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Identifying polyglutamine protein species in situ that best predict neurodegeneration

Nature Chemical Biology volume 7, pages 925–934 (2011)Cite this article

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A Corrigendum to this article was published on 15 February 2012

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Abstract

Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

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Figure 1: The monoclonal antibody 3B5H10 binds low-molecular-weight disease-associated polyQ expansions.
Figure 2: Quantitative binding of Htt-specific antibodies 3B5H10, EM48, MW1 or MW7 to Httex1 is distinguishable and predictable.
Figure 3: New methodology distinguishes which of several simultaneously existing in situ epitopes of diffuse Httex1 best predicts neurotoxicity.
Figure 4: A species of Htt recognized by 3B5H10 best predicts striatal neurodegeneration.
Figure 5: 3B5H10 does not recognize large oligomers of mHtt.
Figure 6: Epitope recognized by 3B5H10 is preferentially present in low-molecular-weight Htt species.
Figure 7: The 'linear lattice' versus 'emergent conformation' hypotheses for expanded polyQ conformation.

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  • 02 December 2011

    In the version of this article initially published, lanes 6 and 8 in Figure 6b had identical blot images. The blot now shown in lane 8 has been confirmed to come from the same gel as those in the other lanes and has been used to replace the incorrect blot image in the HTML and PDF versions of the article.

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Acknowledgements

We thank A. Kazantzev, D. Housman and the Hereditary Disease Foundation (HDF) for pcDNA3.1-Httex1-(Q46, Q97)-GFP plasmids. We also thank R. Truant for eGFP-full-length Htt-β-galactosidase (Q17, Q138) plasmids, M. Diamond for the HA-AR (Q25, Q65) plasmids, J. Burke and CHDI, Inc. for the GST-atrophin-1 (Q19, Q81) plasmids, R. Kopito for Httex1-CFP (Q25, Q97) and Httex1-YFP (Q25, Q97) plasmids, D. Devys for GST-Htt-171 (Q66, Q142) plasmids, R. Pittman for Myc-ataxin-3 (Q27 Q78) plasmids, O. Onodera for Httex1-mCFP (Q17, Q58) and Httex1-mYFP (Q17, Q58) plasmids, P. Bjorkman for the thio-Httex1-Q39-His6 plasmid and P. Daugherty for mammalian codon-optimized CyPet and YPet plasmids. We thank P. Patterson for the monoclonal antibodies MW1, MW7 and MW8 and C. Glabe for the oligomer-specific polyclonal antibody. We thank members of the Finkbeiner lab for useful discussions, S. Ordway and G. Howard for editorial assistance, K. Nelson for administrative assistance and M. Sutherland for her interest and support. Primary support for this work was provided by the Lieberman Award of the HDF and the US National Institute of Neurological Disease and Stroke (S.F.). Additional support was provided by the National Institute of Aging, the High Q Foundation, the Huntington's Disease Society of America, the National Center for Research Resources, the Taube-Koret Center for Huntington's Disease Research, the Hellman Family Foundation Program for Alzheimer's Disease Research and the J. David Gladstone Institutes (S.F.). M.A. and J.M. are supported by the Hillblom Foundation. J.M. and S.M. are supported by the US National Institutes of Health (NIH)–National Institute of General Medical Sciences University of California San Fransisco Medical Scientist Training Program. J.M is supported by a fellowship from the Achievement Rewards for College Scientists Foundation. D.H. is supported by a postdoctoral fellowship from the John Douglas French Alzheimer's Foundation. J.L. and A.O. are supported by the HDF. E.J.M. is supported by a grant from the NIH. The animal care facility was partly supported by an NIH Extramural Research Facilities Improvement Project. The electron microscopy core (E.M.) is supported by a grant from the National Institute of Neurological Disease and Stroke.

Author information

Author notes

  1. Montserrat Arrasate, Elizabeth Brooks, Clare Peters Libeu, Justin Legleiter, Danny Hatters, Kenneth Cheung, Preethi Krishnan, Benjamin A Shaby & Michelle Gray

    Present address: Current addresses: Division of Neuroscience, Center for Applied Medical Research, University of Navarra, Pamplona, Spain (M.A.); Neuroscience Program, University of California School of Medicine, Los Angeles, California, USA (E.B.); Buck Institute for Research on Aging, Novato, California, USA (C.P.L.); The C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia, USA (J.L.); Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia (D.H.); University of California, Davis School of Medicine, Sacramento, California, USA (K.C.); College of Pharmacy, University of Minnesota, Minneapolis, Minnesota, USA (P.K.); Department of Statistical Science, Duke University, Durham, North Carolina, USA (B.A.S.); Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, USA (M.G.).,

  2. Jason Miller and Montserrat Arrasate: These authors contributed equally to this work.

Authors and Affiliations

  1. Gladstone Institute of Neurological Disease, San Francisco, California, USA

    Jason Miller, Montserrat Arrasate, Elizabeth Brooks, Clare Peters Libeu, Justin Legleiter, Danny Hatters, Jessica Curtis, Kenneth Cheung, Preethi Krishnan, Siddhartha Mitra, Kartika Widjaja, Gregor P Lotz, Yvonne Newhouse, Paul J Muchowski, Karl H Weisgraber & Steven Finkbeiner

  2. Taube Koret Center for Huntington's Disease Research, San Francisco, California, USA

    Jason Miller, Montserrat Arrasate, Elizabeth Brooks, Clare Peters Libeu, Justin Legleiter, Danny Hatters, Jessica Curtis, Kenneth Cheung, Preethi Krishnan, Siddhartha Mitra, Kartika Widjaja, Gregor P Lotz, Yvonne Newhouse, Paul J Muchowski, Karl H Weisgraber & Steven Finkbeiner

