Rapamycin reverts TDP-43 splicing defects and mislocalization in human in vitro models of TDP-43 proteinopathy

Aggregates of phosphorylated and ubiquitinated TDP-43 protein in the cytoplasm of neurons are an ALS neuropathological hallmark. Response to stress and formation of stress granules (SG) have been proposed as initiators of TDP-43 pathological aggregation. We previously showed that mild and chronic oxidative stress by arsenite (ARS) induces formation of both SG and phospho-TDP-43 aggregates in primary fibroblasts and iPSC-motor neurons obtained from ALS patients in association to the accumulation of the autophagy receptor P62. Phospho-TDP-43 aggregates resemble those seen in ALS autoptic brains and are more abundant in C9ORF72 than in TARDBP patients’ cells. 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 the autophagy receptor P62. We also observed a defective splicing activity of TDP-43 towards its target genes UNC13A, POLDIP3 and STMN2, a readout of TDP-43 nuclear loss-of-function, upon chronic ARS treatment. We tested candidate drugs involved in promoting autophagy, such as rapamycin and lithium carbonate, and in reducing senescence, such as metformin, in our in vitro model of TDP-43 proteinopathy. Only rapamycin was capable of rescuing ARS-induced loss of TDP-43 splicing activity on its target genes and of reducing TDP-43 cytoplasmic mislocalization and 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 SG formation. In conclusion, we have set up human cell models of TDP-43 pathology in which rapamycin was proved 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.