Neurochemical Research

, Volume 33, Issue 12, pp 2516–2531 | Cite as

Differentiation Increases the Resistance of Neuronal Cells to Amyloid Toxicity

  • Cristina Cecchi
  • Anna Pensalfini
  • Gianfranco Liguri
  • Serena Baglioni
  • Claudia Fiorillo
  • Simone Guadagna
  • Mariagioia Zampagni
  • Lucia Formigli
  • Daniele Nosi
  • Massimo Stefani
Original Paper


A substantial lack of information is recognized on the features underlying the variable susceptibility to amyloid aggregate toxicity of cells with different phenotypes. Recently, we showed that different cell types are variously affected by early aggregates of a prokaryotic hydrogenase domain (HypF-N). In the present study we investigated whether differentiation affects cell susceptibility to amyloid injury using a human neurotypic SH-SY5Y cell differentiation model. We found that retinoic acid-differentiated cells were significantly more resistant against Aβ1-40, Aβ1-42 and HypF-N prefibrillar aggregate toxicity respect to undifferentiated cells treated similarly. Earlier and sharper increases in cytosolic Ca2+ and ROS with marked lipid peroxidation and mitochondrial dysfunction were also detected in exposed undifferentiated cells resulting in apoptosis activation. The reduced vulnerability of differentiated cells matched a more efficient Ca2+-ATPase equipment and a higher total antioxidant capacity. Finally, increasing the content of membrane cholesterol resulted in a remarkable reduction of vulnerability and ability to bind the aggregates in either undifferentiated and differentiated cells.


Amyloid toxicity Prefibrillar aggregates Cell differentiation Oxidative stress Apoptosis 



Bovine serum albumin


Congo Red


2′,7′-Dichlorodihydrofluorescein diacetate, acetyl ester




Dulbecco’s Modified Eagle’s Medium




Enhanced chemiluminescence


Familial Alzheimer disease


Fetal bovine serum




Hanks balanced salt solution


N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)


Horseradish peroxidase


N-Terminal domain of the prokaryotic hydrogenase maturation factor


Lactate dehydrogenase


Mitochondrial permeability transition pore


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


NO synthase


Phosphate buffered saline


Polyoxyetanyl-cholesteryl sebacate




Propidium iodide






Polyvinylidene difluoride


Retinoic acid


Reactive nitrogen species


Reactive oxygen species


Sodium dodecylsulfate polyacrylamide gel electrophoresis




Tapping mode atomic force microscopy



We thank Roberto Caporale for technical advice. This study was supported by grants from the Italian MIUR (project number 2005054147_001 and 2005053998_001) and Compagnia di San Paolo, Torino, Italy (ref. n. 2004.0995).


