Transcriptome-based screening in TARDBP/TDP-43 knock-in motor neurons identifies the NEDD8-activating enzyme inhibitor MLN4924 [miRNA-Seq]

A growing body of knowledge implicates perturbed RNA homeostasis in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that currently has no cure and few available treatments. Dysregulation of the multifunctional RNA-binding protein TDP-43 is increasingly regarded as a convergent feature of this disease, evidenced at the neuropathological level by the detection of TDP-43 pathology in most patient tissues, and at the genetic level by the identification of disease-associated mutations in its coding gene TARDBP. To characterize the transcriptional landscape induced by TARDBP mutations, we performed whole-transcriptome profiling of motor neurons differentiated from two knock-in iPSC lines expressing the ALS-linked TDP-43 variants p.A382T or p.G348C. Our results show that the TARDBP mutations significantly altered the expression profiles of mRNAs and microRNAs of the 14q32 cluster in MNs. Using mutation-induced gene signatures and the Connectivity Map database, we identified compounds predicted to restore gene expression toward wild-type levels. Among top-scoring compounds selected for further investigation, the NEDD8-activating enzyme inhibitor MLN4924 effectively improved cell viability and neuronal activity, highlighting a possible role for protein post-translational modification via NEDDylation in the pathobiology of TDP-43 in ALS. Overall design: To characterized the miRNA profiles induced by TARDBP mutations, we extracted total RNA from motor neurons differentiated from two knock-in iPSC lines expressing the ALS-linked TDP-43 variants p.A382T or p.G348C and their isogenic control iPSC line (AIW002-02) at four weeks post-plating of progenitors cells. We then performed differential expression analysis using data from small RNA-seq of five independent differentiations (i.e., biological replicates) for each cell line. We contrasted the isogenic control against each mutant cell lines individually and against both mutants combined.