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. 1988 Dec 1;107(6):2703–2716. doi: 10.1083/jcb.107.6.2703

Isolation and chemical characterization of Alzheimer's disease paired helical filament cytoskeletons: differentiation from amyloid plaque core protein

PMCID: PMC2115630  PMID: 3060472

Abstract

The paired helical filaments (PHFs) of Alzheimer's disease were purified by a strategy in which the neurons and amyloid plaque cores of protein (APCP) were initially isolated. This was achieved by several steps of isocratic sucrose centrifugations of increasing molarity and a discontinuous isotonic Percoll density gradient. After collagenase elimination of contaminating blood vessels, lysis of neurons was produced by SDS treatment. The released PHF cytoskeletons were separated from contaminating APCP and lipofuscin by sucrose density gradient. A final step consisted in the chemical purification of highly enriched PHFs and APCP components via a formic acid to guanidine hydrochloride transition. PHFs and APCPs were fractionated by size exclusion HPLC and further characterized and quantitated by automatic amino acid analysis. We also present some of the morphological and immunochemical characteristics of PHF polypeptides and APCP. Our studies indicate that apart from differences in localization and morphology, PHF and APCP significantly differ in (a) chemical structure (peptide and amino acid composition); (b) epitope specificity (antiubiquitin, antitau, antineurofilament); (c) physicochemical properties (structural conformation in guanidine hydrochloride); and (d) thioflavine T fluorescence emission. These parameters strongly suggest important differences in the composition and, probably, in the etiopathology of PHF and APCP of Alzheimer's disease.

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Selected References

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  1. Anderton B. H., Breinburg D., Downes M. J., Green P. J., Tomlinson B. E., Ulrich J., Wood J. N., Kahn J. Monoclonal antibodies show that neurofibrillary tangles and neurofilaments share antigenic determinants. Nature. 1982 Jul 1;298(5869):84–86. doi: 10.1038/298084a0. [DOI] [PubMed] [Google Scholar]
  2. Bahmanyar S., Higgins G. A., Goldgaber D., Lewis D. A., Morrison J. H., Wilson M. C., Shankar S. K., Gajdusek D. C. Localization of amyloid beta protein messenger RNA in brains from patients with Alzheimer's disease. Science. 1987 Jul 3;237(4810):77–80. doi: 10.1126/science.3299701. [DOI] [PubMed] [Google Scholar]
  3. Ball M. J., Fisman M., Hachinski V., Blume W., Fox A., Kral V. A., Kirshen A. J., Fox H., Merskey H. A new definition of Alzheimer's disease: a hippocampal dementia. Lancet. 1985 Jan 5;1(8419):14–16. doi: 10.1016/s0140-6736(85)90965-1. [DOI] [PubMed] [Google Scholar]
  4. Ball M. J., Griffin-Brooks S., MacGregor J. A., Nagy B., Ojalvo-Rose E., Fewster P. H. Neuropathological definition of Alzheimer disease: multivariate analyses in the morphometric distinction between Alzheimer dementia and normal aging. Alzheimer Dis Assoc Disord. 1988;2(1):29–37. doi: 10.1097/00002093-198802010-00004. [DOI] [PubMed] [Google Scholar]
  5. Bjarnason J., Carpenter K. J. Mechanisms of heat damage in proteins. 2. Chemical changes in pure proteins. Br J Nutr. 1970 Mar;24(1):313–329. doi: 10.1079/bjn19700031. [DOI] [PubMed] [Google Scholar]
  6. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  7. Chothia C. Principles that determine the structure of proteins. Annu Rev Biochem. 1984;53:537–572. doi: 10.1146/annurev.bi.53.070184.002541. [DOI] [PubMed] [Google Scholar]
