Fig. 2: Df(h15q13)/+ cortical neurons and human patient iNeurons show impairments in synaptic morphology and persistent functional deficits. | Molecular Psychiatry

Fig. 2: Df(h15q13)/+ cortical neurons and human patient iNeurons show impairments in synaptic morphology and persistent functional deficits.

From: Impaired OTUD7A-dependent Ankyrin regulation mediates neuronal dysfunction in mouse and human models of the 15q13.3 microdeletion syndrome

Fig. 2

a Raster plots of MEA recordings of neural network activity from DIV 24 WT and Df(h15q13)/+ mouse cortical neurons. n = 77 wells WT, 65 wells Df(h15q13)/+ from 3 mouse cortical cultures on 3 MEA plates. b Weighted mean firing rate. Repeated measures two-way ANOVA with Sidak’s post hoc test, *p < 0.05, **p < 0.01, ****p < 0.0001; Interaction: F (7, 980) = 3.950, p = 0.0003; DIV: F (7, 980) = 87.98, p < 0.0001; Genotype: F (1, 140) = 30.64, p < 0.0001; Subject: F (140, 980) = 4.436, p < 0.0001. c Network burst frequency. Repeated measures two-way ANOVA with Sidak’s post hoc test, **p < 0.01, ***p < 0.001, ****p < 0.0001; Interaction: F (7, 980) = 4.341, p < 0.0001; DIV: F (7, 980) = 115.2, p < 0.0001; Genotype: F (1, 140) = 40.27; Subject: F (140, 980) = 2.78, p < 0.0001. d Schematic of the human OTUD7A protein showing the location of the L233F variant. e Representative confocal images from co-transfected DIV 14 WT and Df(h15q13)/+ mouse cortical neurons; 20× objective, scale bar 100 μm. f, g Sholl analysis. n = 11 neurons WT + mCherry, 10 neurons [Df(h15q13)/+] + mCherry, 8 neurons [Df(h15q13)/+] + WT OTUD7A-mCherry, 10 neurons [Df(h15q13)/+] + OTUD7A L233F. Samples were taken from 3 mouse cultures. f Two-way ANOVA with Dunnett’s post hoc test; ****p < 0.0001; Interaction: F (57, 700) = 0.505, p = 0.9991; DIV: F (19, 700) = 10.54, p < 0.0001; Distance: F (3, 700) = 57.12, p < 0.0001. g Total number of dendritic intersections. One-way ANOVA with Dunnett’s post hoc test, **p < 0.01, F (3, 35) = 5.775, p = 0.0026. h Representative confocal images of dendritic segments from co-transfected DIV 14 WT and Df(h15q13)/+ mouse cortical neurons; 63× objective, scale bar 2 μm. i Mushroom spine density. n = 8 neurons WT + mCherry, 12 neurons [Df(h15q13)/+] + mCherry, 9 neurons [Df(h15q13)/+] + WT OTUD7A-mCherry, 10 neurons [Df(h15q13)/+] + OTUD7A L233F. Samples were taken from 3 mouse cultures. *p < 0.05, **p < 0.01; one-way ANOVA with Dunnett’s post hoc test; F (3, 35) = 6.423, p = 0.0014. j Pedigree of Family 1 and k the OTUD7A L233F Family. l Representative western blot (left) and quantification (right) of OTUD7A levels in iNeurons; n = 3 separate Ngn2/Rtta transductions per line; one-way ANOVA with Dunnett’s post hoc test, *p < 0.05, F (2.6) = 7.921, p = 0.0207. m Raster plots of MEA recordings of neural network activity from DIV 89 Family 1 and OTUD7AL233F/L233F human iNeurons. Control (Fam 1) n = 29 wells, 15q13.3 HET (Fam 1) n = 29 wells, OTUD7AL233F/L233F n = 30 wells from two separate NGN2/Rtta transductions. n Weighted mean firing rate. Repeated measures two-way ANOVA with Dunnett’s post hoc test; Interaction: F (42, 1785) = 17.59, p < 0.0001; DIV: F (2.925, 248.6) = 108.2, p < 0.0001; Genotype: F (2.85) = 15.96, p < 0.0001; Subject: F (85, 1785) = 16.12, p < 0.0001. o Network burst frequency. Repeated measures two-way ANOVA with Dunnett’s post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p Representative images and q sholl analysis of MAP2-positive Family 1 and OTUD7AL233F/L233F proband human iNeurons; 20× objective, scale bar 100 μm. Control n = 33 neurons, 15q13.3 HET n = 31 neurons, OTUD7AL233F/L233F n = 38; ***p < 0.001, two-way ANOVA with Dunnett’s post hoc test; Interaction: F (68, 3465) = 1.943, p < 0.0001; Distance from soma: F (34, 3465) = 53.30, p < 0.0001; Genotype: F (2, 3465) = 200.2, p < 0.0001.

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