Supplementary Figure 7: NEUROD1 overexpression and Isx-9 increase expression of downregulated transcripts. | Nature Neuroscience

Supplementary Figure 7: NEUROD1 overexpression and Isx-9 increase expression of downregulated transcripts.

From: Developmental alterations in Huntington's disease neural cells and pharmacological rescue in cells and mice

Supplementary Figure 7

(a) NEUROD1 over-expression enhances neuronal gene expression in high CAG repeat lines. Several genes in addition to those depicted in Figure 6 were evaluated in the high CAG repeat HD line 109Q after either human NEUROD1 overexpression lentivirus or pFUGW-eGFP. qPCR demonstrates increased expression of GAD1 (P=0.0002, t=13.20, df=4), ATP2B2 (P=0.0087, t=4.790, df=4) and KCNQ3 (P=0.0121, t=4.352, df=4). (b) Isx-9 enhances expression of NEUROD1 (33Q: P=0.4290, t=0.879, df=4; 60Q: P=0.0173, t=3.92, df=4; 109Q: P=0.0374, t=3.066, df=4) and other neuronal genes POU4F2 (33Q: P<0.0001, t=19.95, df=4; 60Q: P<0.0001, t=16.49, df=4; 109Q: P<0.0001, t=51.96; df=4), CAMK4 (33Q: P=0.0002, t=13.45, df=4; 60Q: P<0.0001, t=24.01, df=4; 109Q: P=0.0004, t=10.98, df=4) and CALB1 (33Q: P<0.0001, t=19.00, df=4; 60Q: P=0.0001, t=15.36, df=4; 109Q: P<0.0001, t=17.18, df=4) in non-disease (33Q) and HD cultures (60Q, 109Q). (c) Additional genes were tested in 21Q and 109Q lines with increased expression of GAD1 (21Q: P<0.0001, t=21.86, df=4; 109Q: P<0.0001, t=27.90, df=4), CALB1 (21Q: P=0.0004, t=10.72, df=4; 109Q P=0.0004, t=11.19; df=4), CAMK4 (21Q: P=0.0007, t=9.628, df=4; 109Q: P=0.0011, t=8.390, df=4), POU4F2 (21Q: P<0.0001, t=16.32, df=4; 109Q: P<0.0001, t=25.26, df=4), KCNQ3 (21Q: P<0.0001, t=21.84, df=4; 109Q: P<0.0001, t=168.1, df=4) and CACNA1A (21Q: P<0.0001, t=50.08, df=4; 109Q: P=0.0005, t=10.19, df=4) in all cultures, as well as increased expression of ATP2B2 in the HD 109Q line (21Q: P=0.0548; t=2.688, df=4; 109Q: P=0.001, t=8.618, df=4). For qPCR analysis, a statistical difference in gene expression between GFP and NEUROD1 overexpression or vehicle and Isx-9 treatment was determined using an unpaired two-tailed t-test in GraphPad Prism. (d) Isx-9 western analysis. 21Q and 109Q cell lines demonstrate increased protein expression of CALB1 and CAMKIV after Isx-9 treatment. WB results (densitometry) were normalized to ACTIN. Statistical significance between Isx-9 and untreated cells (NIM) was determined by one-way ANOVA. CALB1: 21Q plus ISX9 (P=0.0001, n = 5 independent differentiations, df=1, F=123.59). 109Q plus ISX9 (P=0.0001, n=5 independent differentiations, df=1, F=54.11). 109Q plus BDNF (P=0.000457, n=5 independent differentiations, df=1, F=32.43). CAMKIV: 21Q plus ISX9 (P=0.001359, n=3 independent differentiations, df=1, F=63.12). 109Q plus ISX9 (P=0.000337, n=3 independent differentiations, df=1, F=130.03). Full blots in S8.

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