Site-Specific Ubiquitination of Pathogenic Huntingtin Attenuates its Deleterious Effects- PART2

Huntington’s disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a CAG triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine (polyQ) stretch in the protein product which becomes aggregation-prone. The mutant Htt (mHtt) aggregates are associated with components of the Ubiquitin-Proteasome System (UPS), suggesting that mHtt is marked for proteasomal degradation and that for reasons still debated are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and its subsequent cellular response. We identified two lysine residues - 6 and 9 - in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size, but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Furthermore, proteomic analysis indicated that compared to cells expressing the protein that can be modified by ubiquitin, cells expressing the lysine-less protein did not activate protein synthetic pathways that enable the cell to cope with stress. Taken together, the findings suggest a novel role for ubiquitination - attenuation of the pathogenic effect of mHtt.