Identification and evaluation of novel genes promoting axonale regeneration

轴突切开术后神经元生长程序的激活是神经再生克服自身轴突生长能力不足、促进轴突再生的必要前提。无脊椎模式生物的遗传筛选提供了一种利用基因突变、RNAi敲低或cDNA过表达文库来识别神经元再生新调控因子的策略。为了允许再生神经生长的基因解剖，我们最近在果蝇中建立了一个带有荧光标记的节段神经的转基因系统，该系统可以在完整的麻醉动物中进行特定的神经操作和轴突再生成像。在运动神经元特异性Gal4转录因子的控制下，通过跨膜GFP表达，可以通过活体幼虫的半透明角质层观察到不同部位的损伤和单个轴突的再生。神经受压时，突触囊泡在残端近端积聚，损伤部位远端的轴突和神经肌肉连接迅速退化，表明轴突连接完全剥离。通过选择已知的调节细胞生长的候选基因，证明了该筛选系统用于轴突再生调节因子鉴定的适用性。这个初步实验确定了Fat facets，一种去泛素酶，作为一个强大的积极打击。目前的项目在果蝇中提出了基于rnai的功能丧失筛选和基于UAS-ORF的功能获得筛选，以寻找轴突再生的正调控因子和负调控因子。随后，有希望的候选者将在果蝇中进行功能表征。此外，选择的同源基因将被克隆并在哺乳动物细胞培养试验中测试其保守功能。与P6 （Diekmann）和P1 （Fischer/Leibinger）的合作将能够研究这些调节基因在鱼和大鼠/小鼠视神经再生中的作用，潜在地确定促进中枢神经系统再生的新靶点。

Activation of neuronal growth programs following axotomy is an essential prerequisite for nerve regeneration to overcome their insufficient intrinsic capability for axonal growth and to promote axonal regeneration. Genetic screens in invertebrate model organisms offer a strategy to identify new regulators of neuronal regrowth using genetic mutation, RNAi knockdown or cDNA overexpression libraries. To allow the genetic dissection of regenerative nerve growth, we have recently established a transgenic system in Drosophila with fluorescently labeled segmental nerves, which enables specific nerve manipulation and imaging of axon regeneration in intact, anesthetized animals. Injuries can be targeted to different sites and regeneration of single axons visualized through the translucent cuticle of living larvae via transmembrane GFP expression under control of a motoneuron- specific Gal4 transcription factor. Upon nerve crush, synaptic vesicles accumulate at the proximal stump and axons and neuromuscular junctions distal to the lesion site rapidly degenerate, illustrating complete dissection of axonal connections. The applicability of this screening system for the identification of regulators of axonal regeneration was demonstrated using selected candidate genes known to regulate cell growth. This preliminary experiment identified Fat facets, a deubiquinating enzyme, as a strong positive hit. The current project proposes an RNAi-based loss-offunction screen and an UAS-ORF based gain-of-function screen in Drosophila to search for both positive and negative regulators of axonal regeneration. Subsequently, promising candidates will be functionally characterized in Drosophila. Moreover, selected homologous genes will be cloned and tested for conserved functions in mammalian cell culture assays. Collaboration with P6 (Diekmann) and P1 (Fischer/Leibinger) will enable the investigation of the role of these regulatory genes in fish and rat/mouse optic nerve regeneration, potentially identifying new targets for the promotion of CNS regeneration.