Adenosine Kinase Research Articles

Adenosine kinase inhibition attenuates ischemia reperfusion-induced acute kidney injury

Acute kidney injury (AKI) has a high morbidity and mortality, and there is no targeted treatment yet. One of the main causes of AKI is ischemia-reperfusion (IR). Increased release of adenosine under stress and hypoxia exerts anti-inflammatory and antioxidant effects. Adenosine kinase (ADK) is an important enzyme that eliminates adenosine in cells, and can maintain low adenosine concentration in cells. Our previous studies have shown that pretreatment of adenosine kinase inhibitor ABT-702 could markedly attenuate cisplatin-induced nephrotoxicity both in vivo and in vitro. This study is designed to investigate the effect of ADK inhibition on IR-induced AKI. The results showed that ADK expression was positively correlated with the degree of renal tubular injury, which suggested that the degree of ADK inhibition reflected the severity of acute tubular necrosis. In vivo, ADK inhibitor could reduce IR-induced renal injury, which might play a protective role by increasing tissue adenosine level, inhibiting oxidative stress, and reducing cell apoptosis. In HK2 cells, cobaltous dichloride (CoCl2) increased the level of oxidative stress, up-regulated the production of pro-inflammatory factor, and induced apoptosis, ADK inhibition could alleviate the above damaging effects. Moreover, the anti-apoptotic effect exerted by ADK inhibition was independent of inosine. In summary, our results support the idea that ADK inhibition has protective effects on IR-induced AKI. Adenosine kinase inhibition might provide a new target for AKI prevention and treatment.

Adenosine kinase inhibition enhances microvascular dilator function and improves left ventricle diastolic dysfunction.

Inhibition of adenosine kinase (ADK), via augmenting endogenous adenosine levels exerts cardiovascular protection. We tested the hypothesis that ADK inhibition improves microvascular dilator and left ventricle (LV) contractile function under metabolic or hemodynamic stress. In Obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid rats, treatment with the selective ADK inhibitor, ABT-702 (1.5mg/kg, intraperitoneal injections for 8-week) restored acetylcholine-, sodium nitroprusside-, and adenosine-induced dilations in isolated coronary arterioles, an effect that was accompanied by normalized end-diastolic pressure (in mmHg, Lean: 3.4±0.6, Obese: 17.6±4.2, Obese+ABT: 6.6±1.4) and LV relaxation constant, Tau (in ms, Lean: 6.9±1.5, Obese: 13.9±1.7, Obese+ABT: 6.0±1.1). Mice with vascular endothelium selective ADK deletion (ADKVEC KO) exhibited an enhanced dilation to acetylcholine in isolated gracilis muscle (lgEC50 WT: -8.2±0.1, ADKVEC KO: -8.8±0.1, P<.05) and mesenteric arterioles (lgEC50 WT: -7.4±0.2, ADKVEC KO: -8.1±1.2, P<.05) when compared to wild-type (WT) mice, whereas relaxation of the femoral artery and aorta (lgEC50 WT: -7.03±0.6, ADKVEC KO: -7.05±0.8) was similar in the two groups. Wild-type mice progressively developed LV systolic and diastolic dysfunction when they underwent transverse aortic constriction surgery, whereas ADKVEC -KO mice displayed a lesser degree in decline of LV function. Our results indicate that ADK inhibition selectively enhances microvascular vasodilator function, whereby it improves LV perfusion and LV contractile function under metabolic and hemodynamic stress.

Identification of potential dual -targets anti- toxoplasma gondii compounds through structure-based virtual screening and in-vitro studies.

