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Mutation-dependent Polymorphism of Cu,Zn-Superoxide Dismutase Aggregates in the Familial Form of Amyotrophic Lateral Sclerosis*

  • Yoshiaki Furukawa
    Correspondence
    To whom correspondence may be addressed: 3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522, Japan. Fax: 81-45-566-1697;
    Footnotes
    Affiliations
    From the Laboratory for Structural Neuropathology and RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan
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  • Kumi Kaneko
    Affiliations
    From the Laboratory for Structural Neuropathology and RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan
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  • Koji Yamanaka
    Affiliations
    Laboratory for Motor Neuron Disease, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan
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  • Nobuyuki Nukina
    Correspondence
    To whom correspondence may be addressed: 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Fax: 81-48-462-4796;
    Affiliations
    From the Laboratory for Structural Neuropathology and RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan
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  • Author Footnotes
    * This work was supported by Grant-in-aid for Scientific Research on Priority Areas (Research on Pathomechanisms of Brain Disorders) 17025044 (to N. N.), Grants-in-aid 20770130 (to Y. F.) and 21390274 (to K. Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Health and Labour Science Research grant (to K. Y.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. S1–S4.
    2 Present address: Dept. of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522, Japan.
Open AccessPublished:April 19, 2010DOI:https://doi.org/10.1074/jbc.M110.113597
      More than 100 different mutations in Cu,Zn-superoxide dismutase (SOD1) are linked to a familial form of amyotrophic lateral sclerosis (fALS). Pathogenic mutations facilitate fibrillar aggregation of SOD1, upon which significant structural changes of SOD1 have been assumed; in general, however, a structure of protein aggregate remains obscure. Here, we have identified a protease-resistant core in wild-type as well as fALS-causing mutant SOD1 aggregates. Three different regions within an SOD1 sequence are found as building blocks for the formation of an aggregate core, and fALS-causing mutations modulate interactions among these three regions to form a distinct core, namely SOD1 aggregates exhibit mutation-dependent structural polymorphism, which further regulates biochemical properties of aggregates such as solubility. Based upon these results, we propose a new pathomechanism of fALS in which mutation-dependent structural polymorphism of SOD1 aggregates can affect disease phenotypes.

