Abstract
Huntington's disease is a progressive neurodegenerative disease caused by expansion of the polyglutamine domain in the first exon of huntingtin (HttEx1). The extent of expansion correlates with disease progression and formation of amyloid-like protein deposits within the brain. The latter display polymorphism at the microscopic level, both in cerebral tissue and in vitro. Such polymorphism can dramatically influence cytotoxicity, leading to much interest in the conditions and mechanisms that dictate the formation of polymorphs. We examine conditions that govern HttEx1 polymorphism in vitro, including concentration and the role of the non-polyglutamine flanking domains. Using electron microscopy, we observe polymorphs that differ in width and tendency for higher-order bundling. Strikingly, aggregation yields different polymorphs at low and high concentrations. Narrow filaments dominate at low concentrations that may be more relevant in vivo. We dissect the role of N- and C-terminal flanking domains using protein with the former (httNT or N17) largely removed. The truncated protein is generated by trypsin cleavage of soluble HttEx1 fusion protein, which we analyze in some detail. Dye binding and solid-state NMR studies reveal changes in fibril surface characteristics and flanking domain mobility. Higher-order interactions appear facilitated by the C-terminal tail, while the polyglutamine forms an amyloid core resembling those of other polyglutamine deposits. Fibril-surface-mediated branching, previously attributed to secondary nucleation, is reduced in absence of httNT. A new model for the architecture of the HttEx1 filaments is presented and discussed in context of the assembly mechanism and biological activity.
Highlights
Huntington's disease (HD) is one of several heritable diseases that are characterized by the abnormal expansion of a CAG trinucleotide repeat that codes for a polyglutamine stretch or domain in a mutant protein [1]
We presented a model of the HttEx1 fibril architecture, reproduced in Figure 1(d), that integrates information obtained from transmission electron microscopy (TEM), MAS ssNMR and antibody binding assays
The fibrillization temperature is a well-known source of polymorphism for mutant HttEx1 fibrils, with reported impacts on neurotoxicity [2,19]
Summary
Huntington's disease (HD) is one of several heritable diseases that are characterized by the abnormal expansion of a CAG trinucleotide repeat that codes for a polyglutamine (polyQ) stretch or domain in a mutant protein [1]. In HD, polyQ expansion occurs within the first exon (HttEx1) of the huntingtin protein (htt) (Figure 1(a) and (b)), which results in the deposition of htt N-terminal fragments (including HttEx1) as neuronal inclusion bodies. The misfolding and deposition of the mutant protein is generally associated with a toxic gain-of-function that contributes to neuronal degradation in HD [3,4]. Similar to the protein misfolding and amyloid formation processes in those disorders, their HD counterparts are considered disease relevant due to their ability to contribute to disease toxicity, disease creativecommons.org/licenses/by/4.0/).
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