Toxoplasma gondii is the etiologic agent of toxoplasmosis, a disease which can lead to morbidity and mortality of the fetus and immunocompromised individuals. Due to the limited effectiveness or side effects of existing drugs, the search for better drug candidates is still ongoing. In this study, we performed structure-based screening of potential dual-targets inhibitors of active sites of T. gondii drug targets such as uracil phosphoribosyltransferase (UPRTase) and adenosine kinase (AK). First screening of virtual compounds from the National Cancer Institute (NCI) was performed via molecular docking. Subsequently, the hit compounds were tested in-vitro for anti- T. gondii effect using cell viability assay with Vero cells as host to determine cytotoxicity effects and drug selectivities. Clindamycin, as positive control, showed a selectivity index (SI) of 10.9, thus compounds with SI > 10.9 specifically target T. gondii proliferation with no significant effect on the host cells. Good anti- T. gondii effects were observed with NSC77468 (7-ethoxy-4-methyl-6,7-dihydro-5H-thiopyrano[2,3-d]pyrimidin-2-amine) which showed SI values of 25. This study showed that in-silico selection can serve as an effective way to discover potentially potent and selective compounds against T. gondii.

Chronic Morphine-Induced Changes in Signaling at the A3 Adenosine Receptor Contribute to Morphine-Induced Hyperalgesia, Tolerance, and Withdrawal.

Treating chronic pain by using opioids, such as morphine, is hampered by the development of opioid-induced hyperalgesia (OIH; increased pain sensitivity), antinociceptive tolerance, and withdrawal, which can contribute to dependence and abuse. In the central nervous system, the purine nucleoside adenosine has been implicated in beneficial and detrimental actions of morphine, but the extent of their interaction remains poorly understood. Here, we demonstrate that morphine-induced OIH and antinociceptive tolerance in rats is associated with a twofold increase in adenosine kinase (ADK) expression in the dorsal horn of the spinal cord. Blocking ADK activity in the spinal cord provided greater than 90% attenuation of OIH and antinociceptive tolerance through A3 adenosine receptor (A3AR) signaling. Supplementing adenosine signaling with selective A3AR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of A3AR in the spinal cord with an ADK inhibitor or A3AR agonist was associated with reduced dorsal horn of the spinal cord expression of the NOD-like receptor pyrin domain-containing 3 (60%-75%), cleaved caspase 1 (40%-60%), interleukin (IL)-1β (76%-80%), and tumor necrosis factor (50%-60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased twofold. In mice, A3AR agonists prevented the development of tolerance in a model of neuropathic pain and reduced naloxone-dependent withdrawal behaviors by greater than 50%. These findings suggest A3AR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that A3AR agonists may be useful adjunct to opioids to manage their unwanted effects. SIGNIFICANCE STATEMENT: The development of hyperalgesia and antinociceptive tolerance during prolonged opioid use are noteworthy opioid-induced adverse effects that reduce opioid efficacy for treating chronic pain and increase the risk of dependence and abuse. We report that in rodents, these adverse effects are due to reduced adenosine signaling at the A3AR, resulting in NOD-like receptor pyrin domain-containing 3-interleukin-1β neuroinflammation in spinal cord. These effects are attenuated by A3AR agonists, suggesting that A3AR may be a target for therapeutic intervention with selective A3AR agonist as opioid adjuncts.

Compartmentalization of adenosine metabolism in cancer cells and its modulation during acute hypoxia.

Extracellular adenosine mediates diverse anti-inflammatory, angiogenic and vasoactive effects, and has become an important therapeutic target for cancer, which has been translated into clinical trials. This study was designed to comprehensively assess adenosine metabolism in prostate and breast cancer cells. We identified cellular adenosine turnover as a complex cascade, comprising (1) the ectoenzymatic breakdown of ATP via sequential ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1, officially known as ENPP1), ecto-5'-nucleotidase (CD73, also known as NT5E), and adenosine deaminase reactions, and ATP re-synthesis through a counteracting adenylate kinase and members of the nucleoside diphosphate kinase (NDPK, also known as NME/NM23) family; (2) the uptake of nucleotide-derived adenosine via equilibrative nucleoside transporters; and (3) the intracellular adenosine phosphorylation into ATP by adenosine kinase and other nucleotide kinases. The exposure of cancer cells to 1% O2 for 24 h triggered an ∼2-fold upregulation of CD73, without affecting nucleoside transporters, adenosine kinase activity and cellular ATP content. The ability of adenosine to inhibit the tumor-initiating potential of breast cancer cells via a receptor-independent mechanism was confirmed in vivo using a xenograft mouse model. The existence of redundant pathways controlling extracellular and intracellular adenosine provides a sufficient justification for reexamination of the current concepts of cellular purine homeostasis and signaling in cancer.This article has an associated First Person interview with the first author of the paper.

