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Tandem Repeats in Genes, Proteins, and Disease pp 1–20Cite as

Longitudinal Imaging and Analysis of Neurons Expressing Polyglutamine-Expanded Proteins

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1017))

Abstract

Misfolded proteins have been implicated in most of the major neurodegenerative diseases, and identifying drugs and pathways that protect neurons from the toxicity of misfolded proteins is of paramount importance. We invented a form of automated imaging and analysis called robotic microscopy that is well suited to the study of neurodegeneration. It enables the monitoring of large cohorts of individual neurons over their lifetimes as they undergo neurodegeneration. With automated analysis, multiple endpoints in neurons can be measured, including survival. Statistical approaches, typically reserved for engineering and clinical medicine, can be applied to these data in an unbiased fashion to discover whether factors contribute positively or negatively to neuronal fate and to quantify the importance of their contribution. Ultimately, multivariate dynamic models can be constructed from these data, which can provide a systems-level understanding of the neurodegenerative disease process and guide the rationale for the development of therapies.

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References

  1. Saudou F, Finkbeiner S, Devys D, Greenberg ME (1998) Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell 95:55–66

    Article  PubMed  CAS  Google Scholar 

  2. Miller J, Arrasate M, Shaby BA, Mitra S, Masliah E, Finkbeiner S (2010) Quantitative relationships between huntingtin levels, polyglutamine length, inclusion body formation, and neuronal death provide novel insight into huntington’s disease molecular pathogenesis. J Neurosci 30:10541–10550

    Article  PubMed  CAS  Google Scholar 

  3. Arrasate M, Finkbeiner S (2005) Automated microscope system for determining factors that predict neuronal fate. Proc Natl Acad Sci USA 102:3840–3845

    Article  PubMed  CAS  Google Scholar 

  4. Tsvetkov AS, Miller J, Arrasate M, Wong JS, Pleiss MA, Finkbeiner S (2010) A small-molecule scaffold induces autophagy in primary neurons and protects against toxicity in a Huntington disease model. Proc Natl Acad Sci USA 107:16982–16987

    Article  PubMed  CAS  Google Scholar 

  5. Consortium, H. D. i (2012) Induced pluripotent stem cells from patients with Huntington’s disease show CAG-repeat-expansion-associated phenotypes. Cell Stem Cell 11:264–278

    Article  Google Scholar 

  6. Gutekunst CA, Li SH, Yi H, Mulroy JS, Kuemmerle S, Jones R, Rye D, Ferrante RJ, Hersch SM, Li XJ (1999) Nuclear and neuropil aggregates in Huntington’s disease: relationship to neuropathology. J Neurosci 19:2522–2534

    PubMed  CAS  Google Scholar 

  7. Schilling G, Savonenko AV, Klevytska A, Morton JL, Tucker SM, Poirier M, Gale A, Chan N, Gonzales V, Slunt HH, Coonfield ML, Jenkins NA, Copeland NG, Ross CA, Borchelt DR (2004) Nuclear-targeting of mutant huntingtin fragments produces Huntington’s disease-like phenotypes in transgenic mice. Hum Mol Genet 13:1599–1610

    Article  PubMed  CAS  Google Scholar 

  8. Benn CL, Landles C, Li H, Strand AD, Woodman B, Sathasivam K, Li SH, Ghazi-Noori S, Hockly E, Faruque SM, Cha JH, Sharpe PT, Olson JM, Li XJ, Bates GP (2005) Contribution of nuclear and extranuclear polyQ to neurological phenotypes in mouse models of Huntington’s disease. Hum Mol Genet 14:3065–3078

    Article  PubMed  CAS  Google Scholar 

  9. Zala D, Bensadoun JC, Pereira de Almeida L, Leavitt BR, Gutekunst CA, Aebischer P, Hayden MR, Deglon N (2004) Long-term lentiviral-mediated expression of ciliary neurotrophic factor in the striatum of Huntington’s disease transgenic mice. Exp Neurol 185:26–35

    Article  PubMed  CAS  Google Scholar 

  10. Kuemmerle S, Gutekunst CA, Klein AM, Li XJ, Li SH, Beal MF, Hersch SM, Ferrante RJ (1999) Huntington aggregates may not predict neuronal death in Huntington’s disease. Ann Neurol 46:842–849

    Article  PubMed  CAS  Google Scholar 

  11. Slow EJ, Graham RK, Osmand AP, Devon RS, Lu G, Deng Y, Pearson J, Vaid K, Bissada N, Wetzel R, Leavitt BR, Hayden MR (2005) Absence of behavioral abnormalities and neurodegeneration in vivo despite widespread neuronal huntingtin inclusions. Proc Natl Acad Sci USA 102:11402–11407

    Article  PubMed  CAS  Google Scholar 

  12. Warby SC, Chan EY, Metzler M, Gan L, Singaraja RR, Crocker SF, Robertson HA, Hayden MR (2005) Huntingtin phosphorylation on serine 421 is significantly reduced in the striatum and by polyglutamine expansion in vivo. Hum Mol Genet 14:1569–1577

    Article  PubMed  CAS  Google Scholar 

  13. Graham RK, Slow EJ, Deng Y, Bissada N, Lu G, Pearson J, Shehadeh J, Leavitt BR, Raymond LA, Hayden MR (2006) Levels of mutant huntingtin influence the phenotypic severity of Huntington disease in YAC128 mouse models. Neurobiol Dis 21:444–455

