Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control

Abstract

9Mutations that cause Huntington's Disease involve a polyglutamine (polyQ) sequence expansion beyond 10 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key 11 feature of Huntington's disease brain pathology. We previously showed that in cell culture the 12 formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments 13 (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that 14 nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like 15 disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined 16 the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model and 17 examined whether ribosome quality control machinery previously implicated in stalled ribosomes were 18 involved. We found mHttex1 did not appear to stall translation of its own nascent chain and there was 19 no recruitment of RNA into inclusions. However, proteins involved in translation or ribosome quality 20 control were co-recruited into the inclusions (Ltn1 and Rack1) compared to a protein not anticipated to 21 be involved (NACAD). Furthermore, we observed co-aggregation with other proteins previously 22 identified in inclusions, including Upf-1 and chaperone-like proteins Sgta and Hspb1, which also 23 suppressed aggregation at high co-expression levels. The newly formed inclusions contained immobile 24 mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid 25 formation. 26

Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease

“…Htt is proteolytically cleaved by several proteases, resulting in smaller N-terminal poly-Q containing exon 1 and non-poly-Q Htt C-terminal fragments, which are considered important for disease pathogenesis 54,127,128 . Although we found FL-mHtt present on the polysomes and interacts with ribosomal proteins, previous work has shown that exon 1 aggregates could also be associated with ribosomes 129,130 . Therefore, different Htt fragments (e.g., poly-Q exon 1 and HEAT repeat domains) and aggregated forms 54,63,131 may differentially bind to the translating ribosomes.…”