouse strain that has been genetically engineered to express the human FMRP. Dr. Ivan Jeanne Weiler and her students are carrying out the majority of the current work, but I am contributing to the analysis by characterizing the level of human FMR1 mRNA that is present in individual mice.

In addition to the genetically “rescued” mice defined above, I am actively involved in another project funded by FRAXA and designed to rescue FMRP expression in the knockout mouse. In collaboration with Dr. David Bloom at the University of Florida, we have designed experiments to test the ability of viral vectors to express the FMR1 gene in cultured neurons and in mice. Dr. Bloom has finished the construction of the vectors and testing will begin immediately.

I am continuing to investigate the regulation of the FMR1 gene expression in cultured cells. I have been studying the upregulation of FMR1 mRNA expression after cAMP-triggered cellular differentiation in neuroblastoma cell lines. Currently, I am designing experiments to assess the contribution of transcriptional and post-transcriptional mechanisms that may play a role in expression of the gene. These studies will provide important information that will aid our understanding of FMRP expression in both of our “rescue models” described above, as well as any future studies involving FMR1 gene replacement.