Molecular and Chemical Neuropathology

, Volume 14, Issue 3, pp 247–258 | Cite as

Serum from patients with amyotrophic lateral sclerosis induces the expression of B-50/GAP-43 and neurofilament in cultured rat fetal spinal neurons

  • R. Van Der Neut
  • W. H. Gispen
  • P. R. Bär


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons in the spinal cord, brain stem, and cortex. Cultures of fetal rat spinal cord cells were used to test sera from ALS patients (ALS sera) on their ability to influence the expression of the neuron-specific phosphoprotein B-50/GAP-43. Neurons were treated with ALS sera, sera of age-matched controls (CON sera), or sera of patients with autonomic neuropathy (AUTO sera) and fixed after 24 or 96 h. The levels of B-50 and neurofilament (NF) protein were assayed with an enzyme-linked immunoadsorbent assay (ELISA). No toxic effects of the ALS sera were observed. It appeared that after 24 h, both B-50 and NF levels were elevated in the ALS sera-treated cells by 12 and 11%, respectively. After 96 h, the B-50 level was 19% higher than in CON sera-treated neurons, and the NF level was 29% higher. AUTO sera did not differ from CON sera. The stimulating effect of ALS sera was absent if the sera were heated at 56°C for 30 min. We conclude that ALS serum induces the expression of B-50 and the subsequent axonal outgrowth and maturation in vitro. This induction might be a reflection in vitro of the processes underlying the collateral sprouting responses observed in ALS patients.

