Facilitating axon regeneration in the injured CNS by microtubules stabilization

Abstract

Traumatic CNS injuries often cause permanent, devastating disabilities due to a lack of regeneration of damaged axons. Next to an insufficient intrinsic capability of CNS neurons to regrow axons, also inhibitory molecules that are associated with the CNS myelin and the glial scar contribute to the failure of axonal regeneration. Strategies targeting the inhibitory molecules, their receptors or downstream signaling pathways result in little improvement of regeneration in vivo. However, the combination of such approaches together with measures that increase the intrinsic growth potential of neurons reportedly lead to a significantly better outcome. In this mini-review we outline and discuss a novel therapeutic strategy facilitating axon regeneration by directly targeting microtubule dynamics in axonal growth cones and reducing the inhibitory scar formation at the injury site by the anticancer drug Taxol. Moreover, we portray the mechanisms underlying the beneficial effects of Taxol and its potential as an adjuvant drug to accomplish substantial regeneration and functional recovery after CNS injuries in vivo.

Damage of the optic nerve or spinal cord injury can result in devastating disabilities, such as irreversible blindness or paralysis. These disabilities are often due to the insufficient capacity of the central nervous system (CNS) to regenerate injured axons and are partially attributable to a destabilization of growth cones by molecules associated with the CNS white matter (e.g., Nogo, myelin associated glycoprotein and oligodendrocyte-myelin glycoprotein) and/or the glial scar (e.g., chondroitin sulfate proteoglycans, CSPGs).^1,2 Exposure of injured axon tips to central myelin or CSPGs leads via specific receptors and activation of RhoA/ROCK signaling to a depolymerisation of actin filaments in growth cones and subsequent growth inhibition.^3–7 Thus, one common strategy to enhance axon regeneration is stabilizing the growth cones by preventing the actin filament depolymerization by inhibitory molecules.¹ In the visual system, for instance, viral overexpression of a dominant negative form of the Nogo receptor or inactivation of RhoA/ROCK-signaling by the expression of ADP ribosyl transferase in retinal ganglion cells (RGCs) have been shown to overcome myelin inhibition and to promote axon regeneration in the optic nerve.^8,9 However, these studies and others have also shown that overcoming inhibitory signaling alone results only in little regeneration, since mature central neurons, contrary to embryonic or postnatal neurons, possess only a weak intrinsic capacity to regrow axons even in the absence of inhibitors. Therefore, combinatorial treatments aiming to activate the intrinsic growth ability and measures to overcome inhibitory signaling result in significant better axon regeneration than each treatment alone.^8–11

Microtubules Stabilization Facilitates Axonal Growth and Desensitizes Growth Cones Towards Inhibitory Molecules In Vitro

Microtubules polymerization is essential for axonal growth and is modulated by microtubules-actin interaction in the growth cone.^12,13 Thus, myelin inhibitors and CSPGs indirectly compromise microtubules extension. In our recent article we directly targeted microtubules dynamics using the clinically established anticancer drug Paclitaxel (Taxol).¹⁴ Taxol can differentially affect the microtubule dynamics based on the concentrations at which it is applied. At higher concentrations, as typically used for cancer therapy, Taxol hyperstabilizes microtubules, thereby abrogates microtubule extension and inhibits mitotic spindle assembly, which is essential for cell division. However, at lower concentrations Taxol favors the polymerization of microtubules at the plus ends.^15,16 Hence, we hypothesized that at low concentrations Taxol might enhance polymerization of microtubules in growth cones and thereby improve axon growth of mature CNS neurons. Indeed, axon outgrowth of mature RGCs was markedly increased on a growth permissive substrate in culture when Taxol was applied at a concentration of 1–3 nM, whereas at a concentration of 10 nM it had no effect. At a concentration of 50 nM the drug even reduced outgrowth compared to untreated controls. The beneficial effects of Taxol were compromised by nocodazole a compound that increases the catastrophe rate of microtubules, suggesting that Taxol indeed enhanced neurite extension directly by microtubules stabilization. The effects of Taxol on neurite growth were further increased by the coadministration of ciliary neurotrophic factor (CNTF), implying that both molecules acted synergistically through different mechanisms. CNTF mediates its effects via the activation of various signaling pathways in the cell body of neurons.^17,18 In contrast, Taxol treatment stabilized growth cones and completely overcame the neurite outgrowth inhibition by myelin without affecting the myelin-induced activation of RhoA. This disinhibitory effect of Taxol was also found for inhibitory CSPGs, implying that it is not restricted to specific receptors or pathways, but rather mediated by microtubules polymerization. One possible explanation for the disinhibitory effect of Taxol might be the disentanglement of the coupled interaction of actin and microtubules in the growth cone. This makes the microtubule polymerization at the plus ends independent of the state of actin polymerization and thereby stabilizes the overall cytoskeleton structure in the growth cone, including the microtubules in the filopodial extensions.^14,19 Thus, in a defined concentration range Taxol exerts two beneficial effects that are relevant for axon regeneration: it promotes axon extension and decreases the sensitivity of growth cones towards inhibitory molecules.

