Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase

Abstract

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.

Figure 1. Effect of incubation conditions on amyloid formation.

SOD1 at a concentration of 1 mg/ml was incubated with agitation in the Fluoroskan microplate reader for measurement of ThT fluorescence. A) Effect of temperature and agitation with a Teflon ball. Incubation buffer was 50 mM citrate, 0.1 M NaCl, 2 M guanidine, pH 3. Open circles, 37°C with Teflon balls; solid circles, 24°C with Teflon balls; open triangles, 37°C without Teflon balls. B) Effect of guanidine. Incubation buffer was 50 mM sodium citrate, 0.1 M NaCl, pH 3, containing 0 (solid triangles), 0.5 M (solid circles), 1 M (solid squares), 2 M (open inverted triangles), or 3 M (open diamonds) guanidine hydrochloride. C) Effect of pH. Incubation buffer was either 50 mM sodium citrate, pH 3 (solid circles), 50 mM sodium formate, pH 4 (open circles), or 50 mM sodium acetate, pH 5 (solid triangles), plus 0.1 M NaCl and 2 M guanidine hydrochloride. D) Effect of acetonitrile. Incubation buffer was 50 mM sodium citrate, 0.1 N NaCl, pH 3, containing 10% (solid circles), 20% (solid triangles), or 25% (open circles) acetonitrile. No amyloid was formed at 30% acetonitrile (not shown).

Figure 2. Amyloid formation from apo- and reduced WT SOD1 at pH 7.

2A) Apo-SOD1 was incubated in 50 mM MOPS, 0.1 M NaCl, 1 mM EDTA, pH 7, containing zero (solid circles) or 1 M (open circles) guanidine. 2B) Reduced SOD1. The incubation mixtures contained 50 mM MOPS, 0.1 M NaCl with 10 mM TCEP (open circles) or 1 M guanidine hydrochloride (open circles) or 100 mM TCEP (solid circles) or 10 mM TCEP/1 M GuanHCl (solid inverted triangles).

Figure 3. Detection of intermolecular disulfide bonds in SOD1 amyloid prepared under different conditions.

Amyloid produced under each set of conditions was isolated by pelleting and subjected to SDS-PAGE in sample buffers containing 2% mercaptoethanol (“red”) or lacking any reducing agent (“nr”). Lane 1 is MW standards: 66 K, 45 K, 36 K, 29 K, 24 K, 20 K, and 14.2 K. Lanes 2 and 3, metalated SOD1 in 50 mM MOPS, 0.1 M NaCl, 0.1 M TCEP, pH 7; lanes 4 and 5, metalated SOD1 in 50 mM sodium citrate, 0.1 M NaCl, 1 M guanidine hydrochloride, pH 3; lanes 6 and 7, apo-SOD1 in 50 mM MOPS, 0.1 M NaCl, pH 7; lanes 8 and 9, metalated SOD1 in 50 mM MOPS, 0,1 M NaCl, 1 M guanidine hydrochloride, 10 mM TCEP, pH 7.

Figure 4. Electron microscopy of SOD1 amyloid formed at pH 3.

SOD1 was incubated in 1 M guanidine hydrochloride, pH 3 at 37°C (A–B) or 24°C (C–F). Samples are WT SOD1 (A,B,C,F) or G93V (D,E).

Figure 5. Amyloid formation from WT SOD1 and selected mutant SOD1 proteins at pH 5.

SOD1 was incubated as in Figure 1 in 50 mM sodium acetate, 0.1 M NaCl, pH 5, plus 1 M guanidine hydrochloride (A) or 2 M guanidine hydrochloride (B). A: D125H (solid squares), I149T (open circles), H80R (solid circles), G85R (solid triangles), WT (open triangles). B: L38V (open diamonds), E100G (solid triangles), A4V (open triangles), I149T (open circles), G85R (open squares), WT (solid diamonds).

Figure 6. Aggregation assays with mutant SOD1 lacking free sulfhydryl groups.

A) Apo-WT (solid triangles), apo-AS (solid squares), apo-AS/A4V (solid circles), apo-AS/G93A (open circles), and apo-AS/G85R (open triangles) were incubated in 50 mM MOPS, 0.1 M NaCl, 1 mM EDTA, pH 7, at 37°C. B) WT (solid inverted triangles), AS (solid circles), AS/A4V (open circles), AS/G93A (open squares), and AS/G85R (solid squares) were incubated in 50 mM MOPS, 0.1 M NaCl, 0.1 M TCEP, pH 7, at 37°C.