Spinal cord regenerative ability is lost with development, but the mechanisms underlying this loss are still poorly understood. In chick embryos, effective regeneration does not occur after E13, when spinal cord injury induces extensive apoptotic response and tissue damage. As initial experiments showed that treatment with a calcium chelator after spinal cord injury reduced apoptosis and cavitation, we hypothesized that developmentally regulated mediators of calcium-dependent processes in secondary injury response may contribute to loss of regenerative ability. To this purpose we screened for such changes in chick spinal cords at stages of development permissive (E11) and non-permissive (E15) for regeneration. Among the developmentally regulated calcium-dependent proteins identified was PAD3, a member of the peptidylarginine deiminase (PAD) enzyme family that converts protein arginine residues to citrulline, a process known as deimination or citrullination. This post-translational modification has not been previously associated with response to injury. Following injury, PAD3 up-regulation was greater in spinal cords injured at E15 than at E11. Consistent with these differences in gene expression, deimination was more extensive at the non-regenerating stage, E15, both in the gray and white matter. As deimination paralleled the extent of apoptosis, we investigated the effect of blocking PAD activity on cell death and deiminated-histone 3, one of the PAD targets we identified by mass-spectrometry analysis of spinal cord deiminated proteins. Treatment with the PAD inhibitor, Cl-amidine, reduced the abundance of deiminated-histone 3, consistent with inhibition of PAD activity, and significantly reduced apoptosis and tissue loss following injury at E15. Altogether, our findings identify PADs and deimination as developmentally regulated modulators of secondary injury response, and suggest that PADs might be valuable therapeutic targets for spinal cord injury.
Axonal growth cones require an evolutionary conserved repulsive guidance system to ensure proper crossing of the CNS midline. In Drosophila, the Slit protein is a repulsive signal secreted by the midline glial cells. It binds to the Roundabout receptors, which are expressed on CNS axons in the longitudinal tracts but not in the commissural tracts. Here we present an analysis of the genes leak and kuzbanian and show that both genes are involved in the repulsive guidance system operating at the CNS midline. Mutations in leak, which encodes the Roundabout-2 Slit receptor, were first recovered by Nüsslein-Volhard and co-workers based on defects in the larval cuticle. Analysis of the head phenotype suggests that slit may be able to act as an attractive guidance cue while directing the movements of the dorsal ectodermal cell sheath. kuzbanian also regulates midline crossing of CNS axons. It encodes a metalloprotease of the ADAM family and genetically interacts with slit. Expression of a dominant negative Kuzbanian protein in the CNS midline cells results in an abnormal midline crossing of axons and prevents the clearance of the Roundabout receptor from commissural axons. Our analyses support a model in which Kuzbanian mediates the proteolytic activation of the Slit/Roundabout receptor complex.
The chick spinal cord can regenerate following injury until advanced developmental stages. It is conceivable that changes in stem/progenitor cell plasticity contribute to the loss of this capacity, which occurs around E13. We investigated the contribution of proliferation, phenotypic changes in radial glia progenitors, and neurogenesis to spinal cord regeneration. There was no early up-regulation of markers of gliogenic radial glia after injury either at E11 or E15. In contrast, increased proliferation in the grey matter and up-regulation of transitin expression following injury at E11, but not E15, suggested high levels of plasticity within the E11 spinal cord progenitor population that are lost by later stages. Changes in neural progenitors with development were also supported by a higher neurosphere forming ability at E11 than at E15. Co-labelling with doublecortin and neuron-specific markers and BrdU in spinal cord sections and dissociated cells showed that neurogenesis is an ongoing process in E11 chick spinal cords. This neurogenesis appeared to be complete by E15. Our findings demonstrate that the regeneration-competent chick spinal cord is less mature and more plastic than previously believed, which may contribute to its favourable response to injury, and suggest a role for neurogenesis in maintaining regenerative capacity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.