    Article  PubMed  CAS  Google Scholar 

  14. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S (2004) Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431:805–810

    Article  PubMed  CAS  Google Scholar 

  15. Mitra S, Tsvetkov AS, Finkbeiner S (2009) Single neuron ubiquitin-proteasome dynamics accompanying inclusion body formation in huntington disease. J Biol Chem 284:4398–4403

    Article  PubMed  CAS  Google Scholar 

  16. Miller J, Arrasate M, Brooks E, Libeu CP, Legleiter J, Hatters D, Curtis J, Cheung K, Krishnan P, Mitra S, Widjaja K, Shaby BA, Lotz GP, Newhouse Y, Mitchell EJ, Osmand A, Gray M, Thulasiramin V, Saudou F, Segal M, Yang XW, Masliah E, Thompson LM, Muchowski PJ, Weisgraber KH, Finkbeiner S (2011) Identifying polyglutamine protein species in situ that best predict neurodegeneration. Nat Chem Biol 7:925–934

    Article  PubMed  CAS  Google Scholar 

  17. Ortega Z, Diaz-Hernandez M, Maynard CJ, Hernandez F, Dantuma NP, Lucas JJ (2010) Acute polyglutamine expression in inducible mouse model unravels ubiquitin/proteasome system impairment and permanent recovery attributable to aggregate formation. J Neurosci 30:3675–3688

    Article  PubMed  CAS  Google Scholar 

  18. Lang P, Yeow K, Nichols A, Scheer A (2006) Cellular imaging in drug discovery. Nat Rev Drug Discov 5:343–356

    Article  PubMed  CAS  Google Scholar 

  19. Sharma P, Ando DM, Daub A, Kaye JA, Finkbeiner S (2012) High-throughput screening in primary neurons. Methods Enzymol 506:331–360

    Article  PubMed  CAS  Google Scholar 

  20. Barmada SJ, Skibinski G, Korb E, Rao EJ, Wu JY, Finkbeiner S (2010) Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. J Neurosci 30:639–649

    Article  PubMed  CAS  Google Scholar 

  21. Nakamura K, Nemani VM, Azarbal F, Skibinski G, Levy JM, Egami K, Munishkina L, Zhang J, Gardner B, Wakabayashi J, Sesaki H, Cheng Y, Finkbeiner S, Nussbaum RL, Masliah E, Edwards RH (2011) Direct membrane association drives mitochondrial fission by the Parkinson disease-associated protein alpha-synuclein. J Biol Chem 286:20710–20726

    Article  PubMed  CAS  Google Scholar 

  22. Glory E, Murphy RF (2007) Automated subcellular location determination and high-throughput microscopy. Dev Cell 12:7–16

    Article  PubMed  CAS  Google Scholar 

  23. Chiu K, Lau WM, Lau HT, So KF, Chang RCC (2007) Micro-dissection of rat brain for RNA or protein extraction from specific brain region. J Vis Exp (7):e269. doi: 10.3791/269

  24. Preibisch S, Saalfeld S, Tomancak P (2009) Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25:1463–1465

    Article  PubMed  CAS  Google Scholar 

  25. Thevenaz P, Ruttimann UE, Unser M (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7:27–41

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by R01 2NS039746 and 2R01 NS045191 from the National Institute of Neurological Disease and Stroke, P01 2AG022074 from the National Institute on Aging, and the Gladstone Institutes (S.F.), the Milton Wexler Award, and a fellowship from the Hereditary Disease Foundation (A.T.). Gladstone Institutes received support from a National Center for Research Resources Grant RR18928-01. Kelley Nelson provided administrative assistance, and Gary C. Howard and Anna Lisa Lucido edited the manuscript.

Author information

Authors and Affiliations

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

    Andrey S. Tsvetkov

  2. Taube-Koret Center for Huntington’s Disease Research, San Francisco, USA

    Andrey S. Tsvetkov

  3. Gladstone Institute of Neurological Disease, University of California, San Francisco, USA

    D. Michael Ando

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

    D. Michael Ando & Steven Finkbeiner

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

    D. Michael Ando & Steven Finkbeiner

  6. Gladstone Institute of Neurological Disease, Taube-Koret Center for Huntington’s Disease Research, University of California, San Francisco, USA

    Steven Finkbeiner

Authors
  1. Andrey S. Tsvetkov
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  2. D. Michael Ando
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  3. Steven Finkbeiner
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Editor information

Editors and Affiliations

  1. , Dept. of Biochemistry and Molecular Biol, The University of Melbourne, Flemington Road 30, Parkville, 3010, Victoria, Australia

    Danny M. Hatters

  2. Florey Neuroscience Institutes, and Mental Health, The University of Melbourne, Royal Parade, Parkville, 3010, Victoria, Australia

    Anthony J. Hannan

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Tsvetkov, A.S., Ando, D.M., Finkbeiner, S. (2013). Longitudinal Imaging and Analysis of Neurons Expressing Polyglutamine-Expanded Proteins. In: Hatters, D., Hannan, A. (eds) Tandem Repeats in Genes, Proteins, and Disease. Methods in Molecular Biology, vol 1017. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-438-8_1

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  • DOI: https://doi.org/10.1007/978-1-62703-438-8_1

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-437-1

  • Online ISBN: 978-1-62703-438-8

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