Inhibitor-based modulation of huntingtin aggregation reduces fibril toxicity

Abstract

AbstractHuntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of the polyglutamine (polyQ) segment in the exon 1 of the huntingtin (HttEx1) protein. This polyQ expansion leads to protein misfolding and the formation of β-sheet-rich fibrillar aggregates. Several studies have shown that these protein deposits can cause cytotoxicity, suggesting the development of small molecule aggregation inhibitors as potential modulators of HD pathogenesis. This requires a molecular understanding of the impacts of such modulators on the interplay of aggregation, polymorphism and toxic gain-of-function. Here, we study how a polyphenol modulates the HttEx1 aggregation mechanism at sub-stoichiometric ratios. Moreover, we examine how the disrupted aggregation process impacts the protein’s misfolded structure and neurotoxic properties. We combine measurements of aggregation kinetics, electron microscopy, solid-state NMR, cytometry, and cytotoxicity assays. A notable delay of protein aggregation was observed even at sub-stoichiometric ratios of curcumin relative to the HttEx1 protein. Mechanistically, extension of the lag phase indicates an impact on the primary nucleation process that underpins the complex HttEx1 aggregation pathway, with an apparent role for β-hairpin formation. Remarkably, the deposits formed (more slowly) in presence of inhibitor displayed reduced toxicity in cultured neuronal cells, seemingly derived from their modulated structures. Thus, curcumin has a multifaceted effect based on delaying the fibril formation, while also changing the toxic properties of formed fibrils. Our findings highlight the ability of small molecule inhibitors to modulate the protein misfolding landscape, with potential implications for treatment strategies in HD and other protein deposition disorders.Significance StatementHuntington’s disease is an incurable inherited neurodegenerative disorder in which the mutated protein undergoes misfolding and forms fibrillar aggregates. The inhibition of these pathogenic processes represents a major challenge, which also requires an understanding of the complex aggregation mechanism. We observe how small molecule inhibitors can delay formation of toxic deposits by perturbing the aggregation kinetics. Crucially, we find that the modulated aggregation process yields less-toxic fibrils with a different molecular structure. This highlights how inhibition of aggregation process can have unexpected and unintended consequences by yielding novel fibril polymorphs with different biological properties. Analogous multifaceted effects should be considered in the design and testing of aggregation modulators, as they offer both risks and unexpected opportunities as new treatment modalities.