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Relative susceptibility of cytoskeleton-associated and soluble neurofilament subunits to aluminum exposure in intact cells

A possible mechanism for reduction of neurofilament axonal transport during aluminum neurotoxicity
  • Thomas B. Shea
  • Mary Lou Beermann
  • Feng-Song Wang
Original Articles

Abstract

Previous studies have demonstrated the appearance of phosphorylated neurofilament (NF) subunits within perikaryal cytoskeletons following aluminum exposure. In order to examine the mechanisms leading to this altered distribution of NF subunits, we carried out biochemical analyses of NF subunits in Triton-insoluble and-soluble fractions derived from aluminum-treated NB2a/d1 cells. In addition to increases in the Triton-insoluble cytoskeleton, increases in all three NF subunits were also detected within the Triton-soluble fraction of aluminum-treated cells. To address the nature of this increase in Triton-soluble subunits, aluminum-treated and untreated cultures were harvested in the absence of Triton and fractionated by established procedures to yield fractions greatly enriched for perikarya and neurites, respectively. Each of these subcellular fractions was then subjected to further homogenization in the presence of 1% Triton and centrifugation to yield Triton-insoluble cytoskeletons and Triton-soluble material derived from perikarya and axonal neurites, respectively. Resulting Triton-soluble fractions were “clarified” by high-speed centrifugation to eliminate oligomeric assemblies or soluble neurofilaments. Immunoblot analysis demonstrated quantitative recovery of the aluminum-induced increase in Triton-soluble NF subunits in the perikaryal fraction. Additional aluminum-treated and untreated cultures were pulse-chase radiolabeled with [35S]methionine and fractionated into Triton-insoluble and soluble fractions from isolated perikarya and axonal neurites. Autoradiographic analysis of immunoprecipitated NF subunits revealed that aluminum treatment delayed the translocation of newly synthesized subunits into neurites and resulted in the accumulation of radiolabeled subunits within the Triton-soluble fraction of perikarya. These findings suggest that aluminum may exert a relatively greater effect on NF subunits that have not yet undergone axonal transport and/or incorporation into Triton-insoluble structures vs those that have already deposited into axons. This possibility was supported by the observation that a higher concentration of aluminum was required to alter the electrophoretic migration of in vitro reassembled neurofilaments vs that required for unassembled NF subunits. These findings provide possible mechanisms for the accumulation of NF subunits in perikarya during aluminum intoxication.

Index Entries

Aluminum neurofilaments neurotoxicity axonal transport cytoskeleton phosphorylation 

