Self-assembly of mutant huntingtin exon-1 fragments into large complex fibrillar structures involves nucleated branching

Abstract

Huntingtin (HTT) fragments with extended polyglutamine (polyQ) tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies.Here, we performed systematic experimental and theoretical studies to examine the selfassembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). We demonstrate that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent monomers, and (2) a fast secondary fibrildependent nucleation process, which involves branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs primary nucleation and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modelling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates. All results indicate that in vitro, HTT exon-1 fibrillar aggregates are formed by a branching mechanism. KeywordsHuntingtin fibrillogenesis, aggregation mechanism, primary and secondary nucleation, amyloidogenesis, fibril branching peer-reviewed)

The pathobiology of perturbed mutant huntingtin protein–protein interactions in Huntington's disease

“…Besides full‐length mHTT with a pathogenic polyQ tract and a largely α‐helical conformation (Guo et al ), a variety of other conformationally distinct mHTT protein species have been reported to be present in HD patient brains and disease models (Scherzinger et al ; DiFiglia et al ; Sathasivam et al ). This includes monomers and oligomers of full‐length mHTT (Sathasivam et al ; Shirendeb et al ) as well as protein species that are formed of truncated N‐terminal mHTT fragments, such as oligomers or β‐sheet‐rich, ordered fibrils (Wagner et al ). Several lines of evidence indicate efficient proteolytic cleavage of full‐length mHTT resulting in the release of N‐terminal polyQ‐containing fragments (Lunkes et al ; Landles et al ).…”

“…Apart from full‐length mHTT, truncated N‐terminal polyQ‐containing fragments may also play a critical role in HD (Davies et al ; Ast et al ). The mHTTex1 fragment has been reported to efficiently self‐assemble into higher order structures such as spherical oligomers or amyloid fibrils in vitro and in vivo (Sathasivam et al ; Sahoo et al ; Wagner et al ). It has been proposed that such protein assemblies might drive HD pathogenesis (Wanker ).…”

“…While monomers and oligomers are dynamic structures and are assumed to have random coil or α‐helical conformation in vivo (Kang et al ; Kotler et al ), fibrillar mHTTex1 assemblies are rigid and predominantly consist of stable β‐sheets (Hoop et al ; Lin et al ). Antibody binding studies demonstrated that polyQ tracts are exposed on the surface of mHTTex1 monomers and oligomers, but not on the surface of amyloid fibrils (Wagner et al ). Being structurally and morphologically distinct, mHTT fibrils may have very different biological properties and interaction profiles in mammalian cells from those of non‐β‐sheet mHTT species.…”

“…The expansion of the polyglutamine stretch greatly facilitates this process and leads to the rapid formation of insoluble amyloidogenic fibrillar aggregates with cross‐β‐sheet architecture (Scherzinger et al ; Sahoo et al ). Fibril formation follows a nucleated growth mechanism (Bhattacharyya et al ; Wagner et al ). The initial association between HTTex1 molecules is believed to be mediated by coiled‐coil interactions of multiple N17 domains (Thakur et al ; Jayaraman et al ).…”

“…These initial conformational changes (primary nucleation) are slow and rare to occur and therefore manifest in a lag phase, when looking at the aggregation process from a kinetic point of view. Then, fibril formation proceeds to a rapid elongation phase in which additional HTTex1 monomers are recruited to the primary nucleus and adapt a β‐hairpin conformation (Wagner et al ). Within the mature fibre, multiple molecules with a β‐hairpin conformation are stacked atop each other, stabilized by intra‐ and intermolecular hydrogen bonds, thereby forming tightly interconnected β‐sheets (Hoop et al ).…”