The above suggests that alterations in astroglial Ca2+-dependent excitability observed in the epileptic tissue could arise from changes in astrocyte-to-astrocyte signaling, which is mainly mediated by purines in physiological and pathological conditions. In spite of that, how purinergic signaling contributes to astrocyte dysfunction in epilepsy remains unclear. Here, we assessed the possible contribution of P2Y1R as well as pannexin1 and connexin43 hemichannels—both candidates for non-vesicular ATP-release—by performing astroglial Ca2+ imaging and dye uptake experiments in hippocampal slices from control and fully kindled rats. P2Y1R blockade with MRS2179 decreased the mean duration of astroglial Ca2+ oscillations by reducing the frequency of slow Ca2+ transients, and thereby restoring the balance between slow (ST) and fast transients (FT) in the kindled group. The potential contribution of astroglial pannexin1 and connexin43 hemichannels as pathways for purine release  was assessed through dye uptake experiments. Astrocytes from kindled hippocampi exhibit three-fold more EtBr uptake than controls, whereby pannexin1 hemichannels (Panx1 HCs) accounts for almost all dye uptake with only a slight contribution from connexin43 hemichannels (Cx43 HCs). Confirming its functional involvement, Panx1 HCs inhibition decreased the mean duration of astroglial Ca2+ transients and the frequency of slow oscillations in kindled slices, but had no noticeable effects on the control group. As expected, Cx43 HCs blockade did not have any effects over the mean duration of astroglial Ca2+ oscillations. These findings suggest that P2Y1R and Panx1 HCs play a pivotal role in astroglial pathophysiology, which would explain the upregulation of glutamatergic neurotransmission in the epileptic brain and thus represents a new potential pharmacological target for the treatment of drug-refractory epilepsy.Astrocytes from the hippocampus of chronic epileptic rats exhibit an abnormal pattern of intracellular calcium oscillations, characterized by an augmented frequency of long lasting spontaneous Ca All procedures for experimental handling and sacrificing animals were approved by the CIBICA-UV committee.Male Sprague-Dawley rats  were anesthetized and subjected to stereotactic surgery for electrode implantation  developed in our laboratory  and naïve animals; results were pooled together because no significant differences were found in previous reports . Transversal brain slices (300–350 μm) were obtained by using a Vibroslice microtome  and then incubated in regular ACSF solution for 1 h at room temperature (21–24°C), containing (in mM): 124.0 NaCl, 2.7 KCl, 1.25 KH2PO4, 2.0 Mg2SO4, 26.0 NaHCO3, 2.5 CaCl2 y 10.0 glucose. Slices were then transferred to an immersion recording chamber and superfused with carbogen-bubbled ACSF (2 ml/min).Acute hippocampal slices were obtained from control and kindled rats as previously described  for 60–75 min in regular ACSF at room temperature. Under these conditions, most of loaded cells with Fluo4-AM are astrocytes , and images were acquired at 1 Hz for 5 min, regulated by a shutter . Analysis of astrocyte Ca2+ levels was restricted to the cell body and Ca2+ transients were estimated as changes in the fluorescence signal over the baseline (ΔF/F0) after background subtraction. For every individual cell, a baseline was obtained by extracting the fluorescence values of at least 30 consecutive frames were the astrocyte exhibited no spontaneous Ca2+-dependent activity. Two values were obtained from the baseline: an average of the basal fluorescence levels and the standard deviation. Changes in fluorescence were considered as events and automatically picked up by the analysis software when the ΔF/F0 intensity exceeds the fluorescence of the baseline in three or more standard deviations , at least for five consecutive frames. For multi-peak astroglial Ca2+ activity, those events where the ΔF/F0 dropped to half of the maximum fluorescence intensity in relation to the baseline were considered as independent transients.Spontaneous astroglial Cas Figure . The fols Figure  in order10Panx, 10Panx SCR, GAP26, GAP26 SCR) at room temperature in regular carbogen-bubbled ASCF and then incubated with 4 μM ethidium bromide (EtBr) for 10 min. Afterwards, slices were washed three times for 5 min in regular ACSF and fixed for 1 h in 4% paraformaldehyde at 48°C. To quantify dye uptake in a particular cell type, slices were then processed for immunofluorescence detection of a specific molecular marker for the cell type of interest (see below). For immunofluorescence and confocal microscopy imaging, fixed brain slices were incubated for 1 h in blocking solution (PBS containing 0.2% gelatin and 1% Triton X-100) and then incubated overnight at 48°C with the following primary antibodies diluted in blocking solution containing 10% regular donkey serum. The primary antibodies were: Monoclonal mouse anti-GFAP antibody (1:300) for astrocytes, polyclonal rabbit anti-Iba1 antibody (1:100) for microglia and monoclonal mouse anti-NeuN antibody (1:300) for neurons. Afterwards, samples were washed five times with PBS and incubated at room temperate with donkey anti-mouse Alexa Fluor 488 (1:500), donkey anti-rabbit Alexa Fluor 488 (1:500), donkey anti-goat Cy2 (1:300), donkey anti-mouse Cy3 (1:500), donkey anti-rabbit Cy3 (1:500), donkey anti-goat TRITC (1:100), or donkey anti-chicken Cy3 (1:300) antibodies. Samples were washed and mounted with DAPI-flouromount-G and images were taken with a confocal microscope Eclipse Ti-E Nikon. Stacks of consecutive images were taken with a 60× objective at 250 nm interval with three lasers , and Z projections were reconstructed with Leica software. Levels of dye uptake in control and kindled rats were compared as fluorescence intensity, which was digitized in arbitrary units in 65,000 shades of gray using ImageJ software and corrected total cell fluorescence was calculated  of cells counted for each group was approximately 60. At least three fields were selected in every slice.All the procedures and reagents for dye uptake, immunofluorescence and confocal microscopy imaging were described previously  or GAP26  for 30 min at room temperature and then imaged. Mimetic peptides were purchased from SBSBIO .Membrane-permeable fluorescent calcium indicator FLUO4-AM and SR101 were purchased from Molecular Probes (USA). Chemicals were purchased from Sigma-Aldrich (USA) and Tocris (UK). The specific antagonist of P2Y1Rs, 2’-Deoxy-N6-methyladenosine 3’,5’ bisphosphate tetrasodium salt  was added directly to the perfusion system. For Cx43 and Panx1 hemichannel blockade in Cat-test, Mann-Whitney Test as deemed appropriate. For multiple comparisons ANOVA or Kruskal-Wallis test was used as appropriate, both with post hoc Bonferroni correction. Data are expressed as the mean plus standard error (SEM). Differences were considered statistically significant when p < 0.05 (*); p < 0.01 (**) or p < 0.001 (***), as indicated. Blind experiments were not performed but the same criteria were applied to all groups for comparisons. Randomization was not employed.In all cases, the distribution of each population was first determined  before applying statistical comparisons, which were made using parametric Student’s 2+ transients from control and kindled hippocampal slices pre-incubated with the astrocyte-specific marker SR101 and the Ca2+ probe Fluo-4-AM , whereas mean frequencies showed no differences between groups . The duration of Ca2+ transients was distributed among two populations from the 75th percentile of cumulative distribution, corresponding to 17 s and utilized as a cut-off criterion, from which the random events in a same astrocyte were classified into slow (STs >17 s) or fast transients , with the maximum difference between populations above the 75th percentile. Although FTs were the most prevalent events in both groups, ST frequency in KD was higher than in CT , while FT frequency was lower in the KD group . Additionally, 41.70 ± 2.25% of the oscillations corresponded to ST, whereas only 30.50 ± 3.03% was observed in the control groups . Since these differences in FT and ST frequencies between groups could be attributed to a disruption of astroglial excitability, we incorporated the FT/ST frequency ratio as an index of astrocytic hyperexcitability, where a lower index would be associated to greater Ca2+-dependent excitability. In epileptic rats, the FT/ST index value was significantly lower than in the CT group . These findings confirm our previous evidence, and indicate that astrocytes from the epileptic hippocampus exhibit increases in Ca2+-dependent excitability characterized by a higher occurrence of long-lasting transients, expressed as a lower FT/ST frequency ratio.In order to assess the parameter for astroglial hyperexcitability using the modified incubation protocol , we analyzed spontaneous intracellular CaM Figure . The mod2+ transients in the epileptic hippocampus are independent from neuronal activity  and sensitive to purinergic antagonists for P2Y1R . If P2Y1R activation is required to generate both ST and FT, the receptor blockade would diminish the frequency and percentage of both types of oscillations, without changing the FT/ST index. Conversely, if P2Y1R activation is implicated in the higher incidence of ST in the epileptic hippocampus, the antagonist would diminish only frequency and percentage of ST, reaching an FT/ST index similar to that exhibited by the control group. In fact, the average duration of Ca2+ transients diminished in the presence of MRS in kindled rats . This decrease is a consequence of a drop in ST percentage , accompanied by an increase in FT percentage . Interestingly, P2Y1R blockade in the control group did not produce changes in average transient duration , the percentage of ST  and the FT/ST index . Moreover, MRS application did not change the mean frequency in both CT and KD conditions , suggesting that P2Y1R blockade restored the balance between FT and ST without modifying the total amount of astroglial activity. Taken together, these results suggest that slow astroglial Ca2+ transients require P2Y1R activation, which is responsible for the FT/ST imbalance that characterizes abnormal excitability shown by astrocytes from the epileptic brain.Since astroglial slow somatic Ca2+-dependent exocytosis, hemichannels and ligand-gated receptor channels , which showed thicker, elongated and more sinuous processes located in both stratum radiatum and oriens, as well as in pyramidal layers. In basal conditions, reactive astrocytes showed three times higher EtBr uptake compared to the control group . In order to test the contribution of Panx1 and Cx43 HCs in EtBr membrane permeation, we employed the mimetic peptides 10Panx1 and Gap26, which are selective blockers for Panx1 and Cx43 HCs, respectively . In KD slices, 10Panx1 blocked up to 85% of basal EtBr uptake in reactive astrocytes, while the effect of Gap26 reached just 20% . Since there is evidence suggesting a modulatory effect of P2 receptors on the gating of Panx1 HC . The lack of acute effect of MRS in our experimental conditions suggests that the increased Panx1 HC activity in reactive astrocytes is not regulated by G-coupled P2 receptors or the changes in astroglial Ca2+-dependent activity induced by their activation.According with previous descriptions made in our epilepsy animal model , and then spontaneous astroglial Ca2+-signaling was recorded. In the kindling group, Panx1 HC blockade induced a decrease in the average duration of astrocytic Ca2+ transients . Furthermore, the total percentage of the ST component decreased  proportionally to the increase of the FT component . 