Pharmacological Stimulation Research Articles

ADP-ribose transferase PARP16 mediated-unfolded protein response contributes to neuronal cell damage in cerebral ischemia/reperfusion.

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis, leading to impairment of endoplasmic reticulum (ER) function and activating the unfolded protein response (UPR). PARP16 is an active (ADP-ribosyl)transferase known tail-anchored ER transmembrane protein with a cytosolic catalytic domain. Here, we find PARP16 is highly expressed in ischemic cerebral hemisphere and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated immortalized hippocampal neuronal cell HT22. Using an adeno-associated virus-mediated PARP16 knockdown approach in mice, we find PARP16 knockdown decreases infarct demarcations and has a better neurological outcome after ischemic stroke. Our data indicate PARP16 knockdown decreases ER stress and neuronal death caused by OGD/R, whereas PARP16 overexpression promotes ER stress-mediated cell damage in primary cortical neurons. Furthermore, PARP16 functions mechanistically as ADP-ribosyltransferase to modulate the level of ADP-ribosylation of the corresponding PERK and IRE1α arm of the UPR, and such modifications mediate activation of PERK and IRE1α. Indeed, pharmacological stimulation of the UPR using Brefeldin A partly counteracts PARP16 knockdown-mediated neuronal protection upon OGD/R treatment. In conclusion, PARP16 plays a crucial role in post-ischemic UPR and PARP16 knockdown alleviates brain injury after ischemic stroke. This study demonstrates the potential of the PARP16-PERK/IRE1α axis as a target for neuronal survival in ischemic stroke.

Sympathetic Denervation and Pharmacological Stimulation of Parasympathetic Nervous System Prevent Pulmonary Vascular Bed Remodeling in Rat Model of Chronic Thromboembolic Pulmonary Hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) develops in 1.5-2.0% of patients experiencing pulmonary embolism (PE) and is characterized by stable pulmonary artery obstruction, heart failure, and poor prognosis. Little is known about involvement of autonomic nervous system (ANS) in the mechanisms of CTEPH. This study was aimed at evaluation of the effect of vagal and sympathetic denervation, as well as stimulation of the parasympathetic nervous system, on the outcomes of CTEPH in rats. CTEPH was induced by multiple intravenous injections of alginate microspheres. Sympathetic and vagal denervation was performed using unilateral surgical ablation of the stellate ganglion and vagotomy, respectively. Stimulation of the parasympathetic nervous system was carried out by administering pyridostigmine. The effect of neuromodulatory effects was assessed in terms of hemodynamics, histology, and gene expression. The results demonstrated the key role of ANS in the development of CTEPH. Sympathetic denervation as well as parasympathetic stimulation resulted in attenuated pulmonary vascular remodeling. These salutary changes were associated with altered MMP2 and TIMP1 expression in the lung and decreased FGFb level in the blood. Unilateral vagotomy had no effect on physiological and morphological outcomes of the study. The data obtained contribute to the identification of new therapeutic targets for CTEPH treatment.

Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological β-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.

β3 Adrenergic Receptor Agonist Mirabegron Increases AQP2 and NKCC2 Urinary Excretion in OAB Patients: A Pleiotropic Effect of Interest for Patients with X-Linked Nephrogenic Diabetes Insipidus.

We previously reported the novel finding that β3-AR is functionally expressed in the renal tubule and shares its cellular localization with the vasopressin receptor AVPR2, whose physiological stimulation triggers antidiuresis by increasing the plasma membrane expression of the water channel AQP2 and the NKCC2 symporter in renal cells. We also showed that pharmacologic stimulation of β3-AR is capable of triggering antidiuresis and correcting polyuria, in the knockout mice for the AVPR2 receptor, the animal model of human X-linked nephrogenic diabetes insipidus (XNDI), a rare genetic disease still missing a cure. Here, to demonstrate that the same response can be evoked in humans, we evaluated the effect of treatment with the β3-AR agonist mirabegron on AQP2 and NKCC2 trafficking, by evaluating their urinary excretion in a cohort of patients with overactive bladder syndrome, for the treatment of which the drug is already approved. Compared to baseline, treatment with mirabegron significantly increased AQP2 and NKCC2 excretion for the 12 weeks of treatment. This data is a step forward in corroborating the hypothesis that in patients with XNDI, treatment with mirabegron could bypass the inactivation of AVPR2, trigger antidiuresis and correct the dramatic polyuria which is the main hallmark of this disease.

