Enzymes of phospholipid synthesis: axonal versus Schwann cell distribution

Using quantitative EM autoradiography to localize sites of incorporation of tritiated inositol and choline into mouse sciatic nerve, we observed a substantial axon-based phosphatidylinositol synthesis, but no axonal phosphatidylcholine synthesis. In the present communication we provide biochemical evidence for the axonal transport of CDP-diglyceride:inositol transferase (EC 2.7.8.11), the terminal enzyme in de novo phosphatidylinositol biosynthesis. Axonal transport of 1,2-diacyl-glycerol:CDP-choline choline phosphotransferase (EC 2.7.8.2), required for de novo phosphatidylcholine synthesis, was not apparent in these studies. During subcellular fractionation activities for the synthesis of phosphatidylinositol by inositol transferase (IT) and phosphatidylcholine by choline phosphotransferase (CPT) were recovered in crude microsomal fraction of rat sciatic nerve. However, CPT was much more highly enriched in the microsome fraction than IT, which may be an indication of the different subcellular localizations of these enzymes. Following ligation, we detected localized increases in the activities of both enzymes in 5 (and 3) mm segments taken immediately proximal and distal to the ligature. Both activities increased in a linear fashion in the proximal segments over the ensuing 72 h period. It took about 40 h (IT) and 56 h (CPT) for the activities in the segments proximal to the ligature to double compared to unligated contralateral (control) nerves. The time-dependent accumulation of IT was primarily due to axonal transport, while that of CPT was largely a result of increased enzyme activity in local Schwann cells. Evidence came from double ligation studies, where a proximal ligature, acting to restrict orthograde axonal transport, reduced accumulation in a distal ligature by 80% for IT, but only 28% for CPT. Conversely, blockage of the Schwann cell response with actinomycin D, reduced accumulation of CPT by 83% and IT by only 36%. Finally, light microscopic autoradiography was used to show that in the segment proximal to the ligature, tritiated inositol incorporation into lipid was primarily axonal, whereas that of tritiated choline remained primarily associated with Schwann cells.