Gary Bassell, PhD—Emory University School of Medicine
Neuronal Function of FMRP and Signaling Abnormalities in Fragile X

Gary Bassell, PhD, Principal Investigator
Jason Dictenberg, PhD, FRAXA Postdoctoral Fellow (2004)
Ravi Muddashetty, PhD, FRAXA Postdoctoral Fellow
Vijayalaxmi Nalavadi, PhD, FRAXA Postdoctoral Fellow
Laura Antar, Graduate Student (2004)

FRAXA Awards:

$45,000 in 2012
$45,000 in 2011
$40,000 in 2009
$80,000 in 2008
$40,000 in 2007
$15,000 in 2004
$35,000 in 2003
$65,000 in 2002
$30,000 in 2001
$30,000 in 2000


Nerve cells have long processes called axons and dendrites which extend outward to form connections (synapses) with other nerve cells. Dr. Bassell's team has developed powerful molecular genetic techniques to track mRNAs and FMRP particles as they move through these processes in brain tissue from Fragile X knockout mice. Their work indicates an important role for FMRP, the protein lacking in fragile X syndrome. They are studying how FMRP transports mRNAs from a neuron's neucleus to the synapses, and then helps translate those mRNAs into proteins. This information will lead to candidate genes for autism as well as treatment approaches.
Genetic and pharmacologic manipulation of PI3K activity in FXS: assessing the potential therapeutic value

Dr. Bassell's group has shown that a specific intracellular signaling pathway, the PI3K/mTOR pathway, is overactive in the absence of FMRP. This pathway is involved in mediating many neuronal neurotransmitter receptors. This project will test new drugs in development which inhibit an enzyme known as PI3 kinase, a part of the pathway, and have the potential to normalize neuronal function in fragile X.

by Christina Gross, 4/20/2012

Animal models of the fragile X syndrome (FXS) are characterized by excessive and dysregulated signaling through metabotropic glutamate receptors 1 and 5 (mGlu1/5). Recent studies have shown that apart from mGlu1/5, signaling through other neurotransmitter receptors is also exaggerated in FXS, suggesting that the fragile X mental retardation protein (FMRP) might regulate a common downstream signaling molecule shared by multiple pathways. In line with this assumption we and others have shown that a specific intracellular signaling pathway, the phosphoinositide-3 kinase (PI3K)/mTOR pathway, which mediates signaling downstream of many neuronal neurotransmitter receptors, is overactive in the absence of FMRP. Our studies have shown that FMRP associates with and regulates the mRNA of p110ß, a catalytic subunit of PI3K, leading to excessive p110ß mRNA translation, protein expression and enzymatic function at synapses from Fmr1 KO mice (Gross et al., 2010). Additionally, we and others found that FMRP also associates with the mRNA encoding the PI3K enhancer PIKE, and that synaptic PIKE protein expression is increased in Fmr1 knockout mice. Taken together, these studies suggest that FMRP may play an important role in regulating and limiting signaling through the PI3K/mTOR pathway. We thus hypothesize that pharmacologic treatments targeted at the PI3K/mTOR pathway might be a promising therapeutic strategy for patients with FXS. To further explore the PI3K/mTOR signaling complex as a therapeutic target in FXS, we are currently using genetic and pharmacologic rescue approaches to reduce the enzymatic function of specific components of this complex pathway in an FXS mouse model. Results from the first year of FRAXA funding suggest that these PI3K/mTOR pathway-specific rescue models for can reverse several FXS-associated neuronal dysfunctions, including cell-biological and behavioral phenotypes.

Specific drugs targeted at components of the PI3K/mTOR signaling pathway are already being developed and used for cancer research. In this project, we are taking advantage of these advances in cancer research, by testing a specific p110ß-selective antagonist, which is currently used in a clinical trial with human cancer patients, for its potential to rescue or ameliorate FXS-associated phenotypes in the mouse model. We anticipate that in the future, PI3K/mTOR-targeted treatment strategies in patients with FXS could possibly benefit from drugs developed in cancer research.

FMRP as a modulator of dendritic mRNA translation in response to metabotropic glutamate receptors

Dr. Muddashetty and colleagues have discovered FMRP targets at synapses: postsynaptic density-95 (PSD-95) mRNA, AMPA receptor subunits, GluR1 and GluR1 mRNAs, and -CaMKII. The synaptic protein synthesis for each of these targets is dysregulated in response to activation of group 1 metabotropic glutamate receptors (mGluR) in a mouse model of Fragile X syndrome (Muddashetty et al., J. Neuroscience 2007). Each of these targets are known to have critical roles in synaptic plasticity, learning and memory.

