Hypochlorite-induced oxidation promotes aggregation and reduces toxicity of amyloid beta 1-42

Abstract

Exacerbated hypochlorite (OCl^-) production is linked to neurodegenerative processes, but there is growing evidence that lower levels of hypochlorite activity are important to protein homeostasis. In this study we characterise the effects of hypochlorite on the aggregation and toxicity of amyloid beta peptide 1-42 (Aβ_1-42), a major component of amyloid plaques that form in the brain in Alzheimer's disease. Our results demonstrate that treatment with hypochlorite promotes the formation of Aβ_1-42 assemblies ≥100 kDa that have reduced surface exposed hydrophobicity compared to the untreated peptide. This effect is the result of the oxidation of Aβ_1-42 at a single site as determined by mass spectrometry analysis. Although treatment with hypochlorite promotes the aggregation of Aβ_1-42, the solubility of the peptide is enhanced and amyloid fibril formation is inhibited as assessed by filter trap assay, thioflavin T assay and transmission electron microscopy. The results of in vitro assays using SH-SY5Y neuroblastoma cells show that pre-treatment of Aβ_1-42 with a sub-stoichiometric amount of hypochlorite substantially reduces its toxicity. The results of flow cytometry analysis and internalisation assays indicate that hypochlorite-induced modification of Aβ_1-42 reduces its toxicity via at least two-distinct mechanism, reducing the total binding of Aβ_1-42 to the surface of cells and facilitating the cell surface clearance of Aβ_1-42 to lysosomes. Our data is consistent with a model in which tightly regulated production of hypochlorite in the brain is protective against Aβ-induced toxicity.

Keywords: Alzheimer's Disease; Renewable energy; inflammation; oxidative stress; post-translational modification; protein misfolding.

Graphical abstract

Fig. 1

Effect of NaOCl on the solubility and surface hydrophobicity of Aβ_1-42. (A) Western blot image showing Aβ_1-42 following treatment with NaOCl in PBS overnight at ambient room temperature. The molar ratio of NaOCl to Aβ is indicated for each sample. Aβ_1-42 samples were separated on a 10–20% tris-tricine gel and transferred to PVDF membrane. The membrane was probed using monoclonal anti-Aβ_1-42 (WO2) and a relevant secondary antibody before visualisation by enhanced chemiluminescence. (B) (i) Western blot image showing Aβ_1-42 retained on a cellulose acetate membrane after filter trap assay, following treatment of Aβ_1-42 with NaOCl as described in (A) (ii) Corresponding chart shows the relative density of Aβ_1-42. The data points are means (n = 3; ±SD) **p < 0.01; ****p < 0.0001; One-way ANOVA, Tukey's test). (C) Bis-ANS fluorescence (Excitation = 360 nm, Emission = 502 nm) of Aβ_1-42 samples after treatment with NaOCl as in (A). The data are the mean (n = 10; ±SD). **** Control samples are Aβ_1-42 incubated as described, but in PBS alone.

Fig. 2

Effect of NaOCl on Aβ_1-42amyloid fibril formation, as assessed by ThT assay and TEM (A) Graph shows the ThT fluorescence of 5 μM Aβ_1-42 incubated in the presence or absence of NaOCl in PBS at 28 °C with orbital shaking in a Clariostar platereader. All samples contained 25 μM ThT. Data is the mean (n = 4; ±SD) of the background adjusted ThT fluorescence. (B) Graph shows the ThT fluorescence of 4 μM Aβ_1-42, pre-treated in the presence or absence of NaOCl in PBS for 1 h at RT and then supplemented with excess L-Met. Aβ_1-42 was then incubated in the presence of 25 μM ThT at 37 °C with orbital shaking in Clariostar platereader overnight. Data is mean (n = 3; ±SD) of the background adjusted ThT fluorescence. (C) Chart shows the half-time (t_1/2) values for ThT fluorescence for samples shown in (B). Data is mean (n = 3; ±SD) ****p < 0.0001, unpaired t-test. (D) Corresponding TEM images for samples taken at the end point of the assay shown in (B).

Fig. 3

Effect of NaOCl on Aβ_1-42aggregation, as assessed by size exclusion chromatography. Chromatograms show the elution of Aβ_1-42 separated on a Superdex 75 Increase 3.2/300 column at a flow rate of 0.05 ml/min in PBS as measured using the absorbance at 280 nm. Black line shows the elution of Aβ_1-42 in PBS after 1 h incubation at ambient room temperature. Grey line shows the elution of Aβ_1-42 after treatment with a 2-fold molar excess of NaOCl in PBS for 1 h at ambient room temperature.

