Fig. 2: OCN–GPR158 coupling system requires core PC proteins to induce autophagy machinery in hippocampal neurons.
From: A primary cilia–autophagy axis in hippocampal neurons is essential to maintain cognitive resilience
a, Western blot and quantification analysis of TULP3, KIF3A, IFT20, IFT88 and LCB-I/II protein levels. b, Top panels are representative fluorescent images of brain cross-sections 3 weeks after injections with AAV-eSYN-shRNAmir-scramble expressing eGFP. Left panel is a panoramic view; right panels are focus views on the DG, CA3 and cornu ammonis 1 (CA1) regions, respectively. Scale bar, 100 µm. Top right panel is the relative expression of Ift20 mRNA level in the dorsal hippocampus of mice 3 weeks after injection with AAV-eSYN-shRNA-scramble-mir or AAV-eSYN-shRNAmir-Ift20. Bottom panels represent western blot 3 weeks after hippocampal injections with AAV-eSYN-shRNA-scramble-mir or AAV-eSYN-shRNAmir-Ift20 and respective quantification of IFT20 (eSYN-Scrb n = 8 and eSYN-shRNA-Ift20 n = 9) and GPR158 (eSYN-Scrb n = 8 and eSYN-shRNA-Ift20 n = 7). c, Western blot and quantification of IFT20 (eSYN-Scrb+veh n = 4, eSYN-Scrb+OCN n = 5, eSYN-shRNA-Ift20+OCN n = 5), LC3B-I/II (eSYN-Scrb+veh n = 4, eSYN-Scrb+OCN n = 5, eSYN-shRNA-Ift20+OCN, n = 5) and Beclin-1 (eSYN-Scrb+veh n = 4, eSYN-Scrb+OCN n = 4, eSYN-shRNA-Ift20+OCN, n = 5) proteins in hippocampi, 3 weeks after injections with AAV-eSYN-shRNA-scramble-mir or AAV-eSYN-shRNA-Ift20-mir and upon stereotactic injection of vehicle or OCN. Quantification is relative to injection with AAV-eSYN1-shRNA-scramble-mir treated with vehicle. β-actin was used as a loading control for each sample in all western blot analyses. All data are expressed as mean ± s.e.m., and P values were determined by one-tailed (b, top) or two-tailed (a and b, bottom) Student’s t-tests compared to vehicle or eSYN-Scbr. In c, the P values were determined by one-way ANOVA followed by Tukey’s multiple comparison test.
