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Loss of Metal Ions, Disulfide Reduction and Mutations Related to Familial ALS Promote Formation of Amyloid-Like Aggregates from Superoxide Dismutase

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1

Metal levels in oocytes and embryonic and larval stages of zebrafish.

<p>ICP-MS measurements for copper (Cu) (A, D), manganese (Mn) (B, E), and zinc (Zn) (C, F) were performed on oocytes, embryos (2-cell, 512-cell, blastula, 30% epiboly, 50% epiboly, 6, 12, 24, 36, 48, 60, 72 hours post-fertilization (hpf)) (A-C) and larval fish (5, 7, 10, 15, 30 days post-fertilization (dpf)) (D-F). Each bar represents the average and standard deviation of four to five independently collected replicates for a specific developmental stage. Each replicate represents a pooled group of 20 oocytes, 10–20 embryos, or 5–10 larval fish. Metal levels are expressed per oocyte, embryo, or larva. Horizontal brackets above bars indicate a statistically significant difference between bracketed values. (For data in all panels, the Shapiro-Wilk normality test failed, therefore Kruskal-Wallis one-way ANOVA on ranks was performed, with Dunn’s method used for multiple comparison and P<0.05 as cut-off for significance.)</p

Distribution of metals in bodies and yolk sacs of embryos.

<p>(A-C) ICP-MS measurements were collected for copper (Cu) (A), manganese (Mn) (B), and zinc (Zn) (C) in whole embryos, bodies only, and yolk sacs only of embryos at 48 hpf, reared in presence or absence of 5 μM neocuproine, a Cu chelator. Each bar represents the average and standard deviation of three independently collected replicates, with each replicate consisting of a pooled group of 47–50 embryos. Horizontal brackets above bars indicate statistically significant difference between bracketed values. (Data in all panels passed Shapiro-Wilk normality and equal variance tests. One-way ANOVA indicated significant difference between groups, with the Holm-Sidak method used for multiple comparisons and P<0.05 as cut-off for significance.) There was no significant difference in metal levels between neocuproine-treated and–untreated samples of the same sample type. (D, E) Images were taken of zebrafish embryos at 48 hpf, reared in presence or absence of 5 μM neocuproine.</p