AN INHIBITOR OF NEURITE OUTGROWTH PRODUCED BY ASTROCYTES

We have produced a number of astrocytic cell lines, some of which prom ote abundant neurite outgrowth, some of which are poor promoters of ne urite outgrowth. The critical difference between these lines lies in t he extracellular matrix, cell lines that are good promoters of axon gr owth producing a matrix that promotes axon growth, cell lines that are poor promoters of axon growth producing a non-permissive matrix. We w ere unable to find any consistent correlations between promotion of ax on growth and production of proteases, protease inhibitors, N-cadherin , growth cone collapsing activity, and several extracellular matrix mo lecules. In the present study we have compared the least permissive of our cell lines, Neu7, with the most permissive, A7. Medium conditione d by the cell lines has the same properties as the matrix, since dorsa l root ganglia (DRGs) grown in conditioned medium from the Neu7 line g row axons poorly, while DRGs grown in medium conditioned by A7 or prim ary astrocytes grow many long axons. Since matrix produced by all the cell lines contains large amounts of laminin, we looked to see whether the cells were producing laminin-blocking activity. Medium from the N eu7 line blocked laminin, while that from the A7 and primary astrocyte s did not. However, when the conditioned media were heat-treated to re move neurite-promoting activity, they all had laminin-blocking activit y: the blocking activity is heat stable. The neurite-promoting propert ies of the conditioned media therefore probably reflect a balance betw een promoting molecules and blockers. The laminin-blocking activity co uld be reduced by treatment of the heat-treated conditioned media with trypsin, keratanase, chondrointase ABC, but not chondroitinase AC or heparitinase. Fractionation of the conditioned medium on an ion-exchan ge column revealed that the laminin-blocking activity was found in the sulphate-labelled fractions, which are predominantly proteoglycan. Wh ole Neu7 extracellular matrix was treated with enzymes, and its neurit e-promoting activity could be increased by chondroitinase ABC and to a lesser extent by keratinase, but not by heparitinase. We conclude tha t the critical difference between matrix produced by astrocytic cell l ines that promote axon growth and those that do not lies in the level of production of a dermatan/keratan sulphate proteoglycan.