SEQUENTIAL EFFECTS OF ASTROGLIAL-DERIVED FACTORS ON NEURITE OUTGROWTH - INITIATION BY PROTEASE INHIBITORS AND POTENTIATION BY EXTRACELLULAR-MATRIX COMPONENTS

Astroglial-conditioned medium (GCM) induced two distinct, but intimately related, phases of neuritogenesis in NB2a/d1 neuroblastoma cells-a "rapid-out-growth," unstable phase, and a delayed, relatively stable phase, which are apparently regulated by glial-derived protease inhibitors and laminin, respectively. The initial rapid outgrowth (< 4 hr) may be mediated by inhibition of a thrombin-like protease present as a serum component and/or adsorbed to the outer neuronal surface, since (1) a similar effect was obtained by serum removal or by adding the specific thrombin inhibitor, hirudin; (2) exogenous thrombin inhibited the rapid outgrowth of neurites by GCM; and (3) cell-free enzyme assays confirmed the presence of thrombin-inhibitory activity in GCM. Although neurites induced by removal of serum removal or hirudin addition are rapidly resorbed following serum replenishment or hirudin depletion, GCM-induced neurites continued to elongate after GCM removal, indicating that GCM contained additional neurite-promoting factors. Anti-laminin antiserum did not inhibit the initial elaboration of neurites by GCM but prevented their continued elongation. Anti-laminin antiserum had no affect on neurite outgrowth induced by serum deprivation. The more protracted, second phase of neurite outgrowth could also be achieved by the addition of soluble purified laminin to undifferentiated cells. Unlike neurites at 4 hr, neurites at 24 hr were no longer dependent on the protease inhibitors in GCM, since exogenous thrombin no longer caused them to retract. Simultaneous addition of thrombin and anti-laminin antiserum with GCM had identical inhibitory effects on continued neurite elaboration at 24 hr as did anti-laminin antiserum without thrombin. We conclude that protease inhibitors present in GCM initiate the rapid elaboration of unstable neurites and that glial-derived extracellular matrix factors support the continued elaboration of these neurites.