Edited by: Jean-Marc Taymans, KU Leuven, Belgium
Reviewed by: Daniele Bottai, University of Milan, Italy; Darren J. Moore, Swiss Federal Institute of Technology, Lausanne (EPFL), Switzerland
*Correspondence: Peter Vangheluwe, Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, ON1, Campus Gasthuisberg, KU Leuven, Herestraat 49, Box 802, B3000 Leuven, Belgium e-mail:
This article was submitted to the journal Frontiers in Molecular Neuroscience.
†These authors have contributed equally to this work.
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Mutations in
Neurodegenerative diseases, the fourth leading cause of death in developed countries, are characterized by progressive loss of neurons within the central nervous system leading to motor and cognitive dysfunction. Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common neurodegenerative disorders (Lees et al.,
A second hallmark of PD is the accumulation of aggregated α-synuclein into Lewy bodies (LBs) (Polymeropoulos et al.,
α-synuclein is mainly found at the presynaptic terminals of neurons (Maroteaux et al.,
α-synuclein aggregates accumulate in PD and are cleared via various routes, mainly including the ubiquitin-proteasome system, autophagy and lysosomal degradation pathways (Webb et al.,
ATP13A2 is a late endosomal/lysosomal P5-type transport ATPase that is emerging as a critical regulator of lysosomal functions (Ramirez et al.,
This review will focus on ATP13A2 as an orphan member of the family of P-type transport ATPases. P-type ATPases are a large family of evolutionarily related primary transporters present in Archaea, Bacteria and Eukarya (reviewed in Kuhlbrandt,
In the following sections we will give a short description of the architecture and transport mechanism of classical P-type transport ATPases. Then, we will provide an overview of those P-type ATPases that are implicated in neuronal disorders. Finally, we will review the recent literature of ATP13A2 and use available knowledge on P-type ATPase functions to discuss ATP13A2's putative cellular function and pathological role in PD.
P-type ATPases are biological pumps omnipresent in all forms of life, which are recognized by several conserved signature motifs associated with their catalytic mechanism (Axelsen and Palmgren,
According to sequence comparison and phylogenetic analysis, the P-type transport ATPase family can be classified into five distinct subfamilies (P1–P5), which can be further divided into additional subgroups (A, B, etc.) (Axelsen and Palmgren,
Based on the conserved P-type ATPase motifs, 36 human genes are recognized and annotated in databases to encode for P-type ATPases. These include 2 copper-ATPases, 4 Na+/K+-ATPases, 2 H+/K+-ATPases, 9 Ca2+-ATPases, 14 putative lipid flippases and 5 P5-type ATPases with unknown substrate specificity (ATP13A1-5). Figure
P-type ATPases use metabolic energy (ATP) to actively pump substrates against an electrochemical gradient. To prevent backflow of transported ligand(s), P-type ATPases use an alternating access mechanism (Figure
The transport mechanism of P-type ATPases can be described by the model of Post-Albers (Albers,
The transport process can be overall electrogenic if translocation occurs of an unequal amount of charges at both sides of the membrane. Examples are the Na+/K+-ATPase, which transports two Na+ ions out of the cell in exchange for three K+ ions per hydrolyzed ATP, and the SERCA Ca2+-ATPases, which transport two Ca2+ ions from the cytoplasm to the lumen of the ER/SR for two to three H+ in the other direction. Other P-type ATPases transport only in one direction using either the forward E1 to E1P step to bind the transported substrate (P1B copper-ATPase, P3 H+-ATPase) while others use the E2P step for substrate binding (P4 lipid flippases) (Kuhlbrandt,
The two archetypical members of the P-type ATPase family are the sarco(endo)plasmic reticulum (SR/ER) Ca2+-ATPase SERCA1a and the (α1 subunit of) Na+/K+-ATPase for which there is a wealth of structural and kinetic information. SERCA1a was the first P-type ATPase to have its structure solved at high resolution (Toyoshima et al.,
Crystal structures have also been presented for other P-type ATPases including the H+-ATPase of plants (Pedersen et al.,
The N-domain is the least conserved cytoplasmic domain among P-type ATPases and forms the ATP binding pocket. It is situated as a large insert into the P-domain sequence stretch and is connected by a highly flexible hinge region linking the N- and P-domains (Toyoshima et al.,
The M-domain, the largest of the four principal domains, comprises six to twelve α-helices (Bublitz et al.,
P-type ATPases also hold extended N- or C-terminal tails that regulate pump activity by intra-molecular interaction (Vandecaetsbeek et al.,
To translocate substrates across the membrane, P-type pumps undergo extensive conformational changes, which are driven by ATP hydrolysis. In the next section, the general catalytic mechanism of P-type ATPases will be explained based on the Ca2+ transport cycle of the SERCA1a pump (based on references in Toyoshima,
At the start of the catalytic cycle, cytosolic Ca2+ ions interact with the high-affinity binding sites in the M-domain in the E1 conformation. The binding of the two Ca2+ ions is sequential and cooperative. The first Ca2+ proceeds to site I where its binding repositions a critical Asp residue on M6 (D800), which now forms the second Ca2+ binding site II. Upon binding of the second Ca2+, the gating residue E309 on M4 will capture the second Ca2+ ion in site II.
