Henri Tiedge - US grants

Nerve cells extend highly polarized processes, called dendrites and axons, through which they communicate with each other. Repeated physiological stimulation of a neuron may result in long-term changes at the synapse, the site where connections are made. A new theory suggests that nerve cells may synthesize proteins close to the synaptic site, depending on synaptic activity; the majority of proteins in most cells is made by the protein synthetic machinery in the cell body (the soma). The goal of this research project is to assess the extent to which neurons employ extrasomatic protein synthesis. Associate components of the protein synthetic machinery will be identified in extrasomatic domains of neurons, and the accumulation of such components in those domains will be correlated with physiological activity at the synapse. Primary cultures of neurons from both the central and the peripheral nervous system will be used in this approach. In summary, this project seeks to establish the functional significance of decentralized protein synthesis as a potential basis for long-term modulations of protein repertoires in extrasomatic domains of nerve cells.

The phenomenon of dendritic protein synthesis in neurons has been well documented. The specific mRNAs for several proteins with specific dendritic functions or localizations are thought to be targeted to sites of active translation in dendrites. This protein synthesis is associated with the postsynaptic side of synaptic contacts and may provide a mechanism for modulation of postsynaptic functional plasticity. However, the mechanisms for specific transport and targeting of RNA, to dendrites have not been delineated. This application will examine the specific structural elements responsible for localization of BC1, a short, neuron- specific RNA, to dendrites. The specific sequence elements required for targeting to dendrites will be determined and their ability to target foreign chimeric RNA sequences to the dendrite will be evaluated. In the second series of experiments, the role of electrical activity and synaptic transmission on the expression and transport of RNA into dendrites will be assessed. The effects of inhibitors for synaptic transmission, electrical activity, and cytoskeletal organization on various components of the translational apparatus will be examined. It is hoped that these studies will provide new insights into the role of dendritic protein synthesis in neuronal plasticity.

DESCRIPTION (provided by applicant): Non-protein-coding (npc) RNAs are key mediators in the control of eukaryotic gene expression. In brain, npcRNAs have been implicated in adaptive changes that underlie a neuron's capacity for long-term plastic responses to external stimuli. Such capacity, it is suggested, is supported at least in part by the translational regulation of gene expression at the synapse. However, while providing a molecular framework for the input-specific management of synaptic protein repertoires, the concept of synaptic translation is contingent upon molecular tools to ensure stringent control of the translational machinery. What are the functional mechanisms to implement such control, and how are they regulated in neurons? In the research proposed here, it is submitted that small npcRNAs are instrumental in the orchestration of neuronal translational control. Specifically, it is conjectured that synapto-dendritic BC RNAs engage neuronal translation by repressing the initiation mechanism. This conjecture will be experimentally investigated as follows. First, the molecular mechanism of BC-mediated repression will be elucidated by dissecting the functional interplay between BC1 RNA and its target in the translation pathway, eukaryotic initiation factor 4A (eIF4A). This analysis will also probe the role of eIF4B, an eIF4A co-factor, as an effector of BC1-eIF4A interactions. The subsequent step will establish the mode of action of human BC200 RNA in translational control. This effort is prompted by the notion that BC200 RNA, a primate npcRNA that functions as a translational repressor, may be implicated in neurological disease. Neuronal targets of BC-mediated repression will be identified using candidate and unbiased approaches. Because the functional consequences of BC repression appear to intersect with those of the fragile X mental retardation protein (FMRP), it will be necessary to ascertain mode of action and potential convergence of the two repression pathways. In the final objective, the hypothesis will be scrutinized that BC repression, conceivably operating in the MEK/ERK signaling pathway, contributes to a synaptic balance of power vis-[unreadable]-vis translational stimulation resulting from metabotropic glutamate receptor activation. It is the overall goal of the proposed research to establish molecular mechanisms and functional significance of npcRNAs in neuronal translational control pathways. It is anticipated that results from this work will shed light on the role of such pathways in synaptic plasticity maintenance and dysfunction. PUBLIC HEALTH RELEVANCE: In neurons, translational control by synapto-dendritic BC RNAs has been implicated in the maintenance of local stimulation-repression homeostasis at the synapse. Dysregulation of BC1 control precipitates synaptic hyperexcitability and epileptogenic responses that appear to intersect with sequelae resulting from lack of fragile X mental retardation protein. The functional consequences of dysregulated BC control are therefore of direct relevance to the biology of neurological and mental disorders.

DESCRIPTION (provided by applicant): Regulatory RNAs are important mediators of gene expression in eukaryotic cells. BC RNAs, a neuronal subtype of regulatory RNAs, are translational repressors that have been implicated in the control of local protein synthesis at the synapse. On-site translational control of gene expression is now seen as one of the major mechanisms underlying the long-term structural and functional plasticity of synaptic connections. A key requisite for the local translational control pathway in neurons is the selective delivery of various RNA components, in particular of regulatory RNAs, to diverse postsynaptic target sites within neuronal dendritic arborizations. How is such targeted delivery specified by RNAs, and how is it modulated by physiological stimuli and/or man-made drugs? In the research project proposed here, it is hypothesized that neuronal BC RNAs use architectural motifs to convey spatial information. It is suggested that such motifs specify differential destination sites in synapto-dendritic neuronal domains. It is the main objective of the present proposal to test this hypothesis by deciphering spatial information coding in neuronal BC RNAs. In the first part of the project, it is planned to conduct a functional dissection of differential dendritic targeting competence in BC RNAs. RNA motifs will be identified that direct constitutive and conditional dendritic targeting, respectively. The second part of the project will focus on modulation of RNA spatial coding in neurons. It is planned to establish whether dendritic RNA targeting mechanisms are subject to regulation by receptor stimulation, and whether man-made drugs impact and modulate these mechanisms. In summary, the planned research is directed at the physiological significance of spatial information coding in neuronal regulatory RNAs. PUBLIC HEALTH RELEVANCE: Spatial information coding in neuronal npcRNAs, it is hypothesized, is a key prerequisite for local translational control mechanisms that are underlying long-term synaptic plasticity. The planned research is directed at mechanism and modulation of targeted npcRNA delivery to synapto-dendritic destination sites. A molecular understanding of spatial RNA coding in neurons will enable us to appreciate its relevance in neuronal plasticity, drug action, and human disease.