Deletion of the <i>Saccharomyces cerevisiae RACK1</i> homolog, <i>ASC1</i> , enhances autophagy which mitigates TDP-43 toxicity

Cytoplasmic aggregation of nuclear proteins such as TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) is associated with several neurodegenerative diseases. Studies in higher cells suggest that aggregates of TDP-43 and FUS sequester polysomes by binding RACK1 (receptor for activated C kinase 1), a ribosomal protein, thereby inhibiting global translation and contributing to toxicity. However, RACK1 is also a scaffold protein with a role in many other cellular processes including autophagy. Using yeast, we find that deletion of the RACK1 ortholog, ribosomal protein ASC1, reduces TDP-43 toxicity, but not FUS toxicity. TDP-43 foci remain liquid like in the absence of ASC1 but they become smaller. This is consistent with findings in mammalian cells. However, using double label fluorescent tags and co-immunoprecipitation we establish that ASC1 does not co-localize with TDP-43 foci, challenging the polysome sequestration hypothesis. Instead, ASC1 appears to influence toxicity through regulation of autophagy. We previously showed that TDP-43 expression inhibits autophagy and TOROID (TORC1 Organized in Inhibited Domains) formation and that genetic modifiers that rescue yeast from TDP-43 toxicity reverse these effects. Here we show that FUS does not inhibit autophagy. Deletion of ASC1 enhances a non-canonical form of autophagy that effectively counteracts TDP-43 induced autophagy inhibition despite reduced TOROID formation. Our findings highlight autophagy-not polysome sequestration-as a key mechanism underlying ASC1-mediated modulation of TDP-43 toxicity and suggest autophagy as a promising therapeutic target.