Adenosine kinase and adenosine receptors A1R and A2AR in temporal lobe epilepsy and hippocampal sclerosis and association with risk factors for SUDEP

The "adenosine hypothesis of SUDEP" (sudden unexpected death in epilepsy) predicts that a seizure-induced adenosine surge combined with impaired metabolic clearance can foster lethal apnea or cardiac arrest. Changes in adenosine receptor density and adenosine kinase (ADK) occur in surgical epilepsy patients. Our aim was to correlate the distribution of ADK and adenosine A2A and A1 receptors (A2A R and A1 R) in surgical tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE/HS) with SUDEP risk factors. In 75 cases, patients were stratified into high-risk (n=16), medium-risk (n=11) and low-risk (n=48) categories according to the frequency of generalized seizures before surgery. Using whole-slide scanning Definiens image analysis we quantified the labeling index (LI) for ADK, A2A R, and A1 R in seven regions of interest: temporal cortex, temporal lobe white matter, CA1, CA4, dentate gyrus, subiculum, and amygdala and relative to glial and neuronal densities with glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN). A1 R showed predominant neuronal, A2A R astroglial, and ADK nuclear labeling in all regions but with significant variation. Compared with the low-risk group, the high-risk group had significantly lower A2A R LI in the temporal cortex. In HS cases with severe neuronal cell loss and gliosis predominantly in the CA1 and CA4 regions, significantly higher A1 R was present in the amygdala in high-risk than in low-risk cases. There was no significant difference in neuronal loss or gliosis between the risk groups or differences for ADK labeling. Reduced cortical A2A R suggests glial dysfunction and impaired adenosine modulation in response to seizures in patients at higher risk for SUDEP. Increased neuronal A1 R in the high-risk group could contribute to periictal amygdala dysfunction in SUDEP.

NRH salvage and conversion to NAD+ requires NRH kinase activity by adenosine kinase.

Dihydronicotinamide riboside (NRH) was suggested to act as a precursor for the synthesis of NAD+ but the biochemical pathway converting it has been unknown. Here we show that NRH can be converted into NAD+ via a salvage pathway in which adenosine kinase (AK) acts as an NRH kinase. Using isotope labeling approaches, we demonstrate NRH is fully incorporated into NAD+, with NMNH acting as an intermediate. We further show that AK is enriched in fractions from cell lysates with NRH kinase activity and that AK can convert NRH into NAD+. In cultured cells and the mouse liver, pharmacological or genetic inhibition of AK blocks formation of nicotinamide mononucleotide (NMNH) and inhibits NRH-stimulated NAD+ biosynthesis. Finally, we confirm the presence of endogenous NRH in the liver with metabolomics. Our findings establish NRH as a natural precursor of NAD+ and reveal a new route for NAD+ biosynthesis via an NRH salvage pathway involving AK.

Evidence of Progressive Brainstem Pathology after Repeated Seizure Exposure in a Novel Rat Model of SUDEP

