ABSTRACT
Dedicator of cytokinesis (DOCK) proteins play a central role in actin cytoskeleton regulation. This is highlighted by the DOCK2 and DOCK8 deficiencies leading to actinopathies and immune deficiencies. DOCK8 and DOCK11 activate CDC42, a Rho-guanosine triphosphate hydrolases involved in actin cytoskeleton dynamics, among many cellular functions. The role of DOCK11 in human immune disease has been long suspected but, to the best of our knowledge, has never been described to date. We studied 8 male patients, from 7 unrelated families, with hemizygous DOCK11 missense variants leading to reduced DOCK11 expression. The patients were presenting with early-onset autoimmunity, including cytopenia, systemic lupus erythematosus, skin, and digestive manifestations. Patients' platelets exhibited abnormal ultrastructural morphology and spreading as well as impaired CDC42 activity. In vitro activated T cells and B-lymphoblastoid cell lines from patients exhibited aberrant protrusions and abnormal migration speed in confined channels concomitant with altered actin polymerization during migration. Knock down of DOCK11 recapitulated these abnormal cellular phenotypes in monocytes-derived dendritic cells and primary activated T cells from healthy controls. Lastly, in line with the patients' autoimmune manifestations, we also observed abnormal regulatory T-cell (Treg) phenotype with profoundly reduced FOXP3 and IKZF2 expression. Moreover, we found reduced T-cell proliferation and impaired STAT5B phosphorylation upon interleukin-2 stimulation of the patients' lymphocytes. In conclusion, DOCK11 deficiency is a new X-linked immune-related actinopathy leading to impaired CDC42 activity and STAT5 activation, and is associated with abnormal actin cytoskeleton remodeling as well as Treg phenotype, culminating in immune dysregulation and severe early-onset autoimmunity.
Subject(s)
Immune System Diseases , Immunologic Deficiency Syndromes , Humans , Male , Actin Cytoskeleton/metabolism , Autoimmunity , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Immune System Diseases/metabolism , Immunologic Deficiency Syndromes/complications , Immunologic Deficiency Syndromes/genetics , T-Lymphocytes, RegulatoryABSTRACT
Background: The bleeding risk associated with direct oral anticoagulants (DOACs) remains a major concern, and rapid reversal of anticoagulant activity may be required. Although specific and nonspecific hemostatic biotherapies are available, there is a need for small-molecule DOAC reversal agents that are simple and cost-effective to produce, store, and administer. Objectives: To identify and characterize a small molecule with procoagulant activity as a DOAC reversal agent. Methods: We sought to identify a small procoagulant molecule by screening a chemical library with a plasma clotting assay. The selected molecule was assessed for its procoagulant properties and its ability to reverse the effects of the DOACs in a thrombin generation assay. Its activity as a DOAC reversal agent was also evaluated in a tail-clip bleeding assay in mice. Results: The hemostatic molecule (HeMo) dose-dependently promoted thrombin generation in plasma, with dose values effective in producing half-maximum response ranging between 3 and 5 µM, depending on the thrombin generation assay parameter considered. HeMo also restored impaired thrombin generation in DOAC-spiked plasma and reversed DOAC activity in the mouse bleeding model. HeMo significantly reduced apixaban-induced bleeding from 709 to 65 µL (vs 43 µL in controls; P < .01) and dabigatran-induced bleeding from 989 to 155 µL (vs 126 µL in controls; P < .01). Conclusion: HeMo is a small-molecule procoagulant that can counterbalance hemostatic disruption by a thrombin inhibitor (dabigatran) or factor Xa inhibitors (apixaban and rivaroxaban). The compound's effective clot formation and versatility make it a possible option for managing the inherent hemorrhagic risk during DOAC therapy.
ABSTRACT
BACKGROUND: Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that targets factor Xa (FXa) in the presence of protein Z (PZ), and factor XIa (FXIa). In factor-VIII-deficient mice, PZ or ZPI gene knock-out mitigates the bleeding phenotype, and pharmacological inhibition of PZ enhances thrombin generation in plasma from patients with hemophilia. AIMS: To develop a single-domain antibody (sdAb) directed against ZPI to inhibit its anticoagulant activity. METHODS: We screened for anti-ZPI sdAbs in a llama-derived phage display immune library of sdAbs. The sdAbs that bound ZPI were produced and purified for characterization. The binding of sdAbs to ZPI or other serpins was evaluated using ELISAs, and ZPI inhibition was measured in an anti-FXa or anti-FXIa chromogenic assay. The sdAbs's procoagulant activity was assessed in a thrombin generation assay in normal plasma, factor VIII- and FXI-deficient plasma. RESULTS: Of the four sdAbs found to bind to ZPI, one (referred to as ZPI-sdAb2) dose-dependently inhibited ZPI's anti-FXa and anti-FXIa activities with a mean half-maximal inhibitory concentration of 1.8 and 1.3 µM, respectively. ZPI-sdAb2 did not cross-react with other plasma serpins, such as antithrombin and α1-antitrypsin. ZPI-sdAb2 induced a significant increase in thrombin generation in plasma samples from healthy donors, patients with severe hemophilia A, and patients with FXI deficiency. CONCLUSION: ZPI-sdAb2 is the first specific, direct ZPI inhibitor found to exhibit procoagulant activity in plasma. This sdAb might have potential as a treatment for hemophilia or other bleeding disorders.