Fig. 4

Effect of NaOCl on the oxidation of Aβ_1-42. (A) Maximum entropy mass spectrum of (i) control Aβ_1-42 or (ii) Aβ_1-42 incubated with a 5.2-fold molar excess of NaOCl in PBS for 1 h at 4 °C. Masses are shown in daltons. (B) Chart shows the amount of unmodified Aβ_1-42 (4515 Da) remaining after treatment with NaOCl in PBS for 1 h at 4 °C. The molar ratio of [NaOCl]:[Aβ] used is indicated. Data is expressed relative to a control sample that was incubated in the absence of NaOCl.

Fig. 5

Effect of NaOCl on the toxicity of Aβ_1-42against SH-SY5Y neuroblastoma cellsin vitro. (A) Chart shows the percent cell viability of SH-SY5Y cells as measured by MTS assay following treatment with 10 μM Aβ_1-42 or vehicle (PBS) for 48 h. Aβ_1-42 was pre-incubated in Ham's/F12 containing NaOCl at the molar ratios indicated for 4 days at 4 °C and the reaction quenched using excess l-methionine (L-Met) prior to the assay. The data are the mean cell viability (n = 3; ±SD) and are expressed relative to the vehicle control **p < 0.01; ***p < 0.001; One-way ANOVA, Tukey's test. (B) Graph shows the green object count, indicative of dead cells, as measured using an Incucyte live cell imager. Cells were cultured in the presence of 15 μM Aβ_1-42 or vehicle (PBS). Aβ_1-42 (100 μM) was pre-incubated in Ham's/F12 ± 80 μM NaOCl for 2 h at ambient room temperature prior to the assay. (C) Representative images taken at 72 h for the assay shown in (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Effect of NaOCl on the cell-surface binding and internalisation of Aβ_1-42in vitro. (A) Chart shows the Alexa fluor 488 fluorescence of SH-SY5Y cells following incubation with with 5 μM biotinylated Aβ_1-42. Prior to incubation with the cells, biotinylated Aβ_1-42 was pre-incubated with 0–160 μM NaOCl in PBS at ambient temperature overnight. Biotinylated Aβ_1-42 monomer was obtained directly from frozen storage. Aβ_1-42 is the control incubated in PBS in the absence of NaOCl. The molar ratio of NaOCl to Aβ in the remaining samples is indicated on the chart. The data are means (n = 3; ±SD) ****p < 0.0001; One-way ANOVA, Tukey's test). Asterisks denotes significant difference between samples and control. B(i) Representative confocal images of SH-SY5Y cells following incubation with Aβ_1-42-Hilyte-488 (green), Lysotracker Red DND-99 (red) and Hoechst 33452 (blue). Aβ_1-42-Hilyte-488 (50 μM) was pre-incubated ± 40 μM NaOCl in Ham's/F12 for 24 h at RT. SH-SY5Y cells were then cultured in the presence of Aβ_1-42-Hilyte-488 (2.5 μM) for 24 h using standard culture conditions. Cells were stained with Lysotracker Red DND-99 and Hoechst 33452 for 30 min at 37 °C, prior to being fixed and imaged using a Zeiss LSM880 confocal microscope. Scale bar is 20 μm. (ii) The overlap coefficient between Aβ (green) in the lysotracker stained vesicle (red) was analyzed using the ZEN Black software and the Mander's coefficient calculated. The data is mean (n = 5; ±SD). *p < 0.05; unpaired student's t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Effect of oxidizing system on Aβ_1-42neurotoxicity and internalisationin vitro. (A) Chart shows the percent cell viability of SH-SY5Y cells as measured by MTS assay following 48 h treatment with 20 μM Aβ_1-42. Aβ_1-42 was pre-incubated with NaOCl or copper and H₂O₂ in Ham's F12 at 4 °C for 4 days prior to the assay. The data are means (n = 3; ±SD) *p < 0.05; One-way ANOVA, Tukey's test). (B) Representative images of SH-SY5Y cells following incubation with 1 μM Aβ_1-42-Hilyte-488, pre-treated with (i) NaOCl and (ii) copper as in (A) and 100 nM Lysotracker Red DND-99 at 37 °C for 1 h (iii) Chart shows quantified object area of each fluorescent signals and CoA normalised to total cell area in SH-SY5Y cells following treatment with NaOCl-treated Aβ_1-42. Corresponding images are in (i). (iv) Chart shows quantified object area of each fluorescent signals and CoA normalised to total cell area in SH-SY5Y cells following treatment with copper-treated Aβ_1-42. Corresponding images are in (ii). (v) Chart shows the relative CoA of the green and red fluorescent signals shown in (iii) and (iv). The colocalization of Aβ_1-42 (green) in the lysosomes (red) were calculated as the colocalization area (CoA) of the green and red signals. The data are means (n = 27 areas from three wells; ±SD) ****p < 0.05; unpaired student's t-test). Asterisks denotes significant difference between NaOCl and Cu²⁺/H₂O₂. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)