Skip to main content
. Author manuscript; available in PMC: 2016 Nov 29.
Published in final edited form as: Mov Disord. 2015 Sep 4;30(13):1813–1824. doi: 10.1002/mds.26348

FIG. 4.

FIG. 4

PPP2R2B promoter activity and PPP2R2B-induced neurotoxicity. (A) Constructs as depicted in Supporting Figure 1, with luciferase activity normalized to Renilla activity, and all values normalized to the SV40 construct (not shown on graph) to enable comparisons across experiments. This graph represents the mean and SE of five (LA-N-1 cell line and rat primary cortical neurons) or three (N2a and SK-N-AS cell lines) independent experiments, each performed in triplicate. For LA-N-1 cells, there was a strong correlation between repeat length and reporter expression (r2 = 0.90; P = 0.014), with CAG10 significantly different from CAG35, CAG53, and CAG78 (ANOVA: F = 24.51; P < 0.0001; Tukey’s post-hoc test: P < 0.05 for CAG35 and CAG53; P < 0.01 for CAG78). For primary rat cortical neurons, the same effect of increasing repeat length on reporter activity was evident (exponential curve: r2 = 0.81; P = 0.037; ANOVA: F = 58.08; P < 0.0001; post-hoc Tukey’s test: P < 0.05 for CAG35 and CAG53; P < 0.01 for CAG78), but no effect of repeat length was observed in N2a or SK-NAS cell lines. In LA-N-1 and rat primary cortical neurons, the reporter activity induced by the CAG10+(NNN)64 construct was equivalent to the PPP2R2B-CAG10 construct and was significantly less than activity induced by the CAG78 construct (P < 0.01). The reporter activity of the CAG80-HD construct was minimal; activity was significantly less than the CAG10 construct and substantially less than the CAG78 construct in LA-N-1 cells (post-hoc: P < 0.05 and P < 0.01, respectively) and primary rat cortical neurons (P < 0.01 for each comparison). (B and C) Primary cortical neurons cotransfected with DsRed and Bβ1 variant of PPP2R2B, fixed at 72 hours post-transfection, 100x magnification. Neurite outgrowth is markedly greater in cells transfected with vector (B) than in cells transfected with PPP2R2B encoding Bβ1 (C). (D) Sholl analysis demonstrating loss of neurite outgrowth after overexpression of PPP2R2B (ANOVA: F = 541.5; P < 0.0001). Experiment performed in triplicate; N = 25 to 40 neurons in each experiment per condition. ANOVA, analysis of variance.