An ALS Cautionary Tale: Don’t Blame It All on Apo-SOD1

Cells dress up the enzyme SOD1—the cause of some cases of inherited amyotrophic lateral sclerosis—in a variety of ways. They accessorize it with copper and zinc ions. They tie it together with an intramolecular disulfide bond. They link it with another well-dressed SOD1 to form a dimer. All those modifications mean there are many kinds of SOD1 that could lead to ALS—but lately studies have focused on the most immature, naked Apo-SOD1 as the variety most likely to form aggregates and make motor neurons sick. A study posted in the January 21 Proceedings of the National Academy of Sciences USA online questions that focus. Researchers from the University of Waterloo in Ontario, led by Elizabeth Meiering, report that Apo-SOD1 mutants do not aggregate much at all.

More than 100 different SOD1 mutations have been linked to ALS. Some result in rapidly progressing, severe disease; others allow people to survive upwards of a decade following diagnosis. If unstable, aggregated Apo-SOD1 causes ALS, scientists reason; then the more unstable a SOD1 mutant is, the faster it should fell its victims. Meiering and joint first authors Helen Stubbs and Kenrick Vassall (now at the University of Guelph in Ontario) analyzed Apo-SOD1 mutants in vitro and attempted to align their physical properties with the severity of disease they cause in people.

Given Apo-SOD1’s reputation as an unstable aggregator (see ARF related news story on Banci et al., 2009 and ARF related news story on Nordlund et al., 2009), the authors did not expect they could squeeze much data out of the protein using biophysics techniques to analyze protein stability and the formation of small, soluble oligomers. They were pleasantly surprised. “The protein in this most immature form is marginally stable,” Meiering said.

SOD1 contains four cysteines. Two link up in an intramolecular disulfide bridge; the other two, at positions 6 and 111, are usually reduced. But as aggregates form, these free cysteines may hook up with the cysteines on other SOD1 molecules, promoting formation of multiple-SOD1 species. To avoid this confounding factor, Meiering and colleagues used a pseudo-wild-type (pseudo-WT) construct in which those free cysteines are swapped for a serine and alanine. This SOD1 is more stable than normal; while the wild-type Apo-SOD1 is approximately 75 percent folded at equilibrium, the pseudo-WT construct is 95 percent folded.

Some scientists contacted by ARF criticized the use of pseudo-WT SOD1. Nikolay Dokholyan and Rachel Redler, of the University of North Carolina at Chapel Hill, suggested in an e-mail that since the free cysteines are likely involved in aggregation, eliminating them is inappropriate for studies of inclusion formation (see full comment below). Meiering explained that she wanted to examine SOD1 aggregation in a state mimicking the earliest stages of disease, before the protein is likely to encounter oxidative stress and form unnatural disulfide bridges via the cysteines in question (see ARF related news story on Karch et al., 2009).

To analyze the effects of different mutations on stability and aggregation, the researchers piggybacked several kinds of disease-linked SOD1 mutations onto the pseudo-WT construct: the substituted amino acids at the dimer interface (A4V, A4T, A4S, and V148I); in a β-barrel motif (G37R and H43R); in metal-binding regions (H46R and G85R); in other structural elements (G93R, G93S, G93A, G93D); and a salt bridge participant (E100G).

At physiological temperatures, most of the mutants were unstable—more likely to be unfolded than the pseudo-WT. However, H46R and V148I were more likely to be folded than the pseudo-WT, suggesting they are unusually stable. Therefore, not all SOD1 mutations destabilize Apo-SOD1, even though they cause disease. The finding suggests that unstable Apo-SOD1 must not be the cause—at least, not the only cause—for ALS.

Could a greater inclination to aggregate in the Apo form be the common factor that pathogenic SOD1 mutants share? To pursue this hunch, the scientists used ultracentrifugation and sedimentation to examine how large groups of SOD1 molecules grew. The pseudo-WT, in Apo form, tended to hang out by itself with a molecular weight of 15 kDa, monomer size. But some mutations formed larger structures: A4V and E100G weighed in at somewhere between 1 and 2 monomers, and H43R was closer to the size of a dimer. Using light scattering, the scientists determined that, over a course of days, both A4V and H43R grew into aggregates as large as 100 nm across.

