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.
Highlights
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]
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 contribute to early-onset PD
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)
Summary
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]. 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]. 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 contribute to early-onset PD. In contrast to truncating KRS mutations, the mechanism by which missense mutations cause parkinsonism or PD is unclear at present
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