Charge and redox states modulate granulin-TDP-43 coacervation toward phase separation or aggregation

Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other pro-teins. To study the factors that influence the dynamics between these cytoplasmic foci, we investigated the effects of cysteine-rich granulins (GRNs 1???7), which are the proteolytic products of progranulin, a protein implicated in FTLD, on TDP-43. We show that extracellular GRNs, typically generated during inflammation, internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show less likelihood of their presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel-or solid-like aggregates. Using charge patterning and conserved cys-teines among the wild-type GRNs as guides, along with specifically engineered mutants, we discover that the negative charges on GRNs drive LLPS while the positive charges and the redox state of cysteines modulate these phase transitions. Furthermore, RNA and GRNs compete and expel one another from PrLD condensates, providing a basis for GRN???s absence in SGs. Together, the results help uncover potential modulatory mechanisms by which extracellular GRNs, formed during chronic inflam-matory conditions, could internalize and modulate cytoplasmic TDP-43 inclusions in proteinopathies.