Novel SOD1 monoclonal antibodies against the electrostatic loop preferentially detect misfolded SOD1 aggregates

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that leads to motor neuron degeneration and paralysis. Superoxide dismutase (SOD1) mutations are the second most common cause of familial ALS and are responsible for up to 20 % of familial ALS cases. In ALS patients, SOD1 can form toxic misfolded aggregates that deposit in the brain and spinal cord. To better detect SOD1 aggregates and expand the repertoire of conformational SOD1 antibodies, SOD1 monoclonal antibodies were generated by immunizing SOD1 knockout mice with an SOD1 fragment consisting of amino acids 129-146, which make up part of the electrostatic loop. A series of hybridomas secreting antibodies were screened and five different SOD1 monoclonal antibodies (2C10, 2F8, 4B11, 5H5, and 5A10) were found to preferentially detect denatured or aggregated SOD1 by enzyme-linked immunosorbent assay (ELISA), filter trap assay, and immunohistochemical analysis in SOD1 mouse models. The staining with these antibodies was compared to Campbell-Switzer argyrophilic reactivity of pathological inclusions. These new conformational selective SOD1 antibodies will be useful for clinical diagnosis of SOD1 ALS and potentially for passive immunotherapy.

Keywords: Amyotrophic lateral sclerosis; Antibodies; Frontotemporal dementia; Pathology; Silver stain; Superoxide dismutase 1.

Figure 1.. New SOD1 monoclonal antibodies show strong preference for denatured human SOD1 over native human SOD1 by ELISA.

SOD1 antibodies 2C10, 2F8, 4B11, 5H5, and 5A10 were added to ELISA plates coated with denatured SOD1, native SOD1, and uncoated control (plastic). All ELISAs were completed in triplicate. Error bars show standard error of the mean.

Figure 2.. SOD1 antibodies detect SOD1 aggregates by filter trap assay with low nonspecific reactivity.

(A) Filter trap assay for protein aggregates was performed on spinal cord lysates extracted from nTg and transgenic mice expressing human G93A SOD1, and (B) spinal cord lysates from L126Z and H46R/H48Q transgenic mice. The resulting membranes were probed with 2C10, 2F8, 4B11, 5H5, or 5A10 antibodies as described in Materials and Methods.

Figure 3.. New SOD1 monoclonal antibodies detect denatured wild type and G93A SOD1 mutant expressed in transfected cultured cells.

HEK293T cells were either not transfected as a control or transfected to express wild type human SOD1, G93A human SOD1 mutant, or human SOD1 truncated due to the L126Z mutation. Cell lysates in SDS sample buffer were heated to 100°C for 10 minutes to denature the proteins that were separated by SDS-PAGE on 10% polyacrylamide gels. Antibodies 5H5, 5A10, 4B11, 2F8 and 2C10 can specifically react with SDS and heat denatured SOD1 monomers. An anti-serum raised again the whole human SOD1 was also used to confirm expression. The mobilities of molecular mass markers are indicated on the left of each blot.

Figure 4.. New SOD1 monoclonal antibodies detect denatured wild type SOD1 in spinal cord lysate from nTg mice.

20 ug of spinal cord lysates in SDS sample buffer and heated to 100°C for 10 minutes were separated by SDS-PAGE on 15% polyacrylamide gels (shown in duplicate). Antibodies 2C10, 2F8. 4B11 5A10 and 5H5 can specifically react with denatured wild type SOD1. The mobilities of molecular mass markers are indicated on the left of each blot.

Figure 5.. Modified Campbell-Switzer silver staining captures SOD1 aggregates in the brainstem and spinal cord of L126Z, G93A, and H46R/H48Q human SOD1 transgenic mice.

(A) Brainstem and (B) spinal cord from nTg mice, or L126Z, G93A or H46R/H48Q human SOD1 transgenic mice were stained with a modified Campbell-Switzer silver stain protocol as described in Materials and Methods. Scale bar represents 50 μm.

Figure 6.. New SOD1 antibodies effectively react with SOD1 aggregates in the brainstem of G93A and H46R/H48Q human SOD1 transgenic mice.

Brain sections from SOD1 KO mice, nTg mice, and L126Z, G93A or H46R/H48Q human SOD1 transgenic mice were immunostained with SOD1 antibodies (A) 2C10, (B) 4B11, (C) 5A10, or (D) 5H5. The number of mice examined and the number of sections for each animal is provided in Table 2. Scale bar represents 25 μm.

Figure 7.. New SOD1 antibodies immunostain SOD1 aggregates in the ventral horn of spinal cord of G93A and H46R/H48Q human SOD1 transgenic mice.

Spinal cord from SOD1 KO mice, nTg mice, and L126Z, G93A or H46R/H48Q human SOD1 transgenic mice were stained with SOD1 antibodies (A) 2C10, (B) 4B11, (C) 5A10, or (D) 5H5. The number of mice examined and the number of sections for each animal is provided in Table 2. Scale bar represents 25 μm.