**5. Role of cysteine‐mediated conformational change of mutant SOD1 in ALS pathogenesis**

Although we determined the importance of oxidative modification of Cys111 for conforma‐ tional change and copper‐mediated aberrant chemistry of mutant SOD1, it has not been proved whether the modification of Cys111 is actually relevant to the pathogenesis of ALS by mutant SOD1 *in vivo*. Transgenic mice of mutant SOD1 that has simultaneous substitutions of all metal‐ coordinating residues and free cysteines (Cys6 and Cys111) were generated and had no pathology or motor symptoms [51]. However, it is difficult to confirm the direct contribution of Cys111 in these mice. To verify the importance of Cys111 solely, we conducted the study of transgenic mice expressing ALS‐linked mutant SOD1 (H46R) with or without substitution of Cys111 to serine [52]. Both lines of transgenic mice (H46R and H46R/C111S SOD1 mice) were created, respectively, and were observed whether to obtain motor paralysis and the degener‐ ation of motor neurons.

As a result, H46R SOD1 mice developed motor paralysis most quickly at 5 months of age, and reached the lifetime at 6 months of age. On the other hand, the onset of motor paralysis in H46R/C111S SOD1 mice was late at about 12 months of age, and their lifetime was extended to about 14 months after birth (**Figure 2**). Disease duration from the onset to lifetime was also significantly prolonged in H46R/C111S SOD1 mice than that in H46R SOD1 mice. The number of spinal motor neurons was decreased and the tibialis anterior muscle was atrophic at the time of endpoint in H46R SOD1 mice, while these indexes were preserved at the same age of H46R/C111S SOD1 mice. Activation of astrocytes and microglia, the phenomenon seen in the spinal cord of other mutant SOD1 transgenic mice as a modifying factor of neurodegeneration [53, 54], was also observed at endpoint in the spinal cord of H46R SOD1 mice, whereas it was not apparent in H46R/C111S SOD1 mice at the same time point.

**Figure 2.** Kaplan‐Meier curves of the onset and lifespan of H46R and H46R/C111S SOD1 mice.

Both the time at onset and lifespan were significantly extended in H46R/C111S SOD1 mice compared to those in H46R SOD1 mice.

Next, we examined the redox state of Cys111 in SOD1 and the presence of misfolded/insoluble SOD1 in the liver and spinal cord of these mice as well as of wild‐type SOD1 transgenic mice. Cys111‐peroxidized SOD1 was detected in H46R SOD1 mice from the early presymptomatic age regardless of organs, although it was trivial in wild‐type SOD1 transgenic mice. On the other hand, misfolded and insolubly aggregated SOD1 was found only in the spinal cord in parallel to the disease onset of the mice. The SOD1 species was not seen at the same age of H46R/C111S SOD1 mouse; however, it was observed in the same way as H46R SOD1 mice at endpoint. These results indicate that mutant SOD1 is more prone to be attacked at Cys111 by oxidants than wild‐type SOD1 due to a slight structural difference, and peroxidation of Cys111 is important to push the mutant SOD1 into misfolding at the early phase of the disease. However, considering that misfolding and aggregation of Cys111‐peroxidized mutant SOD1 are defined to the spinal cord, other factors may exist to enhance or suppress the misfolding of the SOD1 in the spinal cord or liver, respectively. In fact, the expression of an important protective factor, for example, heat shock factor‐1, is reported to be relatively low in motor neurons [55]. The difference in clearance efficiency of the SOD1 protein in each organ or cell type may also explain the specificity of mutant SOD1 misfolding and aggregation.

As mentioned before, insoluble high molecular weight species of mutant SOD1 is likely to consist of aggregates crosslinked with inter‐subunit disulfide bonds of cysteine residues including Cys111. To verify the significance of this phenomenon in our ALS model, we analyzed the reactivity of the insoluble aggregates to a reducing reagent in the spinal cord of H46R SOD1 mice. The majority of the insoluble aggregates were maintained even in the presence of the reagent. We further examined H46R SOD1 mice by mating with thioredoxin 1 transgenic mice, to see whether the motor symptoms could be alleviated according to inhibi‐ tion of the disulfide bond‐mediated SOD1 crosslinking. Thioredoxin 1 is an antioxidative protein present in the cytoplasm as well as SOD1, and has an effect to revert oxidatively formed protein disulfide bonds to sulfhydryls by reducing reaction. We did not see any change in the course of motor paralysis, decrease of motor neurons, glial activation, or deposition of SOD1 aggregates in H46R SOD1 mice with hemizygous thioredoxin 1 transgenic background. No suppressive effect of the disease was observed even in the SOD1 mice with homozygous thioredoxin 1 transgenic background, excluding the possibility that the expression level of thioredoxin 1 was insufficient to have the effect (Nagano S, unpublished data). It indicates that the involvement of inter‐subunit disulfide bonds of cysteine residues may be limited in our H46R mutant SOD1 disease model. More intense study will be needed in other mutant SOD1 mouse models to know the role of inter‐subunit disulfide crosslinking in the mutant SOD1 neurotoxicity.