10Panx1 reduced ST frequency without changing FT frequency, increasing the FT/ST index to values similar to those observed in the control group . In the CT group, 10Panx1 had no effect on the average duration of astroglial Ca2+ oscillations  or the percentage of FT and ST  and hence, FT/ST index remained without changes . Notably, Panx1 HC blockade did not produce changes in mean frequency in both groups , indicating that the effect of 10Panx1 is restricted to the modulation of the ST component, without affecting the overall activity of the astrocytes. Taken together, these results strongly suggest that the astroglial hyperexcitability observed in the kindled hippocampus require the activation of Panx1 HCs, which likely acts as the release mechanism for the purinergic mediator that activates P2Y1R and increases the duration of Ca2+ oscillations.Since EtBr uptake in KD astrocytes was mostly blocked by  Figures ; p = 0.32+-mediated signals observed in the hippocampus of chronic epileptic rats is likely driven by purinergic astrocyte-astrocyte signaling, and requires the activity of Panx1 HCs and P2Y1R. Panx1 HCs probably acts as the pathway through which purines reach the extracellular medium, which activates P2Y1R and triggers an abnormal pattern of astroglial Ca2+-mediated activity. Those mechanisms could cause an increase in the release of glutamate from astrocytes, which in turn upregulates glutamatergic neurotransmission by modulating the presynaptic component in the epileptic hippocampus  and upregulates the incidence of STs, which is supported by several evidences: (i) TTX-insensitive neurotransmitter release is associated with FTs confined to microdomains in astrocytic processes and not to somatic Ca2+ oscillations (Di Castro et al., 2+ transients as a consequence of P2YR activation (Bowser and Khakh, 1R induces large and long-lasting astrocytic Ca2+ transients that do not require action potentials for their generation (Gallagher and Salter, 1R blockade in the epileptic hippocampus above described (Figure 1R blockade did not change the mean frequency of Ca2+ transients in the epileptic group (Figure 1R activation is associated exclusively with the generation of astroglial STs, which is confirmed by the restoration of the FT/ST index. P2Y1Rs, preferably expressed in astrocytes (Jourdain et al., 1Rs are overexpressed in the hippocampus of two other epilepsy models as well as in hippocampal tissue obtained from patients suffering from temporal lobe epilepsy. In the same work, the functional contribution of P2Y1Rs was also demonstrated by the central injection of the specific P2Y agonist ADP, which exacerbated seizure severity and duration in mice (Alves et al., Although glial purinergic receptors have been implicated in the generation and spread of epileptiform activity during seizures (Kumaria et al., n Figure . The durp Figure , suggest1R activation is one of the key signals for the developing of reactive gliosis in several brain injury conditions (Franke and Illes, 2+ signals do not require P2Y1R activation in the control condition, since their blockade has no effect on Ca2+ oscillations (Figure 1R-dependent Ca2+ signaling may be occurring at the microdomain level, which is outside our range of microscopic detection. In the same sense, the age of the animals employed in this work may explain this discrepancy, since it is highly likely that astrolgial Ca2+ signaling is differentially modulated during the development (Zhu and Kimelberg, 2+ signaling exhibits specific temporal features and dynamics depending on the location of the astrocytes in the CNS (Di Castro et al., It should also be noted that ATP-mediated P2Y1R activation, being these receptors involved in the generation of astroglial Ca2+ ST in epileptic tissue, but not in physiological conditions.Together, the evidence suggests that astroglial hyperexcitability observed in the hippocampus of kindled rats is likely due to an alteration in astrocyte-to-astrocyte signaling that requires P2Y10Panx1 reduces the percentage and frequency of STs in kindled rats (Figure 2+ signals in astrocytes from the control group (Figure Our results show that EtBr dye uptake in reactive astrocytes from the epileptic group was greater than in control slices, being mediated almost exclusively by Panx1 HCs, and with only a mild contribution from Cx43 HC (Figure s Figure . Interesp Figure , suggestp Figure , and thep Figure . Moreovep Figure .2+ oscillation patterns in P2Y1R and Panx1 HC inhibition experiments (Figures in vitro by reducing astrocytic synchronized Ca2+-transients (Kékesi et al., 2+ signals (Notably, we observed the same effects on astroglial Ca Figures . Althoug Figures , which a Figures , their r Figures , and dec signals , our res signals . This pu2+ waves in the astrocyte network (Pascual et al., 2+ transients that propagate at a large scale (Gallagher and Salter, 2+-dependent astroglial signaling represents a new functional hallmark of the epileptic tissue and a mechanism likely contributing to the excitatory loop between astrocytes and neurons that drives neurons to seizure threshold.It was previously suggested that activated microglia release ATP, which in turn stimulate neighboring astrocytes to release more ATP, amplifying CaCB conceived and supervised the experiments and wrote the manuscript. MW performed the experiments, analyzed the data and wrote the manuscript. CÁ-F performed the experiments. CM performed EtBr dye uptake experiments and analyzed the data. JS contributed in experimental design of the EtBr uptake experiments and in the edition of the article.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 globally rising obesity epidemic is associated with a broad spectrum of diseases including atherosclerosis and non-alcoholic fatty liver (NAFL) disease. In the past, research focused on the vasculature or liver, but chronic systemic effects and inter-organ communication may promote the development of NAFL. Here, we investigated the impact of confined vascular endothelial injury, which produces highly inflamed aortic plaques that are susceptible to rupture, on the progression of NAFL in cholesterol fed rabbits.Aortic atherosclerotic inflammation (plaque Gd-enhancement), plaque size , and composition, were measured with in vivo magnetic resonance imaging (MRI) in rabbits fed normal chow or a 1% cholesterol-enriched atherogenic diet. Liver fat was quantified with magnetic resonance spectroscopy (MRS) over 3 months. Blood biomarkers were monitored in the animals, with follow-up by histology.Cholesterol-fed rabbits with and without injury developed hypercholesterolemia, NAFL, and atherosclerotic plaques in the aorta. Compared with rabbits fed cholesterol diet alone, rabbits with injury and cholesterol diets exhibited larger, and more highly inflamed plaques by MRI (P < 0.05) and aggravated liver steatosis by MRS (P < 0.05). Moreover, after sacrifice, damaged  hepatocytes and extensive liver fibrosis were observed by histology. Elevated plasma gamma-glutamyl transferase  and the ratio of liver enzymes aspartate and alanine aminotransferases  indicated the progression of steatosis to non-alcoholic steatohepatitis (NASH).Localized regions of highly inflamed aortic atherosclerotic plaques in cholesterol-fed rabbits may contribute to progression of fatty liver disease to NASH with fibrosis.The online version of this article (10.1186/s12967-018-1587-3) contains supplementary material, which is available to authorized users. One in four Americans, including an increasing number of young adults, develops non-alcoholic fatty liver (NAFL) . After vPorphyromonas gingivalis exposure greatly exacerbates aortic atherosclerosis in cholesterol fed rabbits. Here, we test the hypothesis that progressive atherosclerotic plaque inflammation can promote progression of NASH into a pro-inflammatory phenotype with fibrosis. NASH is associated with increased risk of thrombus formation and pro-coagulation factors as demonstrated by recent clinical cohort  at the end of the 3 months protocol . In vivoMale and female rabbits were fed normal chow and 1.0% CHOL containing chow diet for 3 months . Diffusion-weighted MRI  was used with a diffusion-weighting B value of 3000 s/mm2. The diffusion-weighting B value was selected and optimized based on the protocol developed by Qiao et al., for ex vivo visualization of cholesteryl esters in human and rabbit plaques [High field MRI  was used to study excised aortic segments rinsed in cold saline, fixed in 10% neutral buffered formalin, and then placed in MR-signal inert fomblin oil (Sigma Aldrich). The fomblin oil suppresses background signal, providing high signal-to-noise proportion during visualization of the tissue. Aorta segments were stored in an ice-water bath and allowed to equilibrate to room temperature prior to the ex vivo MRI protocol. All experiments were conducted with high resolution T1W imaging  and plaque gadolinium (Gd) contrast enhancement. Selected segments representing regions with and without plaque rupture and normal vessel areas were compared. For Gd enhancement quantification, pre and post-contrast T1W images were used with a plaque defined as an area of hyperintensity present in at least three consecutive MRI slices (4–8 of these plaque regions were identified in each rabbit). For MRS measurements, the water peak was assigned to 4.7 ppm and lipid signal identified by characteristic peaks between 0.8 and 1.4 ppm . SpectraAt the end of the 3 months protocol Fig. , thrombuBlood was collected prior to MRI scans  in heparin blood collection tubes for quantification of liver enzymes (U/l) and lipids (mg/dl). The biomarker analysis included measurements of the liver enzymes: aspartate aminotransferase (ALT), alanine aminotransferase (AST), and gamma-glutamyl transferase (GGT). The ratio of AST and ALT, also known as the De Ritis ratio , was useFormalin fixed and paraffin embedded liver and aorta sections were stained with Masson’s Trichrome. Fibrotic areas stained blue were quantified after microscopic imaging and in multiple fields of view at the same magnification with ImageJ software using color threshold to quantify liver fibrosis. Lipid deposition in aortic frozen sections was visualized by Oil Red O.Statistical comparison was carried out on data derived from 1% CHOL + injury, 1% CHOL, or normal diet rabbit groups. In addition to comparisons between groups at study time points, baseline and longitudinal measurements