ABSTRACT
BACKGROUND: X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection, and neoplasia (XMEN) disease is a primary immunodeficiency due to loss-of-function mutations in the gene encoding for magnesium transporter 1 (MAGT1). Furthermore, as MAGT1 is involved in the N-glycosylation process, XMEN disease is classified as a congenital disorder of glycosylation. Although XMEN-associated immunodeficiency is well described, the mechanisms underlying platelet dysfunction and those responsible for life-threatening bleeding events have never been investigated. OBJECTIVES: To assess platelet functions in patients with XMEN disease. METHODS: Two unrelated young boys, including one before and after hematopoietic stem cell transplantation, were investigated for their platelet functions, glycoprotein expression, and serum and platelet-derived N-glycans. RESULTS: Platelet analysis highlighted abnormal elongated cells and unusual barbell-shaped proplatelets. Platelet aggregation, integrin αIIbß3 activation, calcium mobilization, and protein kinase C activity were impaired between both patients. Strikingly, platelet responses to protease-activated receptor 1 activating peptide were absent at both low and high concentrations. These defects were also associated with decreased molecular weights of glycoprotein Ibα, glycoprotein VI, and integrin αIIb due to partial impairment of N-glycosylation. All these defects were corrected after hematopoietic stem cell transplantation. CONCLUSION: Our results highlight prominent platelet dysfunction related to MAGT1 deficiency and defective N-glycosylation in several platelet proteins that could explain the hemorrhages reported in patients with XMEN disease.
Subject(s)
Epstein-Barr Virus Infections , Magnesium , Male , Humans , Magnesium/metabolism , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/genetics , Glycosylation , Herpesvirus 4, Human/metabolism , Glycoproteins/genetics , Glycoproteins/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolismABSTRACT
BACKGROUND: Filaminopathies A are rare disorders affecting the brain, intestine, or skeleton, characterized by dominant X-linked filamin A (FLNA) gene mutations. Macrothrombocytopenia with functionally defective platelets is frequent. We have described a filaminopathy A male patient, exhibiting a C-terminal frame-shift FLNa mutation (Berrou et al., Arterioscler Thromb Vasc Biol. 2017;37:1087-1097). Contrasting with female patients, this male patient exhibited gain of platelet functions, including increased platelet aggregation, integrin αIIbß3 activation, and secretion at low agonist concentration, raising the issue of thrombosis risk. OBJECTIVES: Our goal is to assess the thrombotic potential of the patient FLNa mutation in an in vivo model. METHODS: We have established a mutant FlnA knock-in mouse model. RESULTS: The mutant FlnA mouse platelets phenocopied patient platelets, showing normal platelet count, lower expression level of mutant FlnA, and gain of platelet functions: increased platelet aggregation, secretion, and αIIbß3 activation, as well as increased spreading and clot retraction. Surprisingly, mutant FlnA mice exhibited a normal bleeding time, but with increased re-bleeding (77%) compared to wild type (WT) FlnA mice (27%), reflecting hemostatic plug instability. Again, in an in vivo thrombosis model, the occlusion time was not altered by the FlnA mutation, but arteriolar embolies were increased (7-fold more frequent in mutant FlnA mice versus WT mice), confirming thrombus instability. CONCLUSIONS: This study shows that the FlnA mutation found in the male patient induced gain of platelet functions in vitro, but thrombus instability in vivo. Implications for the role of FLNa in physiology of thrombus formation are discussed.
