TDP-43 seeding induces cytoplasmic aggregation heterogeneity and nuclear loss-of-function of TDP-43

Cytoplasmic aggregation and nuclear depletion of TDP-43 are hallmarks of several age-related neurodegenerative disorders. Yet, recapitulating both features in cellular systems has been a major challenge. Here, we produced amyloid-like fibrils from the recombinant low-complexity-domain of TDP-43, and demonstrate that sonicated fibrils trigger TDP-43 pathology in human cell lines and iPSC-derived neurons. Fibril-induced cytoplasmic TDP-43 inclusions acquire distinct biophysical properties, recapitulate pathological hallmarks such as phosphorylation, ubiquitin- and p62-accumulation, and recruit nuclear endogenous TDP-43 leading to nuclear-loss-of-function-driven disease-specific cryptic splicing defects. Cytoplasmic TDP-43 aggregates exhibit, with time, distinct heterogeneous morphologies as in patients including compacted, filamentous or fragmented, through the activation of cellular protein clearance pathways. Cell-specific progressive toxicity is provoked by seeded TDP-43 pathology in human neurons. These findings identify templated aggregation of TDP-43 as a key mechanism driving both cytoplasmic gain- and nuclear-loss-of-function, offering a valuable approach to identify modifiers of sporadic TDP-43 proteinopathies. Overall design: TDP-43-mNLS-Clover expressing U2OS cells (doxycycline induced) were transfected with fibril seeds produced from recombinant TDP-43 LCD (50nM) to induce TDP-43 aggregation. In control conditions, TDP-43-mNLS-Clover expressing U2OS cells were mock transfected with protein storage buffer. Each transfection reaction was performed in triplicates, serving as 3 independent biological replicates. 48 hours after fibril (or control) transfection, cells were collected and image-based FACS sorting was performed using FACSDiscover S8 to isolate cells with TDP-43-Clover aggregates (from fibril transfected condition) or cells with diffuse TDP-43-Clover (from mock transfected condition). FACS-purified cell populations (aggregates vs no_aggregates) were subsequently processed for RNA-Seq.