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In Vitro Aging Revisited: The Longevity of Cultured Neurons

  • Nozomu MoriEmail author

Abstract

Almost half a century ago, Leonard Hayflick reported that normal diploid fibroblasts can grow and age on cultured dishes in vitro and proposed that normal fibroblasts have a limited “replicative” lifespan, which was later termed the Hayflick limit. This limit was a paradigm of the cellular senescence of dividing cells. In contrast, it is not well-known how non-dividing cells, such as neurons and muscles, grow and age in vitro over a long time course in a culture dish. There is growing evidence that neurons dissociated from various brain areas, such as the cerebral neocortex or hippocampus, can survive for several months in culture dishes. Neurons initially proliferate, form mature synapses, and begin to exhibit aging associated with synaptic loss and neuronal elimination. The long-term cultured neurons in vitro seem to represent many similar aspects of physiological and possibly pathological aging that occur in vivo. The usefulness of this system as a new model for the investigation of non-replicative post-mitotic neuronal aging can be discussed.

Keywords

Aging Brain Cellular aging Longevity Neuron Primary culture 

Notes

Acknowledgments

The author would like to thank Drs. Y. Yamaguchi, K. Ohyama, and G. Matsumoto for their contribution to this work, and Ms. K. Onga and Ms. A. Okamoto for their excellent technical assistance. This work was supported by Grants-in-Aid for Scientific Research, Challenging Exploratory Research, and also in part by the Asian CORE Program of the Japan Society of Promotion of Science (JSPS): Asian Aging Core for Longevity .