Mutations in ion channel genes can alter neuronal excitability and have been linked to seizure disorders in humans. We previously established that a knockout rat model of Kcnj16, which encodes potassium channel subunit Kir5.1, on a Dahl SS background (SSKcnj16−/−) exhibits renal and respiratory impairments, in addition to audiogenic reflex epilepsy. While seizures can be considered benign acute events, patients with uncontrolled epilepsy are at high risk for Sudden Unexplained Death in Epilepsy (SUDEP), predicted to be due to cardiorespiratory failure following repeated seizures. The SSKcnj16−/− rat allows intractable epilepsy to be modeled with modifiable SUDEP risk by regulating seizure number and providing a controlled experimental environment with consistent occurrence of seizure‐induced death. During daily seizure exposure for up to 10 days, we observed progressive exacerbation of post‐ictal respiratory rate and heart rate suppression with spontaneous mortality in 33% of SSKcnj16−/− rats within hours of a seizure. We tested the hypothesis that repeated seizures induces neuroinflammation and dysregulation of adenosine (ADO) and serotonin (5HT) within brainstem cardiorespiratory nuclei, which may contribute to progressive cardiorespiratory suppression and subsequent mortalities in SSkcnj16−/− rats. Quantification of brainstem neurochemicals by HPLC revealed increased ADO and decreased 5HT in seizure‐exposed compared to naïve SSkcnj16−/− rats. These findings appeared to be brainstem‐specific as ADO levels in the cortex of seizure exposed rats were decreased. Western blot analyses of whole brainstem lysates showed trends toward decreased expression of ADO kinase (ADK) and A1 receptor (A1R) in seizure‐exposed rats. We used immunofluorescent staining and confocal imaging to label and quantify total 5HT, serotonergic neurons (tryptophan hydroxylase, TPH), microglia (Iba1), astrocytes (GFAP), total neurons (NeuN), and total nuclei (DAPI) in respiratory brainstem nuclei of SSkcnj16−/− rats exposed to 0, 3, or 10 seizures (n=2/group). Consistent with our HPLC data, 5HT staining was decreased in the pre‐Bötzinger Complex (preBötC), Nucleus Ambiguus (NA) and medullary Raphe (MR) of seizure‐exposed rats, which also showed fewer TPH‐positive neurons in MR. Staining also revealed that seizures may induce brainstem inflammation that resulted in transient increases reactive microglia and progressive astrogliosis in these rats. SSkcnj16−/− rats showed increased Iba1 after 3 seizures but no change after 10 seizures in the preBötC, NA, MR and nucleus of the solitary tract (NTS). GFAP progressively increased after 3 and 10 seizures in preBötC, NTS, and MR. Further studies will focus on validating these finding by increasing the number of animals in experimental groups and evaluating additional inflammatory markers. This preliminary data suggests that dysregulation of ADO and 5HT and/or inflammation in respiratory brainstem nuclei may mediate respiratory suppression and subsequent mortality associated with repeated seizure exposure in SSKcnj16−/− rats, which may have central relevance to the pathophysiology of SUDEP which remains poorly understood.Support or Funding InformationR01 HL122358

NAD on the rise again.

Maintaining cellular NAD levels through supplementation with intermediates of NAD synthesis has considerable health benefits. A new study demonstrates that the reduced form of nicotinamide riboside, NRH, can be converted to NAD in a biosynthetic pathway that involves adenosine kinase, thus strongly boosting NAD levels in cells and tissues.

Revealing the complexity of protein abundance in chickpea root under drought-stress using a comparative proteomics approach

Global warming has reached an alarming situation, which led to a dangerous climatic condition. The irregular rainfalls and land degradation are the significant consequences of these climatic changes causing a decrease in crop productivity. The effect of drought and its tolerance mechanism, a comparative roots proteomic analysis of chickpea seedlings grown under hydroponic conditions for three weeks, performed at different time points using 2-Dimensional gel electrophoresis (2-DE). After PD-Quest analysis, 110 differentially expressed spots subjected to MALDI-TOF/TOF and 75 spots identified with a significant score. These identified proteins classified into eight categories based on their functional annotation. Proteins involved in carbon and energy metabolism comprised 23% of total identified proteins include mainly glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, transaldolase, and isocitrate dehydrogenase. Proteins related to stress response (heat-shock protein, CS domain protein, and chitinase 2-like) contributed 16% of total protein spots followed by 13% involved in protein metabolism (adenosine kinase 2, and protein disulfide isomerase). ROS metabolism contributed 13% (glutathione S-transferase, ascorbate peroxidase, and thioredoxin), and 9% for signal transduction (actin-101, and 14-3-3-like protein B). Five percent protein identified for secondary metabolism (cinnamoyl-CoA reductase-1 and chalcone-flavononeisomerase 2) and 7% for nitrogen (N) and amino acid metabolism (glutamine synthetase and homocysteine methyltransferase). The abundance of some proteins validated by using Western blotting and Real-Time-PCR. The detailed information for drought-responsive root protein(s) through comparative proteomics analysis can be utilized in the future for genetic improvement programs to develop drought-tolerant chickpea lines.