  8. Cohen M. L., Golde T. E., Usiak M. F., Younkin L. H., Younkin S. G. In situ hybridization of nucleus basalis neurons shows increased beta-amyloid mRNA in Alzheimer disease. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1227–1231. doi: 10.1073/pnas.85.4.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cork L. C., Sternberger N. H., Sternberger L. A., Casanova M. F., Struble R. G., Price D. L. Phosphorylated neurofilament antigens in neurofibrillary tangles in Alzheimer's disease. J Neuropathol Exp Neurol. 1986 Jan;45(1):56–64. doi: 10.1097/00005072-198601000-00005. [DOI] [PubMed] [Google Scholar]
  10. Corsellis J. A., Bruton C. J., Freeman-Browne D. The aftermath of boxing. Psychol Med. 1973 Aug;3(3):270–303. doi: 10.1017/s0033291700049588. [DOI] [PubMed] [Google Scholar]
  11. Glenner G. G. Alzheimer's disease: its proteins and genes. Cell. 1988 Feb 12;52(3):307–308. doi: 10.1016/s0092-8674(88)80021-7. [DOI] [PubMed] [Google Scholar]
  12. Glenner G. G., Wong C. W. Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun. 1984 May 16;120(3):885–890. doi: 10.1016/s0006-291x(84)80190-4. [DOI] [PubMed] [Google Scholar]
  13. Goedert M. Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer's disease. EMBO J. 1987 Dec 1;6(12):3627–3632. doi: 10.1002/j.1460-2075.1987.tb02694.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldgaber D., Lerman M. I., McBride O. W., Saffiotti U., Gajdusek D. C. Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease. Science. 1987 Feb 20;235(4791):877–880. doi: 10.1126/science.3810169. [DOI] [PubMed] [Google Scholar]
  15. Gray E. G., Paula-Barbosa M., Roher A. Alzheimer's disease: paired helical filaments and cytomembranes. Neuropathol Appl Neurobiol. 1987 Mar-Apr;13(2):91–110. doi: 10.1111/j.1365-2990.1987.tb00174.x. [DOI] [PubMed] [Google Scholar]
  16. Grundke-Iqbal I., Iqbal K., Quinlan M., Tung Y. C., Zaidi M. S., Wisniewski H. M. Microtubule-associated protein tau. A component of Alzheimer paired helical filaments. J Biol Chem. 1986 May 5;261(13):6084–6089. [PubMed] [Google Scholar]
  17. Grundke-Iqbal I., Iqbal K., Tung Y. C., Wang G. P., Wisniewski H. M. Alzheimer paired helical filaments: cross-reacting polypeptide/s normally present in brain. Acta Neuropathol. 1985;66(1):52–61. doi: 10.1007/BF00698295. [DOI] [PubMed] [Google Scholar]
  18. Guiroy D. C., Miyazaki M., Multhaup G., Fischer P., Garruto R. M., Beyreuther K., Masters C. L., Simms G., Gibbs C. J., Jr, Gajdusek D. C. Amyloid of neurofibrillary tangles of Guamanian parkinsonism-dementia and Alzheimer disease share identical amino acid sequence. Proc Natl Acad Sci U S A. 1987 Apr;84(7):2073–2077. doi: 10.1073/pnas.84.7.2073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haas A. L., Bright P. M. The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J Biol Chem. 1985 Oct 15;260(23):12464–12473. [PubMed] [Google Scholar]
  20. Higgins G. A., Lewis D. A., Bahmanyar S., Goldgaber D., Gajdusek D. C., Young W. G., Morrison J. H., Wilson M. C. Differential regulation of amyloid-beta-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1297–1301. doi: 10.1073/pnas.85.4.1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hurrell R. F., Carpenter K. J. Nutritional significance of cross-link formation during food processing. Adv Exp Med Biol. 1977;86B:225–238. doi: 10.1007/978-1-4757-9113-6_16. [DOI] [PubMed] [Google Scholar]
  22. Hussey S., Yates C. M., Fink G. Fluorescence activated cell sorting (FACS) as a separation method for neurofibrillary tangles in Alzheimer's disease. J Neurosci Methods. 1986 Mar;16(1):1–8. doi: 10.1016/0165-0270(86)90002-6. [DOI] [PubMed] [Google Scholar]
