A Cell-Based Assay for Screening Potential Neurodegenerative Disease Therapeutics that Promote Covalent Dimer Formation

2015
Research Category: Health Sciences
Presenter: Jeffrey Agar
PI: Jeffrey Agar

Numerous neurodegenerative disorders are characterized by mutations and aberrant posttranslational modifications leading to destabilization of native protein complex formation and disease onset. For example, one cause of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS) is that mutations in Cu/Zn superoxide dismutase (SOD1) lead to loss of native dimer formation. This causes an accumulation of a toxic species that invariably leads to motor neuron dysfunction, paralysis, and, ultimately, death. Thus, restoration of native dimer formation is a potential strategy for the treatment of ALS. Here, we describe a cell-based assay to screen compounds for their ability to promote dimer formation of SOD1. In developing this assay, which includes immunoblotting for SOD1, it was observed that a widely utilized commercial polyclonal antibody for SOD1 did not recognize the native, folded form of SOD1. A gel-incubation method was developed in order to denature SOD1 so that it could be recognized by this antibody after being natively separated into monomer and dimer forms. A novel polyclonal antibody was also developed that can recognize folded SOD1, alleviating the need for this additional gel-incubation step prior to membrane transfer and antibody detection. Thus, this study provides methods for screening potential therapeutics, as well as illuminating a potential confound regarding previous analyses of SOD1 associated with ALS that may have wide implications.

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