Prevention of ubiquitination at K6 and K9 in mutant huntingtin exacerbates disease pathology in a knock-in mouse model

Huntington disease (HD) is caused by an expansion of the polyglutamine (polyQ) tract in the huntingtin protein (HTT), leading to its misfolding and aggregation. The subcellular localization of mutant HTT (mHTT) aggregates critically influences their neuronal toxicity, with nuclear aggregates contributing more significantly to neurodegeneration than those in the neuropil. Our previous findings demonstrated that site-specific ubiquitination of lysine residues at the positions of K6 and K9 in HTT significantly affect the aggregation properties of mHTT and influence cell viability. However, the in vivo functional relevance of this modification remains elusive. To address this, we generated two HD knock-in (KI) mouse models in which the mouse Htt exon 1 was replaced by human mutant HTT exon 1 containing 134 pure cytosine–adenine–guanine (CAG) repeats. In addition, one of these KI lines carries lysine-to-arginine (K > R) substitutions at residues 6 and 9 to block site-specific ubiquitination (Q134 RR line). Compared to Q134 KK control mice, Q134 RR mice showed a more pronounced accumulation of both soluble and aggregated forms of mHTT. Notably, the K > R substitutions accelerated mHTT aggregation kinetics, resulting in the formation of large inclusion bodies and their exclusive nuclear localization. Furthermore, Q134 RR mice exhibited earlier onset and accelerated progression of motor impairments, brain atrophy, and neuropathological features. Collectively, our findings provide strong in vivo evidence for the crucial role of site-specific ubiquitination at K6 and K9 in modulating mHTT aggregation and HD pathology. These results reinforce the therapeutic potential of targeting these specific ubiquitination sites for clinical translation.