Cavernoscanner: Technique and modelling in erectile dysfunction due to caverno-venous leakage (CVL)

As a result of technical progress in the exploration of erectile dysfunction by caverno-venous leaks, initiated by conventional radiography under artificial erection, authenticated by ultrasound under pharmacological stimulation, cavernoscanner is proving to be an indispensable tool for their precise localization. The study of 337 consecutive examinations carried out over 5 years (2015-2020) with the Vizua platform made it possible to confirm the initial classification proposed by Virag and Paul, distinguishing between no leakage (A), deep leakage (B), superficial leakage (C), and the addition of deep and superficial leakage (D); and to identify two additional locations: opacification of the corpus spongiosum and leakage from the bottom of the corpora cavernosa. A total of 11.57% had no leakage, giving an efficiency of 88.3% for the orientation by pharmacologically stimulated ultrasound; 20.17% had a deep leakage, of which 10.9% were isolated; 15.3% had a superficial leakage, of which 5.4% were isolated; finally 46% had mixed leakage, of which 20.47% had no communication with the corpus spongiosum and leakage through the floor of the corpora cavernosa. CT scan was performed under pharmacological stimulation and contrast medium perfusion with 3 acquisition passages: filling, state phase and emptying. The study of the native and reconstructed images allows the systematized localizations, the evaluation of the severity of the leakage based on the rigidity obtained and the speed of evacuation with or without bladder opacification. Complications are minor (ecchymosis) and the very rare persistence of a rigid erection (n=3) well controlled by injection of the alpha-blocker etilefrine.

S-12-7: THE RENIN ANGIOTENSIN SYSTEM IN BASIC AND CLINICAL RESEARCH

The renin-angiotensin-system (RAS) is one of the oldest known hormonal systems preserved in multiple species over millions of years. A key enzyme of the system, renin, was discovered in 1898, and ever since a huge body of research has continuously revealed physiological and pathophysiological features of the RAS. Classically, these include, via the so-called angiotensin AT1 receptor, powerful vasoconstriction, regulation of the body fluid balance by inducing thirst, stimulation of aldosterone release from the adrenal cortex, renal salt retention, stimulation of the sympathetic nervous system and, more recently discovered, pro-fibrotic and pro-inflammatory effects. Pathophysiological actions of the RAS, with its extension to those of aldosterone (RAAS), include the induction of arterial hypertension, cardiac and vascular hypertrophy, lung and renal fibrosis, vascular atherosclerosis, perivascular fibrosis or chronic inflammation and others. Attempts to inhibit the RAS in hypertension and other cardiovascular diseases first led to the clinical introduction of ACE-inhibitors some forty years ago, followed by selective antagonists of the AT1 receptor, the sartans. After a multitude of evidence-generating trials and global success in clinical practice, both approaches are now firmly established in the therapy of hypertension as well as cardio-metabolic and renal diseases. More recently, research has focused on the so-called protective arm of the RAS, mediated by stimulation of the angiotensin AT2 receptor by or by the Mas receptor stimulated by the angiotensin 1–7 peptide. Activation of these systems has exhibited beneficial effects in numerous preclinical studies via opposing AT1-mediated actions and beyond. Other degradation peptides of the angiotensin precursor, angiotensinogen, may also exhibit protective effects via these receptors. Agonists for the AT2 receptor as well as for the ACE2-Mas system have been developed and are currently under clinical evaluation. It remains to be seen whether the pharmacological stimulation of the protective arm of the RAS will become an effective clinical tool in various cardiovascular and metabolic indications. Theoretically, it would make sense not only to block the harmful RAS but, at the same time, also stimulate the beneficial actions of the system.

