The goal of this work is to define the role of a new gene, Vezatin-like (Vzl)/CG7705, in retrograde axonal transport. This proposal will take advantage of genetic and imaging tools available in Drosophila to test the hypothesis that Vzl/CG7705 is required for the initiation of retrograde axonal transport of endosomes and/or other cargos at terminal presynaptic boutons. In human, defects in axonal transport are associated with neurodegenerative disease. Retrograde axonal transport of endosomes, in particular, is required to convey the receipt of neurotrophins and other signals to the soma and thereby promote neuron survival, differentiation, and growth. Loss of Vzl/CG7705 causes the accumulation of neuronal membrane and pMad, a downstream effector of signaling endosomes, specifically in terminal presynaptic boutons at neuromuscular junction. This defect resembles that caused by disrupting subunits of dynactin, a well-established regulator of retrograde transport. Vertebrate Vezatin is not known to regulate axonal transport, but a fungal Vezatin-like protein has recently been shown to be required for the initiation of retrograde transport of early endosomes in hyphae.
Aim 1 of this proposal will use live imaging of cargos within motorneuron axons and terminal presynaptic boutons in vzlCG7705 mutants to test the hypothesis that Vzl/CG7705 is required for the initiation of retrograde transport of endosomes at terminal boutons and to test whether Vzl/CG7705 might be required for transport of other cargos as well.
Aim 2 will use live imaging of cargos, as well as analysis of fixed images of terminal boutons, to test whether the ability of Vzl/CG7705 to prevent the accumulation of cellular components at terminal boutons is conserved in vertebrate (human) Vezatin. Vertebrate Vezatins bind the GTPase Arf6, which has been shown to affect axonal transport of endosomes. However, complete loss of Drosophila Arf6 does not cause material to accumulate at terminal boutons, suggesting that Vzl/CG7705 does not act through Arf6 in this process.
Aim 3 will therefore use co- immunoprecipation and mass spectroscopy to identify potential Vzl/CG7705 binding partners and will disrupt the corresponding genes either by mutation or UAS-RNAi to test their involvement in Vzl/CG7705-dependent processes. This work will define the role of Vzl/CG7705 and, potentially, Vezatin in axonal transport and will generate preliminary data that will motivate testable models of Vzl/CG7705's molecular mechanism of action.
Defects in axonal transport are associated with the pathogenesis of neurogenerative diseases, including Alzheimer's, Huntington's, and Parkinson's. We have identified a new gene, Vezatin-like (Vzl), that we hypothesize is required for one or more aspects of retrograde axonal transport. We propose to test this hypothesis and carry out a pilot study that will guide future efforts to understand Vzl's molecular function.
