Sean Speese - US grants

[unreadable] DESCRIPTION (provided by applicant): Recent work in our laboratory has uncovered a novel wingless (Wg) signaling pathway that is required for synaptic outgrowth at the Drosophila neuromuscular junction (NMJ). Initial studies have demonstrated that Wg is secreted from the presynaptic motor neuron and that this secretion is required for translocation of a Cterminal fragment of dFrizzled 2 (DFz2), a Wg receptor, into the nucleus. Once translocated into the postsynaptic muscle nuclei, DFz2(C) forms distinct foci. The cleavage of DFz2 to generate the C-terminal fragment and the nuclear import are required for proper synaptic outgrowth. The goal of this proposal is to begin to elucidate how DFz2(C) functions in the nucleus to regulate synapse development. [unreadable] Preliminary microarray experiments indicate the DFz2(C) may regulate synaptic outgrowth by regulating gene expression. In order to uncover the role of DFz2(C) in the nucleus, the first aim will include: 1) Verification of the genes uncovered in the microarray via quantitative PCR 2) Testing if verified genes physically associate with DFz2(C) foci in the nucleus and 3) Determining if DFz2(C) can interact with promoters of genes by performing chromatin immunoprecipitation (ChIP) assays. [unreadable] Additional preliminary data demonstrate that Lamin C (LamC), an A-type lamin, localizes to the DFz2(C) foci. Moreover, lamC null mutants display similar phenotypes to dfz2 mutants and expression of a LamCGFP fusion protein results in changes in the morphology of DFz2(C) foci and specifically disrupts synaptic outgrowth, suggesting that DFz2(C) and LamC may function together in the nucleus to regulate synaptic development. Mutations in A-type lamins in humans lead to a myriad of neuromuscular diseases called laminopathies. The goal of the second aim is to 1) Further test lamC and dfz2 mutants for overlapping phenotypes 2) Look for genetic and biochemical interactions between DFz2(C) and LamC and 3) Determine the function of LamC at DFz2(C) foci. [unreadable] PUBLIC HEALTH RELEVANCE: Misregulation of the Wnt/Wg signaling pathways in humans can lead to a myriad of different cancers and neural diseases. Therefore this study is expected to significantly contribute to our understanding of a novel Wg signaling pathway, and in the development of new strategies to prevent or cure diseases caused by disruptions in this pathway. Preliminary data also suggest that this pathway may interact with A-type lamins, which are mutated in host of muscular dystrophies called laminopathies. Therefore, understanding lamins' role in this signaling will be essential to our understanding of laminopathies. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Glia are the most abundant cells in the human brain and they play key roles in CNS function and health. Glial cells regulate synaptic signaling, ensheath axonal projections and, importantly, protect the brain by serving as the first line of defense against neuronal damage. The adult brain contains a striking array of diverse glial subtypes, but little is known about the unique genetic profiles of distinct classes of glia that alow them to carry out their important and varied functions. Moreover, determining how the transcriptional profile of glial cells are altered in response to neural injury has presented a unique set of challenges, since the process of isolating glia from the brain for transcriptional analysis is, in and of itself, highly stressful to the cells. Recent work has shown that the adult Drosophila melanogaster brain contains a variety of glial subtypes that are strikingly similar to those described in vertebrates. In addition, acute neural injury induces glial immune responses in flies that are highly reminiscent of those triggered in mammalian glia, including upregulation of essential glial immune genes. This project will take advantage of these evolutionarily conserved features of glia and integrate cutting-edge advances in the fields of in vivo RNA labeling and high throughput deep sequencing to generate a comprehensive transcriptome of Drosophila glial cells in the intact adult brain before and after injury. We will use novel genetic drivers that are expressed in discrete glial subtypes in the adult fly brain to genetically label RNA in each class of glia in vivo and then biochemically isolate the labeled RNA to sequence glial subtype transcriptomes by RNA-seq. Using a well-established axotomy assay, we will perform these experiments in uninjured and injured flies to compare the transcriptional profiles of glia before and after acute axon injury. Finally, we will validate the expression of glial genes identified by RNA-seq and begin to characterize the functional role of the newly discovered immune genes that are acutely regulated in glia responding to axotomy. This work (a) will provide critical mechanistic insight into the function of diverse glial subtypes in the adult brain (b) offers a unique opportunity to investigate how gene expression is altered in glia responding to neurodegeneration in the intact CNS and (c) will generate a valuable genetic toolkit for the scientific community to investigate many unexplored aspects of glial cell biology.

The development of new connections between neuronal cells in the brain is a critical process which ultimately underlies our ability to learn and remember. This proposal focuses on a newly-discovered process called Nuclear Envelope Budding (NEB) that allows cells to make targeted changes to the structure of their connections with other cells. Three sets of experiments will be carried out, examining (1) molecular changes that underlie NEB, (2) the mechanical means through which NEB is accomplished, and (3) how NEB actually functions in newly-developing cell-to-cell connections. An exciting and novel aspect of this latter set of experiments is the use of live cell microscopy to document the development of new connections in real time as they form in a living organism. These studies will provide novel and critical insights into how the brain develops and into how our experiences can lead to changes in our neuronal circuitry.

This proposal takes advantage of the well-established Drosophila neuromuscular junction (NMJ) model of synapse outgrowth and maturation to further our understanding of how RNA granules are formed and subsequently trafficked to synaptic sites during development. In particular, these studies will focus on the recently described process of Nuclear Envelope Budding (NEB), a nuclear export pathway by which RNA granules that contain postsynaptic transcripts are released from postsynaptic muscle nuclei. In Aim 1, we will investigate how Pin1, a highly conserved enzyme, functions to regulate NEB via nuclear lamina remodeling. In Aim 2, we will explore the role of a non-canonical Nuclear Pore Complex that we hypothesize loads mRNA transcripts into RNA granules forming at the periphery of the nucleus. Finally, in Aim 3, we will take advantage of a new tool to image trafficking dynamics of RNA granules in postsynaptic muscles at the time of synaptogenesis. This proposal utilizes high resolution imaging modalities including array tomography, immunogold EM and live cell imaging. These approaches, combined with the genetic tractability of the in vivo system, poise this study to make a significant contribution to our understanding of RNA granule biology and provide exciting new mechanistic insight into the bidirectional communication that occurs between the nucleus and the forming synapse during NMJ development.