Tine Holemans

Cellular function and pathological role of ATP13A2 and related P-type transport ATPases in Parkinson's disease and other neurological disorders.

Mutations in ATP13A2 lead to Kufor-Rakeb syndrome, a parkinsonism with dementia. ATP13A2 belongs to the P-type transport ATPases, a large family of primary active transporters that exert vital cellular functions. However, the cellular function and transported substrate of ATP13A2 remain unknown. To discuss the role of ATP13A2 in neurodegeneration, we first provide a short description of the architecture and transport mechanism of P-type transport ATPases. Then, we briefly highlight key P-type ATPases involved in neuronal disorders such as the copper transporters ATP7A (Menkes disease), ATP7B (Wilson disease), the Na+/K+-ATPases ATP1A2 (familial hemiplegic migraine) and ATP1A3 (rapid-onset dystonia parkinsonism). Finally, we review the recent literature of ATP13A2 and discuss ATP13A2s putative cellular function in the light of what is known concerning the functions of other, better-studied P-type ATPases. We critically review the available data concerning the role of ATP13A2 in heavy metal transport and propose a possible alternative hypothesis that ATP13A2 might be a flippase. As a flippase, ATP13A2 may transport an organic molecule, such as a lipid or a peptide, from one membrane leaflet to the other. A flippase might control local lipid dynamics during vesicle formation and membrane fusion events.

A lipid switch unlocks Parkinson's disease- associated ATP13A2

Significance ATP13A2 is a lysosomal transporter that is genetically linked to an autosomal recessive variant of Parkinson’s disease and confers protection against α-synuclein toxicity in neurons. Here we show that an N-terminal hydrophobic domain of ATP13A2 specifically recognizes signaling lipids. Interactions with these signaling lipids enhance cytoprotection to mitochondrial stress. This study provides essential information for establishing the lysosomal function of ATP13A2 and suggests a therapeutic applicability in activating ATP13A2. 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, Mn2+, and Zn2+ 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.

Loss-of-function mutations in the ATP13A2/PARK9 gene cause complicated hereditary spastic paraplegia (SPG78)

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.

Towards defining the substrate of orphan P5A-ATPases.

BACKGROUND P-type ATPases are ubiquitous ion and lipid pumps found in cellular membranes. P5A-ATPases constitute a poorly characterized subfamily of P-type ATPases present in all eukaryotic organisms but for which a transported substrate remains to be identified. SCOPE OF REVIEW This review aims to discuss the available evidence which could lead to identification of possible substrates of P5A-ATPases. MAJOR CONCLUSIONS The complex phenotypes resulting from the loss of P5A-ATPases in model organisms can be explained by a role of the P5A-ATPase in the endoplasmic reticulum (ER), where loss of function leads to broad and unspecific phenotypes related to the impairment of basic ER functions such as protein folding and processing. Genetic interactions in Saccharomyces cerevisiae point to a role of the endogenous P5A-ATPase Spf1p in separation of charges in the ER, in sterol metabolism, and in insertion of tail-anchored proteins in the ER membrane. A role for P5A-ATPases in vesicle formation would explain why sterol transport and distribution are affected in knock out cells, which in turn has a negative impact on the spontaneous insertion of tail-anchored proteins. It would also explain why secretory proteins destined for the Golgi and the cell wall have difficulties in reaching their final destination. Cations and phospholipids could both be transported substrates of P5A-ATPases and as each carry charges, transport of either might explain why a charge difference arises across the ER membrane. GENERAL SIGNIFICANCE Identification of the substrate of P5A-ATPases would throw light on an important general process in the ER that is still not fully understood. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.

Protection against mitochondrial and metal toxicity depends on functional lipid binding sites in ATP13A2

The late endo-/lysosomal P-type ATPase ATP13A2 (PARK9) is implicated in Parkinsons disease (PD) and Kufor-Rakeb syndrome, early-onset atypical Parkinsonism. ATP13A2 interacts at the N-terminus with the signaling lipids phosphatidic acid (PA) and phosphatidylinositol (3,5) bisphosphate (PI(3,5)P2), which modulate ATP13A2 activity under cellular stress conditions. Here, we analyzed stable human SHSY5Y cell lines overexpressing wild-type (WT) or ATP13A2 mutants in which three N-terminal lipid binding sites (LBS1–3) were mutated. We explored the regulatory role of LBS1–3 in the cellular protection by ATP13A2 against mitochondrial stress induced by rotenone and found that the LBS2-3 mutants displayed an abrogated protective effect. Moreover, in contrast to WT, the LBS2 and LBS3 mutants responded poorly to pharmacological inhibition of, respectively, PI(3,5)P2 and PA formation. We further demonstrate that PA and PI(3,5)P2 are also required for the ATP13A2-mediated protection against the toxic metals Mn2+, Zn2+, and Fe3+, suggesting a general lipid-dependent activation mechanism of ATP13A2 in various PD-related stress conditions. Our results indicate that the ATP13A2-mediated protection requires binding of PI(3,5)P2 to LBS2 and PA to LBS3. Thus, targeting the N-terminal lipid binding sites of ATP13A2 might offer a therapeutic approach to reduce cellular toxicity of various PD insults including mitochondrial stress.

