SOD1 and amyotrophic lateral sclerosis: mutations and oligomerization

Abstract

There are about 100 single point mutations of copper, zinc superoxide dismutase 1 (SOD1) which are reported (http://alsod.iop.kcl.ac.uk/Als/index.aspx) to be related to the familial form (fALS) of amyotrophic lateral sclerosis (ALS). These mutations are spread all over the protein. It is well documented that fALS produces protein aggregates in the motor neurons of fALS patients, which have been found to be associated to mitochondria. We selected eleven SOD1 mutants, most of them reported as pathological, and characterized them investigating their propensity to aggregation using different techniques, from circular dichroism spectra to ThT-binding fluorescence, size-exclusion chromatography and light scattering spectroscopy. We show here that these eleven SOD1 mutants, only when they are in the metal-free form, undergo the same general mechanism of oligomerization as found for the WT metal-free protein. The rates of oligomerization are different but eventually they give rise to the same type of soluble oligomeric species. These oligomers are formed through oxidation of the two free cysteines of SOD1 (6 and 111) and stabilized by hydrogen bonds, between beta strands, thus forming amyloid-like structures. SOD1 enters the mitochondria as demetallated and mitochondria are loci where oxidative stress may easily occur. The soluble oligomeric species, formed by the apo form of both WT SOD1 and its mutants through an oxidative process, might represent the precursor toxic species, whose existence would also suggest a common mechanism for ALS and fALS. The mechanism here proposed for SOD1 mutant oligomerization is absolutely general and it provides a common unique picture for the behaviors of the many SOD1 mutants, of different nature and distributed all over the protein.

Figure 1. SOD1 investigated mutations.

Location of the investigated mutations (red spheres) mapped on the (Cu,Zn) WT SOD1 structure (pdb-ID 1l3n).

Figure 2. Formation of ThT-binding structures when apo SOD1 mutants and WT are incubated at 37°C.

Fluorescence due to ThT binding to SOD1 mutants (presented as arbitrary units, A.U.) for apo T54R SOD1 (•); apo L67V SOD1 (Δ), apo D90A SOD1 (∇), apo I113F SOD1 (□), apo V87M SOD1 (▾), apo WT SOD1 (O), apo L144F SOD1 (▪), apo I35T SOD1 (▴), apo V97M SOD1 (▴), apo G93A SOD1 (◊), apo I113T SOD1 (♦), during the incubation of the samples at 37°C. Apo G93D SOD1 mutant is not reported because the oligomeric species formed precipitates after about 30 hours of incubation.

Figure 3. Formation of oligomeric structures when apo I113T SOD1 mutant is incubated at 37°C.

(A) Fluorescence due to ThT binding to SOD1 mutants (presented as arbitrary units, A.U.) for apo I113T SOD1 during the incubation of the samples at 37°C. Panels (B) and ( C ) shows the size exclusion chromatograms on a G2000SW_XL and a G4000SW_XL Tosoh columns respectively, corresponding to the samples analyzed by light scattering. The void volume is labeled V₀. (D) Variation in species distribution during incubation of apo I113T SOD1. Each curve shows the molecular weight distribution detected by light scattering for the sample after different incubation times at 37°C. In all four panels the samples can be identified according to the following colors: before incubation (—), after 16 hours (—), 40 hours (—), 62 hours (—), 4 days (—) and 6 months (—) of incubation at 37°C.

Figure 4. Formation of oligomeric structures when apo T54R SOD1 mutant is incubated at 37°C.

(A) Fluorescence due to ThT binding to SOD1 mutants (presented as arbitrary units, A.U.) for apo T54R SOD1 during the incubation of the samples at 37°C. Panels (B) and ( C ) shows the size exclusion chromatograms on a G2000SW_XL and a G4000SW_XL Tosoh columns respectively, corresponding to the samples analyzed by light scattering. The void volume is labeled V₀. In all four panels the samples can be identified according to the following colors: before incubation (—), after 66 hours (—), 4.8 days (—), 6.8 days (—),7.5 days (—), and 13 months (—) of incubation at 37°C. In panel (B) the zinc reconstituted sample (—) is also reported.

Figure 5. Correlation between percentage of aggregated species and ThT-binding fluorescence.

Percentage of aggregated species (non-dimer), determined by light scattering measurements, vs ThT-binding fluorescence for apo WT SOD1 (O) and apo I113T SOD1 (♦) during the incubation of the samples at 37°C. Error bars are derived from the molecular mass errors of the light scattering experiments.

Figure 6. Mutants SOD1 aggregation.

Formation of soluble oligomers occuring when apo WT SOD1 protein is kept close to physiological conditions for an extended period of time. In the absence of metal ions, SOD1 proteins form abnormal disulfide cross-links though the two free cysteines (Cys 6 and Cys 111) and noncovalent associations with other SOD1 monomers or dimers.