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Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death

Nature volume 431, pages 805–810 (2004)Cite this article

Abstract

Huntington's disease is caused by an abnormal polyglutamine expansion within the protein huntingtin and is characterized by microscopic inclusion bodies of aggregated huntingtin and by the death of selected types of neuron. Whether inclusion bodies are pathogenic, incidental or a beneficial coping response is controversial. To resolve this issue we have developed an automated microscope that returns to precisely the same neuron after arbitrary intervals, even after cells have been removed from the microscope stage. Here we show, by survival analysis, that neurons die in a time-independent fashion but one that is dependent on mutant huntingtin dose and polyglutamine expansion; many neurons die without forming an inclusion body. Rather, the amount of diffuse intracellular huntingtin predicts whether and when inclusion body formation or death will occur. Surprisingly, inclusion body formation predicts improved survival and leads to decreased levels of mutant huntingtin elsewhere in a neuron. Thus, inclusion body formation can function as a coping response to toxic mutant huntingtin.

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Figure 1: PolyQ-expansion-dependent cell death measured with an automated microscope.
Figure 2: Many neurons die without forming IBs.
Figure 3: Levels of diffuse mutant Htt protein predict neuronal death.
Figure 4: IB formation is associated with decreased intracellular levels of diffuse Httex1 and improved neuronal survival.
Figure 5

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Acknowledgements

We thank A. Kazantzev, D. Housman and the Hereditary Disease Foundation for pcDNA3.1-Htt (Q25, Q47, Q72, Q103)-GFP plasmids; R. Truant for the PCR template (GFP–109-17Q-βgal) used to create pGW1-Httex1-Q17-GFP; R. Tsien for mRFP cDNA; D. Bredesen, S. Prusiner, S. Lindquist, R. Edwards, A. Tobin, E. Signer, C. Johnson, P. Muchowski and members of the Finkbeiner laboratory for useful discussions; S. Ordway and G. Howard for editorial assistance; K. Nelson for administrative assistance; and E. Oliver and D. Murphy for their interest and support. Primary support for this work was provided by the National Institute of Neurological Disease and Stroke (S.F). Additional support was provided by the National Institute of Aging and the J. David Gladstone Institutes (S.F.). M.A. is a MECD–Fulbright Fellow and is supported by the Hillblom Foundation. S.M. is supported by the NIH–NIGMS UCSF Medical Scientist Training Program and a fellowship from the UCSF Hillblom Center for the Biology of Aging. E.S. is supported by the National Institute of Neurological Disease and Stroke, the Hereditary Disease Foundation, and the High Q Foundation.

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Authors and Affiliations

  1. Gladstone Institute of Neurological Disease, University of California, San Francisco, California, 94141, USA

    Montserrat Arrasate, Siddhartha Mitra & Steven Finkbeiner

  2. Neuroscience Program, University of California, San Francisco, California, 94141, USA

    Montserrat Arrasate & Steven Finkbeiner

  3. Biomedical Sciences Program, University of California, San Francisco, California, 94141, USA

    Siddhartha Mitra & Steven Finkbeiner

  4. Medical Scientist Training Program, University of California, San Francisco, California, 94141, USA

    Siddhartha Mitra & Steven Finkbeiner

  5. Brain Research Institute, University of California School of Medicine, Los Angeles, California, 90095-1761, USA

    Erik S. Schweitzer

  6. Division of Biostatistics, University of California, California, 94143-0560, San Francisco, USA

    Mark R. Segal

  7. Departments of Neurology and Physiology, University of California, California, 94141, San Francisco, USA

    Steven Finkbeiner

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  1. Montserrat Arrasate
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Correspondence to Steven Finkbeiner.

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Supplementary information

Supplementary Figure 1 (download JPG )

Fluorescence of mRFP provides a measure of neuronal morphology and viability that is independent of httex1-GFP. (JPG 129 kb)

Supplementary Figure 2 (download JPG )

Abrupt loss of fluorescence from transfected mRFP is a sensitive and specific assay of neuronal death. (JPG 56 kb)

Supplementary Figure 3 (download JPG )

Httex1-Q47–GFP increases the risk of death significantly compared with httex1-Q17–GFP. (JPG 29 kb)

Supplementary Figure 4 (download JPG )

Single-neuron levels of GFP estimated by imaging correlate well with levels measured by immunocytochemistry. (JPG 21 kb)

Supplementary Figure 5 (download JPG )

Neurons with similar levels of httex1-Q47-GFP that form IBs on the second day survive better than neurons that do not. (JPG 31 kb)

Supplementary Figure 6 (download JPG )

IB formation is associated with reduced death risk and increased survival among neurons transfected with httex1-Q47-GFP that are alive beginning on the sixth day. (JPG 24 kb)

Supplementary Figure 7 (download JPG )

Neurons with IBs are viable as assessed by annexinV staining. (JPG 143 kb)

Supplementary Figure 8 (download JPG )

IB formation is associated with reduced death risk and increased survival among stably transfected PC12 cells that were induced to express httex1-Q103-GFP and were alive beginning on the third day. (JPG 33 kb)

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Arrasate, M., Mitra, S., Schweitzer, E. et al. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431, 805–810 (2004). https://doi.org/10.1038/nature02998

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