Cell Stress and Chaperones

, Volume 22, Issue 4, pp 589–600 | Cite as

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein

  • Dezerae Cox
  • Heath EcroydEmail author


Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cell-based model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. As such, this work sought to establish a robust model of intracellular α-syn aggregation using Neuro-2a cells. Aggregation of α-syn was found to be sensitive to inhibition of autophagy and the proteasome, resulting in a significant increase in the proportion of cells containing α-syn inclusions. This model was then used to evaluate the capacity of the sHsps, αB-c and Hsp27, to prevent α-syn aggregation in cells. To do so, we used bicistronic expression plasmids to express the sHsps. Unlike traditional fluorescent fusion constructs, these bicistronic expression plasmids enable only individual transfected cells expressing the sHsps (via expression of the fluorescent reporter) to be analysed, but without the need to tag the sHsp, which can affect its oligomeric structure and chaperone activity. Overexpression of both αB-c and Hsp27 significantly reduced the intracellular aggregation of α-syn. Thus, these findings suggest that overexpressing or boosting the activity of sHsps may be a way of preventing amyloid fibrillar aggregation of α-syn in the context of neurodegenerative disease.


Molecular chaperones Amyloid fibrils Protein inclusions Bicistronic vectors Proteostasis 







Bovine serum albumin


Enhanced green fluorescent protein


Foetal bovine serum


Internal ribosome entry site


Small heat shock protein



DC is supported by an Australian Postgraduate Award. HE was supported by an Australian Research Council Future Fellowship (FT110100586). This work was supported by grants from the Australian Department of Health and Ageing and the University of Wollongong. We thank Dr. Tracey Berg (University of Wollongong, Australia) for help in developing the bicistronic constructs used in this work and the Illawarra Health and Medical Research Institute for technical support.

Supplementary material

12192_2017_785_MOESM1_ESM.pdf (278 kb)
ESM 1 (PDF 277 kb)


