FIGURE 2.

Cell-to-cell transmission of α-synuclein to primary astrocytes in co-culture. A and B, primary astrocytes were co-cultured with differentiated SH-SY5Y cells overexpressing α-synuclein. Serial images through z axis were obtained and analyzed to show that transmitted α-synuclein (green) reside inside of astrocytes (red: GFAP). Areas in the white boxes are enlarged (left panels, xy images; right panels, z-stacked images seen through the y axis). C, transfer of neuronal endogenous α-synuclein to astrocytes. Rat primary cortical neurons were co-cultured with astrocytes. Note that the α-synuclein polyclonal antibody used in this experiment also recognizes rat sequences. Top panels: primary astrocytes cultured with neurons. Bottom panels: primary astrocytes only. White perforated lines indicate outlines of astrocyte periphery. D, primary astrocytes were cultured with differentiated SH-SY5Y cells overexpressing α-synuclein and fixed at the indicated times. The control indicates a co-culture with SH-SY5Y cells without α-synuclein overexpression. Arrows: perinuclear inclusions. E, quantitative analyses of α-synuclein transfer (left graph) and glial inclusion formation (right graph). Differentiated SH-SY5Y cells overexpressing human α-synuclein were co-cultured with rat primary astrocytes. The transfer was measured by fluorescence intensities of α-synuclein staining in astrocytes, and the inclusion formation was measured by the percentage of astrocytes with inclusions among the cells with transferred α-synuclein. Experiments were repeated four times, and more than hundred cells were counted per slide. All scale bars, 20 μm (*, p < 0.05; **, p < 0.01; ***, p < 0.001).