were included where possible. Two-sample Student’s t-test for the mean difference per rabbit, one-sided or one sample t-test for comparison with baseline readings, and the F-test for the comparison of variance was carried out in Excel . Alternatively, data were analyzed with the R programming language and Rstudio  for the analysis of variance (ANOVA) in the three groups, or the Wilcoxon rank sum test, a non-parametric alternative to the t-test, with visualization as dot plots.We previously demonstrated the application of non-invasive MRI to quantify the progression of individual atherosclerotic plaques in injured aortas of CHOL-fed rabbits. Plaques that exhibited thrombosis at 3 months were characterized by greatly increasing inflammation between the second and third months , even thConsistent with our previous study using serial MRI , vulneraAfter sacrifice, ex vivo MRI displayed higher vessel wall areas and greater compositional heterogeneity in the 1% CHOL + injury group compared to the 1% CHOL group and the normal diet fed rabbits Fig. . We founAbundant lipids were present in the vessel wall of the 1% CHOL fed rabbits with endothelial injury Fig. . Oil Red) was observed in the blood of the CHOL-fed rabbits compared to rabbits fed normal chow. Free blood-cholesterol and triglycerides increased substantially with 1% CHOL feeding with injury. GGT, AST, and ALT were all increased by 165, 356, and 158%, respectively, although only the increase in GGT was significant. The ratio of AST to ALT increased from 0.63 to 1.11, and this effect was substantial  would provide a better understanding of the impact of atherosclerotic inflammation that adversely affects other tissues such as the liver. Moreover, rabbits fed normal diets with or without injury did not have signs of progressive disease (plaques) in the aorta and lacked signs of liver steatosis or portal fibrosis.The focus of this study was to compare liver disease in 1% CHOL fed rabbits with and without aortic injury and to address whether high vascular inflammation affected the liver. As shown by our in vivo MRI, ex vivo MRI, and histology of aortic atherosclerotic plaques, rabbits Introduction and in [Our rabbit model with CHOL feeding and injury is an established model of human atherosclerosis resembling most of the stages categorized by the American Heart Association (as noted in the n and in ). Rabbitn and in , 39. Then and in  using 1%In addition to representing the human plaques, rabbits have high plasma LDL, VLDL, and other important plasma components, such as CRP –42. PlasOur report is the first study to our knowledge tracking advanced stage plaques that are highly inflamed in relation to liver disease in rabbits. The 1% CHOL + injury rabbits had significantly increased triglyceride deposition in the liver as observed by in vivo MRS at 2 and 3 months compared to 1% CHOL non-injured rabbits at the same time points Fig. . Note thTaken together, above results strongly suggest that vascular inflammation from lipid-rich plaques in the setting of the high cholesterol diet is a major contributor to the liver pathology. After completion of the MRI and MRS studies, the color of the CHOL-fed rabbit’s livers changed from normal deep red to pale or yellowish, most notable for the 1% CHOL + injury group Fig. a. The moSome fibrosis was noted in the 1% CHOL-fed rabbits without injury. In rabbits fed 1% CHOL alone, a mean of 3.65% liver fibrosis by area was found compared to 8.06% liver fibrosis by area in the 1% CHOL + injury rabbits Fig. a. The prwithout periodontitis or psoriasis shows that discrete pathological regions of lipid-rich inflamed pro-thrombotic plaques contribute to the progression of liver disease to advanced stages with high inflammation and fibrosis.Several localized diseases that are characterized by unresolved inflammation have now been linked to cardiovascular disease (CVD). Previous studies in rabbits have demonstrated that periodontitis greatly promotes atherosclerotic plaque inflammatory processes . High ciThis study has demonstrated that progressive atherosclerotic vascular inflammation is associated with worsening liver disease in rabbits. The liver disease appears to synergized between diet, aortic injury, and atherosclerotic inflammation, whereby the injury coupled with local plaque inflammation drives disease progression in both areas. These observations support the emerging broad hypothesis that unresolved inflammation may impart systemic effects. These findings provide a basis for detailed investigation of the mechanisms and molecules released from the plaques that can lead to inflammation and fibrosis in the liver. An important test of our overall hypothesis will be to assess the impact of pro-resolving mediators of inflammation, including lipoxins and resolvins, molecules that have been shown to be potent in treating or possibly preventing many inflammation-associated diseases .Additional file 1: Table S1. Change in liver enzymes in plasma with 1% CHOL diet (N = 3). Table S2. Blood-lipoproteins in rabbits fed 1% CHOL diet with and without injury compared to rabbits fed a normal diet .


Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP3) by activating their membrane-bound receptors. We have previously demonstrated that IP3-mediated sarcoplasmic reticulum (SR) Ca2+ release results in mitochondrial Ca2+ uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP3-mediated SR/mitochondrial feedback in ventricular myocytes.Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca2+ uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates.Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca2+ uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca2+ uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca2+ uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca2+ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca2+ uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca2+ uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions.Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP3-mediated SR Ca2+ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication . Heart failure (HF) is one of the most common chronic conditions associated with aging. Despite advances in HF treatment, it has a poor prognosis with a steady increase in mortality and incidence in the aging population . Current2+) concentration ([Ca2+]m) is thought to be one of the major factors responsible for the activation of Ca2+-dependent dehydrogenases eventually leading to stimulation of the oxidative phosphorylation and ATP generation (2+ uniporter (MCU) knockout (KO) mice  may compensate for the lack of MCU and play a role in the regulation of mitochondrial Ca2+ uptake and ATP generation. The mitochondrial ryanodine receptor (mRyR1) was proposed to be an alternative mitochondrial Ca2+ uptake pathway in cardiac myocytes (2+ released from the sarcoplasmic reticulum (SR) via the inositol 1,4,5-trisphosphate receptor (IP3R) is taken up into the mitochondrial matrix via the mRyR1  or angiotensin (Ang II).An increase in mitochondrial calcium (Caneration . Recent KO) mice  questionKO) mice . These dmyocytes  while thhe mRyR1 , where i2+ release from intracellular stores is the essential signal generated by electrical activation in cardiomyocytes, which is required to initiate cell contraction in the process known as excitation-contraction coupling (ECC). To release Ca2+ from the SR, cardiac myocytes express two different Ca2+ release channels: (1) the ryanodine receptor type 2 (RyR2), which is Ca2+ sensitive and releases Ca2+ from the SR during ECC upon Ca2+ entrance through the voltage-gated L-type Ca2+ channels and (2) the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), which releases Ca2+ after binding of IP3 independently from ECC. IP3 is cleaved from phosphatidylinositol-4,5-bisphosphate (PIP2) by phospholipase C when hormones like ET-1 or Ang II bind to their membrane bound G-protein-coupled receptors. Although IP3Rs are less abundant in ventricular myocytes compared to RyR2s, their activation results in measurable Ca2+ increase during diastole and systole creating areas with highly, localized concentrations of Ca2+, known as microdomains. These microdomains are typically formed at the junction between the SR and mitochondria resulting in elevation of the cytosolic Ca2+ from 0.1 to ~30 μM, which leads to the activation of mitochondrial Ca2+ uptake , interfibrillar (IFM) and perinuclear mitochondria (PNM). All three subgroups differ in enzymatic activity . We found that when the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP3-mediated SR Ca2+ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. We further revealed the difference in Mfn-2 mediated SR-mitochondrial communication depending on mitochondrial location and type of receptor (IP3R vs. Ryanodine receptor), which can explain the existing controversy in the field.In the present study, we aimed to examine the role of Mfn2 in the IPloxp/loxp/cre (Mfn2 KO) mice were obtained from the laboratory of Prof. Gerald W. Dorn, II in St. Louis, Missouri, USA. For induction of Cre-mediated deletion of Mfn2, mice were fed 20 mg tamoxifen/kg body weight/day for 7 days. Littermates of Mfn2 KO mice (heterozygous for Mfn2) were used as control animals and designated as wild-type (WT) group. All experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and approved by the University of Würzburg Institutional Animal Care and Use Committee.Mfn22+-free washing solution followed by enzyme solution containing Liberase TH  as described previously , and protein content was determined using the BCA assay. About 30 μg of protein was separated by 10% SDS-PAGE and transferred onto 0.2 μm PVDF membrane and blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBS-T). Blocked membranes were exposed to primary antibodies toward total Mfn2  and GAPDH . Sheep anti-mouse immunoglobulin G (IgG) antibodies  in 1:4000 dilution were used as secondary antibodies. Protein bands were visualized by enhanced chemiluminescence . Mfn2 bands were normalized to GAPDH bands using the software ScanPack 3.0.Left ventricular tissue was homogenized in ice-cold lysis buffer , cDNA was synthesized from 1 μg of RNA using the iScript™ cDNA synthesis kit . Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed with commercial and customized TaqMan probes . Target gene mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which served as housekeeping gene for comparison.2+]i) and mitochondrial ([Ca2+]m) Ca2+ concentration, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) generation, and mitochondrial ATP generation using specific fluorescent indicators. All fluorescent signals were recorded in electrically stimulated (0.5 Hz) single cardiac myocytes. For measurements, the myocytes were plated on laminin-covered coverslips and incubated in Tyrode solution containing 1 mM Ca2+ at room temperature. The fluorescence image was recorded every 10 s. Fluorescence levels were corrected for background fluorescence and normalized to untreated control (F0) (F/F0).Laser scanning confocal microscopy (Leica SP5 and a Zeiss LSM 780) was used to follow the changes in cytosolic ([Ca2+ concentration ([Ca2+]m) were measured in intact cells using the Ca2+-sensitive fluorescent probe X-Rhod-1/AM . Myocytes were loaded with the dye for 30 min at 37°C. This loading procedure favors mitochondrial localization of X-Rhod-1. To quench the remaining cytosolic fluorescence, the myocytes were incubated in 1 mM CoCl2-containing Tyrode during washout period as published before  was placed directly underneath the sarcolemma and for IFM in the center of the cell excluding the nuclei. Changes in [Ca2+]m levels in perinuclear regions (PNM) were detected in the ROIs located from each side of the nucleus. Fluorescence changes over time were analyzed.For the analysis of m. As shown in 2+ increase induced by ISO (solid blue line) in electrically field-stimulated (0.5 Hz) ventricular myocytes was significantly smaller  compared to the ET-1-induced response (solid green line). Furthermore, in contrast to the data obtained with ET-1, Mfn2 KO had no effect on average ISO-induced mitochondrial Ca2+ uptake  (2+ uptake.We have previously shown that when cytosolic Caobserved . In thisue line) ,B, indic3-mediated mitochondrial Ca2+ uptake in these mitochondrial subfractions. For the analysis of [Ca2+]m levels in SSM, a longitudinal region of interest (ROI) was placed directly underneath the sarcolemma. For [Ca2+]m levels in IFM, ROIs were placed in the center of the cell excluding the nuclei. Changes in [Ca2+]m levels in perinuclear regions (PNM) were detected in the ROIs located from each side of the nucleus. As shown in 2+ than interfibrillar and perinuclear mitochondria following ET-1 stimulation . In Mfn2 KO mice, mitochondrial Ca2+ uptake was significantly inhibited in IFM  and PNM  mitochondria following ET-1 stimulation. There was no difference in [Ca2+]m levels upon ET-1 treatment in Mfn2 KO mitochondria from SSM regions   . These d2+]m levels in the three mitochondrial subgroups was different from ET-1. The highest mitochondrial Ca2+ uptake was detected in subsarcolemmal mitochondria , which gradually declined in IFM  and was the smallest in perinuclear  mitochondria. Furthermore, while Mfn-2 KO had no effect on ISO-induced [Ca2+]m levels in SSM and IFM mitochondrial regions  following ET-1 stimulation using the potentiometric probe TMRM in cardiac myocytes from WT and Mfn2 KO mice , whereas in Mfn-2 KO ΔmΨ decreased upon exposure to ET-1  myocytes, indicating the importance of intact SR-mitochondrial contacts for mitochondrial ΔmΨ maintenance.The CaP levels . Here, w KO mice . In WT mmΨ was different in WT and Mfn2 KO myocytes, we measured TMRM fluorescence at the beginning of the experiment before cell treatment with ET-1. No significant difference in basal ΔmΨ was detected  and ISO (500 nM) were applied to activate the correspondent receptors. As shown in 2+ transfer to mitochondria to mediate ATP production. As summarized in n = 6), whereas in Mfn2 KO myocytes the effect of ET-1 on ATP generation was completely abolished . To the contrary, ISO-treatment caused a significant decrease in ATP levels in WT , while a significant increase in ATP  was observed in Mfn-2 KO myocytes upon ISO stimulation.Next, we monitored changes in [ATP] [Mg2+]i . Therefo2+ uptake. The results are summarized in We further analyzed the effect of ET-1 and ISO on ATP content in mitochondrial subgroups as we did for mitochondrial Ca2+ into the mitochondria, also increased ATP levels significantly higher than IFM and SSM from Mfn2 KO myocytes.A decrease in ATP levels upon ISO exposure was more pronounced in IFM and SSM mitochondrial subgroups but remained unchanged in PNM . In agre2+ uptake and metabolism using mice with a cardiac-specific deletion of Mfn2 . In our previous study , which recovered slowly over the time in a Ca2+-dependent manner  homozygous mice developed by disrupting exon 2 of the ET-1 gene were neonatally lethal , which is associated with a decreased excitability and force generation in cardiac myocytes . ET-1 leeceptors . To deteeceptors . Mice wiy lethal . ET-1−/−elopment , 1995b. elopment , and redelopment . Mice hepressure . These ovia the upregulation of the antioxidant manganese superoxide dismutase through the ETA receptor in cultured cardiomyocytes    . The imp2+ into their matrix following IP3R activation to the same extent. Detailed analysis of mitochondrial subpopulations revealed that in WT cardiac myocytes IFM and PNM take up almost two times more Ca2+ following IP3-mediated SR Ca2+ release compared to the SSM regions . The protocol was approved by the University of Würzburg Institutional Animal Care and Use Committee.LS and ED designed the study and wrote the manuscript. LS, CMag, AB, and MKa performed the experiments. LS, CMag, MKa, and ED analyzed the data. MS and GD provided Mfn 2 KO mice. PE-N and PA-L supported equipment. ED, LS, BG, CMaa, and MKo edited the manuscript. BG and SF provided financial support.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.https://www.frontiersin.org/articles/10.3389/fphys.2019.00733/full#supplementary-materialThe Supplementary Material for this article can be found online at: Click here for additional data file.


Acute myeloid leukemia (AML) is the acute leukemia with highest incidence amongst adults. Despite significant improvements in understanding the genomic landscape and the introduction of novel drugs, long-term outcome remains unsatisfactory. Recently, immunotherapeutic approaches have heralded a new era in cancer treatment. The success of allogeneic hematopoietic stem cell transplantation in AML highlights the disease's immunoresponsiveness. Several immunotherapeutic applications are currently under clinical evaluation and include immune checkpoint blockades, T cell-engaging antibodies, and genetically engineered T cells. However, immunoevasive mechanisms employed by AML blasts severely hamper our endeavors. A better understanding of the underlying mechanisms remains a prerequisite for improving treatment efficacy. One of the hallmarks of the cancer cells is metabolic reprogramming, introduced by Otto Warburg's seminal studies during the beginnings of the last century. Nowadays, it is well established that metabolic adaptation is not just an epiphenomenon during oncogenesis but rather a necessity for tumor development and progression. Furthermore, accumulating data suggest an important role of aberrant tumor cell metabolism for immune escape. AML blasts display a number of metabolic alterations that could be linked to immunoregulation, and these include competition over substrates, abundant release of bioactive metabolites, and an overall microenvironmental metabolic re-modeling that favors the induction or survival of immunoregulatory cell subsets such as regulatory T cells. In this review, we outline the immunoevasive character of the AML blasts' bioenergetics, set it into context with oncogenic mutations, and discuss potentially suitable countermeasures and their limitations. Acute myeloid leukemia (AML) represents the most common form of acute leukemia in adults. Despite advances in AML therapy, treatment outcome remains unsatisfactory. Immunotherapy has heralded a new era in solid and liquid malignancies. Successful usage of allogeneic hematopoietic stem cell transplantation for curing AML suggests its immunoresponsive nature . Severalde novo synthesis of nucleotides, lipids, and proteins) required to meet the biosynthetic demands of highly proliferative cells such as AML blasts (via the pentose phosphate pathway (PPP). AML blasts display elevated levels of reactive oxygen species (ROS) . This phenomenon was first described by Otto Warburg in the 1920s  and is kL blasts . Anotheres (ROS)  and woules (ROS) . In addies (ROS) . Similares (ROS) –16  . Moreovees (ROS) , 15, 17.es (ROS) .Several oncogenic pathways such as HIF-1α or c-Myc have been linked to the neoplasia's glycolytic switch. In AML, an internal tandem duplication (ITD) within the Fms-like tyrosine kinase (FLT3/ITD) represents an unfavorable genetic aberration. Recent findings suggest that FLT3/ITD promotes aerobic glycolysis through an AKT-mediated upregulation of the glycolytic pacemaker enzyme hexokinase 2 (HK2) . PolyploSuch AML-mediated glucose deprivation could substantially impact the functional competence of various immune cells that utilize glucose as an energetic substrate, including monocytes, NK cells, dendritic cells, and, in particular, T cells, which display an exhausted and senescent phenotype in AML patients .Naïve T cells meet most of their energetic demands by use of OXPHOS of fatty acids. However, triggering the T cell receptor (TCR) in conjunction with CD28 co-stimulation leads to an mTOR-orchestrated  rapid upRegs), which are considered important contributors to tumor-induced immunoevasion  such as superoxide or hydrogen peroxide. Those short-lived molecules are hyperpermeable and highly reactive. Oxidative stress is typically found in cancer patients . High ROedoxin-1 , 47. Thiedoxin-1 , 48. Mosedoxin-1 . Those fedoxin-1 .post-hoc analyses revealing that patients with myelomonocytic or monocytic AML might benefit most  can efficiently reduce the NOX2-dependent ROS formation by triggering the histamine type 2 receptor that is expressed on myeloid cells including AML blasts . The abifit most , 53.Increased D-2-hydroxyglutatarate (2-HG) serum levels were recently identified as a novel negative prognostic marker for AML . 2-HG abIDH enzymes convert isocitrate into α-ketoglutarate (α-KG). Beyond its role as an intermediate of the Krebs cycle, α-KG represents a co-substrate for a number of metabolic partners, including >60 mammalian dioxygenases and demethylases. Mutations occur at critical arginine residues of the enzymes' active site (R132 in IDH1 and R140/172 in IDH2). The amino acid substitution prevents its normal catalytic function (“loss-of-function”) and at the same time confers a neomorphic enzymatic activity that facilitates reduction of α-KG to 2-HG (“gain-of-function”). The rate of 2-HG production far exceeds the rate of homeostatic clearance, leading to pathological 2-HG accumulations.Increasing evidence suggests that 2-HG acts as an “onco-metabolite,” driving proliferation and differentiation arrest. Notably, 2-HG and α-KG are structurally similar, except that the oxygen atom linked to C2 in α-KG is replaced by a hydroxyl group in 2-HG. This structural similarity suggests that 2-HG might exert its oncogenic effects through the competitive inhibition of α-KG-dependent enzymes . ExposurIn addition to promoting a pro-glycolytic and ROS-enriched environment, recent studies indicate that 2-HG might directly impact T cell responses. It has been shown that T cells are capable of efficiently taking up 2-HG, further validated by 2-HG-enriched T cells being exclusively found in samples from patients with IDH-mutated AML . In the bona fide representative of metabolic enzymes that exerts dual effects  by simultaneously depleting essential substrates and producing bioactive metabolites   in an IDO-mediated fashion, further potentiating a tolerogenic environment  evaluating the IDO inhibitor indoximod as part of the maintenance regimen after standard induction and consolidation chemotherapy is currently ongoing.Indoleamine-2,3-dioxygenase (IDO) is a abolites . It cataabolites . Tryptopsurvival , 65. Fursurvival . In addipoptosis . The undpoptosis  and supppoptosis , which ipoptosis . Furtherironment . Again, capacity , 69  T cells against AML blasts , a non-essential amino acid, plays an important role in regulating immune responses . T cellsL blasts .