Index Entries

amyotrophic lateral sclerosis B-50/GAP-43 tissue culture fetal spinal neuron B-50 ELISA neurofilament ELISA 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andreasen T. J., Luetje C. W., Heideman W., and Storm D. R. (1983) Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes.Biochemistry 22, 4815–4818.Google Scholar
  2. Appel S. H. (1981) A unifying hypothesis for the cause of amyotrophic lateral sclerosis, parkinsonism, and Alzheimer disease.Ann. Neurol. 10, 499–505.PubMedCrossRefGoogle Scholar
  3. Appel S. H., McManaman J. L., Oppenheim R., Haverkamp L., and Vaca K. (1989) Muscle-derived trophic factors influencing cholinergic neurons in vitro and in vivo.Prog. Brain Res. 79, 251–256.PubMedCrossRefGoogle Scholar
  4. Beach R. L., Rao J. S., Festoff B. W., Reyes E. T., Yanagihara R., and Gajdusek D. C. (1986) Collagenase activity in skin fibroblasts of patients with amyotrophic lateral sclerosis.J. Neurol. Sci. 72, 49–60.PubMedCrossRefGoogle Scholar
  5. Benowitz L. I. and Routtenberg A. (1987) A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity.Trends Neurosci.10, 527–532.CrossRefGoogle Scholar
  6. Bjornskov E. K., Norris F. H., and Mower-Kuby J. (1984) Quantitative axon terminal and end-plate morphology in amyotrophic lateral sclerosis.Arch. Neurol. 41, 527–530.PubMedGoogle Scholar
  7. Bradley W. G., Robison S. H., and Tandan R. (1987) Deficient repair of alkylation damage of DNA in Alzheimer’s disease and amyotrophic lateral sclerosis cells.Adv. Exp. Med. Biol. 209, 2–6.Google Scholar
  8. Brown M. C. (1984) Sprouting of motor nerves in adult muscles: A recapitulation of ontogeny.Trends Neurosci. 7, 10–14.CrossRefGoogle Scholar
  9. Carleton S. A. and Brown W. F. (1979) Changes in motor unit populations in motor neurone disease.J. Neurol. Neurosurg. Psychiatry 42, 42–51.PubMedCrossRefGoogle Scholar
  10. Coërs C., Telerman-Toppet N., and Gérard J. M. (1973) Terminal innervation ratio in neuromuscular disease. II. Disorders of lower motor neuron, peripheral nerve, and muscle.Arch. Neurol. 29, 215–222.PubMedGoogle Scholar
  11. De Koning P., Verhaagen J., Sloot W., Jennekens F. G. I., and Gispen W. H. (1989) Org. 2766 stimulates collateral sprouting in the soleus muscle of the rat after partial denervation.Muscle Nerve 12, 353–359.PubMedCrossRefGoogle Scholar
  12. Dengler R., Konstanzer A., Küther G., Hesse S., Wolf W., and Struppler A. (1990) Amyotrophic lateral sclerosis: Macro-EMG and twitch forces of single motor units.Muscle Nerve 13, 545–550.PubMedCrossRefGoogle Scholar
  13. Digby J., Harrison R., Jehanli A., Lunt G. G., and Clifford-Rose F. (1985) Cultured rat spinal cord neurons: Interaction with motor neuron disease immunoglobulins.Muscle Nerve 8, 595–605.PubMedCrossRefGoogle Scholar
  14. Doherty P., Dickson J. G., Flanigan T. P., and Walsh F. S. (1984) Quantitative evaluation of neurite outgrowth in cultures of human fetal brain and dorsal root ganglion cells using an ELISA for human NF protein.J. Neurochem. 42, 1116–1122.PubMedCrossRefGoogle Scholar
  15. Doherty P., Dickson J. G., Flanigan T. P., Kennedy P. G. E., and Walsh F. S. (1986) Effects of amyotrophic lateral sclerosis serum on cultured chick spinal neurons.Neurology 36, 1330–1334.PubMedGoogle Scholar
  16. Drachman D. B. and Kuncl R. W. (1989) Amyotrophic lateral sclerosis: An unconventional autoimmune disease?.Ann. Neurol. 26, 269–274.PubMedCrossRefGoogle Scholar
  17. Ebendal T., Askmark H., and Aquilonius S. M. (1989) Screening for neurotrophic disturbances in amyotrophic lateral sclerosis.Acta Neurol. Scand. 79, 188–193.PubMedCrossRefGoogle Scholar
  18. Erkman L., Touzeau G., Bertrand D., Bader C. R., and Kato A. C. (1989) Characterization of dissociated monolayer cultures of human spinal cord.Brain Res. Bull. 22, 57–65.PubMedCrossRefGoogle Scholar
  19. Garruto R. M., Shankar S. K., Yanagihara R., Salazar A. M., Amyx H. L., and Gajdusek D. C. (1989) Low-calcium, high-aluminum diet-induced motor neuron pathology in cynomolgus monkeys.Acta Neuropathol.78, 210–219.PubMedCrossRefGoogle Scholar
  20. Gispen W. H., Boonstra J., De Graan P. N. E., Jennekens F. G. I., Oestreicher A. B., Schotman P., Schrama L. H., Verhaagen J., and Margolis F. L. (1990) B-50/GAP-43 in neuronal development and repair.Restor. Neurol. Neurosci.1, 237–244.Google Scholar
  21. Iwasaki Y., Kinoshita M., Ikeda K., Appel S. H., and Stewart S. S. (1989) Trophic effects of skeletal muscle extracts on spinal cord cultures.Int. J. Neurosci. 43, 203–208.CrossRefGoogle Scholar