  23. Iqbal K., Grundke-Iqbal I., Zaidi T., Ali N., Wisniewski H. M. Are Alzheimer neurofibrillary tangles insoluble polymers? Life Sci. 1986 May 5;38(18):1695–1700. doi: 10.1016/0024-3205(86)90414-5. [DOI] [PubMed] [Google Scholar]
  24. Iqbal K., Wiśniewski H. M., Shelanski M. L., Brostoff S., Liwnicz B. H., Terry R. D. Protein changes in senile dementia. Brain Res. 1974 Sep 6;77(2):337–343. doi: 10.1016/0006-8993(74)90798-7. [DOI] [PubMed] [Google Scholar]
  25. Iqbal K., Zaidi T., Thompson C. H., Merz P. A., Wisniewski H. M. Alzheimer paired helical filaments: bulk isolation, solubility, and protein composition. Acta Neuropathol. 1984;62(3):167–177. doi: 10.1007/BF00691849. [DOI] [PubMed] [Google Scholar]
  26. KIDD M. ALZHEIMER'S DISEASE--AN ELECTRON MICROSCOPICAL STUDY. Brain. 1964 Jun;87:307–320. doi: 10.1093/brain/87.2.307. [DOI] [PubMed] [Google Scholar]
  27. KIDD M. Paired helical filaments in electron microscopy of Alzheimer's disease. Nature. 1963 Jan 12;197:192–193. doi: 10.1038/197192b0. [DOI] [PubMed] [Google Scholar]
  28. Kang J., Lemaire H. G., Unterbeck A., Salbaum J. M., Masters C. L., Grzeschik K. H., Multhaup G., Beyreuther K., Müller-Hill B. The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987 Feb 19;325(6106):733–736. doi: 10.1038/325733a0. [DOI] [PubMed] [Google Scholar]
  29. Khachaturian Z. S. Diagnosis of Alzheimer's disease. Arch Neurol. 1985 Nov;42(11):1097–1105. doi: 10.1001/archneur.1985.04060100083029. [DOI] [PubMed] [Google Scholar]
  30. Kirschner D. A., Abraham C., Selkoe D. J. X-ray diffraction from intraneuronal paired helical filaments and extraneuronal amyloid fibers in Alzheimer disease indicates cross-beta conformation. Proc Natl Acad Sci U S A. 1986 Jan;83(2):503–507. doi: 10.1073/pnas.83.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kitaguchi N., Takahashi Y., Tokushima Y., Shiojiri S., Ito H. Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity. Nature. 1988 Feb 11;331(6156):530–532. doi: 10.1038/331530a0. [DOI] [PubMed] [Google Scholar]
  32. Kosik K. S., Duffy L. K., Dowling M. M., Abraham C., McCluskey A., Selkoe D. J. Microtubule-associated protein 2: monoclonal antibodies demonstrate the selective incorporation of certain epitopes into Alzheimer neurofibrillary tangles. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7941–7945. doi: 10.1073/pnas.81.24.7941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kosik K. S., Joachim C. L., Selkoe D. J. Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease. Proc Natl Acad Sci U S A. 1986 Jun;83(11):4044–4048. doi: 10.1073/pnas.83.11.4044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Masters C. L., Multhaup G., Simms G., Pottgiesser J., Martins R. N., Beyreuther K. Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels. EMBO J. 1985 Nov;4(11):2757–2763. doi: 10.1002/j.1460-2075.1985.tb04000.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Masters C. L., Simms G., Weinman N. A., Multhaup G., McDonald B. L., Beyreuther K. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4245–4249. doi: 10.1073/pnas.82.12.4245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Meyer E. M., West C. M., Chau V. Antibodies directed against ubiquitin inhibit high affinity [3H]choline uptake in rat cerebral cortical synaptosomes. J Biol Chem. 1986 Nov 5;261(31):14365–14368. [PubMed] [Google Scholar]