Via the induced fit mechanism of Ca2+ binding, the rearrangement of the TM helices is transmitted to the P-domain creating a Mg2+-binding site near the critical Asp residue (Asp351 in SERCA1a). Presence of Mg2+ is essential as this cofactor decreases the electrostatic repulsion of the γ-phosphate of ATP by the negatively charged Asp and hence, allows phosphate transfer. In this way, ATPase activity of P-type transporters in the cytosolic domains is tightly coupled to the ion binding in the M-domain, preventing unnecessary ATP hydrolysis. The transition toward the intermediate E1~ P phosphorylated state bends the P-domain and tilts the A-domain that rests on the P-domain. This exerts strain on the linkers between the A-domain and M1, M2, and M3 of the M-domain. As a result, M1-M2 is partially lifted out of the membrane forcing E309 in a fixed position, which closes the Ca2+ entry path (occlusion).
Following complete γ-phosphate transfer, the ATP-mediated connection between the N- and P-domains is lost. As a consequence, the pump relaxes and the N-domain moves away from the catalytic site and stretches the linker region between the M3 helix and the A-domain. The generated tension triggers rotation of the A-domain and results in transition to the low-energy E2P state. The significant conformational changes associated with the E1P to E2P transition is the rate-limiting step in the catalytical cycle. The rearrangement of the pump opens the luminal exit pathway for Ca2+ by spreading out M1/M2 and M3/M4 away from M5/M6. In addition, this reduces the affinity of the Ca2+-binding sites promoting the luminal release of Ca2+. In exchange, two to three H+ ions bind with high affinity to the E2P state leading to occlusion of the luminal gate and further rotation of the A-domain. The A-domain rotation also brings the TGE loop closer to the phosphorylation site, shielding the aspartyl-phosphate by restricting the access of ADP or water.
The Ca2+-ATPase pump is reset to E1 by a series of reversal reactions leading to E2P dephosphorylation and proton countertransport. Entry of a water molecule induces a new rotation of the A-domain, which now precisely positions the Glu of the TGE-loop and the water molecule to catalyze an attack on the aspartyl phosphate. The rotation of the A-domain also repositions M1/M2 and the cation-binding site with the protons becomes occluded. Thereafter, the A-domain disengages from the phosphorylation site resulting in transition of the E2 state to the relaxed E1 conformation associated with release of the counterions.
In comparison with the well-studied SERCA1a Ca2+ pump, little is known about the P5-type ATPases to which ATP13A2 belongs. P5-type ATPases are found in all eukaryotic genomes, but are absent in bacterial genomes (Moller et al.,
The P5-ATPase membrane topology is unusual (Sorensen et al.,
Like other P-type ATPases, P5 isoforms, including ATP13A2, contain the key signature motifs KGAPE for ATP coordination (N-domain), DKTG for auto-phosphorylation (P-domain) and TGE for dephosphorylation (A-domain) (Kwasnicka-Crawford et al.,
The presence of a highly conserved M4 region within P5 ATPases indicates that in P5-type ATPases the hydrolysis of ATP may be coupled to the transport of a ligand close to the M4 region (Moller et al.,
The M4 segment of the P5-type ATPase corresponds to the putative substrate binding site and contains a double Pro in (PPxxP) (Moller et al.,
P-type ATPases play important roles in the nervous system, ranging from regulation of Ca2+ homeostasis, osmotic balance, electrical excitability, uptake of trace elements to vesicular transport processes. It is therefore not surprising to see that loss-of-function mutations in many P-type ATPase isoforms are detrimental for neuronal functions. In the following sections we will provide a short overview of those P-type ATPases that according to genetic information are implicated in neurological disorders (Table
P1B | ATP7A | Cu+ | Menkes disease (MD) | [OMIM:309400] |
Occipital horn syndrome (OHS) | [OMIM:304150] | |||
Spinal muscular atrophy, distal, X-linked 3 (SMAX3) | [OMIM:300489] | |||
P1B | ATP7B | Cu+ | Wilson disease (WD) | [OMIM:277900] |