References

  1. Anderson J. P., Carroll Z., Smulowitz M., and Lieberburg I. (1991) A possible mechanism of action of the neurotoxic agent iminodiproprionitrile (IDPN): a selective aggregation of the medium and heavy molecular weight neurofilament polypeptides (NF-M and NF-H).Brain Res. 547, 353–357.PubMedCrossRefGoogle Scholar
  2. Bizzi A. and Gambetti P. (1986) Phosphorylation of neurofilaments is altered in aluminum intoxication.Acta Neuropathol. 71, 154–158.PubMedCrossRefGoogle Scholar
  3. Bizzi A., Crane R. C., Autilio-Gambetti L., and Gambetti P. (1984) Aluminum effect on slow axonal transport.J. Neurosci. 4, 722–731.PubMedGoogle Scholar
  4. Clark A. W., Griffin J. W., and Price D. L. (1980) The axonal pathology in chronic IDPN intoxication.J. Neuropathol. Exp. Neurol. 39, 42–55.PubMedCrossRefGoogle Scholar
  5. Crapper-McLachlan D. R. (1986) Aluminum and Alzheimer’s disease.Neurobiol. Aging 7, 525–532.CrossRefGoogle Scholar
  6. Diaz-Nido J. and Avila J. (1990) Aluminum induces the in vitro aggregation of bovine brain cytoskeletal proteins.Neurosci. Lett. 110, 221–226.PubMedCrossRefGoogle Scholar
  7. Fry K. R., Edwards D. M., Shaw K. A., and Watt C. B. (1991) The rabbit retina: a long-term model system for aluminum-induced neurofibrillary degeneration.Neurosci. Lett. 124, 216–220.PubMedCrossRefGoogle Scholar
  8. Goldstein M. E., Sternberger N. H., and Sternberger L. A. (1987) Phosphorylation protects neurofilaments against proteolysis.J. Neuroimmunol. 14, 149–160.PubMedCrossRefGoogle Scholar
  9. Griffin J. W., Hoffman P. N., Clark A. W., Carrol P. T., and Price D. L. (1978) Slow axonal transport of neurofilament proteins: Impairment by β,β′-iminodiproprionitrile administration.Science 202, 633–635.PubMedCrossRefGoogle Scholar
  10. Hirano A., Donnenfield H., Sasaki S., and Nakano I. (1984) Fine structural observation of neurofilamentous changes in amyotrophich lateral sclerosis.J. Neuropathol. Exp. Neurol. 43, 461–470.PubMedCrossRefGoogle Scholar
  11. Hollosi M., Urge L., Perczel A., Kajtar J., Teplan I., Otvos L. J., and Fasman G. D. (1992) Metal ion-induced conformational changes of phosphorylated fragments of human neurofilament (NF-M) protein.J. Mol. Biol. 223, 673–682.PubMedCrossRefGoogle Scholar
  12. Johnson G. V. W. and Jope R. S. (1988) Phosphorylation of rat brain cytoskeletal proteins is increased after orally administered aluminum.Brain Res. 456, 95–103.PubMedCrossRefGoogle Scholar
  13. Kowall N. W., Pendlebury W. W., Kessler J. B., Perl D. P., and Beal M. F. (1989) Aluminum-induced neurofibrillary degeneration affects a subset of neurons in rabbit cerebral cortex, basal forebrain and upper brainstem.Neuroscience 29, 329–337.PubMedCrossRefGoogle Scholar
  14. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of bacteriophage T4.Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  15. Langui D., Anderton B. H., Brion J.-P., and Ulrich J. (1988) Effects of aluminum chloride on cultured cells from rat brain hemispheres.Dev. Brain Res. 438, 67–76.Google Scholar
  16. Leterrier J. F., Langui D., Probst A., and Ulrich J. (1992) A molecular mechanism for the induction of neurofilament bundling by aluminum ions.J. Neurochem. 58, 2060–2070.PubMedCrossRefGoogle Scholar
  17. Liem R. K. H. and Hutchinson S. B. (1982) Purification of individual components of the neurofilament triplet: filament assembly from the 70,000 dalton subunit.Biochemistry 21, 3221–3226.PubMedCrossRefGoogle Scholar
  18. Martyn C. N., Osmond C., Edwardson J. A., Barker D. J. P., Harris E. C., and Lacey R. F. (1989) Geographical relationship between Alzheimer’s disease and aluminum in drinking water.Lancet. 14, Jan 59.Google Scholar
  19. McDermott J. R., Smith A. I., Iqbal K., Wisniewski H. M. (1979) Brain aluminum in aging and Alzheimer’s disease.Neurology 29, 809.PubMedGoogle Scholar
  20. Miller C. A. and Levine E. M. (1974) Effects of aluminum salts on cultured neuroblastoma cells.J. Neurochem. 22, 751–758.PubMedCrossRefGoogle Scholar
  21. Muma N. A., Troncosco J. C., Hoffman P. N., Koo E. H., and Price D. L. (1988) Aluminum neurotoxicity: altered expression of cytoskeletal genes.Mol. Brain Res. 3, 115–122.CrossRefGoogle Scholar
  22. Nixon R. A., Clarke J. F., Logvinenko K. B., Tan M. K. H., Hoult M., and Grynspan F. (1990) Aluminum inhibits calpain-mediated proteolysis and induces human neurofilament proteins to form protease-resistant high molecular weight complexes.J. Neurochem. 55, 1950–1959.PubMedCrossRefGoogle Scholar
  23. Pant H. C. (1988) Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain.Biochem. J. 256, 665–668.PubMedGoogle Scholar
  24. Parhad I. M., Clark A. W., and Griffin J. W. (1987) Effect of changes in neurofilament content on caliber of small axons: the β,β′-iminodiproprionitrile model.J. Neurosci. 7, 2256–2263.PubMedGoogle Scholar
  25. Parhad I. M., Krekosi C. A., Mathew A., and Tran P. M. (1989) Neuronal gene expression in aluminum myelopathy.Cell Motil. Neurobiol. 9, 123–138.CrossRefGoogle Scholar
