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. 2021 Jun 18:15:680026.
doi: 10.3389/fnins.2021.680026. eCollection 2021.

Squalamine and Its Derivatives Modulate the Aggregation of Amyloid-β and α-Synuclein and Suppress the Toxicity of Their Oligomers

Affiliations

Squalamine and Its Derivatives Modulate the Aggregation of Amyloid-β and α-Synuclein and Suppress the Toxicity of Their Oligomers

Ryan Limbocker et al. Front Neurosci. .

Abstract

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-β peptide (Aβ) and of α-synuclein (αS), which are associated with Alzheimer's and Parkinson's diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aβ and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aβ and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer's and Parkinson's diseases.

Keywords: Alzheimer’s disease; Parkinson’s disease; aminosterols; amyloid-β; oligomers; protein misfolding diseases; small molecule drug discovery; α-synuclein.

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Conflict of interest statement

DB and MZ are inventors in a patent for the use of aminosterols in the treatment of Parkinson’s disease. DB and MZ are co-founders of Enterin Inc. and serve as the President and CSO, respectively, of the company. MV, TPJK, and JH are co-founders, and BM and MP are employees of Wren Therapeutics Ltd., which is independently pursuing inhibitors of protein misfolding and aggregation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Structures of the four aminosterols investigated in this work. Squalamine (SQ, red), des-squalamine (desSQ, green), α-squalamine (αSQ, orange), and trodusquemine (TRO, blue) with unique molecular features indicated.
FIGURE 2
FIGURE 2
Aminosterols accelerate Aβ42 aggregation in vitro. Kinetic profiles of the aggregation of 2 μM Aβ42 in the absence (black) or presence of 0.2 μM (green), 0.4 μM (orange), and 2 μM (red) of TRO (A), SQ (B), αSQ (C), and desSQ (D). Data represent mean ± standard error of the mean (s.e.m.) of three technical replicates. Raw ThT aggregation data are provided in Supplementary Figure 1. (E) AFM images at t = 0 h (top panels, scale bars = 0.2 μm) and t = 4 h (bottom panels, scale bars = 1 μm) of aggregation in the absence (left panels) and presence of an equimolar concentration of SQ (right panels). (F) TEM images of fibrils formed after 4 h of aggregation in the absence (top) or presence of an equimolar concentration of SQ (bottom). Scale bars = 100 nm. AFM and TEM measurements were taken alongside a comparison of the effect of TRO on Aβ42 aggregation, and the untreated fibrils, therefore, have the same population statistics as previously published (Limbocker et al., 2019).
FIGURE 3
FIGURE 3
Aminosterols inhibit the lipid-induced nucleation of αS. Kinetic profiles of the aggregation of 100 μM αS with 100 μM 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) vesicles in the absence (black) or presence of 1 (green), 2.5 (orange), 5 (red), and 10 (dark red) μM concentrations of TRO (A), SQ (B), αSQ (C), and desSQ (D). Data represent mean ± s.e.m. of three technical replicates.
FIGURE 4
FIGURE 4
Aminosterols modify the aggregation processes of Aβ42 and αS. (A) Kinetic profiles of the aggregation of 2 μM Aβ42 in the absence (black) or presence of an equimolar concentration of each aminosterol (represented with various colors). The bar plot shows the half-time of Aβ42 aggregation in the absence or presence of the aminosterols. (B) Kinetic profiles of the aggregation of 100 μM αS in the absence (black) or presence of 10 μM of each aminosterol (represented with various colors). Since trodusquemine has been characterized not to quench ThT or self-assemble into larger species under these conditions (Perni et al., 2018), the decrease in ThT signal for the aminosterols studied is not likely related to ThT artifacts or assemblies of TRO, such as micelles. The bar plot shows the ThT signal quantified after 30 h of αS aggregation in the absence or presence of the aminosterols. In panels (A–D), data represent mean ± s.e.m. of three technical replicates and are the kinetic traces reproduced from Figures 2, 3. (C) Schematic illustration of the effect of aminosterols (AM) on Aβ42 aggregation, where they enhance monomer-dependent secondary nucleation (k2). (D) Schematic illustration of the effect of aminosterols on αS aggregation, where they inhibit lipid-induced primary nucleation.
FIGURE 5
FIGURE 5