  37. Miller C. C., Brion J. P., Calvert R., Chin T. K., Eagles P. A., Downes M. J., Flament-Durand J., Haugh M., Kahn J., Probst A. Alzheimer's paired helical filaments share epitopes with neurofilament side arms. EMBO J. 1986 Feb;5(2):269–276. doi: 10.1002/j.1460-2075.1986.tb04209.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Miller C. C., Haugh M., Anderton B. H., Hudson L., Murphy J. Neurofibrillary tangles from Alzheimer's disease brain purified using a cell sorter. Neurosci Lett. 1986 Jan 30;63(3):247–252. doi: 10.1016/0304-3940(86)90364-2. [DOI] [PubMed] [Google Scholar]
  39. Miyakawa T., Shimoji A., Kuramoto R., Higuchi Y. The relationship between senile plaques and cerebral blood vessels in Alzheimer's disease and senile dementia. Morphological mechanism of senile plaque production. Virchows Arch B Cell Pathol Incl Mol Pathol. 1982 Aug;40(2):121–129. doi: 10.1007/BF02932857. [DOI] [PubMed] [Google Scholar]
  40. Mori H., Kondo J., Ihara Y. Ubiquitin is a component of paired helical filaments in Alzheimer's disease. Science. 1987 Mar 27;235(4796):1641–1644. doi: 10.1126/science.3029875. [DOI] [PubMed] [Google Scholar]
  41. Nukina N., Ihara Y. One of the antigenic determinants of paired helical filaments is related to tau protein. J Biochem. 1986 May;99(5):1541–1544. doi: 10.1093/oxfordjournals.jbchem.a135625. [DOI] [PubMed] [Google Scholar]
  42. Otterburn M., Healy M., Sinclair W. The formation, isolation and importance of isopeptides in heated proteins. Adv Exp Med Biol. 1977;86B:239–262. doi: 10.1007/978-1-4757-9113-6_17. [DOI] [PubMed] [Google Scholar]
  43. Pardridge W. M., Vinters H. V., Yang J., Eisenberg J., Choi T. B., Tourtellotte W. W., Huebner V., Shively J. E. Amyloid angiopathy of Alzheimer's disease: amino acid composition and partial sequence of a 4,200-dalton peptide isolated from cortical microvessels. J Neurochem. 1987 Nov;49(5):1394–1401. doi: 10.1111/j.1471-4159.1987.tb01005.x. [DOI] [PubMed] [Google Scholar]
  44. Perry G., Friedman R., Shaw G., Chau V. Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains. Proc Natl Acad Sci U S A. 1987 May;84(9):3033–3036. doi: 10.1073/pnas.84.9.3033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Perry G., Rizzuto N., Autilio-Gambetti L., Gambetti P. Paired helical filaments from Alzheimer disease patients contain cytoskeletal components. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3916–3920. doi: 10.1073/pnas.82.11.3916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ponte P., Gonzalez-DeWhitt P., Schilling J., Miller J., Hsu D., Greenberg B., Davis K., Wallace W., Lieberburg I., Fuller F. A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors. Nature. 1988 Feb 11;331(6156):525–527. doi: 10.1038/331525a0. [DOI] [PubMed] [Google Scholar]
  47. Robakis N. K., Wisniewski H. M., Jenkins E. C., Devine-Gage E. A., Houck G. E., Yao X. L., Ramakrishna N., Wolfe G., Silverman W. P., Brown W. T. Chromosome 21q21 sublocalisation of gene encoding beta-amyloid peptide in cerebral vessels and neuritic (senile) plaques of people with Alzheimer disease and Down syndrome. Lancet. 1987 Feb 14;1(8529):384–385. doi: 10.1016/s0140-6736(87)91754-5. [DOI] [PubMed] [Google Scholar]
  48. Roher A., Gray E. G., Paula-Barbosa M. Alzheimer's disease: coated vesicles, coated pits and the amyloid-related cell. Proc R Soc Lond B Biol Sci. 1988 Jan 22;232(1269):367–373. doi: 10.1098/rspb.1988.0001. [DOI] [PubMed] [Google Scholar]
  49. Roher A., Wolfe D., Palutke M., KuKuruga D. Purification, ultrastructure, and chemical analysis of Alzheimer disease amyloid plaque core protein. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2662–2666. doi: 10.1073/pnas.83.8.2662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Selkoe D. J., Abraham C. R., Podlisny M. B., Duffy L. K. Isolation of low-molecular-weight proteins from amyloid plaque fibers in Alzheimer's disease. J Neurochem. 1986 Jun;46(6):1820–1834. doi: 10.1111/j.1471-4159.1986.tb08501.x. [DOI] [PubMed] [Google Scholar]
