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

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the presence of aggregates of phosphorylated TDP-43 protein in the cytoplasm of affected neurons. The hypothesized pathomechanism is the loss of TDP-43 nuclear function and the concomitant toxic gain of function of the aggregates. 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 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 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 and POLDIP3, a readout of TDP-43 nuclear loss-of-function, upon chronic ARS treatment. Since autophagy impairment favors TDP-43 pathological aggregation, we first tested the autophagy enhancer rapamycin in our in vitro model of TDP-43 proteinopathy. 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 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.