The formation of cytoplasmic inclusions in neuronal and oligodendroglial cells exposed to α -synuclein. A, Large perikaryal inclusions as well as small aggregates were observed in the SH-SY5Y (a) and KG1C (b) cells exposed to 5 μM recombinant αSYN for 24 hours. The αSYN-positive inclusions and small aggregates were positive for ubiquitin and thioflavin S staining, whereas only the large inclusions co-immunostained positively for phosphorylated serine 129-αSYN. Neither αSYN-positive inclusions nor small aggregates were detected in cells treated with the column flow-through. The crossed lines indicate the positions of the xz- and yz-planes. B, Double-immunolabeling studies demonstrated that the juxtanuclear large inclusions co-localized with γ-tubulin, peripherin, and/or vimentin, which are known markers of the aggresome (a and b). The αSYN-positive inclusion bodies in the KG1C cells (b) were co-localized with TPPP/p25α, a known marker for GCI in MSA. C, The formation of αSYN-positive cytoplasmic inclusions (arrowhead) was also confirmed in primary rat cortical neurons treated under the same condition. No visible αSYN-positive inclusions were detected in the mock-treated cells. D, After αSYN exposure, the incidence of perinuclear inclusions in both the SH-SY5Y and KG1C cells increased in a time-dependent manner, reaching 35% and 24%, respectively, at 24 hours. Data are expressed as the mean ± standard errors. The immunostaining was performed three times and exhibited consistent results. Scale bar: 10.0 μm.