      Introduction

      Misfolding of a protein molecule often causes its insoluble aggregation, and formation of inclusion bodies containing protein aggregates is a major pathological change in conformational diseases such as neurodegenerative disorders (
      • Chiti F.
      • Dobson C.M.
      ). Increasing evidence has suggested that structures/morphologies of protein aggregates affect disease phenotypes and that polymorphism of protein aggregates associates with phenotypic heterogeneity (
      • Bruce M.E.
      • Fraser H.
      ,
      • Jones E.M.
      • Surewicz W.K.
      ,
      • Nekooki-Machida Y.
      • Kurosawa M.
      • Nukina N.
      • Ito K.
      • Oda T.
      • Tanaka M.
      ,
      • Tanaka M.
      • Chien P.
      • Naber N.
      • Cooke R.
      • Weissman J.S.
      ). Distinct molecular structures of protein aggregates will therefore play an important role in expression of different phenotypes observed in conformational diseases.
      Among neurodegenerative disorders, more than a hundred dominant mutations in Cu,Zn-superoxide dismutase (SOD1) have been identified to cause a familial form of amyotrophic lateral sclerosis (fALS)
      The abbreviations used are: fALS
      familial form of amyotrophic lateral sclerosis
      DTT
      dithiothreitol
      ThT
      thioflavin T
      WT
      wild type
      MALDI-TOF
      matrix-assisted laser desorption ionization time-of-flight
      MS/MS
      tandem mass spectrometry
      TCEP
      tris(2-carboxyethyl)phosphine.
      (
      • Rosen D.R.
      • Siddique T.
      • Patterson D.
      • Figlewicz D.A.
      • Sapp P.
      • Hentati A.
      • Donaldson D.
      • Goto J.
      • O'Regan J.P.
      • Deng H.X.
      • et al.
      ). SOD1 binds a copper and a zinc ion and forms an intramolecular disulfide bond (
      • McCord J.M.
      • Fridovich I.
      ), all of which tightly regulate an antioxidant activity of SOD1 that converts superoxide into oxygen and hydrogen peroxide (
      • Furukawa Y.
      • Torres A.S.
      • O'Halloran T.V.
      ). SOD1 knock-out mice, however, show no fALS-like phenotypes, suggesting that an enzymatic role of SOD1 makes a minor contribution to neurodegeneration (
      • Reaume A.G.
      • Elliott J.L.
      • Hoffman E.K.
      • Kowall N.W.
      • Ferrante R.J.
      • Siwek D.F.
      • Wilcox H.M.
      • Flood D.G.
      • Beal M.F.
      • Brown Jr., R.H.
      • Scott R.W.
      • Snider W.D.
      ). Instead, SOD1 has been considered to gain toxic properties by fALS mutations, and one of those is the increased propensity of protein misfolding and aggregation (
      • Bruijn L.I.
      • Miller T.M.
      • Cleveland D.W.
      ). Among all SOD1-related fALS, a common pathological change is accumulation of detergent-insoluble SOD1 aggregates in spinal cords (
      • Bruijn L.I.
      • Miller T.M.
      • Cleveland D.W.
      ).
      So far, much effort has been made to reveal a general mechanism of how >100 fALS mutations promote SOD1 aggregation. Most of fALS mutations destabilize a native structure of SOD1 (
      • Furukawa Y.
      • O'Halloran T.V.
      ,
      • Rodriguez J.A.
      • Shaw B.F.
      • Durazo A.
      • Sohn S.H.
      • Doucette P.A.
      • Nersissian A.M.
      • Faull K.F.
      • Eggers D.K.
      • Tiwari A.
      • Hayward L.J.
      • Valentine J.S.
      ), which retards either metal binding or disulfide formation in SOD1 (
      • Furukawa Y.
      • Kaneko K.
      • Yamanaka K.
      • O'Halloran T.V.
      • Nukina N.
      ,
      • Hayward L.J.
      • Rodriguez J.A.
      • Kim J.W.
      • Tiwari A.
      • Goto J.J.
      • Cabelli D.E.
      • Valentine J.S.
      • Brown Jr., R.H.
      ,
      • Jonsson P.A.
      • Graffmo K.S.
      • Andersen P.M.
      • Brännström T.
      • Lindberg M.
      • Oliveberg M.
      • Marklund S.L.
      ). A pathogenic consequence common to all fALS mutations in SOD1 has hence been proposed to increase an intracellular fraction of a metal-free SOD1 without a disulfide (apo-SOD1SH), which is the most aggregation-prone state (
      • Furukawa Y.
      • Kaneko K.
      • Yamanaka K.
      • O'Halloran T.V.
      • Nukina N.
      ). Overall structures of SOD1 appear to be preserved even after demetallation and disulfide reduction, but a significant structural disorder in the loop regions has been identified in soluble apo-SOD1SH (
      • Hörnberg A.
      • Logan D.T.
      • Marklund S.L.
      • Oliveberg M.
      ). Such increased mobility of the loop regions in the apo-SOD1SH state has been considered to promote non-native interactions between SOD1s (
      • Teilum K.
      • Smith M.H.
      • Schulz E.
      • Christensen L.C.
      • Solomentsev G.
      • Oliveberg M.
      • Akke M.
      ), possibly leading to insoluble aggregation.
      During the aggregate formation, apo-SOD1SH has been predicted to undergo drastic structural changes, which include three-dimensional rearrangement of β-sheets called domain swapping (
      • Khare S.D.
      • Wilcox K.C.
      • Gong P.
      • Dokholyan N.V.
      ). However, there is little experimental evidence to unveil the structure(s) that an SOD1 molecule adopts in the insoluble aggregates. In addition, less attention has been paid so far on any possible differences in structural properties among SOD1 aggregates with different fALS mutations. Given that aggregate polymorphism associates with different disease phenotypes in the other neurodegenerative disorders such as prion diseases (
      • Bruce M.E.
      • Fraser H.
      ,
      • Jones E.M.
      • Surewicz W.K.
      ), an assumption on a “mutation-independent” structure of SOD1 aggregates should now be carefully examined; indeed, phenotypic heterogeneity has been reported in fALS patients with different SOD1 mutations (
      • Andersen P.M.
      • Nilsson P.
      • Keränen M.L.
      • Forsgren L.
      • Hägglund J.
      • Karlsborg M.
      • Ronnevi L.O.
      • Gredal O.
      • Marklund S.L.
      ).
      In this study, we have experimentally identified a protease-resistant core structure in the SOD1 aggregates and found that three different regions (amino acids 1–30, 90–120, and 135–153) within an SOD1 primary sequence form a scaffold of a core in the aggregates. Interestingly, fALS mutations in SOD1 can modulate interactions of those three scaffold regions in a core structure, which produces mutation-dependent structural polymorphism of SOD1 aggregates. Furthermore, such mutation-dependent core structures lead to distinct morphological and biochemical properties of fALS-mutant SOD1 aggregates. Based upon this study, we propose a new pathomechanism of fALS in which mutation-dependent structures of SOD1 aggregates can affect the disease phenotypes.