Possible genetic risk factor for Alzheimer's disease (AD) and parkinsonism | Bull et al., |
|||
P2B | ATP2B3 | Ca2+ | Early onset X-linked spinocerebellar ataxia 1 | [OMIM:300014] |
P2C | ATP1A2 | Na+/K+ | Familial hemiplegic migraine type 2 (FHM2) | [OMIM:602481] |
Alternating hemiplegia of childhood 1 (AHC1) | [OMIM:104290] | |||
P2C | ATP1A3 | Na+/K+ | Rapid-onset dystonia parkinsonism (DYT12, RDP) | [OMIM:128235] |
Alternating hemiplegia of childhood 2 (AHC2) | [OMIM:614820] | |||
P4 | ATP8A2 | PS | Cerebellar ataxia, mental retardation and disequilibrium syndrome 4 (CAMRQ4) | [OMIM:615268] |
P4 | ATP10A | ? | Angelman syndrome (AS) | Blanco-Arias et al., |
P5B | ATP13A2 | ? | Kufor-Rakeb syndrome (KRS) | [OMIM:606693] |
Neuronal ceroid lipofuscinosis (NCL) | ||||
P5B | ATP13A4 | ? | Specific language impairment (SLI) | Gourdon et al., |
autism spectrum disorders (ASD) | Ugolino et al., |
The P1B-type ATPase subfamily consists of the two copper-transporting isoforms in human, ATP7A and ATP7B. ATP7A is ubiquitously expressed in all tissues, including the brain, but except the liver, regulating homeostatic maintenance of cell copper levels. ATP7B is highly expressed in the liver. Mutations in ATP7A are associated with Menkes disease (MD) (Chelly et al.,
Mutations in
ATP7A is targeted to the trans-Golgi network from where it supplies copper to the copper-dependent enzymes as they migrate through the secretory pathway. Under conditions of elevated copper, ATP7A relocalizes to the plasma membrane where it promotes the efflux of copper from cells (Petris et al.,
WD is an autosomal recessive disorder caused by mutations in
A number of single nucleotide polymorphisms in ATP7B are associated with an increased risk of AD (Squitti et al.,
The P2-type ATPases constitute the best characterized subfamily of P-type ATPases. The human P2 isoforms can be subdivided into three groups, P2A, P2B and P2C (P2D is not represented in humans, but consists of eukaryotic Na+-ATPases). The P2A group contains the well-known SERCA and Secretory Pathway/Golgi (SPCA) Ca2+-ATPases, whereas the plasma membrane Ca2+-ATPases (PMCAs) belong to the P2B-ATPases. The P2C-subgroup encompasses the Na+/K+-ATPases and the gastric H+/K+-pumps (Kuhlbrandt,
The Na+/K+-ATPase generates vital Na+ and K+ gradients over the plasma membrane by expelling three Na+ ions in exchange for two K+ ions. This is essential for many physiological functions in the nervous system such as cell volume control, the drive of secondary active transport systems and the support of electrical excitability (reviewed in Benarroch,
The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are autosomal dominant disorders caused by mutations of the Na+/K+-ATPase α2 (FHM2 and AHC1) and α3 (AHC2 and RDP) isoforms. α2 (ATP1A2) is primarily expressed in astrocytes and drives Na+-dependent Glu uptake and removes excess K+ from the extracellular space during neuronal excitation. α3 (ATP1A3) is predominantly expressed in neurons and is involved in post-stimulus recovery (reviewed in Brashear et al.,
According to the crystal structure of Na+/K+-ATPase, the C-terminal tail is inserted within a binding pocket between TM helices (Morth et al.,
PMCA isoforms (ATP2B1-4) remove Ca2+ from the cytosol to the extracellular environment.
The human genome encodes 14 P4-type ATPases, which are putative lipid flippases involved in aminophospholipid transport across membrane bilayers. P4-type ATPases are the first class of P-type transporters that do not transport inorganic ions (reviewed in Graham,
The P4-type ATPase ATP8A2 is a phosphatidylserine (PS) translocase, which is localized to the plasma membrane and highly expressed in retina and brain, particularly in the cerebellum. The P4 lipid flippase ATP8A2 is involved in localization of PS to the inner leaflet of the plasma membrane (Zhu et al.,
ATP10A is a putative aminophospholipid translocase.
Two members of the P5-type ATPases are implicated in neurological disorders. Little is known about the substrate specificity and cellular function of P5-type ATPases, but their putative function is extensively discussed in section 5 of this review.
