Figure 5
From: Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington's disease models
Mapk11 regulates HTT mRNA levels via regulation of HTT mRNA stability. (A) RT-qPCR results in STHdh cells showing that knockdown of Mapk11 lowers Htt mRNA levels in HD (left) but not wild-type cells (right). The X-axis indicates the Mapk11 mRNA expression level, whereas the Y-axis indicates the Htt mRNA level. As for B-D. Each single point exhibits the Htt versus Mapk11 mRNA levels of an independently transfected well, and the numbers are indicated (n). All data were normalized to the average of scrambled siRNA-transfected control samples. Red, scrambled siRNA-transfected control samples; Dark, cells transfected with either of the two Mapk11 siRNAs (Mapk11_si1&Mapk11_si2). The correlation coefficient R and P values on top of each plot were calculated by Spearman correlation analysis. “***” on the right side of each plot indicates that P< 0.001 calculated by unpaired two-tailed t tests for the Htt mRNA levels in Mapk11 siRNA-transfected sample versus the control. (B) Similar to (A), but in HD patient iPSC-derived neurons (Q47). Data plots and statistical analysis were performed in the same way as in (A). (C) Similar to (A), but the mRNAs were extracted from in vivo mouse striata (HdhQ7/Q140 with or without heterozygous knockout of Mapk11, 4-6.5-month-old), and the statistical analyses were performed the same. The “n” numbers indicate the number of mice. (D) The non-radioactive pulse-chase experiment showing that knockdown of Mapk11 does not influence Htt protein degradation. The siRNAs were transfected in STHdhQ7/Q111 cells. Three independently transfected wells for each point were tested. Data plotted as mean and SEM. The statistical analysis was performed by two-way ANOVA to determine if the transfection influences the protein degradation. “ns” on the right side of the plot indicates no statistically significant interaction between siRNA transfection and Htt protein degradation. (E) The Htt lowering by Mapk11 siRNA (Mapk11_si2) was measured in STHdh cells by 2B7/2166 HTRF. The proteasome inhibitor epoxomycin or the autophagy inhibitor bafilomycin A (BafA) did not reduce the lowering effect given by Mapk11 knockdown. Bars represent mean and SEM, and the numbers indicate the number of independently transfected wells. The statistical analysis was performed by one-way ANOVA and Dunnett's post hoc tests. **P< 0.01. (F) The Htt promoter activity was measured in STHdhQ7/Q111 by the luciferase reporter assay (see Materials and Methods). Knockdown of Mapk11 did not significantly influence the Htt promoter activity. Blue lines represent mean and SEM, and 10 independently transfected wells were tested for each condition. The statistical analysis was performed by unpaired two-tailed t tests. (G) Htt mRNA stability measurements in STHdhQ7/Q111 cells (left) and HD patient iPSC-derived neurons Q47 (right). The cells were transfected with indicated siRNAs for 36 h and then treated with actinomycin D to stop new mRNA synthesis. The Htt/HTT mRNA level was then measured at different time points using RT-qPCR, normalized to Hprt/HPRT mRNA levels and plotted as percentage of the initial time point. For HD patient iPSC-derived neurons, β-actin and 18S mRNA were used as controls, which were not influenced by MAPK11 knockdown. Since their degradation is close to HPRT and their levels were normalized to HPRT at each time point, the normalized levels were relatively flat across time points. Data were plotted as mean and SEM. The statistical analysis was performed by two-way ANOVA to determine if the transfection influences the mRNA degradation. ***P< 0.0001 for the effect of MAPK11 siRNA on Htt mRNA degradation.
