Abstract:
In the hippocampus, dentate gyrus granule cells connect to CA3 pyramidal cells via their axons, the mossy fibers (Mf). The synaptic terminals of Mfs (Mf boutons, MfBs) form large and complex synapses with thorny excrescences (TE) on the proximal dendrites of CA3 pyramidal cells (PCs). MfB/TE synapses have distinctive 'detonator' properties due to low initial release probability and large presynaptic facilitation. The molecular mechanisms shaping the morpho-functional properties of MfB/TE synapses are still poorly understood, though alterations in their morphology are associated with Down syndrome, intellectual disabilities, and Alzheimer's disease. Here, we identify the core PCP gene Vangl2 as essential to the morphogenesis and function of MfB/TE synapses. Vangl2 colocalises with the presynaptic heparan sulfate proteoglycan glypican 4 (GPC4) to stabilise the postsynaptic orphan receptor GPR158. Embryonic loss of Vangl2 disrupts the morphology of MfBs and TEs, impairs ultrastructural and molecular organisation, resulting in defective synaptic transmission and plasticity. In adult, the early loss of Vangl2 results in a number of hippocampus-dependent memory deficits including characteristic flexibility of declarative memory, organisation and retention of working / everyday-like memory. These deficits also lead to abnormal generalisation of memories to salient cues and diminished ability to form detailed contextual memories. Together, these results establish Vangl2 as a key regulator of DG-CA3 connectivity and functions.

Abstract:
From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain-of-function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an in vitro model of Mk differentiation from CD34(+) cells and studied step-by-step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 muM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41(+)CD42(+) numbers were reduced by around 1.5-fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA-PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis.

Abstract:
Core planar cell polarity (PCP) genes, which are involved in various neurodevelopmental disorders such as neural tube closure, epilepsy, and autism spectrum disorder, have poorly defined molecular signatures in neurons, mostly synapse-centric. Here, we show that the core PCP protein Prickle-like protein 2 (Prickle2) controls neuronal polarity and is a previously unidentified member of the axonal initial segment (AIS) proteome. We found that Prickle2 is present and colocalizes with AnkG480, the AIS master organizer, in the earliest stages of axonal specification and AIS formation. Furthermore, by binding to and regulating AnkG480, Prickle2 modulates its ability to bundle microtubules, a crucial mechanism for establishing neuronal polarity and AIS formation. Prickle2 depletion alters cytoskeleton organization, and Prickle2 levels determine both axon number and AIS maturation. Last, early Prickle2 depletion produces impaired action potential firing.

Abstract:
Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng(+/+) and Eng(+/-) mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/- TNFalpha and +/- LIM kinase inhibitors (LIMKi). In silico modeling of TNFalpha-Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca(2+)) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin-tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 +/- 15% (p < 0.05) and an increase in pseudo-tube width (41 +/- 4.5%; p < 0.001) compared to control. Upon TNFalpha stimulation, ECFC Eng-siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca(2+) mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFalpha activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC(+/-), and MLEC(+/-) compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 +/- 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFalpha-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFalpha), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.

Abstract:
BACKGROUND: Platelet cytosolic cAMP levels are balanced by synthesis, degradation, and efflux. Efflux can occur via MRP4 (multidrug resistant protein-4, ABCC4) present on dense granule and/or plasma membranes. As lipid rafts have been shown to interfere on cAMP homeostasis, we evaluated the relationships between the distribution and activity of MRP4 in lipid rafts and cAMP efflux. METHODS: Platelet activation and cAMP homeostasis were analyzed in human and wild-type or MRP4-deleted mouse platelets in the presence of methyl-beta-cyclodextrin (MbetaCD) to disrupt lipid rafts, and of activators of the cAMP signalling pathways. Human platelet MRP4 and effector proteins of the cAMP pathway were analyzed by immunoblots in lipid rafts isolated by differential centrifugation. RESULTS: MbetaCD dose-dependently inhibited human and mouse platelet aggregation without affecting per se cAMP levels. An additive inhibitory effect existed between the adenylate cyclase (AC) activator forskolin and MbetaCD that was accompanied by an overincrease of cAMP, and which was significantly enhanced upon MRP4 deletion. Finally, an efflux of cAMP out of resting platelets incubated with PGE1 was observed that was partly dependent on MRP4. Lipid rafts contained a small fraction ( approximately 15%) of MRP4 and most of the inhibitory G-protein Gi, whereas Gs protein, AC3, and phosphodiesterases PDE2 and PDE3A were all present as only trace amounts. CONCLUSION: Our results are in favor of part of MRP4 present at the platelet surface, including in lipid rafts. Lipid raft integrity is necessary for cAMP signalling regulation, although MRP4 and most players of cAMP homeostasis are essentially located outside rafts.