  3. Hellman Family Foundation Program in Alzheimer's Disease Research, San Francisco, California, USA

    Jason Miller, Montserrat Arrasate, Elizabeth Brooks, Clare Peters Libeu, Justin Legleiter, Danny Hatters, Jessica Curtis, Kenneth Cheung, Preethi Krishnan, Siddhartha Mitra, Kartika Widjaja, Gregor P Lotz, Yvonne Newhouse, Paul J Muchowski, Karl H Weisgraber & Steven Finkbeiner

  4. Chemistry and Chemical Biology Program, University of California, San Francisco, California, USA

    Jason Miller

  5. Medical Scientist Training Program, University of California, San Francisco, California, USA

    Jason Miller & Siddhartha Mitra

  6. Neuroscience Program, University of California, San Francisco, California, USA

    Montserrat Arrasate, Elizabeth Brooks, Jessica Curtis, Kenneth Cheung, Preethi Krishnan, Kartika Widjaja, Paul J Muchowski & Steven Finkbeiner

  7. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA

    Clare Peters Libeu, Danny Hatters, Yvonne Newhouse & Karl H Weisgraber

  8. Biomedical Sciences Program, University of California, San Francisco, California, USA

    Siddhartha Mitra, Paul J Muchowski & Steven Finkbeiner

  9. Department of Statistical Science, Cornell University, Ithaca, New York, USA

    Benjamin A Shaby

  10. Department of Biological Chemistry, University of California, Irvine, California, USA

    Emily J Mitchell & Leslie M Thompson

  11. Department of Psychiatry and Human Behavior, University of California, Irvine, California, USA

    Emily J Mitchell & Leslie M Thompson

  12. Department of Medicine, University of Tennessee Graduate School of Medicine, Knoxville, Tennessee, USA

    Alex Osmand

  13. Department of Psychiatry and Biobehavioral Sciences, Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles, California, USA

    Michelle Gray & X William Yang

  14. Bio-Rad Laboratories, Fremont, California, USA

    Vanitha Thulasiramin

  15. Institute Curie, Orsay, France

    Frédéric Saudou

  16. Centre National de la Recherche Scientifique, Unité Mixte de Recherche 3306, Orsay, France

    Frédéric Saudou

  17. Institut National de la Santé et de la Recherche Médicale U1005, Orsay, France

    Frédéric Saudou

  18. Division of Biostatistics, University of California, San Francisco, California, USA

    Mark Segal

  19. Department of Neuroscience, University of California, San Diego, California, USA

    Eliezer Masliah

  20. Department of Pathology, University of California, San Diego, California, USA

    Eliezer Masliah

  21. Department of Neurobiology and Behavior, University of California, Irvine, California, USA

    Leslie M Thompson

  22. Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA

    Paul J Muchowski

  23. Department of Neurology, University of California, San Francisco, California, USA

    Paul J Muchowski & Steven Finkbeiner

  24. Department of Pathology, University of California, San Francisco, California, USA

    Karl H Weisgraber

  25. Department of Physiology, University of California, San Francisco, California, USA

    Steven Finkbeiner

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Contributions

J.M., M.A. and S.F. wrote the manuscript with analytic contributions from S.M. J.M. coordinated data from all authors and performed all immunocytochemistry, FRET, crosslinking and anti-oligomer dot-blot experiments. M.A. optimized 3B5H10 staining conditions and performed all longitudinal survival experiments and the anti-oligomer staining in neurons. J.M. and M.A. cultured neurons for all experiments. J.M., M.A. and M.S. performed initial survival statistics analysis. B.A.S. and J.M. developed final Bayesian survival statistics analysis. E.B., J.C., F.S. and S.F. initiated immunizations and screened hybridomas leading to the identification of 3B5H10. E.B. performed most 3B5H10 western blots and slot blots. P.K., Y.N., K.W., K.C., J.C. and C.P.-L. purified protein, produced Fab, performed size-exclusion experiments and were responsible for some western blots. C.P.-L. and P.K. performed the dynamic light scattering. J.L. performed all atomic force microscopy. D.H. performed all analytical ultracentrifugation. G.P.L. provided advice and assistance for crosslinking and dot-blot experiments. E.J.M. performed agarose gel electrophoresis. A.O. and M.G. performed immunohistochemistry on mouse brains. V.T. performed the synthetic polyQ-peptide binding experiment. E.M. performed electron microscopy. X.W.Y. supervised BACHD immunohistochemistry. L.M.T. supervised agarose gel electrophoresis experiments. P.J.M. supervised AFM, crosslinking and dot blot experiments. K.H.W. supervised aspects of the protein and Fab production as well as size-exclusion chromatography and ultracentrifugation experiments. S.F. supervised the entire project.

Corresponding author

Correspondence to Steven Finkbeiner.

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Competing interests

No research support for this work was provided by any organization that stands to gain or lose financially through this publication. V.T. was employed by Ciphergen, Inc., prior to its acquisition by Bio-Rad, at the time the research was performed. S.F. has served and is expected to continue to serve as an ad hoc paid consultant in the area of neurodegenerative disease therapeutics. A patent covering a series of antibodies, including some (such as 3B5H10 and 4H7H7) used in this study, was issued to the University of California, San Francisco; however, no compensation has been received by any of the authors from the licensing of these antibodies.

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Miller, J., Arrasate, M., Brooks, E. et al. Identifying polyglutamine protein species in situ that best predict neurodegeneration. Nat Chem Biol 7, 925–934 (2011). https://doi.org/10.1038/nchembio.694

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