References

  1. Aksenova MV, Aksenov MY, Markesbery WR, Butterfield DA (1999) Aging in a dish: age-dependent changes of neuronal survival, protein oxidation, and creatine kinase BB expression in long-term hippocampal cell culture. J Neurosci Res 58:308–317CrossRefPubMedGoogle Scholar
  2. Alvarez VA, Sabatini BL (2007) Anatomical and physiological plasticity of dendritic spines. Annu Rev Neurosci 30:79–97CrossRefPubMedGoogle Scholar
  3. Araki T, Sasaki Y, Milbrandt J (2004) Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science 305:1010–1013CrossRefPubMedGoogle Scholar
  4. Aubert G, Lansdorp PM (2008) Telomeres and aging. Physiol Rev 88:557–579CrossRefPubMedGoogle Scholar
  5. Banker GA, Cowan WM (1977) Rat hippocampal neurons in dispersed cell culture. Brain Res 126:397–342CrossRefPubMedGoogle Scholar
  6. Banker GA, Cowan WM (1979) Further observations on hippocampal neurons in dispersed cell culture. J Comp Neurol 187:469–493CrossRefPubMedGoogle Scholar
  7. Bastiani M, Pearson KG, Goodman CS (1984) From embryonic fascicles to adult tracts: organization of neuropile from a developmental perspective. J Exp Biol 112:45–64PubMedGoogle Scholar
  8. Brewer GJ (1995) Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J Neurosci Res 42:674–683CrossRefPubMedGoogle Scholar
  9. Brewer GJ (1997) Isolation and culture of adult rat hippocampal neurons. J Neurosci Methods 71:143–155CrossRefPubMedGoogle Scholar
  10. Burke SN, Barnes CA (2006) Neural plasticity in the ageing brain. Nat Rev Neurosci 7:30–40CrossRefPubMedGoogle Scholar
  11. Campisi J (2001) From cells to organisms: can we learn about aging from cells in culture? Exp Gerontol 36:607–618CrossRefPubMedGoogle Scholar
  12. Clayton DA, Browning MD (2001) Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats. Neurobiol Aging 22:165–168CrossRefPubMedGoogle Scholar
  13. Clayton DA, Grosshans DR, Browning MD (2002) Aging and surface expression of hippocampal NMDA receptors. J Biol Chem 277:14367–14369CrossRefPubMedGoogle Scholar
  14. Cremer H, Chazal G, Goridis C, Represa A (1997) NCAM is essential for axonal growth and fasciculation in the hippocampus. Mol Cell Neurosci 8:323–335CrossRefPubMedGoogle Scholar
  15. Cristofalo VJ (2005) SA beta Gal staining: biomarker or delusion. Exp Gerontol 40:836–838CrossRefPubMedGoogle Scholar
  16. Cristofalo VJ, Stanulis BM (1978) Cell aging: a model system approach. In: Behnke JA, Finch CE, Moment GB (eds) The biology of aging. Plenum Press, New York, pp 19–31CrossRefGoogle Scholar
  17. Cristofalo VJ, Lorenzini A, Allen RG, Torres C, Tresini M (2004) Replicative senescence: a critical review. Mech Ageing Dev 125:827–848CrossRefPubMedGoogle Scholar
  18. Deng Y, Chan SS, Chang S (2008) Telomere dysfunction and tumour suppression: the senescence connection. Nat Rev Cancer 8:450–458PubMedCentralCrossRefPubMedGoogle Scholar
  19. Dickstein DL, Kabaso D, Rocher AB, Luebke JI, Wearne SL, Hof PR (2007) Changes in the structural complexity of the aged brain. Aging Cell 6:275–284PubMedCentralCrossRefPubMedGoogle Scholar
  20. Gan L, Mucke L (2008) Paths of convergence: sirtuins in aging and neurodegeneration. Neuron 58(1):10–14CrossRefPubMedGoogle Scholar
  21. Gray DA, Woulfe J (2005) Lipofuscin and aging: a matter of toxic waste. Sci Aging Knowl Environ 2005(5):re1. doi: 10.1126/sageke.2005.5.re1
  22. Hanson MG, Milner LD, Landmesser LT (2008) Spontaneous rhythmic activity in early chick spinal cord influences distinct motor axon pathfinding decisions. Brain Res Rev 57:77–85PubMedCentralCrossRefPubMedGoogle Scholar
  23. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460CrossRefPubMedGoogle Scholar
  24. Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636CrossRefPubMedGoogle Scholar
  25. Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621CrossRefPubMedGoogle Scholar
  26. Iqbal K, Alonso Adel C, Chen S, Chohan MO, El-Akkad E, Gong CX, Khatoon S, Li B, Liu F, Rahman A, Tanimukai H, Grundke-Iqbal I (2005) Tau pathology in Alzheimer disease and other tauopathies. Biochim Biophys Acta 1739:198–210CrossRefPubMedGoogle Scholar
  27. Jeyapalan JC, Sedivy JM (2008) Cellular senescence and organismal aging. Mech Ageing Dev 129:467–474PubMedCentralCrossRefPubMedGoogle Scholar
  28. Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1:2406–2415CrossRefPubMedGoogle Scholar
  29. Kaur J, Sharma D, Singh R (2001) Acetyl-L-carnitine enhances Na(+), K(+)-ATPase glutathione-S-transferase and multiple unit activity and reduces lipid peroxidation and lipofuscin concentration in aged rat brain regions. Neurosci Lett 301:1–4CrossRefPubMedGoogle Scholar
  30. Kim D, Nguyen MD, Dobbin MM, Fischer A, Sananbenesi F, Rodgers JT, Delalle I, Baur JA, Sui G, Armour SM, Puigserver P, Sinclair DA, Tsai LH (2007) SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer’s disease and amyotrophic lateral sclerosis. EMBO J 26:3169–3179PubMedCentralCrossRefPubMedGoogle Scholar