  1. 1.
    Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297:353–356PubMedCrossRefGoogle Scholar
  2. 2.
    Masters CL, Simms G, Weinman NA et al (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci USA 82:4245–4249PubMedCrossRefGoogle Scholar
  3. 3.
    Lacor PN, Buniel MC, Chang L et al (2004) Synaptic targeting by Alzheimer’s-related amyloid β oligomers. J Neurosci 24:10191–10200PubMedCrossRefGoogle Scholar
  4. 4.
    Lesne S, Koh MT, Kotilinek L et al (2006) A specific amyloid-beta protein assembly in the brain impairs memory. Nature 440:352–357PubMedCrossRefGoogle Scholar
  5. 5.
    Bucciantini M, Giannoni E, Chiti F et al (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416:507–511PubMedCrossRefGoogle Scholar
  6. 6.
    Walsh DM, Klyubin I, Fadeeva JV et al (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416:535–539PubMedCrossRefGoogle Scholar
  7. 7.
    Gharibyan AL, Zamotin V, Yamanandra K et al (2007) Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways. J Mol Biol 365:1337–1349PubMedCrossRefGoogle Scholar
  8. 8.
    Novitskaya V, Bocharova OV, Bronstein I et al (2006) Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons. J Biol Chem 281:13828–13836PubMedCrossRefGoogle Scholar
  9. 9.
    Bokvist M, Lindstrom F, Watts A et al (2004) Two types of Alzheimer’s beta-amyloid (1–40) peptide membrane interactions: aggregation preventing transmembrane anchoring versus accelerated surface fibril formation. J Mol Biol 335:1039–1049PubMedCrossRefGoogle Scholar
  10. 10.
    Kayed R, Head E, Thompson JL et al (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300:486–489PubMedCrossRefGoogle Scholar
  11. 11.
    Stefani M, Dobson CM (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med 81:678–699PubMedCrossRefGoogle Scholar
  12. 12.
    Kourie JI, Henry CL (2002) Ion channel formation and membrane-linked pathologies of misfolded hydrophobic proteins: the role of dangerous unchaperoned molecules. Clin Exp Pharmacol Physiol 29:741–753PubMedCrossRefGoogle Scholar
  13. 13.
    Wakabayashi M, Okada T, Kozutsumi Y et al (2005) GM1 ganglioside-mediated accumulation of amyloid beta-protein on cell membranes. Biochim Biophys Res Com 328:1019–1023CrossRefGoogle Scholar
  14. 14.
    Zhu M, Souillac PO, Ionescu-Zanetti C et al (2002) Surface-catalyzed amyloid fibril formation. J Biol Chem 277:50914–50922PubMedCrossRefGoogle Scholar
  15. 15.
    Montine TJ, Neely MD, Quinn JF et al (2002) Lipid peroxidation in aging brain and Alzheimer’s disease. Free Radic Biol Med 33:620–626PubMedCrossRefGoogle Scholar
  16. 16.
    Mattson MP, Guo Q, Furukawa K et al (1998) Presenilins, the endoplasmic reticulum, and neuronal apoptosis in Alzheimer’s disease. J Neurochem 70:1–14PubMedCrossRefGoogle Scholar
  17. 17.
    Cecchi C, Fiorillo C, Baglioni S et al (2007) Increased susceptibility to amyloid toxicity in familial Alzheimer’s fibroblasts. Neurobiol Aging 28:863–876PubMedCrossRefGoogle Scholar
  18. 18.
    Cecchi C, Fiorillo C, Sorbi S et al (2002) Oxidative stress and reduced antioxidant defenses in peripheral cells from familial Alzheimer’s patients. Free Rad Biol Med 33:1372–1379PubMedCrossRefGoogle Scholar
  19. 19.
    Anderluh G, Gutierrez-Aguirre I, Rabzelj S et al (2005) Interaction of stefin B in the prefibrillar oligomeric form with membranes. Correlation with cellular toxicity. FEBS J 72:3042–3051CrossRefGoogle Scholar
  20. 20.
    Bucciantini M, Calloni G, Chiti F et al (2004) Prefibrillar amyloid protein aggregates share common features of cytotoxicity. J Biol Chem 279:31374–31382PubMedCrossRefGoogle Scholar
  21. 21.
    Cecchi C, Baglioni S, Fiorillo C et al (2005) Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates. J Cell Sci 118:3459–3470PubMedCrossRefGoogle Scholar
  22. 22.
    Dargusch R, Schubert D (2002) Specificity of resistance to oxidative stress. J Neurochem 81:1394–1400PubMedCrossRefGoogle Scholar
  23. 23.
    Benvenuti S, Saccardi R, Luciani P et al (2006) A Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer’s disease-related gene seladin-1. Exp Cell Res 312:2592–2604PubMedCrossRefGoogle Scholar
  24. 24.
    Haughey NJ, Nath A, Chan SL et al (2002) Disruption of neurogenesis by amyloid beta-peptide, and perturbed neural progenitor cell homeostasis, in models of Alzheimer’s disease. J Neurochem 83:1509–1524PubMedCrossRefGoogle Scholar
  25. 25.
    Chiti F, Bucciantini M, Capanni C et al (2001) Solution conditions can promote formation of either amyloid protofilaments or mature fibrils from the HypF N-terminal domain. Protein Sci 10:2541–2547PubMedCrossRefGoogle Scholar
  26. 26.
    Relini A, Torrassa S, Rolandi R et al (2004) Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils. J Mol Biol 338:943–957PubMedCrossRefGoogle Scholar
  27. 27.
