Huntingtin fibrils with different toxicity, structure, and seeding potential can be interconverted

Abstract

The first exon of the huntingtin protein (HTTex1) important in Huntington's disease (HD) can form cross-β fibrils of varying toxicity. We find that the difference between these fibrils is the degree of entanglement and dynamics of the C-terminal proline-rich domain (PRD) in a mechanism analogous to polyproline film formation. In contrast to fibril strains found for other cross-β fibrils, these HTTex1 fibril types can be interconverted. This is because the structure of their polyQ fibril core remains unchanged. Further, we find that more toxic fibrils of low entanglement have higher affinities for protein interactors and are more effective seeds for recombinant HTTex1 and HTTex1 in cells. Together these data show how the structure of a framing sequence at the surface of a fibril can modulate seeding, protein-protein interactions, and thereby toxicity in neurodegenerative disease.

Fig. 1. HTTex1 T and N-fibril types have distinct CD minima, visible appearance, and EM signatures.

a CD spectra of T- and N-fibrils are plotted in black and red, respectively. There is a distinct shift in the minimum of the spectrum as reported previously. b Visual appearance of both fibrils in suspension. The T-fibrils appear translucent whereas the N-fibrils have a cloudy appearance. c The EM image of T-fibrils shows little bundling. d EM image of N-fibrils shows predominantly bundled fibrils. These observations are highly reproducible and have been repeated dozen of times.

Fig. 2. EPR and solid-state NMR spectra indicate a more dynamic PRD in the T-fibrils compared to the N-fibrils.

a Domain structure of HTTex1 highlighting the N17 region in orange, the polyQ domain in blue, the PRD in green, and the C-terminal His-tag in cyan. The residues that were spin-labeled for CW measurements are indicated. b CW EPR spectra of T- and N-fibrils are shown in black and red, respectively. The residue number of the spin label is indicated. The spectra of N- and T-fibrils overlap very well for all positions within the N17 and polyQ region indicating that they have the same structure and dynamics. However, the spectra of the T-fibrils are narrower for the last Q in the polyQ region (63R1) and all sites within the PRD indicating increased dynamics in this region compared to N-fibrils. c The 2D DARR ¹³C-¹³C spectra of N- and T-fibrils overlap very well, indicating that the static fibril core of both fibrils has a very similar structure. The spectra of N- and T-fibrils are shown in red and black, respectively. Both spectra were recorded at 0 °C, 25-kHz MAS with 50-ms mixing time. The spectrum of the T-fibrils was previously published as Fig. 2 by Isas et al.. d 1D ¹³C cross polarization (CP) spectra, which are sensitive to more static residues. Spectra of N-fibrils (red) and T-fibrils (black) were normalized to their Gln Cα intensities. The amino acid assignments of the lines are indicated. The higher intensities of the proline lines in the spectra of the N-fibrils indicate that this domain is more static in this fibril type. e Refocused INEPT spectra, which detect highly dynamic residues, were normalized according to the spectra shown in (d). The assignment of the His resonances that were detected for the T but not for the N-fibrils is indicated.

Fig. 3. Long-range distance inside the PRD of N- and T-fibrils is highly similar.

a Sequence of the PRD indicating the positions of the labels introduced for distance measurements in red and the distance pairs as arrows. The segments of the PRD including the two polyP regions (P11 and P10) and the linker region (L17) and C-terminals region (C12) are also indicated. b Table listing all distances measured via EPR DEER experiment for each spin label pair and fibril type. The distance corresponds to the maximum of the main peak derived from the DEER decay curve (DEER data and fits are shown in Supplementary Fig. 2). In addition, the theoretical distances for a polyP II helix assuming an increase of 3.1 Å per residue and the theoretical radius of gyration (R_G) for a random coil, are given (R_G = R₀ N^ν with R₀ = 1.927 Å, ν = 0.598, and N is the number of residues). Both distances spanning the polyP regions (P11 and P10) correspond nicely to a PPII distance. The distance spanning the L17 region is significantly shorter than a PPII helix and longer than expected for a random coil structure.

Fig. 4. PolyP and HTTex1 difference spectra suggest an increased entanglement of the PRDs in N compared to T-fibrils.

a CD spectra of polyPro at a concentration of 0.1 mg/ml recorded at 25 °C (black) and 90 °C (blue). The spectrum in red was recorded after the spectrum at 90 °C and after removing the liquid from the CD cuvette indicating the formation of a polyP film inside the cuvette. b Difference of N- and T-fibril CD spectra of Fig. 1 (solid line) and polyP CD spectra recorded at 90 and 25 °C (dashed line). The shared maxima at about 206 nm and minima at about 223 nm indicate that the structural differences that occur during heat-induced polyP film formation are comparable to those between HTTex1 N and T-fibrils.