  51. Selkoe D. J., Ihara Y., Salazar F. J. Alzheimer's disease: insolubility of partially purified paired helical filaments in sodium dodecyl sulfate and urea. Science. 1982 Mar 5;215(4537):1243–1245. doi: 10.1126/science.6120571. [DOI] [PubMed] [Google Scholar]
  52. Shapira R., Austin G. E., Mirra S. S. Neuritic plaque amyloid in Alzheimer's disease is highly racemized. J Neurochem. 1988 Jan;50(1):69–74. doi: 10.1111/j.1471-4159.1988.tb13231.x. [DOI] [PubMed] [Google Scholar]
  53. Shaw G., Chau V. Ubiquitin and microtubule-associated protein tau immunoreactivity each define distinct structures with differing distributions and solubility properties in Alzheimer brain. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2854–2858. doi: 10.1073/pnas.85.8.2854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. TERRY R. D., GONATAS N. K., WEISS M. ULTRASTRUCTURAL STUDIES IN ALZHEIMER'S PRESENILE DEMENTIA. Am J Pathol. 1964 Feb;44:269–297. [PMC free article] [PubMed] [Google Scholar]
  55. Tanzi R. E., Gusella J. F., Watkins P. C., Bruns G. A., St George-Hyslop P., Van Keuren M. L., Patterson D., Pagan S., Kurnit D. M., Neve R. L. Amyloid beta protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus. Science. 1987 Feb 20;235(4791):880–884. doi: 10.1126/science.2949367. [DOI] [PubMed] [Google Scholar]
  56. Tanzi R. E., McClatchey A. I., Lamperti E. D., Villa-Komaroff L., Gusella J. F., Neve R. L. Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease. Nature. 1988 Feb 11;331(6156):528–530. doi: 10.1038/331528a0. [DOI] [PubMed] [Google Scholar]
  57. Tanzi R. E., St George-Hyslop P. H., Haines J. L., Polinsky R. J., Nee L., Foncin J. F., Neve R. L., McClatchey A. I., Conneally P. M., Gusella J. F. The genetic defect in familial Alzheimer's disease is not tightly linked to the amyloid beta-protein gene. Nature. 1987 Sep 10;329(6135):156–157. doi: 10.1038/329156a0. [DOI] [PubMed] [Google Scholar]
  58. Terry R. D., Katzman R. Senile dementia of the Alzheimer type. Ann Neurol. 1983 Nov;14(5):497–506. doi: 10.1002/ana.410140502. [DOI] [PubMed] [Google Scholar]
  59. Vaughan D. W., Peters A. The structure of neuritic plaque in the cerebral cortex of aged rats. J Neuropathol Exp Neurol. 1981 Jul;40(4):472–487. doi: 10.1097/00005072-198107000-00009. [DOI] [PubMed] [Google Scholar]
  60. Wisniewski H. M., Vorbrodt A. W., Moretz R. C., Lossinsky A. S., Grundke-Iqbal I. Pathogenesis of neuritic (senile) and amyloid plaque formation. Exp Brain Res. 1982;Suppl 5:3–9. doi: 10.1007/978-3-642-68507-1_1. [DOI] [PubMed] [Google Scholar]
  61. Wiśniewski H. M., Ghetti B., Terry R. D. Neuritic (senile) plaques and filamentous changes in aged rhesus monkeys. J Neuropathol Exp Neurol. 1973 Oct;32(4):566–584. doi: 10.1097/00005072-197310000-00007. [DOI] [PubMed] [Google Scholar]
  62. Wood J. G., Mirra S. S., Pollock N. J., Binder L. I. Neurofibrillary tangles of Alzheimer disease share antigenic determinants with the axonal microtubule-associated protein tau (tau) Proc Natl Acad Sci U S A. 1986 Jun;83(11):4040–4043. doi: 10.1073/pnas.83.11.4040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zain S. B., Salim M., Chou W. G., Sajdel-Sulkowska E. M., Majocha R. E., Marotta C. A. Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex. Proc Natl Acad Sci U S A. 1988 Feb;85(3):929–933. doi: 10.1073/pnas.85.3.929. [DOI] [PMC free article] [PubMed] [Google Scholar]

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