ATP13A2 deficiency disrupts lysosomal polyamine export

Abstract

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...

Mutated ATP10B increases Parkinson’s disease risk by compromising lysosomal glucosylceramide export

“…The eGFP-labeled p.D433N mutant is also well-expressed in the endo-lysosomal compartments of HeLa cells (Fig. S4c, d), and due to its lack of enzymatic activity the p.D433N mutant serves as an important catalytic control for the functional assays to determine the biological transport activity of ATP10B, similar as in [111]. The p.D433N mutant is further used throughout this study as a loss-of-function control for the evaluation of the disease associated mutations.…”

“…Significance was assessed by one-way ANOVA with either Dunnett's post hoc (e, to Rab11; a, c to mirFluc) or Tukey's (g, i) post hoc whereby, 1 mark P < 0.05, 2 marks P < 0.01, 3 marks P < 0.001, 4 marks P < 0.0001 linked to PD [28,90]. As a lysosomal polyamine exporter, ATP13A2 provides protection to rotenone and MnCl 2 [68,111,112], and also maintains lysosomal functionality and membrane integrity [111], pointing to a remarkable synergy between the two lysosomal export systems. Rotenone is a mitochondrial complex I inhibitor, whereas MnCl 2 may inhibit the mitochondrial respiratory chain at the level of complex II [43,102].…”

Genetic perspective on the synergistic connection between vesicular transport, lysosomal and mitochondrial pathways associated with Parkinson’s disease pathogenesis

“…1) [23,128,258,336,345]. Recently, ATP13A2 was identified as a lysosomal polyamine exporter with a high affinity for spermine [348]. The protein is highly expressed in the brain, especially in the substantia nigra.…”

“…Expression of wildtype but not mutant ATP13A2 protects mammalian cell lines and primary rat neuronal cultures against manganese induced cell death, also known as a PD environmental risk factor [336]. High concentrations of polyamines was shown to induce cell toxicity, which exacerbated by ATP13A2 loss due to lysosomal dysfunction [348]. Additionally, ATP13A2 has been shown to be involved in α-synuclein metabolism [205] and lipid homeostasis [212].…”

LRRK2 and the Endolysosomal System in Parkinson’s Disease

“…Additionally, common coding or noncoding variants at several distinct genomic loci have been discovered to modify risk of developing PD (i.e., GBA, LRRK2, MAPT, SNCA, SCARB2, GAK, SH3GL2, TMEM175, ATP6V0A1, GALC, CTSB) [7][8][9][10][11][12][13][14][15][16][17][18]. Many genes linked to PD or parkinsonism play roles in endocytosis (LRRK2, SNCA, DNAJC6, SYNJ1, GAK, SH3GL2) [19][20][21][22][23][24][25][26], vesicular trafficking/sorting (LRRK2, VPS35, RAB39B) [27][28][29], mitophagy (Parkin, PINK1) [30][31][32], or lysosomal function (ATP13A2, GBA, SCARB2, TMEM175, ATP6V0A1, GALC, CTSB) [33][34][35][36][37][38][39][40][41][42], broadly implicating the endolysosomal pathway in PD.…”