Persistent export bias of TDP-43 under native autoregulation links insoluble accumulation to nuclear dysfunction

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• Nuclear depletion and cytoplasmic mislocalization of TDP-43 are key pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).
• TDP-43 protein levels are regulated by its 3' untranslated region (3' UTR), but its protective role during chronic cytoplasmic mislocalization is not fully understood.

Purpose of the Study:

• To investigate whether TDP-43's native autoregulation remains protective when the protein is biased towards the cytoplasm.
• To model the effects of chronic cytoplasmic mislocalization on TDP-43 function and pathology.

Main Methods:

• Engineered full-length human TDP-43 with an N-terminal nuclear export signal (NES) while preserving the 3' UTR autoregulatory module.
• Utilized HEK293T and differentiated SH-SY5Y cells, as well as human induced pluripotent stem cell (iPSC)-derived neurons to assess TDP-43 behavior.
• Performed functional rescue experiments by depleting endogenous TARDBP and comparing wild-type (WT) TDP-43 with NES-TDP-43.

Main Results:

• NES insertion induced cytoplasmic bias and promoted detergent-insoluble TDP-43 species in cell models.
• In neurons, cytoplasmic shift, TDP-43 foci, and splicing defects correlated with the export-biased load.
• While NES-TDP-43 expression increased TARDBP transcripts, the protein accumulated in insoluble fractions, failing to restore nuclear function or normalize splicing.

Conclusions:

• Persistent cytoplasmic export bias of TDP-43 converts its native autoregulation into a maladaptive feedback loop.
• Compensatory TDP-43 output is diverted to insoluble cytoplasmic fractions, uncoupled from nuclear recovery.
• Abundance alone is insufficient to restore function when TDP-43 is export-biased, highlighting the critical role of nuclear localization for preventing pathology in ALS and FTLD.