Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein

Nature Biotechnology volume 28, pages 256–263 (2010)Cite this article

Subjects

Abstract

Huntington's Disease (HD) is a dominantly inherited pathology caused by the accumulation of mutant huntingtin protein (HTT) containing an expanded polyglutamine (polyQ) tract. As the polyglutamine binding peptide 1 (QBP1) is known to bind an expanded polyQ tract but not the polyQ motif found in normal HTT, we selectively targeted mutant HTT for degradation by expressing a fusion molecule comprising two copies of QBP1 and copies of two different heat shock cognate protein 70 (HSC70)–binding motifs in cellular and mouse models of HD. Chaperone-mediated autophagy contributed to the specific degradation of mutant HTT in cultured cells expressing the construct. Intrastriatal delivery of a virus expressing the fusion molecule ameliorated the disease phenotype in the R6/2 mouse model of HD. Similar adaptor molecules comprising HSC70–binding motifs fused to an appropriate structure-specific binding agent(s) may have therapeutic potential for treating diseases caused by misfolded proteins other than those with expanded polyQ tracts.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Inclusion of two HSC70bm sequences within the protein carrying expanded polyQ tracts decreases its aggregation.
Figure 2: RHQ inhibits polyQ aggregation more efficiently than RQ.
Figure 3: RHQ enhances the degradation of tNHTT-60Q-EGFP.
Figure 4: RHQ induces the degradation of tNHTT with expanded polyQ tracts by recruiting the CMA machinery.
Figure 5: Detection of HTT levels and the effect of the rAAV-HQ on inclusion formation in R6/2 mouse brains.
Figure 6: Effect of rAAV-HQ on the HD phenotype in R6/2 mice.

Similar content being viewed by others

References

  1. Zoghbi, H.Y. & Orr, H.T. Glutamine repeats and neurodegeneration. Annu. Rev. Neurosci. 23, 217–247 (2000).

    Article  CAS  Google Scholar 

  2. DiFiglia, M. et al. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277, 1990–1993 (1997).

    Article  CAS  Google Scholar 

  3. Davies, S.W. et al. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90, 537–548 (1997).

    Article  CAS  Google Scholar 

  4. Perutz, M.F., Pope, B.J., Owen, D., Wanker, E.E. & Scherzinger, E. Aggregation of proteins with expanded glutamine and alanine repeats of the glutamine-rich and asparagine-rich domains of Sup35 and of the amyloid beta-peptide of amyloid plaques. Proc. Natl. Acad. Sci. USA 99, 5596–5600 (2002).

    Article  CAS  Google Scholar 

  5. Tanaka, M. et al. Expansion of polyglutamine induces the formation of quasi-aggregate in the early stage of protein fibrillization. J. Biol. Chem. 278, 34717–34724 (2003).

    Article  CAS  Google Scholar 

  6. Dunah, A.W. et al. Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease. Science 296, 2238–2243 (2002).

    Article  CAS  Google Scholar 

  7. Nucifora, F.C. Jr. et al. Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity. Science 291, 2423–2428 (2001).

    Article  CAS  Google Scholar 

  8. Yamanaka, T. et al. Mutant Huntingtin reduces HSP70 expression through the sequestration of NF-Y transcription factor. EMBO J. 27, 827–839 (2008).

    Article  CAS  Google Scholar 

  9. Bauer, P.O. & Nukina, N. The pathogenic mechanisms of polyglutamine diseases and current therapeutic strategies. J. Neurochem. 110, 1737–1765 (2009).

    Article  CAS  Google Scholar 

  10. Yamamoto, A., Lucas, J.J. & Hen, R. Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease. Cell 101, 57–66 (2000).

    Article  CAS  Google Scholar 

  11. Wang, Y.L. et al. Clinico-pathological rescue of a model mouse of Huntington's disease by siRNA. Neurosci. Res. 53, 241–249 (2005).

    Article  CAS  Google Scholar 

  12. DiFiglia, M. et al. Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proc. Natl. Acad. Sci. USA. 104, 17204–17209 (2007).

    Article  CAS  Google Scholar 

  13. Harper, S.Q. et al. RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc. Natl. Acad. Sci. USA. 102, 5820–5825 (2005).

