**2. Conformational change and copper‐mediated oxidative toxicity of mutant SOD1**

SOD1 is a metal‐binding antioxidant enzyme expressed ubiquitously in the body, and it functions to convert prooxidant superoxide anions to hydrogen peroxide and oxygen to remove the oxidative stress [3]. SOD1 forms a homodimer to accomplish its full enzymatic activity. Each subunit binds one atom each of copper and zinc. Copper works for the enzymatic activity, whereas zinc has an important role in maintaining the stable structure of SOD1 protein.

More than 150 different mutations of *SOD1* gene have been found in familial ALS patients so far, and they are scattered throughout the entire coding sequence of the gene regardless of specific functional domains. The enzymatic activity of mutant SOD1 is not necessarily reduced compared to that of wild‐type SOD1 [4]. Moreover, mice deficient of *Sod1* gene do not cause symptoms of motor neuronal dysfunction, whereas transgenic mice that express mutant SOD1, but not wild‐type SOD1, develop progressive motor paralysis and degeneration of motor neurons depending on the expression level of mutant SOD1 in the spinal cord [5, 6]. It means that the pathogenic mechanism of mutant SOD1 for the degeneration of motor neurons is derived from the gain of a specific aberrant function rather than a decrease in the enzyme activity of the protein.

What is the determinant of motor neuronal toxicity by mutant SOD1? Although the nature of mutant SOD1 toxicity has not been fully elucidated, conformational abnormality, that is, misfolding, of mutant SOD1 protein is deeply involved in the pathogenesis of familial ALS [7]. Misfolded SOD1 is subject to dissociate into monomers, which binds together abnormally to form oligomers or further high molecular weight aggregates in cells. Abnormal protein accumulation in neurons can impair their important cellular functions such as axonal transport [8] and degradation machinery of proteins [9]. SOD1‐positive inclusion bodies have been actually detected in degenerating motor neurons in the spinal cords of familial ALS patients and mutant SOD1 transgenic mice [10, 11]. Therefore, it is important to understand the common mode of conformational change in mutant SOD1, which will lead to suppress the onset of ALS derived from SOD1 mutations.

Once SOD1 is misfolded, copper or zinc bound to the subunit of SOD1 is also prone to be exposed from the compact dimer structure. Because copper is catalytically redox‐active, abnormal chemical reactions can occur to generate reactive oxygen (ROS) or nitrogen (RNS) species apart from the original SOD1 activity. Mutant SOD1, unlike wild‐type SOD1, has a potential to generate ROS and RNS such as hydroxyl radicals [12, 13] and peroxynitrite [14] in a copper‐dependent manner *in vitro*, which can be inhibited by copper chelators in cultured cells [15].

mechanism of motor neuronal death by the disease‐causing mutant SOD1. We review the recent concept of neuronal toxicity by mutant SOD1 in relation to the posttranslational modification of SOD1 at cysteine residues, especially at Cys111, which is closely related to its

**2. Conformational change and copper‐mediated oxidative toxicity of**

SOD1 is a metal‐binding antioxidant enzyme expressed ubiquitously in the body, and it functions to convert prooxidant superoxide anions to hydrogen peroxide and oxygen to remove the oxidative stress [3]. SOD1 forms a homodimer to accomplish its full enzymatic activity. Each subunit binds one atom each of copper and zinc. Copper works for the enzymatic activity, whereas zinc has an important role in maintaining the stable structure of SOD1

More than 150 different mutations of *SOD1* gene have been found in familial ALS patients so far, and they are scattered throughout the entire coding sequence of the gene regardless of specific functional domains. The enzymatic activity of mutant SOD1 is not necessarily reduced compared to that of wild‐type SOD1 [4]. Moreover, mice deficient of *Sod1* gene do not cause symptoms of motor neuronal dysfunction, whereas transgenic mice that express mutant SOD1, but not wild‐type SOD1, develop progressive motor paralysis and degeneration of motor neurons depending on the expression level of mutant SOD1 in the spinal cord [5, 6]. It means that the pathogenic mechanism of mutant SOD1 for the degeneration of motor neurons is derived from the gain of a specific aberrant function rather than a decrease in the enzyme

What is the determinant of motor neuronal toxicity by mutant SOD1? Although the nature of mutant SOD1 toxicity has not been fully elucidated, conformational abnormality, that is, misfolding, of mutant SOD1 protein is deeply involved in the pathogenesis of familial ALS [7]. Misfolded SOD1 is subject to dissociate into monomers, which binds together abnormally to form oligomers or further high molecular weight aggregates in cells. Abnormal protein accumulation in neurons can impair their important cellular functions such as axonal transport [8] and degradation machinery of proteins [9]. SOD1‐positive inclusion bodies have been actually detected in degenerating motor neurons in the spinal cords of familial ALS patients and mutant SOD1 transgenic mice [10, 11]. Therefore, it is important to understand the common mode of conformational change in mutant SOD1, which will lead to suppress the

Once SOD1 is misfolded, copper or zinc bound to the subunit of SOD1 is also prone to be exposed from the compact dimer structure. Because copper is catalytically redox‐active, abnormal chemical reactions can occur to generate reactive oxygen (ROS) or nitrogen (RNS) species apart from the original SOD1 activity. Mutant SOD1, unlike wild‐type SOD1, has a potential to generate ROS and RNS such as hydroxyl radicals [12, 13] and peroxynitrite [14] in

conformational change, in ALS pathogenesis.

**mutant SOD1**

88 Update on Amyotrophic Lateral Sclerosis

activity of the protein.

onset of ALS derived from SOD1 mutations.

protein.

We first thought that mutant SOD1 is involved in the degeneration of motor neurons by causing oxidative stress through an adverse enzymatic reaction with copper on the protein, and we examined therapeutic effects of G93A mutant SOD1 transgenic mice by removal of the oxidative stress. As a result, a copper‐chelating agent trientine and an antioxidant ascorbic acid showed a protective effect either alone, yet a higher beneficial effect was achieved by the combined use of these reagents [16, 17]. Furthermore, to confirm the validity of this hypothesis, the mutant SOD1 mice were bred with metallothionein I/II‐deficient mice and the impact of the gene on motor paralysis was analyzed. Metallothionein is an endogenous protein that binds copper to prevent it from being prooxidant in cells. The decrease or halt of metallothionein I/ II expression exacerbated the ALS symptoms in a gene dosage‐dependent manner [18]. Copper‐mediated toxicity in mutant SOD1 was also reinforced with other reports that decreasing intracellular copper, by treatment with copper chelators or by genetic reduction of copper uptake, alleviated ALS phenotype in mutant SOD1 transgenic mice [19–21]. Moreover, the upregulation of metallothionein expression has been shown to attenuate the disease course in mice [22, 23]. These data suggest that copper‐mediated oxidative chemistry underlies the pathogenesis of familial ALS linked to mutations of *SOD1* gene, possibly triggered by misfolding of the mutant protein.