Fig. 5. Over time T-fibrils turn into N-fibrils.

a CD spectra of HTTex1(Q46) fibrils that were spin-labeled at the N-terminus (15R1) and the PRD region (81R1). Spectra of fibrils incubated at 4 °C for 2 days (black), 5 days (red), and 9 days (blue) are shown. The decrease in apparent CD and the blue shift of the minima are compatible with a transition from T- to N-fibrils. b CW EPR spectra of corresponding fibril preparations after 1 day (black) and 7 days of incubaiton at 4 °C (red). The spectrum of the N-terminal 15R1 stays unchanged, whereas the spectrum of 81R1 decreases in intensity over time indicating a decrease in mobility. c Refocused INEPT spectra of HTTex1(Q46) T-fibrils (black), the same fibrils 12 months after fibrillization (blue), and N-fibrils (red). The His resonances, which are detected in freshly prepared T-fibrils, disappear after 12 months and are neither visible in N-fibrils.

Fig. 6. TFA and HFIP can disaggregate T-fibrils to form highly disentangled P-fibrils.

a CD spectra of N-fibrils (black), N-fibrils that were treated with 0.5% TFA in H₂O (orange), and HFIP (purple). The minima of the spectra treated with 0.5% TFA and HFIP are increasingly blue shifted and show an increase in apparent circular dichroims likely due to a decrease in scattering. b EM of highly bundled N-fibrils before HFIP treatment. c EM of same fibrils after HFIP treatment showing the presence of highly unbundled protofibrils. d EM of T-fibrils after HFIP/TFA treatment. Scale bars for (b)–(d) denote 1 μm. e 1D ¹³C spectra of HTTex1(Q46) P- and N-fibrils are shown in violet and black, respectively. CP spectra were normalized to their Gln C_α intensities. The close to perfect overlap of the CP spectra indicates that the static core of these two fibril types is very similar. Refocused INEPT spectra were normalized according to the CP spectra. His side chain carbon resonances are indicated. The much higher relative intensity of the INEPT spectra of the P-fibrils indicates that the PRD is more dynamic. The shift in the His C_ε1 line reflects the low pH used for the protofibril preparation. f Overlap of 2D DARR ¹³C-¹³C spectra of HTTex1(Q46) P-fibrils (violet) and T-fibrils (black) indicates that the static fibril core of both fibrils has a very similar structure. Both spectra were recorded at 0 °C, 25 kHz MAS with 50 ms mixing time. The spectrum of the 4 °C fibrils was previously published as Fig. 2 by Isas et al..

Fig. 7. HTTex1 fibrils with disentangled PRDs are more efficient in seeding soluble HTTex1.

a Time-dependent CW EPR intensities of unseeded HTTex1(Q46) 35R1 (blue) and HTTex1(Q46) 35R1 seeded with N- (red), T- (black), and P-fibrils (purple). b EPR intensity of HTTex1(Q46) 35R1 4 h after initiating fibril formation of three separate experiments (n = 3). The decrease in intensity, which is a reporter of fibril formation, is fastest when seeding with P-fibrils followed by seeding with T-, N-fibrils, and unseeded protein. c Puncta formation in Neuro2a cells transfected with HTTex1 with varying polyQ length, and a C-terminal EGFP tag and EGFP as control. Cells were co-transfected with N-, T-, and P-fibril seeds and BSA fibril seeds as control. Fluorescence microscopy images were taken 48 h after transfection. Representative images for HTTex1Q39 (red) and Q72 (burgundy) are shown. Scale bars denotes 40 μm. Puncta formation was measured in 3–11 biological replicates and was reproducible as illustrated in (d). d Fractions of transfected cells containing puncta depending on HTTex1 polyQ length and nature of co-transfected fibrils seeds. Fractions of separate experiments are reported together with boxplots that show 25th to 75th percentiles, the median in orange, and whiskers from minimum to maximum. Significant P values from a two-sided t-test are indicated. P-fibril seeds led to the strongest induction of puncta in cells transfected with HTTex1Q72 (burgundy) and Q39 (red) followed by T-fibrils. The effect of N-fibril and BSA fibril seeds on puncta formation was roughly the same. Few puncta were observed for HTTex1Q16 (orange) and EGFP (yellow) independent of co-transfected fibril seeds. e Fibril toxicity using STHDH Q7/111 cells. Representative images of DAPI-labeled cells to which fibril types were exogenously added. White arrows point DAPI-labeled brighter small puncta, which are recognized as dead cells. Scale bars denote 20 μm. Data were reproducible as illustrated in (f). f Percent of dead cells detected in ten images coming from three biological replicates. Each individual percentage together with a boxplot is shown. Boxplots indicate 25th to 75th percentiles, the median in orange, and whiskers from minimum to maximum. Significant P values from two tailed t-tests are indicated.

Fig. 8. Model of the N-, T-, and P-fibrils.

The PRDs of the N-fibrils (green) are entangled resulting in bundling and consequently less accessible fibril surfaces. The PRDs of the T-fibrils are keeping the individual fibrils more separate. The P-fibrils are shorter and their PRD are even less restricted and more accessible.