Conformational studies of pathogenic expanded polyglutamine protein deposits from Huntington's disease

Abstract

Huntington’s disease, like other neurodegenerative diseases, continues to lack an effective cure. Current treatments that address early symptoms ultimately fail Huntington’s disease patients and their families, with the disease typically being fatal within 10–15 years from onset. Huntington’s disease is an inherited disorder with motor and mental impairment, and is associated with the genetic expansion of a CAG codon repeat encoding a polyglutamine-segment-containing protein called huntingtin. These Huntington’s disease mutations cause misfolding and aggregation of fragments of the mutant huntingtin protein, thereby likely contributing to disease toxicity through a combination of gain-of-toxic-function for the misfolded aggregates and a loss of function from sequestration of huntingtin and other proteins. As with other amyloid diseases, the mutant protein forms non-native fibrillar structures, which in Huntington’s disease are found within patients’ neurons. The intracellular deposits are associated with dysregulation of vital processes, and inter-neuronal transport of aggregates may contribute to disease progression. However, a molecular understanding of these aggregates and their detrimental effects has been frustrated by insufficient structural data on the misfolded protein state. In this review, we examine recent developments in the structural biology of polyglutamine-expanded huntingtin fragments, and especially the contributions enabled by advances in solid-state nuclear magnetic resonance spectroscopy. We summarize and discuss our current structural understanding of the huntingtin deposits and how this information furthers our understanding of the misfolding mechanism and disease toxicity mechanisms.

Impact statement: Many incurable neurodegenerative disorders are associated with, and potentially caused by, the amyloidogenic misfolding and aggregation of proteins. Usually, complex genetic and behavioral factors dictate disease risk and age of onset. Due to its principally mono-genic origin, which strongly predicts the age-of-onset by the extent of CAG repeat expansion, Huntington’s disease (HD) presents a unique opportunity to dissect the underlying disease-causing processes in molecular detail. Yet, until recently, the mutant huntingtin protein with its expanded polyglutamine domain has resisted structural study at the atomic level. We present here a review of recent developments in HD structural biology, facilitated by breakthrough data from solid-state NMR spectroscopy, electron microscopy, and complementary methods. The misfolded structures of the fibrillar proteins inform our mechanistic understanding of the disease-causing molecular processes in HD, other CAG repeat expansion disorders, and, more generally, protein deposition disease.

Keywords: Neurodegeneration; aggregation; biophysics; nuclear magnetic resonance; proteins; structural biology.

Figure 1.

Genetic aspects of HD and other CAG repeat disorders. (a) Age of onset inversely correlates with the extent of expansion, for lengths beyond a disease-specific threshold. The figure was adapted from Kuiper et al. with permission. (b) Wild-type Htt structure solved by cryoEM (PDB 6EZ8; Guo et al.). (c) Color-coded resolved and invisible domain segments from the Htt cryoEM structure, with the HttEx1 that contains the polyglutamine stretch enlarged below.

Figure 2.

Mutant HttEx1 fibrils in vivo and in vitro. (a) Neuronal inclusions from HD patient; adapted from DiFiglia et al. with permission from AAAS. (b) 3D rendering of tomographic EM showing the interaction zone between an inclusion body and cellular membranes in a HttQ97-transfected HeLa cell. ER membranes (red), ER-bound ribosomes (green), HttQ97 fibrils (cyan), and vesicles inside the IB (white). Adapted from Bauerlein et al. with permission from Cell. (c) Annotated 3D EM tomogram of Q51-HttEx1 fibrils (yellow) in presence of TRiC chaperones (blue/magenta). Adapted with permission from Shahmoradian et al. (d) Negative-stain EM of Q44-HttEx1 fibrils used for ssNMR study. Adapted from Hoop et al. (e) Q44-HttEx1 forms temperature-dependent polymorphs with different widths, which seed polyglutamine protein aggregation (f) and cause neuronal toxicity (g). Panels (e–g) were adapted with permission from Lin et al.

Figure 3.

Mechanisms of aggregation. (a) Expanded polyglutamine without (Q₃₀) and with (htt^NTQ₃₀P₁₀K₂) Htt flanking regions attached show dramatically different aggregation kinetics. Adapted from Sivanandam et al. with permission from the American Chemical Society. (b) Intrinsically disordered HttEx1 can aggregate via both polyglutamine driven (top) and Htt^NT- driven mechanisms (bottom). (c) Both mechanisms yield products with the same ssNMR signals for the polyglutamine fibril core structure (see Figure 4): spectra of Q44-HttEx1 and polyglutamine peptide, D₂Q₁₅K₂. Adapted from Hoop et al.. (d) Length-dependent nucleus size data^– show that fast aggregation of long polyglutamine is facilitated by monomeric nucleation, likely involving β-hairpin formation. (e) Schematic free energy profile of fibril formation and growth, for short and long polyglutamine, based on published nucleation and fiber elongation energy values.^,,,

Figure 4.

SSNMR structural studies of misfolded fibrils. (a) Fibrillar polyglutamine gives an identical ssNMR signature, whether in Q44-HttEx1 fibrils (top), Q46-HttEx1 fibrils (bottom), or polyglutamine peptides with 15 or 30 glutamines. Data from Lin et al. and Sivanandam et al. Data for Q46-HttEx1 were adapted with permission from Isas et al. copyright 2015 American Chemical Society. (b) Tailored ssNMR dihedral angle measurements probe the polyglutamine core structure, and reveal two differently structured β-strand types (adapted from Hoop et al.). (c) A stochastic assembly process of the alternating β-strand structures explains the ssNMR spectral signature of polyglutamine amyloid. Adapted with permission from Hoop et al. (d) Architecture of mutant HttEx1 fibrils derived from ssNMR and EM constraints. (e) SSNMR relaxation measurements show dynamic changes in the solvent-exposed Htt^NT α-helical segment upon solvent freezing. Adapted from Hoop et al., with permission from the American Chemical Society. (f) Schematic illustration of flanking-domain interactions enabling higher order assembly of the wider HttEx1 fibril polymorphs shown in Figure 2(e). Panels (d) and (f) are adapted with permission from Lin et al. (A color version of this figure is available in the online journal.)