+ hematopoietic progenitors  represents a key carbon source fueling OXPHOS in AML blasts, thus supporting their rapid expansion . In factgenitors , 75. In genitors . At the genitors . Howeverimmunity , 79.Reg reduction  is found on AML blasts  while patibodies . Furthertibodies . Immune tibodies , but thetibodies .ex vivo T cell re-modeling with the aim of achieving superior resilience toward detrimental microenvironmental cues  and of enhancing metabolic fitness. Cytokines decisively regulate T cell metabolism; culturing T cells in the presence of IL-15 has shown (in contrast to IL-2) to drive mitochondrial biogenesis, skewing bioenergetic dependency away from aerobic glycolysis and toward fatty acid oxidation (FAO). I has also shown to endow them with an increased antioxidant capacity  with survival advantages, and expression of checkpoint ligands that impair the immune cells' metabolic competence, such as the ability to utilize certain nutrients , which include immune checkpoint blockade, T cell-engaging multispecific antibodies, and genetically modified T cells, it is essential to mechanistically understand the immunometabolic crosstalk for developing the means to improve T cell function. As of today, several mechanisms promoting immunometabolic escape have been described for AML: competition (with immune cells) over critical nutrients such as glucose or amino acids, increased metabolic byproducts such as ROS that negatively impact immune function, microenvironmental metabolic remodeling that endows immunoregulatory subsets (such as Tutrients . For theThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RAF kinase inhibitor protein (RKIP) is an essential regulator of intracellular signaling. A somatic loss of RKIP expression is a frequent event in solid human cancers, and a role of RKIP as metastasis-suppressor is widely accepted nowadays. Recently, RKIP loss has been described in acute myeloid leukemia (AML) and a series of other myeloid neoplasias (MNs). Functional in vitro and in vivo experiments revealed that RKIP is an essential player within the development of these liquid tumors; however, the respective role of RKIP seems to be complex and multi-faceted. In this review, we will summarize the current knowledge about RKIP in myeloid leukemogenesis. We will initially describe its involvement in physiologic hematopoiesis, and will then proceed to discuss its role in the development of AML and other MNs. Finally, we will discuss potential therapeutic implications arising thereof. RAF kinase inhibitor protein  is an essential regulator of intracellular signaling. In more detail, it modulates the signal propagation in many pivotal signal transduction cascades, including the RAS-mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), nuclear factor-κB (NF-κB), G-protein coupled receptor kinase-2 (GRK-2) and Glycogen synthase kinase 3β pathways ,5,6,7. MIn addition to its role as a physiologic regulator of intracellular signaling, RKIP has also been shown to be deregulated in a broad range of human malignancies. A somatic loss of RKIP expression was initially described in 2003 . Fu et aRecently, RKIP loss has been described in acute myeloid leukemia (AML) and a series of other myeloid neoplasias (MNs). Functional in vitro and in vivo experiments revealed that RKIP is an important player within the development of these liquid tumors. However, the respective role of RKIP seems to be complex and multi-faceted. In this review, we will summarize the current knowledge about the role of RKIP in physiologic and malignant myeloid hematopoiesis, with a specific focus on AML. Moreover, we will discuss potential therapeutic implications arising thereof.To discuss the role of RKIP in myeloid leukemogenesis, we initially have to address its relevance in physiologic hematopoiesis. Principally, hematopoiesis arises from hematopoietic stem cells (HSCs), which have the potential for self-renewal on the one hand, and differentiation into all types of blood cells on the other hand. While the complete and complex system of hematopoietic differentiation is reviewed in detail elsewhere ,18,19, t+ hematopoietic stem and progenitor cells (HSPCs). However, its expression is greatly decreased in specimens from whole blood, which contain mainly mature and differentiated blood cells [Rkip expression was high in HSPCs and lymphoid cells, but significantly decreased in mature myeloid cells, including monocytes and granulocytes [Rkip levels remain high during the early stages of myelopoiesis. These include the common myeloid progenitor (CMP) and GMP stages, where Rkip expression is similar to the immature lin−/Sca+/kit+ (LSK) HSPC compartment. Subsequently, Rkip decreases during the terminal stages of granulo-monocytic differentiation [+ HSPCs and performed a knockdown of RKIP by lentiviral transduction. Subsequently, we induced myelomonocytic differentiation by a mix of hematopoietic growth factors, including granulocyte-macrophage colony-stimulating factor (GM-CSF). RKIP knockdown greatly induced the expression of the myelomonocytic surface markers CD11b and CD14, which indicates that RKIP knockdown increases the myelomonocytic differentiation of HSPCs. Interestingly, RKIP knockdown without additional GM-CSF stimulation was insufficient to induce the differentiation process. These data could be corroborated in the above-mentioned HL-60 model. Again, RKIP knockdown increased the Vitamin D3 induced myelomonocytic differentiation, whereas RKIP overexpression inhibited this process. Once more, RKIP knockdown without Vitamin D3 incubation failed to induce HL-60 differentiation. Ultimately, these data were also confirmed in an in vivo setting employing a murine model with a complete knockout of the Rkip gene. As seen in the in vitro experiments, Rkip deletion in these mice increased the GM-CSF induced myelomonocytic differentiation, resulting in increased levels of granulocytes and monocytes in the peritoneal cavity, the peripheral blood, and the bone marrow. As described for the in vitro assays, Rkip deletion alone—without GM-CSF administration—was insufficient to induce myelomonocytic differentiation [A first insight into the role of RKIP in hematopoiesis stems from expression analyses in various hematopoietic cell compartments. Several studies, including work from our laboratory, have shown that RKIP is prominently expressed in CD34od cells ,21,22,23ulocytes . A more ntiation ,25,26,27ntiation . HL-60 intiation . The autntiation ,31,32,33Another interesting observation comes from Schuierer et al., who observed that the decreased expression of RKIP in monocytes starts to rise again when they transform into macrophages . In funcThese data create an interesting picture, where a decrease of RKIP expression in HSPCs is an important step for their commitment into the myelomonocytic lineage and contributes to the formation of monocytes and granulocytes . HoweverAML is an aggressive hematopoietic malignancy that arises from the malignant transformation of HSPCs. Despite the use of intensive treatment strategies, including hematopoietic stem cell transplantation for younger and/or medically fit patients, and the establishment of several non-intensive treatment options for older individuals, the prognosis of AML is still dismal ,38,39,40TP53 [Pathogenetically, almost all AML cases exhibit recurrent genetic aberrations. These include mutational and non-mutational events, the latter comprising gene fusions, copy-number alterations, aberrant methylation signatures, as well as pathologic gene and non-coding RNA expression profiles. ,44,45,46TP53 ,52,53. HThe significance of RKIP for AML has first been highlighted by the observation of an RKIP expression loss in two patients with therapy-related AML (t-AML) . RKIP loImportantly, a loss of RKIP cannot only be observed in the small subgroup of t-AML with CRAF germline mutations but seems to be a more general phenomenon in AML. By studying more than 400 primary patient specimens derived from patients with all different subtypes of AML, we described a loss of RKIP as one of the most frequent molecular aberrations occurring in AML, affecting more than 20% of patients . We coulAs outlined above, RKIP is a negative regulator of RAS-MAPK/ERK signaling. Therefore, it is worth to focus on the interaction between RKIP and other alterations within the RAS-MAPK/ERK pathway in more detail. As mentioned above, RKIP loss correlated with CRAF germline mutations in patients with t-AML. As these alterations also synergized in malignant transformation, one might hypothesize that RKIP loss describes a secondary genetic event in patients with RAS-signaling mutations. In this situation, RKIP would amplify the activating properties of these aberrations and thereby potentiate their oncogenic effects . In agreRkip deletion even aggravated the CMML development in G12DNras mice,[Finally, it is worth to look at the role of RKIP in other myeloid neoplasias (MNs) as well. This is mainly because some general mechanisms of myeloid leukemogenesis are not specific to AML, but also apply to other MN subtypes. In return, studying the mechanisms of RKIP loss in these diseases might also help to learn more about mechanisms that might apply to AML development. Li et al. described a loss of RKIP in chronic myeloid leukemia (CML) . In agre12D mice,. these dA complete or partial loss of RKIP expression is frequently described in MNs, and functional relevance of this RKIP loss for myeloid leukemogenesis could be proven in vitro and in vivo by other groups and us ,56,63. TAnother possibility to use RKIP for optimizing the treatment of AML is not to modulate the expression of RKIP, but rather to use it as a biomarker for the sensitivity to currently used cytotoxic agents. We have previously shown that decreased expression of RKIP correlated with superior survival in intensively treated AML patients. These data are in line with reports of increased sensitivity to cytarabine in AML patients with activated RAS ,87,88,89RKIP plays a major role in physiologic hematopoiesis and myeloid malignancies. In physiologic hematopoiesis, a decrease of RKIP expression in the HSPC pool increases the myelomonocytic lineage commitment of these cells. Importantly, low expression levels of RKIP have been detected in AML and a variety of other MNs, and a functional involvement in myeloid leukemogenesis has been proven. Finally, RKIP expression is of prognostic relevance and might display an exciting candidate for optimizing therapeutic strategies in AML.


