A Brain-Permeable Aminosterol Regulates Cell Membranes to Mitigate the Toxicity of Diverse Pore-Forming Agents

Abstract

The molecular composition of the plasma membrane plays a key role in mediating the susceptibility of cells to perturbations induced by toxic molecules. The pharmacological regulation of the properties of the cell membrane has therefore the potential to enhance cellular resilience to a wide variety of chemical and biological compounds. In this study, we investigate the ability of claramine, a blood-brain barrier permeable small molecule in the aminosterol class, to neutralize the toxicity of acute biological threat agents, including melittin from honeybee venom and α-hemolysin from Staphylococcus aureus. Our results show that claramine neutralizes the toxicity of these pore-forming agents by preventing their interactions with cell membranes without perturbing their structures in a detectable manner. We thus demonstrate that the exogenous administration of an aminosterol can tune the properties of lipid membranes and protect cells from diverse biotoxins, including not just misfolded protein oligomers as previously shown but also biological protein-based toxins. Our results indicate that the investigation of regulators of the physicochemical properties of cell membranes offers novel opportunities to develop countermeasures against an extensive set of cytotoxic effects associated with cell membrane disruption.

Keywords: aminosterols; biotoxin neutralization; cell membranes; cellular resistance; pore-forming agents; steroid polyamines.

Figure 1

Cytotoxicity induced by the pore-forming peptide melittin is attenuated by claramine. (A) Structure of claramine. (B) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays after cells were exposed to 2 μM melittin (MEL) in the absence (red bar) or presence of increasing concentrations of claramine (CL, blue bars) for 20 h. Cells were also exposed to 10 μM claramine alone (gray bar). The physiological range of claramine under these conditions is shown in Figure S1A. n = 60,000 cells per condition corresponding to the six technical replicates shown. Conditions were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test relative to untreated cells or cells treated with melittin, as indicated. Untreated cells and cells treated with 10 μM claramine were analyzed by an unpaired, two-tailed Student’s t-test. Data are representative of n = 3 biologically independent experiments. (C) To study the acute effects of melittin treatment, 0.1 μM melittin was incubated with cells for 5 min in the absence or presence of increasing concentrations of claramine (0.01–10 μM). Cells were also treated with 10 μM claramine in the absence of melittin. The fluorescence of the 6-chloromethyl-2′-7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) general oxidative stress indicator was used to measure the extent of reactive oxygen species (ROS) production in various conditions. A superimposition of 1.0 μm thick sections spanning the height of the entire cell was compiled to generate the shown representative images. Scale bars, 10 μm. Enhanced contrast and brightness images can be seen in Figure S9, which show clearly all cells, including those with a low fluoresce signal. (D) Corresponding semiquantitative values of green fluorescence. All samples were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to untreated cells. Samples containing melittin and claramine were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test relative cells treated with melittin alone. Bars indicate mean ± standard error of the mean (s.e.m.) of at least 130 cells per condition.

Figure 2

The physicochemical properties of hydrophobicity and size for melittin are not changed by claramine. (A) CD spectroscopy measurements for 10 μM melittin in the absence (red trace) and presence of up to 30 μM claramine (CL, blue traces). Smoothed data are shown. (B) BeStSel-quantified secondary structures for the traces shown in (A). Statistically significant differences were not observed for the samples containing claramine relative to melittin alone (P > 0.999 by two-way ANOVA, main row effect). (C) 10 μM melittin was incubated with up to 30 μM concentrations of claramine, after which time 30 μM 8-anilino-1-naphthalenesulfonic acid (ANS) was added to probe the solvent-exposed hydrophobicity of melittin. Free ANS is shown for reference (gray). Error bars indicate the s.e.m. of duplicate technical replicates. Data shown are representative of n = 2 independent experiments. (D) Turbidity absorbance measurements for 10 μM melittin incubated with up to 30 μM claramine for the samples shown in (C). (E) Melittin was incubated in the absence and presence of a 5-fold excess of claramine and measured using atomic force microscopy (AFM). Scale bars, 500 nm. (F) Representative cross-sectional heights are shown (red, melittin; blue, melittin + claramine; n = 500 per condition), as indicated in the color plots of (E). (G) Quantification of the entire sample population. Line and error bars represent mean ± 1 standard deviation. Data were analyzed using an unpaired, two-tailed Student’s t-test. In (A)–(F), all samples were prepared in 20 mM sodium phosphate buffer at pH 7.4.

