Characterizing the inhibition of α-synuclein oligomerization by a pharmacological chaperone that prevents prion formation by the protein PrP

Abstract

Aggregation of the disordered protein α-synuclein into amyloid fibrils is a central feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease. Small, pre-fibrillar oligomers of misfolded α-synuclein are thought to be the key toxic entities, and α-synuclein misfolding can propagate in a prion-like way. We explored whether a compound with anti-prion activity that can bind to unfolded parts of the protein PrP, the cyclic tetrapyrrole Fe-TMPyP, was also active against α-synuclein aggregation. Observing the initial stages of aggregation via fluorescence cross-correlation spectroscopy, we found that Fe-TMPyP inhibited small oligomer formation in a dose-dependent manner. Fe-TMPyP also inhibited the formation of mature amyloid fibrils in vitro, as detected by thioflavin T fluorescence. Isothermal titration calorimetry indicated Fe-TMPyP bound to monomeric α-synuclein with a stoichiometry of 2, and two-dimensional heteronuclear single quantum coherence NMR spectra revealed significant interactions between Fe-TMPyP and the C-terminus of the protein. These results suggest commonalities among aggregation mechanisms for α-synuclein and the prion protein may exist that can be exploited as therapeutic targets.

Keywords: cyclic tetrapyrrole; fluorescence cross-correlation spectroscopy; isothermal titration calorimetry; protein aggregation.

Figure 1

Fe‐TMPyP structure. Iron(III) meso‐tetra (N‐methyl‐4‐pyridyl‐prophine) is a planar molecule with a ferric ion centrally coordinated within a porphyrin ring that is circumscribed peripherally by four positively charged methyl‐pyridinium moieties. Fe‐TMPyP, iron(III) meso‐tetra (N‐methyl‐4‐pyridyl‐prophine)

Figure 2

Fe‐TMPyP decreases α‐synuclein fibrillization extent and increases lag phase as assayed by ThT fluorescence. (a) ThT fluorescence shows concentration‐dependent inhibition of amyloid fibril formation for different molar ratios of Fe‐TMPyP:α‐synuclein: 0:1 (red), 1:10 (orange), 1:5 (brown), 1:2 (green), 1:1 (cyan), 2:1 (blue), 5:1 (pink), and 10:1 (purple). Curve in black shows results using a 10:1 molar ratio of EGCG. (b) The lag phase of fibril formation starts to rise above a 5:1 molar ratio. Error bars show SEM. EGCG, epigallocatechin gallate; Fe‐TMPyP, iron(III) meso‐tetra (N‐methyl‐4‐pyridyl‐prophine)

Figure 3

Fe‐TMPyP suppresses α‐synuclein oligomers monitored by fluorescence cross‐correlation spectroscopy. (a) Large fluorescence fluctuations from Cy5‐ (red) and OG‐labeled (green) α‐synuclein (left panels) are seen more often as the incubation time increases from 0 to 50 h. The corresponding auto‐correlation (Cy5, red; OG, green) and cross‐correlation (orange) curves (right panels) fit to Equations 1, 2, 3 (dashed lines) show increasing cross‐correlation amplitude and diffusion time, indicating the growth of oligomers. (b) Measurements repeated with a fivefold molar excess of Fe‐TMPyP show no large fluorescence fluctuations (left panels) and suppression of the cross‐correlation signal (right panels). Fe‐TMPyP, iron(III) meso‐tetra (N‐methyl‐4‐pyridyl‐prophine)

Figure 4

Fe‐TMPyP decreases oligomer size in FCCS assay. The diffusion time in FCCS assays decreases with increasing Fe‐TMPyP concentration, reflecting smaller oligomers, until oligomers are abolished above 5:1 ligand:protein molar ratios. Red, no ligand; orange, 1:4 molar ratio of Fe‐TMPyP to α‐synuclein; green, 1:2 molar ratio; cyan, 1:1 molar ratio; blue, 2:1 molar ratio; purple, 5:1 molar ratio; and black, 5:1 molar ratio of EGCG to α‐synuclein. Error bars show SEM. EGCG, epigallocatechin gallate; FCCS, fluorescence cross‐correlation spectroscopy; Fe‐TMPyP, iron(III) meso‐tetra (N‐methyl‐4‐pyridyl‐prophine)

Figure 5

Fe‐TMPyP reduces the extent of oligomer formation. The fraction of particles that contain both dye labels in FCCS measurements is reduced with increasing molar ratios of ligand to protein (red, no ligand; orange, 1:4 Fe‐TMPyP:α‐synuclein; green,1:2; cyan, 1:1; blue, 2:1; purple, 5:1; and black, 5:1 molar ratio of EGCG:α‐synuclein. Error bars represent SEM. EGCG, epigallocatechin gallate; FCCS, fluorescence cross‐correlation spectroscopy; Fe‐TMPyP, iron(III) meso‐tetra (N‐methyl‐4‐pyridyl‐prophine)

Figure 6

ITC reveals stoichiometric Fe‐TMPyP binding to α‐synuclein monomers. (a) Two binding sites were found by fitting the baseline‐subtracted binding isotherm (red), one with high affinity (K _D = 8.2 μM) and one with low affinity (K _D = 330 μM). (b) Binding at the high‐affinity site was weakened modestly by increasing [Na⁺] (red), whereas that at the low‐affinity site was weakened significantly (black). Fe‐TMPyP, iron(III) meso‐tetra (N‐methyl‐4‐pyridyl‐prophine); ITC, isothermal titration calorimetry

Figure 7

2D‐[¹H,¹⁵N]‐HSQC NMR spectroscopy of Fe‐TMPyP binding to α‐synuclein. Changes in the spectral peaks for α‐synuclein alone (black) and with fivefold molar excess of Fe‐TMPyP (green) indicate which parts of the protein are most affected by binding. Most of the peaks strongly affected (shifted or absent) by Fe‐TMPyP binding were located in the acidic C‐terminal domain (red), whereas residues in the hydrophobic NAC domain (cyan) or the N‐terminal region (gray) were little changed. Fe‐TMPyP, iron(III) meso‐tetra (N‐methyl‐4‐pyridyl‐prophine); HSQC, two‐dimensional heteronuclear single quantum coherence

Figure 8

Amount of fibril formed is reduced by Fe‐TMPyP binding. The amount of fibril formed, reflected in the ThT fluorescence amplitude at saturation, is reduced roughly linearly (red line: linear fit, r ² = .91) by Fe‐TMPyP binding, suggesting ~65% of Fe‐TMPyP‐bound α‐synuclein cannot form fibrils. Fe‐TMPyP, iron(III) meso‐tetra (N‐methyl‐4‐pyridyl‐prophine); ThT, thioflavin T