Function and regulation of FEZ1, a Kinesin-1 adaptor protein, in transporting presynaptic proteins

轴突运输将蛋白质和膜运送到突触前神经末梢，因此对于活性突触的形成和维持是必不可少的。在神经退行性疾病中发现了影响轴突运输成分的突变，这说明了它的重要性。Kinesin适配器FEZ1似乎运输突触前货物的一个子集，但既不知道货物的范围，也不知道FEZ1依赖的运输的机制和调节。在这里，我计划更多地阐明FEZ1在突触传递突触前功能所需的蛋白质复合体或小泡中的作用。研究的主要目标是：(1)哪些激酶和信号通路调节FEZ1介导的转运？我们观察到FEZ1上的丝氨酸-58的磷酸化是激活突触前SNARE蛋白Synaxin-1的轴突转运所必需的。利用体外激活法，我们鉴定了几个磷酸化丝氨酸-58的FEZ1激酶。在这里，我计划使用培养的哺乳动物神经元和秀丽线虫作为模型系统，进一步表征这些激酶在调节FEZ-1依赖的轴突运输中的作用。我还将调查FEZ1是否有其他信号转导或激动级联途径参与调节。(2)哪些货物由FEZ1/Kinesin-1复合体运输，以及FEZ1功能障碍如何影响突触的发育和功能？已为选定范围的货物确定了各种货物适配器。特别是对两种突触物质进行了详细的研究，即突触小泡蛋白运输小泡(STVS)和短笛巴松管运输小泡(PTVS)。我计划使用免疫分离-定量质谱法来确定FEZ1/Kinesin-1复合体运输的货物的组成。通过与其他轴突运输囊泡的比较，我希望阐明FEZ1-囊泡、STV和PTV之间的关系，并评估FEZ1对突触前发育和功能的贡献。(3)FEZ1缺陷在多大程度上导致突触功能障碍和神经变性？阿尔茨海默病(AD)是一种与突触丢失相关的神经退行性疾病，轴突运输受损可能参与AD的发病。通过降低Kinesin轻链1的水平来损害Kinesin-1的活性足以导致AD患者观察到的运输缺陷。由于FEZ1结合激活了kinesin-1，FEZ1故障可能会导致类似的缺陷。初步数据显示，FEZ1在模型转基因AD小鼠中异常聚集。以此为起点，我计划进一步研究FEZ1在突触功能障碍和神经退行性变中的作用及其在突触货物递送中的作用。我期望这个项目中产生的数据将有助于更好地理解轴突运输及其调控如何参与突触发生和突触维持。

Axonal transport delivers proteins and membranes to presynaptic nerve terminals and is thus essential for formation and maintenance of active synapses. Illustrating its importance, mutations affecting components of axonal transport were identified in neurodegenerative disorders. The Kinesin adaptor FEZ1 appears to transport a subset of presynaptic cargo but neither the range of cargoes nor mechanistic details and regulation of FEZ1-dependent transport are known. Here I plan to shed more light on the role of FEZ1 in the synaptic delivery of protein complexes or vesicles required for presynaptic function. Key objectives of the research proposal are: (1) Which kinases and signaling pathways regulate FEZ1-mediated transport?We observed that phosphorylation of serine-58 on FEZ1 is needed for activating axonal transport of the presynaptic SNARE protein Syntaxin-1. Using in vitro kinase assays, we identified several FEZ1 kinases phosphorylating serine-58. Here I plan to further characterize the role of these kinases in regulating FEZ-1-dependent axonal transport using cultured mammalian neurons and Caenorhabditis elegans as model systems. I will also investigate whether there are additional signaling kinases or kinase cascades involved in the regulation of FEZ1.(2) Which cargoes are transported by FEZ1/Kinesin-1 complexes and how does FEZ1 dysfunction affect synaptic development and function?Various cargo adapters have been identified for a selected range of cargoes. In particular, 2 synaptic cargoes have been studied in detail, namely, synaptic vesicle protein transport vesicles (STVs) and Piccolo-Bassoon transport vesicles (PTVs). I plan to determine the composition of cargoes transported by FEZ1/Kinesin-1 complexes using immunoisolation-quantitative mass spectrometry. By comparing this against other axonal transport vesicles, I hope to clarify the relationships between FEZ1-vesicles, STVs and PTVs and assess the contribution of FEZ1 to presynaptic development and function.(3) To what extent do FEZ1 defects lead to synaptic malfunction and neurodegeneration?Impaired axonal transport occurs in Alzheimers disease (AD), a neurodegenerative disorder associated with synaptic loss, and likely contributes to AD pathogenesis. Impairing Kinesin-1 activity by reducing kinesin light chain 1 levels suffices to induce transport defects observed in AD. As FEZ1 binding activates Kinesin-1, FEZ1 malfunction may cause similar defects. Preliminary data reveal that FEZ1 is abnormally aggregating in model transgenic AD mice. Using this as a starting point, I plan to further investigate the involvement of FEZ1 in synaptic malfunction and neurodegeneration in relation to its function in synaptic cargo delivery. I expect that the data generated in this project will contribute to a better understanding of how axonal transport and its regulation participates in synaptogenesis and synapse maintenance.