Effects of Sodium/Glucose Co-Transporter Inhibitors on Contractility and Ca2+ Signalling In Ventricular Myocytes from Streptozotocin-Induced Diabetic Rats.

The prevalence of diabetes mellitus is increasing at an alarming rate worldwide. Cardiovascular (CV) disease is the major cause of morbidity and mortality in diabetic patients. The search for new treatments has led to developing alternative insulin-independent treatment strategies such as sodium/glucose co-transporter (SGLT) inhibitors. Inhibition of intestinal SGLT1 impairs dietary glucose absorption, while inhibition of renal SGLT2 promotes glucose excretion leading to calorie loss and improved glycemic control. In this study, we hypothesized that inhibiting cardiac SGLTs may alter Ca2+ mobilization in myocytes. The effects of Phlorizin (PHLOR) (non-selective SGLT1 and 2 inhibitor), Quercetin-3-O-glucoside (QUER-3-G) (selective SGLT1 inhibitor), Dapagliflozin (DAPA) (SELECTIVE SGLT2 inhibitor) on ventricular myocyte shortening and intracellular Ca2+ have been investigated in streptozotocin (STZ)-induced diabetic rats and age-matched Controls. Experiments were performed at 35-36°C after 2 months of STZ treatment. Myocyte shortening, intracellular Ca2+ and L-type Ca2+ current were measured by video edge detection in electrically-stimulated (1Hz) myocytes, by fluorescence photometry in Fura-2 loaded myocytes and by whole-cell patch clamp, respectively before and after a 5 minute application of the SGLT inhibitor (10-6M) tested. The amplitude (AMP) of shortening was significantly (P\u3c0.05) reduced by PHLOR in STZ (84.76 ±2.91%, n=20) myocytes and Controls (83.72 ±2.65%, n=23), by QUER-3-G in STZ (79.12 ±2.28%, n=20) myocytes and Controls (76.69 ±1.92%, n=30) and by DAPA in STZ (76.58 ±1.89%, n=42) myocytes and Controls (76.68 ±2.28%, n=37). The AMP of the Ca2+ transient was significantly reduced by PHLOR in STZ (82.37 ±3.16%, n=16) myocytes and Controls (73.94 ±22%, n=21) and by QUER-3-G in STZ (73.62 ±5.83%, n=18) myocytes and Controls (78.32 ±3.54%, n=41). DAPA reduced the AMP of the Ca2+ transient significantly in STZ (71.45 ±5.35%, n=16) myocytes and modestly in Controls (92.01 ±2.72%, n=17). The AMP of L-type Ca2+ current was significantly reduced in myocytes from STZ compared to Control rats across a range of test potentials and was additionally reduced by DAPA. Myofilament sensitivity to Ca2+ and SR Ca2+ were not significantly altered by PHLOR, QUER-3-G or DAPA. The reduction in L-type Ca2+ current in the presence of DAPA may partly underlie its negative inotropic effects. However, further studies are required to investigate the mechanism(s) behind the negative inotropic effects of PHLOR and QUER-3-G

ATP13A3 is a major component of the enigmatic mammalian polyamine transport system

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG

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

Parkinson's disease (PD) is a progressive neurodegenerative brain disease presenting with a variety of motor and non-motor symptoms, loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the occurrence of alpha-synuclein-positive Lewy bodies in surviving neurons. Here, we performed whole exome sequencing in 52 early-onset PD patients and identified 3 carriers of compound heterozygous mutations in the ATP10B P4-type ATPase gene. Genetic screening of a Belgian PD and dementia with Lewy bodies (DLB) cohort identified 4 additional compound heterozygous mutation carriers (6/617 PD patients, 0.97%; 1/226 DLB patients, 0.44%). We established that ATP10B encodes a late endo-lysosomal lipid flippase that translocates the lipids glucosylceramide (GluCer) and phosphatidylcholine (PC) towards the cytosolic membrane leaflet. The PD associated ATP10B mutants are catalytically inactive and fail to provide cellular protection against the environmental PD risk factors rotenone and manganese. In isolated cortical neurons, loss of ATP10B leads to general lysosomal dysfunction and cell death. Impaired lysosomal functionality and integrity is well known to be implicated in PD pathology and linked to multiple causal PD genes and genetic risk factors. Our results indicate that recessive loss of function mutations in ATP10B increase risk for PD by disturbed lysosomal export of GluCer and PC. Both ATP10B and glucocerebrosidase 1, encoded by the PD risk gene GBA1, reduce lysosomal GluCer levels, emerging lysosomal GluCer accumulation as a potential PD driver

