Acta Neuropathologica

, 115:205

A cell culture model for investigation of Hirano bodies

  • Richard C. Davis
  • Ruth Furukawa
  • Marcus Fechheimer
Original Paper


Hirano bodies are paracrystalline F-actin-rich aggregations associated with a variety of conditions including aging, and neurodegenerative diseases. The composition and structure of these inclusions have been described by immunohistochemistry and ultrastructure, respectively. However, studies of the physiological function and dynamics of Hirano bodies have been hindered due to lack of a facile in vitro experimental system. We have developed a model for formation of Hirano bodies in mammalian cell cultures by expression of the carboxy-terminal fragment (CT) of a 34-kDa actin-bundling protein. Expression of the CT protein induces F-actin rearrangement in HEK 293, HeLa, Cos7 cells, neuroblastoma and astrocytic cells, and in primary neurons. We have termed these structures model Hirano bodies, since their composition and ultrastructure is quite similar to that reported in vivo. Model Hirano bodies in cell cultures sometimes appeared to be formed of a number of smaller domains, suggesting that small aggregates are intermediates in the formation of Hirano bodies. Stable lines expressing CT and bearing model Hirano bodies exhibit normal growth, morphology, and motility. This model provides a valuable system for the study of the dynamics of Hirano bodies, and their role in disease processes.


Hirano bodies In vitro model Mammalian cell culture Microscopy Neuropathology Actin cytoskeleton 


