A New Study Fires Up the Debate About Misfolded SOD1 and its Potential Role in Sporadic ALS

SOD1, under the microscope. No misfolded WT SOD1 could be detected in more than 50 patients with sporadic ALS according to a new post-mortem tissue analysis suggesting that misfolded SOD1 may not play a key role in sporadic disease (da Cruz et al., 2017). Image: Hart et al., 1998, PDB.

If there is any misfolded wild-type SOD1 in patients with sporadic ALS, its level is too low to detect with any of seven conformation-specific antibodies in autopsy tissue, according to a new study led by Sandrine da Cruz, Don Cleveland and John Ravits of the University of California in San Diego. The analysis according to Ravits indicates that misfolded SOD1 is not a primary component of sporadic ALS and therefore targeting it is unlikely to be helpful in treating the disease.

The study, which involved the analysis of brain and spinal cord tissue from over 50 patients with sporadic ALS, is the largest to date to rule out misfolded SOD1 as a key contributor to sporadic disease (Liu et al, 2009; Kerman et al., 2010; Ayers et al., 2014).

“It’s an excellent piece of work that will contribute to the debate” said Neil Cashman of the University of British Columbia in Vancouver. “But I don’t believe it is the final word.” Cashman, who was not involved in the study, is developing antibodies against misfolded SOD1 as a potential treatment strategy for the disease.

The study appeared on 28 February 2017 in Acta Neuropathologica.

Misfolded SOD1 and sALS: At C4F6 and 7?

Researchers first suspected in the late 1990s that misfolded SOD1 may contribute to sporadic ALS by investigating modified forms of the enzyme that may arise in the disease. One of these studies, led by Laval University’s Jean-Pierre Julien in Canada, found that the oxidation of SOD1 resulted in motor neuron toxicity in vitro, a post-translational modification that could occur due to oxidative stress and result in its misfolding in both familial and sporadic ALS (Ezzi et al., 2007; Rakhit et al., 2004). Subsequent studies led by Robert Brown of the University of Massachusetts Medical School demonstrated that a similar misfolded form of the enzyme could be detected in post-mortem spinal cord tissue of people with sporadic ALS, and could inhibit axonal transport ex vivo, suggesting a pathogenic mechanism (Bosco et al., 2010).

In more recent years, multiple investigators reported the accumulation of misfolded wild-type (WT) SOD1 in the spinal cord and other tissues of patients with sporadic ALS suggesting it may play a role in the disease (Forsberg et al., 2010; Forsberg, et al., 2011; Pokrishevsky et al., 2012). These studies, however, hinged upon the detection of misfolded WT SOD1 using conformational-specific antibodies including C4F6 and SEDI. The same antibodies according to studies led by University of Florida’s David Borchelt and the University of Toronto’s Janice Robertson and Avi Chakrabartty in Canada were unable to detect WT misfolded SOD1 in post-mortem spinal cord tissue of people with sporadic disease (Liu et al, 2009; Kerman et al., 2010; Ayers et al., 2014).

A prion-like disease? Cytoplasmic TDP-43 induced properly folded SOD1 to misfold and subsequently spread to other cells in vitro according to studies led by Neil Cashman suggesting that misfolded WT SOD1 might play a pathogenic role in other forms of the disease (see January 2016 news; Pokrishevky et al., 2012; Pokrishevky et al., 2016). Image: Pokrishevsky et al., 2016 under CC BY 4.0 license.

With a growing number of potential SOD1 therapies approaching or in the clinic, a research team led by Sandrine da Cruz, John Ravits and Don Cleveland of the University of California in San Diego performed a large, independent analysis in hopes to determine whether misfolded WT SOD1 plays a role in sporadic disease. The team reached out to ALS research laboratories across the globe to obtain seven different conformationally sensitive antibodies against misfolded SOD1 and tested them in high-quality sporadic ALS autopsy tissue from Ravits’s tissue bank. The antibodies included C4F6, B8H10, DSE2 3H1, DSE2 10E11, and 131-153Ra, plus two novel high-affinity antibodies, 4A1 and A5E5.

The da Cruz-Ravits-Cleveland team examined tissue samples from over 50 sporadic ALS patients using a combined immunoprecipitation, immunohistochemistry, and immunofluorescence approach. SOD1 familial ALS and non-neurologic tissues served as controls. As expected, SOD1 familial ALS tissue revealed aggregates of intensely stained mutant SOD1 protein in neurons. In contrast, sporadic ALS tissue contained no aggregates. And, the researchers did not observe cytoplasmic staining in either the lumbar, thoracic or cervical regions of the spinal cords of these patients at a level different from controls. Immunoprecipitation analysis yielded similar conclusions.

“No misfolded SOD1-immunopositive signal was detected,” the authors wrote, “regardless of age/site of onset, disease duration, region of the CNS, or immunodetection approach.”

The results according to Ravits indicate that reducing levels of misfolded SOD1 is therefore unlikely to be therapeutically beneficial in sporadic disease.

“This is a well done paper,” Neil Cashman said, but its conclusion “is a bit of overinterpretation.”

The reason according to Cashman, is that misfolded SOD1 is likely to be soluble and can therefore be more easily washed away during the experimental process. How misfolded SOD1 is detected therefore is critical and these protocols can differ from lab to lab.

To address some of these challenges, in preparation for the study, first author Sandrine da Cruz traveled to the Cashman lab in Canada to be trained to detect misfolded WT SOD1 using their immunoprecipitation protocol. But she nevertheless detected no signs of the misfolded enzyme in any of sporadic ALS samples tested either in the brain or spinal cord.

