Consequently, dysfunctional ATP13A2 sensitizes cells to oxidative stress, which impairs mitochondria, and induces toxicity and cell death. death. ATP13A2-mediated polyamine transport represents a conserved pathway that protects against mitochondrial oxidative stress. The combined protective impact of ATP13A2 on lysosomal health and mitochondrial oxidative stress may explain why ATP13A2 exerts potent neuroprotective effects. (strain deficient in the ATP13A2 ortholog ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export. Loss-of-function mutations in (that lysosomal polyamine export by ATP13A2 effectively lowers ROS levels and promotes mitochondrial health and functionality, pointing to a lysosomal-dependent cell protective pathway that may be implicated in ATP13A2-related neurodegenerative disorders. Results ATP13A2 Protects Cells and Mitochondria against Mitochondrial Toxins. We previously validated stable human neuroblastoma (SH-SY5Y) cell models with either ATP13A2 knockdown (sh-ATP13A2/kd) or wild-type ATP13A2 overexpression (WT-OE), and control cell lines with overexpression or shRNA-mediated knockdown of firefly luciferase (Fluc or sh-Fluc, respectively) (16). ATP13A2 expression offered protection, whereas knockdown exacerbated toxicity against rotenone (17) (Fig. 1and and and and and < 0.05; **< 0.01; ***< 0.001; ****< 0.0001; ns, nonsignificant versus respective untreated unless depicted otherwise; ANOVA post hoc Tukeys multiple comparison test. Loss of ATP13A2 Increases Mitochondrial ROS, Which Activates an ATF4-Dependent Stress Response. Since ATP13A2s transported substrates spermine and spermidine are potent antioxidants (14, 15), and rotenone causes ROS accumulation (28), we examined the impact of ATP13A2 on ROS levels. Using the probes 2?,7?-dichlorofluorescin diacetate (DCFDA) (29) and MitoSOX (30) in the SH-SY5Y cell models, we showed that rotenone, MPP+, and 6-OHDA increased the production of a broad range of ROS (and and < 0.05; **< 0.01; ***< 0.001; ****< 0.0001; ns, nonsignificant versus respective untreated unless depicted otherwise; ANOVA post hoc Tukeys multiple comparison test. It has been reported that MitoROS accumulation leads to increased ATF4 expression, a transcription factor that activates an antioxidant response and IRL-2500 promotes mitochondrial homeostasis (31). Also, ATP13A2 kd cells presented a strong up-regulation of ATF4, peaking at 6 h when challenged with rotenone (Fig. 3< 0.05, **< 0.01, ***< 0.001, ****< 0.0001 versus respective untreated unless depicted otherwise; ####< 0.0001 versus rotenone-treated sh-Fluc; ANOVA post hoc Tukeys multiple comparison test. The mitochondrial phenotype and stress response observed in ATP13A2 kd cells are most likely caused by the induction of MitoROS, rather than as a consequence of cell death. Indeed, kd cells already presented a clear increase in MitoROS (and and < 0.01; ***< 0.001; ****< 0.0001; ns, nonsignificant versus respective untreated unless depicted otherwise, ANOVA post hoc Tukeys multiple comparison test. We previously reported that ATP13A2 activity critically contributes to the native polyamine content as well as the cellular uptake of BODIPY-labeled polyamines spermidine and IRL-2500 spermine (9, 34), reflecting the biochemical activity of ATP13A2 (9). This explains why Fluc cells exhibited higher cellular uptake of the BODIPY-spermine and IRL-2500 -spermidine as compared to ATP13A2 kd (and and and < 0.05, **< 0.01, Mouse monoclonal to IGFBP2 ***< 0.001, ****< 0.0001; ns, nonsignificant versus respective untreated (and and and test (test (strain that is defective in animals were hypersensitive to rotenone (Fig. 6strain, which was further aggravated in the presence of rotenone (Fig. 6and and animals, and further increased by rotenone exposure (Fig. 6and and rescued the MitoROS phenotype, while the catalytically inactive mutant did not, demonstrating the requirement of transport activity (Fig. 6reporter, a marker for expression (40), we observed that mutant worms presented a constitutively induced mitochondrial stress response (Fig. 6and (Fig. 6and and reporter (Fig. 6and is at least partially responsible for the up-regulation in the animals. Of the three orthologs, appears the most relevant isoform that phenocopies mammalian ATP13A2 (39), since the triple null mutant displayed a similar MMP phenotype to the animals (exerts a mitochondrial protective antioxidant function in vivo in and strains carrying a loss-of-function mutation ((WT) or a catalytically inactive mutant (D465N), were exposed to rotenone (Rot, 10 M) (and and reporter. In or with < 0.01, ***< 0.001, ****< 0.0001; ns, nonsignificant versus respective untreated unless otherwise indicated; ##< 0.01, ####< 0.0001 versus rotenone-treated control; ANOVA or KruskalCWallis with post hoc Tukeys (counters mitochondrial oxidative stress in (50) and (51). Also, ATP13A2 KO and KO/D508N cells display a strong reduction in polyamine IRL-2500 levels, indicating that ODC activity is unable to compensate for the loss of polyamine uptake, despite an up-regulation of ODC mRNA expression (wild-type ATP13A2 (WT-OE), catalytically deficient ATP13A2 (D508N-OE) or sh-ATP13A2 (kd) were generated via lentiviral transduction as described previously (16). We made use of two independent polyclonal lines with ATP13A2 knockdown (kd1 and kd2, since two different shRNAs targeting ATP13A2 were used), of which the mean is reported in IRL-2500 the results. Immunoblots show the independent clonal cell lines. KO of ATP13A2 and subsequent lentiviral transduction.