Neuronal deletion of GSK3β increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2.

Márcia A Liz,Torsten Wittmann,Marlene M Morgado,Sílvia Vieira,Calum Sutherland,Telma E Santos,Helena I Pimentel,James R Woodgett,Mónica M Sousa,Ana M Marques,Fernando M Mar,Vera F Sousa,Hayley Pemble

doi:10.1186/1741-7007-12-47

Abstract

BackgroundIn 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.ResultsFollowing 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.ConclusionsOur 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.

Highlights

In the adult central nervous system, axonal regeneration is abortive
No differential phosphorylation of GSK3α was found (Table 1). These observations indicate that following a conditioning lesion there is an overall decrease of glycogen synthase kinase 3β (GSK3β) activity
Overexpression of collapsin response mediator protein 2 (CRMP-2) decreased the ratio of acetylated/tyrosinated microtubules; this effect being stronger when a phospho-resistant mutant (T/ACRMP-2) was used and less pronounced with the phosphomimetic mutant T/D-CRMP-2 (Figure 7H,I). These results demonstrate that in neurons, the binding of CRMP-2 to tubulin heterodimers in the growth cone controls the rate of microtubule assembly and that this effect is negatively regulated by GSK3β phosphorylation

Summary

Introduction

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 the central nervous system (CNS), axons mostly fail to regenerate given the inhibitory environment and the lack of activation of neuronal-intrinsic regenerationassociated pathways. It is, possible to stimulate the intrinsic growth capacity of CNS axons. Pharmacological destabilization of microtubules converts a growth cone into a retraction bulb, and stabilization leads to the formation of growth cone-like structures [2] and increases axonal regeneration in vivo, after spinal cord injury (SCI) [3]. It has been shown that in the PNS, axons have a higher regenerative capacity as they activate a program leading to the activation of histone deacetylase 5 (HDAC5) and to a lower microtubule stability close to the injury site [5]

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Conclusion