Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients

Abstract

Mutations in CuZn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) and are found in 6% of ALS patients. Non-native and aggregation-prone forms of mutant SOD1s are thought to trigger the disease. Two sets of novel antibodies, raised in rabbits and chicken, against peptides spaced along the human SOD1 sequence, were by enzyme-linked immunosorbent assay and an immunocapture method shown to be specific for denatured SOD1. These were used to examine SOD1 in spinal cords of ALS patients lacking mutations in the enzyme. Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only sparsely in 3 of 28 neurodegenerative and 2 of 19 non-neurological control patients. The granular inclusions were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions containing TAR DNA binding protein-43, ubiquitin or markers for endoplasmic reticulum, autophagosomes or mitochondria. Granular inclusions were also found in carriers of SOD1 mutations and in spinobulbar muscular atrophy (SBMA) patients and they were the major type of inclusion detected in ALS patients homozygous for the wild type-like D90A mutation. The findings suggest that SOD1 may be involved in ALS pathogenesis in patients lacking mutations in the enzyme.

Figure 1. Micrographs of spinal cord motoneurons showing SOD1-immunoreactive inclusions.

Using the 4–20Ra-ab, 57–72Ra-ab, and 131–153Ra-ab anti-SOD1 peptide antibodies (0.64, 5 and 0.75 µg/ml, respectively) numerous small granular inclusions could be seen in tissues from sporadic (SALS) and familial (FALS) patients lacking mutations in the SOD1 gene (A, B, D, E, J-L, N and O). Note that the lipofuscin do not stain in B. As a rule motoneurons from the controls lacked inclusions (J, K), but in a few cases were a small number of granular inclusions observed (arrows in C, L). Using a sheep anti-SOD1 antibody against whole SOD1 (Calbiochem), SOD1-immunoreactive inclusions could sometimes be discerned against background staining in ALS patients with abundant small granular inclusions (F). In patients carrying the D90A mutation, small granular inclusions were the major type of inclusion (131–153Ra-ab antibody, 0.75 µg/ml) (G). Using a mutation-specific antibody (10 µg/ml) both larger skein and LBHI-like inclusions (H), as well as small granular inclusions (I), could be seen in an ALS patient carrying the G127X mutation. Sections double-labeled with the the lysosomal marker cathepsin D (M and O; red fluorescence) and the 57–72Ra-ab anti-SOD1 antibody (N and O; green fluorescence; 5 µg/ml). The merged picture of the green and red channel scans shows a partial overlap of green and red fluorescence indicating colocalization of SOD1 and lysosomes (O). Scale bar  = 30 µm (in A–D, H, I), 18 µm (in E–G), 7 µm (in J), 8 µm (in K, M-O) and 11 µm (in L).

Figure 2. Relative reactivities of antibodies towards native and denatured SOD1.

ELISA plates were coated with either native (filled symbols in green) or denatured (unfilled symbols in red) SOD1, and were either reacted with antibodies raised against peptides (A) or whole SOD1 (B). Threefold dilutions were made from a high antibody concentration giving an A₄₉₀ of 0.70–1.6 with either native or denatured SOD1. (A) Reactivity of the 4–20Ra-ab (native  =  ▴, denatured  =  ▵), 57–72Ra-ab (native SOD1  =  , denatured SOD1  =  “) and 131–153Ra-ab (native SOD1  =  ▾, denatured SOD1 =  ▿) anti-SOD1 peptide antibodies. The highest concentrations were 0.1 µg/ml, 0.03 µg/ml, 0.1 µg/ml, respectively. (B) Reactivity of antibodies raised to whole SOD1: Rabbit-1 antibody (native SOD1  =  ;, denatured SOD1  =  “); a sheep antibody from Calbiochem (native  =  ▴, denatured  =  ▵); a sheep antibody from The Binding Site (native SOD1  =  ▾, denatured SOD1 =  ▿); and a mouse monoclonal antibody from Sigma (native SOD1  =  ▪, denatured SOD1  =  ). The highest concentrations were 0.6, 10, 20, and 10 µg/ml, respectively. The data presented are means of 4 wells for each point.

Figure 3. Analysis of SOD1 by western immunoblot.

Homogenates of tissue from the temporal lobe, the precentral gyrus, and the spinal cord ventral horns from 5 control patients, 5 SALS and 4 FALS patients were analyzed by western immunoblots, using the 4–20Ra-ab, 57–72Ra-ab, 100–115Ra-ab and 131–153Ra-ab anti-SOD1 peptide antibodies. Analyses of lumbar spinal cord ventral horns are here shown as examples. All figures depict the same set of extracts and short, intermediate and long exposures are presented. Upon long exposure a weak band at about 28 kDa was seen with the 4–20Ra-ab anti-SOD1 antibody in one of the five SALS samples. The band was probably unspecific since it was not seen with the other antibodies. The intensity was estimated at <0.5% of the SOD1 band.