Cryo-electron tomography provides topological insights into mutant huntingtin exon 1 and polyQ aggregates

Abstract

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags.

Fig. 1. MEx1-Q51 filaments exhibit a large variation in width within and across filaments.

a Schematic of the mEx1-Q51 construct. b Slice parallel to xy (~1.7 nm thick) through a representative 4x downsampled cryoET tomogram of aggregated mEx1-Q51, reconstructed with compressed sensing, lightly filtered to enhance visualization, and corresponding semi-automated 3D annotation. c Selected areas from slices of large mEx1-Q51 aggregates showing individual filament segments, widely varying in width, with the thinnest filaments exhibiting regions down to ~2 nm width, indicated by the red arrows. d Zoomed-in view of a xy slice (~0.4 nm thick) from a selected region of a tomogram without any downsampling, showcasing ultra-thin regions in mEx1-Q51 filaments. e Sections of annotated mEX1-Q51 filamentous aggregates from cryoET tomograms showing relatively narrow branching angles and an example of a thicker laminated sheet-like region (the annotation example in the middle). Scale bars: 100 nm (b), 17 nm (c), 15 nm (d), 50 nm (e).

Fig. 2. Aggregated mEx1-Q51 exhibits lumpy, slab-shaped filaments.

a Pseudo-periodic pattern of repeating lumps (blue arrows) along the length of an mEx1-Q51 filament as seen in an xy slice (4.4 Å thick) from a tomogram of aggregated mEx1-Q51. b Selected regions from semi-automated neural network annotations showing lumpy filaments of various widths, including sheet-like regions (middle region of right-most example). c Subtomogram average of a subpopulation of filament segments (n = 450) exhibiting a lumpy ~7 × 15 nm slab-shaped morphology. Scale bars: 50 nm (a, b), 15 nm and 7 nm (c).

Fig. 3. Lumpy Q51 filaments exhibit a large range of widths.

a Schematic of the Q51 construct, lacking all mEx1 domains except for the polyQ tract. b Slice parallel to the xy plane (~2.1 nm thick) through a representative 4x downsampled cryoET tomogram of aggregated Q51 reconstructed with compressed sensing and corresponding 3D annotation. Zoomed-in views of xy slices (~0.5 nm thick) from selected regions of the tomogram shown in “a” but without any downsampling, exhibiting c a pseudo-periodic pattern of repeating lumps along the length of a Q51 filament (blue arrows), and d regions in thin filaments that are as thin as ~2 nm in width (red arrows). e Examples of 2D xy slices through representative 3D subtomograms of Q51 filament segments showing a wide variation in width, including super-thin regions ~2 nm in width (red arrows). Scale bars: 100 nm (b), 15 nm (c, d), and 17 nm (e).

Fig. 4. Aggregated Q51 exhibits lamination sheets and predominantly lumpy, slab-shaped filaments.

a Representative sections of annotated Q51 filamentous aggregates from cryoET tomograms showing their most common branching/crossover angle (~60°), often in an asterisk-like pattern, and b thicker regions akin to lamination, onside thinner ones. c Subtomogram average of a subpopulation of filament segments (n = 493) exhibiting a lumpy ~7 × 15 nm slab-shaped morphology. Scale bars: 50 nm (a, b), 15 nm, and 7 nm (c).