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Many of the genes whose mutation causes Amyotrophic Lateral Sclerosis (ALS) are RNA-binding proteins which localize to stress granules, while others impact the assembly, stability, and elimination of stress granules. This has led to the hypothesis that alterations in the dynamics of stress granules and RNA biology cause ALS. Genetic mutations in Superoxide Dismutase 1 (SOD1) also cause ALS. Evidence demonstrates that SOD1 harboring ALS-linked mutations is recruited to stress granules, induces changes in alternative splicing, and could be an RNA-binding protein. Whether SOD1 inclusions contain RNA in disease models and whether SOD1 directly binds RNA remains uncertain. We applied methods including cross-linking immunoprecipitation and in vitro gel shift assays to detect binding of SOD1 to RNA in vitro, in cells with and without stress granules, and in mice expressing human SOD1 G93A. We find that SOD1 localizes to RNA-rich structures including stress granules, and SOD1 inclusions in mice contain mRNA. However, we find no evidence that SOD1 directly binds RNA. This suggests that SOD1 may impact stress granules, alternative splicing and RNA biology without binding directly to RNA.
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High levels of familial Amyotrophic Lateral Sclerosis (ALS)-linked SOD1 mutants G93A and G37R were previously shown to mediate disease in mice through an acquired toxic property. We report here that even low levels of another mutant, G85R, cause motor neuron disease characterized by an extremely rapid clinical progression, without changes in SOD1 activity. Initial indicators of disease are astrocytic inclusions that stain intensely with SOD1 antibodies and ubiquitin and SOD1-containing aggregates in motor neurons, features common with some cases of SOD1 mutant-mediated ALS. Astrocytic inclusions escalate markedly as disease progresses, concomitant with a decrease in the glial glutamate transporter (GLT-1). Thus, the G85R SOD1 mutant mediates direct damage to astrocytes, which may promote the nearly synchronous degeneration of motor neurons.
Many patients with amyotrophic lateral sclerosis (ALS; motor neuron disease) use natural or traditional therapies of unproven benefit. One such therapy is ginseng root. However, in some other disease models, ginseng has proven efficacious. Ginseng improves learning and memory in rats, and reduces neuronal death following transient cerebral ischemia. These effects of ginseng have been related to increases in the expression of nerve growth factor and its high affinity receptor in the rat brain, and antioxidant actions, inter alia. Since such actions could be beneficial in ALS as well, we studied the effect of ginseng (Panax quinquefolium), 40 and 80 mg/Kg, in B6SJL-TgN(SOD1-G93A)1Gur transgenic mice. The ginseng was given in drinking water, from age 30d onwards. We measured the time to onset of signs of motor impairment, and survival. There was no difference between the two ginseng groups (n=6, 6) in either measure. However, compared to controls (n=13), there was a prolongation in onset of signs (116d vs. 94d, P
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Eukaryotic Cu, Zn-superoxide dismutase (SOD1) is primarily responsible for cytotoxic filament formation in amyotrophic lateral sclerosis (ALS) neurons. Two cysteine residues in SOD1 form an intramolecular disulfide bond. This study aims to explore the molecular mechanism of SOD1 filament formation by cysteine overoxidation in sporadic ALS (sALS). In this study, we determined the crystal structure of the double mutant (C57D/C146D) SOD1 that mimics the overoxidation of the disulfide-forming cysteine residues. The structure revealed the open and relaxed conformation of loop IV containing the mutated Asp57. The double mutant SOD1 produced more contagious filaments than wild-type protein, promoting filament formation of the wild-type SOD1 proteins. Importantly, we further found that HOCl treatment to the wild-type SOD1 proteins facilitated their filament formation. We propose a feasible mechanism for SOD1 filament formation in ALS from the wild-type SOD1, suggesting that overoxidized SOD1 is a triggering factor of sALS. Our findings extend our understanding of other neurodegenerative disorders associated with ROS stresses at the molecular level.
The wild-type SOD1 protein is converted to the cytotoxic filament under specific but not fully understood environmental stresses in sALS cases8,9,10. One of the leading causes of sALS is oxidative stress, including aberrant free radical metabolism11,12,13,14,15. Notably, extreme physical exercises are a well-known risk factor for sALS16. Previous studies have suggested an increased risk of ALS among athletes and people who engage in intense physical activity17,18,19,20,21,22,23,24,25. To date, the stimuli that trigger filament formation of the wild-type SOD1 proteins at the molecular level remain to be elucidated.
