Fig 2: 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, P < 0.05; 2 marks, P < 0.01; 3 marks, P < 0.001) (ANOVA with Bonferroni post hoc test).

Fig 3: PA and PI(3,5)P2 mediated protection to mitochondrial stress occurs via ATP13A2. SHSY5Y cells stably expressing FLUC, shRNA for KD of ATP13A2, ATP13A2, and full length mutants of ATP13A2 with mutated putative lipid binding sites (LBS1–3) were exposed to rotenone (Rot, 1 μM) in the presence or absence of 1 h pretreatment with the pharmacological inhibitors of PIKfyve lipid kinase (((a) and (b)) YM-201636, 200 nM) or phospholipase D (((c) and (d)) FIPI, 100 nM). Following 24 h exposure to rotenone, cells were assessed for cell viability ((a) and (c)) or death ((b) and (d)) by, respectively, MUH and propidium iodide (PI) assays. Data are the mean of 3 independent experiments ± SD. $ statistical differences between FLUC and WT-OE/sh-ATP13A2/LBS1/LBS2/LBS3/LBS1.2.3, ∗ statistical differences within cell line following treatment with inhibitor (1 mark, P < 0.05; 2 marks, P < 0.01; 3 marks, P < 0.001) (ANOVA with Bonferroni post hoc test).

Fig 4: LBS mutations prevent ATP13A2-mediated cytoprotection. (a) ATP13A2's protective response at 24 h exposure to rotenone (Rot, 1 μM) and the effect of pharmacological inhibition of PIKfyve lipid kinase with YM-201636 (PIK or P, 200 nM) and inhibition of PLD with FIPI (F, 100 nM) on cell death were assessed via a propidium iodide (PI) based assay. $ statistical differences between FLUC and WT-OE/sh-ATP13A2, ∗ statistical differences within cell line following treatment with inhibitor (1 mark, P < 0.05; 2 marks, P < 0.01; 3 marks, P < 0.001) (ANOVA with Bonferroni post hoc test). ((b)–(e)) Stable cell lines were exposed to 1 μM rotenone ((b)-(c)) or 50 μM MPP+ ((d)-(e)) for 24 h and cell death was assessed by PI stained flow cytometry, whereas cell viability was assayed by the MUH protocol. Data are the mean of 3 independent experiments ± SD. ∗ statistical differences between FLUC and WT-OE/sh-ATP13A2/LBS1/LBS2/LBS3/LBS1.2.3 (1 mark, P < 0.05; 2 marks, P < 0.01; 3 marks, P < 0.001) (ANOVA with Bonferroni post hoc test).

Fig 5: The capacity of ATP13A2 to interact with PA/PI(3,5)P2 is essential to protect against heavy metal-induced cytotoxicity. SHSY5Y cell lines stably overexpressing FLUC, ATP13A2 shRNA, ATP13A2 WT, and LBS1–3 mutants were exposed to toxic heavy metal concentrations of Zn2+ (150 μM ((a) and (b))) or Mn2+ (2 μM ((c) and (d))). Cell death induction was assessed by propidium iodide (PI ) based flow cytometry ((a) and (c)) and cell viability by MUH assay ((b) and (d)). Data are the mean of 3 independent experiments ± SD. ∗ statistical differences between FLUC and WT-OE/sh-ATP13A2/LBS1/LBS2/LBS3/LBS1.2.3 (1 mark, P < 0.05; 2 marks, P < 0.01; 3 marks, P < 0.001) (ANOVA with Bonferroni post hoc test).