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Intracellular transport and neuronal activation of phospholipid and glycoprotein synthesis during axonal regeneration of cranio-spinal nerves

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Molecular and Chemical Neuropathology

Abstract

In the present work, the hypothesis that the increased rapid intracellular transport of newly-synthesized material along the axons of a regenerating system is sustained by an alteration of the transport of proteolipid complexes through subcellular compartments of a neuronal cell body was tested by a biochemical methodology. The motoneurons of spinal cord ventral horn, 4 wk after unilateral lesion (crush) of cervico-thoracic nerves of the rabbit at the level of brachial plexus, were chosen as the model system of regeneration. A time-staggered procedure of in vivo and in vitro double labeling with metabolic precursors, such as [3H]-choline, [14C]-choline, [3H]-fucose, and [14C]-fucose, was used. Subcellular fractions (RER, SER, Golgi apparatus, and plasma membranes) of ventral horn tissue, taken from spinal cord hemisections (regenerating and contralateral side), were further isolated. Twenty-eight days after axotomy, we did not observe any change of intracellular transport kinetics (14C/3H ratio) of newly-synthesized choline-phospholipids and glycoproteins in regenerating motoneurons compared to controls. However, associated with regenerating phenomenon in Golgi apparatus, we observed an increase of labeled choline-phospholipid and glycoprotein material that could contribute to the increased fast axonal transport and delivery of membrane proteolipid complexes to plasma membrane and axonal compartments. The increase of glycoprotein labeling was more pronounced in the SER portion (vesicles and elements of smooth membranes). This result is in favor of the hypothesis that membrane-bound proteins are transported from the Golgi to the axon through the perikaryal SER.

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Abbreviations

RER:

rough endoplasmic reticulum

SER:

smooth endoplasmic reticulum

PM:

plasma membrane

PtdCho:

phosphatidylcholine

SM:

sphingomyelin

ACF:

artificial cerebrospinal fluid

References

  • Alberghina M., Viola M., and Giuffrida A. M. (1983) Rapid axonal transport of glycerophospholipids in regenerating hypoglossal nerve of the rabbit.J. Neurochem. 40, 25–31.

    Article  PubMed  CAS  Google Scholar 

  • Alberghina M. (1986) Axonally transported phospholipids and neurite regrowth,Phospholipid Research and The Nervous, System: biochemical and molecular pharmacology (Horrocks L. A., Freysz L., and Taffano G., eds.), Liviana Press, Padua Italy, pp. 251–264.

    Google Scholar 

  • Alberghina M., and Giuffrida Stella A. M. (1988) Changes of phospholipd-metabolizing and lysosomal enzymes in hypoglossal nucleus and ventral horn motoneurons during regeneration of cranio-spinal nerves.J. Neurochem. 51, 15–20.

    Article  PubMed  CAS  Google Scholar 

  • Arienti G., Goracci G., and Porcellati G. (1981) Glycerophospholipid metabolism in neuronal and glial-enriched fractions.Neurochem. Res. 6, 729–742.

    Article  PubMed  CAS  Google Scholar 

  • Barron K. D., Cova J. Scheibly M. E., and Kohberger R. (1982) Morphometric measurements and RNA content of axotomized feline cervical motoneurons.J. Neurocytol 11, 707–720.

    Article  PubMed  CAS  Google Scholar 

  • Benjamins J. A., Miller S. L., and Morell P. (1976) Metabolic relationships between myelin subfractions: entry of galactolipids and phospholipids.J. Neurochem. 27, 565–570.

    Article  PubMed  CAS  Google Scholar 

  • Bennett G., Leblond C. P., and Haddad A. (1974) Migration of glycoprotein from the Golgi apparatus to the surface of various cell types as shown by radio-autography after labeled fucose injection into rats.J. Cell Biol. 60 258–284.

    Article  PubMed  CAS  Google Scholar 

  • Borke R. C. (1982) Perisomatic changes in the maturing hypoglossal nucleus after axon injury.J. Neurocytol. 11, 463–485.

    Article  PubMed  CAS  Google Scholar 

  • Bowe C. M., Yu C. H., and Waxman S. G. (1988) Morphological changes in spinal motor neurons giving rise to long-term regenerated sciatic nerve axons.Brain Res. 463, 69–77.

    Article  PubMed  CAS  Google Scholar 

  • Deshmukh D. S., Vorbrodt A. W., Lee P. H., Bear W. D., and Kuizon S. (1988) Studies on the submicrosomal fractions of bovine oligodendroglia: lipid composition and glycolipid biosynthesis.Neurochem. Res. 13, 571–582.

    Article  PubMed  CAS  Google Scholar 

  • Dunlop D. S., van Elden W., and Lajtha A. (1974) Measurements of rates of protein synthesis in rat brain slices.J. Neurochem. 22, 821–830.