      DISCUSSION

      Mutations in SOD1 have been identified as a cause of fALS, and insoluble aggregation of mutant SOD1 proteins is a common pathological change in this disease (
      • Bruijn L.I.
      • Miller T.M.
      • Cleveland D.W.
      ). We previously proposed a molecular mechanism of SOD1 aggregation, in which either metallation or disulfide formation in SOD1 completely suppresses its fibrillar aggregation (
      • Furukawa Y.
      • Kaneko K.
      • Yamanaka K.
      • O'Halloran T.V.
      • Nukina N.
      ). Furthermore, several groups including us have reported that a role of fALS mutations in facilitating SOD1 aggregation is to decrease the affinity for zinc ions and retard disulfide formation (
      • Furukawa Y.
      • Kaneko K.
      • Yamanaka K.
      • O'Halloran T.V.
      • Nukina N.
      ,
      • Hayward L.J.
      • Rodriguez J.A.
      • Kim J.W.
      • Tiwari A.
      • Goto J.J.
      • Cabelli D.E.
      • Valentine J.S.
      • Brown Jr., R.H.
      ,
      • Jonsson P.A.
      • Graffmo K.S.
      • Andersen P.M.
      • Brännström T.
      • Lindberg M.
      • Oliveberg M.
      • Marklund S.L.
      ). Although significant structural changes of an SOD1 molecule have been assumed upon aggregate formation, it remains an open question how SOD1 changes its structure to form fibrillar aggregates. Here, we have identified a core structure in SOD1 aggregates and extended our SOD1 aggregation model to include a polymorphism of fALS-mutant SOD1 aggregates (Fig. 6).
      Figure thumbnail gr6
      FIGURE 6Our proposed model to describe mutation-dependent structural polymorphism of SOD1 aggregates. In secondary structural representation of SOD1 (left), regions A–C are colored red, blue and green, respectively. Rearrangement of these regions results in the formation of a core, and at least three different combinations of interactions among regions A–C are possible (middle). An exact alignment of β-sheets in the aggregates remains unknown; therefore, alignment of regions A–C in each schematic representation (middle) is still speculative. Interactions among aggregation core regions determine overall morphologies (right) and biochemical properties of SOD1 aggregates. Inset, average disease duration is plotted against average onset of disease. Data were taken from Refs.
      • Prudencio M.
      • Hart P.J.
      • Borchelt D.R.
      • Andersen P.M.
      ,
      • Wang Q.
      • Johnson J.L.
      • Agar N.Y.
      • Agar J.N.
      . Type I/III, II, and IV aggregates are colored by black, red, and blue, respectively.