Comparison of myocardial perfusion between the users of two antiepileptic medications: valproate vs. carbamazepine.

The prevalence of coronary artery disease (CAD) is high in patients with epilepsy using antiepileptic drugs (AED). Epilepsy, AED, or the type and duration of AED use , may contribute to higher CAD risk.In this study, myocardial perfusion imaging (MPI) was compared between patients using carbamazepine and valproate. Out of 73 patients receiving carbamazepine or valproate monotherapy for more than 2 years, visited at a tertiary referral clinic, 32 patients participated in a 2-day stress and rest phases MPI. For each phase, 15-25 mCi 99mTc-MIBI was injected, at peak exercise or by pharmacologic stimulation for the stress phase. SPECT with cardiac gating was done by a dual-head gamma camera and processed and quantified. Scans with at least one definite reversible hypo-perfusion segment were considered abnormal. Seventeen patients received carbamazepine monotherapy and 15 valproates. Age and duration of AED use were similar between the groups. Two scans were abnormal (6.3%) both in valproate group (13.3%). Duration of AED use was higher in patients with abnormal scans. In patients receiving monotherapy >2 years, the frequency of abnormal MPI was similar between groups (P-value=0.12). In patients receiving monotherapy > 5 years, prevalence of abnormal MPI was higher in the valproate group (28.6% vs. 0.0%; P-value=0.042). Considering valproate subgroup, ischemic patients had higher duration of AED use, comparing with the normal patients (17.0±4.2 vs. 6.4±4.8, P-value=0.014). MPIs were abnormal in patients receiving valproate after 5 years compared to patients receiving carbamazepine. Long-term valproate use may increase the risk of CAD.

Stimulation of Liver Fibrosis by N2 Neutrophils in Wilson’s Disease

Wilson's disease is an inherited hepatoneurologic disorder caused by mutations in the copper transporter ATP7B. Liver disease from Wilson's disease is one leading cause of cirrhosis in adolescents. Current copper chelators and zinc salt treatments improve hepatic presentations but frequently worsen neurologic symptoms. In this study, we showed the function and machinery of neutrophil heterogeneity using a zebrafish/murine/cellular model of Wilson's disease. We investigated the neutrophil response in atp7b-/- zebrafish by live imaging, movement tracking, and transcriptional analysis in sorted cells. Experiments were conducted to validate liver neutrophil heterogeneity in Atp7b-/- mice. Invitro experiments were performed in ATP7B-knockout human hepatocellular carcinomas G2 cells and isolated bone marrow neutrophils to reveal the mechanism of neutrophil heterogeneity. Recruitment of neutrophils into the liver is observed in atp7b-/- zebrafish. Pharmacologic stimulation of neutrophils aggravates liver and behavior defects in atp7b-/- zebrafish. Transcriptional analysis in sorted liver neutrophils from atp7b-/- zebrafish reveals a distinct transcriptional profile characteristic of N2 neutrophils. Furthermore, liver N2 neutrophils also were observed in ATP7B-knockout mice, and pharmacologically targeted transforming growth factor β1, DNA methyltransferase, or signal transducer and activator of transcription 3 reduces liver N2 neutrophils and improves liver function and alleviates liver inflammation and fibrosis in ATP7B-knockout mice. Epigenetic silencing of Socs3 expression by transforming growth factor β1 contributes to N2-neutrophil polarization in isolated bone marrow neutrophils. Our findings provide a novel prospect that pharmacologic modulation of N2-neutrophil activity should be explored as an alternative therapeutic to improve liver function in Wilson's disease.

