A human forebrain organoid model phenocopies dysregulated RNA and protein homeostasis in ALS/FTD-associated TDP-43...

TAR DNA-binding protein 43 (TDP-43) proteinopathy is a central hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet current experimental models fail to reproduce the full pathological spectrum without external stress or TDP-43 overexpression. This study aims to establish a human induced pluripotent stem cells (iPSC)-derived system that spontaneously manifests TDP-43 pathology driven by an ALS-associated TDP-43 mutation.

We generated forebrain 3-D organoid cultures from iPSC carrying the TDP-43 K181E patient mutation. Single-cell RNA sequencing was used to define transcriptional alterations across cell types, and enhanced crosslinking immunoprecipitation (eCLIP) was applied to examine the global RNA binding and splicing defects in mutant organoids. We further used immunostaining, RT-PCR and biochemical assays to confirm TDP-43 proteinopathy and validate findings from the multi-omics analyses.

The TDP-43 K181E organoids recapitulated key disease features, including cytoplasmic p-TDP-43 accumulation, RNA dysregulation, and cryptic exon inclusion. Single-cell analysis revealed a population of immature neurons with enhanced neuroinflammation and altered translation capacity. Comparative transcriptomics showed that the ALS mutation-induced transcriptional changes strongly overlap with those in ALS patient-derived brains. eCLIP analysis showed that mutant TDP-43 exhibited altered RNA-binding specificity, resulting in widespread RNA mis-splicing and cryptic exon inclusion. RT-PCR confirmed PRDM2, a gene regulating cell senescence, is mis-spliced in mutant cells. These defects collectively disrupt neuronal homeostasis and cell-cell communications.

Our iPSC-derived forebrain organoid model displays spontaneous TDP-43 proteinopathies and associated molecular dysfunctions without artificial manipulation. The model offers a robust platform for dissecting the mechanisms of TDP-43-mediated neurodegeneration and advancing therapeutic discovery in ALS and FTD.