Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9 ) Associated with Early-Onset Parkinsonism Agata Podhajska, Alessandra Musso, Alzbeta Trancikova, Klodjan Stafa, Roger Moser, Sarah Sonnay, Liliane Glauser, Darren J. Moore* Laboratory of Molecular Neurodegenerative Research, Brain Mind Institute, School of Life Sciences, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Lausanne, Switzerland Abstract Mutations in the ATP13A2 gene (PARK9) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS), a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s) by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and heterozygous missense mutations in ATP13A2 associated with early-onset forms of parkinsonism. Citation: Podhajska A, Musso A, Trancikova A, Stafa K, Moser R, et al. (2012) Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9) Associated with Early-Onset Parkinsonism. PLoS ONE 7(6): e39942. doi:10.1371/journal.pone.0039942 Editor: Charleen T. Chu, University of Pittsburgh, United States of America Received February 8, 2012; Accepted May 29, 2012; Published June 29, 2012 Copyright: 2012 Podhajska et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from the Michael J. Fox Foundation for Parkinson’s Research (D.J.M.), Swiss National Science Foundation (grant no. 310030_127478 to D.J.M.), and the Ecole Polytechnique Fe´de´rale de Lausanne (D.J.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: darren.moore@epfl.ch Introduction In recent years, a number of genes have been identified that are associated with autosomal recessive forms of parkinsonism including parkin (PARK2), DJ-1 (PARK7), PINK1 (PARK6) and ATP13A2 (PARK9) [1,2,3]. Mutations in the ATP13A2 gene cause Kufor-Rakeb syndrome (KRS), a juvenile-onset pallido-pyramidal neurodegenerative disorder characterized by slowly progressive levodopa-responsive parkinsonism often with additional features including supranuclear gaze palsy, pyramidal dysfunction, dysto- nia and dementia [4,5,6]. KRS subjects normally exhibit generalized brain atrophy with evidence of impaired nigrostriatal dopaminergic function [7,8,9,10]. Homozygous or compound heterozygous mutations have been identified in KRS subjects of families from Jordan, Chile, Afghanistan, Pakistan and China that produce frameshift or splicing variants resulting in truncated forms of ATP13A2 protein that are predicted to lead to a loss-of-function [5,8,11,12]. Recent studies have shown that such truncating mutations promote the mislocalization of ATP13A2 to the endoplasmic reticulum (ER) in mammalian cells where they are degraded by the proteasome via the ER-associated degradation (ERAD) pathway [5,11,13]. A number of homozygous (F182L [Japan] [14], G504R [Brazil] [10] and G877R [Italy] [9]) and heterozygous (T12M [Italy] [10], G533R [Italy] [10] and A746T [Taiwan/Singapore] [15]) missense mutations have recently been identified in subjects with early-onset forms of familial or sporadic parkinsonism or Parkinson’s disease (PD) suggesting that ATP13A2 mutations may also contribute to early-onset PD. Of interest, homozygous mutations (F182L, G504R and G877R) typically cause juvenile-onset parkinsonism (10 to 22 years) whereas heterozygous mutations (T12M, G533R and A746T) are associ- ated with early-onset parkinsonism (,50 years) [9,10,14,15], potentially suggesting a gene dosage or graded effect of mutations. In contrast to truncating KRS mutations, the mechanism by which PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39942