title: Neuronal deletion of GSK3β increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2.
abstract: BACKGROUND
  In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity.
  
  
  RESULTS
  Following a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3β (GSK3β) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3β regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3β activity in neurons. Following spinal cord injury, reduced GSK3β levels or complete neuronal deletion of GSK3β led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3β substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3β-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3β-CRMP-2 relevance during axon regeneration.
  
  
  CONCLUSIONS
  Our work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3β-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth.
authors.forename: Márcia A
authors.initials: MA
authors.lastname: Liz
authors.forename: Fernando M
authors.initials: FM
authors.lastname: Mar
authors.forename: Telma E
authors.initials: TE
authors.lastname: Santos
authors.forename: Helena I
authors.initials: HI
authors.lastname: Pimentel
authors.forename: Ana M
authors.initials: AM
authors.lastname: Marques
authors.forename: Marlene M
authors.initials: MM
authors.lastname: Morgado
authors.forename: Sílvia
authors.initials: S
authors.lastname: Vieira
authors.forename: Vera F
authors.initials: VF
authors.lastname: Sousa
authors.forename: Hayley
authors.initials: H
authors.lastname: Pemble
authors.forename: Torsten
authors.initials: T
authors.lastname: Wittmann
authors.forename: Calum
authors.initials: C
authors.lastname: Sutherland
authors.forename: James R
authors.initials: JR
authors.lastname: Woodgett
authors.forename: Mónica M
authors.initials: MM
authors.lastname: Sousa
mesh_terms: D000818:Animals; D001369:Axons / Q000502:physiology*; D005260:Female; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D020439:Growth Cones / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D012038:Regeneration
chemical_list: C506523:CLASP2 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; C095114:collapsin response mediator protein-2; C085284:microtubule-associated protein 1B; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3