Deletion of murine slc29a4 modifies vascular responses to adenosine and 5‐hydroxytryptamine in a sexually dimorphic manner

Equilibrative nucleoside transporter 4 (ENT4), encoded by SLC29A4, mediates the flux of both 5‐hydroxytryptamine (5‐HT) and adenosine across cell membranes. We hypothesized that loss of ENT4 function in mice would modify the effects of these established regulators of vascular function. Male and female wild‐type (WT) and slc29a4‐null (ENT4‐KO) mice were compared with respect to their hemodynamics and mesenteric vascular function. Male ENT4‐KO mice had a complete loss of myogenic tone in their mesenteric resistance arteries. This was accompanied by a decrease in blood flow in the superior mesenteric artery in the male ENT4‐KO mice, and a reduced responsiveness to 5‐HT. In contrast, endothelium‐dependent relaxations of mesenteric arteries from female ENT4‐KO mice were more sensitive to Ca2+‐activated K+ (KCa) channel blockade than WT mice. Female ENT4‐KO mice also demonstrated an enhanced vasodilatory response to adenosine in vivo that was not seen in males. Ketanserin (5‐HT2A inhibitor) and GR55562 (5‐HT1B/1D inhibitor) decreased 5‐HT‐induced tone, but only ketanserin inhibited the relaxant effect of 5‐HT in mesenteric arteries. 5‐HT‐evoked increases in tone were elevated in arteries from ENT4‐KO mice upon block of endothelial relaxant pathways, with arteries from female ENT4‐KO mice showing the greatest increase. Adenosine A2b receptor expression was decreased, while other adenosine transporter subtypes, as well as adenosine deaminase and adenosine kinase were increased in mesenteric arteries from male, but not female, ENT4‐KO mice. These findings indicate that deletion of slc29a4 leads to sex‐specific changes in vascular function with significant consequences for regulation of blood flow and pressure by adenosine and 5‐HT.

Plant regeneration in leaf culture of Centaurium erythraea Rafn. Part 3: de novo transcriptome assembly and validation of housekeeping genes for studies of in vitro morphogenesis

Centaurium erythraea Rafn. (common centaury, Gentianaceae) is a medicinal plant with great regeneration potential and developmental plasticity in vitro. Centaury can be regenerated from leaf explants by both somatic embryogenesis (SE) and shoot development (SD). We believe that its regeneration potential and developmental plasticity rest on high activity of certain genes, which may not be active or present in species recalcitrant to in vitro regeneration. However, there are no sequenced Gentianaceae genomes to support investigation of the molecular events during SE or SD. To this end, we have sequenced six centaury transcriptomes (embryogenic calli, globular somatic embryos, cotyledonary somatic embryos, adventitious buds, leaves and roots of in vitro grown plants) and de novo assembled centaury referent transcriptome comprising 105.726 genes. The high quality and completeness transcriptome was functionally annotated against NCBI nt, Swissprot and PFAM databases with KOG and GO enrichment. In addition, 11 housekeeping and functional genes were validated for expression stability in 27 tissue samples representing the processes of SE and SD, plants from nature and wounded tissues using GeNorm and NormFinder. The most stable genes that can be used for expression studies during SE, SD and in vitro manipulations are Ribosomal protein L2 (RPL2) and TATA binding protein 1 (TBP1) in combination with RAS (Rat Sarcoma)-related Nuclear protein (RAN) or Adenosine kinase (AK). These results comprise a complete framework for the search for genes involved in SE and SD, but may also be useful in identifying genes involved in biosynthesis of C. erythraea secondary metabolites. De novo assembled and fully annotated Centaurim erythraea transcriptome is publically available along with validated housekeeping genes with stable expression during in vitro development and mechanical injury.

Role of Adenosine Kinase Inhibitor in Adenosine Augmentation Therapy for Epilepsy: A Potential Novel Drug for Epilepsy.