  1. Ahmad MF, Raman B, Ramakrishna T, Rao CM (2008) Effect of phosphorylation on αb-crystallin: differences in stability, subunit exchange and chaperone activity of homo and mixed oligomers of αb-crystallin and its phosphorylation-mimicking mutant. J Mol Biol 375:1040–1051CrossRefPubMedGoogle Scholar
  2. Balch WE, Morimoto RI, Dillin A, Kelly JW (2008) Adapting proteostasis for disease intervention. Science 319:916–919CrossRefPubMedGoogle Scholar
  3. Benesch JLP, Ayoub M, Robinson CV, Aquilina JA (2008) Small heat shock protein activity is regulated by variable oligomeric substructure. J Biol Chem 283:28513–28517CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bertoncini CW, Jares-Erijman EA, Jovin TM, Klement R, Roberti MJ (2007) Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged alpha-synuclein. Nat Methods 4:345–351PubMedGoogle Scholar
  5. Bova MP, Ding LL, Horwitz J, Fung BKK (1997) Subunit exchange of αa-crystallin. J Biol Chem 272:29511–29517CrossRefPubMedGoogle Scholar
  6. Bova MP, Mchaourab HS, Han Y, Fung BKK (2000) Subunit exchange of small heat shock proteins: analysis of oligomer formation of alphaa-crystallin and hsp27 by fluorescence resonance energy transfer and site-directed truncations. J Biol Chem 275:1035–1042CrossRefPubMedGoogle Scholar
  7. Brown JWP, Buell AK, Michaels TCT, Meisl G, Carozza J, Flagmeier P, Vendruscolo M, Knowles TPJ, Dobson CM, Galvagnion C (2016) Β-synuclein suppresses both the initiation and amplification steps of α-synuclein aggregation via competitive binding to surfaces. Scientific reports 6:36010CrossRefPubMedPubMedCentralGoogle Scholar
  8. Bruinsma IB, Bruggink KA, Kinast K, Versleijen AAM, Segers-Nolten IMJ, Subramaniam V, Bea Kuiperij H, Boelens W, de Waal RMW, Verbeek MM (2011) Inhibition of alpha-synuclein aggregation by small heat shock proteins. Proteins Struct Funct Bioinformat 79:2956–2967CrossRefGoogle Scholar
  9. Carra S, Rusmini P, Crippa V, Giorgetti E, Boncoraglio A, Cristofani R, Naujock M, Meister M, Minoia M, Kampinga HH, Poletti A (2013) Different anti-aggregation and pro-degradative functions of the members of the mammalian shsp family in neurological disorders. Philos Trans R Soc Lond Ser B Biol Sci 368:20110409Google Scholar
  10. Chai YJ, Sierecki E, Tomatis VM, Gormal RS, Giles N, Morrow IC, Xia D, Götz J, Parton RG, Collins BM, Gambin Y, Meunier FA (2016) Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation. J Cell Biol 214:705–718CrossRefPubMedPubMedCentralGoogle Scholar
  11. Chang E, Kuret J (2008) Detection and quantification of tau aggregation using a membrane filter assay. Anal Biochem 373:330–336CrossRefPubMedGoogle Scholar
  12. Cox D, Carver JA, Ecroyd H (2014) Preventing α-synuclein aggregation: the role of the small heat-shock molecular chaperone proteins. Biochim Biophys Acta (BBA) - Molecular Basis of Disease 1842:1830–1843CrossRefGoogle Scholar
  13. Cox D, Selig E, Griffin MD, Carver JA, Ecroyd H (2016) Small heat shock proteins prevent alpha-synuclein aggregation via transient interactions and their efficacy is affected by the rate of aggregation. J Biol Chem 291:22618–22629Google Scholar
  14. Crivat G, Taraska JW (2012) Imaging proteins inside cells with fluorescent tags. Trends Biotechnol 30:8–16CrossRefPubMedGoogle Scholar
  15. Datskevich PN, Gusev NB (2014) Structure and properties of chimeric small heat shock proteins containing yellow fluorescent protein attached to their c-terminal ends. Cell Stress Chaperones 19:507–518CrossRefPubMedGoogle Scholar
  16. Datskevich PN, Mymrikov EV, Gusev NB (2012a) Utilization of fluorescent chimeras for investigation of heterooligomeric complexes formed by human small heat shock proteins. Biochimie 94:1794–1804CrossRefPubMedGoogle Scholar
  17. Datskevich PN, Mymrikov EV, Sluchanko NN, Shemetov AA, Sudnitsyna MV, Gusev NB (2012b) Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins. Protein Expr Purif 82:45–54CrossRefPubMedGoogle Scholar
