RNA-deficient TDP-43 causes loss of free nuclear TDP-43 by sequestration

Dysfunction and aggregation of the RNA-binding protein, TDP-43, is the unifying hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mechanisms and relative contributions of concurrent TDP-43 nuclear depletion, cytoplasmic accumulation, and post-translational modification to neurodegeneration remain unresolved. We employed CRISPR/Cas9-mediated fluorescent tagging to investigate how disease-associated stressors and pathological TDP-43 alter abundance, localisation, self-assembly, aggregation, solubility, and mobility dynamics of endogenous TDP-43 over time. Oxidative stress stimulated TDP-43 liquid-liquid phase separation into droplets or spherical shell-like anisosomes, which were not formed by over-expressed wild-type TDP-43. Further, nuclear RNA-binding-ablated or acetylation-mimicking TDP-43 rapidly formed anisosomes and inclusions that readily sequestered and depleted free normal nuclear TDP-43. The majority of total endogenous TDP-43 was sequestered into anisosomes, but retained high protein mobility and solubility. However, cytoplasmic RNA-deficient TDP-43 formed large phosphorylated inclusions that occasionally sequestered endogenous TDP-43, rendering it insoluble and immobile, indicating irreversible pathological transition. These findings suggest that post-translational modification and RNA-binding deficiency exacerbate TDP-43 aggregation and dysfunction by driving sequestration, mislocalisation, and depletion of normal nuclear TDP-43 in ALS and FTD.