      Molecular Mechanism to Produce Structurally Distinct SOD1 Aggregates

      Based upon this study, it is likely that SOD1 aggregation occurs through unique and non-native interaction among three major parts of SOD1 (regions A–C, see FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6). As mentioned in our previous study (
      • Furukawa Y.
      • Kaneko K.
      • Yamanaka K.
      • O'Halloran T.V.
      • Nukina N.
      ), a CD spectrum has suggested that SOD1 aggregates are composed mainly of β-sheet structures. Soluble apo-SOD1SH is also rich in β-sheets with an immunoglobulin fold (almost the same with Fig. 1B) but exhibits a CD spectrum different from that of SOD1 aggregates, suggesting significant structural changes of SOD1 upon its aggregation (
      • Furukawa Y.
      • Kaneko K.
      • Yamanaka K.
      • O'Halloran T.V.
      • Nukina N.
      ). Indeed, molecular dynamics simulations of WT SOD1 have proposed a domain swapping model in which drastic rearrangement of β-sheets leads to aggregation (
      • Khare S.D.
      • Wilcox K.C.
      • Gong P.
      • Dokholyan N.V.
      ). In this model, non-native interactions possibly leading to aggregation occur among Ala1–Glu40, Glu100–His120, and the C termini, which correspond to regions A–C in this study, respectively (Fig. 1A). In silico and our in vitro/in vivo results have thus indicated that aggregation of SOD1 occurs not through a simple pile of apo-SOD1SH monomers but through rearrangement of β-sheets in an SOD1 molecule to realize non- native interactions among regions A–C (Fig. 6).
      When a core in the aggregate contains fALS mutations (i.e. A4V, L126X, L144F, and I149T), non-native interactions among regions A–C will be affected. A computer algorithm, TANGO (
      • Fernandez-Escamilla A.M.
      • Rousseau F.
      • Schymkowitz J.
      • Serrano L.
      ), predicts that A4V and L144F mutations increase aggregation propensities of regions A and C, respectively. These changes may describe formation of a distinct aggregation core in A4V (Fig. 2) or significantly high T½ of L144F aggregates (Fig. 5E). In I149T, an aggregation propensity of region C is predicted to significantly decrease (
      • Fernandez-Escamilla A.M.
      • Rousseau F.
      • Schymkowitz J.
      • Serrano L.
      ), which is consistent with no involvement of region C in the core structure of I149T aggregates (Fig. 2). In addition, SOD1 forms type IV fibrils when truncated at Leu126 (L126X), confirming that region C is dispensable for formation of the aggregation core. However, it remains obscure how a distinct aggregation core is realized in the other SOD1s in which fALS mutations are located outside the core regions. Recent studies have suggested mutation-dependent structural dynamics of SOD1 (
      • Molnar K.S.
      • Karabacak N.M.
      • Johnson J.L.
      • Wang Q.
      • Tiwari A.
      • Hayward L.J.
      • Coales S.J.
      • Hamuro Y.
      • Agar J.N.
      ,
      • Shaw B.F.
      • Durazo A.
      • Nersissian A.M.
      • Whitelegge J.P.
      • Faull K.F.
      • Valentine J.S.
      ), which may be an important determinant for unique interactions among the core regions. We thus suppose that fALS mutations can directly or indirectly control non-native interactions among regions A–C and lead to formation of a distinct structure of SOD1 fibrils. Furthermore, in a native state of SOD1, regions A–C contain amino acid residues involved in dimerization, metal binding, and disulfide formation, respectively (Fig. 6). These post-translational processes therefore prohibit non-native interactions among regions A–C and suppress SOD1 aggregation, which is consistent with our previous studies (
      • Furukawa Y.
      • Kaneko K.
      • Yamanaka K.
      • O'Halloran T.V.
      • Nukina N.
      ).

      Potential Roles of Structural Polymorphism of SOD1 Aggregates in Heterogeneous Phenotypes and Pathologies of fALS