    Article  CAS  Google Scholar 

  14. Rodriguez-Lebron, E., Denovan-Wright, E.M., Nash, K., Lewin, A.S. & Mandel, R.J. Intrastriatal rAAV-mediated delivery of anti-huntingtin shRNAs induces partial reversal of disease progression in R6/1 Huntington's disease transgenic mice. Mol. Ther. 12, 618–633 (2005).

    Article  CAS  Google Scholar 

  15. Ravikumar, B. et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36, 585–595 (2004).

    Article  CAS  Google Scholar 

  16. Sarkar, S. et al. Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nat. Chem. Biol. 3, 331–338 (2007).

    Article  CAS  Google Scholar 

  17. Wong, H.K. et al. Blocking acid-sensing ion channel 1 alleviates Huntington's disease pathology via an ubiquitin-proteasome system-dependent mechanism. Hum. Mol. Genet. 17, 3223–3235 (2008).

    Article  CAS  Google Scholar 

  18. Bauer, P.O. et al. Inhibition of rho kinases enhances the degradation of mutant huntingtin. J. Biol. Chem. 284, 13153–13164 (2009).

    Article  CAS  Google Scholar 

  19. Mizushima, N., Ohsumi, Y. & Yoshimori, T. Autophagosome formation in mammalian cells. Cell. Struct. Funct. 27, 421–429 (2002).

    Article  Google Scholar 

  20. Cuervo, A.M. & Dice, J.F. A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273, 501–503 (1996).

    Article  CAS  Google Scholar 

  21. Agarraberes, F.A. & Dice, J.F. A molecular chaperone complex at the lysosomal membrane is required for protein translocation. J. Cell Sci. 114, 2491–2499 (2001).

    CAS  PubMed  Google Scholar 

  22. Nagai, Y. et al. Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening. J. Biol. Chem. 275, 10437–10442 (2000).

    Article  CAS  Google Scholar 

  23. Cuervo, A.M., Stefanis, L., Fredenburg, R., Lansbury, P.T. & Sulzer, D. Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 305, 1292–1295 (2004).

    Article  CAS  Google Scholar 

  24. Cuervo, A.M., Mann, L., Bonten, E.J., d'Azzo, A. & Dice, J.F Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor. EMBO J. 22, 47–59 (2003).

    Article  CAS  Google Scholar 

  25. Nagai, Y. et al. Prevention of polyglutamine oligomerization and neurodegeneration by the peptide inhibitor QBP1 in Drosophila. Hum. Mol. Genet. 12, 1253–1259 (2003).

    Article  CAS  Google Scholar 

  26. Nagai, Y. et al. A toxic monomeric conformer of the polyglutamine protein. Nat. Struct. Mol. Biol. 14, 332–340 (2007).

    Article  CAS  Google Scholar 

  27. Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N. & Miyawaki, A. Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6, 233–238 (2005).

    Article  CAS  Google Scholar 

  28. Rodriguez-Enriquez, S., Kim, I., Currin, R.T. & Lemasters, J.J. Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes. Autophagy 2, 39–46 (2006).

    Article  CAS  Google Scholar 

  29. Weiss, A. et al. Sensitive biochemical aggregate detection reveals aggregation onset before symptom development in cellular and murine models of Huntington's disease. J. Neurochem. 104, 846–858 (2008).

    CAS  PubMed  Google Scholar 

  30. Kotliarova, S. et al. Decreased expression of hypothalamic neuropeptides in Huntington disease transgenic mice with expanded polyglutamine-EGFP fluorescent aggregates. J. Neurochem. 93, 641–653 (2005).

    Article  CAS  Google Scholar 

  31. Beal, M.F. & Ferrante, R.J. Experimental therapeutics in transgenic mouse models of Huntington's disease. Nat. Rev. Neurosci. 5, 373–384 (2004).

    Article  CAS  Google Scholar 

  32. Li, J.Y., Popovic, N. & Brundin, P. The use of the R6 transgenic mouse models of Huntington's Disease in attempts to develop novel therapeutic strategies. NeuroRx 2, 447–464 (2005).

    Article  Google Scholar 

  33. Tanaka, M. et al. Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. Nat. Med. 10, 148–154 (2004).

    Article  CAS  Google Scholar 

  34. Sanchez, I., Mahlke, C. & Yuan, J. Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders. Nature 421, 373–379 (2003).

