Imaging Dendritic mRNA Transport and Translation in Health and Disease

Gary Bassell, Ph.D.

Emory University School of Medicine

Funded in June, 2004: $300000 for 3 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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How Lack of a Specific Regulatory Protein may Result in Mental Retardation

Investigators will examine the normal role of a specific protein that is missing in males who have an inherited form of mental retardation in order to determine how this protein deficiency impairs cognitive development.  The findings may lead to new avenues for preventing this, and perhaps other, mental disabilities.

The researchers hypothesize that males who inherited “Fragile X syndrome” have a gene that failed during their development to produce an essential protein that guides brain cell communication.  The investigators suggest that the “Fragile X protein” ordinarily guides “messenger RNA” on its route to convey DNA instructions to cells in the brain’s hippocampus, where long-term memory and learning occur.  Specifically, the Fragile X protein ordinarily directs messenger RNA to hippocampal brain cell dendrites, the long branches that connect one brain cell to another so that they can communicate.

The DNA message instructs dendrites on how to make and use a neurotransmitter, called glutamate, which is needed for cellular communication.  Without these instructions, according to the investigators, the dendrites never produce and use glutamate to pass messages from one dendrite to another at the junction between the two, called a “synapse.”  The investigators suggest that males who lack this essential protein, therefore, fail to develop the capacity to transmit messages from one brain cell to another to enable them to learn and form memories.

The investigators will test their hypothesis by visualizing the actions of the normal Fragile X protein and the messenger RNA’s that this protein guides to their appropriate hippocampal neurons. They will accomplish this by using new microscopic and florescent techniques to image laboratory cell cultures of live hippocampal neurons.  Then, the researchers will compare these findings to the actions of hippocampal cells cultures taken from an animal model of Fragile X syndrome, in which the protein is missing, to see if their hypothesis is correct.

Significance:  This research to determine the normal role of the Fragile X protein in brain development could produce a better understanding of how this protein guides messenger RNA to deliver its message.  This might lead to means to correct for this lack, and might also have implications for developing approaches to prevent autism and other developmentally acquired mental deficiencies.   
 

ABSTRACT

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Imaging Dendritic mRNA Transport and Translation in Health and Disease

We will develop high resolution fluorescence and live cell imaging methods to test the hypothesis that specific mRNA binding proteins are required for the glutamatergic regulation of mRNA transport in dendrites and their subsynaptic translation. Recent identification of mRNAs that are bound by these proteins now makes it possible to investigate whether specific RNA-protein interactions occur in dendrites and at synapses.

An inherent difficulty in studying RNA-protein interactions in dendrites has been the lack of suitable high resolution microscopic technology to visualize mRNA transport and identify sites of local translation. A new view of the dynamic process toward local translation is made possible by utilizing novel microscopic and imaging technology to visualize mRNAs and their binding proteins in live neurons. Our recent studies have used cultured hippocampal neurons to show that the mRNA binding proteins, ZBP1 and FMRP, are localized in granules within dendrites and spines. ZBP and FMRP granules were induced to traffic in response to activation of NMDA and metabotropic glutmate receptors respectively.

An important objective is to extend our observations of mRNA granule transport in dissociated cultures to organotypic slice cultures. We hypothesize that synaptic activity leading to long-term plasticity involves the trafficking of specific mRNA binding proteins, which serve as adapters to permit the selective localization of mRNAs. We suggest that there may be different classes of RNA granules which respond to specific types of glutamatergic signals.

For the proposed research, we will use hippocampal slice cultures and two-photon microscopy to investigate whether glutamate receptor activation can stimulate trafficking of these mRNA binding proteins, which will be fused to different fluorescent proteins and co-expressed. We also propose to refine the MS2 labeling method, using different color fluorescent proteins, to permit the simultaneous visualization and tracking of mRNA binding proteins and their target mRNAs in dendrites and spines of live neurons. Hippocampal neurons will also be transfected with dominant negative forms of RNPs or cultured from knockout mice to assess whether there are defects in the regulated trafficking of mRNAs in dendrites and spines. Experiments will also be done using fluorescent reporters to visualize glutamate-dependent translation in dendrites and to assess possible defects in the regulated translation of target mRNAs in hippocampal neurons transfected with mutant RNPs or cultured from knockout mice.

These studies will elucidate specific mRNA-protein interactions that are involved in different forms of protein synthesis-dependent synaptic plasticity. Such findings will provide insight into neurological diseases, such as Fragile X syndrome, and efforts to develop potential targets for therapeutic intervention.

HYPOTHESIS

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Hypothesis:
Various forms of long term synaptic plasticity require local protein synthesis, including those in the hippocampus that are dependent on either NMDA or metabotropic glutamate receptor activation.  We hypothesize that NMDA and mGluR signaling pathways can differentially regulate the dendritic trafficking of different mRNA binding proteins, thus providing a mechanism to localize and translate distinct populations of mRNAs at synapses. The proposed experiments will test the hypothesis that the dendritic trafficking of the Fragile X Mental Retardation protein (FMRP) and bound mRNAs is regulated by metabotropic glutamate receptor activation of protein kinase C.  In contrast, we propose that the trafficking of Zipcode Binding Protein is regulated by NMDA receptor activation of either CaMKIIa or MAP kinase pathway.

