Dendrites are master integrators of information flow in the brain and the sites of post-synaptic structural modifications that regulate the plasticity of synapse strength in response to neurotransmission during brain development. Recent data suggest that dendrites are also an important site for the dynamic regulation of localized gene expression in the nervous system, with subsets of synapses possessing the ability to independently alter synapse strength through the local synthesis of proteins that alter plasticity. An important component to this regulation is the active transport of a select group of mRNAs from the cell body into dendrites and targeting of these mRNAs to specific translation sites. Our research goals focus on understanding the mechanisms of mRNA transport and translation within neuronal dendrites, and the role of mRNA binding proteins in the regulation of synapse plasticity during brain development. Several lines of evidence also point to a strong connection between dendritic mRNA localization and synaptic maturation in development. The most common inherited form of mental retardation in humans, Fragile X syndrome (FXS), results from loss of expression of an RNA-binding protein. The phenotype of FXS and many other forms of mental retardation is an abnormal morphology of dendritic spines, and recent work suggests that dynamic actin-rich protrusions early in development, known as filopodia, may play a key role in synaptogenesis. However there is very little known about how filopodial dynamics affect synapse development. Therefore we are interested in how the regulated expression of mRNAs encoding proteins that influence actin dynamics in response to activity affects synapse development and plasticity.
Emphasis is placed on the detection of single mRNAs within individual dendrites in both fixed and living cells, and the visualization of dynamics of mRNA trafficking in dendrites of living hippocampal neurons and the subsequent translation of these mRNAs in response to neuronal stimulation. Our approach is to use primary hippocampal cultures derived from mice bearing mutations in specific mRNA-binding proteins or “knock-ins” of fluorescent mRNAs. Two mRNA-binding proteins are currently used as models for these processes, the Fragile-X mental retardation protein (FMRP) implicated in FXS, and the zipcode-binding protein (ZBP1), which are both involved in the transport and translational control of mRNAs in neurons. These approaches are being developed using quantitative digital microscopic techniques coupled with novel in vivo methods for analysis of single mRNA movements and dynamic trafficking events in living brains. Genetic techniques will used to generate mice expressing fluorescent proteins in the background of these RNA-binding protein knockouts or mRNA-tag “knock-ins” to determine their role in regulating neurite outgrowth and synapse formation in intact brains. Using these unique tools we can visualize mRNA movements in real-time in living cells. A quantification of differences underlying mRNA transport may lead to a greater understanding of the mechanisms contributing to synaptic defects in FXS and other neurologic diseases of mRNA metabolism, such as Rett syndrome and autism. In addition, our work will explore the requirements for specific mRNA localization and translation events in dendritic actin-based protrusive processes that give rise to structural changes during synapse development. We can now visualize translation sites of any gene of interest in living neurons using wide-field microscopy. Ultimately this research will highlight the dynamics and regulation of dendritic mRNAs in the processes of synaptogenesis and long-term synaptic changes that underlie plasticity, and defects in these events that give rise to alterations in learning and memory, and neurological disease. Future work is also aimed at characterizing the structural details that underlie the interaction between these mRNA-binding proteins and the transport machinery using X-ray crystallography. These investigations, combined with bioinformatics approaches, may lead to a greater understanding of how diverse RNA-binding protein domains possess universal structural motifs that are the basis for molecular motor recognition that supports mRNA transport in all cells.