Modeling of TDP-43 proteinopathy by chronic oxidative stress identifies rapamycin as beneficial in ALS patient-derived 2D and 3D iPSC models

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized by TDP-43 proteinopathy for which the hypothesized pathomechanisms are the loss of TDP-43 nuclear function and the concomitant toxic gain of function of cytoplasmic and phosphorylated TDP-43 (P-TDP-43) aggregates. Response to environmental insults and formation of stress granules (SGs) have been proposed as initiators of TDP-43 pathological aggregation. Aim of this study was to generate in vitro models of TDP-43 proteinopathy to be used for drug screening. We first induced a chronic oxidative insult in human SK-N-BE cells by exposure to low doses of sodium arsenite (ARS). Our data showed TDP-43 mislocalization into the cytoplasm, insoluble P-TDP-43 increase, P62 accumulation, and defective splicing activity of TDP-43 towards its target genes UNC13A and POLDIP3 as readouts of TDP-43 nuclear loss-of-function. As autophagy impairment plays a crucial role in ALS, we tested three drugs involved in promoting autophagy, namely rapamycin, lithium carbonate, and metformin: only rapamycin was able to rescue ARS-induced loss of TDP-43 splicing activity on UNC13A. We then tested rapamycin in ALS patient-derived primary fibroblasts, where it reduced P-TDP-43 aggregates and SGs formation. Rapamycin was also tested in motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients, where we confirmed its efficacy in reducing P-TDP-43 aggregates and SGs and rescuing ARS-induced loss of TDP-43 splicing activity. Finally, we tested rapamycin in ALS iPSC-derived brain organoids exposed to chronic ARS, where it rescued loss of TDP-43 splicing activity. In conclusion, we established different human cell models of TDP-43 proteinopathy in which rapamycin was able to rescue chronic oxidative stress-induced alterations in TDP-43 splicing activity and cytoplasmic mislocalization by modulating autophagy. Human SK-N-BE and ALS patient-derived 2D and 3D iPSC models chronically treated with ARS can therefore be exploited as in vitro platforms for future drug screening approaches.