Comparing astrocytic cell lines that are inhibitory or permissive for axongrowth: the major axon-inhibitory proteoglycan is NG2

Astrocytes, oligodendrocytes, and oligodendrocyte/type 2 astrocyte progenit ors (O2A cells) can all produce molecules that inhibit axon regeneration. W e have shown previously that inhibition of axon growth by astrocytes involv es proteoglycans. To identify inhibitory mechanisms, we created astrocyte c ell lines that are permissive or nonpermissive and showed that nonpermissiv e cells produce inhibitory chondroitin sulfate proteoglycans (CS-PGs). We h ave now tested these cell lines for the production and inhibitory function of known large CS-PGs. The most inhibitory line, Neu7, produces three CS-PG s in much greater amounts than the other cell lines: NG2, versican, and the CS-56 antigen. The contribution of NG2 to inhibition by the cells was test ed using a function-blocking antibody. This allowed increased growth of dor sal root ganglion (DRG) axons over Neu7 cells and matrix and greatly increa sed the proportion of cortical axons able to cross from permissive A7 cells onto inhibitory Neu7 cells; CS-56 antibody had a similar effect. Inhibitor y fractions of conditioned medium contained NG2 coupled to CS glycosaminogl ycan chains, whereas noninhibitory fractions contained NG2 without CS chain s. Enzyme preparations that facilitated axon growth in Neu7 cultures were s hown to either degrade the NG2 core protein or remove CS chains. Versican i s present as patches on Neu7 monolayers, but DRG axons do not avoid these p atches. Therefore, NG2 appears to be the major axon-inhibitory factor made by Neu7 astrocytes. In the CNS, NG2 is expressed by O2A cells, which react rapidly after injury to produce a dense NG2-rich network, and by some react ive astrocytes. Our results suggest that NG2 may be a major obstacle to axo n regeneration.