Hanyu, R. | bioRxiv Science

bioRxiv2025

Export-biased, 3'UTR-preserving TDP-43 model links nuclear loss to cytoplasmic aggregation in ALS/FTLD

Nuclear depletion and cytoplasmic mislocalization of TDP-43 are central pathological features of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 protein levels are normally maintained by autoregulation through its native 3' untranslated region (3' UTR). It remains untested whether this feedback is still protective once the perturbation is chronic, and whether the resulting rise in TARDBP transcripts restores the functional nuclear pool or is instead diverted into non-functional species. To address this, we engineered full-length human TDP-43 carrying an N-terminal nuclear export signal (NES) while leaving the native 3' UTR autoregulatory module intact. Expression was titrated so that whole-cell RIPA-soluble exogenous TDP-43 remained [≤]30% of endogenous levels. In HEK293T cells, export bias drove TDP-43 into the cytoplasm and produced detergent-insoluble species. In differentiated SH-SY5Y cells, nuclear splicing defects and autoregulatory changes scaled with export-biased load, and detergent-insoluble accumulation was already present within the same low-load range. Human iPSC-derived neurons showed a comparable cytoplasmic shift, discrete TDP-43-positive foci, and TDP-43-dependent splicing defects. When endogenous TDP-43 was selectively depleted under transgene induction, 3' UTR-coupled autorepression was weakened and transgene-derived TARDBP transcripts rose; however, the additional exogenous TDP-43 did not expand the soluble, splice-competent pool but partitioned into insoluble fractions. Splicing defects in differentiated SH-SY5Y cells persisted even when soluble NES-TDP-43 reached endogenous-equivalent levels. We therefore propose that under native 3' UTR control, sustained cytoplasmic bias uncouples compensatory TARDBP upregulation from recovery of the functional nuclear pool; the extra output is diverted into insoluble, fragmented species rather than restoring nuclear function.

molecular biology↗

bioRxiv2024

ASO-enhancement of TARDBP exitron splicing mitigates TDP-43 proteinopathies

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are fatal neurodegenerative diseases characterized by pathological aggregation and nuclear functional loss of TDP-431,2. Current therapies inadequately address this core pathology3,4, necessitating innovative approaches that target aggregation while preserving TDP-43s essential functions. Here we demonstrate that enhancing the splicing of the TARDBP exitron--a cryptic intron encoding the aggregation-prone intrinsically disordered region (IDR) of TDP-435,6-- effectively mitigates TDP-43 pathology. This exitron splicing event, directly regulated by nuclear TDP-437-9, suppresses the expression of IDR-containing TDP-43 isoforms and generates IDR-spliced-out TDP-43 isoforms7,9,10 (which we term "IDRsTDP"). Our findings reveal that IDRsTDP, known to heterodimerize with full-length TDP-4310, inhibits TDP-43 aggregation by suppressing IDR-mediated clustering and enhances TDP-43 clearance via chaperone-mediated autophagy. In disease states, however, impaired nuclear TDP-43 function disrupts exitron splicing, leading to increased levels of IDR-containing TDP-439,11 and reduced levels of IDRsTDP, exacerbating aggregation and nuclear dysfunction6,12-17. By identifying HNRNPA1 and HNRNPC as key repressors of TARDBP exitron splicing, we designed antisense oligonucleotides (ASOs) to block their binding and restore splicing. These ASOs suppressed TDP-43 pathology and neurodegeneration in both neuronal cell models with impaired nuclear transport and a mouse model of proteasome dysfunction-induced TDP-43 proteinopathy. Our strategy, by rescuing the impaired autoregulatory pathway, inhibits the pathological cycle of TDP-43 aggregation and nuclear dysfunction, offering a promising avenue for treating these currently intractable neurodegenerative diseases.

neuroscience↗