Loss of m6 A on FAM134B promotes adipogenesis in porcine adipocytes through m6 A-YTHDF2-dependent way.

N6 -methyladenosine (m6 A) mRNA modification plays an important role in adipogenesis, but its role on single gene remains unexplored. Family with Sequence Similarity 134, Member B (FAM134B) is a cis-Golgi transmembrane protein that known to be necessary for the long-term survival of nociceptive and autonomic ganglion neurons. Recent work has shown that FAM134B plays a pivotal role in lipid homeostasis and was identified as its significant m6 A level difference between Chinese local Jinhua pigs and Landrace through RNA-sequence. Here, we construct the non-m6 A FAM134B coding sequence (CDS) plasmid (FAM134B-MUT) and found one important m6 A site on its CDS. Expression of FAM134B-MUT was more effective in promoting porcine preadipocytes adipogenic differentiation and lipid deposition than wild-type FAM134B (FAM134B-WT) both in early and ultimate differentiation stage. FAM134B-MUT functions better in promoting fat deposition by upregulating peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBPα) level. The m6 A reader protein YTH m6 A RNA binding protein 2 (YTHDF2) interacts with FAM134B mRNA and down regulated its protein level. These results demonstrate that FAM134B was the target of YTHDF2, which may recognize and binds the m6 A site of FAM134B to reduce its mRNA lifetime and reduce its protein abundance. © 2018 IUBMB Life, 71(5):580-586, 2019.

The t6A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans.

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALysUUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t6A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t6A-deficient yeast derivatives, it is shown that t6A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t6A synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of t6A. However, we describe here a novel and a more sensitive hybridization-based t6A detection method (compared to HPLC) that showed t6A was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans t6A synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t6A modification ratios and of t6A synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.

CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system.

Toxoplasma gondii is an obligate intracellular protozoan pathogen that traffics to the central nervous system (CNS) following invasion of its host. In the CNS, T. gondii undergoes transformation from a rapidly dividing tachyzoite to a long-lived, slow-dividing bradyzoite contained within cysts. The role of extracellular adenosine in T. gondii pathogenesis has not been previously investigated. T. gondii uses host purines such as adenosine for its energy needs, as it is unable to make its own. Here, we show that CD73(-/-) mice, which lack the ability to generate extracellular adenosine, are protected from T. gondii chronic infection, with significantly fewer cysts and reduced susceptibility to reactivation of infection in the CNS independent of host effector function. Parasite dissemination to the brain was unimpaired in CD73(-/-) hosts, suggesting that the reduced cyst number is due to impaired parasite differentiation in the CNS. Confirming this, T. gondii tachyzoites formed fewer cysts following alkaline pH stress in astrocytes isolated from CD73(-/-) mice compared with wild type, and in fibroblasts treated with a CD73 inhibitor. Cyst formation was rescued in CD73(-/-) astrocytes supplemented with adenosine, but not with adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine. Furthermore, mice lacking adenosine receptors had no defect in cyst formation. Based on these findings, we conclude that CD73 expression promotes Toxoplasma bradyzoite differentiation and cyst formation by a mechanism dependent on the generation of adenosine, but independent of adenosine receptor signaling. Overall, these findings suggest that modulators of extracellular adenosine may be used to develop therapies aimed at defending against human toxoplasmosis.

CD73+ regulatory T cells contribute to adenosine-mediated resolution of acute lung injury.

Acute lung injury (ALI) is characterized by alveolar injury and uncontrolled inflammation. Since most cases of ALI resolve spontaneously, understanding the endogenous mechanisms that promote ALI resolution is important to developing effective therapies. Previous studies have implicated extracellular adenosine signaling in tissue adaptation and wound healing. Therefore, we hypothesized a functional contribution for the endogenous production of adenosine during ALI resolution. As a model, we administered intratracheal LPS and observed peak lung injury at 3 d, with resolution by d 14. Treatment with pegylated adenosine-deaminase to enhance extracellular adenosine breakdown revealed impaired ALI resolution. Similarly, genetic deletion of cd73, the pacemaker for extracellular adenosine generation, was associated with increased mortality (0% wild-type and 40% in cd73(-/-) mice; P<0.05) and failure to resolve ALI adequately. Studies of inflammatory cell trafficking into the lungs during ALI resolution revealed that regulatory T cells (Tregs) express the highest levels of CD73. While Treg numbers in cd73(-/-) mice were similar to controls, cd73-deficient Tregs had attenuated immunosuppressive functions. Moreover, adoptive transfer of cd73-deficient Tregs into Rag(-/-) mice emulated the observed phenotype in cd73(-/-) mice, while transfer of wild-type Tregs was associated with normal ALI resolution. Together, these studies implicate CD73-dependent adenosine generation in Tregs in promoting ALI resolution.

