Post‐translational regulation of Crmp in developing and regenerating chick spinal cord

Abstract

It is becoming apparent that regulation at the protein level plays crucial roles in developmental and pathological processes. Therefore, we performed a proteomics screen to identify proteins that are differently expressed or modified at stages of development permissive (E11) and nonpermissive for regeneration (E15) of the chick spinal cord. Proteins regulated either developmentally or in response to spinal-cord injury included collapsin-response-mediator proteins (Crmps), known to modulate microtubule dynamic and axonal growth. No significant changes in Crmp transcripts following injury were observed, indicating regulation mainly at the protein level. Analysis of Crmp-2 protein and its phosphorylated forms, pS522 and pT514, showed that Crmp-2 is developmentally regulated and also expressed in neural progenitors in vivo and in neurospheres. Its cellular localization changed both with development and following spinal-cord injury. In addition, although overall levels of Crmp-2 expression were not affected by injury, abundance of certain phosphorylated forms was altered. pT514 Crmp-2 appeared to be associated with dividing neural progenitors and was greatly reduced at nonpermissive stages for regeneration, whereas it did not seem affected by injury. In contrast, phosphorylation of Crmp-2 at S522 was upregulated early after injury in regenerating spinal cords and the ratio between phosphorylated to total Crmp-2 increased, as indicated by 2D Western blots. Altogether, this study shows highly dynamic regulation of Crmp-2 forms during development and identifies post-translational changes in Crmp-2 as putative contributors to the maintenance of spinal-cord regenerative ability, possibly via a transient stabilization of the neuronal cytoskeleton.

Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway

“…Proteins were extracted from either cell pellets or tissues as previously described [41]. For Western-blot analysis, 20–40 μg of proteins per lane were separated by 10% or 12% SDS-PAGE and electrotransferred to nitrocellulose membranes (GE Healthcare) using a TransBlot-SD (BioRad).…”

Protein deiminases: New players in the developmentally regulated loss of neural regenerative ability

“…PAD3 and Gapdh gene expression was determined by quantitative real time PCR (7500 Fast Real Time PCR System, Applied Biosystems) using the SYBR GreenER reagent kit (Invitrogen)(Gögel et al, 2010). Each sample was run in duplicate.…”

“…Immunoprecipitated protein fractions were analysed by 12% SDS-PAGE and Western blotting. Candidate bands were excised from silver stained gels, and analyzed by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) as previously described (Gögel et al, 2010). …”

Comparative proteomics of spinal cords of rat fetuses with spina bifida aperta

“…Crmps play a role in axon guidance and neuronal growth cone collapse [38]. Crmp-4 is expressed immediately after neuronal birth [39], is induced by peripheral nerve injury, and is suggested to regulate regenerative neurite outgrowth [25,40]. However, Duplan et al [41] found that upregulated Crmp-4 could trigger axonal degeneration and motoneuron death.…”