FIGURE 4.
Amyloid peptide triggers secretion of PAR-4 and ceramide in lipid vesicles that induce apoptosis in astrocytes. A, wild type astrocytes were incubated with 20 μm Aβ25–35 for 48 h in serum-free medium, and secreted vesicles were harvested from the medium by ultracentrifugation (UC). Lane 1, cell lysate; lane 2, medium control (Aβ35–25); lane 3, Aβ25–35-treated astrocytes; lane 4, marker proteins (not stained); lane 5, UC pellet from control astrocytes; lane 6, Aβ-treated astrocytes. B, exosomes harvested by centrifugation with the ExoQuick reagent. Lane 1, control (Aβ35–25); lane 2, Aβ25–35-treated astrocytes. Alix, Alix1. C, UC pellets from wild type and nSMase2-deficient fro/fro astrocytes. Lane 1, cell lysate; lane 2, marker proteins (not stained); lane 3, Aβ35–25-treated fro/fro astrocytes; lane 4, Aβ25–35-treated fro/fro; lanes 5 and 6, UC pellets from wild type astrocytes. D, medium from fro/fro astrocytes treated with C16 and C18 ceramide (Cer) with or without Aβ25–35. Lane 1, cell lysate; lane 2, control: lane 3, C16 + C18 ceramide; lane 4, amyloid peptide; lane 5, amyloid peptide + ceramide. E, as described for D, but exosomes were isolated using ExoQuick reagent and a combination of Aβ25–35 with either C16 (lane 5) or C18 ceramide (lane 6). F, co-immunoprecipitation (IP) assay using lipid vesicles resuspended from the UC pellet of medium conditioned by Aβ25–35-treated wild type astrocytes. Vesicles were subjected to magnetic activated cell sorting, and the flow-through fraction was analyzed by immunoblotting. G, sphingolipidomic analysis of lipid vesicles in the UC pellet wild type astrocytes without or with Aβ25–35 incubation. n = 2; p < 0.05 (*); p < 0.01 (**).