Skip to main content
SpringerLink
Log in

Composition of Soluble Misfolded Superoxide Dismutase-1 in Murine Models of Amyotrophic Lateral Sclerosis

  • Original Paper
  • Published:
NeuroMolecular Medicine Aims and scope Submit manuscript

Abstract

A common cause of amyotrophic lateral sclerosis is mutations in superoxide dismutase-1, which provoke the disease by an unknown mechanism. We have previously found that soluble hydrophobic misfolded mutant human superoxide dismutase-1 species are enriched in the vulnerable spinal cords of transgenic model mice. The levels were broadly inversely correlated with life spans, suggesting involvement in the pathogenesis. Here, we used methods based on antihuman superoxide dismutase-1 peptide antibodies specific for misfolded species to explore the composition and amounts of soluble misfolded human superoxide dismutase-1 in tissue extracts. Mice expressing 5 different human superoxide dismutase-1 variants with widely variable structural characteristics were examined. The levels were generally higher in spinal cords than in other tissues. The major portion of misfolded superoxide dismutase-1 was shown to be monomers lacking the C57–C146 disulfide bond with large hydrodynamic volume, indicating a severely disordered structure. The remainder of the misfolded protein appeared to be non-covalently associated in 130- and 250-kDa complexes. The malleable monomers should be prone to aggregate and associate with other cellular components, and should be easily translocated between compartments. They may be the primary cause of toxicity in superoxide dismutase-1-induced amyotrophic lateral sclerosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Andersen, P. M., & Al-Chalabi, A. (2011). Clinical genetics of amyotrophic lateral sclerosis: What do we really know? Nature Reviews Neurology, 7, 603–615.

    Article  PubMed  CAS  Google Scholar 

  • Andersen, P. M., Nilsson, P., Ala-Hurula, V., Keranen, M. L., Tarvainen, I., Haltia, T., et al. (1995). Amyotrophic lateral sclerosis associated with homozygosity for an Asp90Ala mutation in CuZn-superoxide dismutase. Nature Genetics, 10, 61–66.

    Article  PubMed  CAS  Google Scholar 

  • Andersen, P. M., Sims, K. B., Xin, W. W., Kiely, R., O’Neill, G., Ravits, J., et al. (2003). Sixteen novel mutations in the Cu/Zn superoxide dismutase gene in amyotrophic lateral sclerosis: A decade of discoveries, defects and disputes. Amyotrophic Lateral Sclerosis, 4, 62–73.

    Article  PubMed  CAS  Google Scholar 

  • Bergemalm, D., Forsberg, K., Srivastava, V., Graffmo, K. S., Andersen, P. M., Brannstrom, T., et al. (2010). Superoxide dismutase-1 and other proteins in inclusions from transgenic amyotrophic lateral sclerosis model mice. Journal of Neurochemistry, 114, 408–418.

    Article  PubMed  CAS  Google Scholar 

  • Bergemalm, D., Jonsson, P. A., Graffmo, K. S., Andersen, P. M., Brannstrom, T., Rehnmark, A., et al. (2006). Overloading of stable and exclusion of unstable human superoxide dismutase-1 variants in mitochondria of murine amyotrophic lateral sclerosis models. The Journal of Neuroscience, 26, 4147–4154.

    Article  PubMed  CAS  Google Scholar 

  • Bruijn, L. I., Becher, M. W., Lee, M. K., Anderson, K. L., Jenkins, N. A., Copeland, N. G., et al. (1997). ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron, 18, 327–338.

    Article  PubMed  CAS  Google Scholar 

  • Chattopadhyay, M., Durazo, A., Sohn, S. H., Strong, C. D., Gralla, E. B., Whitelegge, J. P., et al. (2008). Initiation and elongation in fibrillation of ALS-linked superoxide dismutase. Proceedings of the National academy of Sciences of the United States of America, 105, 18663–18668.

    Article  PubMed  CAS  Google Scholar 

  • Forsberg, K., Andersen, P., Marklund, S., & Brännström, T. (2011). Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis. Acta Neuropathologica, 121, 623–634.

    Article  PubMed  CAS  Google Scholar 

  • Forsberg, K., Jonsson, P. A., Andersen, P. M., Bergemalm, D., Graffmo, K. S., Hultdin, M., et al. (2010). Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients. PLoS One, 5, e11552.

