TDP-43 Phosphorylation State Regulates Solubility, Localization, and Cellular Toxicity

The hyperphosphorylated and insoluble TDP-43 inclusions are a pathological hallmark of several neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), frontal temporal dementia (FTD), and Alzheimer's Disease (AD). The mechanism by which phosphorylation regulates TDP-43 physiological functions and its contribution to the observed neuropathology remain unclear. To assess this, we generated recombinant TDP-43 proteins that mimic the hyperphosphorylated and hypophosphorylated condition to study the effect of TDP-43 phosphorylation of the LCD on phase separation, cellular localization, and cytotoxicity. Our studies show that both hyper- and hypo- phosphorylation mimetics promote TDP-43 mislocalization to the cytoplasm , enhance the propensity and stability of the TDP-43 C-terminal domain protein droplets, and disrupt phase separation. However, in all models it is only the phosphodeficient condition that reduces TDP-43 solubility and promotes the formation of cytotoxic aggregates. Furthermore, phosphodeficient TDP-43 induces the sequestration of endogenous TDP-43 to these insoluble aggregates that ultimately diminish TDP-43 splicing capacity. Taken together, our efforts provide further evidence that insoluble, cytoplasmic TDP-43 inclusions are cytotoxic and that phosphorylation might actually play a protective role, inhibiting the formation of insoluble, toxic, cytoplasmic TDP-43 aggregates.