Myelin-associated Glycoprotein Inhibits Microtubule Assembly by a Rho-kinase-dependent Mechanism

Abstract

Myelin-associated glycoprotein (MAG) and Nogo are potent inhibitors of neurite outgrowth from a variety of neurons, and they have been identified as possible components of the central nervous system myelin that prevents axonal regeneration in the adult vertebrate central nervous system. The activation of RhoA and Rhokinase is reported to be an essential part of the signaling mechanism of these proteins. Here, we report that the collapsing response mediator protein-2 (CRMP-2) is phosphorylated by a Rho-kinasedependent mechanism downstream of MAG or Nogo-66. The overexpression of the nonphosphorylated form of CRMP-2 at threonine 555, which is the phosphorylation site for Rho-kinase, counteracts the inhibitory effect of MAG on the postnatal cerebellar neurons. Additionally, the expression of the dominant negative form of CRMP-2 or knockdown of the gene using small interference RNA (siRNA) mimics the effect of MAG in vitro. Consistent with the function of CRMP-2, which promotes microtubule assembly, microtubule levels are down-regulated in the cerebellar neurons that are stimulated with MAG in vitro. Reduction in the density of microtubules is also observed in the injured axons following the spinal cord injury, and this effect depends on the Rho-kinase activity. Our data suggest the important roles of CRMP-2 and microtubules in the inhibition of the axon regeneration by the myelin-derived inhibitors.Several myelin-derived proteins have been identified as components of the central nervous system myelin that prevents axonal regeneration in the adult vertebrate central nervous system. To date, three major inhibitors that are expressed by oligodendrocytes and myelinated fiber tracts have been identified (1). These are Nogo, myelin-associated glycoprotein (MAG), 2 and oligodendrocyte-myelin glycoprotein. All these proteins act on neurons through the p75 receptor (p75) (2-4) in complex with the Nogo receptor. One potential clue to understanding the signal transduction mechanism downstream of p75 and the Nogo receptor is found through observations that demonstrate the small GTPase RhoA as a key intracellular effector for growth inhibitory signaling by myelin. In its active GTP-bound form, RhoA rigidifies the actin cytoskeleton, thereby inhibiting axon elongation and mediating growth cone collapse. RhoA is activated by MAG, Nogo-66, and oligodendrocyte-myelin glycoprotein through a p75-dependent mechanism; thus, inhibiting neurite outgrowth from postnatal sensory neurons and cerebellar neurons (2-5). The regulation of RhoA activity by MAG and Nogo through p75 is mediated by the release of RhoA from Rho GDI, which suppresses the RhoA activity (6, 7).Although RhoA and one of its effectors, Rho-kinase, appear to play a key role in regulating axon growth, the mechanism by which the myelinderived proteins regulate axon outgrowth remains to be elucidated. The outgrowth of axons is based on the dynamic rearrangement of the cytoskeleton (8). Cues that influence the axon outgrowth are sensed by the growth cone, which is a highly mot...

Identification of CRMP4 as a Convergent Regulator of Axon Outgrowth Inhibition

“…This is partly mediated by binding to tubulin heterodimers and promoting microtubule assembly (Fukata et al, 2002b). In fact, CRMP2 undergoes ROCK-dependent phosphorylation in response to Nogo-66, and MAG and has been hypothesized to subsequently regulate microtubule dynamics (Mimura et al, 2006). Although all CRMP members can bind tubulin, CRMP4 is not a substrate for ROCK, and we were unable to detect a change in affinity between CRMP4 and tubulin in response to Nogo-66 stimulation by coimmunoprecipitation analysis in PC12 cells (data not shown).…”

Disorganized Microtubules Underlie the Formation of Retraction Bulbs and the Failure of Axonal Regeneration

“…This suggests that extracellular inhibitory molecules could also induce changes in the dynamics and morphology of the axonal tip by affecting microtubule organization. Indeed, a recent report from Mimura et al (2006) showed that MAG inhibits microtubule assembly and axon outgrowth in postnatal cerebellar granule neurons via Rho kinase activation. Interestingly, they identified CRMP-2 (collapsin response mediator protein-2), a microtubuleassociated protein, as a target for microtubule disassembly both in vitro and in vivo after spinal cord injury.…”

“…Microtubules are regulated by external signals during axon elongation and guidance (Suter et al, 1998;Buck and Zheng, 2002;Dent and Gertler, 2003). In fact, the known inhibitory cues present in the CNS myelin and the glial scar, including myelinassociated glycoprotein (MAG), Nogo, OMgP (oligodendrocytemyelin glycoprotein), and versican, converge downstream of their receptors onto signaling pathways of the Rho GTPases (Dubreuil et al, 2003;Fournier et al, 2003;Schweigreiter et al, 2004;Sivasankaran et al, 2004), which, in addition to the actin cytoskeleton, also affect microtubule stability and dynamics (Etienne-Manneville, 2004;Mimura et al, 2006). This suggests that extracellular inhibitory molecules could also induce changes in the dynamics and morphology of the axonal tip by affecting microtubule organization.…”

Taxol Facilitates Axon Regeneration in the Mature CNS

“…After disruption at the lesion site of the optic nerve, inhibitory factors from the myelin activate RhoA (Mukhopadhyay et al, 1994;Chen et al, 2000;GrandPre et al, 2000;Prinjha et al, 2000;Wang et al, 2002;Atwal et al, 2008) and further downstream cascades lead to a local arrest of axonal microtubule polymerization and axon extension (Mimura et al, 2006;Geraldo and Gordon-Weeks, 2009). The current study demonstrates that neurite growth inhibition of primary RGCs and PC12 cells by CNS myelin in culture is abolished in the presence of Taxol (3 nM).…”