    Article  PubMed  Google Scholar 

  • Furukawa, Y., Kaneko, K., Yamanaka, K., O’Halloran, T. V., & Nukina, N. (2008). Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in the familial form of amyotrophic lateral sclerosis. The Journal of Biological Chemistry, 283, 24167–24176.

    Article  PubMed  CAS  Google Scholar 

  • Furukawa, Y., & O’Halloran, T. V. (2005). ALS mutations have the greatest destabilizing effect on the apo, reduced form of SOD1, leading to unfolding and oxidative aggregation. The Journal of Biological Chemistry, 280, 17266–17274.

    Article  PubMed  CAS  Google Scholar 

  • Graffmo, K. S., Forsberg, K., Bergh, J., Birve, A., Zetterström, P., Andersen, P. M., et al. (2012). Expression of wild-type human superoxide dismutase-1 in mice causes amyotrophic lateral sclerosis. Human Molecular Genetics,. doi:10.1093/hmg/dds399.

    PubMed  Google Scholar 

  • Gurney, M. E., Pu, H., Chiu, A. Y., Dal Canto, M. C., Polchow, C. Y., Alexander, D. D., et al. (1994). Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation. Science, 264, 1772–1775.

    Article  PubMed  CAS  Google Scholar 

  • Hoffman, E. K., Wilcox, H. M., Scott, R. W., & Siman, R. (1996). Proteasome inhibition enhances the stability of mouse Cu/Zn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis. Journal of the Neurological Sciences, 139, 15–20.

    Article  PubMed  CAS  Google Scholar 

  • Ilieva, H., Polymenidou, M., & Cleveland, D. W. (2009). Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. The Journal of Cell Biology, 187, 761–772.

    Article  PubMed  CAS  Google Scholar 

  • Israelson, A., Arbel, N., Da Cruz, S., Ilieva, H., Yamanaka, K., Shoshan-Barmatz, V., et al. (2010). Misfolded mutant SOD1 directly inhibits VDAC1 conductance in a mouse model of inherited ALS. Neuron, 67, 575–587.

    Article  PubMed  CAS  Google Scholar 

  • Johnston, J. A., Dalton, M. J., Gurney, M. E., & Kopito, R. R. (2000). Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis. Proceedings of the National academy of Sciences of the United States of America, 97, 12571–12576.

    Article  PubMed  CAS  Google Scholar 

  • Jonsson, P. A., Ernhill, K., Andersen, P. M., Bergemalm, D., Brannstrom, T., Gredal, O., et al. (2004). Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis. Brain, 127, 73–88.

    Article  PubMed  Google Scholar 

  • Jonsson, P. A., Graffmo, K. S., Andersen, P. M., Brannstrom, T., Lindberg, M., Oliveberg, M., et al. (2006a). Disulphide-reduced superoxide dismutase-1 in CNS of transgenic amyotrophic lateral sclerosis models. Brain, 129, 451–464.

    Article  PubMed  Google Scholar 

  • Jonsson, P. A., Graffmo, K. S., Brannstrom, T., Nilsson, P., Andersen, P. M., & Marklund, S. L. (2006b). Motor neuron disease in mice expressing the wild type-like D90A mutant superoxide dismutase-1. Journal of Neuropathology and Experimental Neurology, 65, 1126–1136.

    Article  PubMed  CAS  Google Scholar 

  • Kabuta, T., Suzuki, Y., & Wada, K. (2006). Degradation of amyotrophic lateral sclerosis-linked mutant Cu, Zn-superoxide dismutase proteins by macroautophagy and the proteasome. The Journal of Biological Chemistry, 281, 30524–30533.

    Article  PubMed  CAS  Google Scholar 

  • Kanning, K. C., Kaplan, A., & Henderson, C. E. (2010). Motor neuron diversity in development and disease. Annual Review of Neuroscience, 33, 409–440.

    Article  PubMed  CAS  Google Scholar 

  • Karch, C. M., Prudencio, M., Winkler, D. D., Hart, P. J., & Borchelt, D. R. (2009). Role of mutant SOD1 disulfide oxidation and aggregation in the pathogenesis of familial ALS. Proceedings of the National academy of Sciences of the United States of America, 106, 7774–7779.

    Article  PubMed  CAS  Google Scholar 

  • Kay, A. (2004). Detecting and minimizing zinc contamination in physiological solutions. BMC Physiology, 4, 4.

