Impact of Amyloid-β on Platelet Mitochondrial Function and Platelet-Mediated Amyloid Aggregation in Alzheimer's Disease

Abstract

Background: Alzheimer's disease (AD) is characterized by an accumulation of amyloid β (Aβ) peptides in the brain and mitochondrial dysfunction. Platelet activation is enhanced in AD and platelets contribute to AD pathology by their ability to facilitate soluble Aβ to form Aβ aggregates. Thus, anti-platelet therapy reduces the formation of cerebral amyloid angiopathy in AD transgenic mice. Platelet mitochondrial dysfunction plays a regulatory role in thrombotic response, but its significance in AD is unknown and explored herein.

Methods: The effects of Aβ-mediated mitochondrial dysfunction in platelets were investigated in vitro.

Results: Aβ40 stimulation of human platelets led to elevated reactive oxygen species (ROS) and superoxide production, while reduced mitochondrial membrane potential and oxygen consumption rate. Enhanced mitochondrial dysfunction triggered platelet-mediated Aβ40 aggregate formation through GPVI-mediated ROS production, leading to enhanced integrin αII_bβ₃ activation during synergistic stimulation from ADP and Aβ40. Aβ40 aggregate formation of human and murine (APP23) platelets were comparable to controls and could be reduced by the antioxidant vitamin C.

Conclusions: Mitochondrial dysfunction contributes to platelet-mediated Aβ aggregate formation and might be a promising target to limit platelet activation exaggerated pathological manifestations in AD.

Keywords: Alzheimer’s disease; Aβ aggregation; GPVI; ROS; cerebral amyloid angiopathy; integrin; mitochondria dysfunction; platelets.

Figure 1

Impact of Aβ on mitochondrial functions in platelets. (A) Washed human platelets were incubated for 24 h with 11.5 µM Aβ40 and 11.5 µM Aβ1-16 (as control). Generation of ROS was reported as mean fluorescence intensity of DCF (n = 3). (B) MitoSOXRed™-loaded platelets were incubated with 5 µM Aβ40 and 5 µM Aβ1-16 for 30 min and the generation of superoxide was measured as mean fluorescence intensity (n = 3). (C) Depolarization of the platelet mitochondrial membrane upon 5 µM Aβ40 and 5 µM Aβ1-16 was observed by decreased TMRM fluorescence intensity (n = 3). (D) The mitochondrial dye, MitoTrackerTM green FM, was added to platelets to determine the release of mitochondria upon Aβ40 stimulation (n = 4–5). Compared to control (resting). (A–D) All samples were measured by flow cytometry. Bar graphs depict mean values ± SEM. All analyses were performed using one-way ANOVA and Dunnett’s multiple comparisons post-hoc test. ** p< 0.01; * p< 0.05. n.s.: not significant.

Figure 2

Determination of oxygen consumption rate (OCR) in human platelets upon treatment with Aβ40. (A) Determination of the oxygen consumption rate (OCR) after injection of indicated chemicals at indicated time points using a Seahorse XF24 analyzer. (B) Basal respiration, (C) respiration after addition of collagen-related peptide (CRP) or vehicle (medium), (D) ATP-linked respiration, (E) maximal respiration, (F) proton leak and (G) non-mitochondrial respiration. (A–G) Data represent mean ± SEM from n = 5–7 donors, two-way ANOVA with Holm-Sidak’s multiple comparisons test, **** = p < 0.0001; ** = p < 0.01; * = p < 0.05. w/o: Without; n.s.: Not significant.

Figure 3

Expression levels of mitochondrial proteins in platelets upon Aβ40 stimulation. (A) Human platelets were stimulated with 5 or 20 µM Aβ40 or 5 µg/mL CRP for 2 h. Using Western blot analysis, the expression levels of mitochondrial proteins were detected as indicated. β-actin served as loading control. (B,C) The intensity of bands was analyzed with ImageJ software. Data represent mean value ± SEM (n = 4). All analyses were performed using one-way ANOVA and Dunnett’s multiple comparisons post-hoc test. * = p < 0.05.

