Huntington's disease age-of-onset linked to polyglutamine aggregation nucleation

Abstract

In Huntington's Disease and related expanded CAG repeat diseases, a polyglutamine [poly(Gln)] sequence containing 36 repeats in the corresponding disease protein is benign, whereas a sequence with only 2-3 additional glutamines is associated with disease risk. Above this threshold range, longer repeat lengths are associated with earlier ages-of-onset. To investigate the biophysical basis of these effects, we studied the in vitro aggregation kinetics of a series of poly(Gln) peptides. We find that poly(Gln) peptides in solution at 37 degrees C undergo a random coil to beta-sheet transition with kinetics superimposable on their aggregation kinetics, suggesting the absence of soluble, beta-sheet-rich intermediates in the aggregation process. Details of the time course of aggregate growth confirm that poly(Gln) aggregation occurs by nucleated growth polymerization. Surprisingly, however, and in contrast to conventional models of nucleated growth polymerization of proteins, we find that the aggregation nucleus is a monomer. That is, nucleation of poly(Gln) aggregation corresponds to an unfavorable protein folding reaction. Using parameters derived from the kinetic analysis, we estimate the difference in the free energy of nucleus formation between benign and pathological length poly(Gln)s to be less than 1 kcal/mol. We also use the kinetic parameters to calculate predicted aggregation curves for very low concentrations of poly(Gln) that might obtain in the cell. The repeat-length-dependent differences in predicted aggregation lag times are in the same range as the length-dependent age-of-onset differences in Huntington's disease, suggesting that the biophysics of poly(Gln) aggregation nucleation may play a major role in determining disease onset.

Figure 1

CD spectra of 0.39 mg/ml K₂Q₄₂K₂ during its aggregation in 10 mM Tris⋅TFA buffer, pH 7.0, 37°C. (a) From bottom to top the spectra are those of the aggregation mixture at : 0, 14.35 (— -), 19.63 (■ ■ ■), 38.35 (– ■ –), 44.68, 62.25, 68.62, 85.83, 91.97, 110.60 (– – –), 136.95, 161.60 (■ ■ ■), and 216.82 h. The spectra are in solid except lines indicated. (b) Spectra of the reaction mixture at 90 h (—), Eppendorf centrifugation supernatant (- - -) and pellet (⋅ ⋅ ⋅), sum of the supernatant and pellet (– - -), and ultracentrifugation supernatant (— ■ —) and pellet (– – –).

Figure 2

(a) Aggregation progress of 66 μM K₂Q₄₂K₂ in 10 mM Tris-TFA, pH 7.0 monitoring by CD ([Θ]₂₀₀) (■), HPLC (●), light scattering (□), and thioflavin T (○) methods and a single curve fit from the four sets of data. (b) Aggregation progress of 20 μM K₂Q₂₈K₂ in PBS, pH 7.4, without seed (—) and with 0.01% (– –), 0.1% (- - -), and 1% (– ■ –) of preformed K₂Q₂₈K₂ aggregates, prepared by the −20°C method as described (14). Aggregation was monitored by Rayleigh light scattering as described (14).

Figure 3

t² plot of K₂Q₃₆K₂ peptide at 36.9 μM (a), 17.1 μM (b), 8.4 μM (c), and 4.5 μM (d).

Figure 4

Linear fits of log (½ k₊² K_n*c^(n*+2)) vs. log c for K₂Q₂₈K₂ (■), K₂Q₃₆K₂ (●), and K₂Q₄₇K₂ (▴). Reference lines with exact slopes of 3 (○) and 4 (□) are shown for comparison. The slope plotted is that from fits such as those shown in Fig. 3.

Figure 5

A model for poly(Gln) aggregate nucleation and extension. In the model, nucleation consists of an unfavorable transition (step a) from an extended, statistical coil state to a compact state corresponding to the aggregation nucleus. The elongation process consists of an initial binding (step b) of the nucleus to an extended conformation monomer, followed by a consolidation of structure (step c) that generates a new binding site for monomer. The resulting dimeric species binds another extended chain monomer (step d) to continue the process.

Figure 6

(a) Simulated aggregation progress of K₂Q₃₆K₂ at 8.4 μM (—), 17.1 μM (⋅ ⋅ ⋅), and 36.9 μM (– ■ –) and the experimental data of 8.4 μM (■), 17.1 μM (●), and 36.9 μM (▴). (b) Simulated aggregation progress of K₂Q₂₈K₂ (■, —), K₂Q₃₆K₂ (●, – ■ –), and K₂Q₄₇K₂ (▴, ⋅ ⋅ ⋅) at a concentration of 0.1 nM.