Normal-repeat-length polyglutamine peptides accelerate aggregation nucleation and cytotoxicity of expanded polyglutamine proteins

Abstract

The dependence of disease risk and age-of-onset on expanded CAG repeat length in diseases like Huntington's disease (HD) is well established and correlates with the repeat-length-dependent nucleation kinetics of polyglutamine (polyGln) aggregation. The wide variation in ages of onset among patients with the same repeat length, however, suggests a role for modifying factors. Here we describe the ability of normal-length polyGln repeat sequences to greatly accelerate the nucleation kinetics of an expanded polyGln peptide. We find that normal-length polyGln peptides enhance the in vitro nucleation kinetics of a Q(47) peptide in a concentration-dependent and repeat-length-dependent manner. In vivo, we show that coexpression of a Q(20) sequence in a Drosophila model of HD expressing Htt exon 1 protein with an Q(93) repeat accelerates both aggregate formation and neurotoxicity. The accelerating effect of short polyGln peptides is attributable to the promiscuity of polyGln aggregate elongation and reflects the intimate relationship between nucleus formation and early elongation events in establishing nucleation kinetics. The results suggest that the overall state of the polyGln protein network in a cellular environment may have a profound effect on the toxic consequences of polyGln expansion and thus may serve as a genetic modifier of age of onset in HD.

Fig. 1.

Nucleation-dependent polymerization mechanism of polyGln aggregation. Formation of nucleus N* from bulk-phase monomer M_a is modeled as a reversible, highly unfavorable reaction. Once formed, the metastable N* can either disintegrate back to the monomer pool or elongate by adding M_b molecules to form N*₊₁, N*₊₂, etc., and by doing so stabilize the system. The term M_b signifies the monomer pool capable of supporting elongation. In most amyloid systems, M_b is identical to M_a, but, as shown in this paper, M_b can be an expanded pool that includes other polyGln sequences. Previously, we showed that the number of molecules of polyGln comprising N* (called critical nucleus, or n*) is equal to 1 for nucleation of polyGln aggregation (15); the general aspects of theory and interpretation discussed in this work, however, are independent of the molecularity of N*.

Fig. 2.

The effect of a short polyGln peptide on the nucleation of a polyGln of pathological length. (a) PolyGln Q₄₇ was incubated at 37°C at 1.8 μM, either alone (○) or in the presence of various concentrations of Q₂₀: 14 μM (◇), 24 μM (◆), 36 μM (△), 44 μM (▲), and 54 μM (■). The disappearance of Q₄₇ from the soluble phase is shown on a relative basis for each reaction mixture. (b) t² plots (14) of the nucleation kinetics of various concentrations of Q₄₇ in the presence of 18 μM Q₂₀. Starting concentrations of Q₄₇ were 13.2 (○), 10.8 (●), 8.6 (△), 6.4 (▲), and 4.5 (◆) μM. (c) Concentration dependence of nucleation kinetics, plotting the logarithm of the slopes of the t² plots from b versus log[Q₄₇]. The slope m of the linear regression fit is 3.2, R² = 0.9881; N* is calculated to be 1.2 (N* = m − 2) (15).

Fig. 3.

The role of repeat length on the augmentation of Q₄₇ aggregation nucleation by shorter polyGln sequences. Samples of 1.5–2.0 μM Q₄₇ were incubated either alone (○) or with 20 μM polyGln peptides of various repeat lengths: Q₁₀ (◇), Q₁₅ (◆), Q₂₀ (△), Q₂₅ (▲), Q₂₉ (□), Q₃₃ (■), or Q₄₀ (●). The disappearance of Q₄₇ from the soluble phase is shown on a relative basis for each reaction mixture.

Fig. 4.

The role of elongation kinetics in aggregation nucleation. (a) Rates of elongation of 2.3 μg/ml Q₄₇ amyloid fibrils by 17 μM monomeric polyGln plotted against the repeat length of the monomeric polyGln. (Inset) Pseudofirst-order rate plot for the elongation of 17 μM Q₂₀; because the molar concentration of fibrils does not change in a simple fibril elongation reaction, elongation kinetics are pseudofirst-order (39), yielding a pseudofirst-order rate constant that is an amalgam of the true second-order rate constant and the molar concentration of fibrils. (b) A plot of the slope of the t² plot for Q₄₇ nucleation (Fig. 3) with respect to the square of the rate constants k₊, describing the elongation of each short polyGln peptide (from a). [We used the square of the rate constant here because of the dependence of nucleation kinetics on the second power of the elongation rate, as shown in the equation Δ = 1/2k₊²K_n* c^(n*+2)t² (14, 15).]

Fig. 5.

Coexpression of Httex1p-Q₂₀p increases formation of nuclear inclusions and toxicity of Httex1p-Q₉₃p. (a) Confocal images of Drosophila eye expressing polyGln transgenes in adult photoreceptor neurons. Htt accumulation is green, rhabdomeres of photoreceptor neurons are red by anti-actin staining, and photoreceptor nuclei are blue by staining with anti-elav antibody. The first four columns of photographs show cross-sections at days 5 and 6. (Scale bar: 10 μM.) The last three columns of photographs show longitudinal sections through the eye at day 6. (Scale bar: 20 μM.) Extensive staining of cytoplasmic Htt is seen at day 5 and is primarily converted to nuclear staining in 6-day-old flies. At each time point, where Q₂₀ is coexpressed with Q₉₃, an increase in the number of visible inclusions is observed. No aggregates are observed with Q₂₀ alone. (b) The number of photoreceptor nuclei with visible inclusions in 5-, 6-, and 10-day-old flies (d5, d6, and d10, respectively). The fraction of nuclei with inclusions at days 5 and 6 is increased when Q₂₀ is coexpressed; however, the percentage of nuclei with inclusions with and without Q₂₀ coexpression plateaus by day 10. The percentage of nuclear accumulation was calculated as Htt-positive nuclei per all nuclei per field [∗, P < 0.025 (0.0239); ∗∗, P < 0.015 (0.0145)]. (c) Pathology is evaluated by comparing the number of rhabdomeres per ommatidium in 8-day-old (d8) and 12-day-old (d12) flies [∗∗∗, P < 0.0015 (0.00141)]. No neurotoxicity is observed up to day 8, but toxicity is evident by day 12 and is increased by the presence of Q₂₀.