Epilepsy, an ancient disease, is defined as an enduring predisposition to generate epileptic seizures and by the neurobiological, cognitive, psychological, and social consequences of this condition. Antiepileptic drugs (AEDs) are currently used as first-line treatment for patients with epilepsy; however, around 36% of patients are diagnosed with refractory epilepsy, which means two or more AEDs have been considered as failed after sufficiently correct usage. Unfortunately, it is unlikely that the improvement of the efficacy of AEDs will be easily achieved, especially since no AEDs show efficacy in ceasing epileptogenesis. Consequently, several endogenous anticonvulsants attract investigators and epileptologists, such as galanin, cannabis, and adenosine. Astrogliosis is a neuropathological hallmark of epilepsy, whatever the etiology is, and astrogliosis is frequently associated with overexpression of adenosine kinase, which means downregulation of synaptic levels of adenosine. Consequently, adenosine is negatively regulated by adenosine kinase through the astrocyte-based cycle. On the other hand, focal adenosine augmentation therapy, using adenosine kinase inhibitor, has been proved to be effective for reducing seizures in both animal models and in vitro human brain tissue resected from a variety of etiology of refractory epilepsy patients. In addition to reducing seizures, adenosine augmentation therapy can also palliate co-morbidities, like sleep, cognition, or depression. Of importance, transgenic mice with reduced ADK were resistant to epileptogenesis induced by acute brain injury. In terms of translation, based on findings of adenosinerelated epileptogenic mechanisms, the application into clinical practice seems to be feasible by molecular strategies that have been already experimentally implemented, including gene and RNA interference. In the present review, we will focus on the evidence of ADK dysfunction in the epileptic brain from human beings and animals, and review the role of ADK inhibitor in adenosine augmentation therapy and the underlying mechanism of prevention of epileptogenesis.

Adenosine kinase is critical for neointima formation after vascular injury by inducing aberrant DNA hypermethylation.

Adenosine receptors and extracellular adenosine have been demonstrated to modulate vascular smooth muscle cell (VSMC) proliferation and neointima formation. Adenosine kinase (ADK) is a major enzyme regulating intracellular adenosine levels but is function in VSMC remains unclear. Here, we investigated the role of ADK in vascular injury-induced smooth muscle proliferation and delineated the mechanisms underlying its action. We found that ADK expression was higher in the neointima of injured vessels and in platelet-derived growth factor-treated VSMCs. Genetic and pharmacological inhibition of ADK was enough to attenuate arterial injury-induced neointima formation due to inhibition of VSMC proliferation. Mechanistically, using infinium methylation assays and bisulfite sequencing, we showed that ADK metabolized the intracellular adenosine and potentiated the transmethylation pathway, then induced the aberrant DNA hypermethylation. Pharmacological inhibition of aberrant DNA hypermethylation increased KLF4 expression and suppressed VSMC proliferation as well as the neointima formation. Importantly, in human femoral arteries, we observed increased ADK expression and DNA hypermethylation as well as decreased KLF4 expression in neointimal VSMCs of stenotic vessels suggesting that our findings in mice are relevant for human disease and may hold translational significance. Our study unravels a novel mechanism by which ADK promotes VSMC proliferation via inducing aberrant DNA hypermethylation, thereby down-regulating KLF4 expression and promoting neointima formation. These findings advance the possibility of targeting ADK as an epigenetic modulator to combat vascular injury.

Adenosine Suppresses Cholangiocarcinoma Cell Growth and Invasion in Equilibrative Nucleoside Transporters-Dependent Pathway

Cholangiocarcinoma (CCA) is a lethal disease with increasing incidence worldwide. Previous study showed that CCA was sensitive to adenosine. Thereby, molecular mechanisms of CCA inhibition by adenosine were examined in this study. Our results showed that adenosine inhibited CCA cells via an uptake of adenosine through equilibrative nucleoside transporters (ENTs), instead of activation of adenosine receptors. The inhibition of ENTs by NBTI caused the inhibitory effect of adenosine to subside, while adenosine receptor antagonists, caffeine and CGS-15943, failed to do so. Intracellular adenosine level was increased after adenosine treatment. Also, a conversion of adenosine to AMP by adenosine kinase is required in this inhibition. On the other hand, inosine, which is a metabolic product of adenosine has very little inhibitory effect on CCA cells. This indicates that a conversion of adenosine to inosine may reduce adenosine inhibitory effect. Furthermore, there was no specific correlation between level of proinflammatory proteins and CCA responses to adenosine. A metabolic stable analog of adenosine, 2Cl-adenosine, exerted higher inhibition on CCA cell growth. The disturbance in intracellular AMP level also led to an activation of 5′ AMP-activated protein kinase (AMPK). Accordingly, we proposed a novel adenosine-mediated cancer cell growth and invasion suppression via a receptor-independent mechanism in CCA.

