Concentration‐dependent cytoplasmic phase separation of TDP ‐43 drives aggregation and proteinopathy

TDP‐43 mislocalization and aggregation are common features of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the mechanisms underlying the transition of nuclear TDP‐43 to cytoplasmic aggregates, and their contribution to disease pathogenesis, remain poorly understood. To address this gap, we present a methodology to chemically control the assembly and disassembly of cytoplasmic TDP‐43 condensates. By fusing TDP‐43 to a phase separation–prone protein scaffold, we can induce the formation of cytoplasmic TDP‐43 condensates or, conversely, promote nuclear localization upon addition of a disassembly molecule. TDP‐43 accumulates into various assemblies, ranging from submicrometric puncta to larger aggregate‐like structures that display hallmarks of proteinopathy in a concentration‐dependent manner. Furthermore, oxidative stress drives the maturation of TDP‐43 assemblies from puncta into aggregates through interactions with stress granule components. Finally, we show that cytoplasmic TDP‐43 aggregates deplete nuclear endogenous TDP‐43 and induce cytotoxicity. Collectively, these findings highlight the local cytoplasmic concentration of TDP‐43 and stress exposure as key determinants in the onset of TDP‐43 proteinopathy, providing a relevant model to study pathological TDP‐43 aggregation.