Loss-of-function mutations in ATP13A2 (PARK9) are a known cause of Kufor-Rakeb syndrome (KRS), an autosomal recessive disorder characterized by juvenile-onset Parkinsonism associated with dementia (Ramirez et al.,
Whereas wild-type ATP13A2 is localized to late endosomal and lysosomal membranes (Ramirez et al.,
In dogs (Farias et al.,
ATP13A4 has been linked to language delay (Kwasnicka-Crawford et al.,
There is very little knowledge concerning the biological role of ATP13A4. In mice, Atp13a4 is mainly expressed in stomach and brain (Schultheis et al.,
A major bottleneck in unraveling the role of ATP13A2 in neurological disorders is the fact that virtually nothing is known concerning the molecular function and substrate specificity of the P5B-type ATPases. In this section, we will discuss the cellular role of ATP13A2 orthologs in different model organisms.
Ypk9p (
Only one gene, TATA-binding protein-associated factor-1,
The link between
The remaining majority of interactors related to
Yeast genes involved in Mn2+ protection mainly belong to categories of vesicle-mediated transport, vacuolar organization and chromatin remodeling (Chesi et al.,
Taken together, the genetic interaction data from yeast suggest a potential role for
CATP-6 locates depending on the tissue type to either cytoplasmic punctae likely corresponding to vesicles associated with the lysosome or to the plasma membrane (Lambie et al.,
The phenotype of a genetic knock-out mouse model of
As mentioned above, the
Twenty to twenty nine months old
In contrast, knocking out
At least three ATP13A2 splice variants are reported (Ugolino et al.,
The promoter region of the human ATP13A2 gene contains hypoxia response elements, which can bind to the transcription factor hypoxia inducible factor 1a (HIF-1a). Hypoxic conditions up-regulate transcription of the
The general accepted view is that ATP13A2 is targeted to acidic compartments,
Mitochondrial dysfunction is tightly linked to the pathogenesis of PD (Auluck et al.,
Studies in KRS patient-derived fibroblasts and ATP13A2-deficient cell lines have revealed that mutations of ATP13A2 or knockdown of the gene transcript lead to several lysosomal alterations. First the number and size of lysosomes is increased (Usenovic et al.,
In parallel, a strong link between ATP13A2 and mitochondrial dysfunction is emerging. Loss of ATP13A2 function impairs mitochondrial maintenance and leads to oxidative stress. ATP13A2 expression protects mammalian cells toward mitochondrial and oxidative stress (Covy et al.,
A role for ATP13A2 in autophagy and mitochondrial clearance has been suggested, but mechanistic details are lacking (Grunewald et al.,
The fact that ATP13A2 expression is upregulated under oxidative stress (Xu et al.,
Together, these observations suggest that ATP13A2 controls mitochondrial maintenance, which would lend further support to converging lysosomal and mitochondrial pathways in PD pathogenesis (Jin and Youle,
The long list of putative physical interactors (Usenovic et al.,
ATP13A2 protects cells toward α-synuclein toxicity. This has been observed in several model systems including yeast,
By regulating lysosomal functions ATP13A2 might control lysosomal α-synuclein degradation and prevent the build-up of α-synuclein aggregates (Dehay et al.,
Alternatively, ATP13A2 may control the delivery of α-synuclein to the lysosomes by regulating different autophagy pathways such as macro-autophagy (Dehay et al.,
α-synuclein may prevent the membrane fusion of the mitochondria resulting in increased mitochondrial fragmentation (Kamp et al.,
ATP13A2 promotes the removal of α-synuclein out of the cell via exosomes reducing the α-synuclein stress in cells (Kong et al.,
As α-synuclein interacts with membranes and the amount of α-synuclein interaction with the membrane seems to correlate with the degree of toxicity (Auluck et al.,
ATP13A2 causes protection toward several heavy metals (Gitler et al.,
Although P5B ATPases have essentially all sequence requirements to act as transporters, their ligands so far remain unidentified. ATP13A2 may regulate lysosomal function by transporting ions (Gitler et al.,
ATP13A2 has been linked to Mn2+, Zn2+, Mg2+, and H+ homeostasis suggesting that ATP13A2 might be implicated in the transport of these cations.