But when the scientists attempted to correlate aggregation with disease duration in people with the mutations, they found no pattern. Having the least stable, most aggregation-prone Apo-SOD1 did not lead to a faster-progressing disease course.

The current analysis of Apo-SOD1 is the most complete to date, wrote Jeffrey Agar of Brandeis University in Waltham, Massachusetts, when asked by ARF (see full comment below). However, the results clearly contradict those of scientists who found that Apo-SOD1 is unstable and aggregates readily, he added.

Meiering and colleagues suggested that the different results come down to whether the protein was shaken or still. “When you shake protein, you can make pretty much any protein aggregate,” she said. Thus, scientists can find aggregation in vitro that would be unlikely to occur in vivo. In these experiments, the Waterloo researchers preferred to mimic natural conditions. “We were trying to be as gentle as possible,” Meiering said—and that gentleness meant less aggregation.

The study suggests Apo-SOD1 is not necessarily the place to look for the cause of ALS. Instead, Meiering advised, researchers should consider all of the protein’s myriad stages of maturation as potential unstable aggregators. Redler and Dokholyan also noted that Apo-SOD1 is not completely off the hook—for example, cellular factors could team up with the protein to promote aggregation in a manner that would not occur in pure solution.—Amber Dance

Comments

  1. In this article, the authors address the contribution of mutant SOD1 stability loss and aggregation to disease severity in ALS. Specifically, they study the propensity of SOD1 mutants to form soluble aggregate species, which have recently been suggested to be primary cytotoxins in ALS. The authors find a weak correlation between the propensity of mutants to form small soluble aggregates and disease severity (duration), which they take as evidence that soluble SOD1 aggregates "[do] not play a dominant role in modulating disease." It is important to note that the evidence presented here does not contradict the hypothesis that soluble SOD1 aggregates are primary contributors to ALS pathogenesis. Rather, the lack of correlation between in vitro aggregation propensity and disease duration underscores the impact of non-genetic factors (e.g., the cellular redox environment) on SOD1 stability and aggregate formation in vivo. In this study, nearly all of the SOD1 mutants studied were in a pseudo-wild-type background in which both free cysteines (at positions 6 and 111) are mutated to serine. Several groups have reported that SOD1's free cysteines stabilize aggregates through the formation of non-native disulfide crosslinking, and our group has found that post-translational modification at Cys-111 destabilizes the native structure. Since free cysteines are apparently involved in SOD1 aggregation, it may not be surprising that the behavior of SOD1 mutants lacking these residues does not correlate with clinical severity of disease.

    View all comments by Rachel Redler

  2. There is comparatively little quantitative biophysical data on the completely immature SOD1 (disulfide reduced and metal deficient). This paper, which characterizes 12 ALS-associated variants, begins to fill in that gap. The authors then compare their biophysical data to patient survival times. The potential benefits of doing so include developing a better model of proteinopathies in general and ALS in particular, and informing structure-based drug design efforts (i.e., to help researchers avoid targeting innocuous structural defects).
    These authors found no correlation between the stability or aggregation of immature SOD1 variants and ALS prognosis.

    These results, which on the one hand represent the most comprehensive analysis to date, on the other hand are acknowledged to conflict with studies by other groups implicating disulfide reduced SOD1 in ALS pathogenesis. So what gives? Fortunately, all of these groups are competent biochemists—when they do measure the same variant in the same way, they get comparable results. For better or for worse, however, the same variants and same techniques are not always employed, nor is the same analysis. The devil may be hiding somewhere in these details. With funding levels as low as they've been in recent history, it's not possible for all of these groups to analyze the ~130 ALS-associated SOD1 variants required for a truly comprehensive study. I would like to think that if they could, a consistent picture would result.

