Identification and evaluation of novel genes promoting axonale regeneration
Final Report Abstract
Severe nerve injuries in the mammalian spinal cord generally have irreparable consequences for the organism as damaged nerves fail to regenerate. In humans, this is associated with severe disease and high socioeconomic costs. Although damaged nerves initiate axonal growth, reestablishment of axonal connections to distant targets usually fail due to myelin-derived inhibitory signals or glial scar formation. In addition, embryonic axon guidance molecules are no longer expressed in adult tissues. Since many basic functions of the nervous system are conserved between vertebrates and invertebrates, genetic model organisms offer the possibility to screen for novel genes regulating axon re-growth and long-term regeneration. For example, mutagen-induced mutations could, in principle, delete inhibitory proteins. Alternatively, overexpression of specific transcription factors might induce groups of downstream genes promoting axonal growth. Here, we used Drosophila melanogaster as a model system to screen for novel regulators of axonal growth and nerve regeneration. In larvae, segmental motor nerves connect the ventral nerve cord to peripheral muscle fibers. Ruptures of these nerves cause posterior paralysis but initiate regenerative growth that can be imaged through the translucent cuticle of intact animals using fluorescent proteins. We labelled peripheral nerves in third instar larvae employing transmembrane GFP markers and induced nerve crush injuries using forceps. Regenerative growth resulted in three types of re-growing tip structures. Nerve stumps showed pyramidal or pointed tips, designated as Type I or Type II regeneration, respectively, and a minor percentage of nerve stumps failed to re-grow entirely. Since overexpression of growth-promoting genes might promote regeneration, we decided to screen a transgenic library of overexpression lines. This FlyORF collection is specifically enriched for growth-regulating genes but also contains a large number of transcription factors. We overexpressed 634 FlyORF genes and visually screened for improved regeneration responses compared to controls. Altogether we identified 36 genes with positively improved growth parameters. From this collection, 10 final candidate genes were selected based on two quantitative parameters. Growth initiation, defined by the number of nerve stumps that initiate re-growth compared to all injured nerve stumps, and growth distance defined by the longest distance of re-growth during a 20h time period. Amongst the most positive factors that initiate growth is the nervous systemspecific zinc finger transcription factor Eagle (Eg), with 93,7% re-growth. In the other phenotypic class, growth distance, i.e. the longest distance of re-growth within 20h, overexpression of the deubiquitinase Fat facets (faf) resulted in longest tip structures. Interestingly, Faf functions in the stabilization of Wallenda, a dual leucine zipper kinase that has been implicated in a nerve injuryinduced retrograde signaling pathway. In upcoming experiments, all candidate genes will be carefully evaluated for their growth promoting functions using fly genetics and molecular analyses.
Publications
- (2015). Hierarchical microtubule organization controls axon caliber and transport and determines synaptic structure and stability. Dev. Cell 33, 5-21
Stephan R., Goellner B., Moreno E., Frank C.A., Hugenschmidt T., Genoud C. Aberle H. and Pielage J.
(See online at https://doi.org/10.1016/j.devcel.2015.02.003) - (2015). Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases. Nat. Commun. 6, 7520
Niehues S., Bussmann J., Steffes G., Erdmann I., Köhrer C., Sun L., Wagner M., Schäfer K., Wang G., Koerdt S.N., Stum M., RajBhandary U.L., Thomas U., Aberle H., Burgess R.W., Yang X.L., Dieterich D. and Storkebaum E.
(See online at https://doi.org/10.1038/ncomms8520) - (2018). Identification and evaluation of novel genes promoting axonal regeneration. NeuroFly, 3.-7. Sept. 2018, Krakow, Poland
Kinold, J.C., Schäfer, C. and Aberle H.
- (2018). Sidestep-induced neuromuscular miswiring causes severe locomotion defects in Drosophila larvae. Development 145, dev163279
Kinold J.C., Pfarr C. and Aberle H.
(See online at https://doi.org/10.1242/dev.163279) - (2019). Axon Guidance and Collective Cell Migration by Substrate-Derived Attractants. Front. Mol. Neurosci. 12, 148
Aberle H.
(See online at https://doi.org/10.3389/fnmol.2019.00148)
