**5. Conclusion**

Numerous studies have revealed that the effects of fALS-associated mutations on the folding, unfolding and aggregation of different forms of SOD1 are highly complex. Mutations can alter both equilibrium stability, in terms of the energetics of dimer dissociation, monomer intermediate stability, and metal binding, and kinetic stability, in terms of the rates of interconversion between various SOD1 species (Section 3). As a consequence, the populations of various aggregation prone species may be increased for different mutations, and this may give rise to different aggregate structures.

There have been a number of attempts to identify the relationships between the effects of the mutations and ALS disease characteristics. In particular, disease duration, which is characteristic for patients carrying a given SOD1 mutation, has been used as a measure of the toxicity of each fALS-associated SOD1 mutation. Early work focused on the loss of superoxide dismutase activity, and increased oxidative stress as the common underlying cause of disease (Valentine et al. 2005). Subsequently, the focus shifted to the toxic gain of function for mutant SOD1, both aberrant enzymatic SOD1 activity, or increased SOD1 aggregation, the latter being the predominant focus of this review. Owing to the high stability and lower aggregation propensity of the holoS-S form, many studies have focused on characterizing the stability and aggregation mechanisms of the more immature, metal deficient SOD1 forms. However, recent work suggests that disease duration does not correlate strongly with the stability of the apoS-S form of mutant SOD1 (Bystrom et al. 2010). This observation was rationalized by considering the role of factors beyond destabilization in modulating aggregation, such as changes in protein net charge and hydrogen bonding. An interesting study by Wang et al. reported that predicted aggregation propensity, based on the physicochemical properties of the polypeptide sequence (Chiti et al. 2003) combined with the stability of mutant apoS-S SOD1 in a summative score and weighted towards mutants with more patient data, correlated fairly well with fALS disease durations (Wang et al. 2008). On the other hand, recent work by Vassall et al. demonstrated that observed aggregation of the apo2SH form is not correlated with disease duration (Vassall et al. 2011). Collectively, these studies demonstrate that multiple factors including protein stability, dynamics, and biophysical characteristics are likely to play a role in modulating SOD1 aggregation, and that fALS phenotypic characteristics are not likely to be fully explained by the aggregation behaviour of any one form of SOD1.

Aggregation studies on holoS-S, apoS-S, and apo2SH SOD1 mutants have identified multiple mechanisms and aggregate morphologies (Section 4 and Figure 4). HoloS-S SOD1, widely thought believed to be much less susceptible to aggregation, has nevertheless been shown to form amorphous aggregates in a nucleation-dependent manner where the lag phase may involve metal loss or monomerization (Hwang et al. 2010). ApoS-S SOD1 may form amyloid- or non-amyloid-like aggregates with or without disulphide cross-linking depending on the solution conditions, and apo2SH SOD1 has been found to adopt the most diverse range of aggregate morphologies, including soluble aggregates under physiologically relevant conditions which may be particularly neurotoxic (Caughey and Lansbury 2003). Considering the influence of SOD1 mutations on the stability, unfolding and folding patterns of all forms of SOD, together with the diverse mechanisms of aggregation, different mutations may be influencing the protein in variable ways, resulting in a wide spectrum of effects. This diversity is likely to play a significant role in the variable disease courses for fALS patients with SOD1 mutations. Ultimately, the role of SOD1 in ALS may be similar to the roles of other globular, oligomeric proteins in misfolding diseases such as: transthyretin in familial amyloidotic polyneuropathy and senile systemic amyloidosis, lysozyme in hereditary non-neuropathic systemic amyloidosis, immunoglobulin light chain in monoclonal protein systemic amyloidosis, prion protein in Kreutzfeld Jakob, and serpins in serpinopathies (Ohnishi and Takano 2004; Harrison et al. 2007). In these diseases mutations are generally destabilizing, but the extent of destabilization of monomer versus subunit interfaces varies widely. The role of SOD1 in disease may be further complicated by the potential aberrant enzymatic activity of misfolded and/or aggregated species which could cause oxidative damage. In addition, it is worth considering the different roles of various types of SOD1 aggregate structures, or contributions of aberrant activity and the effects of these on other cellular components, at different stages throughout the disease course of ALS. For these reasons, it is important that future studies continue to consider the possible roles of multiple forms of SOD1 mutants in modulating the formation of different aggregate structures (Figure 4). A combination of further *in vitro* and *in vivo* studies of folding and aggregation will be critical for untangling the role of toxic aggregation in the syndrome of ALS.

### **6. Acknowledgements**

We thank the ALS Society of Canada, Muscular Dystrophy Canada, and Canadian Institutes of Health Research for their financial support. Furthermore, we would like to express our appreciation to all the members of the Meiering group for their insightful and meaningful discussions, in particular Aron Broom, Martin Smith, Ming Sze Tong and Sarah M. Sullivan.