“If they applied our technique with no deviation, I would have expected the same result,” Cashman said. “[The] safest thing to say is that at the limits of detection, the detection will be variable.”

Detecting misfolded WT SOD1, according to some experts, can be tricky to do. The levels of misfolded WT SOD1 are much lower than mutant misfolded enzyme explained University of Massachusetts’ Medical School’s Daryl Bosco, who characterized the C4F6 antibody. And, according to Bosco, many of these antibodies, which are developed against mutant SOD1, bind to misfolded forms of the wild-type protein with much lower affinity.

“The paper was rigorous,” said Bosco. But small differences in protocols might account for the disparity in results.

David Borchelt of University of Florida at Gainesville agrees. “Immunohistochemistry is still somewhat of an art,” said Borchelt. “There may be nuances that are critical in detecting misfolded WT SOD1 in sporadic ALS cases.”

Reconcilable differences?

A key challenge according to Borchelt is that the study did not test post-mortem tissue from patients previously identified to exhibit misfolded WT SOD1. Therefore, the study leaves open the question of whether these discrepancies are simply a detection issue.

One way to resolve discrepancies according to Neil Cashman is for multiple labs to test blinded samples with identical antibodies.

However, Daryl Bosco, cautions that other approaches may be needed to evaluate its potential role in the disease. “If we use end-stage tissue as a criteria as to whether something is or isn’t involved in ALS, there may be a lot of inconsistency.”

In the meantime, said Bosco, we should keep an open mind about whether misfolded WT SOD1 plays a role in sporadic ALS. “It is a highly abundant protein that is very sensitive to becoming misfolded and toxic.”

Cashman echoed that idea. “The main question is not whether misfolded wild-type SOD1 is detectable, but whether it plays a role in pathogenesis. This possibility is not excluded by the current paper.”

But how to definitively determine whether misfolded SOD1 plays a role in sporadic ALS remains an open question.

“What this paper shows convincingly is that if misfolded wild-type SOD1 is there, it’s not easy to detect.” said Borchelt. “It’s a real challenge to prove a negative.” Therefore, he fully expects the debate to continue. “This is unlikely to be settled until we have a drug that effectively treats familial SOD1 carriers, and we test that drug in sporadic ALS.”

Featured Paper

Da Cruz S, Bui A, Saberi S, Lee SK, Stauffer J, McAlonis-Downes M, Schulte D, Pizzo DP, Parone PA, Cleveland DW, Ravits J. Misfolded SOD1 is not a primary component of sporadic ALS. Acta Neuropathol. 2017 Feb 28. [PubMed].


Liu HN, Sanelli T, Horne P, Pioro EP, Strong MJ, Rogaeva E, Bilbao J, Zinman L, Robertson J. Lack of evidence of monomer/misfolded superoxide dismutase-1 in sporadic amyotrophic lateral sclerosis. Ann Neurol. 2009 Jul;66(1):75-80. [PubMed].

Kerman A, Liu HN, Croul S, Bilbao J, Rogaeva E, Zinman L, Robertson J, Chakrabartty A. Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form. Acta Neuropathol. 2010 Mar;119(3):335-44. [PubMed].

Ayers JI, Xu G, Pletnikova O, Troncoso JC, Hart PJ, Borchelt DR1. Conformational specificity of the C4F6 SOD1 antibody; low frequency of reactivity in sporadic ALS cases. Acta Neuropathol Commun. 2014 May 14;2:55. [PubMed].

Ezzi SA, Urushitani M, Julien JP. Wild-type superoxide dismutase acquires binding and toxic properties of ALS-linked mutant forms through oxidation. J. Neurochem. 2007 Jul;102(1):170-8. Epub 2007 Mar 29. [PubMed].

Rakhit R, Crow JP, Lepock JR, Kondejewski LH, Cashman NR, Chakrabartty A. Monomeric Cu,Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic lateral sclerosis. J Biol Chem. 2004 Apr 9;279(15):15499-504. Epub 2004 Jan 20. [PubMed].

Bosco DA, Morfini G, Karabacak NM, Song Y, Gros-Louis F, Pasinelli P, Goolsby H, Fontaine BA, Lemay N, McKenna-Yasek D, Frosch MP, Agar JN, Julien JP, Brady ST, Brown RH Jr. Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nat Neurosci. 2010 Nov;13(11):1396-403. doi: 10.1038/nn.2660. Epub 2010 Oct 17. [PubMed].

Forsberg K, Jonsson PA, Andersen PM, Bergemalm D, Graffmo KS, Hultdin M, Jacobsson J, Rosquist R, Marklund SL, Brännström T. Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients. PLoS One. 2010 Jul 14;5(7):e11552. doi: 10.1371/journal.pone.0011552. [PubMed].

Forsberg K, Andersen PM, Marklund SL, Brännström T. Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis. Acta Neuropathol. 2011 May;121(5):623-34. Epub 2011 Feb 3. [PubMed].

Pokrishevsky E1, Grad LI, Yousefi M, Wang J, Mackenzie IR, Cashman NR. Aberrant localization of FUS and TDP43 is associated with misfolding of SOD1 in amyotrophic lateral sclerosis. PLoS One. 2012;7(4):e35050. Epub 2012 Apr 6. [PubMed].



disease-als misfolded proteins SOD1 sporadic ALS topic-clinical
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