Subject(s)
Hemostatics , Thrombosis , Male , Female , Mice , Animals , Filamins/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Gain of Function Mutation , Thrombosis/genetics , Thrombosis/metabolism , MutationABSTRACT
Phosphomannomutase 2 (PMM2) deficiency is the most prevalent congenital disorder of glycosylation. It is associated with coagulopathy, including protein C deficiency. Since all components of the anticoagulant and cytoprotective protein C system are glycosylated, we sought to investigate the impact of an N-glycosylation deficiency on this system as a whole. To this end, we developed a PMM2 knockdown model in the brain endothelial cell line hCMEC/D3. The resulting PMM2low cells were less able to generate activated protein C (APC), due to lower surface expression of thrombomodulin and endothelial protein C receptor. The low protein levels were due to downregulated transcription of the corresponding genes (THBD and PROCR, respectively), which itself was related to downregulation of transcription regulators Krüppel-like factors 2 and 4 and forkhead box C2. PMM2 knockdown was also associated with impaired integrity of the endothelial cell monolayer-partly due to an alteration in the structure of VE-cadherin in adherens junctions. The expression of protease-activated receptor 1 (involved in the cytoprotective effects of APC on the endothelium) was not affected by PMM2 knockdown. Thrombin stimulation induced hyperpermeability in PMM2low cells. However, pretreatment of cells with APC before thrombin simulation was still associated with a barrier-protecting effect. Taken as a whole, our results show that the partial loss of PMM2 in hCMEC/D3 cells is associated with impaired activation of protein C and a relative increase in barrier permeability.
Subject(s)
Protein C , Thrombin , Congenital Disorders of Glycosylation , Endothelium , Glycosylation , Humans , Phosphotransferases (Phosphomutases)/deficiencyABSTRACT
Preclinical data support the view that brain-derived neurotrophic factor (BDNF) and serotonergic systems regulate circuits involved in affective disorders. The present study examined neurochemical and behavioural consequences of an acute intrahippocampal injection of BDNF combined with an antidepressant by using in-vivo intracerebral microdialysis in the ventral hippocampus (vHi) in conscious mice and behavioural paradigms predictive of antidepressant and anxiolytic-like effects [the mouse forced swim test (FST), the open-field (OF) paradigm and the elevated plus maze (EPM)]. Neurochemical data revealed that BDNF (100 ng) potentiated the effects of the systemic administration of a serotonin selective reuptake inhibitor (SSRI; paroxetine 4 mg/kg i.p.) and that of a locally applied citalopram perfusion on dialysate 5-HT levels in the vHi. These neurochemical changes correlated with behavioural data since, in the FST, antidepressant-like activity of paroxetine as measured on swimming behaviour was potentiated by BDNF. These data suggest an interesting synergy between BDNF and SSRI on antidepressant-like activity. Furthermore, in both the OF and EPM paradigms BDNF induced an anxiogenic-like activity, whereas paroxetine prevented this effect. Finally, the neurochemical and behavioural effects of BDNF on the serotonergic system might occur at both pre- and post-synaptic levels since by using in-situ hybridization, we showed that TrkB-R mRNA was expressed in the hippocampus and the dorsal raphe nucleus in adult mice. Taken together the neurochemical and behavioural effects of BDNF suggest that these behavioural changes were mediated by increases in 5-HT neurotransmission in vHi. Thus a BDNF+SSRI combination may offer new alternatives to treat mood disorders.
Subject(s)
Antidepressive Agents/administration & dosage , Behavior, Animal/drug effects , Brain-Derived Neurotrophic Factor/administration & dosage , Citalopram/administration & dosage , Depression/drug therapy , Hippocampus/drug effects , Paroxetine/administration & dosage , Selective Serotonin Reuptake Inhibitors/administration & dosage , Analysis of Variance , Animals , Anxiety/chemically induced , Anxiety/metabolism , Anxiety/prevention & control , Anxiety/psychology , Brain-Derived Neurotrophic Factor/toxicity , Depression/metabolism , Depression/physiopathology , Depression/psychology , Disease Models, Animal , Drug Synergism , Hippocampus/metabolism , Hippocampus/physiopathology , In Situ Hybridization , Male , Mice , Microdialysis , Microinjections , Motor Activity/drug effects , RNA, Messenger/metabolism , Receptor, trkB/drug effects , Receptor, trkB/genetics , Serotonin/metabolism , Time FactorsABSTRACT
In obesity, platelets are described as hyperactive, mainly based on increased platelet size and presence of pro-thrombotic plasmatic molecules. We explored platelet functions, including calcium signalling in obesity, and the effect of weight loss. We included 40 obese patients (women with body mass index [BMI] of ≥ 35 kg/m2) who were to undergo gastric bypass surgery and 40 healthy lean subjects (women with BMI of < 25 kg/m2) as a control group. Approximately 1 year after surgery, the obese patients lost weight (75% had a BMI < 35 kg/m2). They were explored a second time with the same healthy control for the same platelet experiments. Compared with controls, obese patients' platelets displayed reduced sensitivity to thrombin (aggregation EC50 increased by 1.9 ± 0.3-fold, p = 0.005) and a lower Ca2+ response (70 ± 7% decrease, p < 10-4). In 17 pairs of patients, we performed additional experiments: in obese patients' platelets, thrombin-induced αIIbß3 activation was significantly lower (p = 0.003) and sarco-endoplasmic reticulum Ca2+ATPase (SERCA3) expression was decreased (48 ± 6% decrease, p < 10-4). These differences were abolished after weight loss. Interestingly, pharmacological inhibition of SERCA3 activity in control group's platelets mimicked similar alterations than in obese patients' platelets and was associated with defective adenosine diphosphate (ADP) secretion. Addition of ADP to agonist restored platelet functions in obese patients and in SERCA3-inhibited control platelets (five experiments) confirming the direct involvement of the SERCA3-dependent ADP secretion pathway. This is the first study demonstrating that platelets from obese patients are hypo-reactive, due to a deficiency of SERCA3-dependent ADP secretion. Weight loss restores SERCA3 activity and subsequent calcium signalling, αIIbß3 activation, platelet aggregation and ADP secretion.
