1,2-Dioctanoyl-s,n-glycerol-induced activation of protein kinase C resultsin striking, but reversible growth cone shape changes and an accumulationof f-actin and serine 41-phosphorylated GAP-43 in the axonal process

摘要

In spinal cord explant cultures from embryonic chicken (E7) we found that both a long-time downregulation of PKC by phorbol-12,13-dibutyrate (PDBu) and an inhibition of PKC by RO-31-8220 strongly reduce neurite outgrowth. Unlike this, in the presence of a high dose of 1,2-dioctanoyl-s,n-glycerol (diC8, 60 mu M), PKC alpha,beta isoforms are not downregulated, but neurite outgrowth appeared reduced up to 37%. A low dose of diC8 (5 mu M), however, was found to stimulate neurite outgrowth up to 25 %. Using this tissue culture system as well as neuronal cell culture we then studied the effects of diC8 on the shapes and actin-based motility of distal axonal processes and growth cones as well as on the spatial distribution of f-actin and serine 41-phosphorylated GAP-43 (neuromodulin, B50). High-resolution microscopy showed that addition of 30-60 mu M diC8 leads within a few minutes to a retraction of filopodia and to an increased protrusion of lamellipodia followed by the formation of club-shaped dense growing tips, axonal varicosities, and a cessation of any actin dynamics. These striking shape changes are completely reversed after replacement of the medium by drug-free medium. Presence of cytochalasins and a panel of different PKC inhibitors prevent or respectively attenuate the diC8 effects. Immuno- and phalloidin-staining confirmed that in control neurons f-actin and serine 41-phosphorylated GAP-43 are confined to and enriched in the growth cones. In parallel with diC8-induced shape changes there is an accretion of f-actin and serine 41-phosphorylated GAP-43 in the entire axonal processes and the rounded growing tips. With respect to the fundamental role of the actin dynamics in growth cone steering and neuronal pathfinding, the data supports the view that in neurons local PKC-regulated phosphorylation of GAP-43 may represent an important mechanism to transduce guiding signals into actin-cytoskeletal responses mediating directed axonal growth.