Parkinson disease related ATP13A2 evolved early in animal evolution

Several human P5-type transport ATPases are implicated in neurological disorders, but little is known about their physiological function and properties. Here, we investigated the relationship between the five mammalian P5 isoforms ATP13A1-5 in a comparative study. We demonstrated that ATP13A1-4 isoforms undergo autophosphorylation, which is a hallmark P-type ATPase property that is required for substrate transport. A phylogenetic analysis of P5 sequences revealed that ATP13A1 represents clade P5A, which is highly conserved between fungi and animals with one member in each investigated species. The ATP13A2-5 isoforms belong to clade P5B and diversified from one isoform in fungi and primitive animals to a maximum of four in mammals by successive gene duplication events in vertebrate evolution. We revealed that ATP13A1 localizes in the endoplasmic reticulum (ER) and experimentally demonstrate that ATP13A1 likely contains 12 transmembrane helices. Conversely, ATP13A2-5 isoforms reside in overlapping compartments of the endosomal system and likely contain 10 transmembrane helices, similar to what was demonstrated earlier for ATP13A2. ATP13A1 complemented a deletion of the yeast P5A ATPase SPF1, while none of ATP13A2-5 could complement either the loss of SPF1 or that of the single P5B ATPase YPK9 in yeast. Thus, ATP13A1 carries out a basic ER function similar to its yeast counterpart Spf1p that plays a role in ER related processes like protein folding and processing. ATP13A2-5 isoforms diversified in mammals and are expressed in the endosomal system where they may have evolved novel complementary or partially redundant functions. While most P5-type ATPases are widely expressed, some P5B-type ATPases (ATP13A4 and ATP13A5) display a more limited tissue distribution in the brain and epithelial glandular cells, where they may exert specialized functions. At least some P5B isoforms are of vital importance for the nervous system, since ATP13A2 and ATP13A4 are linked to respectively Parkinson disease and autism spectrum disorders.status: publishe

ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress.

Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson's disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6 These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.status: publishe

Parkinson disease related ATP13A2 evolved early in animal evolution

<div><p>Several human P5-type transport ATPases are implicated in neurological disorders, but little is known about their physiological function and properties. Here, we investigated the relationship between the five mammalian P5 isoforms ATP13A1-5 in a comparative study. We demonstrated that ATP13A1-4 isoforms undergo autophosphorylation, which is a hallmark P-type ATPase property that is required for substrate transport. A phylogenetic analysis of P5 sequences revealed that ATP13A1 represents clade P5A, which is highly conserved between fungi and animals with one member in each investigated species. The ATP13A2-5 isoforms belong to clade P5B and diversified from one isoform in fungi and primitive animals to a maximum of four in mammals by successive gene duplication events in vertebrate evolution. We revealed that ATP13A1 localizes in the endoplasmic reticulum (ER) and experimentally demonstrate that ATP13A1 likely contains 12 transmembrane helices. Conversely, ATP13A2-5 isoforms reside in overlapping compartments of the endosomal system and likely contain 10 transmembrane helices, similar to what was demonstrated earlier for ATP13A2. <i>ATP13A1</i> complemented a deletion of the yeast P5A ATPase <i>SPF1</i>, while none of <i>ATP13A2-5</i> could complement either the loss of <i>SPF1</i> or that of the single P5B ATPase <i>YPK9</i> in yeast. Thus, ATP13A1 carries out a basic ER function similar to its yeast counterpart Spf1p that plays a role in ER related processes like protein folding and processing. ATP13A2-5 isoforms diversified in mammals and are expressed in the endosomal system where they may have evolved novel complementary or partially redundant functions. While most P5-type ATPases are widely expressed, some P5B-type ATPases (ATP13A4 and ATP13A5) display a more limited tissue distribution in the brain and epithelial glandular cells, where they may exert specialized functions. At least some P5B isoforms are of vital importance for the nervous system, since ATP13A2 and ATP13A4 are linked to respectively Parkinson disease and autism spectrum disorders.</p></div

P5-type ATPase phylogenetic tree.

<p>Simplified view of the phylogenetic tree calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.g001" target="_blank">Fig 1</a>. The P5A group includes only ATP13A1-like sequences (grey), while the P5B group includes subclades belonging to the ATP13A2 (blue), P5B_inv (purple, invertebrate P5B), ATP13A3 (yellow), ATP13A4 (red) and ATP13A5 (green) clades. Sequences from cnidaria, placozoa and ctenophore are marked in white dots. Invertebrate sequences are marked in yellow dots. Sequences from hemichordates and echinoderms are marked with light blue dots and sequences from higher vertebrates are marked in progressively darker shaded blue dots. Yeast Spf1p and Ypk9p are marked with red dots. The yellow numbers indicate the three major P5B gene duplications in vertebrate evolution as discussed in the main text.</p

Genetic complementation in yeast with mammalian P5-type ATPases.