Afatinib Reverses EMT via Inhibiting CD44-Stat3 Axis to Promote Radiosensitivity in Nasopharyngeal Carcinoma

Afatinib, a second-generation tyrosine kinase inhibitor (TKI), exerts its radiosensitive effects in nasopharyngeal carcinoma (NPC). However, the detailed mechanism of afatinib-mediated sensitivity to radiation is still obscure in NPC. Quantitative phosphorylated proteomics and bioinformatics analysis were performed to illustrate the global phosphoprotein changes. The activity of the CD44-Stat3 axis and Epithelial-Mesenchymal Transition (EMT)-linked markers were evaluated by Western blotting. Wound healing and transwell assays were used to determine the levels of cell migration upon afatinib combined IR treatment. Cell proliferation was tested by CCK-8 assay. A pharmacological agonist by IL-6 was applied to activate Stat3. The xenograft mouse model was treated with afatinib, radiation or a combination of afatinib and radiation to detect the radiosensitivity of afatinib in vivo. In the present study, we discovered that afatinib triggered global protein phosphorylation alterations in NPC cells. Further, bioinformatics analysis indicated that afatinib inhibited the CD44-Stat3 signaling and subsequent EMT process. Moreover, functional assays demonstrated that afatinib combined radiation treatment remarkably impeded cell viability, migration, EMT process and CD44-Stat3 activity in vitro and in vivo. In addition, pharmacological stimulation of Stat3 rescued radiosensitivity and biological functions induced by afatinib in NPC cells. This suggested that afatinib reversed the EMT process by blocking the activity of the CD44-Stat3 axis. Collectively, this work identifies the molecular mechanism of afatinib as a radiation sensitizer, thus providing a potentially useful combination treatment and drug target for NPC radiosensitization. Our findings describe a new function of afatinib in radiosensitivity and cancer treatment.

Sulphide donors affect the expression of mucin and sulphide detoxification genes in the mucosal organs of Atlantic salmon (Salmo salar).

Hydrogen sulphide (H2S) is a gas that affects mucosal functions in mammals. However, its detrimental effects are less understood in fish despite being known to cause mass mortality. Here we used explant models to demonstrate the transcriptional responses of Atlantic salmon (Salmo salar) mucosa to the sulphide donor sodium hydrosulphide (NaHS). The study focused on two groups of genes: those encoding for sulphide detoxification and those for mucins. Moreover, we performed pharmacological studies by exposing the organ explants to mucus-interfering compounds and consequently exposed them to a sulphide donor. Exposure to NaHS significantly affected the expression of sulphide:quinone oxidoreductase (sqor1, sqor2) and mucin-encoding genes (muc5ac, muc5b). The general profile indicated that NaHS upregulated the expression of sulphide detoxification genes while a significant downregulation was observed with mucins. These expression profiles were seen in both organ explant models. Pharmacological stimulation and inhibition of mucus production used acetylcholine (ACh) and niflumic acid (NFA), respectively. This led to a significant regulation of the two groups of marker genes in the gills and olfactory rosette explants. Treatment of the mucosal organ explants with the mucus-interfering compounds showed that low dose NFA triggered more substantial changes while a dose-dependent response could not be established with ACh. Pharmacological interference demonstrated that mucins played a crucial role in mucosal protection against H2S toxicity. These results offer insights into how a sulphide donor interfered with mucosal responses of Atlantic salmon and are expected to contribute to our understanding of the least explored H2S-fish interactions-particularly at the mucosa.

