TACE-induced cleavage of NgR and p75NTR in dorsal root ganglion cultures disinhibits outgrowth and promotes branching of neurites in the presence of inhibitory CNS myelin.

After binding, central nervous system (CNS) myelin-derived axon growth inhibitory ligands, the Nogo-66 receptor (NgR), complexes with LINGO-1 and either the low-affinity neurotrophin receptor (p75(NTR)) or TROY to initiate growth cone collapse via a Rho-A inhibitory signaling pathway and/or Ca(2+)-dependent activation of epidermal growth factor receptor (EGFR) through an unknown signaling pathway. We have shown that axon growth through CNS myelin is disinhibited after neurotrophic factor administration by 1) initiating intramembranous proteolysis (RIP) of p75(NTR), leading to cleavage of the extracellular (p75(ECD)) and intracellular domains (p75(ICD)) by alpha- and gamma-secretase, respectively, thereby paralyzing inhibitory signaling; 2) shedding of soluble NgR(ECD), which acts as a competitive antagonist to NgR for binding of inhibitory ligands; and 3) antagonizing NgR/p75(NTR) clustering by competitive p75(ECD)/NgR interaction. Here, we report that TNF-alpha converting enzyme (TACE) (a disintegrin and metalloproteinase 17, ADAM17) induces disinhibition of FGF2-stimulated neurite outgrowth of dorsal root ganglion neurons (DRGN) cultured in the presence of a predetermined concentration of inhibitory CNS myelin-derived ligands. After addition of TACE (which has alpha-secretase activity) to mitotically arrested adult rat mixed DRG cultures, we demonstrate 1) NgR(ECD) shedding; 2) release of p75(ECD) and p75(ICD) by RIP of p75(NTR); 3) blockade of Rho-A activation; 4) reduced EGFR phosphorylation; and 5) increased FGF2-stimulated DRGN neurite outgrowth and branching in the presence of CNS myelin-derived inhibitory ligands. Thus, TACE-induced cleavage of NgR and RIP of p75(NTR) abrogates axon growth inhibitory signaling, thereby disinhibiting CNS axon/neurite growth.

Schwann cell-derived factor-induced modulation of the NgR/p75NTR/EGFR axis disinhibits axon growth through CNS myelin in vivo and in vitro.

When associated with the Nogo receptor (NgR), the transmembrane receptor p75NTR signals growth cone collapse. Arrest of CNS axon growth in vivo is mediated by CNS myelin-derived inhibitory ligands through either an unknown pathway after NgR- and Ca2+-dependent activation of the epidermal growth factor receptor (EGFR), and/or sequential Rho-A/ROCK/LIM-kinase/cofilin phosphorylation leading to actin depolymerization. Paradoxically, rat retinal ganglion cell (RGC) axons regenerate through the CNS myelin-rich transected optic nerve after intravitreal sciatic nerve grafting without inhibitory ligand neutralization. Here, we show that optic nerve regeneration in vivo correlates with Schwann cell-derived factor-induced cleavage of NgR and Nogo-A, and inactivation of p75NTR signalling by the induction of regulated intramembranous proteolysis (RIP) and the release of both extracellular (p75ECD) and intracellular (p75ICD) domains. Hence, Schwann cell-derived factors compromise inhibitory signalling by (i) antagonizing ligand/NgR binding with metalloproteinase-cleaved Nogo-A peptides; (ii) RIP of p75NTR; (iii) competitively blocking NgR/p75NTR clustering with soluble p75ECD; and (iv) consequent reduced downstream EGFR phosphorylation and suppression of Rho-A activation. Moreover, in RGC cultures, exogenous tumour necrosis- converting enzyme (TACE) initiates RIP of p75NTR, reduces EGFR phosphorylation, suppresses activation of Rho-A, cleaves the ECD from both NgR and TROY, and disinhibits neurotrophic factor (NTF) stimulated RGC neurite outgrowth in the presence of CNS myelin. Soluble NgRECD binds all CNS myelin-derived ligands and thus has the potential to act as an inhibitory signalling antagonist, but the role of TROY and its shed ectodomain in growth cone mobility is unknown. siRNA knockdown of p75NTR also inactivates Rho-A and disinhibits NTF-stimulated RGC neurite outgrowth in cultures with added CNS myelin. In all the above experimental paradigms, Schwann cell-derived factor/NTF-induced attenuation of NgR/p75NTR signalling suppresses EGFR activation, thereby potentiating axon growth disinhibition.

Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS myelin by siRNA-mediated knockdown of NgR, p75NTR and Rho-A.

The presence of multiple axon growth inhibitors may partly explain why central nervous system axons are generally incapable of regenerating after injury. Using RNA interference (RNAi) in dorsal root ganglia neurons (DRGN), we demonstrate siRNA-mediated silencing of components of the inhibitory signalling cascade, including p75NTR, NgR and Rho-A mRNA, of 70%, 100% and 100% of the relevant protein, respectively, while changes in neither protein levels nor cellular immunoreactivity were detected using the relevant scrambled siRNA control sequences. Importantly, after 48 h in culture after siRNA-mediated knockdown of Rho-A, neurite outgrowth was enhanced by 30% compared to that after p75NTR and 50% after NgR silencing. By 3 days, a 5-, 3.5- and 6.5-fold increase in betaIII-tubulin protein levels were observed compared to controls without siRNA after knockdown of p75NTR, NgR and Rho-A, respectively. Together, these results suggest that Rho-A knockdown might be the most effective target for a disinhibition strategy to promote CNS axon regeneration in vivo.

Matrix metalloproteases: degradation of the inhibitory environment of the transected optic nerve and the scar by regenerating axons.

After injury to the central nervous system, a glial/collagen scar forms at the lesion site, which is thought to act as a physicochemical barrier to regenerating axons. We have shown that scar formation in the transected optic nerve (ON) is attenuated when robust growth of axons is stimulated. Matrix metalloproteases (MMP), modulated by tissue inhibitors of MMP (TIMP), degrade a wide variety of extracellular matrix components (ECM) and may be activated by growing axons to remodel the ECM to allow regeneration through the inhibitory environment of the glial or collagen scar. Here, we investigate whether MMP levels are modulated in a nonregenerating (scarring) versus a regenerating (nonscarring) model of ON injury in vivo. Western blotting and immunohistochemistry revealed that MMP-1, -2, and -9 levels were higher and TIMP-1 and TIMP-2 levels were lower in regenerating compared to nonregenerating ON and retinae. In situ zymography demonstrated significantly greater MMP-related gelatinase activity in the regenerating model, mainly colocalized to astrocytes in the proximal ON stump and around the lesion site. These results suggest that activation of MMP and coincident down-regulation of TIMP may act to attenuate the inhibitory scarring in the regenerating ON, thus transforming the ON into a noninhibitory pathway for axon regrowth.