Wu, J. J.; Fan, S.-Y.; Chang, T.-H.; Chen, Y.-R.

Amyotrophic lateral sclerosis (ALS) is categorized by TDP-43 proteinopathy, however, the nuclear pathological events remain poorly defined. While cytoplasmic TDP-43 inclusions dominate the late disease stages, accumulating evidence indicates that nuclear TDP-43 assemblies arise earlier and impair RNA splicing. Here, we characterized a single RRM-proximal TDP-43 variant, G148V, designed to disrupt nucleic-acid engagement without altering canonical RNA-binding residues. Structural and biophysical analyses revealed conformational changes and loss of DNA/RNA binding. In mammalian cells, TDP-43 G148V robustly formed nuclear puncta with high penetrance, exhibiting solid-like properties, pathological phosphorylation, splicing dysfunction, and toxicity. Furthermore, we identified molecular chaperone HSC70 as an important regulator of the nuclear puncta assembly. HSC70 redistributed into G148V nuclear puncta to modulate their material state, whereas HSC70 depletion significantly promoted puncta solidification, increased insoluble TDP-43 accumulation, and enhanced cytotoxicity. Disease-associated K181E and K263E mutants also formed nuclear puncta and induced HSC70 nuclear redistribution. These findings establish G148V as a model of early nuclear TDP-43 pathology and highlight HSC70-mediated regulation as a key factor of TDP-43 nuclear assembly.