by Ravi Muddashetty, 5/2/2008

The overall goal of my project is to identify and characterize possible defects in the mGluR dependent translation of FMRP target mRNAs in the mouse model of fragile X syndrome (FXS) using synaptosomal preparation and cultured hippocampal neurons. I have developed a reliable assay to test the translational efficiency of dendritic mRNAs from a synaptoneurosomal preparation. I successfully used this assay to demonstrate translational dysregulation of dendritic mRNAs in Fmr1 knockout mice brain, specifically in the synaptic preparations. These results were published in the Journal of Neuroscience (Muddashetty et al., 2007). During the first year of my FRAXA fellowship, I have used this assay to demonstrate that MPEP, a selective mGluR5 antagonist, can significantly reduce the excess basal translation of FMRP target mRNAs in synaptoneurosomal preparations from Fmr1 knockout mice. I am in the process of extending this study to cultured hippocampal neurons. This will enable us study the translational dysregulation in dendrites of Fmr1 knockout neurons and will also be a useful screening tool for the restorative effect of MPEP and other potential drugs for FXS.
Regulation of dendritic mRNA transport and translation at the synapse by FMRP phosphorylation

by Vijayalaxmi Nalavadi and Gary Bassell, 5/1/2008

Fragile X syndrome is manifested due to excessive immature spines and synapses caused by absence or malfunction of Fragile X Mental Retardation Protein (FMRP), an RNA binding protein that transports and regulates the translation of mRNAs which code for key synaptic proteins. Although it is known that basal levels of translation are elevated in the absence of FMRP, a recent study from our lab suggests that glutamate activated translation of the FMRP-bound mRNAs is in fact reduced in the FMRP null mice. This suggests that there might be a tightly regulated translation of FMRP-bound mRNAs which might be controlled spatially as well as temporally by glutamate stimulation at synapses. One way of controlling the activity of an RNA binding protein is by changing the status of its post-translational modification, like phosphorylation. In a collaborative study with Dr. Stephen Warren's laboratory, Dr. Nalavadi has demonstrated that FMRP exists as a phosphoprotein within neuronal dendrites (Narayan et al., J. Neuroscience 2007). FMRP can be quickly and transiently dephosphorylated in dendrites in response to the Group 1 mGluR agonist DHPG. This rapid dephosphorylation of FMRP appears to stimulate translation. FMRP rephosphorylation is hypothesized to restore translational suppression. The kinases involved in FMRP phosphorylation are unknown. In this study we address the question of whether casein kinase II, GSK 3 beta or S6 kinase are involved in the phosphorylation of FMRP and how they regulate FMRP function, namely in mRNA transport in dendrites and translation at synapses. A combination of methods in high-resolution imaging of neurons together with biochemical assays will be employed to answer these questions. This research will provide new insight into basic mechanisms of local protein synthesis, their function in synaptic plasticity underlying learning and memory, and dysfunction in fragile X syndrome. In addition, a more clear understanding of FMRP phosphorylation changes and their functional significance may lead to the identification of novel kinase and phosphatase based drug candidates, since several kinase inhibitors like GSK 3 beta inhibitors are being considered for treatment of neurodegenerative diseases. In addition, this study will provide possible cell-based assays to test such candidate drugs.
Trafficking of FMRP and Associated mRNAs in Response to Activation of Metabotropic Glutamate Receptors

by Laura Antar, 4/30/2004

A major challenge for Fragile X research is trying to understand the normal function of FMRP in the brain. With this knowledge, one can design pharmacologic treatments that might compensate for the loss of FMRP.

We know that FMRP is an mRNA binding protein, which means that it can transport mRNAs to specific sites in the cell and then regulate the translation of mRNAs at those sites into proteins. We have shown that FMRP is found in the majority of synapses in the brain. Synapses are the sites where two neurons communicate via chemical and electrical signals. At excitatory synapses, one neuron in the brain releases a chemical message called glutamate, onto a second neuron. The second neuron receives this message through its receptors. Excitatory synapses are critically involved in brain development, learning and memory. It is known that these synapses have both structural and functional defects in Fragile X.

The Bassell laboratory has developed microscopic imaging tools to visualize FMRP in live neurons, and we have been able to track the movements of FMRP along dendrites to synapses. This was done first by using recombinant DNA technology to fuse FMRP to a protein called Green Fluorescent Protein (GFP). The fluorescent FMRP protein was then introduced into cultured neurons that taken from embryonic rat brain. Cultured neurons provide a powerful tool to enable visualization of FMRP behavior in response to synapse activation.

Activation of a specific receptor type, metabotropic glutamate receptor (mGluR5), can stimulate the movement of both FMRP and Fmr1 mRNA (an mRNA that FMRP is known to bind) to dendrites. We showed that at synapses, FMRP movement is regulated, but the movement of Fmr1 mRNA is not. This suggests that FMRP may stop regulating the expression of specific mRNAs, such as Fmr1 mRNA, in response to excitatory synaptic activity. It may do this by releasing the mRNA from its grasp. This indicates that different proteins may be expressed in synapses in Fragile X patients (who lack FMRP) as compared with unaffected people, who have the protein. Such a difference in synaptic protein expression could affect cell structure and learning and memory.

This study was published in the Journal of Neuroscience (Antar et al., 2004; 24:2648). Our work has important implications toward understanding how regulation of mRNA localization and translation of mRNAs into proteins may be altered in Fragile X syndrome. Previous research has shown that mGluRs are important for a form of synaptic memory (called mGluR-dependent long term depression (LTD)) which is known to require protein synthesis, yet is abnormally enhanced in mice that do not produce FMRP. Our study showed that a pharmacological agent, MPEP, that reduces the ability of the receptor to receive its chemical signal, is able to change both the dendritic and synaptic movement of FMRP. Our lab, and others, believes that there are important clinical implications in the design of drugs, such as MPEP, that may modulate specific signaling pathways that are imbalanced in Fragile X patients.