LncRNA CASC2 inhibits astrocytic activation and adenosine metabolism by regulating PTEN in pentylenetetrazol-induced epilepsy model

Growing evidence has indicated that long noncoding RNAs (lncRNAs) are closely implicated in the progress of epilepsy. However, the expression profile and potential function of long noncoding RNAs cancer susceptibility candidate 2 (lncRNA CASC2) in epilepsy are poorly studied. The aim of this study was to testify the influence of lncRNA CASC2 on epilepsy in rat and cell models of epileptic seizure. We adopted qRT-PCR on the hippocampus of rats following pentylenetetrazol (PTZ)-stimulated epilepsy. To further examine the correlation between lncRNA CASC2 and Phosphatase and tensin homolog (PTEN), we detected the effects of lncRNA CASC2 on PTEN expression. We found that lncRNA CASC2 and PTEN expression were positively correlated in PTZ-induced epileptic rat. Overexpression of lncRNA CASC2 prolonged the latency and reduced the frequency of epileptic seizure, suppressed the activation of astrocytes and the release of adenosine in epileptic rat, whereas downregulation of lncRNA CASC2 exhibited the opposite effects. Meanwhile, lncRNA CASC2 decreased the adenosine metabolism related proteins expression of p38, Equilibrative nucleoside transporter 1 (ENT1) and Adenosine Kinase (ADK). In PTZ-treated astrocytes, PTEN was found to be a direct target of lncRNA CASC2. Additionally, downregulation of PTEN attenuated the protective effect of lncRNA CASC2 overexpression in epileptic seizure. Our findings manifested the key role of lncRNA CASC2 in the occurrence of epilepsy by targeting PTEN, which provided a novel target for epilepsy therapy.

S-Adenosyl-l-Methionine Salvage Impacts Psilocybin Formation in "Magic" Mushrooms.

Psychotropic Psilocybe mushrooms biosynthesize their principal natural product psilocybin in five steps, among them a phosphotransfer and two methyltransfer reactions, which consume one equivalent of 5′‐adenosine triphosphate (ATP) and two equivalents of S‐adenosyl‐l‐methionine (SAM). This short but co‐substrate‐intensive pathway requires nucleoside cofactor salvage to maintain high psilocybin production rates. We characterized the adenosine kinase (AdoK) and S‐adenosyl‐l‐homocysteine (SAH) hydrolase (SahH) of Psilocybe cubensis. Both enzymes are directly or indirectly involved in regenerating SAM. qRT‐PCR expression analysis revealed an induced expression of the genes in the fungal primordia and carpophores. A one‐pot in vitro reaction with the N‐methyltransferase PsiM of the psilocybin pathway demonstrates a concerted action with SahH to facilitate biosynthesis by removal of accumulating SAH.

Adenosine kinase inhibition promotes proliferation of neural stem cells after traumatic brain injury.

Traumatic brain injury (TBI) is a major public health concern and remains a leading cause of disability and socio-economic burden. To date, there is no proven therapy that promotes brain repair following an injury to the brain. In this study, we explored the role of an isoform of adenosine kinase expressed in the cell nucleus (ADK-L) as a potential regulator of neural stem cell proliferation in the brain. The rationale for this hypothesis is based on coordinated expression changes of ADK-L during foetal and postnatal murine and human brain development indicating a role in the regulation of cell proliferation and plasticity in the brain. We first tested whether the genetic disruption of ADK-L would increase neural stem cell proliferation after TBI. Three days after TBI, modelled by a controlled cortical impact, transgenic mice, which lack ADK-L (ADKΔneuron) in the dentate gyrus (DG) showed a significant increase in neural stem cell proliferation as evidenced by significant increases in doublecortin and Ki67-positive cells, whereas animals with transgenic overexpression of ADK-L in dorsal forebrain neurons (ADK-Ltg) showed an opposite effect of attenuated neural stem cell proliferation. Next, we translated those findings into a pharmacological approach to augment neural stem cell proliferation in the injured brain. Wild-type C57BL/6 mice were treated with the small molecule adenosine kinase inhibitor 5-iodotubercidin for 3 days after the induction of TBI. We demonstrate significantly enhanced neural stem cell proliferation in the DG of 5-iodotubercidin-treated mice compared to vehicle-treated injured animals. To rule out the possibility that blockade of ADK-L has any effects in non-injured animals, we quantified baseline neural stem cell proliferation in ADKΔneuron mice, which was not altered, whereas baseline neural stem cell proliferation in ADK-Ltg mice was enhanced. Together these findings demonstrate a novel function of ADK-L involved in the regulation of neural stem cell proliferation after TBI.

