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.