In vitro evidence consistent with an interaction between wild‐type and mutant SOD 1 protein associated with canine degenerative myelopathy

Canine degenerative myelopathy ( DM ) is a progressive neurological disorder that may be considered to be a large animal model for specific forms of the fatal human disease, familial amyotrophic lateral sclerosis ( fALS ). DM is associated with a c118G>A mutation of the superoxide dismutase 1 ( Sod 1) gene, and a significant proportion of cases are inherited in an autosomal recessive manner in contrast to the largely, but not exclusively, dominant mode of inheritance in fALS . The consensus view is that these Sod 1/ SOD 1 mutations result in a toxic gain of function but the mechanisms remain unclear. Here we used an in vitro neuroblastoma cell line transfection system to monitor wild‐type and mutant forms of SOD 1 fusion proteins containing either a Cherry or an enhanced green fluorescent protein ( EGFP ) tag. These fusion proteins retained SOD 1 enzymatic activity on a native gel assay system. We demonstrate that SOD 1 aggregate density is significantly higher in DM transfectants compared to wild‐type. In addition, we show by co‐immunoprecipitation and confocal microscopy, evidence for a potential interaction between wild‐type and mutant forms of SOD1 in co‐transfected cells. While in vitro studies have shown SOD 1 heterodimer formation in fALS models, this is the first report for DM SOD 1. Therefore, despite for the majority of cases there is a difference in the mode of inheritance between fALS and DM , a similar interaction between wild‐type and mutant SOD 1 forms can occur. Clarifying the role of SOD 1 in DM may also be of benefit to understanding the role of SOD 1 in fALS .