Pathogenic Effect of Missense Mutations in ATP13A2 Figure 5. Homozygous missense mutations disrupt the vesicular membrane localization of ATP13A2. Confocal fluorescence microscopy reveals the cytoplasmic vesicular localization of V5-tagged human ATP13A2 variants transiently expressed in rat primary cortical neurons. WT or missense mutant (T12M, D513N, G533R, A746T or G877R) forms of ATP13A2 adopt a similar localization to large vesicular membranes, whereas F182L and G504R missense mutants or KRS mutants (3057delC, 1632_1653dup22 or 1306+5GRA) abnormally localize to small diffuse punctate structures. Staining for ATP13A2 (anti-V5 antibody, green) and nuclei (DAPI, blue) are indicated. Confocal images are taken from a single z-plane at 0.15 mm thickness. Images are representative of at least two independent transfection experiments. Scale bar: 10 mm. doi:10.1371/journal.pone.0039942.g005 described [23]. The D513N mutant dramatically impairs the ATP hydrolysis activity of ATP13A2 (Figure 7C), consistent with disruption of the phospho-acceptor site in the P1 domain that is critically required for ATPase activity [16,18]. Importantly, microsomes expressing the heterozygous T12M, G533R or A746T mutants display significantly reduced ATPase activity compared to WT ATP13A2 (Figure 7C). Our data suggest that heterozygous missense mutations impair the ATPase activity of ATP13A2. Missense mutations impair the effects of ATP13A2 on neurite outgrowth KRS mutations in ATP13A2 have been shown to induce cellular toxicity, ER stress and sensitize cells to ER stress-induced cell death [11,13]. To initially assess the effects of ATP13A2 missense mutants on cell viability, we examined the effects of overexpressing human ATP13A2 variants on the viability of human SH-SY5Y neural cells using an MTS proliferation assay. The expression of WT or missense mutant forms of ATP13A2 does not appreciably influence the basal viability of SH-SY5Y cells (Figure 8A) demonstrating that ATP13A2 missense mutants are not intrinsically toxic to mammalian cells unlike truncated KRS mutants. We next sought to compare the subtle effects of ATP13A2 variants on neuronal integrity by assessing neurite outgrowth. Rat primary cortical cultures at DIV 3 were transiently co-transfected with V5-tagged human ATP13A2 variants (or empty vector) and EGFP at a 10:1 molar ratio to label transfected neurons. After 72 hours, cultures were fixed and processed for immunocytochemistry to identify MAP2-positive cortical neurons. For each ATP13A2 variant, the length of EGFP+/MAP2+ cortical neurites was determined (Figure 8B). The overexpression of WT and F182L mutant ATP13A2 leads to a non-significant reduction of cortical neurite length compared to control MAP2+ neurons expressing EGFP alone, whereas expression of the T12M, G504R, D513N and G533R variants do not differ from EGFP alone (Figure 8C). Western blot analysis confirmed that the levels of each human ATP13A2 variant in cortical neurons were similar to those achieved in HEK-293T cells (data not shown). Taken together, our data demonstrate that the overexpression of WT ATP13A2 displays a non-significant trend towards impairing neurite PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39942