2008 — 2012 |
Godenschwege, Tanja Angela |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Functional Analyses of Neuroglian/L1 in Synaptogenesis @ Florida Atlantic University
[unreadable] DESCRIPTION (provided by applicant): Drosophila Neuroglian (Nrg), a homolog of vertebrate L1, is a prime example of a multifunctional cell adhesion molecule with a multiplicity of binding partners. Several types of single point mutations at different sites in human L1 have been shown to cause a variety of neurological disorders (CRASH syndrome) including mental retardation, hydrocephalus and spasticity. Nrg/L1 has been shown to be involved in axon pathfinding, neurite extension and cell migration. The role of Nrg/L1 in these developmental processes has been well-characterized in vertebrates and invertebrates but much less is known about potential functions during synapse formation. We have recently shown that Nrg does indeed have an essential function in synaptogenesis. We found that a single missense mutation in the extracellular domain of the nrg849 allele disrupts the assembly and functionality of a central synapse in a well- characterized neuronal circuit, the Giant Fiber System (GFS). Our data suggests that phosphorylation of the intracellular ankyrin binding motif of Nrg/L1 is crucial for giant synapse formation. Interestingly, human L1 is able to completely rescue the phenotype in nrg849 mutants while tested paralogs Neurofascin and NrCAM can not, despite having the same overall domain structure inclusive of the highly conserved ankyrin binding motif. This shows that the GFS is a valid model system for studying L1-specific function. Our preliminary studies indicate that some of the pathological missense mutations identified in L1 affect synapse formation rather than earlier developmental processes. Though a defect in neurite outgrowth, guidance or synapse formation may all result functionally in the same discernable phenotype, a disrupted connection between neurons, the biological process affected is completely different. Our data also suggests that some mutations do not result in a loss of a function phenotype but can also have gain of function and dominant negative consequences as well. Information about the particular biological process being disrupted as well as the protein function being compromised is crucial to find appropriate treatment plans for clinical pathologies associated with different types of mutations in the future. Hence, this grant is designed to further explore Nrg and L1's role in synapse formation as well as to study the effects of identified human mutations in L1 in vivo at a single cell level. We will combine the enormous resource of identified human L1 mutations and the power of genetic and molecular tools in Drosophila to determine which extracellular L1/Nrg interactions and intracellular signaling pathways play a role in synapse formation. We will determine the function of various L1/Nrg constructs in wild type, nrg849 and a temporal loss of function background electrophysiologically and anatomically. The knowledge gained from studies listed in this proposal will enable us to have a better understanding of the mechanisms involved in synaptogenesis as well as the cellular basis of the pathologies underlying L1-related neurological disorders. [unreadable] More than 170 different mutations in the cell adhesion molecule L1 have been identified to result in a variety of human neurological disorders which are associated with mental retardation, hydrocephalus (enlarged head due to collection of fluid on the brain) and spasticity (involuntary contraction of muscles). We intend to study the effects of these various pathological mutations in vivo at a single cell level in a unique model system, which will allow us to identify the particular biological process being affected, as well as the protein function being disrupted by the mutation. This information is essential to find appropriate treatment for the clinical manifestations of these mutations and hence will benefit the health of patients with L1-related disorders in the future. [unreadable] [unreadable]
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2015 |
Godenschwege, Tanja Angela |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
Nuclear Function of L1-Type Cams in the Drosophila Nervous System @ Florida Atlantic University
? DESCRIPTION (provided by applicant): L1-type cell adhesion molecules (CAM) are essential for proper nervous system development. We previously demonstrated that transsynaptic signaling of the sole invertebrate L1-type CAM neuroglian (Nrg) regulates synapse growth and stability and that its function in organizing the cytoskeleton at the synapse is conserved from flies to humans. Preliminary live imaging data reveals that in the adult Nrg is retrogradely transported from the synapse to the soma in a Lissencephaly 1 (Lis1)-dependent manner. This suggests that in the adult L1-type CAMs have a yet uncharacterized function that is distinct from its well-known developmental role at the plasma membrane. Retrogradely transported L1-type CAMs may be degraded or recycled in the soma, be part of a signaling endosome, or be retrograde signals themselves, which translocate to the nucleus. Recently, cytosolic 28kDa and 30KDa L1CAM fragments as well as a 70kDa transmembrane domain- containing fragment were shown to translocate to the nucleus. We have evidence that in addition to fragments, full-length Nrg translocates to the nuclei of Drosophila nervous systems and that Nrg fragments are likely to carry posttranslational modifications distinct from the full-length form. Our hypothesis s that some of the Nrg that is transported from the synapse to the soma will translocate to the nucleus. To test this, we will determine if the nuclear import of Nrg is reduced in Lis1 mutants when compared to wild type background and if the full- length Nrg form and its fragments carry different posttranslational modifications. In addition to nuclear translocation, we propose in aim 1 to determine mechanisms of L1-type CAM retrograde transport. We will use live imaging to determine the type of endosomal compartments in which axonal retrograde transport of Nrg occurs and the intracellular motifs that are required for Nrg retrograde transport. Recombinant expression of the intracellular domain (ICD) of L1CAM in non-neuronal cell lines altered the expression of genes involved in migration, cell cycle control and DNA damage checkpoint responses and in vitro experiments suggest that nuclear L1CAM may have a role in migration, neurite outgrowth and cellular protection against physical damage or genomic and oxidative stress caused by diseases or environmental influences. The main goal of aim 2 is to determine if nuclear L1-type CAMs have a functional role in vivo as well. For this, we will characterize the phenotypes of a mutant that lacks the transmembrane proximal nuclear localization sequence. In addition, we will transgenically express L1-type CAM proteins that mimic nuclear full- length L1-type protein and its fragments in the nervous system of Drosophila to determine if they have distinct functions by analyzing their induced phenotypes in vivo at the single cell and the organismal levels. In summary, the in vivo characterization of the mechanisms and functions of nuclear L1-type CAM signaling in the central nervous system will impact a broad range of research areas such as neuroprotection during stroke, spinal cord regeneration, Alzheimer's disease and cancer.
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