Dissociation of amyloid fibrils of alpha-synuclein and transthyretin by pressure reveals their reversible nature and the formation of water-excluded cavities

Abstract

Protein misfolding and aggregation have been linked to several human diseases, including Alzheimer's disease, Parkinson's disease, and systemic amyloidosis, by mechanisms that are not yet completely understood. The hallmark of most of these diseases is the formation of highly ordered and beta-sheet-rich aggregates referred to as amyloid fibrils. Fibril formation by WT transthyretin (TTR) or TTR variants has been linked to the etiology of systemic amyloidosis and familial amyloid polyneuropathy, respectively. Similarly, amyloid fibril formation by alpha-synuclein (alpha-syn) has been linked to neurodegeneration in Parkinson's disease, a movement disorder characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Here we show that consecutive cycles of compression-decompression under aggregating conditions lead to reversible dissociation of TTR and alpha-syn fibrils. The high sensitivity of amyloid fibrils toward high hydrostatic pressure (HHP) indicates the existence of packing defects in the fibril core. In addition, through the use of HHP we are able to detect differences in stability between fibrils formed from WT TTR and the familial amyloidotic polyneuropathy-associated variant V30M. The fibrils formed by WT alpha-syn were less susceptible to pressure denaturation than the Parkinson's disease-linked variants, A30P and A53T. This finding implies that fibrils of alpha-syn formed from the variants would be more easily dissolved into small oligomers by the cellular machinery. This result has physiological importance in light of the current view that the pathogenic species are the small aggregates rather the mature fibrils. Finally, the HHP-induced formation of fibrils from TTR is relatively fast (approximately 60 min), a quality that allows screening of antiamyloidogenic drugs.

Fig. 1.

(A–F) HHP induces dissociation of fibrils of WT and V30M TTR. (A) WT TTR (3.5 μM, •) or V30M (1 μM, ▪) was compressed at 3,000 bar (+p) at pH 5 and 37°C for 30 min. Then, pressure was released (–p), and the light scattering (LS) was recorded for 2 h (B). When the LS increase leveled off, pressure (+) was applied again (C). (D–F) Another round of decompression (–), compression (+), and decompression (–), respectively. LS was measured at 320 nm. (G and H) The species rescued from the WT fibrils is partially folded and binds bis-ANS. An experiment similar to that described in A–F was performed in the presence of bis-ANS (2 μM WT TTR and 20 μM bis-ANS) but measuring its fluorescence intensity (G, left ordinate) and maximum emission wavelength (H, right ordinate). Consecutive cycles of compression (+p)/decompression (–p) are shown, and the bars in G and H represent the following: 1, sample at atmospheric pressure (soluble, native tetramer); 2, sample after 60 min at 3,000 bar (partially folded state); 3, after returning to atmospheric pressure (fibril formation); 4 and 5, another cycle of compression (partially folded state) and decompression (fibril formation), respectively; and 6, the binding of bis-ANS to the urea-unfolded WT protein. (G) The binding of bis-ANS under different conditions is normalized to its binding to the native tetramer (A/A₀). (I) Diclofenac inhibits WT TTR fibrillogenesis. Wt TTR (3.5 μM) was compressed at 3,000 bar in absence of diclofenac at 37°C. After 60 min, the decompression was performed at 1°C to avoid aggregation. The high-pressure cuvette was opened on ice, and 70 μM diclofenac was added to the sample. Then, the temperature was raised to 37°C triggering aggregation, and the LS was recorded (□). A control curve (absence of diclofenac) is presented for comparison (•).

Fig. 2.

Pressure titration of WT (•) and V30M (▪) fibrils. The soluble proteins were subjected to a cycle of compression–decompression under aggregating conditions (pH 5, 37°C) to induce fibril formation. Then, pressure was applied in steps and the light scattering was recorded as in Fig. 1. The initial light scattering value of the fibrils was taken as 100% aggregation.

Fig. 3.

(A) Pressure titration of α-syn fibrils from WT and variant proteins. Fibrils were produced at atmospheric pressure (see text) and then subjected to increasing pressure at 37°C while recording the light scattering (LS) as in Fig. 1. Proteins were: WT (▪), A53T (•), and A30P (▴). The isolated symbols on the left represent the LS values obtained after decompression. (B) Kinetics of the pressure-induced dissociation of WT and variant fibrils of α-syn. The fibrils solutions were compressed at 1,700 bar at 37°C. The extent of fibril dissociation was recorded at intervals as a decrease in LS in comparison to the initial value (LS₀). Symbols are same as in A.

Fig. 4.

Characterization of the species rescued from α-syn fibrils dissociated by HHP. (A) CD spectra of the soluble WT α-syn (continuous line); fibrils (long-dashed line); and the species rescued from HHP treatment (dashed line). (B) Binding of Congo red (filled bars) and thioflavin T (empty bars) to WT (Left) and A53T α-syn (Right) in the form of soluble protein, fibrils, and species rescued from the fibrils (after pressure). (C) Time course of fibril formation (see text) is expressed as the OD at 330 nm for the virgin, soluble protein (open symbols) and the soluble species rescued from the fibrils by HHP (filled symbols). Squares, WT protein; circles, A53T variant. (Inset) TEM images of the WT species rescued from the fibrils by HHP treatment (Upper) and of the fibrils reformed from this sample (Lower) after incubation under appropriate conditions (37°C, gentle agitation for 120 h). (Bar = 150 nm.)

Fig. 5.

Schematic representation of the effects of HHP on the fibrils of WT TTR and α-syn. The core of the amyloid fibrils is not perfectly packed, creating cavities that render the fibrils susceptible to HHP. Upon compression (+p), water infiltrates and dissociation takes place, forming either a partially folded species, as in the case of WT TTR, or a native, unfolded monomer, as in the case of α-syn. In the case of TTR fibrils, Trp and bis-ANS present a blue-shifted emission, suggesting that both fluorophores lie in nonpolar environments. Upon fibril dissociation, the partially folded species of TTR is formed, the Trps are exposed to solvent (red-shifted emission), and the bis-ANS binding pocket is more hydrophilic than in the fibrils. Upon pressure release (–p), the partially folded species of TTR reforms fibrils in <1 h, whereas the native, unfolded α-syn has lost none of its amyloidogenic properties and can aggregate as fast as the virgin, soluble protein.