Photoactive chlorin e6 is a multifunctional modulator of amyloid-β aggregation and toxicity via specific interactions with its histidine residues

Abstract

The self-assembly of Aβ to β-sheet-rich neurotoxic oligomers is a main pathological event leading to Alzheimer's disease (AD). Selective targeting of Aβ oligomers without affecting other functional proteins is therefore an attractive approach to prevent the disease and its progression. In this study, we report that photodynamic treatment of Aβ in the presence of catalytic amounts of chlorin e6 can selectively damage Aβ and inhibit its aggregation and toxicity. Chlorin e6 also reversed the amyloid aggregation process in the dark by binding its soluble and low molecular weight oligomers, as shown by thioflavin T (ThT) fluorescence and photoinduced cross-linking of unmodified protein (PICUP) methods. Using HSQC NMR spectroscopy, ThT assays, amino acid analysis, SDS/PAGE, and EPR spectroscopy, we show that catalytic amounts of photoexcited chlorin e6 selectively damage the Aβ histidine residues H6, H13, and H14, and induce Aβ cross-linking by generating singlet oxygen. In contrast, photoexcited chlorin e6 was unable to cross-link ubiquitin and α-synuclein, demonstrating its high selectivity for Aβ. By binding to the Aβ histidine residues, catalytic amounts of chlorin e6 can also inhibit the Cu²⁺-induced aggregation and toxicity in darkness, while at stoichiometric amounts it acts as a chelator to reduce the amount of free Cu²⁺. This study demonstrates the great potential of chlorin e6 as a multifunctional agent for treatment of AD, and shows that the three N-terminal Aβ histidine residues are a suitable target for Aβ-specific drugs.

Fig. 1. The inhibitory activity of Ce6 on Aβ aggregation. (a) Increasing amounts of Ce6 (0.2–2 μM) were incubated in PBS (50 mM, pH 7.4) with monomeric Aβ40 (20 μM) in darkness or photoirradiated for 1 h. Samples were incubated with constant shaking for further 72 h, and the extent of Aβ aggregation was then determined by the ThT assay. Results are mean ± SD of three experiments (n = 3 each). (b and c) Effect of photoexcited Ce6 on the secondary structure of Aβ. Time dependent far-UV CD spectra of freshly prepared Aβ40 monomer (10 μM) incubated in the absence or presence of photoexcited Ce6 (10 μM) in phosphate buffer (50 mM, pH 7.4) and analyzed after (b) 0 and (c) 72 h of aging. (d) TEM images of Aβ (20 μM) incubated in the absence or presence of Ce6 (2 μM) in darkness or after 1 h photoirradiation following 72 h incubation in darkness. Negatively stained samples are shown. (e) Effect of Ce6 and photoexcited Ce6 on antibody binding to Aβ at different pH values. Aβ40 (20 μM) was aged for 0 or 72 h in the absence or presence of Ce6 or photoexcited Ce6 (20 μM) at different pH values, spotted onto nitrocellulose membranes, and probed with either the A11 or 6E10 antibody.

Fig. 2. The mechanism of action of Ce6 in darkness and after irradiation. (a) Ce6 disassembles aggregated Aβ under dark and light conditions. Aβ (50 μM) was aged for 3 days to generate Aβ aggregates with high ThT fluorescence. Ce6 (5 or 50 μM) was then added and the mixtures were kept in the dark or photoirradiated for 1 h. The amount of Aβ fibrils was then determined after 3 days by the ThT assay. The data represent mean ± SD values of an experiment out of four carried in duplicate. (b) Effect of Ce6 on oligomer distribution of Aβ. Treatment of Aβ with Ce6 significantly reduces its self-assembly under dark conditions. Aβ (50 μM) was aged in the dark for 0, 24, or 48 h in the absence (lanes 1–3) or presence (lanes 4–6) of Ce6 (50 μM) and analyzed by the Tris–tricine SDS–PAGE PICUP method. (c) Tris–tricine SDS–PAGE separation of Aβ (50 μM) before (lane 1) and after PICUP cross-linking using Ru²⁺ complex (lane 2). Lane 3 represents Tris–tricine SDS–PAGE separation of Aβ (50 μM) incubated with Ce6 (50 μM) and photoirradiated for 1 min in the absence of Ru²⁺. (d) Effect of Aβ40 on photogeneration of singlet oxygen from Ce6. EPR spectra obtained from photoirradiation of Ce6 (40 μM) and TEMP (40 μM) in the absence or presence of Aβ (40 μM) in PBS/D₂O (1 : 9). EPR conditions: microwave power, 20 mW; modulation amplitude, 1.0 G; receiver gain, 4 × 10⁵; time constant, 0.64 s; scan range, 100 G; center of field, 3300 G; temperature, 25 °C. (e) Effect of pH on antiamyloidogenic activity of Ce6 in darkness and after photoirradiation, using the ThT assay. Details are identical to those described in Fig. 1a legend. (f) SDS–PAGE separation of α-synuclein (6 μM, lane 1) treated either with photoexcited Ce6 (33 μM, lane 2) or Ru²⁺ catalyst (33 μM, lane 3). Lanes 4–6 demonstrate respectively SDS–PAGE separation of ubiquitin (30 μM, lane 4) treated either with photoexcited Ce6 (33 μM, lane 5) or Ru²⁺ catalyst (33 μM, lane 6).