HPB O05 Identifying pathways involved in post-hepatectomy liver failure

Abstract Background The liver is the only visceral organ with regenerative capabilities. This ensures maintenance of the liver-to-bodyweight ratio required for homeostasis. Failure of this mechanism can be fatal. Precipitants to liver failure include infection, drug toxicity and surgical resection. Post-hepatectomy liver failure (PHLF) is associated with small for size syndrome i.e., insufficient post hepatectomy liver volume. The mechanisms by which liver failure occurs on a molecular basis are not yet fully understood, but theories include uncontrolled apoptosis from unrivalled superoxide radical formation causing irreparable oxidative stress. A major research focus of liver regeneration has been modulating the anti-oxidative response to any form of liver injury, but identifying pathways involved in dysfunctional liver regeneration after surgery have yet to be elucidated. Nuclear factor erythoid 2-related factor 2 (Nrf2) is an anti-oxidant transcription factor which promotes liver regeneration. Using a murine partial hepatectomy model, we have shown that pharmacological stimulation of Nrf2 increases the rate of post hepatectomy liver regeneration. Furthermore glutathione (GSH – a downstream target of Nrf2), has been linked with impaired liver regeneration when deficient. In this study, we present data on GSH analysis in liver tissue taken from patients undergoing hepatectomy. Methods Liver parenchyma was collected ex-vivo from patients undergoing liver resection (National Research Ethics Service approval 11/NW/0327). Additionally, isolated RNA from hepatic parenchyma from patients undergoing liver resections was received from collaborators at the Medical University of Vienna, Austria. From liver tissue, protein was isolated using a standard protocol and immunoblotting performed; RNA was isolated using a Monarch RNA preparation kit (NEB, UK), and quantitative PCR was performed. GSH levels in liver tissue were analysed using a standard enzyme recycling method. Relevant targets were identified based on their response to anti-oxidative stress as defined in literature. Primers were designed based on internally validated sequences and antibodies were purchased externally. qPCR was performed using the fluorescent dye, SYBR Green with a ViiA 7 Real-Time PCR system (ThermoFisher). Immunoblotting consisted of transferring separated by weight proteins onto a membrane which was then blocked in 5% milk, with primary and then secondary antibody incubation. Chemiluminescence was applied and bands were visualised using a ChemiDoc (BioRad). Bands were quantified using ImageJ software, and expressed in relation to actin. PHLF was classified according to the ISGLS criteria. Control patients (with no PHLF) were matched for demographic and clinico-pathologic variables (major resection as per the Brisbane criteria). Results Protein expression and GSH expression was measured in liver tissue from 23 patients. Of these, 10 had no PHLF, 4, 3 and 6 were classified as having Grades A, B, and C PHLF respectively. Additional isolated RNA samples yielded 11 with no PHLF, and 14 PHLF (4, 7 and 3, grade A, grade B and grade C, respectively) patients. A GSH assay showed no change in expression between those without PHLF and those with grade A and grade B, but patients who had grade C PHLF had significantly lower levels of GSH (p=0.0040). With respect to gene expression, GCLC, encoding the enzyme glutamate cysteine ligase catalytic subunit, the first rate limiting enzyme of glutathione synthesis, was also significantly lower in patients with grade C compared to those without PHLF of grade A/B (p=0.0206). Protein expression showed significantly reduced levels of GCLC when comparing grade C with patients without PHLF. Furthermore, samples taken at 2 timepoints (pre-parenchymal transection, and then 2 hours later from the resection margin) have shown a decrease in GCLC gene expression in patients who have grade C PHLF. Conclusions Our results have shown that patients who have grade C PHLF (who all died from acute liver failure post-operatively), have significantly lower levels of GSH in liver tissue taken at the time of resection. There is already a weight of evidence showing activation of the transcription factor Nrf2 (encoding GSH) enhances liver regeneration, however this is not without off target effects. Although not known to promote oncogenesis, in vitro studies have shown that persistent overstimulation of Nrf2 promotes cancer cell growth and proliferation, in part by improving the chemoresistance of cancer cells. This effect is theorised to be due to metabolic reprogramming of cancer cells, resulting in elevated levels of metabolic enzymes involved in cell proliferation.Whilst it is appreciated that liver failure is multi-factorial, targeting genes that promote liver regeneration may reduce the risk of failure. Identifying and targeting individual genes which are involved in liver regeneration may help to overcome potentially deleterious off target effects. This data suggests that GSH is a potential therapeutic target reducing the risk of PHLF.

Loss of soluble guanylyl cyclase in platelets contributes to atherosclerotic plaque formation and vascular inflammation

Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr−/− mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. Importantly, pharmacological sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr−/− mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD.

