Loss of P-type ATPase ATP13A2/PARK9 function induces general lysosomal deficiency and leads to Parkinson disease neurodegeneration

Parkinson disease (PD) is a progressive neurodegenerative disorder pathologically characterized by the loss of dopaminergic neurons from the substantia nigra pars compacta and the presence, in affected brain regions, of protein inclusions named Lewy bodies (LBs). The ATP13A2 gene (locus PARK9) encodes the protein ATP13A2, a lysosomal type 5 P-type ATPase that is linked to autosomal recessive familial parkinsonism. The physiological function of ATP13A2, and hence its role in PD, remains to be elucidated. Here, we show that PD-linked mutations in ATP13A2 lead to several lysosomal alterations in ATP13A2 PD patient-derived fibroblasts, including impaired lysosomal acidification, decreased proteolytic processing of lysosomal enzymes, reduced degradation of lysosomal substrates, and diminished lysosomal-mediated clearance of autophagosomes. Similar alterations are observed in stable ATP13A2-knockdown dopaminergic cell lines, which are associated with cell death. Restoration of ATP13A2 levels in ATP13A2-mutant/depleted cells restores lysosomal function and attenuates cell death. Relevant to PD, ATP13A2 levels are decreased in dopaminergic nigral neurons from patients with PD, in which ATP13A2 mostly accumulates within Lewy bodies. Our results unravel an instrumental role of ATP13A2 deficiency on lysosomal function and cell viability and demonstrate the feasibility and therapeutic potential of modulating ATP13A2 levels in the context of PD.     

Protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial Ca2+ stress by melatonin's cascade metabolites C3‐OHM and AFMK in RBA1 astrocytes

Cyclic 3‐hydroxymelatonin (C3‐OHM) and N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK) are two major cascade metabolites of melatonin. We previously showed melatonin provides multiple levels of mitochondria‐targeted protection beyond as a mitochondrial antioxidant during ionomycin‐induced mitochondrial Ca²⁺ (mCa²⁺) stress in RBA1 astrocytes. Using noninvasive laser scanning fluorescence coupled time‐lapse digital imaging microscopy, this study investigated whether C3‐OHM and AFMK also provide mitochondrial levels of protection during ionomycin‐induced mCa²⁺ stress in RBA1 astrocytes. Interestingly, precise temporal and spatial dynamic live mitochondrial images revealed that C3‐OHM and AFMK prevented specifically mCa²⁺‐mediated mitochondrial reactive oxygen species (mROS) formation and hence mROS‐mediated depolarization of mitochondrial membrane potential (△Ψ_m) and permanent lethal opening of the MPT (p‐MPT). The antioxidative effects of AFMK, however, were less potent than that of C3‐OHM. Whether C3‐OHM and AFMK targeted directly the MPT was investigated under a condition of “oxidation free‐Ca²⁺ stress” using a classic antioxidant vitamin E to remove mCa²⁺‐mediated mROS stress and the potential antioxidative effects of C3‐OHM and AFMK. Intriguingly, two compounds still effectively postponed “oxidation free‐Ca²⁺ stress”‐mediated depolarization of △Ψ_m and p‐MPT. Measurements using a MPT pore‐specific indicator Calcein further identified that C3‐OHM and AFMK, rather than inhibiting, stabilized the MPT in its transient protective opening mode (t‐MPT), a critical mechanism to reduce overloaded mROS and mCa²⁺. These multiple layers of mitochondrial protection provided by C3‐OHM and AFMK thus crucially allow melatonin to extend its metabolic cascades of mitochondrial protection during mROS‐ and mCa²⁺‐mediated MPT‐associated apoptotic stresses and may provide therapeutic benefits against astrocyte‐mediated neurodegeneration in the CNS.     

Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2

P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na⁺,K⁺-ATPase and Ca²⁺-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase.Members of the P4 subfamily of P-type ATPases are known as flippases, because they transport (flip) specific phospholipids from the exoplasmic to the cytoplasmic leaflet of the plasma membrane bilayer (1–3). Thus, they establish and maintain a physiologically essential asymmetry between the two leaflets, with phosphatidylserine (PS) and phosphatidylethanolamine (PE) being concentrated in the cytoplasmic leaflet and phosphatidylcholine (PC) and sphingomyelin in the exoplasmic leaflet. There are 14 human P4-ATPases, and mutations in many of these are linked to severe disorders (4–7). On the basis of amino acid sequence alignment, the P4-ATPases are predicted to structurally resemble the classic P-type ATPase cation pumps Na⁺,K⁺-ATPase and Ca²⁺-ATPase, possessing a transmembrane domain with 10 helices (M1–M10) and three cytoplasmic domains, P (phosphorylation), N (nucleotide binding), and A (actuator) (Fig. 1A), known from crystal structures to undergo large movements during the catalytic cycle (8, 9). Recently, we showed that the P4-ATPase ATP8A2, like the cation pumps, forms an aspartyl-phosphorylated intermediate and undergoes a catalytic cycle involving the conformations E₁, E₁P, E₂P, and E₂ similar to the Post-Albers scheme originally proposed for the Na⁺,K⁺-ATPase (Fig. 1 B and C) (10, 11). We found that the dephosphorylation of E₂P is activated by the transported substrates PS and PE, similar to K⁺ activation of dephosphorylation in Na⁺,K⁺-ATPase (Fig. 1C), and that K873, located in transmembrane helix M5 of ATP8A2 at a position equivalent to that of a K⁺-binding serine in Na⁺,K⁺-ATPase, is critical for the sensitivity of the phosphoenzyme to PS and PE (12), which seems to argue for a high degree of mechanistic similarity of the P4-ATPases to Na⁺,K⁺-ATPase and Ca²⁺-ATPase, not only in relation to the catalysis of ATP hydrolysis, but also in the transduction of the liberated energy into substrate movement. The M5 lysine might function directly or indirectly in binding of the phospholipid (12). It is, however, an open question how the flippase is able to move a phospholipid, which is ∼10-fold larger than the ions transported by Na⁺,K⁺-ATPase and Ca²⁺-ATPase and which has to reorient during the translocation. This enigma has been referred to as the “giant substrate problem” (13, 14). On the other hand, it is now clear that no protein other than the P4-ATPase catalytic chain in association with its small CDC50 subunit (β-subunit) is required for the flippase function, because the flipping of specific phospholipids is retained after purification and reconstitution of these proteins in lipid vesicles (2, 3, 12). In Na⁺,K⁺-ATPase and Ca²⁺-ATPase, the central helices, M4, M5, and M6, play major roles in cation binding and occlusion. However, in recent studies using mutagenesis to exchange residues between the two yeast flippases Dnf1 and Drs2 of differing phospholipid head group specificity, side-chains critical to the PS specificity of Drs2 have been pinpointed at two interfacial regions flanking transmembrane helices M1–M3, outside the central core region of the protein, thus leading to the suggestion that the transport pathway for the lipid is unique compared with the canonical pathway used by the P-type cation pumps (13, 15). From these results, it also appears that a tyrosine at the cytoplasmic border of M4 (not present in Drs2 or mammalian PS flippases) participates in selection against PS in Dnf1 (13, 16).Open in a separate windowFig. 1.Topology and reaction schemes of flippase and Na⁺,K⁺-ATPase. (A) Diagram of P4-ATPase topology indicating P-type ATPase domains and residues studied here by mutagenesis, as well as K873 of M5 studied previously (12). (B) Proposed flippase reaction cycle. (C) Reaction cycle of Na⁺,K⁺-ATPase [Post-Albers scheme (10, 11)]. E₁, E₁P, E₂P, and E₂ are the major enzyme forms, the P indicating phosphorylation.Transmembrane segment M4 is a key element in the mechanism of the cation-transporting P-type ATPases. In the well-documented catalytic cycles of Na⁺,K⁺-ATPase and Ca²⁺-ATPase, the glutamate of M4 binds Na⁺/K⁺ and Ca²⁺/H⁺ and is alternately exposed to the two sides of the membrane during the cycle (9, 17, 18). Mutations of nearby located residues in M4 have been shown to block the E₁P→E₂P or E₂P→E₂ transition (19–21). Crystal structures of the Ca²⁺-ATPase indicate that the E₁P→E₂P conformational change involves a vertical movement of M4, like a pump rod, corresponding to a whole turn of the helix, allowing delivery of Ca²⁺ bound at the M4 glutamate to the lumen. During the E₂→E₁ transition of the dephosphoenzyme, M4 moves a similar distance in the opposite direction toward the cytosol (8, 9). In ATP8A2, the residue located at the position equivalent to the ion binding glutamate in M4 of Ca²⁺-ATPase and Na⁺,K⁺-ATPase is an isoleucine, which is highly conserved among P4-ATPases (Fig. S1). Intriguingly, a missense mutation of this isoleucine to methionine was recently identified as the cause of cerebellar ataxia, mental retardation, and dysequilibrium (CAMRQ) syndrome in a Turkish family (22).Here, we investigated the functional consequences of mutations of isoleucine-364 (I364) and adjacent residues of ATP8A2 (see Fig. 1A for overview). The specific interaction with phospholipids and the individual partial reactions of the enzyme cycle were studied, and we provide evidence that I364 is crucial to the lipid transport, with mutations affecting the affinity for the phospholipid or the dissociation of the translocated lipid toward the cytoplasmic leaflet. In addition, N359 of M4 appears to play an important functional role. Molecular modeling suggests a mechanistic explanation of our observations, showing a remarkable movement of large, water-filled cavities during the transition between the E₂P and E₂ conformations. Moreover, the water flow appears to be gated by a central hydrophobic cluster composed of the side-chains of I364 and nearby conserved hydrophobic residues of M1, M2, and M6. Among these, I115 of M2 is found to be as mutation sensitive as I364 and N359. These data suggest a unique water-mediated transport pathway for the phospholipid head group along a groove outlined by M1, M2, M4, and M6, which could allow the lipid tail to follow passively, still in the membrane lipid phase, jutting out between M2 and M6. In such a mechanism, movement of M4 and in particular I364 would be as crucial to energy transduction and movement of the transported substrate as in the cation-translocating P-type ATPases.