Anti-Ganglioside Antibody-Mediated Activation of RhoA Induces Inhibition of Neurite Outgrowth

Abstract

Anti-ganglioside antibodies (Abs) are strongly associated with axonal forms of Guillain Barré syndrome (GBS). Some studies indicate that these Abs, including those with GD1a reactivity, are associated with poor prognosis and/or incomplete recovery. We recently demonstrated that a disease-relevant anti-ganglioside Ab with GD1a reactivity inhibits axon regeneration after PNS injury in an animal model (Lehmann et al., 2007). An implication of these findings is that anti-GD1a Abs can mediate inhibition of axon regeneration and limit recovery in some patients with GBS. The downstream inhibitory intracellular signaling that mediates anti-ganglioside Ab-induced axon inhibition remains unclear. In the current study, we show that disease-relevant and GBS patient's anti-ganglioside Abs can inhibit neurite outgrowth in dissociated primary neuronal cultures. Activation of small GTPase RhoA and its key downstream effector Rho kinase (ROCK) are critical mediators of growth cone and neurite outgrowth inhibition. Therefore, we examined the role of these intracellular signaling molecules in our primary neuronal cultures by molecular and pharmacologic approaches. Our results show that the Ab-mediated inhibition of neurite outgrowth involves the activation of RhoA and ROCK pathway and this activation is through the engagement of specific cell-surface gangliosides by Abs. In summary, these studies directly link patient autoantibodies to an intracellular inhibitory signaling pathway involved in anti-ganglioside Abmediated inhibition of neurite outgrowth.

Autoantobodies activate small GTPase RhoA to modulate neurite outgrowth

“…Ab treatment for 24 h significantly increased the number of collapsed growth cones. 41 These observations supported the notion that Ab-mediated inhibition of neurite outgrowth is at least partly mediated at the level of growth cones. This idea was further tested in Xenopus spinal neurons in which localized growth cone collapse leads to repulsive response of growth cones.…”

“…A gradient of anti-GD1a reactive Abs induced repulsive turning responses of growth cones in this model. 41 Overall, cell cultures and growth cone studies showed that anti-ganglioside Abs mimicked the Animal studies. Our initial studies focused on establishing an animal model in which effects of anti-ganglioside Abs on axon regeneration could be examined.…”

“…This issue was examined in p75-null mice and our studies showed that p75-null mice are susceptible to anti-ganglioside Ab-mediated inhibition of axon regeneration indicating that p75 is not involved in inhibitory signaling in our animal model. 41 Two unresolved issues of critical importance include: (a) whether RhoA and ROCK activation is involved in Ab-mediated inhibition in animal model; and (b) whether a transmembrane adaptor molecule is necessary for Ab-mediated activation of intracellular signaling, if so, then the identity of such molecule(s), and if not, then mechanisms involved in intracellular signaling resulting from perturbation of cell surface glycolipids confined to the outer leaflet of plasma membranes. Our group is in the process of examining these issues.…”

“…We established neuronal culture models to study inhibitory effects of anti-ganglioside Abs on neurite growth and growth cone behavior. 41 Development of cell culture models also allowed us to study the downstream inhibitory intracellular signaling that mediates anti-ganglioside Ab-induced axon inhibition. We found that disease-relevant and GBS patient's anti-ganglioside Abs can inhibit neurite outgrowth in dissociated primary sensory and motor neuron cultures.…”

“…The Ab-mediated inhibitory effects were seen in embryonic, post-natal and adult neuron cultures. We performed Schwann cell depleted cultures to show that Ab-mediated inhibition is at the level of axon and not due to indirect injury of glial/Schwann cells; 41 an issue that could not be addressed in animal studies. That Ab-mediated inhibition was through gangliosides was again confirmed in cell cultures studies as neurons obtained from mutant mice lacking complex gangliosides were not susceptible to antiganglioside Abs.…”