Human in vitro models of TDP-43 proteinopathy for drug screening approaches

Aggregates of phosphorylated TDP-43 protein in the cytoplasm of neurons are an ALS neuropathological hallmark. Response to stress and formation of stress granules (SGs) have been proposed as initiators of TDP-43 pathological aggregation. We previously showed that chronic oxidative stress by arsenite (ARS) induces the formation of SGs and phospho-TDP-43 aggregates in primary fibroblasts and iPSC-motor neurons from ALS patients. Aim of our study was to generate a robust and reproducible in vitro model of TDP-43 pathology to be used for drug screening. We induced a chronic oxidative insult in human neuroblastoma SK-N-BE cells by exposure to low doses of ARS for 9-24 hours. Our data showed TDP-43 mislocalization from the nucleus to the cytoplasm in both a dose- and time-dependent manner and increase of P62. We also observed a defective splicing activity of TDP-43 towards its target genes UNC13A and POLDIP3 a readout of TDP-43 nuclear loss-of-function, upon chronic ARS treatment. We tested candidate drugs involved in promoting autophagy, namely rapamycin, lithium carbonate and metformin in our in vitro model of TDP-43 proteinopathy. Only rapamycin was able to rescue ARS-induced loss of TDP-43 splicing activity on its target genes and to reduce P62 accumulation. We then tested rapamycin in C9ORF72 patient-derived fibroblasts and iPSC-motor neurons, where its efficacy in rescuing ARS-induced loss of TDP-43 splicing activity was confirmed. Rapamycin also significantly reduced ARS-induced phospho-TDP-43 aggregates and SGs formation. In conclusion, we have set up human cell models of TDP-43 pathology in which rapamycin was proven to be beneficial in rescuing chronic oxidative stress-induced alterations in TDP-43 splicing activity and cytoplasmic mislocalization by modulating autophagy. Human SK-N-BE and ALS patient-derived cells chronically treated with ARS can therefore be exploited as valuable in vitro platforms for future drug screening approaches.