Cytoplasmic Colocalization of Granulins and TDP-43 Prion-like Domain Involves Electrostatically Driven Coacervation Tuned by the Redox State of Cysteines

Abstract

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 proteins. We were interested in understanding if and how cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of a genetic risk factor in FTLD called progranulin, interact with TDP-43. We show that extracellular GRNs internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show no presence in SGs. In addition, GRNs and PrLD undergo liquid-liquid phase separation (LLPS) by complex coacervation, or form aggregates via liquid-solid phase separation (LSPS); the dynamics in these phase transitions appear to be driven by the negative charges on GRNs and fine-tuned by the positive charges and the redox state of cysteines. Furthermore, RNA competes with and expunges GRNs from GRN-PrLD condensates, providing a basis for GRN's absence in SGs. Together, the results bring to bear unique mechanisms by which GRNs could modulate TDP-43 proteinopathies.