Phospholipase A2 in nerve injury and axonal outgrowth
Abstract: This thesis deals with processes coupled to injury in the peripheral nervous system (PNS), with a general aim to investigate the role of phospholipase A2 (PLA2) enzymes in axonal outgrowth. The axonal outgrowth of dorsal root ganglia (DRG) neurons in vitro was reduced by several different inhibitors of PLA2 activity and enhanced by an activator of this enzyme. The PLA2 inhibitors acted locally in the outgrowth region and the effect only comprised the axonal elongation stage. Time-lapse recording of growing axons showed a rapid retraction of filopodia and a reduction in growth cone motility at exposure to the drugs. The PLA2 activity was upregulated in the DRG and nerve after a sciatic nerve injury in vivo, most profoundly in the crush region of the nerve. The upregulated activity was strongly Ca2+-dependent, acid sensitive and reduced by an inhibitor of type IV cytosolic (c) PLA2 (methyl arachidonyl fluorophosphonate, MAFP) and the role of this PLA2 form in axonal outgrowth was further investigated in vitro. MAFP reduced the axonal outgrowth length with ~50%, and caused rapid collapse of the growth cone, an effect which was counteracted by addition of arachidonic acid (AA). Control experiments ruled out a major role of the Ca2+-independent type VI iPLA2 as well as diacylglycerol-lipase (DAG-lipase). An inhibitor of 5-lipoxygenase (LOX) (AA861) reduced axonal outgrowth and inhibited growth cone motility. By immunocytochemistry cPLA2 was visualised in neurons, Schwann cells and macrophages and found to be upregulated in the crush region after injury in vivo. cPLA2 also accumulated in the cut axonal endings. Whole-mount immunostaining demonstrated cPLA2 and 5-LOX in the growing axons and their growth cones. Interestingly, elevated levels of cPLA2 was also found in the cell bodies of degenerating neurons, as detected by TUNEL- and Fluoro-Jade staining. The results of this thesis show the importance of continuous PLA2 activity for growth cone motility and axonal outgrowth in the adult peripheral nerve. The data strongly link cPLA2 activity to axonal outgrowth and growth cone function via mechanisms involving AA and leukotrienes. However, the role of cPLA2 activity after injury might be more complex, involving perhaps also harmful effects of this enzyme form.
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