    Article  PubMed  Google Scholar 

  • Kayatekin, C., Zitzewitz, J. A., & Matthews, C. R. (2008). Zinc binding modulates the entire folding free energy surface of human Cu, Zn superoxide dismutase. Journal of Molecular Biology, 384, 540–555.

    Article  PubMed  CAS  Google Scholar 

  • Lindberg, M. J., Normark, J., Holmgren, A., & Oliveberg, M. (2004). Folding of human superoxide dismutase: Disulfide reduction prevents dimerization and produces marginally stable monomers. Proceedings of the National academy of Sciences of the United States of America, 101, 15893–15898.

    Article  PubMed  CAS  Google Scholar 

  • Mondola, P., Annella, T., Santillo, M., & Santangelo, F. (1996). Evidence for secretion of cytosolic CuZn superoxide dismutase by Hep G2 cells and human fibroblasts. The International Journal of Biochemistry and Cell Biology, 28, 677–681.

    Article  CAS  Google Scholar 

  • Mourelatos, Z., Gonatas, N. K., Stieber, A., Gurney, M. E., & Dal Canto, M. C. (1996). The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu, Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease. Proceedings of the National academy of Sciences of the United States of America, 93, 5472–5477.

    Article  PubMed  CAS  Google Scholar 

  • Mulligan, V. K., Kerman, A., Ho, S., & Chakrabartty, A. (2008). Denaturational stress induces formation of zinc-deficient monomers of Cu, Zn superoxide dismutase: Implications for pathogenesis in amyotrophic lateral sclerosis. Journal of Molecular Biology, 383, 424–436.

    Article  PubMed  CAS  Google Scholar 

  • Nordlund, A., Leinartaitė, L., Saraboji, K., Aisenbrey, C., Gröbner, G., Zetterström, P., et al. (2009). Functional features cause misfolding of the ALS-provoking enzyme SOD1. Proceedings of the National academy of Sciences of the United States of America, 106, 9667–9672.

    Article  PubMed  CAS  Google Scholar 

  • Pasinelli, P., Belford, M. E., Lennon, N., Bacskai, B. J., Hyman, B. T., Trotti, D., et al. (2004). Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria. Neuron, 43, 19–30.

    Article  PubMed  CAS  Google Scholar 

  • Rosen, D. R., Siddique, T., Patterson, D., Figlewicz, D. A., Sapp, P., Hentati, A., et al. (1993). Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 362, 59–62.

    Article  PubMed  CAS  Google Scholar 

  • Rumfeldt, J. A. O., Lepock, J. R., & Meiering, E. M. (2009). Unfolding and folding kinetics of amyotrophic lateral sclerosis-associated mutant Cu, zn superoxide dismutases. Journal of Molecular Biology, 385, 278–298.

    Article  PubMed  CAS  Google Scholar 

  • Urushitani, M., Sik, A., Sakurai, T., Nukina, N., Takahashi, R., & Julien, J. P. (2006). Chromogranin-mediated secretion of mutant superoxide dismutase proteins linked to amyotrophic lateral sclerosis. Nature Neuroscience, 9, 108–118.

    Article  PubMed  CAS  Google Scholar 

  • Uversky, V. N., & Ptitsyn, O. B. (1996). Further evidence on the equilibrium “Pre-molten Globule State”: Four-state guanidinium chloride-induced unfolding of carbonic anhydrase b at low temperature. Journal of Molecular Biology, 255, 215–228.

    Article  PubMed  CAS  Google Scholar 

  • Williamson, T. L., & Cleveland, D. W. (1999). Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motor neurons. Nature Neuroscience, 2, 50–56.

    Article  PubMed  CAS  Google Scholar 

  • Wooley, C. M., Sher, R. B., Kale, A., Frankel, W. N., Cox, G. A., & Seburn, K. L. (2005). Gait analysis detects early changes in transgenic SOD1(G93A) mice. Muscle and Nerve, 32, 43–50.

    Article  PubMed  CAS  Google Scholar 

  • Wroe, R., Wai-Ling Butler, A., Andersen, P. M., Powell, J. F., & Al-Chalabi, A. (2008). ALSOD: The amyotrophic lateral sclerosis online database. Amyotrophic Lateral Sclerosis, 9, 249–250.