  26. Pyle S. J., Amarnath V., Graham D. G., and Anthony D. (1992) The role of pyrrole formation in the alteration of neurofilament transport induced during exposure to 2,5-hexanedione.J. Neuropathol. Exp. Neurol. 51, 451–458.PubMedCrossRefGoogle Scholar
  27. Selkoe D. J., Liem R. K. H., Yen S.-H., and Shelanski M. L. (1979) Biochemical and immunological characterization of neurofilaments in experimental neurofibrillary degeneration.Brain Res. 163, 235–252.PubMedCrossRefGoogle Scholar
  28. Shea T. B. (1994) Leupeptin enhances phosphorylated neurofilament immuno-reactivity in Triton-insoluble cytoskeletons of mouse NB2a/d1 neuroblastoma cells.Neuro. Res. Comm. 15, 143–148.Google Scholar
  29. Shea T. B., Sihag R., and Nixon R. A. (1988) Neurofilament triplet proteins of NB2a/d1 neuroblastoma: posttranslational modification and incorporation into the cytoskeleton during differentiation.Dev. Brain Res. 41, 97–109.CrossRefGoogle Scholar
  30. Shea T. B., Beermann M. L., and Nixon R. A. (1992a) Aluminum alters the electrophoretic properties of neurofilament proteins: Role of phosphorylation state.J. Neurochem. 58, 542–547.PubMedCrossRefGoogle Scholar
  31. Shea T. B., Beermann M. L., and Nixon R. A. (1992b) Aluminum inhibits neurofilament protein degradation by multiple cytoskeleton-associated proteases.FEBS Lett. 307, 195–198.PubMedCrossRefGoogle Scholar
  32. Shea T. B., Beermann M. L., and Wang F.-S. (1993a) Multiple interaction of aluminum ions with neurofilament subunits: Regulation by phosphate-dependent interactions between C-terminal extensions of the high and middle molecular weight subunits.J. Neurosci. Res. 38, 160–166.CrossRefGoogle Scholar
  33. Shea T. B., Paskevich P. A., and Beermann M. L. (1993b) The protein phosphatase inhibitor okadaic acid increases axonal neurofilaments and neurite caliber, and decreases axonal microtubules in NB2a/d1 cells.J. Neurosci. Res. 35, 507–521.PubMedCrossRefGoogle Scholar
  34. Shea T. B., Beermann M. L., Nixon R. A. (1994) Aluminum treatment of NB2a/d1 cells alters neurofilament phosphorylation and solubility.Trans. Am. Soc. Neurochem. 25, 117.Google Scholar
  35. Shea T. B. and Beermann M. L. (1994) Multiple interactions of aluminum with neurofilament subunits: Regulation by phosphate-dependent interactions between C-terminal extensions of the high and middle molecular weight subunits.J. Neurosci. Res. 38, 160–166.PubMedCrossRefGoogle Scholar
  36. Shea T. B., Clarke J. F., Wheelock T. R., Paskevich P., and Nixon R. A. (1989) Aluminum salts induce the accumulation of neurofilaments in perikarya of NB2a/d1 neuroblastoma.Brain Res. 492, 53–64.PubMedCrossRefGoogle Scholar
  37. Terry R. D. and Pena C. (1965) Experimental production of neurofibrillary degeneration. 2. Electron microscopy, phosphate histochemistry and electron probe analysis.J. Neuropathol. Exp. Neurol. 24, 200–210.PubMedGoogle Scholar
  38. Tokutake S., Hutchinson S. B., Patcher J. S., Liem R. K. H. (1983) A batchwise filtration procedure of neurofilament proteins.Anal. Biochem. 135, 102–105.PubMedCrossRefGoogle Scholar
  39. Towbin H., Staehelin T., and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Nat. Acad. Sci. USA 76, 4350–4354.PubMedCrossRefGoogle Scholar
  40. Troncosco J. C., March J. L., Haner M., and Aebi U. (1990) Effect of aluminum and other multivalent cations on neurofilaments in vitro: an electromicroscopic study.J. Struct. Biol. 103, 2–12.CrossRefGoogle Scholar
  41. Troncosco J. C., Hoffman P. N., Griffin J. W., Hess-Koslow K. M., and Price D. L. (1985) Aluminum neurotoxicity: a disorder of neurofilament transport in motor neurons.Brain Res. 342, 172–175.CrossRefGoogle Scholar
  42. Troncosco J. C., Sternberger N. H., Sternberger L. A., Hoffman P. N., and Price D. L. (1986) Immunocytochemical studies of neurofilament antigens in the neurofibrillary pathology produced by aluminum.Brain Res. 364, 295–300.CrossRefGoogle Scholar
  43. Wisniewski H. M., Moretz R. C., and Iqbal K. (1986) No evidence for aluminum in etiology and pathogenesis of Alzheimer’s disease.Neurobiol. Aging 7, 532–535.CrossRefGoogle Scholar
  44. Yano I., Yoshida S., Uebayashi Y., Yoshimasu F., and Yase Y. (1989) Degenerative changes in the central nervous system of Japanese monkeys induced by oral administration of aluminum salt.Biomed. Res. 10, 33–41.Google Scholar

Copyright information

© Humana Press Inc. 1995

Authors and Affiliations

  • Thomas B. Shea
    • 1
    • 2
    • 3
  • Mary Lou Beermann
    • 1
    • 2
  • Feng-Song Wang
    • 1
    • 2
  1. 1.Laboratories for Molecular Neuroscience, Mailman Research CenterMcLean HospitalBelmont
  2. 2.Department of PsychiatryHarvard Medical SchoolBoston
  3. 3.Center for Cellular Neurobiology and Neurodegeneration Research, Department of Biological SciencesUniversity of Massachusetts at LowellLowell

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