Pathogenic Effect of Missense Mutations in ATP13A2 Figure 9. Overexpression of ATP13A2 sensitizes cortical neurons to heavy metal-induced neurite shortening. A, Rat primary cortical neurons were 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 treated without or with cadmium (Cd2+, 30 mM) or nickel (Ni2+, 50 mM) for 24 h and then fixed at DIV 6. Representative fluorescent micrographs reveal the co-labeling of MAP2+ cortical neurons with EGFP for each condition. Axonal processes are indicated with arrows. Scale bar: 400 mm. B, Analysis of the length of EGFP+/MAP2+ neurites reveals that the overexpression of WT or F182L ATP13A2 sensitizes cortical neurons to PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e39942