Fibril polymorphism affects immobilized non-amyloid flanking domains of huntingtin exon1 rather than its polyglutamine core

Abstract

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells.

Figure 1. Htt exon1 sequence and domain structure.

(a) The domain structure and sequence of htt exon1 is shown at the top. The locations of PTMs, as well as the binding sites of various antibodies and other htt-binding proteins are indicated. (b) Design of previously studied HNTF peptide htt^NTQ₃₀P₁₀K₂. (c) Design of the MBP fusion protein, with the sequence of the Factor Xa cleavage site in the linker shown below.

Figure 2. Cleavage and aggregation of mutant htt exon1.

(a) SDS–PAGE gels showing time-dependent factor Xa cleavage at 22 °C. (b,c) Fibril width derived from negative-stain TEM on the mature fibrils formed at 37 °C (597 measurements over 99 fibrils) and 22 °C (219 measurements over 73 fibrils). (d) Second-derivative FTIR of htt exon1 fibrils formed at 37 °C and (e) 22 °C, for fibrils dispersed in either H₂O or D₂O. The coloured arrows mark the most notable differences between the fibril types. (f) Reference data on fibrillar K₂Q₃₁K₂, HNTF (htt^NTQ₃₀P₁₀K₂) fibrils, and aggregated α-helical htt^NT in PBS buffer, adapted with permission from ref. , Copyright 2011 American Chemical Society. (g) Resonance frequencies of different secondary structure elements.

Figure 3. 1D ¹³C ssNMR spectra of uniformly ¹³C- and ¹⁵N-labelled htt exon1 fibrils.

(a,d,g) Fibrils were formed at 37 °C, or (b,e,h) 22 °C, and studied using (a–c) cross-polarization (rigid residues), (d–f) direct polarization and (g–i) INEPT-based (mobile residues) MAS ssNMR. (c,f,i) Overlaid aliphatic regions, with assignments indicating the random coil (P_rc) and PPII-helical Pro (P_II). The NMR measurements were performed at 275 K on a 600 MHz (¹H frequency) spectrometer.

Figure 4. MAS ssNMR identifies the immobilized parts of htt exon1 fibrils.

(a) 2D ¹³C–¹³C CP/DARR spectrum on U-¹³C,¹⁵N htt exon1 fibrils prepared at 22 °C. Signals from the polyQ domain are boxed, and underlined peak assignments are from residues in htt^NT. (b) Analogous 2D spectra of HNTF fibrils with site-specific U-¹³C,¹⁵N-labelling of the indicated htt^NT residues and the first Gln of the Q₃₀ repeat (Q18). The blue and black contours are for samples labelled in residues A10/F11/L14/Q18, or A2/L7/F17, respectively. (c,d) Detection of α-helical secondary structure (blue bars) in htt^NT residues of htt exon1 (c) and HNTF (d) fibrils, based on the secondary chemical shifts Δδ(Cα–Cβ). Error bars reflect the s.d. in the chemical shift (see Supplementary Table 3). The NMR measurements were performed at 267–275 K on a 600 MHz (¹H) spectrometer. Panel (b) was adapted with permission from ref. , Copyright 2011 American Chemical Society.

Figure 5. SSNMR dipolar recoupling curves reveal exon1 fibril domain motion.

(a) Reference PDSD buildup profiles for one-bond Cα–Cβ cross-peaks of the crystalline dipeptide N-acetyl-Val-Leu, reflecting an example of a fully rigid molecule. The dashed line illustrates the lack of buildup for a fully mobile (for example, dissolved) molecule. Intermediate motion is expected to lead to build-up curves in-between these extremes. (b) PDSD buildup profiles for Cα–Cβ peaks of type-‘a' Gln in the polyQ core, (c) α-helical A10 in htt^NT, random coil Ala (A_RC) and (d) the random coil (RC) and PPII-structured Pro in the PRD of htt exon1 fibrils formed at 22 °C. Pale grey lines show the reference curves from a. Error bars indicate the s.d., as described in the Methods. The NMR measurements were performed at 275 K on a 600 MHz (¹H) spectrometer.