Glutathione levels are associated with methotrexate resistance in acute lymphoblastic leukemia cell lines.

Methotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use. To better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX. MTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested. Altogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.

BCL7A‐containing SWI/SNF/BAF complexes modulate mitochondrial bioenergetics during neural progenitor differentiation

Mammalian SWI/SNF/BAF chromatin remodeling complexes influence cell lineage determination. While the contribution of these complexes to neural progenitor cell (NPC) proliferation and differentiation has been reported, little is known about the transcriptional profiles that determine neurogenesis or gliogenesis. Here, we report that BCL7A is a modulator of the SWI/SNF/BAF complex that stimulates the genome‐wide occupancy of the ATPase subunit BRG1. We demonstrate that BCL7A is dispensable for SWI/SNF/BAF complex integrity, whereas it is essential to regulate Notch/Wnt pathway signaling and mitochondrial bioenergetics in differentiating NPCs. Pharmacological stimulation of Wnt signaling restores mitochondrial respiration and attenuates the defective neurogenic patterns observed in NPCs lacking BCL7A. Consistently, treatment with an enhancer of mitochondrial biogenesis, pioglitazone, partially restores mitochondrial respiration and stimulates neuronal differentiation of BCL7A‐deficient NPCs. Using conditional BCL7A knockout mice, we reveal that BCL7A expression in NPCs and postmitotic neurons is required for neuronal plasticity and supports behavioral and cognitive performance. Together, our findings define the specific contribution of BCL7A‐containing SWI/SNF/BAF complexes to mitochondria‐driven NPC commitment, thereby providing a better understanding of the cell‐intrinsic transcriptional processes that connect metabolism, neuronal morphogenesis, and cognitive flexibility.

Adrenal insufficiency in patients with Prader-Willi syndrome.

The generalized dysfunction of the hypothalamic-pituitary axis in patients with Prader-Willi syndrome (PWS) is the most likely cause of hypogonadism, inadequate growth hormone secretion, excessive appetite and associated obesity, impaired body temperature regulation, and hypothyroidism. The syndrome is also related to an increased risk of central adrenal insufficiency, although its prevalence remains unknown. The results of the studies in which different methods of pharmacological stimulation were used do not provide conclusive outcomes. As a result, there are no clear guidelines with regard to diagnosis, prevention, or long-term care when adrenal insufficiency is suspected in patients with PWS. Currently, most patients with PWS are treated with recombinant human growth hormone (rhGH). It has been confirmed that rhGH therapy has a positive effect on growth, body composition, body mass index (BMI), and potentially on psychomotor development in children with PWS. Additionally, rhGH may reduce the conversion of cortisone to cortisol through inhibition of 11β-hydroxysteroid dehydrogenase type 1. However, its influence on basal adrenal function and adrenal stress response remains unexplained in children with PWS. This paper reviews the literature related to the hypothalamic-pituitary-adrenal axis dysfunction in the PWS patient population with a focus on children.

Targeting HIF-Hydroxylases Compromises Disease Initiation and Propagation, and Synergises with Current Therapies to Eliminate Cancer Stem Cells in AML