Measuring Ca2+-Dependent Ca2+-Uptake Activity in the Mouse Heart

The apparent Ca(2+) affinity of the isoforms of the sarco/endoplasmic reticulum Ca(2+) ATPase SERCA2 is controlled primarily by two proteins, phospholamban (PLB) and sarcolipin (SLN). The rate of ATP-driven Ca(2+) uptake into sarcoplasmic reticulum (SR)-derived vesicles can be monitored by a technique in which the net uptake of (45)Ca(2+) in the form of an intravesicular calcium oxalate precipitate is recorded. Here, we present details of a modification of such a protocol for determining the apparent Ca(2+) affinity of the Ca(2+) pump, and its control by various regulators, in crude homogenates of mouse heart.

ATP13A2/PARK9 regulates endo-/lysosomal cargo sorting and proteostasis through a novel PI(3, 5)P2-mediated scaffolding function

&NA; ATP13A2 (also called PARK9), is a transmembrane endo‐/lysosomal‐associated P5 type transport ATPase. Loss‐of‐function mutations in ATP13A2 result in the Kufor‐Rakeb Syndrome (KRS), a form of autosomal Parkinsons disease (PD). In spite of a growing interest in ATP13A2, very little is known about its physiological role in stressed cells. Recent studies suggest that the N‐terminal domain of ATP13A2 may hold key regulatory functions, but their nature remains incompletely understood. To this end, we generated a set of melanoma and neuroblastoma cell lines stably overexpressing wild‐type (WT), catalytically inactive (D508N) and N‐terminal mutants, or shRNA against ATP13A2. We found that under proteotoxic stress conditions, evoked by the proteasome inhibitor Bortezomib, endo‐/lysosomal associated full‐length ATP13A2 WT, catalytically‐inactive or N‐terminal fragment mutants, reduced the intracellular accumulation of ubiquitin‐conjugated (Ub) proteins, independent of autophagic degradation. In contrast, ATP13A2 silencing increased the intracellular accumulation of Ub‐proteins, a pattern also observed in patient‐derived fibroblasts harbouring ATP13A2 loss‐of function mutations. In treated cells, ATP13A2 evoked endocytic vesicle relocation and increased cargo export through nanovesicles. Expression of an ATP13A2 mutant abrogating PI(3,5)P2 binding or chemical inhibition of the PI(3,5)P2‐generating enzyme PIKfyve, compromised vesicular trafficking/nanovesicles export and rescued intracellular accumulation of Ub‐proteins in response to proteasomal inhibition. Hence, our study unravels a novel activity‐independent scaffolding role of ATP13A2 in trafficking/export of intracellular cargo in response to proteotoxic stress.

Unlocking ATP13A2/PARK9 activity

ATP13A2 (OMIM 610513) is a lysosomal P-type ATPase implicated in neurological disorders including Kufor-Rakeb Syndrome and Parkinsons disease, providing protection against stressors such as α-synuclein and heavy metal toxicity incited by Mn2+ and Zn2+. To date, the molecular function underlying ATP13A2s protection remains uncertain, despite significant advances in its cellular contributions, with notable roles of ATP13A2 in lysosomal function, autophagy, mitochondrial maintenance and exosome release (reviewed in1).

Measuring Ca2+ pump activity in overexpression systems and cardiac muscle preparations

Sarco-/endoplasmic reticulum (SR/ER) Ca(2+) pumps (SERCAs) build up vital Ca(2+) gradients across the intracellular SR/ER membrane, helping to control cell function, proliferation, growth, differentiation, and death. We describe two techniques to measure the SERCA activity either in mammalian culture cells overexpressing SERCAs or in muscle tissue containing high levels of endogenous SERCAs. As Ca(2+) transport is tightly coupled to ATP hydrolysis, it is possible to determine the rate of Ca(2+)-dependent ATP hydrolysis and use it as a measure for SERCA activity or, in a second approach, to quantify ATP-stimulated uptake of radioactive (45)Ca(2+). Here, we first provide an overview of the mechanism of Ca(2+)-transport ATPases and show how this can be taken advantage of in protocols for measuring Ca(2+) pump activity.

High-Throughput Measurement of the Ca2+-Dependent ATPase Activity in COS Microsomes

We provide a detailed procedure to determine the Ca(2+)-dependent ATPase activity in COS or HEK293 cells overexpressing a Ca(2+) pump. The ATPase activity is determined by the Baginsky method, which allows measurement of the steady-state production of inorganic phosphate (Pi). We have adapted this widely applied method into a sensitive, fast, and semi-high-throughput protocol suitable for use in a 96-well plate format.