TDP‐43 dysregulation and STMN‐2 mis‐splicing upon proteasomal inhibition in potential iPSC‐derived neuronal ALS model

Abstract Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons. Many ALS cases are associated with nuclear‐to‐cytosolic mislocalization and phosphorylation of the transactivation response DNA binding protein (TDP)‐431. This is caused by several mutations such as M337V in the TAR DNA binding protein (TARDBP) gene1. In addition, proteasomal degradation is often affected, resulting in aggregation of TDP‐432. TDP‐43 is involved in many mRNA processes, and its depletion results in mis‐splicing of the neuronal growth associated factor, stathmin 2 (STMN‐2)3. This mis‐splicing contributes to axonal degeneration. To model the ALS phenotypes in vitro, proteasomal inhibition is often used to trigger TDP‐43‐associated phenotypes2. Methods In this report we assessed if homozygous or heterozygous CRISPR‐edited TPD‐43 M337V mutations in induced pluripotent stem cell (iPSC)‐derived glutamatergic neurons, generated by bit.bio using the opti‐ox technology4‐5, alone, or in combination with an MG‐132 proteasomal inhibition trigger, would result in TDP‐43 mislocalization and/or STMN‐2 splicing, and could therefore be used as an ALS‐relevant disease model. This was assessed in highly mature (DIV21), and less mature (DIV7) neurons. Results Immunocytochemistry, high‐content imaging, and automated image analysis were used for visualization, localization, and quantification of proteins of interest in individual cells. Here, increase in TDP‐43 mislocalization, phosphorylation (and aggregation) was observed upon acute proteasomal inhibition in all cell lines compared to their respective untreated sample, with more extreme phenotypes on DIV7. More interestingly, the homozygous M337V cell line D5 showed showed TDP‐43 associated phenotypes in untreated conditions at DIV7. Digital droplet PCR was used to evaluate mRNA expression, using absolute quantification of nucleic acid molecules. This showed decrease of full length STMN‐2 and the generation of truncated STMN‐2 upon MG‐132 treatment in most cell lines. Additionally, preliminary functional data generated using Multi Electrode Arrays (MEA) showed that all neuronal lines demonstrated formation of synchronous activity at late stage of maturation, suggesting these neurons are electrophysiologically active and amenable to functional studies. Conclusion These results suggest that wildtype and mutant clones show TDP‐43 dysregulation and mis‐splicing of STMN‐2 upon acute proteasomal inhibition, thus establishing an ALS relevant phenotype that can be used for in vitro drug discovery.