Acute myeloid leukaemia (AML) is an aggressive disorder of haematopoietic stem and progenitor cells (HSPCs), in which they acquire mutations, resulting in generation of disease fuelling, and ultimately treatment-resistant, leukaemic stem cells (LSCs). Given the hypoxic nature of the bone marrow microenvironment, the role, and pharmacological manipulation of the Hypoxia Inducible Factor (HIF) pathway in normal and malignant haematopoiesis is an area of significant interest. We, and others, previously found that HIF-α, the master regulator of the pathway, is not essential for normal haematopoiesis and its deletion promotes leukaemogenesis. Here we reveal through genetic, pharmacological and omics-based investigations that HIF stabilisation, through inhibition of its regulator PHD2, is a novel potent therapeutic strategy in AML. First, to genetically dissect to role of PHD2 in the initiation of AML, we transformed Phd2cKO HSPCs with AML-associated oncogenes and found that AML cells lacking Phd2 were compromised in vitro, established a weaker leukemic burden and initiated AML with a significantly longer latency in vivo. Moreover, employing our iMLL-AF9;shPhd2 mice, which upon DOX activate MLL-AF9 and a short-hairpin targeting Phd2, we found that Phd2 knockdown in vivo significantly delayed the onset of AML. Next, to investigate acute Phd2 depletion in newly diagnosed disease, we transplanted transformed shPhd2 cells, and upon AML detection induced expression of shPhd2. Notably, Phd2 knockdown in established AML cells compromised disease progression and significantly improved survival of leukaemic mice. Following this, we examined the impact of Phd2 inactivation on HSC function and multilineage haematopoiesis. Phd2cKO mice displayed no adverse phenotypes, and Phd2-deficient HSCs were able to successfully sustain multilineage haematopoiesis upon transplantation. Moreover, global DOX-inducible Phd2 knockdown had little effect on haematopoiesis. Thus, PHD2 inactivation compromises AML development and maintenance, but does not impact normal haematopoiesis, suggesting an ample therapeutic window for its inhibition. Given our genetic evidence that PHD2 is a promising target in AML, we explored the potential of its inhibition. We employed a commercially available clinical trial grade PHD inhibitor, as well as our own novel, selective, and potent PHD inhibitor, both of which work through distinct mechanisms. Significantly, both inhibitors compromised the growth and viability of murine AML cells, as well as multiple established human AML cell-lines and primary patient samples with diverse mutational backgrounds. Moreover, we have also discovered that PHD inhibition markedly synergises with AML treatments currently used in the clinic, thus enhancing their anti-leukaemic effect. Investigating the molecular mechanisms behind our findings, we found HIF-α stabilisation across multiple AML human cell lines following PHD inhibition. Furthermore, we utilised our HifαcKO leukaemic cells which, unlike control cells, were not affected by PHD inhibition, indicating that the anti-leukaemic effect of PHD inhibition is indeed HIF-dependent. Next, we performed RNA-seq and proteomic analyses following genetic and pharmacological PHD inhibition. These experiments revealed increased expression of key myeloid differentiation genes, glycolytic pathway, and a consistent upregulation of HIF target genes, including the pro-apoptotic BNIP3. Finally, pharmacological stimulation of apoptosis combined with PHD inhibition displayed a further and profound anti-leukaemic effect. Taken together, our data strongly indicate that genetic deletion and specific potent pharmacological inhibition of PHD2 can upregulate HIF-dependent pathways, eradicating AML disease in vitro and in vivo, thus unveiling a novel promising therapeutic target.

CyclinD2-mediated regulation of neurogenic output from the retinal ciliary margin is perturbed in albinism

In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. Here, we focus on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ generates predominantly ipsilaterally projecting RGCs, but this output is altered in the albino visual system because of CyclinD2 downregulation and disturbed timing of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit diminished ipsilateral retinogeniculate projection and poor depth perception. In albino mice, pharmacological stimulation of calcium channels, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit.

Adipocyte IRE1α promotes PGC1α mRNA decay and restrains adaptive thermogenesis.

Adipose tissue undergoes thermogenic remodeling in response to thermal stress and metabolic cues, playing a crucial role in regulating energy expenditure and metabolic homeostasis. Endoplasmic reticulum (ER) stress is associated with adipose dysfunction in obesity and metabolic disease. It remains unclear, however, if ER stress-signaling in adipocytes mechanistically mediates dysregulation of thermogenic fat. Here we show that inositol-requiring enzyme 1α (IRE1α), a key ER stress sensor and signal transducer, acts in both white and beige adipocytes to impede beige fat activation. Ablation of adipocyte IRE1α promotes browning/beiging of subcutaneous white adipose tissue following cold exposure or β3-adrenergic stimulation. Loss of IRE1α alleviates diet-induced obesity and augments the anti-obesity effect of pharmacologic β3-adrenergic stimulation. Notably, IRE1α suppresses stimulated lipolysis and degrades Ppargc1a messenger RNA through its RNase activity to downregulate the thermogenic gene program. Hence, blocking IRE1α bears therapeutic potential in unlocking adipocytes' thermogenic capacity to combat obesity and metabolic disorders.

