Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to hypoactivity and synaptic abnormalities in human iPSC-derived motor neurons

Abstract

Cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore, while mutations in the TARDBP gene (encoding TDP-43) have been associated with ALS, the pathogenic consequences of these mutations remain poorly understood. Using CRISPR/Cas9, we engineered two homozygous knock-in iPSC lines carrying mutations in TARDBP encoding TDP-43A382Tand TDP-43G348C, two common yet understudied ALS TDP-43 variants. MNs differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization, phosphorylation, solubility, or aggregation compared with isogenic control MNs. However, our results highlight synaptic impairments in both TDP-43A382Tand TDP-43G348CMN cultures, as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively, our findings argue that MN dysfunction precedes the occurrence of TDP-43 pathology and neurodegeneration in ALS, and further implicates synaptic and excitability defects in the pathobiology of this disease.

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

“…Given that, to our knowledge, similar cross-linking experiments characterizing TDP-43-bound transcripts have not yet been performed in human spinal cord specimens or spinal MN cultures, additional RNA-protein interaction studies may be needed to reveal cell type-specific TDP-43 binding targets. In line with this idea, recent studies have shown that different cell environments modulate the RNA processing functions of TDP- 43 49,50.…”

“…We have recently reported the generation of two homozygous knock-in iPSC lines with mutations in TARDBP coding for TDP-43 A382T or TDP-43 G348C , two frequent ALS variants of TDP-43. 43 To characterize the transcriptomic profiles of MNs differentiated from those cells, we performed next-generation RNA sequencing (RNA-seq) on total RNA extracted from mutant and isogenic control iPSC-derived MN cultures at 4-weeks post-plating ( Figure 1A and Supplemental Table S1 ). Analysis of normalized counts confirmed the expression of the MN markers ISL1 , MNX1 (Hb9), CHAT , and SLC18A3 (VAChT) ( Supplemental Figure 1A ).…”

“…45 Importantly, the expression levels of cell type markers did not significantly differ between mutant and control samples, indicating a similar cell composition and differentiation propensity, as previously described. 43…”

“…In previous work, we showed that removal of neurotrophic factors (NFs) supplementation from the differentiation medium significantly decreased the viability of MNs regardless of their genotype. 43 Thus, we treated MNs cultured without NFs with two concentrations of the candidate compounds (0.1 μM and 1.0 μM) for 6 days to screen for compounds with neuroprotective properties ( Figure 5E ). We also added to our panel of compounds three FDA-approved drugs for the treatment of ALS, namely riluzole, edaravone, and the combination of sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) (i.e., AMX0035).…”

“…Positing that shared alterations may be reflective of underlying disease mechanisms, we queried the Connectivity Map (CMap) database 42 for compounds predicted to normalize gene expression changes toward wild-type levels. Selected top-scoring compounds identified in silico were tested experimentally for their ability to ameliorate previously identified disease-relevant readouts 43 in two phenotypic screens for viability and neuronal activity. This approach led to the identification of the NEDD8-activating enzyme inhibitor MLN4924.…”