Comparative screening of glial cell types reveals extracellular matrix that inhibits retinal axon growth in a chondroitinase ABC-resistant fashion

Abstract

Glial cells provide an optimal surface for attachment, migration, and growth of CNS neurons. This study was designed to investigate the ability of four glia cell types, retinal Müller Glia (MG), cortical astrocytes (CA), A7, and Oli-neu to support the outgrowth of embryonic day 18 rat retinal explants. Extracellular matrix (ECM) prepared by water lysis of monolayers of A7, CA, and MG cells significantly increased, whereas Oli-neu-derived ECM inhibited fiber growth. Analogous results were obtained with explants on live monolayers. The inhibitory effect of Oli-neu matrix, but not of live cells, could be neutralized with the Rho-kinase inhibitor Y27632. Studies on the message and protein level revealed the expression of a range of ECM glycoproteins and the major chondroitin sulfate proteoglycans (CSPGs). Oli-neu produced large amounts of tenascin-C (TN-C), DSD-1-PG/phosphacan, and NG2, correlating with its inhibitory properties. Upon treatment with chondroitinase ABC (ChABC), retinal axon growth was increased on CA, MG, and A7, in accordance with the degradation of inhibitory CSPGs. In contrast, inhibition exerted by Oli-neu or postnatal oligodendrocytes was not abolished. When the neurite growth promoting properties of TN-C were neutralized by the monoclonal antibody J1/tn2, retinal axon growth was clearly diminished on MG, indicating a dominance of the FNIII domain D of TN-C in this cell type. The results suggest that glial cells construct complex ECM structures with distinct properties ranging from promotion to inhibition of retinal axons, depending on their composition. Furthermore, inhibitory ECM may resist ChABC treatment in some situations.