  1. 1.
    Adamec E, Yang F, Cole GM, Nixon RA (2001) Multiple-label immunocytochemistry for the evaluation of nature of cell death in experimental models of neurodegeneration. Brain Res Brain Res Protoc 7:193–202PubMedCrossRefGoogle Scholar
  2. 2.
    Asai DJ, Thompson WC, Wilson L, Dresden CF, Schulman H, Purich DL (1985) Microtubule-associated proteins (MAPs): a monoclonal antibody to MAP 1 decorates microtubules in vitro but stains stress fibers and not microtubules in vivo. Proc Natl Acad Sci USA 82:1434–1438PubMedCrossRefGoogle Scholar
  3. 3.
    Atsumi T, Yamamura Y, Sato T,Ikuta F (1980) Hirano bodies in the axon of peripheral nerves in a case with progressive external ophthalmoplegia with multisystemic involvements. Acta Neuropathol (Berl) 49:95–100CrossRefGoogle Scholar
  4. 4.
    Cartier L, Galvez S, Gajdusek DC (1985) Familial clustering of the ataxic form of Creutzfeldt-Jakob disease with Hirano bodies. J Neurol Neurosurg Psychiatry 48:234–238PubMedCrossRefGoogle Scholar
  5. 5.
    David-Ferreira JF, David-Ferreira KL, Gibbs CJJ, Morris JA (1968) Scrapie in mice. Ultrastructural observations in the cerebral cortex. Proc Soc Exp Biol Med 127:313–320PubMedGoogle Scholar
  6. 6.
    Dhingra V, Li Q, Allison AB, Stallknecht DE, Fu ZF (2005) Proteomic profiling and neurodegeneration in West-Nile-Virus-Infected neurons. J Biomed Biotechnol 2005(3):271–279PubMedCrossRefGoogle Scholar
  7. 7.
    Dravid SM, Murray TF (2004) Spontaneous synchronized calcium oscillations in neocortical neurons in the presence of physiological [Mg(2+)]: involvement of AMPA/kainite and metabotropic glutamate receptors. Brain Res 1006:8–17PubMedCrossRefGoogle Scholar
  8. 8.
    Fernandez R, Fernandez JM, Cervera C, Teijeira S, Teijeiro A, Dominguez C, Navarro C (1999) Adult glycogenosis II with paracrystalline mitochondrial inclusions and Hirano bodies in skeletal muscle. Neuromuscul Disord 9:136–143PubMedCrossRefGoogle Scholar
  9. 9.
    Field EJ, Mathews JD, Raine CS (1969) Electron microscopic observations on the cerebellar cortex in kuru. J Neurol Sci 8:209–224PubMedCrossRefGoogle Scholar
  10. 10.
    Field EJ, Narang HK (1972) An electron-microscopic study of scrapie in the rat: further observations on “inclusion bodies” and virus-like particles. J Neurol Sci 17:347–364PubMedCrossRefGoogle Scholar
  11. 11.
    Fulga TA, Elson-Schwab I, Khurana V, Steinhilb ML, Spires TL, Hyman BT, Feany MB (2007) Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal degeneration in vivo. Nat Cell Biol 9:139–148PubMedCrossRefGoogle Scholar
  12. 12.
    Furukawa R, Butz S, Fleischmann E, Fechheimer M (1992) The Dictyostelium discoideum 30,000 dalton protein contributes to phagocytosis. Protoplasma 169:18–27CrossRefGoogle Scholar
  13. 13.
    Furukawa R, Fechheimer M (1997) The structure, function, and assembly of actin filament bundles. Int Rev Cytol 175:29–90PubMedGoogle Scholar
  14. 14.
    Galloway PG, Perry G, Gambetti P (1987) Hirano body filaments contain actin and actin-associated proteins. J Neuropathol Exp Neurol 46:185–199PubMedGoogle Scholar
  15. 15.
    Galloway PG, Perry G, Kosik KS, Gambetti P (1987) Hirano bodies contain tau protein. Brain Res 403:337–340PubMedCrossRefGoogle Scholar
  16. 16.
    Galvan V, Gorostiza OF, Banwait S, Ataie M, Logvinova AV, Sitaraman S, Carlson E, Sagi SA, Chevallier N, Jin K, Greenberg DA, Bredesen DE (2006) Reversal of Alzheimer’s-like pathology and behavior in human APP transgenic mice by mutation of asp 664. Proc Natl Acad Sci USA 103:7130–7135PubMedCrossRefGoogle Scholar
  17. 17.
    Galvin JE, Lee VM, Schmidt ML, Tu PH, Iwatsubo T, Trojanowski JQ (1999) Pathobiology of the Lewy body. Adv Neurol 80:313–324PubMedGoogle Scholar
  18. 18.
    Gambetti P, Schecket G, Ghetti B, Hirano A, Dahl D (1983) Neurofibrillary changes in human brain: an immunocytochemical study with a neurofilament antiserum. J Neuropathol Exp Neurol 42:69–79PubMedGoogle Scholar
  19. 19.
    Gessaga EC,Anzil AP (1975) Rod-shaped filamentous inclusions and other ultrastructural features in a cerebellar astrocytoma. Acta Neuropathol (Berl) 33:119–127CrossRefGoogle Scholar
  20. 20.
    Gibson PH, Tomlinson BE (1977) Numbers of Hirano bodies in the hippocampus of normal and demented people with Alzheimer’s disease. J Neurol Sci 33:199–206PubMedCrossRefGoogle Scholar
  21. 21.
    Goedert M (1999) Filamentous nerve cell inclusions in neurodegenerative diseases: tauopathies and alpha-synucleinopathies. Philos Trans R Soc Lond B Biol Sci 354:1101–1118PubMedCrossRefGoogle Scholar
