In the adult central nervous system, axons can not spontaneously regenerate through the lesion site after injury. Regenerating axons display growth cones at their tips and elongate under the normal condition. At glial scar in injury sites, growth cones are forced to swell and stop. These abnormal growth cones are known as dystrophic growth cones. The formation of dystrophic growth cones is a hallmark of regenerative failure of axons. So far, it has been well established that an extracellular gradient of proteoglycans at the glial scar is responsible for the dystrophic growth cone formation. In vitro, the dystrophic growth cone is formed in adult mouse dorsal root ganglion neurons cultured on a gradient of increasing concentration of chondroitin sulfate proteoglycans. Although dystrophic growth cones display bizarre morphology and contain numerous vacuoles, molecular mechanisms and intracellular events underlying the dystrophic growth cone formation remain elusive. Here, we show that disruption of autophagic flux is involved in the dystrophic growth cone formation by employing an in vitro model of a proteoglycan gradient. Using electron microscopy, we have identified a significant number of vacuoles in dystrophic growth cones as autophagosomes, which contain cytosolic fractions. It has been also verified that a number of vacuoles are positive for LC3, a marker of autophagosomes. Moreover, gene knockdown of SNARE proteins, which play the central role in the fusion of autophagosomes and lysosomes, led to the dystrophic growth cone-like phenotype such as axonal outgrowth inhibition and accumulation of autophagosomes at axonal tips. Finally, we confirmed accumulation of autophagosomes in dystrophic growth cones in vivo. Our study demonstrates that (i) vacuoles in dystrophic endball are autophagosome in both in vitro and in vivo, (ii) in dystrophic growth cone, autophagosomes do not fuse with lysosomes, (iii) gene knockdown of SNARE proteins induce the dystrophic growth cone-phenotypes. These results suggest that sugar chain signaling may interrupt autophagic flux and induce the dystrophic growth cone formation at axonal tips. Our data provide new therapeutic targets to axonal regeneration failure.

研究助成：Research funds : KAKENHI　（23110001）