Soluble polyglutamine oligomers formed prior to inclusion body formation are cytotoxic

Abstract

Expanded polyglutamine (polyQ) repeats cause neurodegenerative disorders, but their cytotoxic structures remain to be elucidated. Although soluble polyQ oligomers have been proposed as a cytotoxic structure, the cytotoxicity of soluble polyQ oligomers, not inclusion bodies (IBs), has not been proven in living cells. To clarify the cytotoxicity of soluble polyQ oligomers, we carried our fluorescence resonance energy transfer (FRET) confocal microscopy and distinguished oligomers from monomers and IBs in a single living cell. FRET signals were detected when donor and acceptor fluorescent proteins were attached to the same side, not the opposite side, of polyQ repeats, which agrees with a parallel beta-sheet or a head-to-tail cylindrical beta-sheet model. These FRET signals disappeared in semi-intact cells, indicating that these polyQ oligomers are soluble. PolyQ monomers assembled into soluble oligomers in a length-dependent manner, which was followed by the formation of IBs. Notably, survival assay of neuronally differentiated cells revealed that cells with soluble oligomers died faster than those with IBs or monomers. These results indicate that a length-dependent formation of oligomers is an essential mechanism underlying neurodegeneration in polyQ-mediated disorders.

Firefly luciferase mutants as sensors of proteome stress

“…This indicates that collapse of proteostasis might have occurred before the appearance of detectable Htt-97Q aggregates. This observation is in line with other studies that suggest a role of the oligomeric species of the mutant Htt protein in proteostasis imbalance and cellular toxicity (Arrasate et al, 2004;Bodner et al, 2006;Miller et al, 2010;Saudou et al, 1998;Takahashi et al, 2008). However, after the mutant Htt formed visible aggregates, the aggregation of FlucDM-EGFP became more severe and rapid which may suggest that aggregation of Htt continues to impose a challenge on the proteostasis machinery of cells.…”

“…A considerable body of experimental evidence suggests that formation of Htt inclusions is a protective mechanism, employed by cells to sequester potentially dangerous soluble oligomers of misfolded Htt (Arrasate et al, 2004;Bodner et al, 2006;Miller et al, 2010;Takahashi et al, 2008). This sequestration may reduce the number of exposed regions in mutant Htt that otherwise would titrate away cellular folding factors, putting the folding of the metastable proteome in jeopardy (Gidalevitz et al, 2006;Satyal et al, 2000).…”

“…At the moment this question is difficult to answer. While several lines of indirect evidence support a role of Htt inclusion bodies in cellular toxicity DiFiglia et al, 1997;Yang et al, 2002), other studies indicate a role of misfolded monomers or oligomers in neuronal cell death (Arrasate et al, 2004;Bodner et al, 2006;Miller et al, 2010;Saudou et al, 1998;Takahashi et al, 2008). To address this question using Fluc sensors, we performed live-cell imaging in HEK 293T cells that were transiently cotransfected with equal amounts of DNA encoding Fluc sensors and mutant Htt (Figure 31).…”

p62 Plays a Protective Role in the Autophagic Degradation of Polyglutamine Protein Oligomers in Polyglutamine Disease Model Flies

“…Although several studies reported that the amount of polyQ protein inclusions does not correlate with neurodegeneration or that these inclusions are even protective, oligomeric species of misfolded polyQ proteins have been suggested as the principal culprit of neurodegeneration (6,35). In other protein-misfolding neurodegenerative diseases, soluble oligomers composed of A␤ or ␣-synuclein have also been shown to exert cytotoxicity, suggesting the intrinsic toxicity of oligomeric structures, regardless of their primary amino acid sequences (36).…”

“…PolyQ proteins are prone to misfold, oligomerize, and form aggregates and eventually accumulate as inclusion bodies in affected neurons (2,3). Whereas the formation of polyQ protein inclusion bodies is believed to be protective, by sequestering the toxic polyQ proteins (4), the intermediate structures formed during the aggregation process, such as monomers or oligomers, are reported to be more toxic for the cells, leading to neuronal dysfunction or neuronal cell death (5,6). The polyQ diseases are thus considered as one of the protein-folding diseases, together with Alzheimer disease, Parkinson disease, and ALS.…”