  22. 22.
    Goldman JE (1983) The association of actin with Hirano bodies. J Neuropathol Exp Neurol 42:146–152PubMedGoogle Scholar
  23. 23.
    Hirano A, Dembitzer HM, Kurland LT, Zimmerman HM (1968) The fine structure of some intraganlionic alterations. J Neuropathol Exp Neurol 27:167–182PubMedGoogle Scholar
  24. 24.
    Hirano A, Demblitzer HM (1976) Eosinophilic rod-like structure in myelinated fibres of hamster spinal roots. Neuropathol Appl Neurobiol 2:225–232Google Scholar
  25. 25.
    Hirano A (1994) Hirano bodies and related neuronal inclusions. Neuropathol Appl Neurobiol 20:3–11PubMedGoogle Scholar
  26. 26.
    Hirano A (2005) The role of electron microscopy in neuropathology: a personal historical note. Acta Neuropathol (Berl) 109:115–123CrossRefGoogle Scholar
  27. 27.
    Izumiyama N, Ohtsubo K, Tachikawa T, Nakamura H (1991) Elucidation of three-dimensional ultrastructure of Hirano bodies by the quick-freeze, deep-etch and replica method. Acta Neuropathol (Berl) 81:248–254CrossRefGoogle Scholar
  28. 28.
    Johns JA, Brock AM, Pardee JD (1988) Colocalization of F-actin and 34-kilodalton actin bundling protein in Dictyostelium and cultured fibroblasts. Cell Motil Cytoskeleton 9:205–218PubMedCrossRefGoogle Scholar
  29. 29.
    Johnston JA, Ward CL, Kopito RR (1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143:1883–1898PubMedCrossRefGoogle Scholar
  30. 30.
    Jordan-Sciutto K, Dragich J, Walcott D, Bowser R (1998) The presence of FAC1 protein in Hirano bodies. Neuropathol Appl Neurobiol 24:359–366PubMedCrossRefGoogle Scholar
  31. 31.
    Kopito RR (2000) Aggresomes, inclusion bodies, and protein aggregation. Trends Cell Biol 10:524–530PubMedCrossRefGoogle Scholar
  32. 32.
    Laas R, Hagel C (1994) Hirano bodies and chronic alcoholism. Neuropathol Appl Neurobiol 20:12–21PubMedGoogle Scholar
  33. 33.
    Lee H-g, Ueda M, Miyamoto Y, Yoneda Y, Perry G, Smith MA, Zhu X (2006) Aberrant localization of importin a1 in hippocampal neurons in Alzheimer disease. Brain Res 1124:1–4PubMedCrossRefGoogle Scholar
  34. 34.
    Lee SC, Zhao ML, Hirano A, Dickson DW (1999) Inducible nitric oxide synthase immunoreactivity in the Alzheimer disease hippocampus: association with Hirano bodies, neurofibrillary tangles, and senile plaques. J Neuropathol Exp Neurol 58:1163–1169PubMedGoogle Scholar
  35. 35.
    Lim RWL, Furukawa R, Eagle S, Cartwright RC, Fechheimer M (1999) Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein. Biochemistry 38:800–812PubMedCrossRefGoogle Scholar
  36. 36.
    Lim RWL, Furukawa R, Fechheimer M (1999) Evidence of intramolecular regulation of the Dictyostelium discoideum 34,000 dalton F-actin Bundling Protein. Biochemistry 38:16323–16332PubMedCrossRefGoogle Scholar
  37. 37.
    Maciver SK, Harrington CR (1995) Two actin binding proteins, actin depolymerizing factor and cofilin, are associated with Hirano bodies. Neuroreport 6:1985–1988PubMedCrossRefGoogle Scholar
  38. 38.
    Maselli AG, Davis R, Furukawa R, Fechheimer M (2002) Formation of Hirano bodies in Dictyostelium and mammalian cells induced by expression of a modified form of an actin cross-linking protein. J Cell Sci 115:1939–1952PubMedGoogle Scholar
  39. 39.
    Maselli AG, Furukawa R, Thomson SAM, Davis RC, Fechheimer M (2003) Formation of Hirano bodies induced by expression of an actin cross-linking protein with a gain of function mutation. Eukaryot Cell 2:778–787PubMedCrossRefGoogle Scholar
  40. 40.
    Mitake S, Ojika K, Katada E, Otsuka Y, Matsukawa N, Fujimori O (1995) Accumulation of hippocampal cholinergic neurostimulating peptide (HCNP)- related components in Hirano bodies. Neuropathol Appl Neurobiol 21:35–40PubMedGoogle Scholar
  41. 41.
    Mitake S, Katada E, Otsuka Y, Matsukawa N, Iwase T, Tsugu T, Fujimori O, Ojika K (1996) Possible implication of hippocampal cholinergic neurostimulating peptide (HCNP)-related components in Hirano body formation. Neuropathol Appl Neurobiol 22:440–445PubMedGoogle Scholar
  42. 42.
    Mitake S, Ojika K, Hirano A (1997) Hirano bodies and Alzheimer’s disease. Kaohsiung J Med Sci 13:10–18PubMedGoogle Scholar
  43. 43.
    Munoz DG, Wang D, Greenberg BD (1993) Hirano bodies accumulate C-terminal sequences of beta-amyloid precursor protein (beta-APP) epitopes. J Neuropathol Exp Neurol 52:14–21PubMedGoogle Scholar
  44. 44.
    Nagara H, Yajima K, Suzuki K (1980) An ultrastructural study on the cerebellum of the brindled mouse. Acta Neuropathol (Berl) 52:41–50CrossRefGoogle Scholar