Subject(s)
Adenosine Diphosphate/blood , Blood Platelets/metabolism , Gastric Bypass , Obesity/surgery , Platelet Activation , Sarcoplasmic Reticulum Calcium-Transporting ATPases/blood , Weight Loss , Adult , Calcium Signaling , Female , Humans , Obesity/blood , Obesity/diagnosis , Obesity/physiopathology , Paris , Platelet Aggregation , Platelet Function Tests , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Secretory Pathway , Time Factors , Treatment OutcomeABSTRACT
Whereas SSRIs produce rapid blockade of the serotonin transporter (SERT) in vitro and in vivo, the onset of an observable clinical effect takes longer to occur and a variety of pharmacological effects caused by antidepressants have been speculated to be involved either in initiating antidepressant effects and/or enhancing their effects on serotonergic transmission so as to cause clinical improvement. Among such secondary factors is increased activity of brain-derived neurotrophic factor (BDNF), which requires the Tropomyosine-related kinase B receptor (TrkB) for its effects. To begin an analysis of the influence of BDNF on serotonergic activity, we studied the acute effects of BDNF on SERT activity. A single BDNF injection (either intracerebroventricularly or directly into the CA3 region of hippocampus) decreased the signal amplitude and clearance rate produced by exogenously applied 5-HT compared to what was measured in control rats, shown using in vivo chronoamperometry. It also reduced the ability of a locally applied SSRI to block the clearance of 5-HT. In awake freely moving mice, acute intrahippocampal injection of BDNF decreased extracellular levels of 5-HT in the hippocampus, as measured using microdialysis. In addition, perfusion with BDNF decreased KCl-evoked elevations of 5-HT. These effects of BDNF were blocked by the non-selective antagonist of TrkB receptors, K252a. Overall, it may be inferred that in the hippocampus, through TrkB activation, a single injection of BDNF enhances SERT function. Such acute effects of BDNF would be expected to counter early effects of SSRIs, which might, in part, account for some delay in therapeutic effect.
Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Hippocampus/drug effects , Serotonin/physiology , Synaptic Transmission/drug effects , Animals , Autoradiography , Electrodes , Electrophysiology , Enzyme Inhibitors/pharmacology , Hippocampus/metabolism , Male , Mice , Microdialysis , Proadifen/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, trkB/antagonists & inhibitors , Serotonin Plasma Membrane Transport Proteins/metabolismABSTRACT
In vivo intracerebral microdialysis is an important neurochemical technique that has been applied extensively in genetic and pharmacological studies aimed at investigating the relationship between neurotransmitters. Among the main interests of microdialysis application is the infusion of drugs through the microdialysis probe (reverse dialysis) in awake, freely moving animals. As an example of the relevance of intracerebral microdialysis, this review will focus on our recent neurochemical results showing the impact of Brain-Derived Neurotrophic Factor (BDNF) on serotonergic neurotransmission in basal and stimulated conditions. Indeed, although the elevation of 5-HT outflow induced by chronic administration of selective serotonin reuptake inhibitors (SSRIs) causes an increase in BDNF protein levels and expression (mRNA) in the hippocampus of rodents, the reciprocal interaction has not been demonstrated yet. Thus, the neurochemical sight of this question will be addressed here by examining the consequences of either a constitutive decrease or increase in brain BDNF protein levels on hippocampal extracellular levels of 5-HT in conscious mice.
