Fig. 8: Effects of AAV-USP22 administration on cognitive function, the K48-Ub and methylation of β-catenin, and the expressions of β-catenin and survivin in CA1 after tGCI with or without hypoxia.

A Schematic diagram of the experimental design of the Morris water maze (MWM) test after tGCI with or without hypoxia. B Representative swimming path tracings in each group during the training period (learning) and probe trial (memory). C, D The escape latency and path length were recorded among AAV-Con/USP22 treated rats in Sham, tGCI and HPC group. With Mauchly’s test of sphericity, p = 0.001 for the escape latency, p = 0.009 for the path length. *p < 0.05 versus the Sham group with AAV-Con (For the escape latency, in the tGCI+AAV-Con groups, p = 0.016 at day1, p < 0.001 at day2, p = 0.001 at day3, p < 0.001 at day4 and day5, in the HPC + AAV-Con groups, p = 0.648 at day1, p = 1.000 at day2-5; For the path length, in the tGCI+AAV-Con groups, p = 0.008 at day1, p = 0.001 at day2, p < 0.001 at day3 and day 4, p = 0.004 at day5, in the HPC + AAV-Con groups, p = 0.264 at day1, p = 1.000 at day2-5, MANOVA Test followed by Bonferroni post-test), ^#p < 0.05 represents HPC groups versus tGCI groups with AAV-Con (For the escape latency, p = 1.000 at day1, p = 0.020 at day2, p = 0.009 at day3, p = 0.001 at day4, p = 0.002 at day5; For the path length, p = 1.000 at day1, p = 0.056 at day2, p = 0.004 at day3, p = 0.006 at day4, p = 0.029 at day5, MANOVA Test followed by Bonferroni post-test), and ^&р<0.05 versus the same group with AAV-Con (For the escape latency, p = 1.000 at day1-5 in the Sham and the HPC groups, p = 0.416 at day1, p = 0.057 at day2, p = 0.086 at day3, p = 0.004 at day4, p = 0.014 at day5 in the tGCI groups; For the path length, p = 1.000 at day1-5 in the Sham and HPC groups, p = 1.000 at day1, p = 0.180 at day2, p = 0.032 at day3, p = 0.007 at day4, p = 0.067 at day5 in the tGCI groups; n = 6-8, MANOVA Test followed by Bonferroni post-test). E The percentage of time spent in the target quadrant was recorded at day 7 after tGCI when the total time of probe trial was 30 s. *p < 0.05 versus Sham group with AAV-Con (p < 0.001 in the tGCI group with AAV-Con, p = 0.041 in the HPC group with AAV-Con, unpaired t-test), ^#p < 0.05 versus tGCI group with AAV-Con (p = 0.016, unpaired t-test) and ^&р<0.05 versus the same group with AAV-Con (p = 0.264 in the Sham group, p < 0.001 in the tGCI group, p⁼0.015 in the HPC group, n = 7-10, unpaired t-test). F, G Representative immunoblots and quantitative data show USP22, β-catenin and survivin in cytoplasmic and nuclear fractions, separately. Lamin B1 and GAPDH are used as loading controls to normalize the relative expressions of cytoplasmic and nuclear proteins of interest, respectively. Data are expressed as percentage of value of sham-operated animals. *p < 0.05 versus Sham group with AAV-Con (for cytoplasmic USP22, p < 0.001 in the tGCI group with AAV-Con, p < 0.001 in the HPC group with AAV-Con, n = 5; for cytoplasmic β-catenin, p = 0.179 in the tGCI group with AAV-Con, p = 0.546 in the HPC group with AAV-Con, n = 4; for nuclear USP22, p = 0.001 in the tGCI group with AAV-Con, p = 0.397 in the HPC group with AAV-Con, n = 8-15; for nuclear β-catenin, p = 0.017 in the tGCI group with AAV-Con, p = 0.941 in the HPC group with AAV-Con, n = 8–14; for nuclear survivin, p = 0.003 in the tGCI group with AAV-Con, p = 0.545 in the HPC group with AAV-Con, n = 8, unpaired t-test), ^#p < 0.05 versus tGCI group with AAV-Con (p = 0.474 for cytoplasmic USP22, p = 0.175 for cytoplasmic β-catenin, p = 0.026 for nuclear USP22, p = 0.008 for nuclear β^-catenin, p = 0.001 for nuclear survivin, unpaired t-test), ^&р<0.05 versus the same group with AAV-Con (For cytoplasmic USP22, p = 0.089 in the Sham group, p = 0.269 in the tGCI group, p = 0.169 in the HPC group; For cytoplasmic β^-catenin, p = 0.148 in the Sham group, p = 0.862 in the tGCI group, p = 0.923 in the HPC group; For nuclear USP22, p = 0.030 in the Sham group, p = 0.030 in the tGCI group, p = 0.005 in the HPC group; For nuclear β-catenin, p = 0.038 in the Sham group, p = 0.040 in the tGCI group, p = 0.018 in the HPC group; For nuclear survivin, p = 0.309 in the Sham group, p = 0.021 in the tGCI group, p = 0.313 in the HPC group, unpaired t-test). H Immunoprecipitation assay showing the level of nuclear K48-ubiquitinated β-catenin and me-β-catenin in CA1 of Sham, tGCI and HPC groups with AAV-Con/USP22. IgG antibody was used as a negative control. Data are expressed as percentage of value of sham-operated animals. Each bar represents the mean ± SD. *p < 0.05 versus Sham group with AAV-Con (for K48-Ub/β-catenin, p = 0.002 in the tGCI group with AAV-Con, p = 0.904 at the HPC group with AAV-Con; for methyl-K/β-catenin, p = 0.073 in the tGCI group with AAV-Con, p = 0.698 in the HPC group with AAV-Con, unpaired t-test), ^#p < 0.05 versus tGCI group with AAV-Con (p = 0.002 for K48-Ub/β-catenin, p = 0.040 for methyl-K/β-catenin, unpaired t-test), ^&р<0.05 versus the same group with AAV-Con (for K48-Ub/β-catenin, p⁼0.454 in the Sham group, p = 0.041 in the tGCI group, p = 0.442 in the HPC group; for methyl-K/β-catenin, p = 0.703 in the Sham group, p⁼0.012 in the tGCI grou, p = 0.430 in the HPC group, n = 4-6, unpaired t-test). MWM,Morris water maze; S, second; cm, centimeter; d, day. Each bar represents the mean ± SD (error bars).