    View all comments by Jeffrey Agar

  3. The paper by Vassall et al. reminds us that finding a common, toxic characteristic (e.g., aggregation propensity) amongst ALS-linked SOD1 mutants is not straightforward. Moreover, the extrapolation of in vitro aggregation kinetics to human disease pathogenesis may fall short because it is difficult to fully recapitulate a complex, in vivo environment within a test tube. The lack of correlation between mutant SOD1 aggregation propensity and ALS disease severity may also reflect that the production of insoluble SOD1 aggregates does not contribute to disease pathogenesis. In fact, much focus has been placed on investigating pre-aggregated forms of neurodegenerative disease associated proteins, such as misfolded soluble species and oligomeric species that are believed to be on-pathway to the large, insoluble aggregates observed in end-stage autopsy patient tissues. Soluble forms (i.e., not higher-order aggregates) of mutant and modified SOD1 proteins have been shown to form aberrant protein interactions, mislocalize within the cell, and inappropriately activate cellular pathways. Perhaps a correlation between one of these parameters and ALS disease severity will be revealed upon further analysis. The results of Vassall et al. underscore the need to consider factors other than in vitro protein aggregation propensities in the context of neurodegeneration. As the authors note, "additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS."

    View all comments by Daryl Bosco

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References

News Citations

  1. No Metal, No Stability: Structure of Apo SOD1
  2. Frustrated ALS Enzyme: SOD1 Sacrifices Structural Stability for Function
  3. Following SOD1 Biochemistry in ALS from Start to Finish

Paper Citations

  1. Banci L, Bertini I, Boca M, Calderone V, Cantini F, Girotto S, Vieru M. Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants. Proc Natl Acad Sci U S A. 2009 Apr 28;106(17):6980-5. PubMed.
  2. Nordlund A, Leinartaite L, Saraboji K, Aisenbrey C, Gröbner G, Zetterström P, Danielsson J, Logan DT, Oliveberg M. Functional features cause misfolding of the ALS-provoking enzyme SOD1. Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9667-72. PubMed.
  3. Karch CM, Prudencio M, Winkler DD, Hart PJ, Borchelt DR. Role of mutant SOD1 disulfide oxidation and aggregation in the pathogenesis of familial ALS. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7774-9. PubMed.

Further Reading

Papers

  1. 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. Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nat Neurosci. 2010 Nov;13(11):1396-403. PubMed.
  2. Chattopadhyay M, Durazo A, Sohn SH, Strong CD, Gralla EB, Whitelegge JP, Valentine JS. Initiation and elongation in fibrillation of ALS-linked superoxide dismutase. Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18663-8. PubMed.
  3. Karch CM, Borchelt DR. A limited role for disulfide cross-linking in the aggregation of mutant SOD1 linked to familial amyotrophic lateral sclerosis. J Biol Chem. 2008 May 16;283(20):13528-37. PubMed.
  4. Soto C, Estrada LD. Protein misfolding and neurodegeneration. Arch Neurol. 2008 Feb;65(2):184-9. PubMed.
  5. Zetterström P, Stewart HG, Bergemalm D, Jonsson PA, Graffmo KS, Andersen PM, Brännström T, Oliveberg M, Marklund SL. Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in spinal cords throughout life in murine ALS models. Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):14157-62. PubMed.
  6. Tiwari A, Hayward LJ. Familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase are susceptible to disulfide reduction. J Biol Chem. 2003 Feb 21;278(8):5984-92. PubMed.
  7. Oztug Durer ZA, Cohlberg JA, Dinh P, Padua S, Ehrenclou K, Downes S, Tan JK, Nakano Y, Bowman CJ, Hoskins JL, Kwon C, Mason AZ, Rodriguez JA, Doucette PA, Shaw BF, Selverstone Valentine J. Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase. PLoS One. 2009;4(3):e5004. PubMed.
  8. Wang J, Farr GW, Zeiss CJ, Rodriguez-Gil DJ, Wilson JH, Furtak K, Rutkowski DT, Kaufman RJ, Ruse CI, Yates JR, Perrin S, Feany MB, Horwich AL. Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS. Proc Natl Acad Sci U S A. 2009 Feb 3;106(5):1392-7. PubMed.
  9. Furukawa Y, Kaneko K, Yamanaka K, O'Halloran TV, Nukina N. Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in the familial form of amyotrophic lateral sclerosis. J Biol Chem. 2008 Aug 29;283(35):24167-76. PubMed.
  10. Khare SD, Caplow M, Dokholyan NV. The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2004 Oct 19;101(42):15094-9. PubMed.

Primary Papers

  1. Vassall KA, Stubbs HR, Primmer HA, Tong MS, Sullivan SM, Sobering R, Srinivasan S, Briere LA, Dunn SD, Colón W, Meiering EM. Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS. Proc Natl Acad Sci U S A. 2011 Feb 8;108(6):2210-5. PubMed.

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