Fig. 3. NMR investigations of the molecular interactions between the monomeric Aβ40 and Ce6 in darkness and following 1 h photoirradiation. (a) ¹H, ¹⁵N-HSQC NMR spectrum of Aβ40 (75 μM) in PB (20 mM, pH 7.3). (b) ¹H, ¹⁵N-HSQC NMR spectra of Aβ40 (75 μM) after addition of Ce6 (1 mg) under dark conditions (blue peaks), and following 1 h of photoirradiation (red peaks). (c) ¹H, ¹⁵N-HSQC NMR spectrum of Aβ40 (75 μM) in PB (20 mM, pH 5.0). (d) ¹H, ¹⁵N-HSQC NMR spectra of Aβ40 (75 μM) in PB after addition of Ce6 (1 mg) under dark conditions (blue peaks), and after 1 h exposure to light (red peaks). (e) ¹H, ¹³C-HSQC NMR spectra of Aβ40 (75 μM) and Ce6 (1 mg) in PB (20 mM, pH 7.3) before (blue peaks) and after 1 h exposure to light (red peaks).

Fig. 4. Effect of Ce6 and photoexcited Ce6 on the toxicity of Aβ40 to rat pheochromocytoma PC12 cells. Freshly prepared Aβ40 (100 μM) was aged for 48 h with Ce6 under either dark or light conditions (100 and 1000 μM) in PBS. The samples were then diluted 10 times in the wells containing 10 000 cells. Cell viability was then determined after 48 h of incubation by MTT. *p < 0.005 compared to the same treatment in darkness and with Aβ only. All samples contained 0.25% DMSO, which was found to have no effect on cell survival (100% cell survival).

Fig. 5. Effect of Ce6 on Cu²⁺-induced Aβ aggregation and toxicity. (a) Monomeric Aβ (5 μM) was incubated in the dark with Cu²⁺ (1.5 μM) in the absence or presence of a catalytic amount of Ce6 (0.125 μM) in HEPES buffer (50 mM, 160 mM NaCl, pH 7.4), and the extent of amyloid formation was monitored over time using the ThT fluorescence. (b) Ce6 counters the effect of Cu²⁺ on Aβ-induced PC12 toxicity. Aβ (10 μM) was aged for 48 h with Cu²⁺ in absence or presence of increasing amounts of Ce6 and exposed to PC12 cells for a further 4 days. Cell viability was then determined by the MTT assay. All samples contained 0.25% of DMSO, which was found to be harmless (100% cell survival). The results are presented as mean ± SD of two experiments (n = 3–5 each). Significance (*,**p < 0.05) were calculated relative to Aβ₄₀ (10 μM) or Aβ + Cu²⁺ (10 : 1 μM) toxicity, respectively.

Fig. 6. Effect of Cu²⁺ and Ce6 on the Aβ NMR spectrum. (a) ¹H, ¹⁵N-HSQC NMR spectrum of Aβ₄₀ (80 μM) in HEPES buffer (40 mM, pH 7.3). (b) NMR spectra of Aβ₄₀ (80 μM) in HEPES buffer, before (blue) and after (red) addition of Cu²⁺ (40 μM). Most N-terminal Aβ crosspeaks have completely disappeared. (c) NMR spectrum of Aβ (80 μM) and Cu²⁺ (40 μM) in HEPES buffer, before (blue) and after (red) addition of Ce6 (800 μM). Many N-terminal Aβ crosspeaks have partially returned, indicating that Ce6 can compete with Cu²⁺ ions for binding to the Aβ N-terminal segment.