bioRxiv. 2025 Sep 05. pii: 2025.09.05.674500. [Epub ahead of print]
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 these 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. But RACK1 is also a scaffold protein with many other roles including a role in autophagy. Using yeast we find that deletion of the RACK1 ortholog, ASC1 , reduces TDP-43 toxicity, but not FUS toxicity. TDP-43 foci remain liquid like in the presence asc1Δ but they become smaller. This is consistent with the findings in cell culture. However, using double label tags we establish that ASC1 does not co-localize with TDP-43 foci, arguing against the sequestration hypothesis. Instead, ASC1 appears to influence toxicity through autophagy. We previously showed that expression of TDP-43 inhibits autophagy and TOROID (TORC1 Organized in Inhibited Domains) formation and that modifiers that rescue yeast from TDP-43 toxicity reverse these inhibitions. Here we show that FUS does not inhibit autophagy. This autophagy enhanced by asc1Δ is non-canonical, marked by reduced TOROID formation, and effectively counteracts the autophagy inhibition caused by TDP-43. Our findings suggest that ASC1 influences TDP-43 toxicity through autophagy regulation rather than polysome sequestration, highlighting autophagy as a key therapeutic target.
Summary: TDP-43 and FUS aggregates are linked to neurodegenerative diseases. RACK1, a ribosomal protein, was previously thought to contribute to toxicity by co-localizing with these aggregates and sequestering polysomes. In yeast, deletion of ASC1 -the RACK1 homolog-reduces TDP-43 toxicity but not FUS toxicity. TDP-43 foci remain liquid-like, but ASC1 does not co-localize with them, challenging the sequestration hypothesis. Instead, asc1Δ enhances autophagy, rescuing cells from the autophagy inhibition caused by TDP-43. Unlike TDP-43, FUS does not inhibit autophagy. These findings highlight autophagy, rather than polysome sequestration, as the key mechanism of TDP-43 toxicity and its mitigation via ASC1/RACK1 reduction.