Local Application of Taxol Facilitates Axon Regeneration In Vivo

The optic nerve is a widely used model system to study the regenerative failure of CNS axons. In vivo mature RGCs can be transformed into a regenerative state by lens injury or intravitreal application of zymosan or the toll-like receptor 2 agonist Pam₃Cys.^20–24 The transformation of RGCs into a regenerative state is associated with a dramatic change in gene expression, which helps RGCs to survive optic nerve injury and also extend axons beyond the optic nerve injury site.⁸ Astrocyte-derived CNTF and leukemia inhibitory factor (LIF) have been identified as the essential key factors mediating both the neuroprotective and axon growth-promoting effects after lens injury.^17,25,26 Nevertheless, the regeneration of stimulated RGCs is still limited by the inhibitory environment of the glial scar and myelin.

Our recent paper demonstrates that locally applied Taxol, although not affecting the regenerative state or survival of RGCs, markedly augmented initial growth of axons beyond the lesion site of the optic nerve.¹⁴ These effects may be due to its disinhibitory and axon growth-promoting effects observed in cell culture. When evaluated 14 days after injury, local Taxol application only moderately improved axonal regeneration in the optic nerve. However, regeneration was dramatically enhanced when Taxol treatment was combined with a stimulation of the regenerative state of RGCs by lens injury. The regeneration of axons 1 mm beyond the lesion site was 15 times higher with the combinatorial treatment compared to lens injury alone. Notably, although Taxol exerted beneficial effects at a broad concentration range in vivo, its effects were, as observed in culture, concentration dependent with strongest effects measured at low concentrations. This finding may be relevant in view of a potential therapeutic use for nerve repair in humans, since it may lower the risk for known adverse side effects associated with the application of much higher dosages of the drug in cancer therapy. In contrast to locally applied Taxol at the injury site, intravitreal injections of Taxol did not facilitate axon regeneration, suggesting that the growth promoting effects were due to an interaction of the drug with the microtubules in growth cones and, as discussed below, to the delayed glial scar formation.

In accordance to our results, another concurrently published study has reported that the continuous application of low concentrations of Taxol into the lesion site of the spinal cord combined with an activation of the regenerative state of dorsal root ganglion neurons through a peripheral conditioning lesion improved axon regeneration and functional recovery.²⁷ These data demonstrate that microtubules stabilization by Taxol is a suitable approach to successfully facilitate axon regeneration in different areas of the CNS.

Taxol Treatment Delays Glial Scar Formation

Glial scar formation is one of the major impediments that hinders axon regeneration after CNS injury. The events that occur around the lesion site include the proliferation of astrocytes, which leads to astrocytic hypertrophy and expression of inhibitory molecules such as CSPGs.² In addition, the activation of microglia and recruitment of peripheral macrophages from the blood stream are also involved in glial scar formation. The latter ones have been recently proposed to function as another significant barrier for axon regeneration.^28,29 Since Taxol is an anti-proliferative drug and also known to affect cell migration,^30,31 it was plausible that locally applied Taxol might also influence the glial scar formation. Indeed, even at low concentrations Taxol transiently delayed the proliferation of astrocytes and the infiltration of macrophages around the lesion site after optic nerve injury. Notably, Taxol suppressed the expression of inhibitory CSPGs in the injured optic nerve a finding that was also reported in the injured spinal cord.^14,27 One explanation for the CSPG suppression could be an inhibition of transforming growth factor beta (TGFβ) signaling by Taxol, which is necessary for the inhibitory scar formation.^32,33 Previous reports proposed that microtubules destabilization modulates TGFβ signaling by favoring phosphorylation of Smads and translocation of the microtubule bound-transcription factors from the cytoplasm to the nucleus.³⁴ Thus, microtubule stabilization by Taxol enhances the binding of Smad to microtubules, thereby diminishing the translocation of Smads to the nucleus and abrogating TGFβ signaling.²⁷ Another explanation could be that the expression of TGFβ per se is reduced by the Taxol treatment, since Taxol delays the proliferation and migration of glial cells and macrophages, which are known sources of TGFβ at the lesion site. Regardless of the underlying mechanism, the delay in glial scar formation and reduction of macrophage invasion by Taxol treatment may have also contributed to the augmented regeneration observed after optic nerve injury.

Perspectives

Current reports demonstrate that Taxol, when locally applied at the injury site fosters axon regeneration by microtubules stabilization in the axonal growth cone and delays the inhibitory glial scar formation.^14,27 Moreover, Taxol exerts its beneficial effects on axon regeneration at low concentrations, which reduces the risk of potential adverse side effects. Taxol is also a clinically approved drug for humans which makes this compound a promising candidate as an adjuvant drug for the treatment of patients suffering from traumatic CNS injuries or stroke. Additional optimization of modalities of drug application may further improve the beneficial outcome on axon regeneration and be necessary for a potential application in humans. In fact, a previous study has reported that the systemic application of Taxol supports functional recovery of rats after spinal cord injury, although it is not clear whether these effects were the result of improved axonal regeneration or caused by other mechanisms.³⁵ Nevertheless, very recently published results from both, optic nerve and spinal cord injury models lend encouragement to the possibility that microtubule-stabilizing compounds such as Taxol may be suitable adjuvant drugs for the treatment of CNS injuries particularly when combined with interventions stimulating the intrinsic regenerative state of neurons.