### **7. References**

288 Amyotrophic Lateral Sclerosis

Numerous studies have revealed that the effects of fALS-associated mutations on the folding, unfolding and aggregation of different forms of SOD1 are highly complex. Mutations can alter both equilibrium stability, in terms of the energetics of dimer dissociation, monomer intermediate stability, and metal binding, and kinetic stability, in terms of the rates of interconversion between various SOD1 species (Section 3). As a consequence, the populations of various aggregation prone species may be increased for

There have been a number of attempts to identify the relationships between the effects of the mutations and ALS disease characteristics. In particular, disease duration, which is characteristic for patients carrying a given SOD1 mutation, has been used as a measure of the toxicity of each fALS-associated SOD1 mutation. Early work focused on the loss of superoxide dismutase activity, and increased oxidative stress as the common underlying cause of disease (Valentine et al. 2005). Subsequently, the focus shifted to the toxic gain of function for mutant SOD1, both aberrant enzymatic SOD1 activity, or increased SOD1 aggregation, the latter being the predominant focus of this review. Owing to the high stability and lower aggregation propensity of the holoS-S form, many studies have focused on characterizing the stability and aggregation mechanisms of the more immature, metal deficient SOD1 forms. However, recent work suggests that disease duration does not correlate strongly with the stability of the apoS-S form of mutant SOD1 (Bystrom et al. 2010). This observation was rationalized by considering the role of factors beyond destabilization in modulating aggregation, such as changes in protein net charge and hydrogen bonding. An interesting study by Wang et al. reported that predicted aggregation propensity, based on the physicochemical properties of the polypeptide sequence (Chiti et al. 2003) combined with the stability of mutant apoS-S SOD1 in a summative score and weighted towards mutants with more patient data, correlated fairly well with fALS disease durations (Wang et al. 2008). On the other hand, recent work by Vassall et al. demonstrated that observed aggregation of the apo2SH form is not correlated with disease duration (Vassall et al. 2011). Collectively, these studies demonstrate that multiple factors including protein stability, dynamics, and biophysical characteristics are likely to play a role in modulating SOD1 aggregation, and that fALS phenotypic characteristics are not likely to be fully explained by

Aggregation studies on holoS-S, apoS-S, and apo2SH SOD1 mutants have identified multiple mechanisms and aggregate morphologies (Section 4 and Figure 4). HoloS-S SOD1, widely thought believed to be much less susceptible to aggregation, has nevertheless been shown to form amorphous aggregates in a nucleation-dependent manner where the lag phase may involve metal loss or monomerization (Hwang et al. 2010). ApoS-S SOD1 may form amyloid- or non-amyloid-like aggregates with or without disulphide cross-linking depending on the solution conditions, and apo2SH SOD1 has been found to adopt the most diverse range of aggregate morphologies, including soluble aggregates under physiologically relevant conditions which may be particularly neurotoxic (Caughey and Lansbury 2003). Considering the influence of SOD1 mutations on the stability, unfolding and folding patterns of all forms of SOD, together with the diverse mechanisms of aggregation, different mutations may be influencing the protein in variable ways, resulting in a wide spectrum of effects. This diversity is likely to play a significant role in the variable disease courses for fALS patients with SOD1 mutations. Ultimately, the role of SOD1 in ALS

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**5. Conclusion** 


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**12** 

*Japan* 

Seiichi Nagano

**Oxidative Modifications of Cu, Zn-Superoxide** 

*Department of Peripheral Nervous System Research, National Institute of Neuroscience,* 

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative disease of motor neurons. About 10 % of ALS cases are affected in a familial trait, a subset of which is caused by the mutation of *Cu, Zn-superoxide dismutase (SOD1)* gene (Rosen et al., 1993). Since the identification of the gene for familial ALS, research emphasis for ALS has been placed on uncovering the pathogenic mechanism of motor neuronal death by the disease-causing mutant SOD1. So far over 150 different mutations of *SOD1* gene have been found in familial ALS patients, and they are scattered throughout the entire sequence of the gene regardless of specific functional domains. Transgenic mice that express mutant SOD1, but not wild type SOD1 nor SOD1 knockout mice, develop motor neuron disease, often while retaining normal dismutase activity (Gurney et al., 1994; Reaume et al., 1996). It means that mutant SOD1 gains a new aberrant toxic function apart from the primary enzymatic function of the protein, which has remained uncertain to date. Although the nature of mutant SOD1 toxicity has not been fully determined, conformational abnormalities of mutant SOD1 protein are deeply involved in the pathogenesis of familial ALS (Chattopadhyay & Valentine, 2009). Moreover, recent studies suggest that the phenotype of sporadic ALS also might be regulated by the conformational change of wild type SOD1 (Bosco et al., 2010). I review the recent concept of neuronal toxicity by oxidatively-modified SOD1, which is closely related

SOD1 is a major metal-binding enzyme expressed constitutively in tissues, and converts pro-oxidant superoxide radicals to hydrogen peroxide and oxygen (Valentine et al., 2005). In a metal-coordinated state, SOD1 forms homodimer to accomplish its full enzymatic activity. Each subunit coordinates one atom each of Cu and Zn. Cu is necessary for the enzymatic activity, whereas Zn mainly works to maintain the stable structure of the protein. Because Cu is catalytically redox-active and has a potential to oxidize proteins including SOD1 itself, inappropriate reactivity of Cu coordinated in SOD1 can underlie the conformational change of mutant SOD1. Inversely, conformational change in mutant SOD1 may increase the

**1. Introduction** 

to its conformational change, in ALS pathogenesis.

**2. Cu-mediated oxidative toxicity by mutant SOD1** 

**Dismutase (SOD1) – The Relevance to** 

**Amyotrophic Lateral Sclerosis (ALS)** 

*National Center of Neurology and Psychiatry* 

