Under hyperglycemic conditions, SCO cells secrete WNT5a, which interacts with ependymal cell cilia.

(A) Immunohistochemical staining of Wnt5a (red) and SCO-Spondin (green) in frontal brain sections from normoglycemic animals. Focal Wnt5a immunoreactivity was detected most in the basal area of the cells. N = 3. Ependymal cells were negative for Wnt5a and SCO-spondin. Scale bar: 20 μm. (B) Immunohistochemical staining of Wnt5a (red) and SCO-spondin (green) in frontal brain sections from hyperglycemic animals (CSF glucose concentration of 5 mM glucose). Wnt5a immunoreactivity was mainly observed in the apical part of the cells (yellow arrows). N = 3. Immunoreactivity was also observed by DIC microscopy (Bˋ). Ependymal cell cilia were positive for Wnt5a and very weakly positive for SCO-spondin (B`). Scale bar: 20 μm. (C) Immunohistochemical staining of Wnt5a (red) and SCO-spondin (green) in frontal brain sections from hyperglycemic animals (CSF glucose concentration of 10 mM). Wnt5a immunoreactivity was observed mainly in the apical area of the cells. N = 3. Ependymal cell cilia were strongly positive for Wnt5a (Cˋ). Immunoreactivity was also observed by DIC microscopy. Ependymal cell cilia were strongly positive for Wnt5a and SCO-spondin (Cˋˋ). Scale bar: 20 μm. (D) Immunofluorescence staining of SCO-spondin and Wnt5a in ependymal cells cilia. Increased colocalization was observed (white arrows). Scale bar: 20 μm. (E) No immunoreactivity was detected when the primary antibody was omitted. Scale bar: 20 μm. (F) Quantitative analysis of Wnt5a immunoreactivity under different glycemic conditions. The graph shows data from 4 biologically independent samples. The error bars represent the SD; ***P t test). (G) Quantification of Mander’s overlap coefficient for SCO-spondin and Wnt5a in SCO cells and ependymal cell cilia under different glycemic conditions. The graph shows data from 4 biologically independent samples (Normo in ependymal cells, N = 3). The error bars represent the SD; ***P t test). (H) TEM analysis. Ependymal cells with aggregated secretions in the apex of cilia. Exosome-like vesicles were also detected (arrowheads and inset). Scale bars: H, 0.3 μm; I, inset, 0.08 μm. Data used to generate graphs can be found in S1 Data. CSF, cerebrospinal fluid; DIC, differential interference contrast; d3v, dorsal third ventricle; SCO, subcommissural organ; TEM, transmission electron microscopy.