    Article  PubMed  CAS  Google Scholar 

  • Ellisman M. H. and Lindsey J. D. (1982) Organization of axoplasm-membranous and fibrillar components possibly involved in fast axonal transport,Axoplasmic transport (Weiss D. G. ed.), Raven, New York, pp 55–63.

    Google Scholar 

  • Engh C. A., Schofield B. H., Doty S. B. M., and Robinson R. A. (1971) Perikaryal synthetic function following reversible and irreversible peripheral axon injuries as shown by radioautography.J. Comp. Neurol. 142, 465–480.

    Article  PubMed  CAS  Google Scholar 

  • Fleischer B., Fleischer S., and Ozawa H. (1969) Isolation and characterization of Golgi membranes from bovine liver.J. Cell Biol. 43, 59–79.

    Article  PubMed  CAS  Google Scholar 

  • Frizell M. and Sjöstrand J. (1974) The axonal transport of [3H]fucose labelled glycoproteins in normal and regenerating peripheral nerves.Brain Res. 78, 109–123.

    Article  PubMed  CAS  Google Scholar 

  • Gilmore S. A. and Walls R. C. (1981) Patterns of labeling of intraspinal reactive cells in rats injected with [3H]thymidine prior to or following sciatic axotomy.Brain Res.,218, 1–13.

    Article  PubMed  CAS  Google Scholar 

  • Giulian D., Les Ruisseaux H., and Cowburn D. (1980) Biosynthesis and introaxonal transport of proteins during neuronal regeneration.J. Biol. Chem. 255, 6494–6501.

    PubMed  CAS  Google Scholar 

  • Gonatas J. O., Gonatas N. K., Stieber A., and Fleischer B. (1985) Isolation and characterization of an enriched Golgi fraction from neurons of developing rat brains.J. Neurochem. 45, 497–507.

    Article  PubMed  CAS  Google Scholar 

  • Goodrum J. F. and Morell P. (1982) Axonal transport, deposition, and metabolic turnover of glycoproteins in the rat optic pathway.J. Neurochem. 38, 696–704.

    Article  PubMed  CAS  Google Scholar 

  • Gould R. M., Spivack W. D., Sinatra R. S., Lindquist T. D., and Ingoglia N. A. (1982) Axonal transport of choline lipids in normal and regenerating rat sciatic nerve.J. Neurochem. 39, 1569–1578.

    Article  PubMed  CAS  Google Scholar 

  • Graeber M. B., Tetzlaff W., Streit W. J., and Kreutzberg G. W. (1988) Microglial cells but not astrocytes undergo mitosis following rat facial nerve axotomy.Neurosci. Lett. 85, 317–321.

    Article  PubMed  CAS  Google Scholar 

  • Griffin J. W., Price D. L., Drachman D. B., and Morris J. (1981) Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration.J. Cell Biol. 88, 205–214.

    Article  PubMed  CAS  Google Scholar 

  • Hammerschlag R., Stone G. C., Bolen F. A., Lindsey J. D., and Ellisman M. H. (1982) Evidence that all newly-synthesized proteins destined for fast axonal transport pass through the Golgi apparatus.J. Cell Biol. 93, 568–575.

    Article  PubMed  CAS  Google Scholar 

  • Havton L. and Kellerth J. O. (1987) Regeneration by supernumerary axons with synaptic terminals in spinal motoneurons of cats.Nature 325, 711–714.

    Article  PubMed  CAS  Google Scholar 

  • Hirschberg C. B. and Snider M. D. (1987) Topography of glycosylation in the endoplasmic reticulum and Golgi apparatus.Ann. Rev. Biochem. 56, 63–87.

    Article  PubMed  CAS  Google Scholar 

  • Hymes A. J. and Mullinax F. (1984) Assay of galactosyltransferase by high-performance liquid chromatography.Anal. Biochem. 139, 68–72.

    Article  PubMed  CAS  Google Scholar 

  • Ipata P. L. (1967): A coupled optical enzyme assay for 5′-nucleotidase.Anal. Biochem. 20, 30–36.

    Article  PubMed  CAS  Google Scholar 

  • Kerns J. M. and Hinsman E. I. (1973) Neurological response to sciatic neurectomy. I. Light microscopy and autoradiography.J. Comp. Neurol. 151, 237–253.

    Article  PubMed  CAS  Google Scholar 

  • Kurihara T. and Tsukada Y. (1967) The regional and subcellular distribution of 2′, 3′-cyclic nucleotide 3′-phosphohydrolase in the central nervous system.J. Neurochem. 14 1167–1174.

    Article  PubMed  CAS  Google Scholar 

  • Lieberman A. R. (1971) The axon reaction: a review of the principal features of perikaryal responses to axon injury.Int. Rev. Neurobiol. 14, 49–124.

    Article  PubMed  CAS  Google Scholar 

  • Lipsky N. G. and Pagano R. E. (1985) Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane.J. Cell Biol. 100, 27–34.

    Article  PubMed  CAS  Google Scholar 

  • Longo F. and Hammerschlag R. (1980) Relation of somal lipid synthesis to the fast-axonal transport of protein and lipid.Brain Res. 193, 471–485.

