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

Mislocalization of TDP-43 with nuclear loss of function is a defining feature of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), yet the earliest, low-amplitude events that shift the system from homeostasis to pathology have been difficult to model. We engineered full-length human TDP-43 bearing an N-terminal nuclear export signal while preserving the native 3′UTR, thereby maintaining N-terminal domain multimerization and physiological autoregulation. Titration to a low-load regime allowed interrogation of nucleocytoplasmic balance near homeostasis in HEK293T cells, differentiated SH-SY5Y neurons, and human iPSC-derived neurons. A minimal increase in export produced an exogenous pool that was disproportionately cytoplasmic and detergent-insoluble, while total cellular TDP-43 remained near baseline. This subtle cytoplasmic bias initiated a cascade: co-recruitment of endogenous TDP-43, formation of cytoplasmic puncta, progressive impairment of TDP-43–dependent splicing—including STMN2 —and C-terminal fragmentation. Targeted reduction of endogenous nuclear TDP-43 relieved 3′UTR-mediated autoregulatory repression. The resulting increase in exogenous transcript and protein further amplified detergent-insoluble and fragmented species, quantitatively delineating a maladaptive positive-feedback loop in which nuclear loss both drives and is reinforced by cytoplasmic accumulation. By imposing a defined, minimal perturbation while preserving physiological control, this export-biased, 3′UTR-preserving platform unifies the three defining axes of TDP-43 proteinopathy—nuclear loss of function, cytoplasmic accumulation of insoluble/fragmented species, and 3′UTR-based autoregulation—within a single, tunable circuit. Conceptually, it identifies a threshold at which modestly increased cytoplasmic residency becomes pathogenic; practically, it provides early, human-relevant readouts for screening interventions that strengthen 3′UTR-dependent autoregulation or normalize nucleocytoplasmic transport. These findings recast the initiation of TDP-43 pathology as a measurable, targetable failure of nucleocytoplasmic homeostasis.