Here, we compared wildtype Doc2b with two Ca2+ binding site mutants named DN and 6A, previously classified as gain- and loss-of-function mutants. They carry the substitutions D218,220N or D163,218,220,303,357,359A respectively. We found that both mutants bound phospholipids at low Ca2+ concentrations and were membrane-associated in resting neurons, thus mimicking a Ca2+-activated state. Their overexpression in hippocampal primary cultured neurons had similar effects on spontaneous and evoked release, inducing high mEPSC frequencies and increased short-term depression. Together, these data suggest that the DN and 6A mutants both act as gain-of-function mutants at resting conditions.Communication between neurons involves presynaptic neurotransmitter release which can be evoked by action potentials or occur spontaneously as a result of stochastic vesicle fusion. The Ca Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (SNARE) proteins, Ca2+-sensors and a number of accessory proteins1. Neurotransmitter release is either triggered by action potentials (APs)5 or occurs spontaneously at resting membrane potential6. Evoked release consists of synchronous and asynchronous release components8. Fast, synchronous release triggered by local Ca2+ influx (nano & micro-domain) occurs in less than a millisecond9 and is governed by the fast Ca2+ sensors Syt-1, 2 or 910. Another class of high affinity Ca2+ sensors with slow kinetics such as Syt-7 mediates asynchronous release13 and synaptic plasticity14. In synapses lacking the fast sensor, diminished synchronous release is accompanied by increased asynchronous release as shown in Syt-117 and Syt-219 mutant mice.Regulated exocytosis is strictly dependent on 2+ 22. Spontaneous release is important for nervous system functioning as it is involved in synapse maturation, maintenance and synaptic plasticity26. Like asynchronous release, its frequency is suppressed by Syt-1 and Syt228 and stimulated by double C2 (Doc2) proteins29.Unlike evoked release, spontaneous release is AP-independent and occurs as a stochastic process with a probability that appears to be partially regulated by intracellular Ca31. Both Doc2a and –b contribute to spontaneous release as shown in knockout and knock-down models29. A recent study suggested that glutamatergic and GABAergic events are driven by the expression of Doc2a and –b respectively, although both isoforms are functionally redundant and can rescue both miniature excitatory and inhibitory post-synaptic current (respectively mEPSC and mIPSC) frequencies27.Doc2a, -b and –c isoforms together constitute the Doc2 protein family. Doc2a is mainly expressed in the adult brain while Doc2b is more widely expressed in the nervous system and various neuroendocrine tissues32. The polybasic sequence also enables Doc2b to bind PIP2, a phospholipid enriched on the cytoplasmic leaflet of the plasma membrane33. On the opposite site of the C2 domain structures, negatively charged residues 41. Phorbol esters potentiate exocytosis in a Ca2+-independent way relying on the Doc2/Munc-13 interaction42. Consistently, Doc2 overexpression causes a Ca2+-independent, Munc-13 dependent release increase upon phorbol ester stimulation38 and conversely, blockade of the Doc2-Munc13 interaction by synthetic peptides abolishes phorbol ester potentiation39. Munc13-1 is necessary for the Doc2b-induced priming of secretory granules in chromaffin cells43. However, alteration of this interaction by mutations in the MID domain has no effect on Ca2+-induced Doc2b migration to the membrane44. Hence, Doc2b could support exocytosis by both of two mechanisms: (i) together with Munc-13 for vesicle priming or superpriming; (ii) Ca2+-dependently by enhancing membrane fusion.The N-terminal domain of Doc2a/b interacts with Munc13 via a Munc13 interacting domain , this was taken to support a role as Ca2+ sensor45. Another Ca2+-ligand mutant designated Doc2b6A, in which six aspartates were substituted by alanines  tag used for the protein purification was preserved to enable C2A self-association. Phospholipid binding causes liposome clustering which can be measured as an absorbance increase at 350 nm. Addition of a GST-C2AWT protein fragment to a Ca2+-containing solution caused rapid liposome clustering . In contrast, both GST-C2ADN and GST-C2A3A showed a similar strong phospholipid binding at low [Ca2+]free  neurons cultured on glial micro-islands Fig. . DKO neuing Fig. . Westernons Fig. . Viral tons Fig. .Figure 5WT increased the average mini frequency from 10 ± 1.4 to 13 ± 1.6 Hz in DKO cells , consistent with previous observations21. Note that in neuronal networks, spontaneous release was measured in presence of 1 µM tetrodotoxin (TTX) to block voltage-gated sodium channels.Expression of Doc2blls Fig. . This trncy Fig.  while thncy Fig.  were unaups Fig. . In bothDN and Doc2b6A have similar gain of function effects on the spontaneous release frequency, which confirms previous observations29. The effect of mutant Doc2b on spontaneous mEPSC frequency parallels the alteration at rest in in vitro plasma membrane binding and phospholipid clustering (i.e. a gain of function in resting conditions). We conclude that Doc2bDN and Doc2b6A both act as dominant positive mutants that increase the probability of quantal release at rest as those mutants have no effect on synaptic density  induced by a single action potential (AP) or paired APs at varying intervals in DKO autaptic neurons. Expression of Doc2bude Fig. . We thenude Fig. . The paiant Fig. , most noant Fig. . The sinant Fig. .Figure 6vr), although alternative explanations exist. Unlike in DKO neurons, WT cells expressing Doc2bDN or Doc2b6A mutant showed a higher 1st evoked EPSC charge 53. To test if a similar mechanism occurs in neurons we compared the EPSC amplitudes induced by a single AP and a subsequent treatment with calcimycin in wildtype autaptic neurons expressing either eGFP control or overexpressing Doc2bWT. Calcimycin is a Ca2+ ionophore which bypasses VGGCs, and triggers exocytosis by an artificial Ca2+ influx. No significant change was observed in the EPSC amplitude, nor in the charge transfer induced by calcimycin  and high (40 Hz) frequency  and Doc2b6A expressing cells (48 ± 1.8%) recovered more slowly to similar levels as GFP-expressing control group (52 ± 2.5%). These data suggest that wildtype Doc2b contributes to synaptic recovery in a manner that requires Ca2+ binding.During repetitive stimulation at 5 Hz, the expression of mutant Doc2b did not affect the balance between synchronous and asynchronous release Fig. . Howeverins Fig. . Doc2bWTWT or mutants  size, calculated by linear interpolation of the cumulative synchronous release from 40 Hz trains Fig. , appearents Fig.  but the  6A Fig. .WT potentiated the EPSC recovery and this effect was absent in mutants  mimic an activated state at low [Ca2+] resulting in constitutive membrane enrichment and increased spontaneous release rates; (ii) induce faster depression during repetitive neuronal stimulation .To shed light on Doc2b protein function in synaptic release, we studied the CaDN has been interpreted as a gain-of-function mutant based on the increased phospholipid binding at rest (i.e. the mutations were considered to mimic Ca2+ binding). Doc2b6A was described to be a loss-of-function mutant based on the loss of Ca2+ binding capacity. This classification as gain- and loss-of-function mutants had important implications: the enhanced spontaneous release in Doc2bDN overexpression was taken as evidence that Doc2b functions as a Ca2+ sensor21, while the very similar phenotype in Doc2b6A overexpressing neurons was taken to demonstrate a Ca2+-independent function29.In previous studies, Doc2b6A shows constitutive membrane binding, as supported by other studies43. Thus, the Doc2b6A is not a loss-of-function mutant. At the same time however, the Doc2bDN mutant is also not a pure gain-of-function mutant, as indicated by the incapacity to potentiate release after repetitive neuronal activity. In fact, both mutants share very similar phenotypes in enhancing lipid and membrane association, neurotransmitter release at rest and during stimulation but also impaired EPSC potentiation after intense stimulation. The dual effect of Ca2+ binding site mutations could be attributed to the different surface charge distribution of the aspartates in the Ca2+- and membrane binding site of the C2 domains. In Doc2b wildtype protein, the aspartates residues are neutralized by binding of Ca2+ ions. In the mutants, the neutralization of the aspartates could support membrane binding at rest, but this binding may not reach the same affinity as with Ca2+-bound aspartates. Alternatively, the mutations could slightly misplace the membrane-inserting residues in the loops surrounding the aspartates.Our data demonstrate that Doc2b2+-dependent membrane association of wildtype and mutants seems slightly more complex than might be expected. At high (>500 nM) [Ca2+]free, isolated C2AB fragments showed a loss of liposome clustering activity using synthetic membranes composed of 75% DOPC and 25%DOPS. Recent investigations, revealed a similar effect for the Doc2b6A mutant (termed 6x) as its liposome binding capacity dropped in presence of Ca2+ 27. In our hands a similar reduction in lipid binding occurred at high (>700 nM) [Ca2+]free for Doc2bWT-C2AB. Syt-7 was also reported to have a reduced lipid binding activity at high [Ca2+]free, effect that was associated with an inhibitory effect on norepinephrine secretion in PC12 cells54. Another similar observation is the negative effect of high [Ca2+]free on Syt-1 in vitro fusion ability55, together suggesting that most Ca2+-sensors function in a limited [Ca2+] window and that higher [Ca2+] might have a negative effect.The Ca2+ binding activity in a recombinant C2AB fragment named C2ACLMB 44 likely due to its functional C2B domain. In our hands, Doc2bDN specifically shifted the synchronous/asynchronous release balance in favor of fast release  completely abolishes Doc2b translocation and this mutant does not rescue spontaneous release27, confirming the idea that Doc2b acts as a Ca2+-sensor. Considering all these mutants, there is a striking correlation between the Ca2+-dependent phospholipid association of Doc2b under resting [Ca2+] and its function in spontaneous neurotransmission.In presence of phospholipids, the negatively charged head groups may stabilize bound CaDN and 6A mutants affect synaptic plasticity during repetitive activity. In chromaffin granule secretion, wildtype Doc2b serves both positive and negative roles43, a feature that is shared with other exocytotic proteins such as synaptotagmins, complexins and munc18s. In this system, Doc2bDN and Doc2b6A favour immediate chromaffin granule fusion but impair sustained release at high [Ca2+]. This phenotype might also be a collateral effect of constant vesicle fusion at rest, exhausting the immediate releasable pool (IRP). In WT neurons, we also observed a modification of first evoked release by Ca2+ binding mutants, but this effect was not significant in Doc2a,b DKO neurons. Despite the alteration