Aminosterols modify the hydrophobicity and size of αS, Aβ40 stabilized by Zn2+, and HypF-N oligomers. (A–F) 8-Anilinonaphthalene-1-sulfonate (ANS) binding measurements to probe the hydrophobicity of oligomers (5 μM in monomer equivalents) of αS (A), Aβ40 (B), and HypF-N (C) with corresponding size changes monitored by turbidity absorbance (D–F) in the absence and presence of a 10-fold excess of SQ, TRO, αSQ, and desSQ (represented in different colors). ANS alone is shown for reference in (A–C) (purple). Data represent mean ± s.e.m. of two technical replicates. Dose-dependent measurements for ANS fluorescence and turbidity absorbance can be found in Supplementary Figures 3–5 ranging from 0 to 50 μM of each aminosterol. A 50 μM concentration of each aminosterol in the absence of oligomers did not noticeably impact ANS fluorescence or turbidity absorbance relative to the effect observed for oligomers and the respective concentrations of each molecule (Supplementary Figures 3–5). (G) The indicated oligomers in the absence and presence of a fivefold excess of SQ visualized using high-resolution, phase-controlled AFM after sample preparation using a microfluidic device (Ruggeri et al., 2018).
FIGURE 6
FIGURE 6
Squalamine suppresses the toxicity associated with Aβ42 oligomeric species to neuroblastoma cells. (A) Aβ-derived diffusible ligands (ADDLs) of Aβ42 (denoted Aβ42 oligomers) were resuspended in cell culture medium at a concentration of 1 μM (in monomer equivalents) and incubated with or without increasing concentrations (0.1, 0.33, 1, 3, and 10 μM) of SQ (red bars) for 1 h at 37°C and then added to the cell culture medium of SH-SY5Y cells for 24 h. The cells were also treated with the same concentrations of SQ pre-incubated in the absence of oligomers for 1 h at 37°C (white bars). (B) Representative confocal scanning microscopy images of the apical planes of cells treated for 15 min with Aβ42 oligomers (1 μM in monomer equivalents) in the absence or presence of 10 μM SQ. Red and green fluorescence indicates the cell membranes and the Aβ42 oligomers, respectively. Scale bar = 10 μm. (C) The histogram shows the percentage of colocalization on regions of interest (12–22 cells). In all panels, data represent mean ± s.e.m. of three independent experiments, the symbols ** and *** indicate p < 0.01 and 0.001, respectively, relative to untreated cells, and the symbol °°° indicates p < 0.001 relative to cells treated with oligomers. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) data were analyzed by one-way ANOVA followed by Bonferroni’s post comparison test. Cell binding data were analyzed using an unpaired, two-tailed Student’s t-test.
FIGURE 7
FIGURE 7
Squalamine reduces the membrane binding affinity and related toxicity of Aβ40 stabilized by Zn2+ and HypF-N oligomers to cultured human neuroblastoma cells. (A) HypF-N oligomers (6 μM in monomer equivalents) were resuspended in cell culture medium in the absence and presence of 0.1, 0.33, 1, and 3 molar equivalents of SQ (blue), incubated (1 h, 37°C), and subsequently added to the cell culture medium of SH-SY5Y cells for 24 h. Cells were treated under the same conditions with 18 μM SQ in the absence of oligomers (white bar). Similarly, Zn2+-stabilized Aβ40 oligomers (5 μM in monomer equivalents) were resuspended in cell culture medium in the absence and presence of a 1:3 ratio of Aβ40-to-SQ (red). (B) Representative confocal scanning microscopy images of the apical sections of SH-SY5Y cells treated for 15 min with HypF-N oligomers (6 μM in monomer equivalents, left panels) and Zn2+-stabilized Aβ40 oligomers (5 μM in monomer equivalents, right panels) in the absence and presence of a 1:3 ratio of oligomers-to-SQ. Red and green fluorescence indicates the cell membranes and the oligomers, respectively. Scale bar = 10 μm. (C) Histograms show the percentage of colocalization between membranes and oligomers in the regions of interest (12–22 cells in total). In all panels, data represent mean ± s.e.m. of three independent experiments, the symbols * and *** indicate p < 0.05 and 0.001, respectively, relative to untreated cells, and the symbols °, °°, and °°° indicate p < 0.05, 0.01, and 0.001, respectively, relative to cells treated with oligomers. MTT data were analyzed by one-way ANOVA followed by Bonferroni’s post comparison test. Cell binding data were analyzed using an unpaired, two-tailed Student’s t-test.
FIGURE 8
FIGURE 8
Schematic representation of the effect of aminosterols on isolated or stabilized oligomers of αS, Aβ40 stabilized by Zn2+, and HypF-N. After oligomer isolation or stabilization, the addition of aminosterols (AM) to the reaction mixture can induce the non-physiological clustering of the aggregates at high concentrations (shown as gray species), and the physiologically relevant displacement of oligomers from cell membranes at low concentrations (toxic oligomers are shown as red species). For the latter scenario, trodusquemine exhibited comparable effects with squalamine at 3–10× lower concentrations of the molecule, signaling its enhanced efficacy at displacing protein misfolded oligomers from cell membranes.

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