Goals:
To develop multiple labeling techniques for live cell imaging, using confocal and two-photon microscopy, to permit the simultaneous visualization of the dynamics of mRNAs and their binding proteins in dendrites and spines in response to glutamatergic activation of hippocampal neurons in slice cultures. Experiments in Aim-1 will visualize the regulated movements of mRNAs and mRNA binding proteins in dendrites and spines of slice cultures. Experiments in Aim-2 will use fluorescent reporters to visualize glutamate-dependent translation  in dendrites. Experiments will also assess possible defects in the regulated transport or translation of mRNAs in hippocampal neurons following perturbation of the expression or function of these mRNPs.

Methods:
Rat and mouse hippocampal slices will be transfected with CFP-FMRP and YFP-ZBP constructs using the biolistics Gene-Gun. An important objective will  be to extend our past observations of FMRP and mRNA transport in dissociated cultures to organotypic slice cultures. Both stimulus and chemical induced forms of LTD and LTP will be induced and the trafficking of RNPs will be examined using FRAP analysis. In addition, the MS2-GFP tagging method will be adapted to examine the co-transport of mRNAs and RNP proteins. Hippocampal neurons will also be transfected with dominant negative forms of RNPs or cultured from knockout mice to assess whether there are defects in the regulated trafficking of mRNAs in dendrites and spines. Experiments will also be done using fluorescent reporters to visualize glutamate-dependent translation  in dendrites and to assess possible defects in the regulated translation of target mRNAs in hippocampal neurons transfected with mutant RNPs or cultured from knockout mice.

 

FINDINGS

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Lay Results:
Fragile X syndrome (FXS) is the most common form of inherited mental retardation, with genetic links to autism and epilepsy. FXS is caused by the lack of a single protein, the Fragile X Mental Retardation protein (FMRP). A major challenge for research is to understand the normal function of FMRP in the brain. FMRP is an mRNA binding protein that appears to play an important role at synapses, which are the junctions where neurons communicate with each other. Brain synapses have structural and functional defects in Fragile X. The objective of this research project was to use state-of-the-art methods in brain imaging to visualize the FMRP protein in the nerve cells of normal mice and to try to determine its function at synapses.  Imaging experiments were also done on a mouse model for Fragile X syndrome that was previously developed using molecular genetic methods to delete the gene encoding for FMRP.  During this project, Dr. Bassell’s laboratory developed microscopic imaging tools that permitted the direct visualization of FMRP and they were able to track the movements of FMRP along the dendrites to the site of the synapse.

Experimental findings demonstrated a role FMRP as a dendritic shuttle bus that carries specific mRNAs to the synapse to permit local synthesis of the encoded proteins. Several mRNA and protein molecules were discovered to have altered expression in dendrites and at synapses in the FXS mouse model. This study allows us to propose a dynamic process of “on-site” and “on-demand” protein synthesis at the synapse that is critical to brain development and learning, and is altered in FXS. Our research findings will have important clinical applications in the design of drugs that may modulate specific brain signaling pathways that are imbalanced in Fragile X, and possibly other brain disorders such as autism, epilepsy, and mental illness.

Scientific Results:
The goal of this project was to further develop fluorescence imaging methods to permit high-resolution analysis of the localization of the Fragile X Mental Retardation protein and associated mRNAs in neurons. It was anticipated that the refinement of this technology would lead to the discovery of novel dendritically localized mRNAs, and their possible dysregulation in a mouse model of fragile x syndrome. 

Toward aim-1, we discovered a new function for the fragile X mental retardation protein (FMRP) in the rapid delivery of several novel dendritic mRNAs important for synaptogenesis and synapse plasticity that are implicated in fragile X syndrome (FXS). Specific mRNAs in neurons from FMR1 KO mice were deficient in glutamatergic signaling-induced dendritic localization, and direct observation of mRNA granules in live neurons revealed impaired transport dynamics. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a novel mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to developmental morphologic plasticity.

Toward aim-2, we discovered that GluR1/2 and PSD-95 mRNAs are localized to dendrites in vivo and associated with FMRP. The steady state levels of GluR1/2 and PSD-95 mRNAs in dendrites did not differ between wild type and FMR1 knockout mice. In contrast, GluR1/2 and PSD-95 mRNAs were translated in excess at basal states in FMR1, yet were dysregulated at synapses in response to mGluR activation. In addition, FMR1 KO mice exhibited an overall increase in the rate of basal protein synthesis at synapses; yet showed loss of mGluR-stimulated protein synthesis. These findings reveal a novel mechanism whereby FMRP regulates the local synthesis of AMPAR subunits and PSD-95 downstream of mGluR-activation. Collectively, our findings implicate a diverse role for FMRP in both the regulation of dendritic mRNA transport and synaptic protein synthesis. In addition, we have made advancements in the FISH technology suitable for discovery and analysis of other dendritic mRNAs in cultured neurons and brain sections.

 

SELECTED PUBLICATIONS

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Muddashetty R., Kelic S., Gross C., Xu M., and Bassell G.J.  Dysregulated metabotropic glutamate receptor-dependent translation of AMPA receptor and postsynaptic density-95 mRNAs at synapses in a mouse model of fragile X syndrome. Journal of Neuroscience, 2007, 27(20):5338-5348.

Kiebler M. and Bassell G.J.  Neuronal RNA granules: movers and makers. Neuron, 2006, 51(6):685-690.

Antar L.N., Li C., Zhang H., Carroll R., and Bassell G.J.  Local functions for FMRP in axon growth cone motility and activity-dependent regulation of filopodia and spine synapses. Molecular and Cellular Neuroscience, 2006, 32:37-48.

Antar L.N., Dictenberg J.B., and Bassell G.J.  Localization of FMRP-associated mRNA granules and requirement of microtubules for activity-dependent trafficking in hippocampal neurons. Genes, Brain and Behavior, 2005; 4:350-359.