Subject(s)
Brain-Derived Neurotrophic Factor/physiology , Hippocampus/metabolism , Microdialysis/methods , Serotonin Plasma Membrane Transport Proteins/genetics , Serotonin/metabolism , Animals , Antidepressive Agents/pharmacology , Brain-Derived Neurotrophic Factor/pharmacology , Citalopram/metabolism , Mice , Paroxetine/pharmacology , RNA, Messenger/analysis , Serotonin Plasma Membrane Transport Proteins/physiologyABSTRACT
Preclinical studies suggest that substance P (SP) neurokinin 1 (NK1) receptor antagonists are efficient in the treatment of anxiety and depression. This therapeutic activity could be mediated via stimulation of serotonin (5-HT) neurons located in the dorsal raphe nucleus (DRN), which receive important SP-NK1 receptor immunoreactive innervations. The present study examined the effects of intraraphe injection of SP on extracellular 5-HT levels in the frontal cortex, ventral hippocampus, and DRN by using intracerebral microdialysis in conscious mice. Intraraphe SP injection dose dependently decreased cortical 5-HT release, whereas no effects were detected in the ventral hippocampus. Cortical effects were blocked by the selective NK1 receptor antagonist N-[[2-methoxy-5-[5-(trifluoromethyl)tetrazol-1-yl]phenyl]methyl]-2-phenylpiperidin-3-amine (GR205171) and completely dampened in mice lacking NK1 receptors. Furthermore, genetic (in knockout 5-HT1A(-/-) mice) or pharmacological inactivation of 5-HT1A autoreceptors blocked cortical responses to SP. Contrasting with its cortical effects, intraraphe SP injection increased 5-HT outflow in the DRN in wild-type mice; this effect was potentiated by a local perfusion of the selective 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY100635). Finally, SP-induced changes in frontal cortex and DRN dialysate 5-HT levels were blocked by the DRN perfusion of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate ionotropic receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). These data support the hypothesis that SP-induced over-activation of 5-HT1A autoreceptors within the DRN limits cortical 5-HT release. A better knowledge of the complex relationship between tachykininergic, serotonergic, and glutamatergic systems within the DRN might help better understand the pathophysiology and subsequent treatment of depression.
Subject(s)
Frontal Lobe/metabolism , Raphe Nuclei/metabolism , Receptors, Neurokinin-1/metabolism , Serotonin/metabolism , Substance P/physiology , Animals , Injections, Intraventricular , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Raphe Nuclei/drug effects , Raphe Nuclei/physiology , Receptors, Neurokinin-1/physiology , Substance P/administration & dosage , Substance P/metabolismABSTRACT
Selective serotonin reuptake inhibitors like paroxetine (Prx) often requires 4-6 weeks to achieve clinical benefits in depressed patients. Pindolol shortens this delay and it has been suggested that this effect is mediated by somatodendritic 5-hydroxytryptamine (5-HT) 1A autoreceptors. However clinical data on the beneficial effects of pindolol are conflicting. To study the effects of (+/-)-pindolol-paroxetine administration, we used genetical and pharmacological approaches in 5-HT1A knockout mice (5-HT1A-/-). Two assays, in vivo intracerebral microdialysis in awake mice and the forced swimming test (FST), were used to assess the antidepressant-like effects of this drug combination. Basal levels of extracellular serotonin, 5-HT ([5-HT]ext) in the frontal cortex (FCX) and the dorsal raphe nucleus (DRN) did not differ between the two strains of mice, suggesting a lack of tonic control of 5-HT1A autoreceptors on nerve terminal 5-HT release. Prx (1 and 4 mg/kg) dose-dependently increased cortical [5-HT]ext in both genotypes, but the effects were greater in mutants. The selective 5-HT1A receptor antagonist, WAY-100635 (0.5 mg/kg), or (+/-)-pindolol (5 and 10 mg/kg) potentiated the effects of Prx (4 mg/kg) on cortical [5-HT]ext in 5-HT1A+/+, but not in 5-HT1A-/- mice. Similar responses were obtained following local intra-raphe perfusion by reverse microdialysis of either WAY-100635 or (+/-)-pindolol (100 microM each). In the FST, Prx administration dose-dependently decreased the immobility time in both strains of mice, but the response was much greater in 5HT1A-/- mice. In contrast, (+/-)-pindolol blocked Prx-induced decreases in the immobility time while WAY-100635 had no effect in both genotypes. These findings using 5-HT1A-/- mice confirm that (+/-)-pindolol behaves as an antagonist of 5-HT1A autoreceptor in mice, but its blockade of paroxetine-induced antidepressant-like effects in the FST may be due to its binding to other neurotransmitter receptors.
Subject(s)
Behavior, Animal/drug effects , Cerebral Cortex/drug effects , Pindolol/pharmacology , Receptor, Serotonin, 5-HT1A/deficiency , Serotonin Antagonists/pharmacology , Serotonin/metabolism , 8-Hydroxy-2-(di-n-propylamino)tetralin/pharmacology , Analysis of Variance , Animals , Area Under Curve , Dose-Response Relationship, Drug , Drug Interactions , Immobility Response, Tonic/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microdialysis/methods , Paroxetine/pharmacology , Raphe Nuclei/drug effects , Serotonin Receptor Agonists/pharmacology , Selective Serotonin Reuptake Inhibitors/pharmacology , SwimmingABSTRACT
RATIONALE: Evidence links alterations in α5-containing nicotinic receptors (α5*-nAChRs) to nicotine addiction. Notably, the rs16969968 polymorphism in the α5 gene (α5SNP) increases the risk for heavy smoking and impairs nicotine-rewarding properties in mice. Additional work is needed to understand how native and polymorphic α5*-nAChRs contribute to processes associated with the risk for nicotine addiction. OBJECTIVES: We aimed at understanding the contribution of α5*-nAChRs to endophenotypes like increased responses to novelty and anxiety, known to promote vulnerability to addiction, and to the response of the dopamine and serotonin systems to nicotine. METHODS: Behavioural phenotypes were investigated in mice lacking the α5 gene (α5(-/-)). Nicotine injections were performed to test the consequences of nicotine exposure on the phenotypes identified. Dopamine and serotonin signalling were assessed using in vivo microdialysis and electrophysiology. We used lentiviral vectors to compare the consequences of re-expressing either the α5 wild-type allele or the α5SNP in specific brain areas of α5(-/-) mice. RESULTS: α5(-/-) mice did not exhibit high responses to novelty but showed decreased novelty-induced rearing behaviour together with high anxiety. Exposure to high doses of nicotine rescued these phenotypes. We identified altered spontaneous and nicotine-elicited serotonin and dopamine activity in α5(-/-) mice. Re-expression of α5 in the ventral tegmental area and hippocampus rescued rearing and anxiety levels in α5(-/-) mice, respectively. When expressing the α5SNP instead, this resulted in a knockout-like phenotype for both behaviours. CONCLUSIONS: We propose that altered α5*-nAChR cholinergic signalling contributes to emotional/behavioural impairments that may be alleviated by nicotine consumption.
Subject(s)
Anxiety/genetics , Brain/drug effects , Exploratory Behavior/drug effects , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Receptors, Nicotinic/genetics , Animals , Behavior, Animal/drug effects , Brain/metabolism , Dopamine/metabolism , Exploratory Behavior/physiology , Hippocampus/metabolism , Male , Mesencephalon/metabolism , Mice , Mice, Knockout , Microdialysis , Polymorphism, Single Nucleotide , Reward , Serotonin/metabolism , Tobacco Use Disorder/genetics , Ventral Tegmental Area/metabolismABSTRACT
von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.
Subject(s)
von Willebrand Disease, Type 2/blood , von Willebrand Disease, Type 2/pathology , Amino Acid Substitution , Animals , Disease Models, Animal , Female , Genetic Engineering , Hemostasis/genetics , Humans , Inflammation/genetics , Inflammation/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis, Site-Directed , Mutant Proteins/blood , Mutant Proteins/chemistry , Mutant Proteins/genetics , Platelet Adhesiveness , Platelet Count , von Willebrand Disease, Type 2/genetics , von Willebrand Factor/chemistry , von Willebrand Factor/genetics , von Willebrand Factor/metabolismABSTRACT
Vortioxetine (Lu AA21004) is an investigational novel antidepressant with multimodal activity that functions as a 5-HT3, 5-HT7 and 5-HT(1D) receptor antagonist, 5-HT(1B) receptor partial agonist, 5-HT(1A) receptor agonist and inhibitor of the 5-HT transporter in vitro. Here we explore its anxiolytic and antidepressant potential in adult mice. Vortioxetine was assessed in BalB/cJ@RJ mice using the open-field and forced-swim tests (acute: p.o. 1 h, repeated: daily p.o. 21 days), and in 129S6/SvEvTac mice using the novelty suppressed feeding paradigm (acute: p.o. 1 h, sustained: daily p.o. 14 or 21 days). Fluoxetine and diazepam were controls. Acute and repeated dosing of vortioxetine produced more pronounced anxiolytic- and antidepressant-like activities than fluoxetine. Vortioxetine significantly increased cell proliferation and cell survival and stimulated maturation of immature granule cells in the subgranular zone of the dentate gyrus of the hippocampus after 21 days of treatment. After 14 days, a high dose of vortioxetine increased dendritic length and the number of dendrite intersections, suggesting that vortioxetine accelerates the maturation of immature neurons. Vortioxetine displays an antidepressant and anxiolytic profile following repeated administration associated with increased neurogenesis at several stages. Vortioxetine effects were observed at low levels of 5-HT transporter occupancy, suggesting an alternative mechanism of action to 5-HT reuptake inhibition.
Subject(s)
Anti-Anxiety Agents/pharmacology , Antidepressive Agents/pharmacology , Neurogenesis/drug effects , Piperazines/pharmacology , Sulfides/pharmacology , Animals , Dentate Gyrus/cytology , Dentate Gyrus/drug effects , Diazepam/pharmacology , Drug Evaluation, Preclinical , Exploratory Behavior/drug effects , Fluoxetine/pharmacology , Immobility Response, Tonic/drug effects , Male , Mice , Motor Activity/drug effects , VortioxetineABSTRACT
RATIONALE: Nicotine is the main addictive component of tobacco and modifies brain function via its action on neuronal acetylcholine nicotinic receptors (nAChRs). The mesolimbic dopamine (DA) system, where neurons of the ventral tegmental area (VTA) project to the nucleus accumbens (ACb), is considered a core site for the processing of nicotine's reinforcing properties. However, the precise subtypes of nAChRs that mediate the rewarding properties of nicotine and that contribute to the development of addiction remain to be identified. OBJECTIVES: We investigated the role of the nAChRs containing the α7 nicotinic subunit (α7 nAChRs) in the reinforcing properties of nicotine within the VTA and in the nicotine-induced changes in ACb DA outflow in vivo. METHODS: We performed intra-VTA self-administration and microdialysis experiments in genetically modified mice lacking the α7 nicotinic subunit or after pharmacological blockade of α7 nAChRs in wild-type mice. RESULTS: We show that the reinforcing properties of nicotine within the VTA are lower in the absence or after pharmacological blockade of α7 nAChRs. We also report that nicotine-induced increases in ACb DA extracellular levels last longer in the absence of these receptors, suggesting that α7 nAChRs regulate the action of nicotine on DA levels over time. CONCLUSIONS: The present results reveal new insights for the role of α7 nAChRs in modulating the action of nicotine within the mesolimbic circuit. These receptors appear to potentiate the reinforcing action of nicotine administered into the VTA while regulating its action over time on DA outflow in the ACb.
Subject(s)
Dopamine/metabolism , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Receptors, Nicotinic/metabolism , Animals , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Microdialysis , Neurons/metabolism , Nicotine/administration & dosage , Nicotinic Agonists/administration & dosage , Nucleus Accumbens/metabolism , Receptors, Nicotinic/genetics , Reinforcement, Psychology , Reward , Self Administration , Time Factors , Ventral Tegmental Area/metabolism , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
Selective serotonin (5-HT) re-uptake inhibitors (SSRIs) are commonly used in the treatment of generalized anxiety disorder in Humans. However, because only few animal models display overt anxious-like behavior, detailed preclinical studies of the anxiolytic properties of antidepressants are still lacking. Here, we studied the neurochemical and behavioral effects of a double 5-HT(1A/1B) receptor knockout in mice (5-HT(1A/1B)-/-) as compared to their wild-type littermates (5-HT(1A/1B)+/+). It is known that single deletion of either 5-HT(1A) or 5-HT(1B) receptor induces behavioral changes that are not correlated with differences in brain serotonergic tone. Deletion of both receptors resulted in (i) higher emotionality of animals, as observed in three unconditioned paradigms of anxiety (open field, elevated plus maze and novelty suppressed feeding tests); (ii) a ≈200% increase in the mean spontaneous firing rate of 5-HT neurons in the dorsal raphe nucleus (DRN) compared to 5-HT(1A/1B)+/+ mice; (iii) elevated basal dialysate levels of 5-HT in the DRN and frontal cortex; (iv) an exaggerated response to acute paroxetine administration in microdialysis experiments, and (v) increased basal core body temperature. These findings suggest that the deletion of both autoreceptors induces a strong anxious-like behavioral state associated with increased 5-HT neurotransmission. Interestingly, 5-HT(1A/1B)-/- mice are still sensitive to the acute administration of diazepam. Moreover, while deletion of both receptors impacted on the response to acute SSRI treatment in the forced swim test, anxiolytic-like effects of a chronic SSRI treatment were still observed in 5-HT(1A/1B)-/- mice. Thus, the 5-HT(1A/1B)-/- mouse model could be of great interest to unveil the mechanisms of action of the anxiolytic effects of SSRIs.
Subject(s)
Anti-Anxiety Agents/therapeutic use , Anxiety/drug therapy , Disease Models, Animal , Receptor, Serotonin, 5-HT1A/physiology , Receptor, Serotonin, 5-HT1B/physiology , Animals , Anxiety/metabolism , Anxiety/physiopathology , Behavior, Animal/drug effects , Body Temperature Regulation , Frontal Lobe/drug effects , Frontal Lobe/metabolism , Gene Expression , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/drug effects , Neurons/metabolism , Paroxetine/therapeutic use , RNA, Messenger/metabolism , Raphe Nuclei/drug effects , Raphe Nuclei/metabolism , Receptor, Serotonin, 5-HT1A/genetics , Receptor, Serotonin, 5-HT1B/genetics , Serotonin/metabolism , Serotonin Plasma Membrane Transport Proteins/genetics , Serotonin Plasma Membrane Transport Proteins/metabolism , Selective Serotonin Reuptake Inhibitors/therapeutic use , Synaptic Transmission/drug effectsABSTRACT
The onset of a therapeutic response to antidepressant treatment exhibits a delay of several weeks. The present study was designed to know whether extracellular serotonin (5-HT) levels need to be increased in territories of 5-HT innervation in order to obtain beneficial effects from a chronic treatment with a serotonin-selective reuptake inhibitor (SSRI). Thus, we performed a longitudinal study of a chronic fluoxetine treatment in a model of highly emotional mice (BALB/cJ). The function of the 5-HT system in the raphe nuclei and hippocampus, was assessed by using repeated in vivo microdialysis sessions in awake freely moving mice, then studying its relation with behavior, analyzed mainly with open field paradigm. One of the neural mechanisms underlying such delay has been proposed to be the functional status of 5-HT1A autoreceptors in raphe nuclei. Thus, we also assessed the degree of 5-HT1A autoreceptor desensitization by using a local infusion in the raphe of the antagonist, WAY 100635 via reverse microdialysis. We report that the anxiolytic-like effects of fluoxetine correlate in time and amplitude with 5-HT1A autoreceptor desensitization, but neither with the extracellular levels of 5-HT in the raphe nuclei, nor in the hippocampus. Our study suggests that the beneficial anxiolytic/antidepressant-like effects of chronic SSRI treatment indeed depend on 5-HT1A autoreceptor internalization, but do not require a sustained increase in extracellular 5-HT levels in a territory of 5-HT projection such as hippocampus.
Subject(s)
Antidepressive Agents/pharmacology , Behavior, Animal/drug effects , Emotions , Fluoxetine/administration & dosage , Fluoxetine/pharmacology , Microdialysis/methods , Serotonin/metabolism , Administration, Oral , Animals , Antidepressive Agents/administration & dosage , Antidepressive Agents/therapeutic use , Anxiety/drug therapy , Anxiety/metabolism , Anxiety/pathology , Depressive Disorder/drug therapy , Depressive Disorder/metabolism , Depressive Disorder/pathology , Extracellular Space/drug effects , Extracellular Space/metabolism , Fluoxetine/therapeutic use , Longitudinal Studies , Male , Mice , Mice, Inbred BALB C , Receptor, Serotonin, 5-HT1A/metabolism , Time FactorsABSTRACT
We studied the effects of 1 mg/kg doses of nicotine and the alpha4beta2* nicotinic acetylcholine receptor (nAChR) partial agonist, varenicline, on extracellular dopamine (DA) levels in the nucleus accumbens (NuAcc) of lentivirally vectorized male mice. Three separate experimental groups were injected with a lentiviral vector transducing the ventral tegmental area (VTA): wild-type C57BL/6J mice with a vector expressing eGFP only, beta2 knock-out mice (beta2KO) with the eGFP-only vector, and beta2KO mice with a bicistronic vector reintroducing beta2 and eGFP into the VTA as recently described (Maskos et al., 2005). Our results suggest that the neurochemical effects of varenicline as measured by using microdialysis in awake, freely moving mice are mainly mediated via beta2* nAChR subunits localized in the VTA.