Figure 6. Dynamic residues in the polymorphic htt exon1 fibrils identified via 2D INEPT-based ssNMR.

¹³C–¹³C INEPT/TOBSY spectra for fibrils prepared at 22 °C (a), and 37 °C (b). Observed residue types are from the very C-terminal tail of the PRD, indicating that this part of the fibrillized protein is highly dynamic. Spectra acquired at 600 MHz (¹H) and 8.33 kHz MAS, at a temperature of 275 K.

Figure 7. Accessibility of the htt exon1 fibril PRDs probed by solvent-filtered ssNMR and antibody binding.

(a,b) Solvent accessibility of htt exon1 fibrils prepared at (a) 37 °C and (b) 22 °C probed by ssNMR. Peak intensities after 7 ms ¹H–¹H diffusion from the solvent into the fibrils (blue) are compared to the ¹³C CP spectrum in absence of T₂-based solvent filtering (grey). Each spectrum was normalized to the highest peaks to highlight the relative solvent exposures. Up/down arrows indicate sites with high/low solvent accessibility. The NMR measurements were performed at 275 K on a 600 MHz (¹H) spectrometer. (c) Dot blot analysis shows that in the monomeric protein the polyQ domain, oligoPro segments and PRD tail are all accessible for binding by MW1, MW7 and MW8, respectively (Fig. 1). Upon aggregation at 22 or 37 °C, MW1 binding to the polyQ is largely abolished, while the PRD tail is still strongly recognized by MW8. OligoPro binding by MW7 is weaker in the 22 °C fibrils compared to the 37 °C polymorph.

Figure 8. PolyQ protein recruitment and neuronal toxicity assay results.

(a) Aggregation kinetics at 22 °C in the absence (solid black and grey lines) and presence (dashed lines) of pre-made seed aggregates, detected as ThT fluorescence at indicated time points after complete trypsin cleavage of the htt exon1 fusion protein. Dark blue and magenta dashed lines reflect the aggregation in presence of 20 mol-% htt exon1 aggregates formed at 22 and 37 °C, respectively. The unseeded reactions have lag phases exceeding 4 h, which are eliminated by the seeds. Error bars indicate s.d., with n=2–3, as described in the Methods section. (b) Enlargement of the first 500 min. (c,d) Results of a single (n=1) HPLC measurement of the residual monomer concentration after aggregate sedimentation, applied to the same samples, as a complementary measure of aggregation. Error bars reflect the estimated peak integration error as described in the Methods. (e) Cellular viability of human dopaminergic neuronal cells upon exposure to varying concentrations of pre-formed fibrils prepared at 22 and 37 °C. The data reflect MTT reduction assays performed after 24 h (n=2; two biological replicates with three technical replicates each—shown is the mean with s.d. compared to non-treated controls set at 100%). (f) Cell viability assay data for a 24 h exposure of immortalized HT-22 neurons (n=2; two biological replicates with 6 technical replicates each–shown is the mean with s.d. compared to non-treated controls set at 100%; *P<0.05, Mann–Whitney non-parametric test).

Figure 9. Schematic proposed model of htt exon1 fibrils.

(a) The htt^NT α-helices (dark blue) and PRD PPII helices (light blue) are immobilized and tightly clustered on the perimeter of the rigid amyloid core (green β-strands). C-terminal domains show increased dynamics, either in the form of the unstructured C-terminal tail or a subpopulation of more exposed PRDs (top right; red). An individual protein monomer with its β-hairpin-based polyQ core is shown with lighter (yellow) β-strands. (b) Schematic illustration of interfilament flanking domain interactions that we propose to explain the larger TEM-based widths of the fibrils formed at 22 °C, as well as the observed differences in accessibility and immobilization of the PRD.