Absence of overexpression of KIR3DL2 on CD4+ T cells and NK cells in patients with axial spondyloarthritis.

Dear Editor, Chan et al. described an overexpression of KIR3DL2, a receptor for HLA-B27 homodimer, on NK cells and CD4+ T cells in patients with axial SpA (axSpA) [1, 2]. HLA-B27:05 expressing cells have also been shown to promote KIR3DL2+ NK cells, with more NK cells and CD4+ T cells expressing KIR3DL2 in patients with axSpA [3]. However the overexpression of KIR3DL2 was not confirmed in another population of patients with active axSpA [4]. All these studies were conducted in patients carrying the HLA-B27 antigen. We aimed to assess the expression of KIR3DL2 in patients with axSpA, according to the presence or not of HLA B27. We included 20 patients with axSpA [16 with AS and 4 with non-radiographic axSpA (nr-axSpA)] fulfilling the Assessment of SpondyloArthritis international Society (ASAS) classification criteria [5] and 5 healthy controls. Stratification according to the presence or not of HLA-B27 was applied. The axSpA main characteristics were: 11 HLA-B27+, 9 HLA-B27-, 14 males, mean disease duration 15.2 years and mean BASDAI of 4.7/10; all were synthetic and biologic DMARD naïve. We analysed by flow cytometry whole blood and peripheral blood mononuclear cell samples using fluorescent antibodies against CD45, CD14, CD3, CD19, CD4, CD8, gamma-delta-TCR, CD56, CD16, CD45RA, CXCR3, CCR6, KIR3DL1, KIR3DL2, IL-17A, IL-22, IL-2 and IFN-gamma. Positivity thresholds were set using control isotypes. Overnight pharmacological stimulation was performed with PMA/Ionomycin. All statistical analyses were performed using Graph Pad Prism (GraphPad Software, San Diego, CA, USA). Paired data were analysed with Wilcoxon test, and unpaired data with Mann–Whitney test. P-values <0.05 were considered to be significant. The study was approved by local ethics committee and conducted in accordance with the Declaration of Helsinki.

Phosphatidic acid inhibits inositol synthesis by inducing nuclear translocation of kinase IP6K1 and repression of myo-inositol-3-P synthase

Inositol is an essential metabolite that serves as a precursor for structural and signaling molecules. Although perturbation of inositol homeostasis has been implicated in numerous human disorders, surprisingly little is known about how inositol levels are regulated in mammalian cells. A recent study in mouse embryonic fibroblasts demonstrated that nuclear translocation of inositol hexakisphosphate kinase 1 (IP6K1) mediates repression of myo-inositol-3-P synthase (MIPS), the rate-limiting inositol biosynthetic enzyme. Binding of IP6K1 to phosphatidic acid (PA) is required for this repression. Here, we elucidate the role of PA in IP6K1 repression. Our results indicate that increasing PA levels through pharmacological stimulation of phospholipase D (PLD) or direct supplementation of 18:1 PA induces nuclear translocation of IP6K1 and represses expression of the MIPS protein. We found that this effect was specific to PA synthesized in the plasma membrane, as endoplasmic reticulum–derived PA did not induce IP6K1 translocation. Furthermore, we determined that PLD-mediated PA synthesis can be stimulated by the master metabolic regulator 5′ AMP-activated protein kinase (AMPK). We show that activation of AMPK by glucose deprivation or by treatment with the mood-stabilizing drugs valproate or lithium recapitulated IP6K1 nuclear translocation and decreased MIPS expression. This study demonstrates for the first time that modulation of PA levels through the AMPK-PLD pathway regulates IP6K1-mediated repression of MIPS.