Adenosine dysfunction in epilepsy.

Extracellular levels of the brain's endogenous anticonvulsant and neuroprotectant adenosine largely depend on an astrocyte-based adenosine cycle, comprised of ATP release, rapid degradation of ATP into adenosine, and metabolic reuptake of adenosine through equilibrative nucleoside transporters and phosphorylation by adenosine kinase (ADK). Changes in ADK expression and activity therefore rapidly translate into changes of extracellular adenosine, which exerts its potent anticonvulsive and neuroprotective effects by activation of pre- and postsynaptic adenosine A(1) receptors. Increases in ADK increase neuronal excitability, whereas decreases in ADK render the brain resistant to seizures and injury. Importantly, ADK was found to be overexpressed and associated with astrogliosis and spontaneous seizures in rodent models of epilepsy, as well as in human specimen resected from patients with hippocampal sclerosis and temporal lobe epilepsy. Several lines of evidence indicate that overexpression of astroglial ADK and adenosine deficiency are pathological hallmarks of the epileptic brain. Consequently, adenosine augmentation therapies constitute a powerful approach for seizure prevention, which is effective in models of epilepsy that are resistant to conventional antiepileptic drugs. The adenosine kinase hypothesis of epileptogenesis suggests that adenosine dysfunction in epilepsy undergoes a biphasic response: an acute surge of adenosine that can be triggered by any type of injury might contribute to the development of astrogliosis via adenosine receptor-dependent and -independent mechanisms. Astrogliosis in turn is associated with overexpression of ADK, which was shown to be sufficient to trigger spontaneous recurrent electrographic seizures. Thus, ADK emerges as a promising target for the prediction and prevention of epilepsy.

Adenosine activation of A(2B) receptor(s) is essential for stimulated epithelial ciliary motility and clearance.

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A(1), A(2A), A(2B), or A(3)). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A(1), A(2A), A(2B), or A(3), respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 µM) and/or procaterol (10 µM), in the presence or absence of 5'-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A(1), A(2A), A(3)); 10 µM (A(2B))], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 µM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 µM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ~3 Hz. Likewise, CBF significantly increased ~2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A(1), A(2A), and A(3) knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A(2B) knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A(2B) adenosine receptor activation, likely via a PKA-dependent pathway.

Methylxanthines, seizures, and excitotoxicity.

Clinical evidence, in particular the wide use of theophylline as a bronchodilator, suggests that methylxanthines can cause seizures in patients without known underlying epilepsy. Theophylline is also known to be an added risk factor for seizure exacerbation in patients with epilepsy. The proconvulsant activity of methylxanthines can best be explained by their antagonizing the brain's own anticonvulsant adenosine. Recent evidence suggests that adenosine dysfunction is a pathological hallmark of epilepsy contributing to seizure generation and seizure spread. Conversely, adenosine augmentation therapies are effective in seizure suppression and prevention, whereas adenosine receptor antagonists such as methylxanthines generally exacerbate seizures. The impact of the methylxanthines caffeine and theophylline on seizures and excitotoxicity depends on timing, dose, and acute versus chronic use. New findings suggest a role of free radicals in theophylline-induced seizures, and adenosine-independent mechanisms for seizure generation have been proposed.

Dopamine/adenosine interactions involved in effort-related aspects of food motivation.

