Assessing Autophagic Activity and Aggregate Formation of Mutant Huntingtin in Mammalian Cells

  • Eleanna Stamatakou
  • Ye Zhu
  • David C. RubinszteinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1780)


The accumulation of mutant aggregate-prone proteins is a hallmark of the majority of neurodegenerative disorders, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Autophagy, a cytosolic bulk degradation system, is the major clearance pathway for several aggregate-prone proteins, such as mutant huntingtin. The autophagosome-associated protein LC3-II is a specific marker of autophagic flux within cells, whereas aggregate formation of mutant huntingtin represents a good readout for studying autophagy modulation. Here we describe the method of assessing autophagic flux using LC3-II western blotting and substrate clearance by expressing the N-terminal fragment of huntingtin (htt exon 1) containing an expanded polyglutamine tract in mammalian cells.


Autophagy LC3-II Autophagic flux Bafilomycin A1 Huntingtin aggregates 



We thank the Tau consortium (D.C.R.), Wellcome Trust (Principal Research Fellowship to 095317/Z/11/Z), a Wellcome Trust Strategic Grant to Cambridge Institute for Medical Research (100140/Z/12/Z), and NIHR Biomedical Research Unit in Dementia at Addenbrooke’s Hospital, for funding. Ye Zhu is supported by CSC Cambridge Scholarship from Cambridge Trust and China Scholarship Council.


  1. 1.
    Imarisio S, Carmichael J, Korolchuk V et al (2008) Huntington’s disease: from pathology and genetics to potential therapies. Biochem J 412:191–209CrossRefPubMedGoogle Scholar
  2. 2.
    Rubinsztein DC (2002) Lessons from animal models of Huntington’s disease. Trends Genet 18(4):202–209CrossRefPubMedGoogle Scholar
  3. 3.
    Sathasivam K, Neueder A, Gipson TA et al (2013) Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease. Proc Natl Acad Sci U S A 110:2366–2370CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Scherzinger E, Lurz R, Turmaine M, Mangiarini L et al (1997) Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell 90:549–558CrossRefPubMedGoogle Scholar
  5. 5.
    Landles C, Sathasivam K, Weiss A et al (2010) Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem 285:8808–8823CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Zuccato C, Valenza M, Cattaneo E (2010) Molecular mechanisms and potential therapeutical targets in Huntington’s disease. Physiol Rev 90:905–981CrossRefPubMedGoogle Scholar
  7. 7.
    Wang Y, Mandelkow E (2012) Degradation of tau protein by autophagy and proteasomal pathways. Biochem Soc Trans 40:644–652CrossRefPubMedGoogle Scholar
  8. 8.
    Lee MJ, Lee JH, Rubinsztein DC (2013) Tau degradation: the ubiquitin-proteasome system versus the autophagy-lysosome system. Prog Neurobiol 105:49–59CrossRefPubMedGoogle Scholar
  9. 9.
    Ravikumar B, Duden R, Rubinsztein DC (2002) Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum Mol Genet 11:1107–1117CrossRefPubMedGoogle Scholar
  10. 10.
    Ravikumar B, Vacher C, Berger Z et al (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36:585–595CrossRefPubMedGoogle Scholar
  11. 11.
    Sarkar S, Floto RA, Berger Z et al (2005) Lithium induces autophagy by inhibiting inositol monophosphatase. J Cell Biol 170:1101–1111CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Sarkar S, Ravikumar B, Floto RA et al (2009) Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell Death Differ 16:46–56CrossRefPubMedGoogle Scholar
  13. 13.
    Webb JL, Ravikumar B, Atkins J et al (2003) Alpha-synuclein is degraded by both autophagy and the proteasome. J Biol Chem 278:25009–25013CrossRefPubMedGoogle Scholar
  14. 14.
    Kabeya Y, Mizushima N, Ueno T et al (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kabeya Y, Mizushima N, Yamamoto A et al (2004) LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci 117:2805–2812CrossRefPubMedGoogle Scholar
  16. 16.
    Klionsky DJ, Elazar Z, Seglen PO et al (2008) Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy 4:849–850CrossRefPubMedGoogle Scholar
  17. 17.
    Narain Y, Wyttenbach A, Rankin J et al (1999) A molecular investigation of true dominance in Huntington’s disease. J Med Genet 36:739–746CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Li XJ, Li H, Li S (2010) Clearance of mutant huntingtin. Autophagy 6(5):663–664. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Ortega Z, Lucas JJ (2014) Ubiquitin-proteasome system involvement in Huntington’s disease. Front Mol Neurosci 7:77CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bento CF, Ashkenazi A, Jimenez-Sanchez M et al (2016) The Parkinson’s disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway. Nat Commun 7:11803CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Klionsky DJ, Abdelmohsen K, Abe A et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1–222CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kaizuka T, Morishita H, Hama Y et al (2016) An autophagic flux probe that releases an internal control. Mol Cell 64:835–849CrossRefPubMedGoogle Scholar
  23. 23.
    Korolchuk VI, Mansilla A, Menzies FM et al (2009) Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates. Mol Cell 33:517–527CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Eleanna Stamatakou
    • 1
  • Ye Zhu
    • 1
  • David C. Rubinsztein
    • 1
    Email author
  1. 1.Department of Medical Genetics, Cambridge Institute for Medical ResearchUniversity of CambridgeCambridgeUK

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