    Datki Z, Papp R, Zadori D et al (2004) In vitro model of neurotoxicity of Abeta 1–42 and neuroprotection by a pentapeptide: irreversible events during the first hour. Neurobiol Dis 17:507–515PubMedCrossRefGoogle Scholar
  28. 28.
    Negre-Salvayre A, Auge N, Duval C et al (2002) Detection of intracellular reactive oxygen species in cultured cells using fluorescent probes. Methods Enzymol 352:62–71PubMedCrossRefGoogle Scholar
  29. 29.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  30. 30.
    Saborido A, Delgado J, Megias A (1999) Measurement of sarcoplasmic reticulum Ca2+-ATPase activity and E-type Mg2+-ATPase activity in rat heart homogenates. Anal Biochem 268:79–88PubMedCrossRefGoogle Scholar
  31. 31.
    Petronilli V, Miotto G, Canton M et al (1999) Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 76:725–754PubMedGoogle Scholar
  32. 32.
    Datki Z, Juhasz A, Galfi M et al (2003) Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells. Brain Res Bull 62:223–229PubMedCrossRefGoogle Scholar
  33. 33.
    Arispe N, Doh M (2002) Plasma membrane cholesterol controls the cytotoxicity of Alzheimer’s disease AbetaP (1-40) and (1-42) peptides. FASEB J 16:1526–1536PubMedCrossRefGoogle Scholar
  34. 34.
    Butterfield DA (2004) Proteomics: a new approach to investigate oxidative stress in Alzheimer’s disease brain. Brain Res 1000:1–7PubMedCrossRefGoogle Scholar
  35. 35.
    Demuro A, Mina E, Kayed R et al (2005) Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. J Biol Chem 280:17294–17300PubMedCrossRefGoogle Scholar
  36. 36.
    Loo DT, Copani A, Pike CJ et al (1993) Apoptosis is induced by beta-amyloid in cultured central nervous system neurons. Proc Natl Acad Sci USA 90:7951–7955PubMedCrossRefGoogle Scholar
  37. 37.
    Hensley K, Carney JM, Mattson MP et al (1994) A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease. Proc Natl Acad Sci USA 91:3270–3274PubMedCrossRefGoogle Scholar
  38. 38.
    Yip CM, Elton EA, Darabie AA et al (2001) Cholesterol, a modulator of membrane-associated Abeta-fibrillogenesis and neurotoxicity. J Mol Biol 311:723–734PubMedCrossRefGoogle Scholar
  39. 39.
    Olesen OF, Dago L, Mikkelsen JD (1998) Amyloid beta neurotoxicity in the cholinergic but not in the serotonergic phenotype of RN46A cells. Brain Res Mol Brain Res 57:266–274PubMedCrossRefGoogle Scholar
  40. 40.
    Subasinghe S, Unabia S, Barrow CJ et al (2003) Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes. J Neurochem 84:471–479PubMedCrossRefGoogle Scholar
  41. 41.
    Cecchi C, Pensalfini A, Stefani M, Baglioni S, Fiorillo C, Cappadona S, Caporale R, Nosi D, Ruggiero M, Liguri G (2008) Replicating neuroblastoma cells in different cell-cycle phases display different vulnerability to amyloid toxicity. J Mol Med 86:197–209PubMedCrossRefGoogle Scholar
  42. 42.
    Cecchi C, Rosati F, Pensalfini A, Formigli L, Nosi D, Liguri G, Dichiara F, Morello M, Danza G, Pieraccini G, Peri A, Serio M, Stefani M (2008) Seladin-1/DHCR24 protects neuroblastoma cells against Aβ toxicity by increasing membrane cholesterol content. J Cell Mol Med 12 [Epub ahead of print]Google Scholar
  43. 43.
    Ischiropoulos H, Gow A, Thom SR et al (1999) Detection of reactive nitrogen species using 2,7-dichlorodihydrofluorescein and dihydrorhodamine 123. Methods Enzymol 301:367–373PubMedCrossRefGoogle Scholar
  44. 44.
    Ferreiro E, Resende R, Costa R et al (2006) An endoplasmic-reticulum-specific apoptotic pathway is involved in prion and amyloid-beta peptides neurotoxicity. Neurobiol Dis 23:669–678PubMedCrossRefGoogle Scholar
  45. 45.
    Orrenius S, Zhivotovsky B, Nicotera P (2003) Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4:552–565PubMedCrossRefGoogle Scholar
  46. 46.
    Squier TC (2001) Oxidative stress and protein aggregation during biological aging. Exp Gerontol 36:1539–1550PubMedCrossRefGoogle Scholar
  47. 47.
    Chan CS, Guzman JN, Ilijic E et al (2007) ‘Rejuvenation’ protects neurons in mouse models of Parkinson’s disease. Nature 447:1081–1086PubMedCrossRefGoogle Scholar
  48. 48.
    Resende R, Pereira C, Agostinho P et al (2007) Susceptibility of hippocampal neurons to Abeta peptide toxicity is associated with perturbation of Ca2+ homeostasis. Brain Res 1143:11–21PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Cristina Cecchi
    • 1
    • 2
  • Anna Pensalfini
    • 1
  • Gianfranco Liguri
    • 1
    • 2
  • Serena Baglioni
    • 1
  • Claudia Fiorillo
    • 1
  • Simone Guadagna
    • 1
  • Mariagioia Zampagni
    • 1
  • Lucia Formigli
    • 3
  • Daniele Nosi
    • 3
  • Massimo Stefani
    • 1
    • 2
  1. 1.Department of Biochemical SciencesUniversity of FlorenceFlorenceItaly
  2. 2.Research Centre on the Molecular Basis of NeurodegenerationUniversity of FlorenceFlorenceItaly
  3. 3.Department of Anatomy, Histology and Forensic MedicineUniversity of FlorenceFlorenceItaly

Personalised recommendations