Tracking Mutant Huntingtin Aggregation Kinetics in Cells Reveals Three Major Populations That Include an Invariant Oligomer Pool

Abstract

Huntington disease is caused by expanded polyglutamine sequences in huntingtin, which procures its aggregation into intracellular inclusion bodies (IBs). Aggregate intermediates, such as soluble oligomers, are predicted to be toxic to cells, yet because of a lack of quantitative methods, the kinetics of aggregation in cells remains poorly understood. We used sedimentation velocity analysis to define and compare the heterogeneity and flux of purified huntingtin with huntingtin expressed in mammalian cells under non-denaturing conditions. Non-pathogenic huntingtin remained as hydrodynamically elongated monomers in vitro and in cells. Purified polyglutamine-expanded pathogenic huntingtin formed elongated monomers (2.4 S) that evolved into a heterogeneous aggregate population of increasing size over time (100-6,000 S). However, in cells, mutant huntingtin formed three major populations: monomers (2.3 S), oligomers (mode s(20,w) = 140 S) and IBs (mode s(20,w) = 320,000 S). Strikingly, the oligomers did not change in size heterogeneity or in their proportion of total huntingtin over 3 days despite continued monomer conversion to IBs, suggesting that oligomers are rate-limiting intermediates to IB formation. We also determined how a chaperone known to modulate huntingtin toxicity, Hsc70, influences in-cell huntingtin partitioning. Hsc70 decreased the pool of 140 S oligomers but increased the overall flux of monomers to IBs, suggesting that Hsc70 reduces toxicity by facilitating transfer of oligomers into IBs. Together, our data suggest that huntingtin aggregation is streamlined in cells and is consistent with the 140 S oligomers, which remain invariant over time, as a constant source of toxicity to cells irrespective of total load of insoluble aggregates.

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo

“…Furthermore, recently both simulations and models have also predicted a heterogeneity of oligomer and aggregate species (75,76). Studies on Htt aggregation in cells have also described an oligomer population reaching an equilibrium, suggesting that oligomers are the rate-limiting event in inclusion body formation (73,77). Our results indicate that, in both aged animals at the threshold length, Q35, and for Q37 and Q40, an equilibrium is reached that maintains oligomers in a specific populational distribution, perhaps through interactions with cellular factors and quality control machinery, such as chaperones and clearance machines, and by the continual regeneration of monomers.…”

“…While some in vitro studies have suggested a predominant oligomer species (30,71,72), others indicate the appearance of a heterogeneous distribution of oligomers (63,73,74). Furthermore, recently both simulations and models have also predicted a heterogeneity of oligomer and aggregate species (75,76).…”

Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation

“…The roles of various molecular chaperones in polyQcontaining protein aggregation have been subject to active investigations, but their modes of action remain elusive. Various and sometimes contradictory effects have been reported in cellular or animal models (15)(16)(17)(18)(19). In addition, the existence of a direct interaction between the chaperones and the polyQ stretch per se is subject to debate (19 -22) because the interaction between the chaperones and the hydrophilic polyQ stretch is a priori unfavorable (23,24).…”

Spontaneous self-assembly of pathogenic huntingtin exon 1 protein into amyloid structures

“…sheet-rich polypeptide, which forms typical amyloid fibrils in in vitro aggregation assays, was shown to be highly toxic for mammalian cells [23]. This polypeptide is thought to cause cellular toxicity through the formation of stable β-sheet-rich protein aggregates that can sequester several important, metastable proteins.…”

“…This polypeptide is thought to cause cellular toxicity through the formation of stable β-sheet-rich protein aggregates that can sequester several important, metastable proteins. PolyQ aggregates in cells potentially cause dysfunction and toxicity by binding essential cellular proteins, thereby preventing their ability to perform their normal cellular tasks [23]. However, the relevance of different aggregate species to pathogenesis needs further elucidation.…”