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.

  • Technical Report
  • Published:

An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS

Nature Medicine volume 13, pages 754–759 (2007)Cite this article

Abstract

Misfolding of Cu/Zn-superoxide dismutase (SOD1) is emerging as a mechanism underlying motor neuron degeneration in individuals with amyotrophic lateral sclerosis (ALS) who carry a mutant SOD1 gene (SOD1 ALS). Here we describe a structure-guided approach to developing an antibody that specifically recognizes monomer-misfolded forms of SOD1. We raised this antibody to an epitope that is normally buried in the SOD1 native homodimer interface. The SOD1 exposed dimer interface (SEDI) antibody recognizes only those SOD1 conformations in which the native dimer is disrupted or misfolded and thereby exposes the hydrophobic dimer interface. Using the SEDI antibody, we established the presence of monomer-misfolded SOD1 in three ALS mouse models, with G37R, G85R and G93A SOD1 mutations, and in a human individual with an A4V SOD1 mutation. Despite ubiquitous expression, misfolded SOD1 was found primarily within degenerating motor neurons. Misfolded SOD1 appeared before the onset of symptoms and decreased at the end stage of the disease, concomitant with motor neuron loss.

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: Design and validation of SEDI antibody, which selectively recognizes monomer-misfolded SOD1, but not native dimeric SOD1.
Figure 2: Misfolded SOD1 is deposited predominantly on the periphery of vacuoles and aggregates in motor neurons of ALS mice.
Figure 3: Subcellular distribution of monomer-misfolded SOD1.
Figure 4: Age-dependent accumulation and decrease of misfolded SOD1 with concomitant loss of motor neurons in mouse models of ALS.
Figure 5: Monomer-misfolded SOD1 in a human individual with SOD1 ALS.

Similar content being viewed by others

References

  1. Bruijn, L.I., Miller, T.M. & Cleveland, D.W. Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu. Rev. Neurosci. 27, 723–749 (2004).

    Article  CAS  Google Scholar 

  2. Majoor-Krakauer, D., Willems, P.J. & Hofman, A. Genetic epidemiology of amyotrophic lateral sclerosis. Clin. Genet. 63, 83–101 (2003).

    Article  CAS  Google Scholar 

  3. Batulan, Z. et al. High threshold for induction of the stress response in motor neurons is associated with failure to activate HSF1. J. Neurosci. 23, 5789–5798 (2003).

    Article  CAS  Google Scholar 

  4. Urushitani, M., Kurisu, J., Tsukita, K. & Takahashi, R. Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis. J. Neurochem. 83, 1030–1042 (2002).

    Article  CAS  Google Scholar 

  5. Liu, J. et al. Toxicity of familial ALS-linked SOD1 mutants from selective recruitment to spinal mitochondria. Neuron 43, 5–17 (2004).

    Article  CAS  Google Scholar 

  6. Pasinelli, P. et al. Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria. Neuron 43, 19–30 (2004).

    Article  CAS  Google Scholar 

  7. Estevez, A.G. et al. Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficient superoxide dismutase. Science 286, 2498–2500 (1999).

    Article  CAS  Google Scholar 

  8. Forman, H.J. & Fridovich, I. On the stability of bovine superoxide dismutase. The effects of metals. J. Biol. Chem. 248, 2645–2649 (1973).

    CAS  PubMed  Google Scholar 

  9. Borchelt, D.R. et al. Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity. Proc. Natl. Acad. Sci. USA 91, 8292–8296 (1994).

    Article  CAS  Google Scholar 

  10. Wood, J.D., Beaujeux, T.P. & Shaw, P.J. Protein aggregation in motor neurone disorders. Neuropathol. Appl. Neurobiol. 29, 529–545 (2003).

    Article  CAS  Google Scholar 

  11. Wang, J., Xu, G. & Borchelt, D.R. High molecular weight complexes of mutant superoxide dismutase 1: age-dependent and tissue-specific accumulation. Neurobiol. Dis. 9, 139–148 (2002).