  22. Liu Y. and Storm D. R. (1990) Regulation of free calmodulin levels by neuromodulin: Neuron growth and regeneration.Trends Pharmacol. Sci. 11, 107–111.PubMedCrossRefGoogle Scholar
  23. Maher I., Pouplard-Barthelaix A., and Emile J. (1987) Cytotoxicity of serum from amyotrophic lateral sclerosis patients on spinal cord cells in culture.Adv. Exp. Med. Biol. 209, 75–77.PubMedGoogle Scholar
  24. Mosmann T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.J. Immunol. Meth. 65, 55–63.CrossRefGoogle Scholar
  25. Nunn P. B., Seelig M., Zagoren J. C., and Spencer P. S. (1987) Stereospecific acute neuronotoxicity of ‘uncommon’ amino acids linked to human motorsystem diseases.Brain Res.410, 375–379.PubMedCrossRefGoogle Scholar
  26. Nurcombe V., Hill M. A., Eagleson K. L., and Bennett M. R. (1984) Motor neuron survival and neuritic extension from spinal cord explants induced by factors released from denervated muscle.Brain Res. 291, 19–28.PubMedCrossRefGoogle Scholar
  27. Oestreicher A. B. Van Dongen C. J., Zwiers H., and Gispen W. H. (1983) Affinity-purified anti-B-50 protein antibody: Interference with the function of the phosphoprotein B-50 in synaptic plasma membranes.J. Neurochem.41, 331–340.PubMedCrossRefGoogle Scholar
  28. Patten B. M. and Pages M (1984) Severe neurological disease associated with hyperparathyroidism.Ann. Neurol. 15, 453–456.PubMedCrossRefGoogle Scholar
  29. Politis M. J. and Spencer P. S. (1983) An in vivo assay of neurotrophic activity.Brain Res. 278, 229–231.PubMedCrossRefGoogle Scholar
  30. Provinciali L., Laurenzi M. A., Vesprini L., Giovagnoli A. R., Bartocci C., Montroni M., Bagnarelli P., Clementi M., and Varaldo P. E. (1988) Immunity assessment in the early stages of amyotrophic lateral sclerosis: A study of virus antibodies and lymphocyte subsets.Acta Neurol. Scand. 78, 449–554.PubMedCrossRefGoogle Scholar
  31. Roisen F. J., Bartfeld H., Donnenfeld H., and Baxter J. (1982) Neuron specific in vitro cytotoxicity of sera from patients with amyotrophic lateral sclerosis.Muscle Nerve 5, 48–53.PubMedCrossRefGoogle Scholar
  32. Skene J. H. P. (1989) Axonal growth-associated proteins.Ann. Rev. Neurosci. 12, 127–156.PubMedCrossRefGoogle Scholar
  33. Skene J. H. P. and Willard M. (1981) Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems.J. Cell Biol. 89, 96–103.PubMedCrossRefGoogle Scholar
  34. Slack J. R. and Pockett S. (1982) Motor neurotrophic factor in denervated adult skeletal muscle.Brain Res. 247, 138–140.PubMedCrossRefGoogle Scholar
  35. Stålberg E. (1982) Electrophysiological studies of reinnervation in ALS, inHuman Motor Neuron Diseases, Adv. Neurol. 36 (Rowland L. P., ed.) pp. 47–59, Raven Press, New York.Google Scholar
  36. Touzeau G. and Kato A. C. (1983) Effects of amyotrophic lateral sclerosis sera on cultured cholinergic neurons.Neurology 33, 317–322.PubMedGoogle Scholar
  37. Touzeau G. and Kato A. C. (1986) ALS serum has no effect on three enzymatic activities in cultured human spinal cord neurons.Neurology 36, 573–576.PubMedGoogle Scholar
  38. Väänänen H. K., Takala T. E., Tolonen U., Vuori J., and Myllyla V. V. (1988) Muscle-specific carbonic anhydrase III is a more sensitive marker of muscle damage than creatine kinase in neuromuscular disorders.Arch. Neurol. 45, 1254–1256.PubMedGoogle Scholar
  39. Van der Neut R., Bär P. R., Sodaar P., and Gispen W. H. (1988) Trophic effects of αMSH and ACTH4−10 on neuronal outgrowth in vitro.Peptides 9, 1015–1020.PubMedCrossRefGoogle Scholar
  40. Van der Neut R., Oestreicher A. B., Gispen W. H., and Bär P. R. (1990) The expression B-50/GAP-43 during development of rat spinal neurons in culture is regulated by interneuronal contact.Neurosci. Lett. 109, 36–41.PubMedCrossRefGoogle Scholar
  41. Van der Zee C. E. E. M., Brakkee J. H., and Gispen W. H. (1988) αMSH and Org. 2766 in peripheral nerve regeneration: Different routes of delivery.Eur. J. Pharmacol.147, 351–357.PubMedCrossRefGoogle Scholar
  42. Verhaagen J., Edwards P. M., Jennekens F. G. I., and Gispen W. H. (1987) Pharmacological aspects of the influence of melanocortins on the formation of regenerative peripheral nerve sprouts.Peptides 8, 581–584.PubMedCrossRefGoogle Scholar
  43. Wood J. N. and Anderton B. H. (1981) Monoclonal antibodies to mammalian neurofilaments.Biosci. Rep. 1, 263–268.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 1991

Authors and Affiliations

  • R. Van Der Neut
    • 1
  • W. H. Gispen
    • 2
  • P. R. Bär
    • 1
    • 2
  1. 1.Research Laboratory of NeurologyUniversity Hospital of UtrechtUtrechtThe Netherlands
  2. 2.Rudolf Magnus InstituteUniversity of UtrechtUtrechtThe Netherlands

Personalised recommendations