Nucleus accumbens dopamine (DA) is involved in effort-related aspects of food motivation. Accumbens DA depletions reduce the tendency of rats to work for food, and alter effort-related choice, but leave other aspects of food motivation and appetite intact. DA and adenosine receptors interact to regulate effort-related processes. Adenosine A(2A) antagonists can reverse the effects of DA D(2) antagonists on effort-related choice, and intra-accumbens injections of a adenosine A(2A) agonist produce effects that are similar to those produced by accumbens DA depletion or antagonism. These studies have implications for understanding the neurochemical interactions that underlie activational aspects of motivation.

Adenosine Depletion as A New Strategy to Decrease Glioblastoma Stem-Like Cells Aggressiveness.

Glioblastoma is the brain tumor with the worst prognosis. This is mainly due to a cell subpopulation with an extremely aggressive potential, called glioblastoma stem-like cells (GSCs). These cells produce high levels of extracellular adenosine, which are increased even more under hypoxic conditions. Under hypoxia, adenosine signaling is related to HIF-2α expression, enhancing cell aggressiveness. Adenosine can be degraded using recombinant adenosine deaminase (ADA) to revert its pathological effects. The aim of this study was to degrade adenosine using ADA in order to decrease malignancy of GSCs. Adenosine depletion was performed using recombinant ADA. Migration and invasion were measured by transwell and matrigel-coated transwell assay, respectively. HIF-2α-dependent cell migration/invasion decreased in GSCs treated with ADA under hypoxia. MRPs-mediated chemoresistance and colony formation decreased in treatment with ADA. In conclusion, adenosine depletion using adenosine deaminase decreases GSCs aggressiveness.

The Inhibition of CD39 and CD73 Cell Surface Ectonucleotidases by Small Molecular Inhibitors Enhances the Mobilization of Bone Marrow Residing Stem Cells by Decreasing the Extracellular Level of Adenosine.

We have recently demonstrated that purinergic signaling in bone marrow (BM) microenvironment regulates mobilization of hematopoietic stem progenitor cells (HSPCs), mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic like stem cells (VSELs) into the peripheral blood (PB). While extracellular adenosine triphosphate (ATP) promotes mobilization, its metabolite extracellular adenosine has an opposite effect. Since ATP is processed in extracellular space to adenosine by ectonucleotidases including cell surface expressed CD39 and CD73, we asked if inhibition of these enzymes by employing in vivo small molecular inhibitors ARL67156 and AMPCP of CD39 and CD73 respectively, alone or combined could enhance granulocyte stimulating factor (G-CSF)- and AMD3100-induced pharmacological mobilization of stem cells. Herein we report that pre-treatment of donor mice with CD39 and CD73 inhibitors facilitates the mobilization of HSPCs as well as other types of BM-residing stem cells. This data on one hand supports the role of purinergic signaling in stem cell trafficking, and on the other since both compounds are not toxic against human cells, they could be potentially employed in the clinic to enhance the mobilization of BM residing stem cells for clinical purposes.

Is adenosine associated with sudden death in schizophrenia? A new framework linking the adenosine pathway to risk of sudden death.

Schizophrenia is associated with an increased mortality from cardiovascular disease. Relatively few studies have assessed the putative association of schizophrenia pathophysiology with sudden death. Low adenosine levels have been associated with schizophrenia. In cardiology, increased mortality among patients with congestive heart failure has been associated with genetic polymorphisms that potentially lead to lower adenosine levels. Thus, we hypothesize that adenosine could link schizophrenia and cardiovascular mortality, with decreased adenosine levels leading to increased vulnerability to hyperexcitability following hypoxic insults, increasing the odds of fatal arrhythmias. Low adenosine levels might also lead to a small increase in overall mortality rates and a major increase in the sudden death rate. This hypothesis paves the way for further investigation of the increased cardiac mortality associated with schizophrenia. Potentially, a better characterization of adenosine-related mechanisms of sudden death in schizophrenia could lead to new evidence of factors leading to sudden death in the general population.

Knock-down of AHCY and depletion of adenosine induces DNA damage and cell cycle arrest.

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.

S-adenosyl methionine: A connection between nutritional and genetic risk factors for neurodegeneration in Alzheimer's disease.

