Evaluation of TTR toxicity in supernatants of FAP-derived hepatocyte-like cells

Familial amyloid polyneuropathy (FAP) is a neurodegenerative disease caused by mutations of the transthyretin (TTR) gene, which is primarily secreted by the liver. Mutations in TTR cause misfolding of the protein ultimately leading to extracellular deposition of amyloidogenic TTR in tissues and organs, disrupting regular function. Typically, amyloid deposits are observed in biopsies of FAP patients. Due to the variety of the clinical findings and the rare incidence of the disease, many patients can only be diagnosed several years after onset of the disease. Besides genetic testing of TTR there is no single biochemical assay to confirm FAP diagnosis. In vitro, purified TTR derived from E. coli was reported to be toxic when incubated with tissue culture cells. We addressed the question whether supernatants derived from hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem (iPS) cells of FAP patients induce cellular toxicity on the IMR-32 neuronal cell line. After three to seven days of incubation, the cell viability was determined by an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and the respective absorbance was determined. TTR protein levels were determined by ELISA and quantified by western blot analysis. Furthermore, stability of TTR was assessed by treatment with the small-molecule tafamidis. Our preliminary data indicate that supernatants of FAP patients exhibit toxicity that is related to variant TTR in contrast to supernatants derived from other cell lines. The assessment of toxicity observed in supernatants of FAP-HLCs could be valuable to support the tedious diagnostic evaluation of patients to monitor efficacy of treatment as well as to investigate TTR-related amyloidogenesis.