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

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.

siRNA-Mediated Knockdown of the mTOR Inhibitor RTP801 Promotes Retinal Ganglion Cell Survival and Axon Elongation by Direct and Indirect Mechanisms

“…82 Several reports provide evidence that GSK3b inhibition is associated with enhanced neuronal growth. [83][84][85] In summary, siRTP801-dependent potentiation of PI3K pathway signaling in RGCs via paracrine secretion of neurotrophins by activated glia combined with siRTP801-mediated activation of both mTORC1 and mTORC2 in RGCs themselves may be responsible for the observed increased survival and longer axon/neurite growth seen after siRTP801 treatment both in vitro (mixed retinal cultures) and in vivo. The challenge remains for the development of novel neuroprotective and axon regenerative therapies that can be used for the benefit of patients.…”

Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons

“…Downstream to Akt is glycogen synthase kinase 3 beta (GSK-3b) which plays a central role in axonal growth. Activated Akt phosphorylates GSK-3b at Ser9, inactivating its constitutive kinase activity (Grimes and Jope 2001), a process required for neuronal polarization (Jiang et al 2005;Yoshimura et al 2005), axon growth (Zhou et al 2004), regeneration (Liz et al 2014) and branching (Garrido et al 2007). GSK-3b also regulates microtubule extension at the growth cone (Fukata et al 2002;Cole et al 2004;Zhou et al 2004Yoshimura et al 2005.…”

Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration

“…Several signaling pathways converge onto microtubules to promote axon regeneration in the mammalian system (4). The GSK3-␤ pathway regulates the activity of microtubule-binding proteins such as cytoplasmic linker associated protein (CLASP), adenomatous polyposis coli, and CRMP2 to coordinate the stability, growth speed, and configuration of microtubules in growth cones and regulates axon growth (5,6). In addition to microtubule-binding proteins, posttranslational modifications of tubulin are believed to correlate with the dynamic properties of microtubules (7).…”