    Article  PubMed  CAS  Google Scholar 

  • Zetterstrom, P., Andersen, P. M., Brannstrom, T., & Marklund, S. L. (2011a). Misfolded superoxide dismutase-1 in CSF from amyotrophic lateral sclerosis patients. Journal of Neurochemistry, 117, 91–99.

    Article  PubMed  Google Scholar 

  • Zetterstrom, P., Graffmo, K. S., Andersen, P. M., Brannstrom, T., & Marklund, S. L. (2011b). Proteins that bind to misfolded mutant superoxide dismutase-1 in spinal cords from transgenic ALS model mice. The Journal of Biological Chemistry, 286, 20130–20136.

    Article  PubMed  Google Scholar 

  • Zetterstrom, P., Stewart, H. G., Bergemalm, D., Jonsson, P. A., Graffmo, K. S., Andersen, P. M., et al. (2007). Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in spinal cords throughout life in murine ALS models. Proceedings of the National academy of Sciences of the United States of America, 104, 14157–14162.

    Article  PubMed  Google Scholar 

  • Zhang, F., Strom, A. L., Fukada, K., Lee, S., Hayward, L. J., & Zhu, H. (2007). Interaction between familial amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants and the dynein complex. The Journal of Biological Chemistry, 282, 16691–16699.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Eva Bern, Karin Hjertkvist, Ann-Charloth Nilsson, Ulla-Stina Spetz, and Agneta Öberg for excellent technical assistance. This work was supported by the Swedish Science Council, the Swedish Brain Fund/Hållsten Fund, the Swedish Medical Society including the Björklund Fund for ALS Research, the ALS Association, the Swedish Association of Persons with Neurological Disabilities, Västerbotten County Council, the Kempe Foundations, and the King Gustaf V and Queen Victoria Foundation.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Medical Biosciences, Clinical Chemistry, Umeå University, 901 85, Umeå, Sweden

    Per Zetterström & Stefan L. Marklund

  2. Department of Medical Biosciences, Pathology, Umeå University, 901 85, Umeå, Sweden

    Karin S. Graffmo & Thomas Brännström

  3. Department of Pharmacology and Clinical Neuroscience, Umeå University, 901 85, Umeå, Sweden

    Peter M. Andersen

Authors
  1. Per Zetterström
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Karin S. Graffmo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter M. Andersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas Brännström
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stefan L. Marklund
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan L. Marklund.

Electronic supplementary material

Below is the link to the electronic supplementary material.

12017_2012_8204_MOESM1_ESM.eps

Supplementary material Fig. S1 Oxidation of hSOD1 without protection. Three pieces of brain from a G93A mouse were homogenized in PBS, PBS supplemented with 40 mM IAM, or PBS supplemeted with 0.5 mM DTT. After centrifugation, the extracts were passed over NAP5 columns (GE Healthcare) into the respective buffers. Eluted samples were incubated at 4ºC and 37ºC and samples taken after the times indicated. The disulfide status of SOD1 was investigated by non-reducing western immunoblots (EPS 15796 kb)

12017_2012_8204_MOESM2_ESM.eps

Supplementary material Fig. S2 Superdex 200 SEC pattern. The spinal cord extract from the G93A mouse used for cross-linking experiments (see Fig. 7) was separated by SEC using a Superdex 200 column, and misfolded hSOD1 was analyzed with misELISA. The letters indicate the elution positions of molecular weight markers in the SEC: a, 2000 kDa; b, 669 kDa; c, 440 kDa; d, 158 kDa; e, 75 kDa; f, 69 kDa; g, 44 kDa; h, 32 kDa; i, 13.7 kDa (EPS 843 kb)

12017_2012_8204_MOESM3_ESM.docx

Supplementary material Table 1 Comparison of EDTA or IAM in homogenization buffer. CNS homogenates containing EDTA or IAM from hSOD1 transgenic mice were analyzed for misfolded hSOD1 with misELISA (DOCX 14 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zetterström, P., Graffmo, K.S., Andersen, P.M. et al. Composition of Soluble Misfolded Superoxide Dismutase-1 in Murine Models of Amyotrophic Lateral Sclerosis. Neuromol Med 15, 147–158 (2013). https://doi.org/10.1007/s12017-012-8204-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12017-012-8204-z

Keywords

Search

Navigation