Rasmussen’s encephalitis: mechanisms update and potential therapy target

Rasmussen’s encephalitis (RE) is rare neurological diseases characterized as epilepsia partialis continua, invariably hemiparesis, and cognitive impairment. This disease is encountered frequently in childhood and presents with progressive atrophy of the unilateral hemisphere, and there are also sustained neurological complications. Owing to uncertain pathogenesis, the most effective way to limit the influence of seizures currently is cerebral hemispherectomy. In this review, we focus on four main lines of pathogenesis: virus infection, antibody-mediated, cell-mediated immunity, and microglia activation. Although one or more antigenic epitopes may give rise to infiltrating T cell responses in RE brain tissue, no exact antigen was confirmed as the definite cause of the disease. On the other hand, the appearance of antibodies related with RE seem to be a secondary pathological process. Synthetic studies have suggested an adaptive immune mechanism mediated by CD8+ T cells and an innate immune mechanism mediated by activated microglia and neuroglia. Accordingly, opinions have been raised that immunomodulatory treatments aimed at initial damage to the brain that are induced by cytotoxic CD8+ T cell lymphocytes and microglia in the early stage of RE slow down disease progression. However, systematic exploration of the theory behind these therapeutic effects based on multicenter and large sample studies are needed. In addition, dysfunction of the adenosine system, including the main adenosine removing enzyme adenosine kinase and adenosine receptors, has been demonstrated in RE, which might provide a novel therapeutic target for treatment of RE in future.

Dysregulation of adenosine kinase isoforms in breast cancer.

Dysregulated adenosine signaling pathway has been evidenced in the pathogenesis of breast cancer. However, the role of adenosine kinase (ADK) in tumorigenesis remains unclear while it crucially regulates the removal and availability of adenosine. ADK has two isoforms that localize to discrete subcellular spaces: i.e., nuclear, long-isoform (ADK-L) and cytosolic, short-isoform (ADK-S). We hypothesized that these two ADK isoforms would be differentially expressed in breast cancer and may contribute to divergent cellular actions in cancer. In this study, we examined the expression profiles of ADK isoforms in breast cancer tissues from 46 patient and followed up with an in vitro investigation by knocking down the expression of ADK-L or ADK-S using CRISPR gene editing to evaluate the role of ADK isoform in cancer progression and metastasis of cultured triple-negative breast cancer cell line MDA-MB-231. We demonstrated that (i) ADK-L expression level was significantly increased in breast cancer tissues versus paired normal tissues adjacent to tumor, whereas the ADK-S expression levels were not significantly different between cancerous and normal tissues; (ii) CRISPR/Cas9-mediated downregulation of ADK isoforms, led to suppressed cellular proliferation, division, and migration of cultured breast cancer cells; (iii) ADK-L knockdown significantly upregulated gene expression of matrix metalloproteinase (ADAM23, 9.93-fold; MMP9, 24.58-fold) and downregulated expression of cyclin D2 (CCND2, -30.76-fold), adhesive glycoprotein THBS1 (-8.28-fold), and cystatin E/M (CST6, -16.32-fold). Our findings suggest a potential role of ADK-L in mitogenesis, tumorigenesis, and tumor-associated tissue remodeling and invasion; and the manipulation of ADK-L holds promise as a therapeutic strategy for aggressive breast cancer.