ATP13A2 was first suggested to be a lysosomal Mn2+ transporter (Gitler et al.,
A role of ATP13A2 in Mn2+ homeostasis and Mn2+ toxicity has been proposed based on the following observations. Loss of
For instance, instead of possibly transporting Mn2+ directly, ATP13A2 might influence other Mn2+ removal pathways which can depend on vesicular transport and/or other Mn2+ carriers. It is clear that efficient Mn2+ resistance in yeast depends on all steps in the secretory pathway involving proteins of vesicle-mediated transport, vacuolar organization and chromatin remodeling (Chesi et al.,
Several Mn2+ transport routes were studied in neuronal cells. Mn2+ is sequestered into the Golgi/secretory pathway compartments by SPCA1/PMR1, which also belongs to the P-type ATPase family (P2-type) (Vangheluwe et al.,
The ubiquitous ZIP14 is present in the plasma membrane and promotes Mn2+ uptake in SHSY5Y neuroblastoma cells. Conversely, SLC30A10 controls Mn2+ secretion in SHSY5Y cells, which is thought to be a Mn2+ transporter (Quadri et al.,
Besides Mn2+, Ypk9p protects yeast against other heavy metals, including Zn2+, Cd2+, Ni2+, and Se2+ (Gitler et al.,
Several recent studies underscored a strong link between ATP13A2 and Zn2+ homeostasis (Kong et al.,
Whether ATP13A2 pumps Zn2+ directly or rather affects other Zn2+ transporters or vesicular transport remains to be clarified. In the hONs, the majority of known secondary Zn2+ transporters were upregulated in
Nevertheless, the result of impaired ATP13A2 activity is a rise in cytosolic Zn2+ concentrations. Zn2+ dyshomeostasis has been associated with a variety of neurological disorders (Sensi et al.,
Zn2+ is not involved in redox reactions and therefore does not generate oxidative stress by itself. However, excessive mitochondrial Zn2+ uptake in conditions of high Zn2+ exposure inhibits several enzymes and complexes of the mitochondria leading to the production of ROS. This imposes oxidative stress, which can induce cell death (Park et al.,
In conclusion, it appears that the Zn2+ phenotype of
The ATP13A2 homologue Kil2 in
ATP13A2 is present in acidic compartments. Because loss of ATP13A2 leads to an elevated lysosomal pH, ATP13A2 might be involved in organellar acidification (Dehay et al.,
Establishing conclusively that ATP13A2 is a cation transporter will depend on a biochemical characterization of the purified pumps reconstituted in lipid vesicles that allow for measurements of transport. So far, we can only question whether the ATP13A2 protein supports Zn2+, Mn2+, Mg2+, or H+ transport. As explained before, the M4 region in P-type ATPases is critically involved in substrate coordination and specific M4 sequence motifs correlate well with substrate specificity. We first compared the sequence of ATP13A2 with SPCA, an established P-type Mn2+ transport ATPase (Figure
Coordination of Zn2+ typically involves His and/or Cys residues (Simonson and Calimet,
In conclusion, at least some residues in the M-region of ATP13A2 might support ion binding, but the overall composition significantly differs from other P-type Mn2+ and Zn2+ transporters. So until strong biochemical evidence becomes available, we may need to consider other possibilities.
Among the P-type ATPases the 14 members of the P4 ATPases transport other ligands than inorganic cations. Members of this subfamily flip phospholipids from one membrane leaflet to the other. Because P5 ATPases phylogenetically are more related to P4 ATPases than to any other P-type ATPase subfamily (Figure
Amongst other possible hypotheses, we should consider the possibility that ATP13A2 might be a flippase that transports a lipid or another organic molecule from one membrane leaflet to the other. Such an activity might in turn control vesicular dependent processes that regulate ion homeostasis, exosome formation, mitophagy/autophagy and α-synuclein clearance.
As a putative (lipid) flippase, ATP13A2 might alter membrane curvature, alter lipid dynamics, organize lipid microdomains or expose/remove important signaling molecules at one or the other membrane leaflet (Graham,
Although the cell biological context in which ATP13A2 is involved is gradually emerging, studying the molecular function and substrate specificity of ATP13A2 using biochemical methods and isolated systems will be required to unravel the substrate specificity and transport properties of ATP13A2. Understanding ATP13A2 at the molecular level will reveal its link to KRS, NCL, dystonia, and PD. This might open new therapeutic possibilities to treat this spectrum of disorders.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors appreciate the helpful discussions with Dr. F. Wuytack, Dr. P. Agostinis and Dr. V. Baekelandt (KU Leuven, Belgium). This work has been funded by the Rapid Response Innovation Award of the Michael J. Fox Foundation, the Jake's Ride Award of the Bachmann-Strauss Foundation, the Flanders Research Foundation FWO, the KU Leuven (OT/13/091) and the Danish National Research Foundation.
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