Dysfunction of NMDA receptors in neuronal models of an autism spectrum disorder patient with a DSCAM mutation and in Dscam-knockout mice - PubMed

Fig. 3. NMDA-R-mediated currents are compromised in ASD iN cells.

a Voltage dependency of NMDA-R-mediated currents (I_NMDA) in control and ASD iN cells. Note the significantly lower I_NMDA at +40 and +60 mV in ASD iN cells (Control: n = 73, ASD: n = 92 from ten independent cultures; two-way repeated-measures ANOVA, interaction, F_{(6, 228)} = 6.200, p < 0.0001; Bonferroni post hoc test, +40 mV, ****p < 0.0001; +60 mV, ****p < 0.0001). b Quantitative RT-PCR analysis of normalized NR1/NR2A/NR2B gene expression in control and ASD iN cells. The expression of NR1, NR2A, and NR2B was significantly reduced in ASD iN cells. For NR1 [Control: n = 51, ASD: n = 31 (Control#1: n = 13, Control#2: n = 13, Control#3: n = 13, Control#4: n = 12, ASD#3: n = 13, ASD#4: n = 10, ASD#5: n = 8), Kruskal–Wallis test for left panels (p < 0.0001) followed by Dunn’s multiple comparisons test (Control#1 vs. Control#4, ****p < 0.0001, Control#1 vs. ASD#3, ***p < 0.001, Control#1 vs. ASD#4, ***p < 0.001, Control#1 vs. ASD#5, ***p < 0.001, Control#2 vs. Control#4, *p < 0.05, Control#2 vs. ASD#4, *p < 0.05, Control#2 vs. ASD#5, *p < 0.05, Control#3 vs. Control#4, *p < 0.05, Control#3 vs. ASD#4, *p < 0.05, Control#3 vs. ASD#5, *p < 0.05), unpaired t-test for right panels (****p < 0.0001)]. For NR2A [Control: n = 32, ASD: n = 22 (Control#1: n = 8, Control#2: n = 8, Control#3: n = 8, Control#4: n = 8, ASD#3: n = 8, ASD#4: n = 6, ASD#5: n = 8), Kruskal–Wallis test for left panels (p < 0.01) followed by Dunn’s multiple comparisons test (ns, not significant), unpaired t-test for right panels (**p < 0.01)]. For NR2B [Control: n = 32, ASD: n = 21 (Control#1: n = 8, Control#2: n = 8, Control#3: n = 8, Control#4: n = 8, ASD#3: n = 8, ASD#4: n = 5, ASD#5: n = 8), Kruskal–Wallis test for left panels (p < 0.01) followed by Dunn’s multiple comparisons test (ns, not significant), unpaired t-test for right panels (*p < 0.05)]. c Immunocytochemical analysis of NR1 expression in control and ASD iN cells. Scale bar: 50 μm. d The expression of NR1 was significantly reduced in ASD iN cells [Control: n = 51, ASD: n = 31 (Control#1: n = 13, Control#2: n = 13, Control#3: n = 13, Control#4: n = 12, ASD#3: n = 13, ASD#4: n = 10, ASD#5: n = 8), Kruskal–Wallis test for left panel (p < 0.0001) followed by Dunn’s multiple comparisons test (Control#1 vs. Control#3, **p < 0.01, Control#2 vs. ASD#3, ****p < 0.0001, Control#3 vs. Control#4, *p < 0.05, Control#3 vs. ASD#3, ****p < 0.0001, Control#3 vs. ASD#4, **p < 0.01), Mann–Whitney test for right panel (****p < 0.0001)]. e, f Western blot analysis of NR1 expression from control and ASD iN cells. e Representative Western blot images of NR1 expression. f Quantitative analysis of normalized NR1 expression in control and ASD iN cells. Note that the NR1 expression was significantly reduced in ASD iN cells as compared to control iN cells [Control: n = 24, ASD: n = 15 (Control#1: n = 6, Control#2: n = 6, Control#3: n = 6, Control#4: n = 6, ASD#3: n = 5, ASD#4: n = 6, ASD#5: n = 4), one-way ANOVA for left panel (p < 0.0001, F_(6,32) = 10.99) followed by Tukey’s multiple comparisons test (Control#1 vs. Control#2, ***p < 0.001, Control#2 vs. Control#4, ***p < 0.001, Control#2 vs. ASD#3, ****p < 0.0001, Control#2 vs. ASD#4, ****p < 0.0001, Control#2 vs. ASD#5, ***p < 0.001, Control#3 vs. ASD#3, **p < 0.01, Control#3 vs. ASD#4, **p < 0.01, Control#3 vs. ASD#5, *p < 0.05), unpaired t-test for right panel (***p < 0.001)]. g Exogenous N-terminal DSCAM expression in control iN cells led to significantly decreased I_NMDA at +40 and +60 mV (Control + mRuby: n = 11, Control + N-terminal DSCAM: n = 22; two-way repeated-measures ANOVA, interaction, F_{(6, 186)} = 7.892, p < 0.0001; Bonferroni post hoc test, +40 mV, p = 0.0054; +60 mV, p < 0.0001). Exogenous overexpression of human WT DSCAM in ASD iN cells significantly increased I_NMDA at +40 and +60 mV (ASD + Full DSCAM: n = 25, ASD + mRuby: n = 15; two-way repeated-measures ANOVA, interaction, F_{(6, 228)} = 6.200, p < 0.0001; Bonferroni post hoc test, +40 mV, p < 0.05; +60 mV, p < 0.0001). h Exogenous N-terminal DSCAM expression in control iN cells led to significantly decreased NR1 density (Control + mRuby: n = 86; Control#1 + mRuby: n = 37, Control#2 + mRuby: n = 17, Control#3 + mRuby: n = 27, Control#4 + mRuby: n = 5, Control + N-terminal DSCAM: n = 65; Control#1 + N-terminal DSCAM: n = 31, Control#2 + N-terminal DSCAM: n = 11, Control#3 + N-terminal DSCAM: n = 16, Control#4 + N-terminal DSCAM: n = 7) and exogenous overexpression of WT full-length DSCAM in ASD iN cells significantly increased NR1 expression (ASD + mRuby: n = 112; ASD#3 + mRuby: n = 69, ASD#4 + mRuby: n = 33, ASD#5 + mRuby: n = 10, ASD + Full-length DSCAM: n = 93; ASD#3 + Full-length DSCAM: n = 36, ASD#4 + Full-length DSCAM: n = 45, ASD#5 + Full-length DSCAM: n = 12). Kruskal–Wallis test for left panel (p < 0.0001) followed by Dunn’s multiple comparisons test (Control#3 + mRuby vs. Control#3 + N-terminal DSCAM, **p < 0.01, ASD#3 + mRuby vs. ASD#3 + N-terminal DSCAM, *p < 0.05, ASD#4 + mRuby vs. ASD#4 + N-terminal DSCAM, **p < 0.01), Kruskal–Wallis test for right panel (p < 0.0001) followed by Dunn’s multiple comparisons test (Control + mRuby vs. Control + N-terminal DSCAM, ****p < 0.0001, Control + mRuby vs. ASD + mRuby, ****p < 0.0001, Control + mRuby vs. ASD + Full-length DSCAM, *p < 0.05, ASD + mRuby vs. ASD + Full-length DSCAM, ****p < 0.0001).