    Article  CAS  Google Scholar 

  35. Karpuj, M.V. et al. Transglutaminase aggregates huntingtin into nonamyloidogenic polymers, and its enzymatic activity increases in Huntington's disease brain nuclei. Proc. Natl. Acad. Sci. USA 96, 7388–7393 (1999).

    Article  CAS  Google Scholar 

  36. Karpuj, M.V. et al. Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nat. Med. 8, 143–149 (2002).

    Article  CAS  Google Scholar 

  37. Dedeoglu, A. et al. Therapeutic effects of cystamine in a murine model of Huntington's disease. J. Neurosci. 22, 8942–8950 (2002).

    Article  CAS  Google Scholar 

  38. Ferrante, R.J. et al. Histone deacetylase inhibition by sodium butyrate chemotherapy ameliorates the neurodegenerative phenotype in Huntington's disease mice. J. Neurosci. 23, 9418–9427 (2003).

    Article  CAS  Google Scholar 

  39. Smith, K.M. et al. Dose ranging and efficacy study of high-dose coenzyme Q10 formulations in Huntington's disease mice. Biochim. Biophys. Acta 1762, 616–626 (2006).

    Article  CAS  Google Scholar 

  40. Nguyen, T., Hamby, A. & Massa, S.M. Clioquinol down-regulates mutant huntingtin expression in vitro and mitigates pathology in a Huntington's disease mouse model. Proc. Natl. Acad. Sci. USA 102, 11840–11845 (2005).

    Article  CAS  Google Scholar 

  41. Ona, V.O. et al. Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease. Nature 399, 263–267 (1999).

    Article  CAS  Google Scholar 

  42. Jin, K. et al. FGF-2 promotes neurogenesis and neuroprotection and prolongs survival in a transgenic mouse model of Huntington's disease. Proc. Natl. Acad. Sci. USA 102, 18189–18194 (2005).

    Article  CAS  Google Scholar 

  43. Peng, Q. et al. The antidepressant sertraline improves the phenotype, promotes neurogenesis and increases BDNF levels in the R6/2 Huntington's disease mouse model. Exp. Neurol. 210, 154–163 (2008).

    Article  CAS  Google Scholar 

  44. Ferrante, R.J. et al. Chemotherapy for the brain: the antitumor antibiotic mithramycin prolongs survival in a mouse model of Huntington's disease. J. Neurosci. 24, 10335–10342 (2004).

    Article  CAS  Google Scholar 

  45. Ryu, H. et al. ESET/SETDB1 gene expression and histone H3 (K9) trimethylation in Huntington's disease. Proc. Natl. Acad. Sci. USA 103, 19176–19181 (2006).

    Article  CAS  Google Scholar 

  46. Popiel, H.A., Nagai, Y., Fujikake, N. & Toda, T. Delivery of the aggregate inhibitor peptide QBP1 into the mouse brain using PTDs and its therapeutic effect on polyglutamine disease mice. Neurosci. Lett. 449, 87–92 (2009).

    Article  CAS  Google Scholar 

  47. Southwell, A.L., Ko, J. & Patterson, P.H. Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease. J. Neurosci. 29, 13589–13602 (2009).

    Article  CAS  Google Scholar 

  48. Harper, S.Q. et al. RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc. Natl. Acad. Sci. USA 102, 5820–5825 (2005).

    Article  CAS  Google Scholar 

  49. Rodriguez-Lebron, E., Denovan-Wright, E.M., Nash, K., Lewin, A.S. & Mandel, R.J. Intrastriatal rAAV-mediated delivery of anti-huntingtin shRNAs induces partial reversal of disease progression in R6/1 Huntington's disease transgenic mice. Mol. Ther. 12, 618–633 (2005).

    Article  CAS  Google Scholar 

  50. DiFiglia, M. et al. Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proc. Natl. Acad. Sci. USA 104, 17204–17209 (2007).

    Article  CAS  Google Scholar 

  51. Huang, B. et al. High-capacity adenoviral vector-mediated reduction of huntingtin aggregate load in vitro and in vivo. Hum. Gene Ther. 18, 303–311 (2007).

    Article  CAS  Google Scholar 

  52. Zhang, Y., Engelman, J. & Friedlander, R.M. Allele-specific silencing of mutant Huntington's disease gene. J. Neurochem. 108, 82–90 (2009).

