Tandem detergent-extraction and immunoprecipitation of proteinopathy: Scalable enrichment of ALS-associated TDP-43 aggregates
Baggio Evangelista, Shannon R. Cahalan, Joey V. Ragusa, Angie L. Mordant, Julie Necarsulmer, Robert J. Perna, Tejazaditya Ajit, Kristen White, Natalie K. Barker, Xu Tian, Sarah Cohen, Rick B. Meeker, Laura E. Herring, Todd J. Cohen
Abstract
Transactive response DNA-binding protein of 43 kDa (TDP-43) is a highly conserved, ubiquitously expressed nucleic acid-binding protein that regulates DNA/RNA metabolism. Genetics and neuropathology studies have linked TDP-43 to several neuromuscular and neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Under pathological conditions, TDP-43 mislocalizes to the cytoplasm where it forms insoluble, hyper-phosphorylated aggregates during disease progression. Here, we optimized a scalable in vitro immuno-purification strategy referred to as tandem detergent-extraction and immunoprecipitation of proteinopathy (TDiP) to isolate TDP-43 aggregates that recapitulate those identified in postmortem ALS tissue. Moreover, we demonstrate that these purified aggregates can be utilized in biochemical, proteomics, and live-cell assays. This platform offers a rapid, accessible, and streamlined approach to study ALS disease mechanisms, while overcoming many limitations that have hampered TDP-43 disease modeling and therapeutic drug discovery efforts.