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...

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders characterized by progressive spasticity of the lower limbs due to degeneration of the corticospinal motor neurons. In a Bulgarian family with three siblings affected by complicated hereditary spastic paraplegia, we performed whole exome sequencing and homozygosity mapping and identified a homozygous p.Thr512Ile (c.1535C > T) mutation in ATP13A2. Molecular defects in this gene have been causally associated with Kufor-Rakeb syndrome (#606693), an autosomal recessive form of juvenile-onset parkinsonism, and neuronal ceroid lipofuscinosis (#606693), a neurodegenerative disorder characterized by the intracellular accumulation of autofluorescent lipopigments. Further analysis of 795 index cases with hereditary spastic paraplegia and related disorders revealed two additional families carrying truncating biallelic mutations in ATP13A2. ATP13A2 is a lysosomal P5-type transport ATPase, the activity of which critically depends on catalytic autophosphorylation. Our biochemical and immunocytochemical experiments in COS-1 and HeLa cells and patient-derived fibroblasts demonstrated that the hereditary spastic paraplegia-associated mutations, similarly to the ones causing Kufor-Rakeb syndrome and neuronal ceroid lipofuscinosis, cause loss of ATP13A2 function due to transcript or protein instability and abnormal intracellular localization of the mutant proteins, ultimately impairing the lysosomal and mitochondrial function. Moreover, we provide the first biochemical evidence that disease-causing mutations can affect the catalytic autophosphorylation activity of ATP13A2. Our study adds complicated hereditary spastic paraplegia (SPG78) to the clinical continuum of ATP13A2-associated neurological disorders, which are commonly hallmarked by lysosomal and mitochondrial dysfunction. The disease presentation in our patients with hereditary spastic paraplegia was dominated by an adult-onset lower-limb predominant spastic paraparesis. Cognitive impairment was present in most of the cases and ranged from very mild deficits to advanced dementia with fronto-temporal characteristics. Nerve conduction studies revealed involvement of the peripheral motor and sensory nerves. Only one of five patients with hereditary spastic paraplegia showed clinical indication of extrapyramidal involvement in the form of subtle bradykinesia and slight resting tremor. Neuroimaging cranial investigations revealed pronounced vermian and hemispheric cerebellar atrophy. Notably, reduced striatal dopamine was apparent in the brain of one of the patients, who had no clinical signs or symptoms of extrapyramidal involvement.

ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor-Rakeb syndrome and Parkinson's disease (PD), providing protection against α-synuclein, Mn 2+ , and Zn 2+ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders.mitochondria | lysosome | flippase | α-synuclein | P5-type ATPase N euronal fitness depends on optimal lysosomal function and efficient lysosomal delivery of proteins and organelles by autophagy for subsequent breakdown (1, 2). Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of Parkinson's disease (PD) associated with dementia, which is caused by mutations in ATP13A2/PARK9 (3). Mutations in or knockdown (KD) of ATP13A2 lead to lysosomal dysfunctions, including reduced lysosomal acidification, decreased degradation of lysosomal substrates (4), impaired autophagosomal flux (4, 5), and accumulation of fragmented mitochondria (5, 6). By contrast, overexpression (OE) of Ypk9p (i.e., the yeast ATP13A2 ortholog) protects yeast against toxicity of α-synuclein (7), which is the major protein in Lewy bodies, the abnormal protein aggregates that develop inside nerve cells in PD. This protective effect of ATP13A2 on α-synuclein toxicity is conserved in yeast, Caenorhabditis elegans, and rat neuronal cells (7). Because ATP13A2 imparts resistance to Mn 2+ (7-9) and Zn 2+ (10-12), it was proposed that ATP13A2 may function as a Mn 2+ (7-9) and/or Zn 2+ transporter (10-12).ATP13A2 belongs to the P5 subfamily of the P-type ATPase superfamily, which comprises five subfamilies (P1-5) of membrane transporters. P-type ATPases hydrolyze ATP to actively transport inorganic ions across membranes or lipids between membrane leaflets (reviewed in ref. 13). During the transport cycle, a phosphointermediate is formed on a conserved aspartate residue (14). The human P5-type ATPases are divided into two groups, P5A (ATP13A1) and P5B (ATP13A2-5), but their transport specificity has not been established (14-16).P-type ATPases comprise a membrane-embedded core of six transmembrane (TM) helices (M1-6) that form the substrate binding site(s) and entrance/exit pathways for the transp...