Pathogenic Effect of Missense Mutations in ATP13A2 Figure 8. Overexpression of ATP13A2 impairs neurite outgrowth of cortical neurons. A, Viability of SH-SY5Y cells transiently expressing V5-tagged human ATP13A2 variants at 48 h post-transfection assessed by MTS assay. Bars represent mean6SEM (n = 4 experiments). B, Rat primary cortical neurons co-transfected at DIV 3 with V5-tagged human ATP13A2 variants (or control empty vector) and EGFP plasmids at a DNA molar ratio of 10:1. Cultures were fixed at DIV 6. Representative fluorescent micrographs reveal the co-labeling of cortical neurons with EGFP and MAP2 for each condition. EGFP images representing ATP13A2-positive cortical neurons were pseudo-colored with ICA to improve the contrast of neuritic processes for length measurements. Axonal processes are indicated with arrows. Scale bar: 400 mm. C, Analysis of the length of EGFP+/MAP2+ neurites from rat primary cortical neurons expressing V5-tagged human ATP13A2 variants (or control empty vector). Expression of WT or F182L ATP13A2 produces a non-significant shortening of cortical axonal processes compared to control neurons expressing EGFP alone. Bars represent the length of MAP2+ neurites in mm (mean6SEM; n = 67–88 neurons from three independent cultures). Non-significant (ns) compared to control (-) by one-way ANOVA with Newman-Keuls post-hoc analysis. doi:10.1371/journal.pone.0039942.g008 PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39942