Fig. 5: Ankyrin-G displays decreased levels and altered protein stability and ubiquitination in human 15q13.3 microdeletion and OTUD7A^L233F/L233F patient iNeurons.

a SIM imaging of Venus-transfected PNI day 28 Family 1 and OTUD7A^L233F/L233F iNeurons stained for Ankyrin-G. Top: Scale bar = 10 µm, Middle: Scale bar = 5 µm, Bottom Scale bar = 1 µm. b Ankyrin-G intensity in the dendrites (**p < 0.01; one-way ANOVA with Dunnett’s post hoc test; F (2, 63) = 5.261, p = 0.0077), c spine morphology (**p < 0.01, ****p < 0.0001; Mushroom: Kruskal–Wallis test with Dunn’s post hoc test, p < 0.0001 (approximate), Kruskal–Wallis statistic = 19.20; Filopodia: one-way ANOVA with Dunnett’s post hoc test, F (2, 63) = 0.9011, p = 0.4113; Stubby: Kruskal–Wallis test with Dunn’s post hoc test, p = 0.5212 (approximate), Kruskal–Wallis statistic = 1.303. Control (Fam 1) n = 20 dendrites, 15q13.3 HET (Fam 1) n = 27 dendrites, OTUD7A^L233F/L233F n = 19 dendrites. d mushroom spine head area (one-way ANOVA with Bonferroni’s post hoc test; F (2, 148) = 0, 0 = 0.9658. e number of Ankyrin-G puncta in mushroom spine heads (Kruskal–Wallis test with Dunn’s post hoc test; p = 0.0167 (approximate Kruskal–Wallis statistic: 8.189) and f Ankyrin-G nanodomain area in mushroom spine heads. One-way ANOVA with Dunnett’s post hoc test; F (2, 149) = 10.06, p < 0.0001); Control (Fam 1): n = 84 spines, 15q13.3 HET (Fam 1) n = 53 spines, OTUD7A^L233F/L233F n = 15 spines *p < 0.05, **p < 0.01, ***p < 0.001. g Confocal images of Family 1 and OTUD7A^L233F/L233F PNI day 28 iNeurons stained for MAP2 and Ankyrin-G. Scale bar = 50 µm. h Ankyrin-G intensity (mean gray value) in the AIS. ****p < 0.0001, two-way ANOVA with Dunnett’s post hoc test; Interaction: F (160, 6196) = 1.891, p < 0.0001; Distance from soma: F (80, 6196) = 8.481, p < 0.0001; Genotype: F (2, 6196) = 69.18, p < 0.0001. i Western blot and j analysis of Ankyrin-G in PNI day 7 human iNeurons from Family 1 and OTUD7A^L233F/L233F. n = 5 separate Ngn2/Rtta transductions per line, *p < 0.05, one-way ANOVA with Dunnett’s post hoc test, F (2, 12) = 3.317, p = 0.0713). k Western blot of time-course of Ankyrin-G levels after cycloheximide (20 µg/mL) treatment. l Kinetics of Ankyrin-G protein stability in Family 1 and OTUD7A^L233F/L233F induced neurons. n = 3 NGN2 transductions per condition; ***p < 0.001; simple linear regression followed by comparison of slopes by one-way ANOVA with Dunnett’s post hoc test; F (2, 30) = 10.99, p = 0.0003. m TUBE pulldown from Family 1 and OTUD7A^L233F/L233F human iNeurons probed for Ankyrin-G and Ubiquitin. n Quantification of ubiquitinated Ankyrin-G from TUBE pulldown, normalized to the levels of Ankyrin-G in the input (whole lysate). n = 6 wells Control, 5 wells 15q13.3 microdeletion, 5 wells OTUD7A^L233F/L233F; *p < 0.05, one-way ANOVA with Dunnett’s post hoc test, F (2, 13) = 5.238, p = 0.0215.