The Role Of HnRNP K And Its Underlying Mechanisms During Optic Nerve Regeneration In Xenopus Laevis

In contrast to mammals, axotomized optic axons of Xenopus and other anamniotes successfully regenerate functional connections throughout their whole life. To better understand the molecular basis for this successful regeneration, we focused on the role of an RNA-binding protein, heterogeneous nuclear ribonucleoprotein (hnRNP) K, because it is required for axonogenesis during development and because several of its mRNA targets are under strong post-transcriptional control during regeneration. We fist successfully built the optic crush model in Xenopus. At11d after optic nerve crush, hnRNP K underwent significant translocation from the cytoplasm into the nucleus of retinal ganglion cells (RGCs), indicating that the protein became activated during regeneration. To suppress its expression, we intravitreously injected an antisense Vivo-Morpholino (VMO) oligonucleotide targeting hnRNP K. The results showed that VMO efficiently inhibited hnRNP K expression with an optimal reduction of80%of its immunofluorescence. Moreover, the suppression of hnRNP K expression was extensively restricted to the retinal ganglion cells (RGCs), inducing neither an axotomy response nor axon degeneration. The establishment of such in vivo knock down model in RGCs made it possible for us to study the role of hnRNP K with the underlying mechanisms during optic nerve regeneration in Xenopus. After optic nerve crush, staining for multiple markers of regenerating axons showed no regrowth of axons beyond the lesion site with hnRNP K knockdown. RGCs nonetheless responded to the injury by increasing expression of multiple growth-associated RNAs and experienced no additional neurodegeneration above that normally seen with optic nerve injury. At the molecular level, hnRNP K knockdown during regeneration inhibited protein, but not mRNA, expression of several known hnRNP K RNA targets (NF-M, GAP-43) by compromising their efficient nuclear transport and disrupting their loading onto polysomes for translation, and thereby significantly reduced the synthesis of multiple cytoskeletal associated factors required to rebuild the axon. Our study therefore provides evidence of a novel post-transcriptional regulatory pathway orchestrated by hnRNP K that is essential for successful CNS axon regeneration.