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

FEZ1（一种驱动蛋白-1 接头蛋白）在运输突触前蛋白中的功能和调节

• 批准号: 249372430
• 负责人: Dr. John Jia En Chua
• 金额: --
• 依托单位: Abteilung Neurobiologie (aufgelöst)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/249372430?language=en
• 关键词: Function; regulation; FEZ1; Kinesin; adaptor

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.

轴突运输将蛋白质和膜递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需的。在神经退行性疾病中发现了影响轴突运输组分的突变，这说明了其重要性。驱动蛋白适配器FEZ 1似乎运输突触前货物的一个子集，但既不货物的范围，也不机械细节和FEZ 1依赖运输的监管是已知的。在这里，我计划阐明FEZ 1在突触传递突触前功能所需的蛋白质复合物或囊泡中的作用。该研究提案的主要目标是：（1）哪些激酶和信号通路调节FEZ 1介导的转运？我们观察到FEZ 1上丝氨酸-58的磷酸化是激活突触前SNARE蛋白Syntaxin-1的轴突运输所必需的。使用体外激酶测定，我们确定了几个FEZ 1激酶磷酸化丝氨酸-58。在这里，我计划进一步表征这些激酶在调节FEZ-1依赖的轴突运输的作用，培养的哺乳动物神经元和秀丽隐杆线虫作为模型系统。我还将研究是否有额外的信号激酶或激酶级联参与FEZ 1的调节。(2)FEZ 1/Kinesin-1复合物转运哪些货物，FEZ 1功能障碍如何影响突触发育和功能？已经为选定的一系列货物确定了各种货物适配器。特别地，已经详细研究了2种突触货物，即突触囊泡蛋白转运囊泡（STV）和Piccolo-Bassoon转运囊泡（PTV）。我计划使用免疫隔离-定量质谱法来确定FEZ 1/Kinesin-1复合物运输的货物的组成。通过与其他轴突运输囊泡进行比较，我希望澄清FEZ 1囊泡，STV和PTV之间的关系，并评估FEZ 1对突触前发育和功能的贡献。(3)FEZ 1缺陷在多大程度上导致突触功能障碍和神经退行性变？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，轴突运输受损可能导致AD发病。通过降低驱动蛋白轻链1水平来损害驱动蛋白-1活性足以诱导在AD中观察到的转运缺陷。由于FEZ 1结合激活驱动蛋白-1，FEZ 1功能障碍可能导致类似的缺陷。初步数据显示，FEZ 1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FEZ 1在突触功能障碍和神经退行性变中的作用，以及它在突触货物传递中的功能。我希望这个项目产生的数据将有助于更好地理解轴突运输及其调节如何参与突触发生和突触维持。