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. 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.
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