Increased Nuclear Localization of Engineered Hsp104 Variants Mitigates aS, FUS, and TDP-43 Toxicity in Yeast.

The cytoplasmic mislocalization and aggregation of aberrantly folded proteins is a hallmark of human neurodegenerative diseases, including Parkinson's disease (PD) and frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS). Endogenous human disaggregase machineries are insufficient to combat pathological protein aggregates. However, the yeast protein Hsp104 has shown enhanced activity in antagonizing oligomers and aggregates of the PD-associated protein alpha-Synuclein (aS) and the FTD-ALS-associated proteins Fused-In-Sarcoma (FUS) and TAR DNA binding protein 43 (TDP-43). Hsp104 is a hexameric AAA+ ATPase and protein disaggregase which, in collaboration with the Hsp70/Hsp40 chaperone system, can extract peptides from higher-order protein structures and mediate their proper refolding and cellular localization. Strategic missense mutations potentiate the ability of Hsp104 to mitigate aS, FUS, and TDP-43 toxicity in yeast. However, on their own, some of the potentiated Hsp104 variants demonstrate off-target toxicity which impairs yeast growth. Here, yeast survival assays show that increasing the nuclear localization of these already potentiated Hsp104 mutants reduces their off-target toxicity and improves yeast growth. Additionally, fluorescence microscopy indicates that the resolution of cytoplasmic foci and the proper relocalization of disease-associated proteins often corresponds to increased yeast survival. Together, these data suggest that increasing the nuclear localization of already potentiated Hsp104 mutants further enhances their rescue activity, in part through the increased resolution of cytoplasmic aS, FUS, and TDP-43 foci. Ultimately, the delivery of these potent nuclear Hsp104 mutants to first animal models and then humans could represent a novel therapy for a variety of neurodegenerative diseases.