Critical impact of lysine 136 in TDP-43 phase separation, compartmentalization, and aggregation in living vertebrates.

TDP-43 is a nuclear RNA-binding protein that undergoes liquid-liquid phase separation (LLPS) and forms insoluble aggregates in neurodegenerative diseases. By studying TDP-43 in living vertebrates, we confirmed that TDP-43 undergoes LLPS and forms dynamic biomolecular condensates in spinal motor neurons. We validated that interfering with the lysine residue at position 136 altered the phase separation behavior of TDP-43 by reducing cytoplasmic mislocalization and aggregation. These alterations were post-translational modification (PTM) independent, highlighting that residue 136 is a key structural regulator of TDP-43 function. We further established an adeno-associated virus (AAV)-mediated expression approach in mice that confirmed altered nuclear condensation characteristics of lysine-modified TDP-43. These assessments exposed the formation of dynamic nuclear TDP-43 condensates and emphasize the important role of lysine 136 in maintaining TDP-43 function. Altogether, we establish lysine 136 as a molecular regulator for phase separation and TDP-43 aggregation in amyotrophic lateral sclerosis (ALS) in two platforms.