A system for in vivo structure-function analysis of kinesin in neurodegeneration

描述(申请人提供)：本研究项目的主要目标是开发一种强大的新的分子遗传学方法，使我们和其他研究人员能够将分子马达蛋白Kinesin的体外机械力化学与其相对较少了解的体内运输功能以及运动神经元疾病的病理学联系起来。肌动蛋白重链(KHC)是肌动蛋白-1(kinesin-1)的动力产生亚基，它是一种丰富的微管马达，驱动许多细胞质运动过程，包括线粒体、小泡和RNA复合体的运输。它的功能在神经元中尤其重要，它将新合成的成分运送到轴突中，并沿着轴突走向突触终末。Kinesin-1功能缺陷可以造成严重的健康后果，事实证明，KIF5A(人类KHC同源物)的20个不同的突变氨基酸变化可导致遗传性痉挛截瘫(HSP)和Charcot-Marie-Tooth(CMT2)神经退行性疾病。为了能够在一个完整的模式生物中测试KHC结构和生物物理的特定变化对其功能的影响，我们将在果蝇中创建一个同源基因替换工具集，该工具集将允许在本地位置快速插入KHC基因的体外工程版本。这一战略需要 一种基因上复杂且耗时的同源重组，用酶激活的attP“着陆垫”(？khc-attP)取代天然的KHC编码序列。将构建一个含有缺失的KHC序列和同源attB整合位点(KHC-attB)的“传递质粒”，它将允许快速、直接地插入修饰的KHC基因。同源重组的步骤虽然有些艰巨，但只需完成一次。随后插入修饰的KHC基因将是简单和快速的。着陆垫/递送质粒方法将首先用正常的KHC基因替换进行仔细的测试，以确保它们重现本地表达模式、线粒体的正常轴突运输以及生物体的正常发育生命周期。然后，该系统将用于一个试点项目，以解决有关KIF5A环12中的一组强烈聚集的突变变化如何导致HSP/CMT2神经变性的问题。新的疾病模型系将用于详细分析这些突变对活体动物的线粒体和神经分泌囊泡轴突运输行为的影响。这些结果应该允许我们开始填补关于Loop 12-微管结合关系的结构信息、Kinesin-1的体内运输功能和HSP/CMT2样轴突变性机制之间的空白。通过这个试点项目对KHC基因替换工具集的验证，将使我们和其他人能够将其应用扩展到许多其他问题，如Kinesin如何在体内完成其功能。

DESCRIPTION (provided by applicant): The primary objective of this research project is to develop a powerful new molecular-genetic methodology that will allow us and other researchers to connect the well characterized in vitro mechanochemistry of the molecular motor protein kinesin to its relatively poorly understood in vivo transport functions and to the pathology of motor neuron diseases. Kinesin heavy chain (Khc) is the force producing subunit of kinesin-1, which is an abundant microtubule motor that drives many cytoplasmic motility processes, including the transport of mitochondria, vesicles and RNA complexes. Its functionality is particularly important in neurons for delivering newly synthesized components into and along axons toward their synaptic terminals. Defective kinesin-1 function can have serious health consequences, as evidenced by the fact that 20 different mutant amino acid changes in KIF5A, a human Khc homolog, have been identified as causing hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth (CMT2) neurodegenerative diseases. To allow tests of the effects of specific changes in Khc structure and biophysics on its functions in an intact model organism, we will create a homologous gene replacement tool set in Drosophila that will allow rapid insertion of in vitro engineered versions of the Khc gene at the native locus. The strategy entails a genetically complicated and time consuming homologous recombination to replace the native Khc coding sequence with an enzyme-actuated attP "landing pad" (¿Khc-attP). A "delivery plasmid" bearing the missing Khc sequence and a cognate attB integration site (Khc-attB) will be constructed that will allow fast straightforward insertion of modified Khc genes. The homologous recombination step, while somewhat arduous, will need to be accomplished just once. Subsequent insertion of modified Khc genes will be simple and fast. The landing pad/delivery plasmid approach will first be carefully tested with a normal Khc gene replacement to ensure that they recapitulate native expression patterns, normal axonal transport of mitochondria, and a normal developmental life-cycle of the organism. The system will then be used in a pilot project to address questions about how an intensely clustered set of mutant changes in Loop 12 of KIF5A cause HSP/CMT2 neurodegeneration. The new disease model lines will be used for detailed analysis of the effects of those mutations on the axonal transport behavior of mitochondria and neurosecretory vesicles in live animals. The results should allow us to start filling the gaps between structural information on the Loop 12-microtubule binding relationship, the in vivo transport function of kinesin-1, and the mechanisms of HSP/CMT2-like axon degeneration. Validation of the Khc gene replacement tool set by this pilot project will allow us and others to expand its use into many other questions about how kinesin accomplishes its functions in vivo.