Thermodynamic stability modulates chaperone-mediated disaggregation of α-synuclein fibrils

Abstract

Aggregation of the intrinsically disordered protein alpha-synuclein into amyloid fibrils and their subsequent intracellular accumulation are hallmark features of several neurodegenerative disorders, including Parkinson's disease, for which no curative treatments currently exist. In this study, we investigate the relationship between fibril morphology, thermodynamic stability, and susceptibility to disaggregation by the human chaperone system comprising HSP70, DNAJB1, and Apg2. By varying assembly conditions and incubation times, we generated alpha-synuclein fibrils with diverse morphological and biochemical properties, including a broad range of thermodynamic stabilities, which we quantified using a chemical depolymerization assay. The chaperone system effectively disaggregated three of the four fibril types, with efficiencies that correlated with their thermodynamic stabilities. One fibril type resisted disaggregation despite exhibiting a comparable stability to those that were disaggregated, suggesting that additional structural features influence chaperone susceptibility. Our findings establish a quantitative link between fibril stability and chaperone-mediated disaggregation for three in vitro αSyn fibril types as well as fibrils amplified from brain extracts of PD but not MSA patients, highlighting the importance of fibril thermodynamics in biologically relevant disaggregation processes and disease pathology.

Fig. 1. Overview of the experimental design. Left panel: αSyn monomer was brought to four different sets of solution conditions – physiological (Fm, blue), low salt (Ri, orange), mildly acidic (‘low’) pH (F65, green), and mildly basic (‘high’) pH (F91, violet). Middle panel: sample aliquots (0.5 mL, 100 µM for each timepoint and condition) were incubated at 37 °C and 600 rpm for 3, 6, 14, 28, 56, and 84 days. All samples were analyzed simultaneously by the indicated assays described in detail in the materials and methods section. Right panel: schematic overview of the relation of fibril stability and susceptibility to the chaperone system Hsp70, DNAJB1 and Apg2. The energy diagram illustrates that highly disordered monomeric αSyn with high energy assembles into the thermodynamically more favorable fibrillar state. Different fibrils can have different thermodynamic stability, indicated by the purple and green states, and can be depolymerized by tri-chaperone system to varying degrees. The stability and chaperone disaggregation of the fibrils from each sample were analyzed using established methods.

Fig. 2. Thermodynamic stability of fibrils formed in conditions Fm, Ri, F65 and F91 and measured in 50 mM HEPES, pH 7.5, 50 mM KCl, 5 mM MgCl₂, 2 mM DTT. (a) Fitting of the isodesmic model to depolymerization curves of amyloid fibrils formed under condition F65 at various time points (color-coded as indicated in the legend) using Bayesian analysis. The prior distribution for the m-values was defined by a mean of 3.5 and a standard deviation of 2. Opaque lines represent 100 random samples from a Hamiltonian Monte Carlo (HMC) analysis, drawn from at least 2000 total samples. The area under each curve (AUC) corresponds to the monomer concentration measured by FIDA. (b) Joint probability distributions of ΔG and m-values obtained from the fitting in (a), highlighting the correlation between these two fitting parameters. (c) Thermodynamic stability (ΔG) of fibrils formed at different incubation times, determined from urea-induced depolymerization experiments. Depolymerization curves were fitted to the isodesmic model using Bayesian analysis as demonstrated in (a and b). Error bars represent the uncertainty (mean ± standard deviation) derived from the HMC analysis (n = 2000 samples). Repeats with significant (p < 0.05) negative slopes are marked with a red asterisk. Parameter values (ΔG and m-values) are listed in SI Tables 1–3.

Fig. 3. Kinetics of αSyn fibril disaggregation by the chaperone system HSP70, DNAJB1 and Apg2 in the presence of ATP. (a) Normalized ThT signal over time in the presence of chaperone system HSP70 (4 µM), DNAJB1 (2 µM) and Apg2 (0.2 µM), ATP (2 mM) and amyloid fibrils (2 µM) formed in either condition Fm, F65 or F91. Data corresponds to the first repeat in case of Fm and F65 and to the second repeat in case of F91. (b) Disaggregation efficiency of the chaperone system of amyloid fibrils formed in condition Fm, F65 or F91 at different time points of two repeats. A degree of disaggregation of 1 represents the complete loss of ThT signal. A degree of disaggregation of 0 represents no change of the ThT signal during the measurement. Error bars represent the propagated uncertainties based on the standard error of the mean from duplicate (3rd repeat) or triplicate (1st and 2nd repeat) measurements. Fibrils formed in condition Ri were not disaggregated (with a single exception in the third repeat) (SI Fig. 24).

Fig. 4. Thermodynamic stability and disaggregation by chaperones of fibrils amplified from brain extracts of PD and MSA patients. (a) Thermodynamic stability. Upper panel: SDS-PAGE analysis of soluble fraction of 20 µM PD or MSA-amplified fibrils incubated at varying urea concentrations for 4 days at 25 °C. Soluble fractions of samples marked with a red asterisk contained fibrils (SI Fig. 28) and were excluded in the analysis of thermodynamic stability. Lower panel: fitting of the isodesmic model to depolymerization curves of fibrils amplified from brain extracts of PD and MSA patients using Bayesian analysis. The points correspond to the normalized gel band intensities obtained from image analysis of the gels shown on top. A uniform prior was used for the m-value. Opaque lines represent 100 random samples from a Hamiltonian Monte Carlo (HMC) analysis, drawn from at least 2000 total samples. Inset: joint probability distributions of ΔG and m-values obtained from the fitting, highlighting the correlation between these two fitting parameters. (b) Disaggregation of 0.8 µM fibrils amplified from brains of PD and MSA patients by the chaperone system HSP70, DNAJB1 and Apg2 in the presence of ATP. The disaggregation was followed by ThT over time and residual monomer concentration at the endpoint of the reaction (16 h) was determined using western blotting (insets).

Fig. 5. Correlation of the thermodynamic stability of αSyn fibrils and their disaggregation by the chaperone system HSP70, DNAJB1 and Apg2. Stars mark amyloid fibrils amplified from brains of PD and MSA patients. Fibrils formed in condition Ri are not disaggregated (with a single exception in the third repeat). Their degree of disaggregation (as well as data point F65 56 days in the third repeat) was set to 0 for comparison. Pearson's correlation coefficients (r and p-values) are provided as measures of the correlation strength and the statistical significance, respectively. Data point F65 56 days in the third repeat was set to 0 since the numerical value was negative.

Fig. 6. Change in the free energy of ATP as a function of its depletion. The Gibbs free energy of ATP hydrolysis to ADP and phosphate (P) (ΔG, black line) was calculated for conditions of the disaggregation assay (150 mM ionic strength, pH 7.5, 5 mM Mg²⁺) using the standard values from. The range of free energy corresponding to the fibril stabilities measured here are highlighted in red and green.