<p>The genome of <i>S</i>. <i>cerevisiae</i> contains a single P5A ATPase gene (<i>SPF1</i>) and a single P5B ATPase gene (<i>YPK9</i>). <b>A.</b> Gene knockout of <i>SPF1</i> results in increased sensitivity to caffeine which can be rescued by expression of untagged Spf1p and mATP13A1 respectively, but not by the untagged catalytically dead SPF1 D487N or by untagged mammalian ATP13A2-5. (e.v.–empty vector) <b>B.</b> Deletion of <i>ypk9</i>^<i>-</i> results in increased sensitivity to MnCl₂, which can be rescued by expression of untagged Ypk9p, but not by the untagged and catalytically dead Ypk9p D781N. None of the untagged mammalian P5 ATPase genes showed rescue of <i>ypk9</i>^<i>-</i> (not shown). <b>C.</b> ATP13A1 complements the caffeine phenotype of the <i>spf1</i>^<i>-</i> knockout strain, whereas the catalytically dead mutant D530N fails to complement. <b>D.</b> Expression of Ypk9p and catalytically dead Ypk9p D781N proteins was confirmed by both N-terminal GFP fusion constructs. Ypk9p tagged with GFP show vacuolar localization. <b>E.</b> Expression of mammalian ATP13A1-5 proteins was similarly confirmed by N- and C-terminal GFP fusion constructs. N- or C-terminal positioning of GFP showed no apparent difference in localization patterns. ATP13A1 shows ER localization and is absent in vacuoles, ATP13A2 and ATP13A4 show vacuolar localization, ATP13A3 and ATP13A5 show localization to cytosolic spots which could represent a pre-vacuolar compartment or early endosomes. Scale bars represent 2,5 μm.</p

Auto-phosphorylation of the P5-type ATPases.

<p><b>A-C.</b> Microsomal fractions of COS-8 cells, transiently overexpressing WT of dead mutants of the P5-isoforms (N-mCherry-tag) were analyzed on immunoblot or radiogram (EP³²) to examine respectively expression levels and autophosphorylation. As a control, non-transfected cells (NT) were included. <b>A.</b> Immunoblot with mCherry antibody shows overexpression for ATP13A1 and ATP13A2 WT and dead mutants. GAPDH levels were determined as loading control. The radiogram (EP³²) demonstrates phosphorylation for WT ATP13A1 and ATP13A2, but not for dead mutants. <b>B.</b> Radiogram indicates that ATP13A1 and ATP13A2 are both sensitive towards hydroxylamine (HA), a hallmark of P-type ATPases. HA quenches the phosphorylated Asp residues, but leaves phosphorylated Thr, Ser or Tyr residues intact. The lower panel shows a coomassie stained gel that depicts the equal loading for all lanes in the upper panel. <b>C.</b> Immunoblot and radiogram of the autophosphorylated intermediates (EP³²) of the five P5 isoforms demonstrate overexpression of all isoforms, whereas phosphorylation was only seen for ATP13A1-4. <b>D.</b> Graph showing the autophosphorylated levels (EP³² panel C) normalized to the relative expression levels of each isoform (mCherry immunoblot panel C). Results indicate strongest autophosphorylation for ATP13A1, ATP13A2 and ATP13A3. ATP13A4 displays low autophosphorylation levels, while ATP13A5 levels are negligible. Data are represented as average ± SD (n = 4). *, P ≤ 0.05 <i>versus</i> ATP13A1 and ATP13A2; #, P ≤ 0.05 <i>versus</i> ATP13A1, ATP13A2 and ATP13A3 (one-way ANOVA, Bonferroni post-hoc). <b>E.</b> Immunoblot demonstrating the expression of the N-terminal 10xHis-FLAG tagged versions of the yeast Ypk9p and Ypk9p (D781N) in yeast total membrane fractions. Antibody against the FLAG part of the tag was used to visualize protein expression and GAPDH was used as a loading control. The wildtype but not catalytic dead protein was able to complement loss of the native <i>YPK9</i> gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.g006" target="_blank">Fig 6B</a>). Both Ypk9p variants were successfully purified using the 10xHis part of the tag (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.s003" target="_blank">S3 Fig</a>). Proteins are expressed from the galactose inducible promotor when grown in rich media supplemented with galactose (YP-Gal) while glucose repress expression (YP-Glu). <b>F.</b> Quantification of Ypk9p autophosphorylation using scintillation counting, normalized to μg of purified Ypk9p. Purified Ypk9p was able to undergo autophosphorylation, which was abolished by mutating the Asp residue in the catalytic motif (D781N).</p

Phylogenetic analysis of the P5-type ATPases.

<p>Phylogenetic tree of 146 protein sequences of P5-type ATPases as inferred from the combined output of two independent statistical methods of measurement (Bayesian inference and maximum likelihood analysis, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#sec002" target="_blank">materials and methods</a> for a detailed description). Yellow and blue shades represent sequences from protostomia and deuterostomia, respectively. Black dots represent Bayesian inference values of 1.00 and numbers noted at key nodes are represented with Bayesian inference statistical values on the <i>left</i> and maximum likelihood statistical values on the <i>right</i> of the dash (<i>left</i>/<i>right</i>). Accession numbers of named sequences can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.s009" target="_blank">S1 Table</a>. Positions of human and model animal organisms (<i>C</i>. <i>elegans</i>, <i>D</i>. <i>melanogaster</i>, <i>D</i>. <i>rerio</i>, <i>M</i>. <i>musculus</i>) are marked by # and *, respectively. Yeast sequences are marked by ‡. The meaning of the colored arrows is provided in the description of the main text. The yellow numbers indicate the three major P5B gene duplications in vertebrate evolution as discussed in the main text.</p