    Article  CAS  Google Scholar 

  12. Rakhit, R. et al. Oxidation-induced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis. J. Biol. Chem. 277, 47551–47556 (2002).

    Article  CAS  Google Scholar 

  13. Rakhit, R. et al. Monomeric Cu,Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic lateral sclerosis. J. Biol. Chem. 279, 15499–15504 (2004).

    Article  CAS  Google Scholar 

  14. Kayed, R. et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489 (2003).

    Article  CAS  Google Scholar 

  15. Paramithiotis, E. et al. A prion protein epitope selective for the pathologically misfolded conformation. Nat. Med. 9, 893–899 (2003).

    Article  CAS  Google Scholar 

  16. Deng, H.X. et al. Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science 261, 1047–1051 (1993).

    Article  CAS  Google Scholar 

  17. Gurney, M.E. et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264, 1772–1775 (1994).

    Article  CAS  Google Scholar 

  18. Wong, P.C. et al. An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 14, 1105–1116 (1995).

    Article  CAS  Google Scholar 

  19. Bruijn, L.I. et al. ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron 18, 327–338 (1997).

    Article  CAS  Google Scholar 

  20. Tiwari, A., Xu, Z. & Hayward, L.J. Aberrantly increased hydrophobicity shared by mutants of Cu,Zn-superoxide dismutase in familial amyotrophic lateral sclerosis. J. Biol. Chem. 280, 29771–29779 (2005).

    Article  CAS  Google Scholar 

  21. Jonsson, P.A. et al. Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis. Brain 127, 73–88 (2004).

    Article  Google Scholar 

  22. Furukawa, Y., Torres, A.S. & O'Halloran, T.V. Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS. EMBO J. 23, 2872–2881 (2004).

    Article  CAS  Google Scholar 

  23. Jaarsma, D. et al. Human Cu/Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondrial vacuolization, axonal degeneration, and premature motoneuron death and accelerates motoneuron disease in mice expressing a familial amyotrophic lateral sclerosis mutant SOD1. Neurobiol. Dis. 7, 623–643 (2000).

    Article  CAS  Google Scholar 

  24. Deng, H.X. et al. Conversion to the amyotrophic lateral sclerosis phenotype is associated with intermolecular linked insoluble aggregates of SOD1 in mitochondria. Proc. Natl. Acad. Sci. USA 103, 7142–7147 (2006).

    Article  CAS  Google Scholar 

  25. Higgins, C.M., Jung, C. & Xu, Z. ALS-associated mutant SOD1G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes. BMC Neurosci. 4, 16 (2003).

    Article  Google Scholar 

  26. Bergemalm, D. et al. Overloading of stable and exclusion of unstable human superoxide dismutase-1 variants in mitochondria of murine amyotrophic lateral sclerosis models. J. Neurosci. 26, 4147–4154 (2006).

    Article  CAS  Google Scholar 

  27. Kikuchi, H. et al. Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model. Proc. Natl. Acad. Sci. USA 103, 6025–6030 (2006).

    Article  CAS  Google Scholar 

  28. Atkin, J.D. et al. Induction of the unfolded protein response in familial amyotrophic lateral sclerosis and association of protein disulfide isomerase with superoxide dismutase 1. J. Biol. Chem. 281, 30152–30165 (2006).

    Article  CAS  Google Scholar 

  29. Boillee, S. et al. Onset and progression in inherited ALS determined by motor neurons and microglia. Science 312, 1389–1392 (2006).

