Involvement of axonal phospholipase A2 activity in the outgrowth of adult mouse sensory axons in vitro

The effect on axonal outgrowth of inhibition of phospholipase A₂ activity was studied in a recently developed in vitro model, where dorsal root ganglia with attached spinal roots and nerve stumps from young adult mice were cultured in an extracellular matrix material (Matrigel). The phospholipase A₂ inhibitors 4-bromophenacyl bromide and oleyloxyethyl phosphorylcholine dose-dependently reduced axonal outgrowth from the sciatic nerve stump. A similar inhibitory effect was seen when only the cut nerve end was exposed to the inhibitors in a compartmental culture system. The local effect of phospholipase A₂ inhibition was further investigated on axons established in culture, using time-lapse recording. Exposure to phospholipase A₂ inhibitors caused the retraction of filopodia extensions and a reduction in growth cone motility within a few minutes. After removal of inhibition, normal growth cone motility and axonal growth were regained. Nerve cell bodies and axons, in contrast to Schwann cells, showed immunoreactivity after staining with an antiserum against secretory phospholipase A₂, and elevated levels of the enzyme could be detected after culture for 24 h. The immunoreactive protein was of approximately 170,000 molecular weight (phospholipase A₂-170) as determined by sodium dodecyl sulphate- polyacrylamide gel electrophoresis and immunoblotting. The localization of phospholipase A₂-170 in axons growing into the Matrigel was also demonstrated by use of a whole-mount technique. The results of this study show the importance of continuous phospholipase A₂ activity for growth cone motility and axonal outgrowth in the mammalian peripheral nerve, and suggest the involvement of an axonally localized enzyme.