Clinical manifestation of Alzheimer's disease may depend upon interaction among its risk factors. Apolipoprotein E-deficient mice undergo oxidative damage and cognitive impairment when deprived of folate. We demonstrate herein that these mice were depleted in the methyl donor S-adenosyl methionine (SAM), which inhibited glutathione S-transferase, since this enzyme requires methylation of oxidative species prior to glutathione-dependent reduction. Dietary supplementation with SAM alleviated neuropathology. Since SAM deficiency promotes presenilin-1 overexpression, which increases gamma-secretase expression and Abeta generation, these findings directly link nutritional deficiency and genetic risk factors, and support supplementation with SAM for Alzheimer's therapy.

Abnormal regulation of methylthioadenosine and polyamine metabolism in methylthioadenosine phosphorylase-deficient human leukemic cell lines.

Approximately 30 to 40% of human leukemic cell lines are completely deficient in the purine catabolic enzyme 5'-methylthioadenosine phosphorylase. Using two 5'-methylthioadenosine phosphorylase-negative leukemias, we have for the first time been able to measure the synthesis and biological effects of 5'-methylthioadenosine (MTA) in intact mammalian tumor cells. Malignant cells lacking this enzyme, unlike enzyme-positive cells, excreted MTA into the culture medium at a rate of 0.58 to 0.70 nmol/hr/mg protein. The production of the nucleoside was inhibited effectively by nontoxic concentrations of methylglyoxal bis(guanylhydrazone), a putrescine-depending S-adenosylmethionine decarboxylase inhibitor, and also by spermidine and spermine but was enhanced by putrescine. In a reciprocal fashion, MTA at low concentrations progressively increased both the synthesis and concentration of putrescine but suppressed spermine production. The unique alterations in polyamine metabolism induced by elevated MTA levels could offer a selective growth advantage to the 5'-methylthioadenosine phosphorylase-deficient cells and thus may be related to the high frequency of this enzyme deficiency among human leukemic cell lines.

Dependence of adenine production upon polyamine synthesis in cultured human lymphoblasts.

The exact source of de novo adenine produced by mammalian cells remain poorly understood, and this prompted the present study. Using a human lymphoblastoid cell line (WI-L2) deficient in adenine phosphoribosyltransferase (EC 2.4.2.7), we have quantitated the rate of adenine synthesis and the relative importance of the phosphorolysis of 5'-methylthioadenosine versus adenosine or 2'-deoxyadenosine in adenine generation. Dividing adenine phosphoribosyltransferase-deficient WI-L2 cells produced adenine at a rate of 0.27 nmol/mg protein/h. This represented approximately 10% of the rate of hypoxanthine production by WI-L2 cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) but was equivalent to the rate of 5'-methylthioadenosine synthesis by human lymphoblastoid CCRF-CEM deficient in 5'-methylthioadenosine, phosphorylase (5'-methylthioadenosine: orthophosphate methylthioribosyltransferase). Up to 97% of adenine, but not hypoxanthine, synthesis was inhibited dose-dependently by the S-adenosylmethionine decarboxylase-inhibitor methylglyoxal bis(guanylhydrazone) and also by spermidine and spermine, but was enhanced by putrescine. The addition of 2-fluoroadenine, a potent competitive inhibitor of methylthioadenosine phosphorylase (Ki = 0.43 microM) to adenine phosphoribosyl-transferase-deficient cells resulted in a progressive accumulation of 5'-methylthioadenosine in the culture medium, and up to an 85% decrease in adenine production at non-toxic concentrations. These results show that de novo adenine synthesis by dividing human cells is considerable, and that 85-97% derives from the cleavage of 5'-methylthioadenosine and hence from polyamine synthesis.

Adenosine and epilepsy: from therapeutic rationale to new therapeutic strategies.

Adenosine, as the brain's endogenous anticonvulsant, is considered to be responsible for seizure arrest and postictal refractoriness. On the other hand, deficiencies within the adenosine-based neuromodulatory system may contribute to epileptogenesis. Based on these natural mechanisms and on findings that adenosine and its analogs can suppress pharmacoresistant seizures, a new field of adenosine-based therapies has emerged, including the use of adenosine receptor agonists and adenosine transport inhibitors, or the inhibition of adenosine kinase, which is thought to be the key enzyme for the regulation of intra- and extracellular adenosine levels. However, most of these pharmacological approaches are limited by strong systemic side effects ranging from a decrease of heart rate, blood pressure, and body temperature to sedation. Recently, new strategies have been developed aimed at the local reconstitution of the inhibitory adenosinergic tone by intracerebral implantation of cells engineered to release adenosine. Adenosine-releasing cells or devices implanted into or near a seizure focus offer new hopes for a side effect-free therapy for pharmacoresistant epilepsy.