Eukaryotic SOD1 is a 32-kDa homodimeric metalloenzyme containing Cu and Zn ions at the active site in the active state. The protomer of SOD1 forms an eight-stranded Greek-key β-barrel fold with four conserved cysteine residues and three long loops (loops IV, VI, and VII). The Cu and Zn ions are bound at the active site by loop IV, whose conformation is stabilized by the highly conserved intramolecular disulfide bond between Cys57 in loop IV and Cys146 in 8th β-strand (β8). The cleavage of the intramolecular disulfide bond and the loss of metal ions resulted in the metal-free denatured monomer, which is the entry point to aggregation for filament formation in pathological states26. Without a reducing agent, the SOD1 protein rarely forms oligomers in vitro.
The human copper chaperone for SOD1 (hCCS) forms a transient complex with monomeric SOD1, catalyzing copper binding to SOD1 and mediating disulfide bond formation between Cys57 and Cys146 within the subunit, leading to the formation of dimeric holo-SOD1. This hCCS-dependent disulfide bond formation of SOD1 requires molecular oxygen; thus, hypoxic conditions hinder hCCS functions27,28. A recent study showed that hypoxic stress on the cells induced a disulfide reduction, thereby increasing the structural disorders of SOD128. Furthermore, other studies have shown that the disulfide-forming cysteine in SOD1, especially Cys146, is prone to be overoxidized. A previous study has shown that the Cys146 that is mutated in familial ALS29, is oxidized to cysteine-sulfonic acid in AD and PD brains30. In addition, a high-resolution mass spectrometric analysis also revealed that yeast Sod1 is oxidized at Cys146 and His71 upon sustained expression under oxidative conditions31.
Cysteine is oxidized into three different structures depending on the oxidation states: cysteine-sulfenic acid, cysteine-sulfinic acid, and cysteine-sulfonic acid (Fig. 1a and supplementary Fig. 1). Cysteine-sulfenic acid can be reduced back to cysteine in the presence of reducing agent or makes a disulfide with cysteine. Cysteine-sulfinic acid can be reduced only by special enzymes, such as sulfiredoxin, requiring ATP hydrolysis32. It has not been found that cysteine-sulfonic acid cannot be reduced under the normal conditions33,34. Aspartic acid mimics cysteine-sulfinic acid in terms of the electrostatic charge and the atomic arrangement. Both have one negative charge and two oxygen atoms attached to the central atom (C or S) in the triangular plane geometry. We previously confirmed that the mutation of Cys to Asp well mimics the cysteine-sulfinic acid or cysteine-sulfonic acid in the high-resolution crystal structures of the bacterial redox sensor OxyR35.
To investigate the molecular mechanism for sALS instances with the wild-type SOD1 gene, we focused on overoxidation at Cys57 and Cys146 in facilitating the nucleation and growth of the filament formation of SOD1. The crystal structure and biochemical studies of the overoxidation-mimicking mutant (C57D/C146D) SOD1 proposed a new mechanism linking physiological stimuli to filament formation at the molecular level.
Cysteines can be oxidized into disulfide, which is highly resistant to further oxidation in the physiological state. However, cysteines are also vulnerable to overoxidation into the cysteine-sulfinic or cysteine-sulfonic acid forms under certain oxidative environments. To investigate the possible roles of the overoxidation of cysteine residues in SOD1, a mutant SOD1 protein that could mimic the overoxidized Cys57 and Cys146 was generated. The C57D/C146D mutant SOD1 proteins were well expressed in the E. coli expression system as the wild-type SOD1 protein.
To biochemically characterize the overoxidation-mimicking mutant SOD1, we first compared the metal contents of the as-isolated mutant and wild-type SOD1 proteins purified using the same procedure. The ICP-MS analysis revealed that the mutant SOD1 proteins almost lost the bound Cu2+ and Zn2+ compared to the wild-type SOD1 proteins (Table 1). This result showed that the protein sample contained only a small portion of the holoenzyme containing Zn2+ and Cu2+ and further indicates that the C57D/C146D mutant SOD1 protein has lower affinities for Zn2+ and Cu2+ than the wild-type SOD1 protein. 2ff7e9595c
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