      Increasing evidence has suggested that a molecular structure of protein aggregates is tightly linked to a phenotype (
      • Tanaka M.
      • Chien P.
      • Naber N.
      • Cooke R.
      • Weissman J.S.
      ). For example, in transmissible spongiform encephalopathies, multiple strains with distinct disease phenotypes exist (
      • Bruce M.E.
      • Fraser H.
      ), and the emergence of such strains has been described by structural differences in fibrillar aggregates of a prion protein (
      • Jones E.M.
      • Surewicz W.K.
      ). Another example can be found in a neurodegenerative disease collectively called tauopathies, in which a microtubule-binding protein, Tau, forms insoluble fibrillar aggregates (
      • Lee V.M.
      • Goedert M.
      • Trojanowski J.Q.
      ). Tauopathies include Alzheimer disease, Pick disease, and others; in each of these tauopathies, Tau forms fibrils with distinct morphologies such as paired helical filaments in Alzheimer disease and straight filaments in Pick disease (
      • Lee V.M.
      • Goedert M.
      • Trojanowski J.Q.
      ). Importantly, albeit with less attention, different fALS mutations in SOD1 also associate with distinct disease phenotypes such as severity and age of disease onset (
      • Andersen P.M.
      • Nilsson P.
      • Keränen M.L.
      • Forsgren L.
      • Hägglund J.
      • Karlsborg M.
      • Ronnevi L.O.
      • Gredal O.
      • Marklund S.L.
      ,
      • Prudencio M.
      • Hart P.J.
      • Borchelt D.R.
      • Andersen P.M.
      ,
      • Wang Q.
      • Johnson J.L.
      • Agar N.Y.
      • Agar J.N.
      ). For example, duration of the disease depends upon fALS mutations and ranges from less than a year to more than 20 years (
      • Wang Q.
      • Johnson J.L.
      • Agar N.Y.
      • Agar J.N.
      ). Because symptomatic changes follow appearance of SOD1 inclusions in fALS patients and rodent models (
      • Turner B.J.
      • Talbot K.
      ), a possible mutation dependence of SOD1 aggregate structures may be relevant in understanding phenotypic heterogeneity in fALS.
      Among SOD1 mutations examined here, there seems no correlation of disease onset with disease duration (Fig. 6, inset; Table 1) (
      • Prudencio M.
      • Hart P.J.
      • Borchelt D.R.
      • Andersen P.M.
      ,
      • Wang Q.
      • Johnson J.L.
      • Agar N.Y.
      • Agar J.N.
      ). Based upon our structural classification of SOD1 aggregates, type IV aggregates seem to associate with relatively early onset and short duration of disease (Fig. 6, inset). Also, later disease-onset would be expected in type I/III mutant (L144F and G85R) and WT, but obviously the effects of the structural polymorphism of aggregates on disease phenotypes still need further investigations. For example, A4V and H46R mutations are well known to exhibit very severe and mild progression of the disease, respectively (
      • Wang Q.
      • Johnson J.L.
      • Agar N.Y.
      • Agar J.N.
      ), but these two mutant SOD1 aggregates are categorized into the same type (type II) in our classification. Many biophysical factors such as affinity for metal ions (
      • Furukawa Y.
      • Kaneko K.
      • Yamanaka K.
      • O'Halloran T.V.
      • Nukina N.
      ) as well as protein structural stability (
      • Furukawa Y.
      • O'Halloran T.V.
      ) regulate the kinetics of SOD1 aggregation, which would further affect some of the disease phenotypes (
      • Lindberg M.J.
      • Tibell L.
      • Oliveberg M.
      ). Our in vitro and in vivo data therefore support roles of fALS mutations in structural and biochemical properties of SOD1 aggregates but also show that aggregate structure is not the only decisive factor regulating disease phenotypes.
      Furthermore, it is notable that morphologies of SOD1 inclusions are variable among transgenic mice expressing human SOD1 with different fALS mutations (
      • Turner B.J.
      • Talbot K.
      ). For example, formation of SOD1 inclusions is a major change in H46R and G85R SOD1 transgenic mice, although in G37R and G93A SOD1 severe mitochondrial damage is observed with lesser amounts of SOD1 inclusions (
      • Nagai M.
      • Aoki M.
      • Miyoshi I.
      • Kato M.
      • Pasinelli P.
      • Kasai N.
      • Brown Jr., R.H.
      • Itoyama Y.
      ,
      • Watanabe M.
      • Dykes-Hoberg M.
      • Culotta V.C.
      • Price D.L.
      • Wong P.C.
      • Rothstein J.D.
      ). In addition, thioflavin S, a fluorescent dye upon binding with amyloid-like fibrils, can stain SOD1 inclusions in G37R, G85R, and G93A (
      • Furukawa Y.
      • Kaneko K.
      • Yamanaka K.
      • O'Halloran T.V.
      • Nukina N.
      ,
      • Wang J.
      • Xu G.
      • Gonzales V.
      • Coonfield M.
      • Fromholt D.
      • Copeland N.G.
      • Jenkins N.A.
      • Borchelt D.R.
      ) but not L126X transgenic mice (
      • Wang J.
      • Xu G.
      • Li H.
      • Gonzales V.
      • Fromholt D.
      • Karch C.
      • Copeland N.G.
      • Jenkins N.A.
      • Borchelt D.R.
      ). Also in a Caenorhabditis elegans model, morphologically and biophysically distinct classes of mutant SOD1 aggregates have been found (
      • Gidalevitz T.
      • Krupinski T.
      • Garcia S.
      • Morimoto R.I.
      ); FRAP analysis shows higher mobility of L126X aggregates than those of G85R and G93A aggregates in the body wall muscle cells. All these in vivo studies have thus suggested that fALS mutations affect structural properties of pathological SOD1 aggregates, and this study will provide a molecular basis to describe mutation-dependent structural and biochemical properties of SOD1 aggregates.
      In summary, we have revealed mutation-dependent structural polymorphism of SOD1 aggregates. Multiple regions in a single SOD1 protein are responsible for formation of a core upon its fibrillar aggregation. We have further found that morphologies as well as biochemical properties of SOD1 aggregates are affected by a combination of interactions among the aggregate core regions (Fig. 6). In analogy with a protein folding process, we suppose that variable structures of protein aggregates are possible by alternatively or non-natively “folding” the peptide fragments involved in the core of aggregates. Disease-causing mutations would regulate such non-native folding pathways to form a distinct structure of protein aggregates and exert distinct cytotoxicity. Given that several regions within a single protein sequence are often predicted to have high aggregation propensities (
      • Fernandez-Escamilla A.M.
      • Rousseau F.
      • Schymkowitz J.
      • Serrano L.
      ), our proposing model (Fig. 6) would be a general mechanism producing structural polymorphism in the aggregates of many other proteins.

      Acknowledgments

      We thank Dr. Shoji Watanabe for fruitful discussions. We also thank the Support Unit for Bio-material Analysis; RIKEN BSI Research Resources Center, especially Masaya Usui and Kaori Otsuki, for mass analysis; and Yuriko Sakamaki for electron micrographic observations.

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