Identification and evaluation of novel genes promoting axonale regeneration

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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.