    Article  CAS  Google Scholar 

  53. Cho, S.R. et al. Induction of neostriatal neurogenesis slows disease progression in a transgenic murine model of Huntington disease. J. Clin. Invest. 117, 2889–2902 (2007).

    Article  CAS  Google Scholar 

  54. Southwell, A.L. et al. Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity. J. Neurosci. 28, 9013–9020 (2008).

    Article  CAS  Google Scholar 

  55. Emadi, S., Barkhordarian, H., Wang, M.S., Schulz, P. & Sierks, M.R. Isolation of a human single chain antibody fragment against oligomeric alpha-synuclein that inhibits aggregation and prevents alpha-synuclein-induced toxicity. J. Mol. Biol. 368, 1132–1144 (2007).

    Article  CAS  Google Scholar 

  56. Lynch, S.M., Zhou, C. & Messer, A. An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity. J. Mol. Biol. 377, 136–147 (2008).

    Article  CAS  Google Scholar 

  57. Wang, G.H. et al. Caspase activation during apoptotic cell death induced by expanded polyglutamine in N2a cells. Neuroreport 10, 2435–2438 (1999).

    Article  CAS  Google Scholar 

  58. Wang, G.H., Sawai, N., Kotliarova, S., Kanazawa, I. & Nukina, N. Ataxin-3, the MJD1 gene product, interacts with the two human homologs of yeast DNA repair protein RAD23, HHR23A and HHR23B. Hum. Mol. Genet. 9, 1795–1803 (2000).

    Article  CAS  Google Scholar 

  59. Peters, M.F. & Ross, C.A. Preparation of human cDNas encoding expanded polyglutamine repeats. Neurosci. Lett. 275, 129–132 (1999).

    Article  CAS  Google Scholar 

  60. Zemskov, E.A. et al. Pro-apoptotic protein kinase C delta is associated with intranuclear inclusions in a transgenic model of Huntington's disease. J. Neurochem. 87, 395–406 (2003).

    Article  CAS  Google Scholar 

  61. Madden, E.A., Wirt, J.B. & Storrie, B. Purification and characterization of lysosomes from Chinese hamster ovary cells. Arch. Biochem. Biophys. 257, 27–38 (1987).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (Research on Pathomechanisms of Brain Disorders) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17025044) and by a Grant-in-Aid for the Research on Measures for Intractable Diseases from the Ministry of Health, Welfare and Labor, Japan. This work was supported partly by a grant from the Japan Society for the Promotion of Science (JSPS). P.O.B. was a JSPS postdoctoral fellow. We thank D.W. Chapmon for stylistic revision of the manuscript. Monomeric KikGR (mKikGR) was a kind gift of A. Miyawaki, RIKEN BSI Japan.

Author information

Authors and Affiliations

  1. Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan.,

    Peter O Bauer, Anand Goswami, Hon Kit Wong, Misako Okuno, Masaru Kurosawa, Mizuki Yamada, Haruko Miyazaki, Gen Matsumoto, Yoshihiro Kino & Nobuyuki Nukina

  2. Department of Degenerative Neurological Diseases, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.,

    Yoshitaka Nagai

Authors
  1. Peter O Bauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anand Goswami
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hon Kit Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Misako Okuno
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masaru Kurosawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mizuki Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Haruko Miyazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gen Matsumoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yoshihiro Kino
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yoshitaka Nagai
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Nobuyuki Nukina
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.O.B. raised the hypothesis and designed the experiments. Experimental work was performed by P.O.B., A.G., H.K.W., H.M., M.O., M.K. and M.Y. The tNHTT-62Q-kikGR Neuro2a cell line system was prepared by G.M. The EGFP-AR constructs were prepared by Y.K. The manuscript was written by P.O.B. and N.N. Y.N. provided some information about treatment using QBP1. N.N. supervised the project. All authors discussed results and commented on the manuscript.

Corresponding author

Correspondence to Nobuyuki Nukina.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–13 and Supplementary Table 1 (PDF 13292 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bauer, P., Goswami, A., Wong, H. et al. Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein. Nat Biotechnol 28, 256–263 (2010). https://doi.org/10.1038/nbt.1608

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1608

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing