The redox-active defensive Selenoprotein T as a novel stress sensor protein playing a key role in the pathophysiology of heart failure.

Maladaptive cardiac hypertrophy contributes to the development of heart failure (HF). The oxidoreductase Selenoprotein T (SELENOT) emerged as a key regulator during rat cardiogenesis and acute cardiac protection. However, its action in chronic settings of cardiac dysfunction is not understood. Here, we investigated the role of SELENOT in the pathophysiology of HF: (i) by designing a small peptide (PSELT), recapitulating SELENOT activity via the redox site, and assessed its beneficial action in a preclinical model of HF [aged spontaneously hypertensive heart failure (SHHF) rats] and against isoproterenol (ISO)-induced hypertrophy in rat ventricular H9c2 and adult human AC16 cardiomyocytes; (ii) by evaluating the SELENOT intra-cardiomyocyte production and secretion under hypertrophied stimulation. Results showed that PSELT attenuated systemic inflammation, lipopolysaccharide (LPS)-induced macrophage M1 polarization, myocardial injury, and the severe ultrastructural alterations, while counteracting key mediators of cardiac fibrosis, aging, and DNA damage and restoring desmin downregulation and SELENOT upregulation in the failing hearts. In the hemodynamic assessment, PSELT improved the contractile impairment at baseline and following ischemia/reperfusion injury, and reduced infarct size in normal and failing hearts. At cellular level, PSELT counteracted ISO-mediated hypertrophy and ultrastructural alterations through its redox motif, while mitigating ISO-triggered SELENOT intracellular production and secretion, a phenomenon that presumably reflects the extent of cell damage. Altogether, these results indicate that SELENOT could represent a novel sensor of hypertrophied cardiomyocytes and a potential PSELT-based new therapeutic approach in myocardial hypertrophy and HF.

To what extent may aminochrome increase the vulnerability of dopaminergic neurons in the context of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder that progresses over time and is characterized by preferential reduction of dopaminergic neurons in the substantia nigra. Although the precise mechanisms leading to cell death in neurodegenerative disorders, such as PD, are not fully understood, it is widely accepted that increased oxidative stress may be a prevalent factor contributing to the deterioration of the nigrostriatal dopaminergic fibers in such conditions. Aminochrome, generated from dopamine (DA) metabolism, plays an important role in multiple pathogenic mechanisms associated with PD. Its capacity to induce a gradual reduction in dopaminergic neurons is due to its endogenous neurotoxicity. The formation of aminochrome results in the production of various reactive oxygen species (ROS), including pro-inflammatory factors, superoxide, nitric oxide, and hydroxyl radicals. This, in turn, causes loss of dopaminergic neurons, reducing DA uptake, and reduced numbers and shortened dendrites. Notably, o-quinones, which are more cytotoxic, arise from the oxidation of DA and possess a higher capacity to impede cellular defense mechanisms, thereby resulting in the death of neuronal cells. Aminochrome potentially contributes to the pathophysiology of PD by forming adducts with various proteins. All of the aforementioned effects suggest that aminochrome may play a crucial role in the pathophysiology of PD. Thus, aminochrome may serve as a more relevant preclinical model for PD, facilitating a better understanding of its pathophysiological processes and identification of novel therapeutic strategies aimed at preventing or slowing disease progression.

The novel non-hallucinogenic compound DM506 (3-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole) induces sedative- and anxiolytic-like activity in mice by a mechanism involving 5-HT2A receptor activation.

The anxiolytic and sedative-like effects of 3-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole (DM506), a non-hallucinogenic compound derived from ibogamine, were studied in mice. The behavioral effects were examined using Elevated O-maze and novelty suppressed feeding (NSFT) tests, open field test, and loss of righting reflex (LORR) test. The results showed that 15 mg/kg DM506 induced acute and long-lasting anxiolytic-like activity in naive and stressed/anxious mice, respectively. Repeated administration of 5 mg/kg DM506 did not cause cumulative anxiolytic activity or any side effects. Higher doses of DM506 (40 mg/kg) induced sedative-like activity, which was inhibited by a selective 5-HT2A receptor antagonist, volinanserin. Electroencephalography results showed that 15 mg/kg DM506 fumarate increased the transition from a highly alert state (fast γ wavelength) to a more synchronized deep-sleeping activity (δ wavelength), which is reflected in the sedative/anxiolytic activity in mice but without the head-twitch response observed in hallucinogens. The functional, radioligand binding, and molecular docking results showed that DM506 binds to the agonist sites of human 5-HT2A (Ki = 24 nM) and 5-HT2B (Ki = 16 nM) receptors and activates them with a potency (EC50) of 9 nM and 3 nM, respectively. DM506 was relatively less potent and behaved as a partial agonist (efficacy <80%) for both receptor subtypes compared to the full agonist DOI (2,5-dimethoxy-4-iodoamphetamine). Our study showed for the first time that the non-hallucinogenic compound DM506 induces anxiolytic- and sedative-like activities in naïve and stressed/anxious mice in a dose-, time-, and volinanserin-sensitive manner, likely through mechanisms involving 5-HT2A receptor activation.

Palmitate-Induced Cardiac Lipotoxicity Is Relieved by the Redox-Active Motif of SELENOT through Improving Mitochondrial Function and Regulating Metabolic State.

Cardiac lipotoxicity is an important contributor to cardiovascular complications during obesity. Given the fundamental role of the endoplasmic reticulum (ER)-resident Selenoprotein T (SELENOT) for cardiomyocyte differentiation and protection and for the regulation of glucose metabolism, we took advantage of a small peptide (PSELT), derived from the SELENOT redox-active motif, to uncover the mechanisms through which PSELT could protect cardiomyocytes against lipotoxicity. To this aim, we modeled cardiac lipotoxicity by exposing H9c2 cardiomyocytes to palmitate (PA). The results showed that PSELT counteracted PA-induced cell death, lactate dehydrogenase release, and the accumulation of intracellular lipid droplets, while an inert form of the peptide (I-PSELT) lacking selenocysteine was not active against PA-induced cardiomyocyte death. Mechanistically, PSELT counteracted PA-induced cytosolic and mitochondrial oxidative stress and rescued SELENOT expression that was downregulated by PA through FAT/CD36 (cluster of differentiation 36/fatty acid translocase), the main transporter of fatty acids in the heart. Immunofluorescence analysis indicated that PSELT also relieved the PA-dependent increase in CD36 expression, while in SELENOT-deficient cardiomyocytes, PA exacerbated cell death, which was not mitigated by exogenous PSELT. On the other hand, PSELT improved mitochondrial respiration during PA treatment and regulated mitochondrial biogenesis and dynamics, preventing the PA-provoked decrease in PGC1-α and increase in DRP-1 and OPA-1. These findings were corroborated by transmission electron microscopy (TEM), revealing that PSELT improved the cardiomyocyte and mitochondrial ultrastructures and restored the ER network. Spectroscopic characterization indicated that PSELT significantly attenuated infrared spectral-related macromolecular changes (i.e., content of lipids, proteins, nucleic acids, and carbohydrates) and also prevented the decrease in membrane fluidity induced by PA. Our findings further delineate the biological significance of SELENOT in cardiomyocytes and indicate the potential of its mimetic PSELT as a protective agent for counteracting cardiac lipotoxicity.

SELENOT Deficiency in the Mouse Brain Impacts Catecholaminergic Neuron Density: An Immunohistochemical, in situ Hybridization and 3D Light-Sheet Imaging Study.

BACKGROUND: Selenoprotein T (SELENOT), a PACAP-regulated thioredoxin-like protein, plays a role in catecholamine secretion and protects dopaminergic neurons. However, the role of SELENOT in the establishment of the catecholaminergic (CA) neuronal system is not known yet. METHODS: We analyzed by immunohistochemistry and RNAscope in situ hybridization the distribution of SELENOT and the expression of its mRNA, respectively. In addition, 3D imaging involving immunostaining in toto, clearing through the iDISCO+ method, acquisitions by light-sheet microscopy, and processing of 3D images was performed to map the CA neuronal system. A semi-automatic quantification of 3D images was carried out. RESULTS: SELENOT protein and mRNA are widely distributed in the mouse brain, with important local variations. Three-dimensional mapping, through tyrosine hydroxylase (TH) labeling, and semi-automated quantification of CA neurons in brain-specific SELENOT knockout mice showed a significant decrease in the number of TH-positive neurons in the area postrema (AP-A2), the A11 cell group (A11), and the zona incerta (ZI-A13) of SELENOT-deficient females, and in the hypothalamus (Hyp-A12-A14-A15) of SELENOT-deficient females and males. CONCLUSION: These results showed that SELENOT is diffusely expressed in the mouse brain and that its deficiency impacts CA neuron distribution in different brain areas including Hyp-A12-A14-A15, in both male and female mice.

Emerging roles of ER-resident selenoproteins in brain physiology and physiopathology.

The brain has a very high oxygen consumption rate and is particularly sensitive to oxidative stress. It is also the last organ to suffer from a loss of selenium (Se) in case of deficiency. Se is a crucial trace element present in the form of selenocysteine, the 21st proteinogenic amino acid present in selenoproteins, an essential protein family in the brain that participates in redox signaling. Among the most abundant selenoproteins in the brain are glutathione peroxidase 4 (GPX4), which reduces lipid peroxides and prevents ferroptosis, and selenoproteins W, I, F, K, M, O and T. Remarkably, more than half of them are proteins present in the ER and recent studies have shown their involvement in the maintenance of ER homeostasis, glycoprotein folding and quality control, redox balance, ER stress response signaling pathways and Ca2+ homeostasis. However, their molecular functions remain mostly undetermined. The ER is a highly specialized organelle in neurons that maintains the physical continuity of axons over long distances through its continuous distribution from the cell body to the nerve terminals. Alteration of this continuity can lead to degeneration of distal axons and subsequent neuronal death. Elucidation of the function of ER-resident selenoproteins in neuronal pathophysiology may therefore become a new perspective for understanding the pathophysiology of neurological diseases. Here we summarize what is currently known about each of their molecular functions and their impact on the nervous system during development and stress.

The SELENOT mimetic PSELT promotes nerve regeneration by increasing axonal myelination in a facial nerve injury model in female rats.

Peripheral nerve injury (PNI) is frequent and many patients suffer lifelong disabilities in severe cases. Although the peripheral nervous system is able to regenerate, its potential is limited. In this study, we tested in a nerve regeneration model in rat the potential beneficial effect of a short mimetic peptide, named PSELT, which derives from SELENOT, an essential thioredoxin-like selenoprotein endowed with neuroprotective and antioxidant activities. For this purpose, the right facial nerve of female Long-Evans rats was axotomized then bridged with a free femoral vein interposition graft. PSELT (1 µM) was injected into the vein immediately and 48 h after the injury, and the effects observed were compared to those found after an end-to-end suture used as a gold standard treatment. Whisking behavior, electrophysiological potential, and histological analyses were performed 3 months after injury to determine the effects of these treatments. These analyses revealed that PSELT-treated animals exhibit a better motor recovery in terms of protraction amplitude and velocity of vibrissae compared to control and end-sutured nerve animal groups. Moreover, administration of PSELT following injury enhanced muscle innervation, axonal elongation, and myelination of newly formed nerve fibers. Altogether, these results indicate that a PSELT-based treatment is sufficient to enhance facial nerve myelination and regeneration and could represent a new therapeutic tool to treat PNI.

The 26RFa (QRFP)/GPR103 neuropeptidergic system in mice relays insulin signalling into the brain to regulate glucose homeostasis.

AIMS/HYPOTHESIS: 26RFa (pyroglutamilated RFamide peptide [QRFP]) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localised in the hypothalamus. In this study we investigated whether 26RFa neurons are involved in the hypothalamic regulation of glucose homeostasis. METHODS: 26Rfa+/+, 26Rfa-/- and insulin-deficient male C57Bl/6J mice were used in this study. Mice received an acute intracerebroventricular (i.c.v.) injection of 26RFa, insulin or the 26RFa receptor (GPR103) antagonist 25e and were subjected to IPGTTs, insulin tolerance tests, acute glucose-stimulated insulin secretion tests and pyruvate tolerance tests (PTTs). Secretion of 26RFa by hypothalamic explants after incubation with glucose, leptin or insulin was assessed. Expression and quantification of the genes encoding 26RFa, agouti-related protein, the insulin receptor and GPR103 were evaluated by quantitative reverse transcription PCR and RNAscope in situ hybridisation. RESULTS: Our data indicate that i.c.v.-injected 26RFa induces a robust antihyperglycaemic effect associated with an increase in insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26Rfa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26Rfa are mainly localised in the lateral hypothalamic area, that they co-express the gene encoding the insulin receptor and that insulin induces the expression of 26Rfa in these neurons. Concurrently, the central antihyperglycaemic effect of insulin is abolished in the presence of a GPR103 antagonist and in 26RFa-deficient mice. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production. CONCLUSION/INTERPRETATION: We have identified a novel mechanism in the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system, and we provide evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.

The Antioxidant Selenoprotein T Mimetic, PSELT, Induces Preconditioning-like Myocardial Protection by Relieving Endoplasmic-Reticulum Stress.

Oxidative stress and endoplasmic reticulum stress (ERS) are strictly involved in myocardial ischemia/reperfusion (MI/R). Selenoprotein T (SELENOT), a vital thioredoxin-like selenoprotein, is crucial for ER homeostasis and cardiomyocyte differentiation and protection, likely acting as a redox-sensing protein during MI/R. Here, we designed a small peptide (PSELT), encompassing the redox site of SELENOT, and investigated whether its pre-conditioning cardioprotective effect resulted from modulating ERS during I/R. The Langendorff rat heart model was employed for hemodynamic analysis, while mechanistic studies were performed in perfused hearts and H9c2 cardiomyoblasts. PSELT improved the post-ischemic contractile recovery, reducing infarct size and LDH release with and without the ERS inducer tunicamycin (TM). Mechanistically, I/R and TM upregulated SELENOT expression, which was further enhanced by PSELT. PSELT also prevented the expression of the ERS markers CHOP and ATF6, reduced cardiac lipid peroxidation and protein oxidation, and increased SOD and catalase activities. An inert PSELT (I-PSELT) lacking selenocysteine was ineffective. In H9c2 cells, H2O2 decreased cell viability and SELENOT expression, while PSELT rescued protein levels protecting against cell death. In SELENOT-deficient H9c2 cells, H2O2 exacerbated cell death, that was partially mitigated by PSELT. Microscopy analysis revealed that a fluorescent form of PSELT was internalized into cardiomyocytes with a perinuclear distribution. Conclusions: The cell-permeable PSELT is able to induce pharmacological preconditioning cardioprotection by mitigating ERS and oxidative stress, and by regulating endogenous SELENOT.

Acute but Not Chronic Central Administration of the Neuropeptide 26RFa (QRFP) Improves Glucose Homeostasis in Obese/Diabetic Mice.

INTRODUCTION: The aim of the study is to investigate whether acute or chronic central administration of the hypothalamic neuropeptide 26RFa may ameliorate the glycemic control of obese/diabetic mice. METHODS: Mice were treated for 4 months with a high-fat (HF) diet and received a single i.c.v. injection of 26RFa (3 µg) or a chronic i.c.v. administration of the peptide during 28 days via osmotic minipumps (25 µg/day). i.p. and oral glucose (GLU) tolerance tests, insulin (INS) tolerance test, glucose-stimulated insulin secretion (GSIS), food/water intake, horizontal/vertical activity, energy expenditure, meal pattern, and whole-body composition were monitored. In addition, 26RFa and GPR103 mRNA expressions as well as plasma 26RFa levels were evaluated by RT-QPCR and radioimmunoassay. RESULTS: Acute administration of 26RFa in HF mice induced a robust antihyperglycemic effect by enhancing INS secretion, whereas chronic administration of the neuropeptide is unable to improve glucose homeostasis in these obese/diabetogenic conditions. By contrast, chronic 26RFa treatment induced an increase of the body weight accompanied with an enhanced food intake and a decreased energy expenditure. Finally, we show that the HF diet does not alter the hypothalamic expression of the 26RFa/GPR103 neuropeptidergic system nor the levels of circulating 26RFa. CONCLUSION: Our data indicate that the central beneficial effect of 26RFa on glucose homeostasis, by potentiating GSIS, is preserved in HF mice. However, chronic administration of the neuropeptide is unable to balance glycemia in these pathophysiological conditions, suggesting that the hypothalamic 26RFa/GPR103 neuropeptidergic system mainly affects short-term regulation of glucose metabolism.

Prediction methods for microRNA targets in bilaterian animals: Toward a better understanding by biologists.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level. Because of their wide network of interactions, miRNAs have become the focus of many studies over the past decade, particularly in animal species. To streamline the number of potential wet lab experiments, the use of miRNA target prediction tools is currently the first step undertaken. However, the predictions made may vary considerably depending on the tool used, which is mostly due to the complex and still not fully understood mechanism of action of miRNAs. The discrepancies complicate the choice of the tool for miRNA target prediction. To provide a comprehensive view of this issue, we highlight in this review the main characteristics of miRNA-target interactions in bilaterian animals, describe the prediction models currently used, and provide some insights for the evaluation of predictor performance.

Cell-penetrating, antioxidant SELENOT mimetic protects dopaminergic neurons and ameliorates motor dysfunction in Parkinson's disease animal models.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor dysfunction for which there is an unmet need for better treatment options. Although oxidative stress is a common feature of neurodegenerative diseases, notably PD, there is currently no efficient therapeutic strategy able to tackle this multi-target pathophysiological process. Based on our previous observations of the potent antioxidant and neuroprotective activity of SELENOT, a vital thioredoxin-like selenoprotein, we designed the small peptide PSELT from its redox active site to evaluate its antioxidant properties in vivo, and its potential polyfunctional activity in PD models. PSELT protects neurotoxin-treated dopaminergic neurons against oxidative stress and cell death, and their fibers against neurotoxic degeneration. PSELT is cell-permeable and acts in multiple subcellular compartments of dopaminergic neurons that are vulnerable to oxidative stress. In rodent models of PD, this protective activity prevented neurodegeneration, restored phosphorylated tyrosine hydroxylase levels, and led to improved motor skills. Transcriptomic analysis revealed that gene regulation by PSELT after MPP+ treatment negatively correlates with that occurring in PD, and positively correlates with that occurring after resveratrol treatment. Mechanistically, a major impact of PSELT is via nuclear stimulation of the transcription factor EZH2, leading to neuroprotection. Overall, these findings demonstrate the potential of PSELT as a therapeutic candidate for treatment of PD, targeting oxidative stress at multiple intracellular levels.

Tolerant/Persister Cancer Cells and the Path to Resistance to Targeted Therapy.

The capacity of cancer to adapt to treatment and evolve is a major limitation for targeted therapies. While the role of new acquired mutations is well-established, recent findings indicate that resistance can also arise from subpopulations of tolerant/persister cells that survive in the presence of the treatment. Different processes contribute to the emergence of these cells, including pathway rebound through the release of negative feedback loops, transcriptional rewiring mediated by chromatin remodeling and autocrine/paracrine communication among tumor cells and within the tumor microenvironment. In this review, we discuss the non-genetic mechanisms that eventually result in cancer resistance to targeted therapies, with a special focus on those involving changes in gene expression.

Selenoprotein T: An Essential Oxidoreductase Serving as a Guardian of Endoplasmic Reticulum Homeostasis.

Significance: Selenoproteins incorporate the essential nutrient selenium into their polypeptide chain. Seven members of this family reside in the endoplasmic reticulum (ER), the exact function of most of which is poorly understood. Especially, how ER-resident selenoproteins control the ER redox and ionic environment is largely unknown. Since alteration of ER function is observed in many diseases, the elucidation of the role of selenoproteins could enhance our understanding of the mechanisms involved in ER homeostasis. Recent Advances: Among selenoproteins, selenoprotein T (SELENOT) is remarkable as the most evolutionarily conserved and the only ER-resident selenoprotein whose gene knockout in mouse is lethal. Recent data indicate that SELENOT contributes to ER homeostasis: reduced expression of SELENOT in transgenic cell and animal models promotes accumulation of reactive oxygen and nitrogen species, depletion of calcium stores, activation of the unfolded protein response and impaired hormone secretion. Critical Issues: SELENOT is anchored to the ER membrane and associated with the oligosaccharyltransferase complex, suggesting that it regulates the early steps of N-glycosylation. Furthermore, it exerts a selenosulfide oxidoreductase activity carried by its thioredoxin-like domain. However, the physiological role of the redox activity of SELENOT is not fully understood. Likewise, the nature of its redox partners needs to be further characterized. Future Directions: Given the impact of ER stress in pathologies such as neurodegenerative, cardiovascular, metabolic and immune diseases, understanding the role of SELENOT and developing derived therapeutic tools such as selenopeptides to improve ER proteostasis and prevent ER stress could contribute to a better management of these diseases.

Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction.

OBJECTIVE: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. CONCLUSIONS: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis.

Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.

Coronaridine congeners potentiate GABAA receptors and induce sedative activity in mice in a benzodiazepine-insensitive manner.

To determine whether (+)-catharanthine induces sedative- or anxiolytic/anxiogenic-like activity in male mice, proper animal paradigms were used. The results showed that (+)-catharanthine induces sedative-like activity in the 63-72 mg/Kg dose range in a flumazenil-insensitive manner, but neither this effect nor anxiolytic/anxiogenic-like activity was observed at lower doses. To determine the underlying molecular mechanism of the sedative-like activity, electrophysiological and radioligand binding experiments were performed with (+)-catharanthine and (±)-18-methoxycoronaridine [(±)-18-MC] on GABAA (GABAARs) and glycine receptors (GlyRs). Coronaridine congeners both activated and potentiated a variety of human (h) GABAARs, except hρ1. (+)-Catharanthine-induced potentiation followed this receptor selectivity (EC50's in µM): hα1ß2 (4.6 ± 0.8) > hα2ß2γ2 (12.6 ± 3.8) ~ hα1ß2γ2 (14.4 ± 4.6) indicating that both α1 and α2 are equally important, whereas γ2 is not necessary. (+)-Catharanthine was >2-fold more potent and efficient than (±)-18-MC at hα1ß2γ2. (+)-Catharanthine also potentiated, whereas (±)-18-MC inhibited, hα1 GlyRs with very low potency. Additional [3H]-flunitrazepam competition binding experiments using rat cerebellum membranes clearly demonstrated that these ligands do not bind to the benzodiazepine site. This is supported by the observed activity at hα1ß2 (lacking the BDZ site) and similar effects between α1- and α2-containing GABAARs. Our study shows, for the first time, that (+)-catharanthine induced sedative-like effects in mice, and coronaridine congeners potentiated human α1ß2γ2, α1ß2, and hα2ß2γ2, but not ρ1, GABAARs, both in a benzodiazepine-insensitive fashion, whereas only (+)-catharanthine slightly potentiated GlyRs.

Glucose homeostasis is impaired in mice deficient in the neuropeptide 26RFa (QRFP).

INTRODUCTION: 26RFa (pyroglutamyl RFamide peptide (QRFP)) is a biologically active peptide that has been found to control feeding behavior by stimulating food intake, and to regulate glucose homeostasis by acting as an incretin. The aim of the present study was thus to investigate the impact of 26RFa gene knockout on the regulation of energy and glucose metabolism. RESEARCH DESIGN AND METHODS: 26RFa mutant mice were generated by homologous recombination, in which the entire coding region of prepro26RFa was replaced by the iCre sequence. Energy and glucose metabolism was evaluated through measurement of complementary parameters. Morphological and physiological alterations of the pancreatic islets were also investigated. RESULTS: Our data do not reveal significant alteration of energy metabolism in the 26RFa-deficient mice except the occurrence of an increased basal metabolic rate. By contrast, 26RFa mutant mice exhibited an altered glycemic phenotype with an increased hyperglycemia after a glucose challenge associated with an impaired insulin production, and an elevated hepatic glucose production. Two-dimensional and three-dimensional immunohistochemical experiments indicate that the insulin content of pancreatic ß cells is much lower in the 26RFa-/- mice as compared with the wild-type littermates. CONCLUSION: Disruption of the 26RFa gene induces substantial alteration in the regulation of glucose homeostasis, with in particular a deficit in insulin production by the pancreatic islets. These findings further support the notion that 26RFa is an important regulator of glucose homeostasis.

Three-dimensional mapping of tyrosine hydroxylase in the transparent brain and adrenal of prenatal and pre-weaning mice: Comprehensive methodological flowchart and quantitative aspects of 3D mapping.

BACKGROUND: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step for the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline. Although its distribution in different organs, species and stages of development has been the subject of numerous studies, the recent emergence of 3D imaging techniques has created the potential to shed new light on the dynamics of TH expression during the development of the mammalian central and peripheral nervous systems. NEW METHOD: Here, we describe a flowchart summarizing different protocols adapted to developmental stage-specific tissues to generate a 3D atlas of the catecholaminergic system in the brain and peripheral nervous system in mice from embryonic to pre-weaning stages. The procedures described allowed a quantitative assessment of developing TH-positive neuronal populations and pathways, previously understudied due to dimensional limitations. RESULTS: Our approach allowed us to reveal in 3D the dynamics of the onset and the establishment of the catecholaminergic system in embryonic and developing central and peripheral nervous system. Quantitative analyses applied to 3D images yielded accurate measurements of neuron population volumes and numbers, and tract pathway dimensions for selected TH-positive brain structures. COMPARISON WITH EXISTING METHODS: We applied a set of different protocols to yield a comprehensive flowchart for 3D imaging and a precise quantitative assessment of specific neuronal populations during the course of their development up to adulthood in mice. CONCLUSION: The procedures described and the extensive 3D mapping of TH immunoreactivity at early embryonic and postnatal stages provide a comprehensive view of the onset and development of the catecholaminergic system in the mouse brain and sympathoadrenal nervous system.

Chromogranin A in the early steps of the neurosecretory pathway.

Chromogranin A (CgA) is a soluble glycoprotein stored with hormones and neuropeptides in secretory granules (SG) of most (neuro)endocrine cells and neurons. Since its discovery in 1967, many studies have reported its structural characteristics, biological roles, and mechanisms of action. Indeed, CgA is both a precursor of various biologically active peptides and a granulogenic protein regulating the storage and secretion of hormones and neuropeptides. This review emphasizes the findings and theoretical concepts around the CgA-linked molecular machinery controlling hormone/neuropeptide aggregation and the interaction of CgA-hormone/neuropeptide aggregates with the trans-Golgi membrane to allow hormone/neuropeptide targeting and SG biogenesis. We will also discuss the intriguing alteration of CgA expression and secretion in various neurological disorders, which could provide insights to elucidate the molecular mechanisms underlying these pathophysiological conditions.