of several synaptic release parameters by Doc2b mutants, the overexpression of Doc2bWT  and changes the balance in vesicle recruitment from a reserve pool as recently claimed in a peer investigation43.Additionally, a subtle but significant shift in favour of asynchronous release during repetitive stimulation appeared in DKO neurons rescued with Doc2bWT Fig.  but not DN and 6A mutant overexpression caused an increase in the spontaneous release frequency and short-term depression during trains and paired pulse stimulation. These effects may be attributable to a change in the vesicular release probability (Pvr), an idea supported by Figs 58. When SV recruitment is reduced, short-term facilitation will turn into depression59. Molecular perturbations of Ca2+-sensors like Syt-1 can lead to labile primed states affecting short-term plasticity60. Therefore, an alternative explanation for the fast rundown would be that Doc2b mutants induce labile docking of newly recruited SVs, causing RRP depletion and synaptic fatigue.Doc2bDN and Doc2b6A did not enhance delayed release, as was previously observed for another Ca2+-ligand mutant  in locally stimulated neuronal networks47. Also, we did not observe increased RRP sizes in presence of Doc2bDN and 6A which were reported for that mutant44. These differences could be explained by different cell culture conditions, stimulation method or by the different point mutations used. However, our results are compatible with a study in Syt-1 KO neurons47 where Doc2b Ca2+-binding site mutants enhanced the remaining asynchronous release component triggered by a single AP.In our hands, Doc2bDN and Doc2b6A mutants do not represent divergent gain- and loss-of-function mutants but show similar behavior, characterized by increased neurotransmitter release at rest and during the early phase of neuronal activity. Moreover, the strict correlation for the wildtype protein between plasma membrane association and spontaneous release frequency supports a direct role as a Ca2+ sensor. Our results provide more insight in the various functional properties of Doc2b and its Ca2+ binding site mutants, which fit published conflicting data.We conclude that the Doc2b21. To dissociate brain tissue from DKO mice, hippocampi were isolated at postnatal day 1 (P1). For wildtype mice, E18–stage embryos were used. In this case, pregnant females were sacrificed by cervical dislocation, embryos were obtained by caesarian section, decapitated and used for dissection.Animals were housed, bred and handled in accordance with Dutch and EU governmental guidelines. Protocols were approved by the VU University Animal Ethics and Welfare Committee . Wildtype C57BL/6J mice were obtained from Charles River Laboratories. Doc2 a & b double knockout mice (DKO), maintained on the same C57BL/6J genetic background, were previously described61.To isolate mouse neurons, brains were placed in Hanks buffered salt solution  buffered with 1 mM HEPES (Invitrogen). After meninges removal, hippocampi and cortices were dissociated and separately treated. The tissue was incubated with 0.25% trypsin (Invitrogen) for 20 min at 37 °C and washed in DMEM. Cells were dissociated by trituration with a fire-polished Pasteur pipette and counted in a Fuchs‐Rosenthal chamber. Neurons were plated in warmed Neurobasal medium supplemented with 2% B-27, 1.8% 1 M HEPES, 0.25% glutamax and 0.1% Pen-strep  as previously established61. For autaptic cultures, 1.5K cells per 12-well or 3K per 6-well were plated on coverslips with astrocyte micro-islands stamps61. For live imaging, hippocampal neurons were plated on rat astrocytes in low density networks (10K per 12-well)61. For western blotting, cortical neurons were plated at 300K per well in 6-well plates without coverslips, coated overnight with 0.0005% poly-L-Ornithine (Sigma) and 2 µg/ml laminin (Sigma) in PBS and washed with sterile water.Electrophysiology experiments were performed in network or autaptic cultures. For network cultures, hippocampal neurons were plated at a density of 25K cells per well in 12-wells plates on etched glass coverslips containing a confluent layer of rat astrocytesWT (LIP#1984) was compared to Doc2bDN carrying the D218, 220N mutation (LIP#1985)41 and Doc2b6A carrying the D163, 218, 220, 303, 357, 359A mutation (LIP#1986)29. Lentiviral infectious particles were packaged in HEK293T human embryonic kidney cells with a passage number lower than 25, maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 50 U/ml penicillin-streptomycin and 1x non-essential amino acids (Gibco). At 2 days in vitro (DIV2) the cells were transfected at 50% confluence with three plasmids: p.MDG2 , pCMVΔR8.2 (encoding packaging factors) and a p156RRL-derived plasmid encoding Doc2b. LIP#1984 encoded Doc2bWT, LIP#1985 Doc2bDN and LIP#1986 Doc2b6A. At DIV3 the medium was changed to Optimem + 50 U/ml penicillin-streptomycin without fetal bovine serum. At DIV4, the supernatant containing infectious particles was centrifuged at 1000 × g to remove cell debris. The supernatant was concentrated by ultrafiltration using a 100 kDa cutoff membrane  to achieve a final volume of 150 µl. The LIPs were diluted to 1 ml with phosphate-buffered saline, filtered through 0.45 µm and stored in aliquots at −80 °C until use. Neurons were infected at DIV1 to induce Doc2b expression. To investigate subcellular protein localization, Doc2b was C-terminally tagged with EGFP. Neurons were transduced with Semliki infectious particles 10 to 12 hours before experimentation  as described43.For functional assays, Doc2b and EGFP were expressed as separate proteins from a single mRNA using an IRES2 internal ribosome entry site. Wildtype rat Doc2b2, 4 mM MgCl2, 10 mM HEPES, 10 mM glucose, 300 mOsm, pH 7.3. Our standard intracellular (patch pipette) solution was EGTA free to prevent Ca2+ buffering; it constituted 125 mM K-gluconate, 10 mM NaCl, 4.6 mM MgCl, 4 mM K2-ATP, 15 mM creatine phosphate and 10 U/ml phosphocreatine kinase, 300 mOsm, pH 7.3. To record spontaneous excitatory events in network cultures, 1 µM tetrodotoxin  and 20 µM gabazine (Sigma Aldrich) were added to the extracellular medium. In autapses, only gabazine was added. Where indicated, the Ca2+ ionophore calcimycin  was used at a final concentration of 10 µM and applied by puff for 100 seconds through a barrel placed in the vicinity of the soma.For electrophysiology, both continental and island cells were used between DIV 14 to 21. Doc2b-expressing cells were identified by monitoring EGFP fluorescence. The standard extracellular medium included 140 mM NaCl, 2.4 mM KCl, 4 mM CaCls) exceeding 15 MOhm or with an Rs increase beyond 20% of the initial value were excluded. Rs was compensated to 70%. EPSCs were elicited by depolarizing the cell to 0 mV for 1 ms. Standard stimulation paradigms comprised spontaneous activity recording, paired pulse stimuli with intervals from 20 ms to 1 s, two trains of each 100 action potentials at 5 Hz and 40 Hz. Each train was followed by a single stimulus at 2 s after the last depolarization to test synaptic recovery.The patch pipettes were made of borosilicate and pulled using a multi-step filament pulling  to achieve a pipette resistance of 3 to 5 MOhm. In whole-cell configuration, neurons were voltage clamped at −70 mV with an Axopatch 200B or Multiclamp 700B amplifier (Molecular Devices). Signal was low-pass filtered at 1 kHz and digitized at 10 kHz with a Digidata 1440 A or 1550 (Molecular Devices). Neurons with a series resistance (R® (Mathworks) environment62 to calculate the paired pulse ratio, the EPSCs charge and amplitude. The paired-pulse ratio was calculated by dividing the 2nd EPSC from paired response to the 1st EPSC. The 1st EPSC quantal content was estimated by dividing the mEPSC charge by the single EPSC charge from the same cell. Total, synchronous and asynchronous EPSC charge components were calculated using a homemade Matlab® routine. Our program uses 2 different baselines: first a straight line connecting the current before the train to the baseline after full recovery. This baseline is used to calculate the total charge. To separate the synchronous and asynchronous charge components, the second baseline connects the EPSC values immediately before each pulse following a previously described method7. Both components were calculated using cubic interpolation. To calculate the pool size, the cumulative synchronous charge was used. In order to determine the Y-intercept, data were fitted in their entirety by an exponential and a linear component in the form of the function beside Miniature EPSCs (mEPSCs) were detected using Mini Analysis 6.0 (Synaptosoft Inc.), using thresholds of 7 pA for event amplitude and 15 pC for area. Evoked release events (paired pulse stimulations) were analyzed using an in-house routine in the MATLABWT-EGFP and either Doc2bDN-mCherry or Doc2b6A-mCherry at DIV15. The two SIP stocks were first mixed in a 1:1 volume ratio and then added to each coverslip. Live imaging was performed 8–11 h post infection using a Nikon A1R confocal laser microscope controlled by NIS-elements AR software version 4.30 (Laboratory Imaging). Extracellular solutions for the chamber perfusion was similar to electrophysiology experiments. Neurons were stimulated after 15 seconds of rest by puff application for 30 seconds of a depolarizing solution containing 82.4 mM NaCl, 60 mM KCl, 4 mM CaCl2, 4 mM MgCl2, 10 mM HEPES, 10 mM glucose, 300 mOsm, pH 7.3. The puff was triggered from a Master 8 connected to a valve opening system (WPI type A385). ImageJ was used for data analysis. First, 2 to 6 ROIs depending on cell size and morphology were drawn as lines with a thickness of 10 pixels spanning a neurite or soma. Line scans were obtained from the 4 first stacks each in the resting condition  were made by predicting [Ca2+]free with MaxChelator (http://maxchelator.stanford.edu/CaEGTA-TS.htm). Actual [Ca2+]free was verified in each solution by recording fluorescence excitation spectra of fura-2  at an emission wavelength of 510 nm on a LS55 fluorescence spectrophotometer (Perkin Elmer). [Ca2+]free was calculated as Kd × [(R − Rmin)/(Rmax − R)] × (Fmax380/Fmin380), where F380 is the fluorescence intensity at ʎexcit. = 380 nm and R is the ratio F340/F380. KdEGTA was measured at 34.906 nM.Chelated Casn-glycero-3-phospho-L-serine  and 75% 1,2-dioleoyl-sn-glycero-3-phosphocholine under a nitrogen stream. The phospholipids were resuspended in 50 mM HEPES, 100 mM KCl, pH 7.4 to a final concentration of 1 mg/ml, sonicated 5 times for 10 seconds and centrifuged for 90 min at 21,000 × g to clear the liposomes from large aggregates as described previously48.Liposomes were formed by drying chloroform solutions containing 25% 1,2-dioleoyl-2A (aa125-255) and C2AB (115-412) fragment of Doc2bWT, Doc2bDN and Doc2b6A were expressed as glutathione-S-transferase (GST) fusion proteins in the E. coli strain BL21 and purified as described63. GST-C2A was eluted from glutathione-agarose beads by glutathione, leaving the GST tag attached and allowing GST dimerization. Each C2AB batch was incubated with 10 U thrombin  diluted in 200 µl of