Analyse der Evolution und die funktionelle Rolle von nogo/rtn4 Gen während der axonalen Regeneration im Zebrafisch

In contrast to fish, mammals are unable to regenerate lesioned fiber tracts in the central nervous system (CNS). Two major factors are postulated to be responsible: intrinsic neuronal properties and the glial cell environment. Upon injury several types of molecules produced by glial cells elicit inhibitory effect on neurite outgrowth. One of these, oligodendrocyte-derived RTN4/Nogo was demonstrated to act as a potent inhibitor of axon regeneration and to block neurite extension via two domains: the Nogo-A-specific region and Nogo-66 located in N- and C-terminal parts of the protein, respectively.The present work focuses on the evolution of RTN4/Nogo and its inhibitory domains in chordates as well as their functional characterization in fish. In order to comprehend the origin of the Nogo-A-specific region (NSR) all available genomes of chordates were analyzed. Our data indicate that the common ancestor of fish and tetrapods had an NSR-coding rtn4 gene, which underwent duplication and divergent evolution in bony fish. Thus, in the zebrafish, the NSR was lost in rtn4 but retained in its duplicate rtn6, whereas in the pufferfish, the NSR was retained in rtn4 and the entire rtn6 gene was deleted. Distant homology screening in combination with protein architecture analysis reveals the relation of this region to CSPG neurocan on the levels of domain organization and sequence similarity, such as shared presence of the putative integrin-binding motifs. Therefore, Nogo-A most likely originated from the insertion of a neurocan DNA sequence into an ancestral rtn4 gene. Notably, the proposed timing of this event coincides with the acquisition of jaws and myelin by vertebrates. These results not only shed light on the evolution of Nogo-A, but may facilitate the identification of its molecular receptor(s).Although the NSRs in fish and mammals share only 18% identity on the primary structure level, zebrafish Nogo-66 is 66% identical and more than 80% similar to its rat homologue. This notion raises the question whether the fish peptide is able to exert neurite outgrowth inhibition. Surprisingly, in the outgrowth, collapse and contact assays zebrafish Nogo-66 appeared to be growth-permissive for fish and mammalian neurons, quite in contrast to its rat Nogo-66 homologue which inhibits growth. Upon binding to their common receptor NgR1, the rat peptide in contrast to zebrafish Nogo-66 elicits phosphorylation of the downstream effector cofilin which leads to actin filament disassembly. These data are in agreement with the apparent absence of neurite outgrowth inhibitors in fish CNS. However, it is not clear how so similar peptides can exert different responses. Thus, we have analyzed Nogo-66/NgR1 interaction combining coevolutionary and structure modeling approaches. Our results demonstrate that both proteins are already present in cephalochordates but began to coevolve only after fish-tetrapod split. Based on conservation analysis of primary and tertiary structures of these molecules and on published functional data we were able to reconstruct the receptor-ligand complex and model Nogo-66/NgR1 interactions in mammals and fish. The obtained results are in agreement with the previous notion that Nogo-66/NgR1-induced signal transduction becomes inhibitory during/after the fish-tetrapod transition.These extensive analyses of evolution of both inhibitory domains of RTN4/Nogo may help to understand why the ability to regenerate lesioned axons in the CNS became restricted during vertebrate evolution. Moreover, the combination of structural and functional approaches can provide the necessary information which molecular changes during RTN4 evolution are responsible for the acquisition of its inhibitory properties