    Article  PubMed  CAS  Google Scholar 

  • Lowry O. H., Rosebrough N. J., Farr A. L., and Randall R. J. (1951) Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193, 265–275.

    PubMed  CAS  Google Scholar 

  • Mercurio A. M. and Holtzman E. (1982) Ultrastructural localization of glycerolipid synthesis in rod cells of the isolated frog retina.J. Neurocytol. 11, 259–322.

    Google Scholar 

  • Miani N., Rizzoli A., and Bucciante G. (1961) Metabolic and chemical changes in regenerating neurons. II.In vitro rate of incorporation of amino acids into proteins of the nerve cell perikaryon of the C.8 spinal ganglion of rabbit.J. Neurochem. 7, 161–173.

    Article  CAS  Google Scholar 

  • Pfeffer S. R. and Rothman J. E. (1987) Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi.Ann. Rev. Biochem. 56, 829–852.

    Article  PubMed  CAS  Google Scholar 

  • Price D. L. and Porter K. R. (1972) The response of ventral horn neurons to axonal transection.J. Cell. Biol. 53, 24–37.

    Article  PubMed  CAS  Google Scholar 

  • Reisert I., Wildemann G., Grab D., and Pilgrim C. H. (1984) The glial reaction in the course of axon regeneration: a stereological study of the rat hypoglossal nucleus.J. Comp. Neurol. 229, 121–128.

    Article  PubMed  CAS  Google Scholar 

  • Schwartz A., Bachelard H. S., and Mc Ilwain H. (1962) The sodium-stimulated adenosine-triphosphatase activity and other properties of cerebral microsomal fractions and subfractions.Biochem. J. 84, 626–637.

    PubMed  CAS  Google Scholar 

  • Sottocasa G. L., Kuylenstierna B., Ernster L., and Bergstrand A. (1967) An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study.J. Cell. Biol. 32, 415–438.

    Article  PubMed  CAS  Google Scholar 

  • Tessler A., Autilio-Gambetti L., and Gambetti P. (1980) Axonal growth during regeneration: A quantitative autoradiographic study.J. Cell Biol. 87, 197–203.

    Article  PubMed  CAS  Google Scholar 

  • Toews A. D., Horrocks L. A., and King J. S. (1976) Simultaneous isolation of purified microsomal and myelin fraction from rat spinal cord.J. Neurochem. 27, 25–31.

    Article  PubMed  CAS  Google Scholar 

  • Tsay G. Chen and Dawson G. (1977) A sensitive spectrophotometric method for detection of L-fucose.Anal. Biochem. 78, 423–427.

    Article  PubMed  CAS  Google Scholar 

  • Tsukada Y., Nagai K., and Suda H. (1980) A rapid micromethod for 2′, 3′-cyclic nucleotide 3′-phosphohydrolase assay using micro high performance liquid chromatography.J. Neurochem. 34, 1019–1022.

    Article  PubMed  CAS  Google Scholar 

  • Veraa R. P. and Grafstein B. (1981) Cellular mechanisms for recovery from nervous system injury: A conference report.Exp. Neurol. 71, 6–75.

    Article  PubMed  CAS  Google Scholar 

  • Voelker D. R. and Kennedy E. P. (1982) Cellular enzymatic synthesis of sphingomyelin.Biochemistry 21, 2753–2759.

    Article  PubMed  CAS  Google Scholar 

  • Wallick E. T., Dowd F., Allen J. C., and Schwartz A. (1974) The nature of the transport adenosine triphosphate-digitalis complex. VII. Characteristics of ouabagenin-Na+, K+-adenosine triphosphatase interaction.J. Pharmacol. Exp. Ther. 189, 434–444.

    PubMed  CAS  Google Scholar 

  • Watson W. E. (1972) Some quantitative observations upon the responses of neuroglial cells which follow axotomy of adjacent neurons.J. Physiol. 225, 415–435.

    PubMed  CAS  Google Scholar 

  • Whitnall M. H. and Grafstein B. (1982) Perikaryal routing of newly-synthesized proteins in regenerating neurons: quantitative electron microscopic autoradiography.Brain Res. 239, 41–56.

    Article  PubMed  CAS  Google Scholar 

  • Zatz M. and Barondes S. H. (1970) Fucose incorporation into glycoproteins of mouse brain.J. Neurochem. 70, 157–163.

    Article  Google Scholar 

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A preliminary report of these results was presented previously at the Satellite Symposium of European Neurological Society (ENS), held in Sanremo, Italy, June 24–25, 1988.

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Alberghina, M., Viola, M. Intracellular transport and neuronal activation of phospholipid and glycoprotein synthesis during axonal regeneration of cranio-spinal nerves. Molecular and Chemical Neuropathology 10, 137–155 (1989). https://doi.org/10.1007/BF03159725

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  • DOI: https://doi.org/10.1007/BF03159725

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