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DOI: 10.1039/D4DT02188F (Communication) Dalton Trans., 2024, 53, 14966-14970

Fighting fire with fire: remodeling Aβ aggregation with H-aggregates of a europium(III) complex†

Yuancun Zhou a, Jiacheng Zhu a, Furong Gao a, Ming Hu *a, Chengyuan Qian b, Xin Wang a and Xiaohui Wang *a
aInstitute of Chemical Biology and Functional Molecules, State Key Laboratory of Materials-Oriented Chemical Engineering, School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 211816, P. R. China. E-mail: wangxhui@njtech.edu.cn; huming@njtech.edu.cn
bSchool of Life Sciences, Nanjing University, Nanjing 210093, P. R. China

Received 31st July 2024 , Accepted 15th August 2024

First published on 22nd August 2024

Abstract

We herein report a “Fight Aggregation with Aggregation” (FAA) approach for redirection of amyloid-β peptide (Aβ) aggregation using a europium(III) complex (EuL3) that can undergo H-aggregation in aqueous solution under physiological conditions. The H-aggregates of EuL3 may serve as scaffolds that can facilitate the accumulation of Aβ to form non-fibrillar co-assemblies. As a result, the Aβ aggregation-induced cytotoxicity was inhibited.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder characterized by aberrant protein aggregation, particularly amyloid plaques generated from amyloid-β peptide (Aβ) self-assembly.1 Although the exact pathogenesis of AD is still unclear, Aβ aggregation plays a pivotal role in neurodegeneration,2 which generates neurotoxic aggregates, particularly low molecular weight (LMW) Aβ oligomers, e.g., dimers and trimers, which can cause neuronal dysfunction and loss.3 Hence, mitigating oligomers’ toxicity could be a promising strategy for AD therapy.4 Currently, various anti-oligomer therapies have been developed through modulation of Aβ aggregation,5 including inhibition of oligomerization,6 disaggregation of oligomers,7 and promotion of fibril formation.8 However, the metastable nature and unknown high-resolution structures of oligomers make it challenging for them to target oligomers,9 leading to low therapeutic efficacy. Moreover, there is a risk that modulating Aβ aggregation could cause recurring toxicity due to the possibility of generating Aβ monomers or fibrils that re-enter the aggregation process to form oligomers.10

Remodeling the Aβ oligomers into off-pathway aggregates would be an effective strategy to irreversibly eliminate the oligomers’ toxicity.11 Following this strategy, several agents have been reported to redirect Aβ aggregation into non-toxic amorphous species via co-assembly.12 For example, multivalent macromolecules containing multiple binding sites can efficiently capture Aβ species to form discrete nanostructures.13 Unfortunately, their binding selectivity for oligomers is modest. Our group has developed a europium complex (EC) that can selectively co-assemble with low molecular weight Aβ oligomers to form non-fibrillar, non-toxic co-aggregates through binding to hydrophobic regions of oligomers.14 Therefore, multivalent macromolecules with binding selectivity for oligomers would be excellent candidates for remodeling the Aβ oligomers via multivalent co-assembly, which, unfortunately, are rare in the literature.

Recently, we found that the bis(azobenzene)-functionalized lanthanide complexes TbL3 and EuL3 can self-assemble into H-aggregates through non-covalent interactions in aqueous solutions.15 The rigid lanthanide chelating center guarantees the aggregates’ stability. More importantly, considering their similar structural features to the oligomer probe EC, the planar hydrophobic N,N-dimethylaminoazoaniline (DAA) moieties and dimethylamino groups with high binding affinity to Aβ may endow the complexes with binding selectivity to Aβ oligomers. These findings encouraged us to try out the H-aggregates of such complexes on remodeling Aβ oligomers via multivalent co-assembly. As a proof of concept, EuL3 can significantly promote Aβ accumulation into non-fibrillar aggregates, which reduce the Aβ-induced cytotoxicity in PC12 cells (Scheme 1). Thus, EuL3 would provide a prototypical design of molecular aggregates for anti-Aβ management of AD.


image file: d4dt02188f-s1.tif
Scheme 1 EuL3 aggregates remodel Aβ oligomers into non-fibrillar co-assemblies.

