Pathogenic Effect of Missense Mutations in ATP13A2 Figure 6. Homozygous F182L and G504R mutations induce the mislocalization of ATP13A2 to the endoplasmic reticulum. Confocal fluorescence microscopy reveals the enhanced co-localization of F182L and G504R mutant ATP13A2 with the endoplasmic reticulum marker, calreticulin, and reduced co-localization with lysosomes (LAMP1-RFP), compared to WT or missense mutant (T12M, D513N, G533R, A746T or G877R) ATP13A2 transiently expressed in human SH-SY5Y neural cells. Staining for V5-tagged human ATP13A2 (V5, green), LAMP1-RFP (red), calreticulin (gray) and nuclei (DAPI, blue) are indicated. Cytofluorograms and correlation coefficients (Rcoloc) indicate the extent of co-localization of ATP13A2 variants with lysosomal (LAMP1) and ER (calreticulin) markers. 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.g006 outgrowth whereas missense mutations in ATP13A2, with the exception of the F182L variant, disrupt this neurite effect consistent with a loss-of-function mechanism of action. Our data further suggest that the F182L variant may act through both a loss-of-function (i.e. impaired protein stability) and a gain-of- function (i.e. impaired neurite outgrowth) mechanism similar to KRS truncation mutations [11,13]. ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by heavy metals Since the overexpression of human ATP13A2 tends to impair the neurite outgrowth of primary cortical neurons, we next assessed the impact of exposure to heavy metals on this neuronal phenotype to assess the potential functional interaction between ATP13A2 and metal cations. Primary cortical neurons were co- transfected with V5-tagged human ATP13A2 variants (or empty vector) and EGFP plasmids at a 10:1 molar ratio, treated after 48 hours with or without Cd2+ (30 mM) or Ni2+ (50 mM) for 24 hours, and subjected to immunocytochemistry to label MAP- positive neurons. As before, we determined the length of EGFP+/ MAP2+ neurites for each condition (Figure 9A). In control neurons expressing EGFP alone, treatment with 30 mM Cd2+ results in a ,33% reduction of neurite length compared to untreated neurons (Figure 9B). In contrast, Cd2+ treatment produces a marked ,47% reduction of neurite length in neurons expressing WT ATP13A2, a ,59% reduction with the F182L mutant, and a ,39% reduction with the G504R mutant (Figure 9B). Treatment with 50 mM Ni2+ induces a ,7% reduction of neurite length of control neurons expressing EGFP alone, whereas neurons expressing WT, F182L or G504R ATP13A2 exhibit a ,19%, ,20% or ,3% reduction of neurite length, respectively, compared to untreated neurons (Figure 9B). Mn2+ treatment fails to induce neurite shortening in the presence or absence of ATP13A2 and was not studied further (data not shown). Collectively, our data demonstrate that the overexpression of ATP13A2 sensitizes neurons to impairments of neurite outgrowth induced by exposure to cadmium or nickel ions. The F182L mutation enhances the effects of cadmium compared to WT ATP13A2 further suggesting enhanced neuronal toxicity of this mutant protein, whereas the G504R mutation acts in a loss-of- Figure 7. Heterozygous missense mutations impair the ATPase activity of ATP13A2. A, Microsomal fractions prepared by ultracentrifugation from HEK-293T cells transiently expressing V5-tagged human ATP13A2 variants (or empty vector control) following treatment for 24 hours with 5 mM MG132 and 50 mM NH4Cl to inhibit proteasomal and lysosomal degradation. Microsomes or total homogenates were probed with anti-V5 antibody to monitor ATP13A2 levels, or with anti-LAMP1 and anti-Giantin antibodies to detect enrichment of lysosomal and Golgi membranes, respectively. Homozygous mutants (F182L, G504R and G877R) fail to localize to microsomes. B, Microsomal fractions from HEK-293T cells expressing WT or mutant V5-tagged ATP13A2 or HA-tagged human SPCA1 were probed with anti-V5 or anti-HA antibodies to monitor each protein variant, or with anti-LAMP1 and anti-Giantin antibodies to detect membranes. C, ATP hydrolysis activity of ATP13A2 variants or SPCA1 was determined in microsomal fractions by measuring the concentration of free phosphate (Pi) released from ATP. The levels of ATP13A2 in each microsomal fraction were assessed by Western blot analysis with anti-V5 antibody, quantified by densitometry, and used for normalization of Pi release between ATP13A2 variants. ATP hydrolysis activity is expressed as Pi release as a percent of WT ATP13A2 activity. Bars represent the mean6SEM (n = 4 experiments). **P,0.01 compared to WT ATP13A2 by one-way ANOVA with Newman-Keuls post-hoc analysis. doi:10.1371/journal.pone.0039942.g007 PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39942