## **6. Conclusions**

Oxidation of Cys111 leads mutant SOD1 dimers to dissociate into monomers, which causes

**5. Role of cysteine‐mediated conformational change of mutant SOD1 in**

Although we determined the importance of oxidative modification of Cys111 for conforma‐ tional change and copper‐mediated aberrant chemistry of mutant SOD1, it has not been proved whether the modification of Cys111 is actually relevant to the pathogenesis of ALS by mutant SOD1 *in vivo*. Transgenic mice of mutant SOD1 that has simultaneous substitutions of all metal‐ coordinating residues and free cysteines (Cys6 and Cys111) were generated and had no pathology or motor symptoms [51]. However, it is difficult to confirm the direct contribution of Cys111 in these mice. To verify the importance of Cys111 solely, we conducted the study of transgenic mice expressing ALS‐linked mutant SOD1 (H46R) with or without substitution of Cys111 to serine [52]. Both lines of transgenic mice (H46R and H46R/C111S SOD1 mice) were created, respectively, and were observed whether to obtain motor paralysis and the degener‐

As a result, H46R SOD1 mice developed motor paralysis most quickly at 5 months of age, and reached the lifetime at 6 months of age. On the other hand, the onset of motor paralysis in H46R/C111S SOD1 mice was late at about 12 months of age, and their lifetime was extended to about 14 months after birth (**Figure 2**). Disease duration from the onset to lifetime was also significantly prolonged in H46R/C111S SOD1 mice than that in H46R SOD1 mice. The number of spinal motor neurons was decreased and the tibialis anterior muscle was atrophic at the time of endpoint in H46R SOD1 mice, while these indexes were preserved at the same age of H46R/C111S SOD1 mice. Activation of astrocytes and microglia, the phenomenon seen in the spinal cord of other mutant SOD1 transgenic mice as a modifying factor of neurodegeneration [53, 54], was also observed at endpoint in the spinal cord of H46R SOD1 mice, whereas it was

not apparent in H46R/C111S SOD1 mice at the same time point.

**Figure 2.** Kaplan‐Meier curves of the onset and lifespan of H46R and H46R/C111S SOD1 mice.

compared to those in H46R SOD1 mice.

Both the time at onset and lifespan were significantly extended in H46R/C111S SOD1 mice

oxidative stress through the copper and aggregate formation of the monomers.

**ALS pathogenesis**

92 Update on Amyotrophic Lateral Sclerosis

ation of motor neurons.

We have shown that Cys111 drives the pathogenicity of mutant SOD1 by demonstrating that the substitution of a single residue in mutant SOD1 significantly reduces the disease phenotype of ALS model mice. Cys111 of mutant SOD1 is peroxidized and promotes misfolding of the protein to generate reducing reagent‐resistant, high molecular weight insoluble SOD1 species. It is promising to create a new therapeutic strategy for mutant SOD1‐related ALS by devel‐ oping reagents that inhibit the modification of Cys111 or subsequent monomerization of the mutant SOD1. Dimedone, a trapping reagent of sulfenylated (‐SOH) cysteines to block further peroxidation [56], or bis‐maleimidoethane, a crosslinker that is shown to crosslink between Cys111 of each SOD1 subunit to inhibit monomerization [49], may be candidates, but the problem is that these reagents have no specificity for SOD1. The reagent that binds specifically to a pocket of SOD1 dimer interface was developed by in silico drug screening approach and had a suppressive effect for monomerization of mutant SOD1 *in vitro* [57]. Clinical application will be achieved by developing a drug having higher effect and permeability into the central nervous system using the reagent as a lead compound. Alternatively, considering that misfolded wild‐type or mutant SOD1 is likely to be propagated to the neighboring neurons [58] to cause further misfolding of SOD1 and the spread of the disease, antibody therapy targeting Cys111 or dimer interface of SOD1 may also be effective to inhibit the progress of ALS.