We first validated the water-triggered self-assembly of EuL3 in DMSO/water solution by absorption spectroscopy. As shown in Fig. 1A, the absorption of the EuL3 monomer at 429 nm gradually decreased and the blue-shifted peak emerged at 357 nm with an increase of water fraction (fw) from 20% to 90%. EuL3 exhibited the same water-responsive absorption profile as TbL3,15 confirming its H-aggregation. We next examined whether the H-aggregates of EuL3 could bind to Aβ with high binding affinity in Tris buffer containing 2.5% v/v DMSO. First, the steady-state fluorescence of EuL3 aggregates increased with increasing concentration of Aβ40 (Fig. S1†), indicating that the interactions may exist between DAA and Aβ. Thioflavin T (ThT) fluorescence competition assay was next employed, which is a commonly used method for evaluating the binding affinity of agents to Aβ using ThT as a fluorescent probe for Aβ species. ThT preferentially interacts with central hydrophobic regions of β-sheet-rich Aβ species, resulting in significant enhancement of fluorescence.16 However, the addition of EuL3 to the solution of ThT–Aβ adducts significantly reduced the Aβ-enhanced ThT fluorescence (Fig. 1B). Given the much weaker influence of EuL3 on the fluorescence of ThT per se (Fig. S2A†), the fluorescence decrease of ThT–Aβ was mainly attributed to the binding of EuL3 to Aβ40 by replacing ThT at similar binding sites with higher affinity than that of ThT. Furthermore, the addition of ThT to the solution of EuL3–Aβ led to weaker fluorescence than that of ThT–Aβ with the addition of EuL3 (Fig. S2B†), confirming that the binding of EuL3 to Aβ would prevent the binding of ThT to Aβ. Compared with its ligand L3–3H that cannot self-assemble in the aqueous solution,15 EuL3 aggregates exhibited a similar profile in competition with ThT (Fig. S3†). The inhibition constants of EuL3 and L3-3H were calculated to be 0.36 and 0.29 μM, respectively. The results indicate that the self-assembly of DAA groups hardly weaken its binding to Aβ. The binding of EuL3 aggregates to Aβ was further validated by fluorescence competition with 8-anilinonaphthalene-1-sulfonate (ANS), which is a “turn-on” fluorescent probe for Aβ oligomers through binding to the solvent-exposed hydrophobic patches of oligomers.17 Despite the minimum quenching effect on ANS fluorescence (Fig. S4†), the addition of EuL3 caused a remarkable decrease of Aβ-bound ANS emission and a red-shift to that of ANS per se (Fig. 1C), indicating the higher binding affinity of EuL3 to the central hydrophobic region of Aβ than that of ANS. These results consolidate that the H-aggregates of EuL3 possess high binding affinity to Aβ through interactions between DAA and Aβ.


image file: d4dt02188f-f1.tif
Fig. 1 (A) Absorption spectra of EuL3 (40 μM) in DMSO/H2O mixed solvents with different water fractions (fw). (B) Fluorescence spectra of ThT (20 μM, λex = 440 nm) in the presence of Aβ40 with varying concentrations of EuL3 (0–95 μM). (C) Fluorescence spectra of ANS (20 μM, λex = 380 nm) in the presence of Aβ40 with different concentrations of EuL3 (0–75 μM). All fluorescence spectra were recorded in Tris buffer (20 mM Tris-HCl, 150 mM NaCl, 2.5% v/v DMSO, pH 7.4).

Given that Cu2+ could accelerate Aβ aggregation and stabilize oligomers in AD,18 we next investigated the influence of EuL3 aggregates on both Cu2+-free and Cu2+-induced Aβ40 aggregation by sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting using the anti-Aβ antibody 6E10. In Fig. 2A, a band (∼10 kDa) corresponding to LMW oligomers (dimers and trimers) is clearly observed on the membrane for the Aβ40 sample in the absence of EuL3 and Cu2+ after 1 h of incubation (Fig. 2A). At that time, LMW oligomers would be the dominant species, because Aβ would enter into its lag phase of aggregation according to the growth profile of Aβ self-aggregation (Fig. S5†). Similar results of western blotting can be found in the literature.19 However, the band intensity decreased with increasing incubation time for 4 h due to conversion of oligomers into fibrils in the stationary phase (Fig. S5†). A weaker band of LMW oligomers was found on the membrane for the Cu2+–Aβ40 sample (1 h), owing to the promotion effect of Cu2+ on Aβ aggregation. Upon incubation for 4 h, the band seemed slightly darker than that of Aβ, probably due to the stabilizing effect of Cu2+ on oligomers.18b In contrast, EuL3 aggregates can remarkably decrease LMW oligomers of Aβ in both the absence and presence of Cu2+, suggesting that EuL3 aggregates can promote the assembly of Aβ oligomers. Dot blotting using the 6E10 antibody was further performed by measuring soluble Aβ40 species in the supernatant of incubated samples after centrifugation. The weaker darkness of dots means a higher degree of aggregation. As shown in Fig. 2B and C, compared with Cu2+-free and Cu2+-induced Aβ aggregation, EuL3 reduced soluble Aβ levels in samples at varied incubation times, consistent with the result of western blotting. Quantitative analysis of the dots by using ImageJ consolidated the above observation (Fig. S6A†). Furthermore, the influence of EuL3 on the concentration of soluble Aβ species in the supernatant was also confirmed by BCA assay (Fig. S6B†). The ability of EuL3 aggregates to promote Aβ aggregation can be visually observed with the naked eye. After a long-term (12 h) incubation, EuL3-treated Aβ40 samples only produced insoluble species (Fig. 2D), which were identified as Aβ aggregates by dot blot using the 6E10 antibody (Fig. S7†). Both the EuL3-induced precipitates in the absence and presence of Cu2+ clearly showed the dot signals of Aβ. On the other hand, no insoluble aggregates were seen in EuL3 or Aβ. Interestingly, dot blotting assay showed that the ligand L3-3H can marginally inhibit Aβ aggregation under the same conditions as EuL3 (Fig. S8A†), and no insoluble species appeared after 12 h of incubation (Fig. S8B†). Hence, the formation of H-aggregates is indispensable for promotion of Aβ aggregation. The aggregates may provide multiple binding sites for raising the payload of Aβ, resulting in off-pathway co-assemblies.


image file: d4dt02188f-f2.tif
Fig. 2 Western blot (A), dot blot (B and C) analysis of the time-dependent effect of EuL3 (40 μM) on Aβ40 (20 μM) aggregation in the absence or presence of Cu2+ (20 μM) using the anti-Aβ antibody 6E10. (D) Images of Aβ40 (20 μM) samples with or without EuL3 (20 μM) in the absence or presence of Cu2+ (20 μM) after incubation at 37 °C for 12 h.

