mutations, to model the cellular consequences of disease-associ- ated ATP13A2 mutations [17,25,26,27]. ATP13A2 knockdown has been suggested to impair neuronal integrity and/or induce neuronal cell death through a number of potential mechanisms, including abnormal cation homeostasis [17], decreased macro- autophagy [26], lysosomal dysfunction [27], a-synuclein accumu- lation [27], and/or alterations in mitochondrial quality control [17,25,26]. Which of these cellular abnormalities are directly responsible for mediating neuronal damage is not yet clear but they most likely result as a consequence of impaired lysosomal activity. Therefore, lysosomal dysfunction potentially underlies the pathogenic effects of ATP13A2 loss-of-function in neurons. We find that the overexpression of ATP13A2 missense mutants in human SH-SY5Y neural cells does not influence cell viability suggesting that they are not intrinsically toxic. KRS mutants have been shown to induce cell death in HeLa cells where they also sensitize to ER stress-induced cell death [13]. However, another study reports the lack of cell death induced by the same KRS mutants overexpressed in rat hippocampal neurons [24]. Further- more, in the context of Mn2+-induced cell death in cell lines and hippocampal neurons, WT ATP13A2 protects from cell death whereas KRS mutants lack this ability [24]. Additional toxicity induced by KRS mutants may not therefore play a major role in disease and could relate to overexpression at non-physiological levels, especially given that such mutations lead to a dramatic destabilization and degradation of ATP13A2 [5,13]. It will be important to determine whether the expression of KRS mutants at physiological levels in patient-derived cells and tissues similarly induces cellular toxicity. A recent study has shown that human olfactory neurospheres harboring compound heterozygous muta- tions in ATP13A2 (3253delC and L1059R) exhibit signs of ER stress although neuronal viability was not assessed [11]. In primary cortical neurons, we find that the overexpression of WT ATP13A2 tends to impair neurite outgrowth. Consistent with a loss-of- function effect for missense mutations, the disease-associated T12M, G504R and G533R variants fail to influence neurite outgrowth compared to WT ATP13A2. These effects were similar to the effects of an ATPase-deficient D513N variant further supporting a loss-of-function mechanism. Similar to certain unstable KRS truncation mutants [13], the F182L variant appears to induce additional toxicity when overexpressed since it causes a trend of reduced neurite outgrowth of cortical neurons similar to WT ATP13A2 despite its markedly impaired protein stability. It is likely that these effects are exacerbated by overexpression at non- physiological levels in neurons with the primary pathogenic effect of the F182L mutation at physiological expression levels being a loss-of-function due to reduced protein stability. Of note, the heterozygous T12M and G533R variants which have no obvious effects on protein stability or subcellular localization, have no effect on neurite outgrowth suggesting that they may also act as a pathogenic loss-of-function mutations albeit through an alternative mechanism such as via reduced ATPase activity. In support of a role for ATP13A2 in regulating the transport of Cd2+ and Ni2+, we find that the overexpression of WT ATP13A2 sensitizes cortical neurons to neurite shortening induced by exposure to these heavy metal ions. This finding was somewhat unexpected as we would anticipate that ATP13A2 might protect against Cd2+ and Ni2+-induced neuronal toxicity similar to the reported neuroprotective effects of ATP13A2 against Mn2+ and a- synuclein toxicity [18,24]. Consistent with a loss-of-function effect for the G504R mutation, the overexpression of this mutant exhibited a reduced sensitizing effect on neurite length following Cd2+ and Ni2+ exposure compared to WT ATP13A2. In contrast, the F182L mutation enhanced the sensitizing effect of ATP13A2 Pathogenic Effect of Missense Mutations in ATP13A2 following Cd2+ exposure consistent with the enhanced neuronal toxicity of this unstable protein following its overexpression. The significance of neurite shortening induced by ATP13A2 is unclear at present and could indicate impaired neuronal integrity due to toxicity or a physiological effect of ATP13A2 in regulating neuronal process morphology similar to the PD-associated protein LRRK2 [28]. Nevertheless, our data support a specific functional interaction of human ATP13A2 with Cd2+ and Ni2+ suggesting a potential role in the transport of these metal cations in post-mitotic mammalian neurons. Previous studies demonstrate that deletion of the ATP13A2 ortholog, ykp9, impairs yeast cell growth in the presence of Cd2+, Ni2+, Mn2+ and Se2+ [18,19]. Furthermore, the expression of WT but not KRS mutant ATP13A2 in mammalian cell lines or rat hippocampal neurons may protect from Mn2+- induced apoptotic cell death [24]. Our recent studies have shown that the overexpression of human ATP13A2 in primary cortical neurons delays mitochondrial fragmentation induced by acute exposure to cadmium [17]. Collectively, these studies support a functional interaction between ATP13A2 and heavy metal cations, particularly cadmium. Our future studies will continue to clarify whether ATP13A2 can directly transport heavy metal cations across vesicular membranes, the substrate selectivity of ATP13A2, and the functional significance of cation transport [17]. Collectively, the present study demonstrates that disease- associated homozygous and heterozygous missense mutations in ATP13A2 exhibit differential pathogenic effects on protein stability, subcellular localization, ATPase activity and neuronal phenotypes, thereby providing support for a pathogenic loss-of- function mechanism for these mutations in precipitating early- onset parkinsonism. Materials and Methods Animals All animal experiments were approved by the SCAV (Service de la consommation et des affaires veterinaires) in the Canton de Vaud (Animal authorization No. 2293), and conducted in strict accordance with the European Union directive (2010/63/EU) for the care and use of laboratory animals. Animals were maintained in a pathogen-free barrier facility and exposed to a 12 h light/dark cycle with food and water provided ad libitum. Pregnant female Sprague-Dawley rats were obtained from Charles River Labora- tories (L’Arbresle Cedex, France) and resulting P0 rats were used for preparation of post-natal primary cortical cultures. Expression plasmids and antibodies Mammalian expression plasmids containing full-length V5- tagged human ATP13A2 (WT, 1306+5GRA, 1632_1653dup22 and 3057delC) were kindly provided by Dr. Christian Kubisch (University of Cologne, Germany) [5]. Disease-associated or functional missense mutations (T12M, F182L, G504R, D513N, G533R, A467T and G788R) were introduced into V5-tagged WT ATP13A2 by site-directed mutagenesis using the Stratagene QuickChange II XL kit (Agilent Technologies, La Jolla, CA, USA) according to manufacturer’s instructions. The integrity of all constructs was confirmed by DNA sequence analysis. As plasmid controls, a pEGFP-N1 plasmid was obtained from Clontech (Mountain View, CA, USA) and a pcDNA3.1 plasmid was obtained from Invitrogen (Basel, Switzerland). A plasmid express- ing HA-tagged human SPCA1 was kindly provided by Dr. Adam Linstedt (Carnegie Mellon University, Pittsburgh, PA) [29]. Expression plasmids containing human EGFP-LC3B (plasmid #11546; [30]), rat LAMP1-RFP (plasmid #1817, [31]), human RFP-Rab5A (plasmid #14437, [32]), human GFP-Rab7A (plas- PLoS ONE | www.plosone.org 13 June 2012 | Volume 7 | Issue 6 | e39942