The adenosine antagonist theophylline impairs p50 auditory sensory gating in normal subjects.

In the p50 suppression paradigm, when two auditory stimuli are presented 500 ms apart, the amplitude of the second response (S2), compared with the first (S1), is markedly attenuated in healthy subjects. This is an index of sensory gating. Most schizophrenic patients fail to inhibit the p50 response to the second stimulus, which is assumed to reflect an inhibitory deficit. Adenosine is a neuromodulator with mostly inhibitory activity which is released by physiological stimuli. Since this inhibitory pattern resembles the phenomenon of sensory gating, the contribution of adenosine to p50 suppression was investigated in normal volunteers after treatment with the adenosine antagonist theophylline or placebo. P50 recordings were conducted in thirteen healthy subjects at baseline and 5, 30, 60, and 90 min after oral administration of theophylline (0.66 mg/kg, maximum dose of 500 mg) or placebo in a cross-over design. Baseline results from 17 drug-treated schizophrenic patients were included for comparison. Compared with placebo, theophylline treatment significantly increased P50 ratio (S2/S1) from 0.28 +/- 0.03 to 0.82 +/- 0.11 at 30 min and 0.61 +/- 0.07 at 60 min (mean +/- SEM), which were not significantly different from the schizophrenia group (0.74 +/- 0.05). The increased p50 ratio by theophylline was due to a combined decrease in S1 and increase in S2 amplitude. The impairment of p50 suppression by theophylline in normal subjects suggests a modulatory role of adenosine in sensory gating, which may be related to p50 suppression deficit in schizophrenia and is in agreement with a hypoadenosinergic model of schizophrenia.

Adenosine modulation of [Ca2+]i in cerebellar granular cells: multiple adenosine receptors involved.

Elimination of adenosine by addition of adenosine deaminase (ADA) to the media leads to alterations in intracellular free calcium concentration ([Ca(2+)](i)) in cerebellar granular cells. Adenosine deaminase brings about increases or decreases in [Ca(2+)](i) depending on the previous activation state of the cell. These effects are dependent on the catalytic activity of adenosine deaminase, since its previous catalytic inactivation with Hg(2+) prevents the above-mentioned changes in intracellular calcium. Extracellular calcium is required for the increase in [Ca(2+)](i) promoted by ADA. This rise is insensitive to thapsigargin, but sensitive to micromolar concentrations of Ni(2+). Toxins specific for L, N and P/Q calcium channels do not overtly reduce this effect. N(6)-Cyclopentyl adenosine (CPA), an A(1) receptor agonist, produces a partial reversion of ADA effects, while CGS21680, A(2A)/A(2B) receptor agonist, slightly enhances them. Expression of A(1), A(2A), A(2B) and A(3) adenosine receptor mRNAs was detected in cerebellar granular cell cultures. These results suggest that adenosine modulate [Ca(2+)](i) in cerebellar granule cells through different adenosine receptor subtypes which, at least in part, seem to act through R-type calcium channels.

Glucose and erythrocyte ATP: distinctive effects of dipyridamole and of ticlopidine.

This experiment suggests the following points: 1. Erythrocytes in control patients and in atherosclerosis patients seem to have a variable grade of affinity for adenosine and for plasma glucose. This variable grade seems to fix the level of the adenosine triphosphate (ATP) reserves and induces the erythrocytes' deformability. 2. The drop in the level of ATP reserves that induces the poor deformability of the erythrocytes in atherosclerosis patients would appear to be caused by two consecutive shortages: first a shortage that seems to be related to a deficiency of erythrocyte adenosine as the ATP shortage disappears with dipyridamole treatment and then a shortage induced by the lack of erythrocyte glucose and suppressed by the addition of ticlopidine to the dipyridamole treatment.