  45. 45.
    Novak KD, Peterson MD, Reedy MC, Titus MA (1995) Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis. J Cell Biol 131:1205–1221PubMedCrossRefGoogle Scholar
  46. 46.
    Ogata J, Budzilovich GN, Cravioto H (1972) A study of rod-like structures (Hirano bodies) in 240 normal and pathological brains. Acta Neuropathol (Berl) 21:61–67CrossRefGoogle Scholar
  47. 47.
    Okamoto K, Hirai S, Hirano A (1982) Hirano bodies in myelinated fibers of hepatic encephalopathy. Acta Neuropathol (Berl) 58:307–310CrossRefGoogle Scholar
  48. 48.
    Orr HT (2001) Beyond the Q’s in the polyglutamine diseases. Genes Dev 15:925–932PubMedCrossRefGoogle Scholar
  49. 49.
    Peress NS, Perillo E (1995) Differential expression of TFG Beta 1, 2, and 3 isotypes in Alzheimer’s disease: a comparative immunohistochemical study with cerebral infarction, aged human and mouse control brains. J Neuropathol Exp Neurol 54:802–811PubMedGoogle Scholar
  50. 50.
    Peterson C, Kress Y, Vallee R, Goldman JE (1988) High molecular weight microtubule-associated proteins bind to actin lattices (Hirano bodies). Acta Neuropathol (Berl) 77:168–174Google Scholar
  51. 51.
    Previll LA, Crosby ME, Castellani RJ, Bowser R, Perry G, Smith MA, Zhu X (2007) Increased Expression of p130 in Alzheimer Disease. Neurochem Res 32:639–644PubMedCrossRefGoogle Scholar
  52. 52.
    Renkawek K, Bosman GJ, de Jong WW (1994) Expression of small heat shock protein hsp 27 in reactive gliosis in Alzheimer disease and other types of dementia. Acta Neuropathol (Berl) 87:511–519CrossRefGoogle Scholar
  53. 53.
    Rodriguez LG, Wu X, Guan JL (2005) Wound-healing assay. Methods Mol Biol 294:23–29PubMedGoogle Scholar
  54. 54.
    Rossiter JP, Anderson LL, Yang F, Cole GM (2000) Caspase-cleaved actin (fractin) immunolabelling of Hirano bodies. Neuropathol Appl Neurobiol 26:342–346PubMedCrossRefGoogle Scholar
  55. 55.
    Saganich MJ, Schroeder BE, Galvan V, Bredesen DE, Koo EH, Heinemann SF (2006) Deficits in synaptic transmission and learning in amyloid precursor protein (APP) transgenic mice require c-terminal cleavage of APP. J Neurosci 26:13428–13436PubMedCrossRefGoogle Scholar
  56. 56.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  57. 57.
    Schmidt ML, Lee VM, Trojanowski JQ (1989) Analysis of epitopes shared by Hirano bodies and neurofilament proteins in normal and Alzheimer’s disease hippocampus. Lab Invest 60:513–522PubMedGoogle Scholar
  58. 58.
    Schochet SS Jr, Lampert PW, Lindenberg R (1968) Fine structure of the Pick and Hirano bodies in a case of Pick’s disease. Acta Neuropathol (Berl) 11:330–337CrossRefGoogle Scholar
  59. 59.
    Schochet SS Jr, McCormick WF (1972) Ultrastructure of Hirano bodies. Acta Neuropathol (Berl) 21:50–60CrossRefGoogle Scholar
  60. 60.
    Selkoe DJ (1998) The cell biology of b-amyloid precursor protein and presenilin in Alzheimer’s disease. Trends Cell Biol 8:447–453PubMedCrossRefGoogle Scholar
  61. 61.
    Setoguti T, Esumi H, Shimizu T (1974) Specific organization of intracytoplasmic filaments in the dog testicular interstitial cell. Cell Tissue Res 148:493–497PubMedCrossRefGoogle Scholar
  62. 62.
    Shao CY, Crary JF, Rao C, Sacktor TC, Mirra SS (2006) Atypical protein kinase C in neurodegnerative disease II: PKC i/l in tauophathies and a-synucleinophathies. J Neuropathol Exp Neurol 65:327–335PubMedCrossRefGoogle Scholar
  63. 63.
    Sima AA, Hinton D (1983) Hirano-bodies in the distal symmetric polyneuropathy of the spontaneously diabetic BB-Wistar rat. Acta Neurol Scand 68:107–112PubMedGoogle Scholar
  64. 64.
    Soriano Z, Pardee JD (2004) M34 Actin regulatory protein is a sensitive diagnostic marker for early- and late-stage mammary carcinomas. Clin Cancer Res 10:4437–4443PubMedCrossRefGoogle Scholar
  65. 65.
    Tomonaga M (1974) Ultrastructure of Hirano bodies. Acta Neuropathol (Berl) 28:365–366CrossRefGoogle Scholar
  66. 66.
    Tomonaga M (1983) Hirano body in extraocular muscle. Acta Neuropathol (Berl) 60:309–313CrossRefGoogle Scholar
  67. 67.
    Yamamoto T, Hirano A (1985) Hirano bodies in the perikaryon of the Purkinje cell in a case of Alzheimer’s disease. Acta Neuropathol (Berl) 67:167–169CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Richard C. Davis
    • 1
  • Ruth Furukawa
    • 1
  • Marcus Fechheimer
    • 1
  1. 1.Department of Cellular BiologyUniversity of GeorgiaAthensUSA

Personalised recommendations