  31. Kiselyov K, Jennigs JJ Jr, Rbaibi Y, Chu CT (2007) Autophagy, mitochondria and cell death in lysosomal storage diseases. Autophagy 3:259–262PubMedCentralCrossRefPubMedGoogle Scholar
  32. Lesuisse C, Martin LJ (2002) Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death. J Neurobiol 51:9–23CrossRefPubMedGoogle Scholar
  33. Linskens MH, Harley CB, West MD, Campisi J, Hayflick L (1995) Replicative senescence and cell death. Science 267:17CrossRefPubMedGoogle Scholar
  34. Magnusson KR (2000) Declines in mRNA expression of different subunits may account for differential effects of aging on agonist and antagonist binding to the NMDA receptor. J Neurosci 20:1666–1674PubMedGoogle Scholar
  35. Magnusson KR, Nelson SE, Young AB (2002) Age-related changes in the protein expression of subunits of the NMDA receptor. Brain Res Mol Brain Res 99:40–45CrossRefPubMedGoogle Scholar
  36. Mattson MP, Kater SB (1988) Isolated hippocampal neurons in cryopreserved long-term cultures: development of neuroarchitecture and sensitivity to NMDA. Int J Dev Neurosci 6:439–452CrossRefPubMedGoogle Scholar
  37. Mattson MP, Duan W, Maswood N (2002) How does the brain control lifespan? Ageing Res Rev 1(2):155–165CrossRefPubMedGoogle Scholar
  38. McBurney MW, Reuhl KR, Ally AI, Nasipuri S, Bell JC, Craig J (1988) Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture. J Neurosci 8:1063–1073PubMedGoogle Scholar
  39. Mielke JG, Comas T, Woulfe J, Monette R, Chakravarthy B, Mealing GA (2005) Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development. Brain Res Dev Brain Res 160:275–286CrossRefPubMedGoogle Scholar
  40. Mori N, Morii H (2002) SCG10-related neuronal growth-associated proteins in neural development, plasticity, degeneration, and aging. J Neurosci Res 70:264–273CrossRefPubMedGoogle Scholar
  41. Morii H, Shiraishi-Yamaguchi Y, Mori N (2006) SCG10, a microtubule destabilizing factor, stimulates the neurite outgrowth by modulating microtubule dynamics in rat hippocampal primary cultured neurons. J Neurobiol 66:1101–1114CrossRefPubMedGoogle Scholar
  42. Morrison JH, Hof PR (1997) Life and death of neurons in the aging brain. Science 278:412–419CrossRefPubMedGoogle Scholar
  43. Oka T, Akisada M, Okabe A, Sakurai K, Shiosaka S, Kato K (2002) Extracellular serine protease neuropsin (KLK8) modulates neurite outgrowth and fasciculation of mouse hippocampal neurons in culture. Neurosci Lett 321:141–144CrossRefPubMedGoogle Scholar
  44. Okamoto A, Taguchi H, Onga K, Ohyama K, Mori N (2013) An in vitro aging model of long-term cultured hippocampal neurons. In: Cold Spring Harbor Asia Conference “Molecular basis of aging and disease”, p 33Google Scholar
  45. Pettmann B, Louis JC, Sensenbrenner M (1979) Morphological and biochemical maturation of neurones cultured in the absence of glial cells. Nature 281:378–380CrossRefPubMedGoogle Scholar
  46. Qin W, Yang T, Ho L, Zhao Z, Wang J, Chen L, Zhao W, Thiyagarajan M, MacGrogan D, Rodgers JT, Puigserver P, Sadoshima J, Deng H, Pedrini S, Gandy S, Sauve AA, Pasinetti GM (2006) Neuronal SIRT1 activation as a novel mechanism underlying the prevention of Alzheimer disease amyloid neuropathology by calorie restriction. J Biol Chem 281:21745–21754CrossRefPubMedGoogle Scholar
  47. Rosenzweig ES, Barnes CA (2003) Impact of aging on hippocampal function: plasticity, network dynamics, and cognition. Prog Neurobiol 69(3):143–179CrossRefPubMedGoogle Scholar
  48. Shiraishi Y, Mizutani A, Mikoshiba K, Furuichi T (2003) Coincidence in dendritic clustering and synaptic targeting of homer proteins and NMDA receptor complex proteins NR2B and PSD95 during development of cultured hippocampal neurons. Mol Cell Neurosci 22:188–201CrossRefPubMedGoogle Scholar
  49. Shiraishi-Yamaguchi Y, Mori N (2008) In vitro aging of the primary cultured hippocampal neurons. Biomed Gerontol (Tokyo) 32(3):27–30, in JapaneseGoogle Scholar
  50. Sulzer D, Mosharov E, Talloczy Z, Zucca FA, Simon JD, Zecca L (2008) Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease. J Neurochem 106:24–36CrossRefPubMedGoogle Scholar
  51. Ueda K, Masliah E, Saitoh T, Bakalis SL, Scoble H, Kosik KS (1990) Alz-50 recognizes a phosphorylated epitope of tau protein. J Neurosci 10:3295–3304PubMedGoogle Scholar
  52. Vucic S, Kiernan MC (2007) Pathophysiologic insights into motor axonal function in Kennedy disease. Neurology 69:1828–1835CrossRefPubMedGoogle Scholar
  53. Wenk GL, Barnes CA (2000) Regional changes in the hippocampal density of AMPA and NMDA receptors across the lifespan of the rat. Brain Res 885:1–5CrossRefPubMedGoogle Scholar
  54. Wolfer DP, Henehan-Beatty A, Stoeckli ET, Sonderegger P, Lipp HP (1994) Distribution of TAG-1/axonin-1 in fibre tracts and migratory streams of the developing mouse nervous system. J Comp Neurol 345:1–32CrossRefPubMedGoogle Scholar

Copyright information

© Springer Japan 2015

Authors and Affiliations

  1. 1.Department of Anatomy and Neurobiology, Graduate School of Biomedical SciencesNagasaki UniversityNagasakiJapan

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