Guillain Barré Syndrome-associated anti-glycan antibodies alter growth cone cytoskeleton from growing DRGs neurons

ROZÉS SALVADOR, V.1; WOJNACKI, J. 1; HEREDIA, F.1 ; PALANDRI, A. 1; SHEIKH, KAZIN A.3; CACERES, A1; LOPEZ, P.H.H.1,2

Saint Malo

Congreso; Peripheral Nerve Society Meeting 2013; 2013

Peripheral Nerve Society

High titers of anti-ganglioside antibodies have been associated with delayed and/or incomplete clinical recovery in Guillain-Barré Syndrome (GBS) patients. In most cases incomplete recovery is associated with the extent and location of the lesion in the peripheral nerves. Passive transfer of GBS patients-derived anti-ganglioside abs targeting GD1a/GT1b (clone-1B7) can halt axon regeneration in mice. Inhibition of axon regeneration was associated with the presence of end-bulb like structures characteristic of dystrophic growth cones in the nerve. Unmasking the molecular mechanisms underlying antibody-mediated disruption of nerve repair is critical to develop new therapeutic strategies to enhance axon growth. We found that treatment of dissociated dorsal root ganglion neurons-DRGn cultures with mAb 1B7 inhibited neurite outgrowth by altering cytoskeleton components of the growth cone including changes in stable and dynamic microtubules as well as actin network. In addition DRGn were nucleofected with LifeAct-mCherry and Tubulin-GFP and subject to time-lapse microscopy to analyze spatial/temporal changes of these components. Treatment with mAb 1B7 induced first a rapid loss of actin filopodia and then retraction of microtubules from growth cones in the first three hours of treatment. Further studies included analysis of spatio-temporal dynamics of small GTPase RhoA activity, a known downstream effector of antibody-triggered signaling, by using a biosensor based on fluorescence resonance energy transfer-FRET. We next attempted to develop a model where dystrophic growth cones with similar morphology to the observed in mice treated with mAb 1B7 could be reproduced and studied. DRG explants were co-cultured with peripheral nerve, treated with mAb 1B7 and axon regeneration analyzed in distal portions of the nerve. Treatment of nerves with 1B7 resulted in robust inhibition of axon regeneration associated with formation of end-bulb like shaped dystrophic growth cones. Infection of DRGs with lentiviral particles carrying shRNA sequence against St3gal2, a key enzyme in the biosynthesis of GD1a/GT1b gangliosides, reversed axon inhibition. Thus, this model could represent a novel opportunity to study the inhibitory role of anti-ganglioside Abs on axon regeneration. Overall this data provides knowledge about the molecular mechanisms determining impaired nerve repair in GBS which could suggest new pharmacological targets for promoting axon regeneration.