    Article  CAS  Google Scholar 

  30. Rudiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501–1507 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Strong (University of Western Ontario) for providing human spinal cord sections and P.H. Chan (Stanford University) for providing human wild-type SOD1 rats. We also thank N. Ng and members of the Chakrabartty lab and the Centre for Research in Neurodegenerative Diseases for helpful comments and review; V. Mulligan, J. Kim and W. Zhou for technical assistance; and C. Accardi for assistance with animal care. This project was funded by the Neuromuscular Research Partnership—the Canadian Institutes of Health Research (CIHR), the ALS Society (Canada) and the Muscular Dystrophy Association (Canada) (J.R. and A.C.); the ALS Association (US) and MND Association (UK) (J.R.); and CIHR and the Temerety Family Trust (N.R.C). C.V.V. is funded by the Muscular Dystrophy Association and D.W.C. receives salary support from the Ludwig Institute. R.R. is the recipient of a Doctoral Research Award from the CIHR Institute of Neuroscience, Mental Health and Addiction (CIHR-INMHA) and the ALS Society (Canada). J.R. holds a Canada Research Chair in Molecular Mechanisms of ALS. N.R.C. holds a Canada Research Chair in Neurodegeneration and Protein Misfolding Diseases.

Author information

Author notes

  1. Neil R Cashman

    Present address: Present address: Department of Medicine (Neurology) and Brain Research Centre, University of British Columbia Hospital, University of British Columbia, 2211 Wesbrook Mall, Vancouver, British Columbia V6T 2B5, Canada.,

Authors and Affiliations

  1. Departments of Biochemistry and Medical Biophysics, University of Toronto and Ontario Cancer Institute, 101 College Street, Toronto, M5G 1L7, Ontario, Canada

    Rishi Rakhit & Avijit Chakrabartty

  2. and Department of Laboratory Medicine and Pathobiology, Centre for Research in Neurodegenerative Diseases, University of Toronto, 6 Queen's Park Crescent West, Toronto, M5S 3H2, Ontario, Canada

    Janice Robertson, Patrick Horne, Deborah M Ruth, Jennifer Griffin & Neil R Cashman

  3. Ludwig Institute for Cancer Research and University of California at San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA

    Christine Vande Velde & Don W Cleveland

Authors
  1. Rishi Rakhit
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Janice Robertson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christine Vande Velde
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patrick Horne
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Deborah M Ruth
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jennifer Griffin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Don W Cleveland
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Neil R Cashman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Avijit Chakrabartty
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.R., J.R., D.W.C., N.R.C. and A.C. designed the research. R.R., J.R., C.V.V., P.H., D.M.R. and J.G. performed the research. R.R., J.R., C.V.V., D.W.C., N.R.C. and A.C. analyzed the data. R.R., J.R., C.V.V., D.W.C., N.R.C. and A.C. wrote the manuscript.

Corresponding author

Correspondence to Avijit Chakrabartty.

Ethics declarations

Competing interests

R.R., N.R.C. and A.C. are named as inventors on patent applications relating to this technology. The University Health Network has licensed this patent to a private-sector company that provides research funding; however, no funding from this company was used in executing the work described in this paper.

Supplementary information

Supplementary Fig. 1

SOD1 unfolding was followed by changes to tryptophan fluorescence (ex. = 280nm, em. = 350nm). (PDF 640 kb)

Supplementary Fig. 2

Western blots were performed to further demonstrate the specificity of the SEDI antibody. (PDF 1011 kb)

Supplementary Fig. 3

Comparison of immunoprecipitation reactions using SEDI antibody or pre-immune IgG. (PDF 937 kb)

Supplementary Fig. 4

SEDI antibody immunohistological labeling is specific and can be saturated by competition with the antigenic peptide. (PDF 1516 kb)

Supplementary Fig. 5

SEDI labeling of a mutant SOD1-transgenic mouse spinal cords. (PDF 3472 kb)

Supplementary Fig. 6

Our immunohistochemistry experiments show that monomer/misfolded SOD1 accumulates around vacuoles (Fig. 3b). (PDF 1420 kb)

Supplementary Fig. 7

Putative Hsp70 binding sites in SOD1 – 4-5 hydrophobic residues flanked by basic residues. (PDF 961 kb)

Supplementary Methods (PDF 42 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rakhit, R., Robertson, J., Velde, C. et al. An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS. Nat Med 13, 754–759 (2007). https://doi.org/10.1038/nm1559

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm1559

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