Figure 3

Cytotoxicity induced by the pore-forming peptide α-hemolysin is attenuated by claramine. (A) MTT viability assays after cells were exposed to 50 μg/mL α-hemolysin (α-HEM) in the absence (red) or presence of increasing concentrations of claramine (CL, blue bars) for 20 h. Cells were also exposed to 10 μM claramine alone (gray). n = 60,000 cells per condition corresponding to the shown six technical replicates. Conditions were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test relative cells treated with α-hemolysin or Student’s t-test, as indicated. Data shown are representative of n = 3 biologically independent experiments. (B) 50 μg/mL α-hemolysin was incubated with cells for 1 min in the absence or presence of 10 μM claramine. The fluorescence of the CM-H₂DCFDA general oxidative stress indicator was used to measure the extent of ROS production. A superimposition of 1.0 μm thick sections spanning the height of the entire cell was compiled to generate the shown representative images. Scale bars, 10 μm. Enhanced contrast and brightness images can be seen in Figure S9, which show clearly all cells, including those with a low fluoresce signal. (C) Corresponding semiquantitative values of green fluorescence. Conditions were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test relative cells treated with α-hemolysin or Student’s t-test, as indicated. Bars indicate mean ± s.e.m. of at least 130 cells per condition.

Figure 4

Claramine attenuates melittin binding to cell membranes. SH-SY5Y cells were treated for 5 min with 0.2 μM melittin (MEL) in the absence (red bar) or presence of 0.1, 1.0, or 10 μM claramine (CL, blue bars). Untreated cells exposed only to cell culture media are shown for comparison (black bar). Red and green fluorescence correspond to the cell membrane labeled with wheat germ agglutinin (WGA) and the Alexa 488-labeled melittin, respectively. The bar plot shows the colocalization of melittin with the cell membrane. Scale bars, 10 μm. All samples were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to untreated cells. Samples containing melittin and claramine were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test relative cells treated with melittin alone. Bars indicate mean ± s.e.m. of at least 200 cells per condition. Data shown are representative of n = 3 biologically independent experiments.

Figure 5

Claramine also reduces α-hemolysin binding to cell membranes. SH-SY5Y cells were treated for 15 min with 5 μg/mL α-hemolysin (α-HEM) in the absence (red bar) or presence of 0.1 or 10 μM claramine (CL, blue bars). Untreated cells exposed only to cell culture media are shown for comparison (black bar). Red and green fluorescence correspond to the cell membrane labeled with wheat germ agglutinin (WGA) and the α-hemolysin protein, respectively. Scale bars, 10 μm. The bar plot shows the colocalization of α-hemolysin with the cell membrane. All samples were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to untreated cells. Samples containing α-hemolysin and claramine were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test relative cells treated with α-hemolysin alone. Bars indicate mean ± s.e.m. of at least 300 cells per condition. Data shown are representative of n = 2 biologically independent experiments.

Figure 6

Schematic for the mechanism of action by which the folded toxins melittin (A) and α-hemolysin (B) disrupt and create pores in the cell membranes. The illustration shows how claramine (green) incorporates into the cell membrane to prevent the pore-forming toxins from docking, analogous to the case for protein misfolded oligomers observed in neurodegenerative diseases.^,,,, The graphic was generated from the knowledge that aminosterols localize within the hydrophilic region of the lipid bilayer and extend to the interface between the hydrophilic and hydrophobic regions with a well-defined oblique angle (about 55°) for the major axis of the molecule with respect to the normal to the bilayer plane and with superficial positioning of its positively charged spermine tail. As a result, the membrane becomes less negatively charged, and it causes a redistribution of cholesterol and ganglioside GM1 molecules and acquires resistance to indentation.