missense mutations cause parkinsonism or PD is unclear at present. The human ATP13A2 gene encodes an 1180 amino acid protein of ,130 kDa belonging to the P5 subfamily of P-type transport ATPases that are predicted to contain ten transmem- brane-spanning domains [16]. Disease-associated missense muta- tions are located within the intracellular and extracellular regions of the protein with a particular cluster in the second intracellular loop region containing the catalytic ATPase domain. The physiological function of ATP13A2 in mammalian cells is unknown. ATP13A2 is highly expressed in the mammalian brain with particular enrichment in the substantia nigra [5]. In PD brains, ATP13A2 expression is up-regulated in surviving substan- tia nigra dopaminergic neurons [5,17]. ATP13A2 is localized at least in part to lysosomal membranes in mammalian cells where it is predicted to participate in the active transport of cations across vesicular membranes in an ATP-dependent manner [5,16]. However, at present, the cation transporting activity or selectivity of ATP13A2 has not been directly demonstrated. In yeast, deletion of the ATP13A2 ortholog, ykp9 (YOR291W), confers sensitivity to growth in the presence of heavy metals, including cadmium (Cd2+), manganese (Mn2+), nickel (Ni2+) and selenium (Se2+) [18,19], implicating Ykp9 in sequestering heavy metals. ATP13A2 orthologs have been reported to protect against cellular toxicity induced by expression of the PD-associated protein a-synuclein in yeast, nematode worm and primary midbrain dopaminergic neurons [18]. KRS mutations produce unstable truncated forms of ATP13A2 but are also directly toxic to cells, induce ER stress and sensitize cells to ER stress-induced cell death [5,11,13]. Thus, truncating KRS mutations most likely cause disease through a loss- of-function mechanism but they may also induce additional cellular toxicity. It is not yet clear how homozygous missense mutations (F182L, G504R and G877R) in ATP13A2 associated with parkinsonism potentially cause disease as they are likely to have subtle effects compared to truncations [9,10,14]. At this juncture it is not clear whether heterozygous mutations in ATP13A2 (T12M, G533R and A746T) are truly disease-causing since they could represent rare polymorphic variants, or pathogenic variants that contribute to disease in combination with unidentified mutations, in a dominant or haploinsufficient manner, or as risk factors [10,15]. A complete understanding of the effects of disease-associated missense mutations on the basic properties of ATP13A2 will help to clarify their mechanism of action and whether they are likely to represent authentic pathogenic variants. Here, we comprehen- sively examine the effects of parkinsonism-associated homozygous and heterozygous missense mutations on the protein stability, subcellular localization and ATPase activity of ATP13A2, and their effects on neuronal integrity. Our data demonstrate that homozygous mutations commonly impair the protein stability of ATP13A2 and lead to its enhanced proteasomal degradation, whereas heterozygous mutations commonly impair the ATPase activity of ATP13A2. We further demonstrate that overexpression of wild-type ATP13A2 impairs neurite outgrowth whereas the majority of heterozygous and homozygous missense mutations lack this ability. Finally, the overexpression of ATP13A2 sensitizes to neurite shortening induced by exposure to the heavy metal ions, cadmium or nickel, and missense mutations influence this effect. Our data support a common loss-of-function mechanism for missense mutations associated with early-onset parkinsonism. Pathogenic Effect of Missense Mutations in ATP13A2 Results Disease-associated missense mutations reduce the steady-state levels of ATP13A2 To begin to explore the potential pathogenic effects of missense mutations in ATP13A2 associated with early-onset parkinsonism, we generated expression constructs for V5-tagged human ATP13A2 harboring homozygous (F182L, G504R and G877R) or heterozygous (T12M, G533R and A746T) missense mutations (Figure 1A). We also introduced a synthetic D513N mutation that ablates a critical P1 domain phospho-acceptor site required for ATPase activity that is predicted to be non-functional (Figure 1A) [16,18]. To initially explore the biochemical properties of ATP13A2, the solubility of these variants in different extraction buffers at ambient temperature was determined by Western blotting from HEK-293T cell extracts transiently expressing V5- tagged ATP13A2. ATP13A2 is most soluble in 1% Triton X- 100 buffer and Laemmli sample buffer consistent with being a transmembrane-spanning protein (Figure 1B). We also assessed the effects of temperature on the solubility of ATP13A2 derived from HEK-293T cells. Increasing temperature from 60 to 90uC decreases the solubility of ATP13A2 in 1% Triton X-100 buffer (Figure 1C). Therefore, for all subsequent experiments we routinely employed buffer containing 1% Triton X-100 without heating to efficiently extract ATP13A2 from cells. Frameshift and splicing mutations in ATP13A2 associated with KRS (i.e. 3057delC, 1632_1653dup22 and 1306+5GRA) produce truncat- ed proteins that are unstable and degraded by the proteasome [5,13]. To explore the effects of missense mutations on protein stability, we examined the steady-state levels of ATP13A2 variants in HEK-293T cells by Western blotting. The homozygous mutations, F182L, G504R and G877R, produce a significant decrease in the steady-state levels of Triton-soluble ATP13A2 compared to WT protein (Figure 1D). The heterozygous T12M, G533R and A746T mutations or the non-functional variant, D513N, do not significantly influence the levels of ATP13A2 protein (Figure 1D). Similar observations were made for the steady-state levels of Triton-insoluble ATP13A2 thereby demon- strating that missense mutations do not influence the detergent solubility of ATP13A2 (Figure 1D). The homozygous mutations, F182L, G504R and G877R, similarly reduce the steady-state levels of ATP13A2 in the Triton-soluble fraction of human SH- SY5Y neural cells (Figure 1E). For comparison, we also assessed the stability of ATP13A2 harboring KRS truncation mutations. The 3057delC, 1632_1653dup22 and 1306+5GRA mutations markedly reduce the steady-state levels of ATP13A2 to levels comparable to the effects of the F182L, G504R and G877R mutations (Figure 1F). To determine the effects of missense mutations on the steady-state mRNA expression levels of human ATP13A2 we conducted quantitative RT-PCR for each ATP13A2 variant expressed in HEK-293T cells. With the exception of the G877R variant, WT and mutant ATP13A2 mRNAs are expressed at similar levels (Figure S1). G877R ATP13A2 exhibits a non-significant reduction in mRNA expres- sion levels suggesting that this mutation could potentially influence mRNA transcription and/or stability, which contributes in part to the markedly reduced steady-state protein levels observed for this variant (Figure 1D and 1E). Collectively, our data demonstrate that homozygous missense mutations associated with early-onset parkinsonism commonly reduce the steady-state levels of ATP13A2 consistent with reduced protein stability. PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39942