Pathogenic Effect of Missense Mutations in ATP13A2 neurite shortening induced by treatment with 30 mM Cd2+ or 50mM Ni2+ for 24 h, compared to neurons expressing G504R ATP13A2 or EGFP alone (control). Data represent neurite length (mean6SEM; control, n = 73–156 neurons; cadmium, n = 39–119 neurons; nickel, n = 32–72 neurons; from three independent cultures) expressed as a % of untreated neurons (control or ATP13A2 overexpression). *P,0.05 or ***P,0.001 as indicated by one- way ANOVA with Newman-Keuls post-hoc analysis. doi:10.1371/journal.pone.0039942.g009 function manner. These data support a functional interaction of ATP13A2 with heavy metal ions in post-mitotic neurons. Discussion Homozygous and heterozygous missense mutations in ATP13A2 are associated with early-onset forms of parkinsonism [9,10,14,15]. The pathogenic mechanism(s) by which these mutations may cause disease have not been elucidated. For heterozygous mutations, it unclear whether they represent rare benign polymorphic variants or authentic disease-causing variants. Here, we demonstrate that homozygous missense mutations (F182L and G504R) commonly exhibit reduced steady-state levels, increased turnover, and enhanced proteasomal degradation of ATP13A2 consistent with impaired protein stability. The homozygous G877R mutation exhibits a combination of reduced protein stability and reduced mRNA expression levels. In contrast, heterozygous mutations, T12M, G533R and A746T, have small or minimal effects on protein stability. We further demonstrate that ATP13A2 is normally localized to cytoplasmic acidic vesicular membranes in mammalian neurons, including lysosomes and early and late endosomes. Similar to truncating KRS mutations [5,11], the highly unstable F182L and G504R mutations disrupt the cytoplasmic vesicular localization of ATP13A2 and promote its mislocalization to the ER. Heterozygous mutations (T12M, G533R and A746T) impair the ATPase activity of microsome- localized ATP13A2 whereas the homozygous F182L, G504R and G877R mutations fail to appreciably localize within microsomes. In contrast to KRS mutations in ATP13A2 [11,13], missense mutants are not intrinsically toxic to human cell lines. However, in primary cortical neurons, the overexpression of wild-type ATP13A2 compromises neuronal integrity by impairing neurite outgrowth whereas missense mutations (with the exception of F182L) lack this toxic effect consistent with a loss-of-function mechanism for these mutations. ATP13A2 overexpression further sensitizes neurons to neurite shortening induced by exposure to the heavy metal ions, cadmium and nickel, with the F182L or G504R mutations enhancing or reducing this sensitizing effect, respec- tively. Collectively, our study reveals differential pathogenic effects of missense mutations on ATP13A2 protein stability, subcellular localization, ATPase activity and neuronal phenotypes consistent with a common loss-of-function mechanism for the actions of homozygous and heterozygous mutations. Homozygous missense mutations (i.e. F182L and G504R) markedly impair the protein stability of ATP13A2. Similar to truncating KRS mutations (i.e. 1632_1653dup22, 3057delC, 1306+5GRA and 3253delC) that mislocalize to the ER [5,13], the F182L and G504R mutations impair the normal localization of ATP13A2 to vesicular membranes and facilitate mislocalization to the ER suggesting that these mutant proteins are potentially misfolded and may be subjected to degradation by the ERAD pathway. The G877R mutation, although partly unstable, can normally localize to vesicular membranes suggesting that it may not be a major substrate of the ERAD pathway. The F182L and G504R mutations appear to enhance the turnover of ATP13A2 by promoting its proteasomal degradation. The F182L, G504R and G877R mutations are localized to the first extracellular (F182L) and second intracellular loop (G504R and G877R) regions of ATP13A2 and most likely cause conformational alterations to these loops that destabilize the protein. Two of these mutations result in the non-conservative substitution of a neutral glycine residue for a positively charged arginine residue perhaps suggesting that simply altering the charge of this loop or increasing the side chain length has a destabilizing conformational effect that varies in magnitude depending on the location of this residue within the loop i.e. G504R.G877R. The effect of substituting a hydrophobic phenylalanine residue for a similarly hydrophobic leucine residue at position 182 is less obvious but may relate to the reduced bulk of the side chain due to the absence of an aromatic benzene ring. Potentially, disease-associated missense mutations may produce a non-functional protein that is selectively targeted for degradation. Of note, the G504R variant is located adjacent to the P1 domain whereas the G877R variant occupies the P2 domain, with both domains implicated to play a role in the ATPase activity of ATP13A2 and may therefore produce a non- functional protein. Three heterozygous mutations localized to the intracellular N-terminal tail (T12M) and the intracellular catalytic loop (G533R and A746T) fail to have obvious effects on protein stability or subcellular localization suggesting that they may act through an alternate mechanism. Accordingly, heterozygous mutations (T12M, G533R and A746T) commonly impair the ATPase activity of microsomal-localized ATP13A2 whereas the homozygous F182L, G504R and G877R mutants fail to appre- ciably localize to microsomes and were thus refractory to analysis of ATPase activity. Missense mutations therefore appear to commonly act through a loss-of-function mechanism by exerting differential effects on the protein stability, subcellular localization or ATPase activity of ATP13A2. A potential caveat of the present study is that it relies on the transient overexpression of human ATP13A2 variants in human cell lines and cultured primary neurons at non-physiological expression levels. Consistent with our study, recent studies of KRS truncation mutants using similar overexpression approaches in a range of mammalian cells have revealed a destabilizing effect on the ATP13A2 protein resulting from its mislocalization to the ER and enhanced proteasomal degradation [5,11,13,24]. Collectively, there is strong agreement that PD-associated missense mutations and KRS mutations act through similar mechanisms to produce a loss-of-function effect. It will be important to confirm the destabilizing effects of missense mutations and truncating KRS mutations on the ATP13A2 protein in physiologically-relevant systems such as patient-derived cells or tissues, or knockin mouse models. Along these lines, cultured olfactory neurospheres harboring compound heterozygous ATP13A2 mutations (i.e. 3253delC frameshift and L1059R missense mutations) reveal a reduction in endogenous ATP13A2 mRNA levels primarily due to nonsense-mediated decay of the 3523delC mutant mRNA although ATP13A2 protein levels were not assessed in this model [11]. The impact of disease-associated mutations on the stability of the endogenous ATP13A2 protein awaits confirmation in future studies pending the availability of appropriate cellular models or tissues. PD-associated and KRS mutations share similar biochem- ical effects suggesting that ATP13A2 loss-of-function is primarily responsible for precipitating neurodegeneration. Recent studies have employed ATP13A2 knockdown in cultured cell lines or primary neurons, or patient-derived fibroblasts bearing ATP13A2 PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e39942