Extracellular Vesicles Slow Down Aβ(1-42) Aggregation by Interfering with the Amyloid Fibril Elongation Step

Abstract

Formation of amyloid-β (Aβ) fibrils is a central pathogenic feature of Alzheimer's disease. Cell-secreted extracellular vesicles (EVs) have been suggested as disease modulators, although their exact roles and relations to Aβ pathology remain unclear. We combined kinetics assays and biophysical analyses to explore how small (<220 nm) EVs from neuronal and non-neuronal human cell lines affected the aggregation of the disease-associated Aβ variant Aβ(1-42) into amyloid fibrils. Using thioflavin-T monitored kinetics and seeding assays, we found that EVs reduced Aβ(1-42) aggregation by inhibiting fibril elongation. Morphological analyses revealed this to result in the formation of short fibril fragments with increased thicknesses and less apparent twists. We suggest that EVs may have protective roles by reducing Aβ(1-42) amyloid loads, but also note that the formation of small amyloid fragments could be problematic from a neurotoxicity perspective. EVs may therefore have double-edged roles in the regulation of Aβ pathology in Alzheimer's disease.

Keywords: Alzheimer’s disease; EVs; amyloid kinetics; amyloid-β; extracellular vesicles; protein aggregation.

Figure 1

Size and molecular identity of EVs. (a) Size distributions and particle concentrations of EVs isolated from SH-SY5Y (black) and HEK293-T (blue), as determined by NTA. Mean EV diameters ± standard deviations are given in the legend. (b) Western blots showing the presence of different protein markers in the EV samples and whole cell lysates. Abbreviations: SH = SH-SY5Y and HEK = HEK293-T.

Figure 2

Aβ(1–42) aggregation kinetics in the presence of EVs. (a,b) Change in ThT fluorescence as a function of time, representing the aggregation kinetics of 2 μM Aβ(1–42) into amyloid fibrils in the presence of increasing concentrations of EVs purified from (a) SH-SY5Y and (b) HEK293-T cells. The EV concentrations are given in particles/mL, as indicated by the legend in (a). Three replicate kinetic curves are overlaid for each condition. (c) Change in end-point ThT fluorescence (defined as the mean ThT signal over the final 3 h of the plateau phase, which corresponds to 37 data points) as a function of increasing EV concentration. (d) Reaction half-times and (e) reaction growth-times, extracted from the data in (a,b). The error bars represent standard deviation (n = 3).

Figure 3

Effect of EVs on the seeded aggregation of Aβ(1–42). (a–f) Normalized Aβ(1–42) aggregation kinetic curves showing the effects of EVs in the absence (a,d) and presence (b,c and e,f) of 5 or 25% preformed Aβ(1–42) fibril seeds. Panels (a–c) and (d–f) show data for SH-SY5Y and HEK293-T EVs, respectively. The solid lines were fitted to the data using a multistep secondary nucleation model of amyloid formation setting the rate constant for elongation (k₊) as a free parameter as described in the main text. The parameters underlying these fits are given in Tables S4 and S5. (g,h) Reaction half-times as a function of EV and seed concentration, derived from the data in, respectively, (a–c and d–f). The error bars represent the standard deviation (n = 3). (i) Change in the elongation rate constant (k₊) as a function of EV concentration, as determined by the fitting of the data in a–f. The elongation rates are reported relative to that of 2 μM Aβ(1–42) aggregating in the absence of EVs.

Figure 4

Morphological characterization of Aβ(1–42) fibrils formed in the absence and presence of EVs. (a–c) AFM images of Aβ(1–42) fibrils formed (a) in phosphate buffer with DPBS (see Methods) and (b,c) in the presence of SH-SY5Y and HEK293-T EVs. Scale bars = 2 μm. (d,e) AFM-based analysis of the distributions of (d) fibril lengths (e) and cross-sectional heights of the Aβ(1–42) fibrils formed in the absence and presence of EVs (n = 100–120 per condition, *** denotes p < 0.001 by one-way ANOVA). (f–k) Cryo-TEM images of Aβ(1–42) fibrils formed in the absence of EVs (f,g) and in the presence of EVs from, respectively, SH-SY5Y(h,i) and HEK293-T (j,k) cells. The Aβ(1–42) fibrils formed in the presence of EVs contained small dark dots, indicated by the white arrows in (i) and (k), suggestive of the dense association of EV components. Scale bars = 250 nm. All analyses have an EV concentration of 7.2 × 10⁹ particles/mL.