To clarify the working mode of EuL3 with Aβ, transmission electron microscopy (TEM) was used to analyze the morphology of EuL3-induced Aβ40 aggregates. As shown in Fig. 3A, the intertwined filament-like fibrils were observed in the sample of Aβ after self-incubation. Compared with the flake-like structure of EuL3 aggregates, amounts of spherical aggregates were found in the EuL3-treated Aβ sample under the same conditions, indicating that EuL3 can completely convert the amyloidogenic aggregation of Aβ into a non-amyloidogenic pathway. Circular dichroism (CD) was further employed to test the influence of EuL3 aggregates on the secondary structure of Aβ (Fig. 3B). The typical β-sheet structure of Aβ fibrils characterized with a minimum around 218 nm can be observed for Aβ self-aggregation. The treatment of EuL3 aggregates can cause disappearance of the negative peak around 218 nm; meanwhile, a new minimum at 212 nm appeared. This result consolidates the ability of EuL3 to remodel Aβ aggregates into non-fibrillar assemblies. To verify whether EuL3 could co-assemble with Aβ, the time-resolved luminescence of EuL3-induced insoluble Aβ aggregates was measured in the presence of Na2S2O4 that can activate the luminescence of Eu(III) through cleavage of the N[double bond, length as m-dash]N double bonds of DAA groups.15 The addition of Na2S2O4 resulted in intense luminescence (Fig. S9†), suggesting the presence of substantial EuL3 aggregates in the insoluble deposit. Taking all the results into account, the H-aggregates of EuL3 would serve as scaffolds that can promote the accumulation of Aβ to form non-fibrillar co-assemblies.


image file: d4dt02188f-f3.tif
Fig. 3 (A) TEM images of Aβ40 (20 μM) aggregates in the absence or presence of EuL3 (40 μM). Scale bar = 500 nm. (B) CD spectra of Aβ40 (20 μM) in the absence or presence of EuL3 (40 μM) at different incubation time points (0 and 12 h) in Tris-HCl buffer (20 mM Tris-HCl/150 mM NaCl, 1% v/v DMSO, pH 7.4) at 37 °C.

Given that Aβ aggregation can be completely redirected by EuL3, we further tested the influence of EuL3 on Aβ40-induced cytotoxicity to PC12 cells using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The nontoxicity of EuL3 aggregates was initially proved (Fig. 4A). In contrast, incubation of the cells with Aβ reduced the cell viability to ∼73%, owing to the neurotoxicity of Aβ aggregates (Fig. 4B). However, the cell viability was obviously recovered through the treatment of EuL3 aggregates in a concentration-dependent manner. More than 83.2% cells survived in the presence of 40 μM EuL3. The results imply that the non-fibrillar co-assemblies would be non-toxic, thereby reducing the cytotoxicity of the EuL3-treated group.


image file: d4dt02188f-f4.tif
Fig. 4 (A) Viability (%) of PC12 cells upon incubation with different concentrations of EuL3. (B) Viability (%) of PC12 cells upon incubation with Aβ40 (20 μM) in the absence and presence of EuL3 (10, 20, 30, and 40 μM). The data were normalized and calculated as a percentage of untreated cells only containing 2% of DMSO as a control. Error bars indicate ± s.d. (n = 5 independent experiments).

In summary, we have reported a FAA strategy to modulate Aβ aggregation by using H-aggregates of a europium(III) complex EuL3. EuL3 can self-assemble into its H-aggregates in aqueous solution under physiological conditions. The EuL3 aggregates can completely remodel Aβ oligomers into non-fibrillar co-assemblies. As a result, the Aβ-induced cytotoxicity can be irreversibly reduced. EuL3 exhibits several benefits over previously reported multivalent macromolecule-based modulators of Aβ aggregation, including ease of preparation and structural characterization, high selectivity for Aβ oligomers, and efficient aggregation manipulation. This work would offer a promising direction for designing self-assembling metal complexes to remodel Aβ aggregation in AD.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We appreciate the financial support from the National Natural Science Foundation of China (Grant: 21771105), the Natural Science Foundation of Jiangsu Province (Grant: BK20170103), and the Natural Science Foundation of the Jiangsu Higher Education Institutions (Grant: 23KJB150012).

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and additional figures. See DOI: https://doi.org/10.1039/d4dt02188f
This journal is © The Royal Society of Chemistry 2024