Effects of Macromolecular Cosolutes on the Kinetics of Huntingtin Aggregation Monitored by NMR Spectroscopy

Abstract

The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hht^ex1) with a 35 polyglutamine repeat, htt^ex1Q₃₅, are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis for quantitative analysis of htt^ex1Q₃₅ aggregation, monitored by measuring cross-peak intensities in a series of 2D ¹H-¹⁵N NMR correlation spectra acquired during the course of aggregation. The primary effects of the two cosolutes are associated with shifts in the prenucleation tetramerization equilibrium resulting in substantial changes in concentration of "preformed" htt^ex1Q₃₅ tetramers. Similar effects of the two cosolutes on the tetramerization equilibrium observed for a shorter, nonaggregating huntingtin variant with a 7-glutamine repeat, htt^ex1Q₇, lend confidence to the conclusions drawn from the fits to the htt^ex1Q₃₅ aggregation kinetics.

Figure 1.

Schematic representation of the unified model of htt^ex1Q₃₅ aggregation. The prenucleation tetramerization equilibrium (shown at the top) is coupled with monomer-independent conversion, chain elongation and fibril surface-mediated secondary nucleation stages (see text). The concentration of tetramers, [T], as a function of monomer concentration, [m], is expressed through the product of equilibrium constants, $K_{\mathrm{T}}={\left(K_{\text{eq},1}\right)}^2{K}_{\text{eq,}2}$.

Figure 2.

Effects of cosolutes on the tetramerization equilibrium for htt^ex1Q₇. (A) Examples of concentration-dependent chemical shift changes (¹⁵N/¹H_N-δ_ex) and effective transverse relaxation rates (¹⁵N-R_2,eff; RF spin-lock field strength of 750 Hz) obtained for htt^ex1Q₇ in the absence of cosolutes (red) and in the presence of 200 mg/mL dextran-20 (blue) and 100 mg/mL hen egg white lysozyme (green). Experimental data are shown with open circles; continuous solid lines represent the global best-fit to the 3-state exchange model in panel (B). The data in the presence of lysozyme were not best-fit, and the dashed green lines are drawn solely to guide the eye. The reduced χ² of the best-fit was 4.6. The full sets of concentration-dependent ¹⁵N/¹H_N-δ_ex and ¹⁵N-R_2,eff data obtained for htt^ex1Q₇ and best-fit to the model in panel (B), are provided in the SI, Figures S1 and S2, respectively. (B) The model of oligomerization used to fit the htt^ex1Q₇ data. The populations of each species (in monomer units; %) are reported for [htt^ex1Q₇] = 1.0 mM, in the absence of cosolutes (red) and in the presence of 200 mg/mL dextran-20 (blue). See SI, “Materials and Methods”, for NMR acquisition parameters and details of the fitting procedures. The NMR experiments were carried out at 5 °C and pH 6.5.

Figure 3.

Aggregation profiles and temporal changes in the concentration of tetramers T (in monomer units) for htt^ex1Q₃₅ in the absence of cosolutes (A), in the presence of 200 mg/mL dextran-20 (B), and 100 mg/mL lysozyme (C). The experimental data, recorded at 5 °C (800 MHz) and normalized to the first time point (t = 0), are shown with open circles. The best-fit curves are shown as black continuous lines and were obtained from a global fit to the kinetic scheme in Figure 1 and the model described by eqs 1.1 and 1.2. Initial concentrations of each sample, m_tot, are indicated on the plots. The following sets of initial conditions P(0) were used in the fits (in nanomolar, nM, units): {2; 4; 6} for htt^ex1Q₃₅ in the absence of cosolutes; {0.5; 1.0; 10} for htt^ex1Q₃₅ in the presence of dextran-20; and {0.5; 12; 60} in the presence of lysozyme. See SI, “Materials and Methods” for the details of the fitting procedure.

Figure 4.

Time dependence of mature fibrils M, nuclei P, and the M/P ratios, simulated for htt^ex1Q₃₅ aggregation (5 °C) using the optimized parameters of the kinetic model in eqs 1.1 and 1.2 in the absence of cosolutes (A), and in the presence of 200 mg/mL dextran-20 (B) and 100 mg/mL lysozyme (C).