  18. de Jong WW, Leunissen JA, Voorter CE (1993) Evolution of the alpha-crystallin/small heat-shock protein family. Mol Biol Evol 10:103–126PubMedGoogle Scholar
  19. de Lau LML, Breteler MMB (2006) Epidemiology of Parkinson’s disease. Lancet Neurol 5:525–535CrossRefPubMedGoogle Scholar
  20. Dorsey ER, Constantinescu R, Thompson JP, Biglan KM, Holloway RG, Kieburtz K, Marshall FJ, Ravina BM, Schifitto G, Siderowf A, Tanner CM (2007) Projected number of people with parkinson disease in the most populous nations, 2005 through 2030. Neurology 68:384–386CrossRefPubMedGoogle Scholar
  21. Fioriti L, Dossena S, Stewart LR, Stewart RS, Harris DA, Forloni G, Chiesa R (2005) Cytosolic prion protein (prp) is not toxic in n2a cells and primary neurons expressing pathogenic prp mutations. J Biol Chem 280:11320–11328CrossRefPubMedGoogle Scholar
  22. Földi I, Tóth AM, Szabó Z, Mózes E, Berkecz R, Datki ZL, Penke B, Janáky T (2013) Proteome-wide study of endoplasmic reticulum stress induced by thapsigargin in n2a neuroblastoma cells. Neurochem Int 62:58–69CrossRefPubMedGoogle Scholar
  23. Gui M-c, Chen B, Yu S-s, Bu B-t (2014) Effects of suppressed autophagy on mitochondrial dynamics and cell cycle of n2a cells. Journal of Huazhong University of Science and Technology [Medical Sciences] 34:157–160CrossRefGoogle Scholar
  24. Guo F, He X-B, Li S, Le W (2016) A central role for phosphorylated p38α in linking proteasome inhibition-induced apoptosis and autophagy. Mol Neurobiol:1–13. doi: 10.1007/s12035-016-0260-1
  25. Hoyer W, Cherny D, Subramaniam V, Jovin TM (2004) Impact of the acidic c-terminal region comprising amino acids 109−140 on α-synuclein aggregation in vitro. Biochemistry 43:16233–16242CrossRefPubMedGoogle Scholar
  26. Jehle S, Rajagopal P, Bardiaux B, Markovic S, Kuhne R, Stout JR, Higman VA, Klevit RE, van Rossum BJ, Oschkinat H (2010) Solid-state nmr and saxs studies provide a structural basis for the activation of alphab-crystallin oligomers. Nat Struct Mol Biol 17:1037–1042CrossRefPubMedPubMedCentralGoogle Scholar
  27. Jehle, S, Vollmar, BS, Bardiaux, B, Dove, KK, Rajagopal, P, Gonen, T, Oschkinat, H and Klevit, RE (2011). N-terminal domain of αb-crystallin provides a conformational switch for multimerization and structural heterogeneity. Proc Natl Acad Sci U S A 108:6409–6414Google Scholar
  28. Juenemann K, Wiemhoefer A, Reits EA (2015) Detection of ubiquitinated huntingtin species in intracellular aggregates. Front Mol Neurosci 8:1CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kampinga H, Hageman J, Vos M, Kubota H, Tanguay R, Bruford E, Cheetham M, Chen B, Hightower L (2009) Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperon 14:105–111CrossRefGoogle Scholar
  30. Kanda S, Bishop JF, Eglitis MA, Yang Y, Mouradian MM (2000) Enhanced vulnerability to oxidative stress by α-synuclein mutations and c-terminal truncation. Neurosci 97:279–284CrossRefGoogle Scholar
  31. Klucken J, Shin Y, Masliah E, Hyman BT, McLean PJ (2004) Hsp70 reduces α-synuclein aggregation and toxicity. J Biol Chem 279:25497–25502CrossRefPubMedGoogle Scholar
  32. Krishnan J, Lemmens R, Robberecht W, Van Den Bosch L (2006) Role of heat shock response and hsp27 in mutant sod1-dependent cell death. Exp Neurol 200:301–310CrossRefPubMedGoogle Scholar
  33. Lelj-Garolla B, Mauk AG (2006) Self-association and chaperone activity of hsp27 are thermally activated. J Biol Chem 281:8169–8174CrossRefPubMedGoogle Scholar
  34. Li W, West N, Colla E, Pletnikova O, Troncoso JC, Marsh L, Dawson TM, Jäkälä P, Hartmann T, Price DL, Lee MK (2005) Aggregation promoting c-terminal truncation of α-synuclein is a normal cellular process and is enhanced by the familial Parkinson’s disease-linked mutations. Proc Natl Acad Sci U S A 102:2162–2167CrossRefPubMedPubMedCentralGoogle Scholar