Figure 4

Effects of antimycin A and the antioxidant vitamin C on platelet-mediated Aβ aggregate formation. (A) Representative images of congo red-stained Aβ deposits in platelet culture. Platelets were incubated with 5 µM of soluble synthetic Aβ40 and different concentrations of antimycin A for three days at 37 °C and 5% CO₂. EtOH served as the control (vehicle). Scale bar, 50 µm. (B) Quantification of remaining soluble Aβ40 in the supernatant of platelet culture using Western blot. (C) Isolated platelets were incubated in the absence or presence of antimycin A (500 ng/mL) for 24 h at 37 °C. EtOH (0.0015%) served as solvent control for antimycin A. Aβ40 levels were determined using ELISA (n = 4). (D) Platelet culture after incubation with soluble, synthetic Aβ40 for three days. Where indicated, platelets were incubated with antimycin A (500 ng/mL) or antimycin A (500 ng/mL) and different concentrations of vitamin C (100 µM or 1 mM). EtOH served as control (vehicle). Scale bar, 50 µm. (E) Quantification of remaining soluble Aβ40 in the supernatant of platelet culture using Western blot (n = 3).

Figure 5

Impact of complex III inhibition on platelet functions following Aβ40 stimulation. (A) Detection of intracellular ATP levels after incubation of platelets with Aβ40 (5 µM), antimycin A (12.5 µM) and EtOH (as control, vehicle) for 90 min using Luminescence intensity (n = 4, *** = p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test). (B) Measurement of ATP release from antimycin A-pretreated platelets (EtOH was used in controls as vehicle) following Aβ40 (20 µM) or CRP (1 µg/mL) (n = 5–6). Analyses were performed using one-way ANOVA and Dunnett’s multiple comparisons post-hoc test. ** p < 0.01; n.s.: not significant. (C) Aggregation of antimycin A-pretreated platelets upon Aβ40 (10 µM) or CRP (1µg/mL). EtOH was used as control (vehicle) (n = 5–6). (D) Measurement of reactive oxygen species (ROS) generation with DCF-DA in GPVI-deficient platelets upon Aβ40 and CRP (n = 6). Data represent mean value ± SEM; two-way ANOVA with Sidak’s multiple comparisons test. *** p < 0.001; ** p < 0.01; * p < 0.05. (E) Flow cytometric analysis of integrin activation at the surface of platelets using PAC-1 antibody upon stimulation with Aβ40 (11.5 µM) and ADP (5µM). Where indicated, samples were pre-incubated with vitamin C (1 mM) for 30 min at RT (n = 4). Data represent mean value ± SEM; two-way ANOVA with Tukey’s multiple comparisons test. *** p < 0.001; ** p < 0.002.

Figure 6

Analysis of mitochondrial function in platelets from APP23 mice. (A,B) Platelets from one- and two-year-old WT and APP23 mice were incubated with 5 and 20 µM Aβ40 for 30 min. Formation of superoxide was examined using MitoSOX™ Red and depolarization of platelet mitochondrial membrane was observed by decrease in TMRM fluorescence intensity. Data show the mean value ± SEM (n = 3–4), two-way ANOVA with Tukey’s multiple comparisons test. *** p < 0.001; ** p < 0.01; * p < 0.05; vs. basal or as indicated. (C) Expression levels of mitochondrial proteins in platelets from one- and two-year-old WT and APP23 mice were determined by Western blot analysis. β-actin served as loading control (n = 4). (D) Western blot analysis of the mitochondrial protein OPA1 in platelets from two-year-old WT and APP23 mice after stimulation with Aβ40 and CRP. β-actin served as loading control (n = 3). (E) Representative images of congo-red stained amyloid aggregates in the culture of platelets after incubation with soluble, synthetic Aβ40 (5 µM) for three days in the presence or absence of antimycin A (500 ng/mL) and vitamin C (100 µM). Platelets from one-year-old WT and APP23 mice were used (n = 3). Scale bar, 50 µm.