Protection against Mitochondrial and Metal Toxicity Depends on Functional Lipid Binding Sites in ATP13A2

Figure 4

PA and PI(3,5)P2 provide ATP13A2-mediated protection to PD-related metal toxicity. ((a)-(b)) The SHSY5Y cell models stably overexpressing FLUC, WT-OE, or sh-ATP13A2 were exposed for 24 h to rotenone (Rot, 1 μM, positive control) and MPP+ (50 μM), as well as the heavy metals Zn²⁺ (150 μM), Mn²⁺ (2 μM), and Fe³⁺ (1.5 mM). Cell viability was evaluated by the MUH assay (a), whereas cell death was assessed by propidium iodide (PI) stained flow cytometry (b). To test whether the protective role of ATP13A2 depends on the signaling lipids PI(3,5)P2 and PA, the SHSY5Y cell lines were pretreated for 1 h with the PIKfyve inhibitor (200 nM; P or PIK) and/or the phospholipase D inhibitor (100 nM; F or FIPI) prior to the addition of the heavy metals Zn²⁺ (c), Mn²⁺ (d), and Fe³⁺ (e). The level of cellular protection was evaluated by PI stained flow cytometry. Data are the mean of 3 independent experiments ± SD. statistical differences between FLUC and WT-OE, statistical differences within cell line following treatment with inhibitor (1 mark, ; 2 marks, ; 3 marks, ) (ANOVA with Bonferroni post hoc test).

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