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 neuropathologically by TDP-43 proteinopathy with loss of TDP-43 nuclear splicing activity and formation of cytoplasmic TDP-43 aggregates. The lack of suitable experimental models of TDP-43 proteinopathy has hampered the discovery of effective therapies. We already showed that chronic and mild oxidative insult by sodium arsenite (ARS) triggered TDP-43 cytoplasmic aggregation and stress granules (SGs) formation in ALS patient-derived fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs). However, whether this insult induces a reduction of TDP-43 splicing activity in the nucleus, thus recapitulating both gain and loss of function pathomechanisms, still remains to be determined.

In this study we first showed that chronic ARS in human neuroblastoma cells triggered TDP-43 cytoplasmic mislocalization, SGs formation and defective splicing of TDP-43 target genes UNC13A and POLDIP3 as functional readouts of TDP-43 proteinopathy. Additionally, a dysregulation of autophagy and senescence markers was observed in this condition. In a preliminary drug screening approach with autophagy-promoting drugs, namely rapamycin, lithium carbonate and metformin, only rapamycin prevented ARS-induced loss of TDP-43 splicing activity. We then demonstrated that, in addition to TDP-43 cytoplasmic aggregation, chronic ARS triggered TDP-43 loss of splicing activity also in ALS patient-derived primary fibroblasts and iPSC-MNs and that rapamycin was beneficial to reduce these TDP-43 pathological features. By switching to a neuro-glial 3D in vitro model, we observed that treatment of ALS iPSC-brain organoids with chronic ARS also induced a defective TDP-43 splicing activity which was prevented by rapamycin.

Collectively, we established different human cell models of TDP-43 proteinopathy which recapitulate TDP-43 gain and loss of function, prevented by rapamycin administration. Human neuroblastoma cells and patient-derived fibroblasts and 2D- and 3D-iPSC models exposed to chronic oxidative stress represent therefore suitable in vitro platforms for future drug screening approaches in ALS.