ATP13A2 (PARK9) is a late endo-lysosomal transporter of unknown function that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome, a parkinsonism with dementia 1 and early-onset Parkinson's disease (PD) 2. ATP13A2 offers protection against genetic and environmental risk factors of PD, whereas loss of ATP13A2 compromises lysosomal function 3. The lysosomal transport function of ATP13A2 remained unclear, but here, we establish ATP13A2 as a lysosomal polyamine exporter with highest affinity for spermine. Polyamines stimulate the activity of purified ATP13A2, while disease mutants are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes cellular polyamine uptake via endocytosis and transports polyamines into the cytosol, which highlights a role for endo-lysosomes in cellular polyamine uptake. At high concentrations, polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with loss of ATP13A2 or its orthologues. Thus, defective lysosomal polyamine export is a new mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration. Our findings further shed light on the molecular identity of the elusive mammalian polyamine transport system. ATP13A2 is a P5B-ATPase belonging to the family of P-type ATPases, which couple ATP hydrolysis to substrate transport while transiently forming a catalytic phospho-intermediate 4. ATP13A2 is generally described as a heavy metal transporter 5 , but Ca 2+ 6 and the polyamine spermidine (SPD) 7,8 were also proposed. To screen for the transported substrate(s) of ATP13A2, we measured ATPase activity in the presence of various candidate substrates in solubilized microsomal membrane fractions of SH-SY5Y cells that overexpress human ATP13A2 wild type (WT) (WT-OE) or comparable levels of the catalytically dead D508N mutant (D508N-OE) 9,10. ATPase activity of ATP13A2 WT was significantly stimulated by the polyamines SPD and spermine (SPM) (Fig. 1a), whereas SPM had no effect on the D508N mutant (Extended Data Fig. 1a). MnCl2, ZnCl2, FeCl3, CaCl2, diamines, monoamines and amino acids exerted no effect (Extended Data Fig. 1a-3 d). The polyamines SPM, N 1-acetylspermine and SPD were able to stimulate ATPase activity in a concentration-dependent manner (Fig. 1b, Extended Data Fig. 1e) with the highest apparent affinity for SPM (Extended Data Table 1). The catalytic auto-phosphorylation and/or dephosphorylation reactions of P-type ATPases occur in response to binding of the transported substrate 4. ATP13A2 forms a phospho-intermediate on the D508 residue in the absence of SPM supplementation 9,10 , whereas SPM leads to a dose-dependent reduction in ATP13A2 phospho-enzyme levels (Fig. 1c), which is not seen with ornithine (Extended Data Fig. 1f). The dephosphorylation rate following a chase with non-radioactive ATP increased in the presence of...
Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson’s disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6. These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans 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.
P5B ATPases are present in the genomes of diverse unicellular and multicellular eukaryotes, indicating that they have an ancient origin, and that they are important for cellular fitness. Inactivation of ATP13A2, one of the four human P5B ATPases, leads to early-onset Parkinson’s disease (Kufor-Rakeb Syndrome). The presence of an invariant PPALP motif within the putative substrate interaction pocket of transmembrane segment M4 suggests that all P5B ATPases might have similar transport specificity; however, the identity of the transport substrate(s) remains unknown. Nematodes of the genus Caenorhabditis possess three paralogous P5B ATPase genes, catp-5, catp-6 and catp-7, which probably originated from a single ancestral gene around the time of origin of the Caenorhabditid clade. By using CRISPR/Cas9, we have systematically investigated the expression patterns, subcellular localization and biological functions of each of the P5B ATPases of C. elegans. We find that each gene has a unique expression pattern, and that some tissues express more than one P5B. In some tissues where their expression patterns overlap, different P5Bs are targeted to different subcellular compartments (e.g., early endosomes vs. plasma membrane), whereas in other tissues they localize to the same compartment (plasma membrane). We observed lysosomal co-localization between CATP-6::GFP and LMP-1::RFP in transgenic animals; however, this was an artifact of the tagged LMP-1 protein, since anti-LMP-1 antibody staining of native protein revealed that LMP-1 and CATP-6::GFP occupy different compartments. The nematode P5Bs are at least partially redundant, since we observed synthetic sterility in catp-5(0); catp-6(0) and catp-6(0) catp-7(0) double mutants. The double mutants exhibit defects in distal tip cell migration that resemble those of ina-1 (alpha integrin ortholog) and vab-3 (Pax6 ortholog) mutants, suggesting that the nematode P5Bs are required for ina-1and/or vab-3 function. This is potentially a conserved regulatory interaction, since mammalian ATP13A2, alpha integrin and Pax6 are all required for proper dopaminergic neuron function.
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