Accumulation of Mutant Huntingtin Fragments in Aggresome-like Inclusion Bodies as a Result of Insufficient Protein Degradation

TLDR

Huntingtin exon 1 proteins with pathogenic polyglutamine repeats (51 or 83 Q) form aggresome‑like perinuclear inclusions in human 293 Tet‑Off cells, whereas normal‑range repeats (20 Q) do not. Immunofluorescence and immunogold electron microscopy show that proteasome subunits (20S, 19S, 11S), chaperones (BiP/GRP78, Hsp70, Hsp40), TIA‑1, 14‑3‑3, and α‑synuclein colocalize with these inclusions. The inclusions contain aggregated, ubiquitinated huntingtin exon 1 with fibrillar morphology, are associated with centrosomal truncated protein surrounded by vimentin, increase two‑fold when proteasome activity is inhibited, and their formation induces cell toxicity, nuclear envelope disruption, mitochondrial clustering, and cytoplasmic vacuolation, supporting the ubiquitin–proteasome system as a therapeutic target for glutamine‑repeat disorders.

Abstract

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin–proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14–3-3, and α-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin–proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.