  35. Massano J, Bhatia KP (2012) Clinical approach to Parkinson’s disease: features, diagnosis, and principles of management. Cold Spring Harb Perspect Med 2:a008870CrossRefPubMedPubMedCentralGoogle Scholar
  36. Matsuzaki M, Hasegawa T, Takeda A, Kikuchi A, Furukawa K, Kato Y, Itoyama Y (2004) Histochemical features of stress-induced aggregates in α-synuclein overexpressing cells. Brain Res 1004:83–90CrossRefPubMedGoogle Scholar
  37. McLean PJ, Kawamata H, Hyman BT (2001) Α-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons. Neurosci 104:901–912CrossRefGoogle Scholar
  38. Nasir I, Linse S, Cabaleiro-Lago C (2015) Fluorescent filter-trap assay for amyloid fibril formation kinetics in complex solutions. ACS Chem Neurosci 6:1436–1444CrossRefPubMedPubMedCentralGoogle Scholar
  39. Ojha J, Masilamoni G, Dunlap D, Udoff RA, Cashikar AG (2011) Sequestration of toxic oligomers by hspb1 as a cytoprotective mechanism. Mol Cell Biol 31:3146–3157CrossRefPubMedPubMedCentralGoogle Scholar
  40. Olshina MA, Angley LM, Ramdzan YM, Tang J, Bailey MF, Hill AF, Hatters DM (2010) Tracking mutant huntingtin aggregation kinetics in cells reveals three major populations that include an invariant oligomer pool. J Biol Chem 285:21807–21816CrossRefPubMedPubMedCentralGoogle Scholar
  41. Ormsby AR, Ramdzan YM, Mok Y-F, Jovanoski KD, Hatters DM (2013) A platform to view huntingtin exon 1 aggregation flux in the cell reveals divergent influences from chaperones hsp40 and hsp70. J Biol Chem 288:37192–37203CrossRefPubMedPubMedCentralGoogle Scholar
  42. Ostrerova-Golts N, Petrucelli L, Hardy J, Lee JM, Farer M, Wolozin B (2000) The a53t α-synuclein mutation increases iron-dependent aggregation and toxicity. J Neurosci 20:6048–6054PubMedGoogle Scholar
  43. Outeiro TF, Klucken J, Strathearn KE, Liu F, Nguyen P, Rochet J-C, Hyman BT, McLean PJ (2006) Small heat shock proteins protect against α-synuclein-induced toxicity and aggregation. Biochem Biophys Res Commun 351:631–638CrossRefPubMedPubMedCentralGoogle Scholar
  44. Paxinou E, Chen Q, Weisse M, Giasson BI, Norris EH, Rueter SM, Trojanowski JQ, Lee VM, Ischiropoulos H (2001) Induction of alpha-synuclein aggregation by intracellular nitrative insult. J Neurosci 21:8053–8061PubMedGoogle Scholar
  45. Ramdzan YM, Polling S, Chia CPZ, Ng IHW, Ormsby AR, Croft NP, Purcell AW, Bogoyevitch MA, Ng DCH, Gleeson PA, Hatters DM (2012) Tracking protein aggregation and mislocalization in cells with flow cytometry. Nat Methods 9:467–470CrossRefPubMedGoogle Scholar
  46. Van Montfort R, Slingsby C, Vierling E (2002) Structure and function of the small heat shock protein/alpha-crystallin family of molecular chaperones. Adv Protein Chem 59:105–156CrossRefGoogle Scholar
  47. Wan OW, Chung KKK (2012) The role of alpha-synuclein oligomerization and aggregation in cellular and animal models of Parkinson’s disease. PLoS One 7:e38545CrossRefPubMedPubMedCentralGoogle Scholar
  48. Waudby CA, Knowles TP, Devlin GL, Skepper JN, Ecroyd H, Carver JA, Welland ME, Christodoulou J, Dobson CM, Meehan S (2010) The interaction of alphaB-crystallin with mature alpha-synuclein amyloid fibrils inhibits their elongation. Biophys J 98:843–851Google Scholar
  49. Yerbury JJ, Stewart EM, Wyatt AR, Wilson MR (2005) Quality control of protein folding in extracellular space. EMBO Rep 6:1131–1136CrossRefPubMedPubMedCentralGoogle Scholar
  50. Zourlidou A, Payne Smith MD, Latchman DS (2004) Hsp27 but not hsp70 has a potent protective effect against α-synuclein-induced cell death in mammalian neuronal cells. J Neurochem 88:1439–1448CrossRefPubMedGoogle Scholar

Copyright information

© Cell Stress Society International 2017

Authors and Affiliations

  1. 1.Illawarra Health and Medical Research InstituteUniversity of WollongongWollongongAustralia
  2. 2.School of Biological SciencesUniversity of WollongongWollongongAustralia

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