thrombin elution buffer  at 4 °C for 16 h, thus removing the GST tag  and polyclonal guinea pig anti-Synaptophysin1  both diluted 1000-fold. After washing, cells were incubated overnight at 4 °C with Alexa-546- and -647-conjugated secondary antibodies , washed again and mounted with Mowiol.After lentiviral expression, hippocampal neurons expressing Doc2b® (Mathworks) routine65.Images were acquired with a confocal microscope LSM 510 (Carl Zeiss) with 488 nm, 543 nm, 633 nm lasers, using a 40x oil immersion objective and a scan resolution of 1024 × 1024 pixels. Stacks of images with an optical thickness of 0.4 µm were obtained. Neuronal morphology characteristics were analyzed with an automated image analysis MATLABCortical neuron cultures from WT and Doc2a, b DKO mice were plated to 200 K cells per well, infected at DIV 1 with lentiviral Doc2b constructs  and harvested at DIV 17. Cells were washed 2 times with PBS, lysed in Laemmli sample buffer, loaded with 50% or the totality of each well for WT and DKO cells respectively, separated by SDS-PAGE and blotted on PVDF membrane (Biorad). Membranes were blocked in 2% skim milk powder (Merck) and 0.5% FCS (Gibco) in PBS with 0.01% Tween-20 (Sigma-Aldrich). Doc2B polyclonal antibody 13.2 was used as primary antibody (1:500) for incubation overnight at 4 °C. Goat anti rabbit alkaline phosphatase (Jackson lab) was used as secondary and Attophos (Promega) as substrate for 30 min incubation at RT. Reprobing was made for actin immunostaining with monoclonal anti-actin antibody C4  and Goat anti mouse alkaline phosphatase as secondary Ab (1:10000).All statistical analysis was performed using SPSS v.25.00 . All data are reported as mean ±SEM, except when specified. The number of measurements “n”, indicate the number of cells per group and “N” the number of independent observations, meaning the number of experimental weeks. Data were checked for normality using Shapiro-Wilk and Kolmogorov-Smirnov tests. Homogeneity of the variance was assessed with Levene’s test and the sphericity assumption was tested with Mauchly’s test. Parametric or non-parametric tests were run depending on homogeneity assumption respect or violation. Moreover, if the sphericity assumption was not met the Greenhouse-Geisser correction was used to adjust the degree of freedom. Considering that groups were independently acquired from one another, independent samples tests were performed: i) For two groups a Mann-Whitney U test was performed; ii) For more than 2 groups, one-way repeated measure ANOVA or Friedman’s ANOVA or Kruskall-Wallis test were used followed by pairwise post-hoc. For each experiment, the p-value alpha significance threshold was adjusted for multiple testing. The effect size was calculated for the independent samples Mann-Whitney U test as Supplementary Information


The correct names are: Minh Duc Pham, Van Hoang Pham and Thang Chien Nguyen. The correct citation is: Pham MD, Byrkit M, Pham VH, Pham T, Nguyen TC (2013) Improving Pharmacy Staff Knowledge and Practice on Childhood Diarrhea Management in Vietnam: Are Educational Interventions Effective? PLoS ONE 8(10): e74882.


This initial change is then amplified into downstream-signaling changes that include increased levels of reactive oxygen species and nitric oxide, which ultimately lead to major changes in the growth rate, defense responses, and leaf stomatal apertures of plants. This review presents and discusses the evidence that links receptor activation to increased [Ca2+]cyt and, ultimately, to growth and diverse adaptive changes in plant development. It also discusses the evidence that increased [Ca2+]cyt also enhances the activity of apyrase (nucleoside triphosphate diphosphohydrolase) enzymes that function in multiple subcellular locales to hydrolyze ATP and ADP, and thus limit or terminate the effects of these potent regulators.Among the most recently discovered chemical regulators of plant growth and development are extracellular nucleotides, especially extracellular ATP (eATP) and extracellular ADP (eADP). Plant cells release ATP into their extracellular matrix under a variety of different circumstances, and this eATP can then function as an agonist that binds to a specific receptor and induces signaling changes, the earliest of which is an increase in the concentration of cytosolic calcium ([Ca There is evidence to suggest that some eATP responses in plants may be initiated by a receptor other than P2K1, and this hypothesis is also discussed.The growth and development of plants are controlled by a remarkably diverse array of endogenous chemical regulators beyond the hormones typically featured in textbooks, and new discoveries add to the list of these regulatory compounds every year. Among the most recently discovered of these compounds are extracellular nucleotides, especially extracellular ATP (eATP) and extracellular ADP (eADP) . Althoug2+]cyt ,5,6,7, a2+]cyt, because two of the biochemically characterized plant ectoapyrases bind to and are stimulated by calmodulin (CaM) cyt [2+]cyt, and the positive results that were observed in cells of Arabidopsis root and shoot tissue cyt . Thus, wt tissue ,11 initi+cyt . Th2+]cyt response to applied eATP and eADP cyt [2+]cyt that was very different from the way eATP induces this change by either of the receptors characterized in animals, because the P2X receptor induces increased [Ca2+]cyt via opening a plasma membrane-localized ion channel, and the P2Y receptor does this by releasing Ca2+ from internal stores via G-protein and phosphoinositide mediation. However, two studies by Demidchik et al. [2+]cyt differed depending on whether eATP or eADP was the extracellular nucleotide agonist. The rapidity of change induced by eADP (<3 s) more resembled the direct mechanism of cation channel opening when P2X is activated than an indirect mechanism that would require the intermediate steps of phosphorylation and synthesis of ROS.The recent report by Chen et al.  was espeCa2+]cyt , this pak et al. ,6 sugges2+ uptake into the roots, occurs in dorn1-1 and dorn1-3 null mutants, indicating some other eATP receptor, as yet unidentified, is the initiator. Interestingly, this eATP response was dependent on the mediation of the Gα subunit of the heterotrimeric G-protein. Because this root-avoidance response was also blocked by EGTA and Gd3+, and because Gα activation is typically upstream of increased [Ca2+]cyt, this report implies a signaling pathway that begins with eATP activation of a non-P2K1 receptor, and proceeds through Gα activation and the opening of a plasma-membrane Ca2+ channel to a root-bending response. Like other root-bending responses, it was also dependent on asymmetric distribution of auxin and PIN2. This report is the first to document a P2K1-independent response to eATP, so discovering the receptor that mediates this response would be a novel and important contribution to the field. Although there are other reports of G-protein mediation of, or involvement in, the responses of plant cells to eATP cyt [2+]cyt can be observed as oscillations due to the transient opening of channels in the PM and/or on internal organelles, and, in Arabidopsis, these oscillations differ depending on whether the nucleotide agonist is ATP, GTP or CTP cyt cyt ,11,22. JP or CTP . After tpathways  and can  [2+]cyt  and the  [2+]cyt . Moreove [2+]cyt ,26,27. T [2+]cyt ,29.Psuedomonas syringae [2+]cyt. There are ROS-activated Ca2+ channels in plants cyt cyt . NO can Ca2+]cyt . In bothCa2+]cyt ,53. WhilS-nitrosylation induced by NO is calmodulin. Regulation of calmodulin activity in this way would provide an avenue for cross-talk between NO and Ca2+ [2+ connection is also suggested by evidence that NO generation can lead to an increase in [Ca2+]cyt. The precise mechanism of this response and identity of Ca2+ channels involved remains unclear, but the regulation may occur by NO induction of cGMP cyt and calmodulin activation are known to have major effects on stress-induced changes in gene expression [2+]cyt. Choi et al. cyt both precedes and is necessary for downstream signaling and physiological changes in plants. 2+ channels, and an increase in the [Ca2+]cyt. The model speculates there could be an alternative pathway whereby P2K1 receptor activation leads to the release of Ca2+ from internal stores, such as vacuoles and mitochondria. Based on the Zhu et al. [α-dependent pathway to induce an increase in [Ca2+]cyt that is required for the root-avoidance response to eATP.In this section, we reviewed publications that linked the early effects of receptor activation by eATP to downstream-signaling changes, revealing that the eATP-induced increase in cyt and ROS levels [2+ ionophore ionomycin induces both an increase in [Ca2+]cyt and ROS levels, and, like eATP, has been linked to the induction of stress responses in plants cyt because of the Ca2+ dependence of enzymes that generate NO and ROS [2+]cyt and downstream signaling and gene-expression changes in response to eATP [2+]cyt and downstream cellular responses, Song et al. cyt above the 100 nM level needed to activate CaM cyt induced by eATP would differently impact CaM-regulated apyrases, like psNTP9 and AtAPY1, depending on their subcellular locale. There is CaM in the wall domain of plants [2+]cyt would not be expected to directly alter the activity of calmodulins in the ECM, where [Ca2+] would typically be above the threshold needed to activate CaM [2+]cyt, however, would likely increase the [Ca2+] of the nucleus, since the [Ca2+] between these two cell compartments appears to rapidly equilibrate cyt are characteristically early steps in the signaling pathways induced by most plant growth regulators, so it is not surprising that it is also one of the first signaling changes induced by eATP. This review describes evidence that eATP-induced changes in [Ca2+]cyt both precede and are necessary for the downstream signaling changes that promote the physiological responses to eATP. Turning off signaling pathways is as important as turning them on and, in peas and Arabidopsis, the same Δ [Ca2+]cyt that turns on the downstream increases in ROS, NO, and MAP kinase activity also, through CaM, activates an apyrase that removes the eATP agonist, thus inactivating the receptor. Although the connection between receptor activation and a rapid increase in [Ca2+]cyt is well understood in mammalian cells, we note in this review that more research is needed to resolve this connection in plants.Changes in [CaArabidopsis thaliana, a member of the Brassicaceae family, which includes less than 1% of the flowering plant species. Other families of plants may have other kinds of receptors, and certainly some plants may not have any CaM-regulated apyrases. The discovery of the P2K1 receptor for eATP has spurred increased interest in purinergic signaling, and this new wave of interest will surely lead to broadening the scope of research to include other model plants, both vascular and nonvascular.Another significant limitation in the current state of knowledge about eATP signaling is that too much of this knowledge is based on research on only one plant,