Abstract
Compartmented cultures of sympathetic neurons from newborn rats were employed to test the hypothesis that the lipids required for maintenance and growth of axonal membranes must be synthesized in the cell body and transported to the axons. In compartmented cultures the distal axons grow into a compartment separate from that containing the cell bodies and proximal axons, in an environment free from other contaminating cells such as glial cells and fibroblasts. There is virtually no bulk flow of culture medium or small molecules between the cell body and axonal compartments. When [methyl-3H]choline was added to the cell body-containing compartment the biosynthesis of [3H]-labeled phosphatidylcholine and sphingomyelin occurred in that compartment, with a gradual transfer of lipids (less than 5% after 16 h) into the axonal compartment. Surprisingly, addition of [methyl-3H]choline to the compartment containing only the distal axons resulted in the rapid incorporation of label into phosphatidylcholine and sphingomyelin in that compartment. Little retrograde transport of labeled phosphatidylcholine and sphingomyelin (less than 15%) into the cell body compartment occurred. Moreover, there was minimal transport of the aqueous precursors of these phospholipids (e.g., choline, phosphocholine and CDP-choline) between cell compartments. Similarly, when [3H]ethanolamine was used as a phospholipid precursor, the biosynthesis of phosphatidylethanolamine occurred in the pure axons, and approximately 10% of the phosphatidylethanolamine was converted into phosphatidylcholine. Experiments with [35S]methionine demonstrated that proteins were made in the cell bodies, but not in the axons. We conclude that axons of rat sympathetic neurons have the capacity to synthesize membrane phospholipids. Thus, a significant fraction of the phospholipids supplied to the membrane during axonal growth may be synthesized locally within the growing axon.
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- Campenot R. B. Development of sympathetic neurons in compartmentalized cultures. Il Local control of neurite growth by nerve growth factor. Dev Biol. 1982 Sep;93(1):1–12. doi: 10.1016/0012-1606(82)90232-9. [DOI] [PubMed] [Google Scholar]
- Campenot R. B., Draker D. D. Growth of sympathetic nerve fibers in culture does not require extracellular calcium. Neuron. 1989 Dec;3(6):733–743. doi: 10.1016/0896-6273(89)90242-0. [DOI] [PubMed] [Google Scholar]
- Campenot R. B. Independent control of the local environment of somas and neurites. Methods Enzymol. 1979;58:302–307. doi: 10.1016/s0076-6879(79)58146-4. [DOI] [PubMed] [Google Scholar]
- Campenot R. B., Walji A. H., Draker D. D. Effects of sphingosine, staurosporine, and phorbol ester on neurites of rat sympathetic neurons growing in compartmented cultures. J Neurosci. 1991 Apr;11(4):1126–1139. doi: 10.1523/JNEUROSCI.11-04-01126.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dennis E. A., Kennedy E. P. Intracellular sites of lipid synthesis and the biogenesis of mitochondria. J Lipid Res. 1972 Mar;13(2):263–267. [PubMed] [Google Scholar]
- FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
- Gould R. M., Pant H., Gainer H., Tytell M. Phospholipid synthesis in the squid giant axon: incorporation of lipid precursors. J Neurochem. 1983 May;40(5):1293–1299. doi: 10.1111/j.1471-4159.1983.tb13569.x. [DOI] [PubMed] [Google Scholar]
- Gould R. M., Spivack W. D., Robertson D., Poznansky M. J. Phospholipid synthesis in the squid giant axon: enzymes of phosphatidylinositol metabolism. J Neurochem. 1983 May;40(5):1300–1306. doi: 10.1111/j.1471-4159.1983.tb13570.x. [DOI] [PubMed] [Google Scholar]
- Hawrot E., Patterson P. H. Long-term culture of dissociated sympathetic neurons. Methods Enzymol. 1979;58:574–584. doi: 10.1016/s0076-6879(79)58174-9. [DOI] [PubMed] [Google Scholar]
- KENNEDY E. P., WEISS S. B. The function of cytidine coenzymes in the biosynthesis of phospholipides. J Biol Chem. 1956 Sep;222(1):193–214. [PubMed] [Google Scholar]
- Krystal G. A silver-binding assay for measuring nanogram amounts of protein in solution. Anal Biochem. 1987 Nov 15;167(1):86–96. doi: 10.1016/0003-2697(87)90136-9. [DOI] [PubMed] [Google Scholar]
- Pritchard P. H., Vance D. E. Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes. Biochem J. 1981 Apr 15;196(1):261–267. doi: 10.1042/bj1960261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tanaka T., Yamaguchi H., Kishimoto Y., Gould R. M. Lipid metabolism in various regions of squid giant nerve fiber. Biochim Biophys Acta. 1987 Oct 31;922(1):85–94. doi: 10.1016/0005-2760(87)90248-7. [DOI] [PubMed] [Google Scholar]
- Vance D. E. Boehringer Mannheim Award lecture. Phosphatidylcholine metabolism: masochistic enzymology, metabolic regulation, and lipoprotein assembly. Biochem Cell Biol. 1990 Oct;68(10):1151–1165. doi: 10.1139/o90-172. [DOI] [PubMed] [Google Scholar]
- Vance D. E., Ridgway N. D. The methylation of phosphatidylethanolamine. Prog Lipid Res. 1988;27(1):61–79. doi: 10.1016/0163-7827(88)90005-7. [DOI] [PubMed] [Google Scholar]
- Vance D. E., Weinstein D. B., Steinberg D. Isolation and analysis of lipoproteins secreted by rat liver hepatocytes. Biochim Biophys Acta. 1984 Jan 17;792(1):39–47. doi: 10.1016/0005-2760(84)90280-7. [DOI] [PubMed] [Google Scholar]