Abstract:
Blood platelets are essential for controlling hemostasis. They are released by megakaryocytes (MKs) located in the bone marrow, upon extension of cytoplasmic protrusions into the lumen of bone marrow sinusoids. Their number increases in postpulmonary capillaries, suggesting a role for oxygen gradient in thrombopoiesis (ie, platelet biogenesis). In this study, we show that initiation of thrombopoiesis from human mature MKs was enhanced under hyperoxia or during pro-oxidant treatments, whereas antioxidants dampened it. Quenching mitochondrial reactive oxygen species (mtROS) with MitoTEMPO decreased thrombopoiesis, whereas genetically enhancing mtROS by deacetylation-null sirtuin-3 expression increased it. Blocking cytosolic ROS production by NOX inhibitors had no impact. Classification according to the cell roundness index delineated 3 stages of thrombopoiesis in mature MKs. Early-stage round MKs exhibited the highest index, which correlated with low mtROS levels, a mitochondrial tubular network, and the mitochondrial recruitment of the fission activator Drp1. Intermediate MKs at the onset of thrombopoiesis showed high mtROS levels and small, well-delineated mitochondria. Terminal MKs showed the lowest roundness index and long proplatelet extensions. Inhibiting Drp1-dependent mitochondrial fission of mature MKs by Mdivi-1 favored a tubular mitochondrial network and lowered both mtROS levels and intermediate MKs proportion, whereas enhancing Drp1 activity genetically had opposite effects. Reciprocally, quenching mtROS limited mitochondrial fission in round MKs. These data demonstrate a functional coupling between ROS and mitochondrial fission in MKs, which is crucial for the onset of thrombopoiesis. They provide new molecular cues that control initiation of platelet biogenesis and may help elucidate some unexplained thrombocytopenia.

Abstract:
Lowe syndrome (LS) is an oculocerebrorenal syndrome of Lowe (OCRL1) genetic disorder resulting in a defect of the OCRL protein, a phosphatidylinositol-4,5-bisphosphate 5-phosphatase containing various domains including a Rho GTPase-activating protein (RhoGAP) homology domain catalytically inactive. We previously reported surgery-associated bleeding in patients with LS, suggestive of platelet dysfunction, accompanied with a mild thrombocytopenia in several patients. To decipher the role of OCRL in platelet functions and in megakaryocyte (MK) maturation, we conducted a case-control study on 15 patients with LS (NCT01314560). While all had a drastically reduced expression of OCRL, this deficiency did not affect platelet aggregability, but resulted in delayed thrombus formation on collagen under flow conditions, defective platelet spreading on fibrinogen and impaired clot retraction. We evidenced alterations of the myosin light chain phosphorylation (P-MLC), with defective Rac1 activity and, inversely, elevated active RhoA. Altered cytoskeleton dynamics was also observed in cultured patient MKs showing deficient proplatelet extension with increased P-MLC that was confirmed using control MKs transfected with OCRL-specific small interfering(si)RNA (siOCRL). Patients with LS also had an increased proportion of circulating barbell-shaped proplatelets. Our present study establishes that a deficiency of the OCRL protein results in a defective actomyosin cytoskeleton reorganisation in both MKs and platelets, altering both thrombopoiesis and some platelet responses to activation necessary to ensure haemostasis.

Abstract:
Ticagrelor, an antiplatelet adenosine diphosphate (ADP)-P2Y12 receptor antagonist, increases the risk of bleeding. Its management is challenging because platelet transfusion is ineffective and no specific antidote is currently available. Epinephrine, a vasopressor catecholamine prescribed during shock, restores platelet functions inhibited by ticagrelor through stimulation of alpha2A-adrenoreceptors. It subsequently inhibits cyclic adenosine monophosphate (cAMP) pathway and PI3K signaling. However, since epinephrine may expose a patient to deleterious hemodynamic effects, we hypothesized that other alpha2-adrenoreceptor agonist drugs used in clinical practice with fewer side effects could reverse the antiplatelet effects of ticagrelor. We compared in vitro the efficacy of clonidine, dexmedetomidine, brimonidine, and norepinephrine with epinephrine to restore ADP- and PAR-1-AP-induced washed platelet aggregation inhibited by ticagrelor, as well as resulting platelet cAMP levels. In ticagrelor-free samples, none of the alpha2-adrenoreceptor agonists induced aggregation by itself but all of them potentiated ADP-induced aggregation. Compared with epinephrine, norepinephrine, and brimonidine partially restored ADP- and fully restored PAR-1-AP-induced aggregation inhibited by ticagrelor while clonidine and dexmedetomidine were ineffective. Indeed, this lack of effect resulted from a lower decrease in cAMP concentration elicited by these partial alpha2-adrenoreceptor agonists, clonidine, and dexmedetomidine, compared with full alpha2-agonists. Our results support the development of specific full and systemic alpha2-adrenoreceptor agonists for ticagrelor reversal.

Abstract:
Ticagrelor, an antagonist of the platelet adenosine diphosphate (ADP)-P2Y12 receptor is recommended for patients with acute coronary syndromes. However, ticagrelor exposes to a risk of bleeding, the management of which is challenging because platelet transfusion is ineffective, and no antidote is yet available. We hypothesized that the vasopressor drug epinephrine could counter the antiplatelet effects of ticagrelor and restore platelet functions. We assessed in vitro the efficiency of epinephrine in restoring platelet aggregation inhibited by ticagrelor and investigated the underlying mechanisms. Washed platelet aggregation and secretion were measured upon stimulation by epinephrine alone or in combination with ADP, in the presence or absence of ticagrelor. Mechanistic investigations used P2Y1 and phosphoinositide 3-kinase (PI3K) inhibitors and included vasodilator-stimulated phosphoprotein (VASP) and Akt phosphorylation assays as well as measurement of Ca(2+) mobilisation. We found that epinephrine restored ADP-induced platelet aggregation, but not dense granule release. Epinephrine alone failed to induce aggregation whereas it fully induced VASP dephosphorylation and Akt phosphorylation regardless of the presence of ticagrelor. In the presence of ticagrelor, blockage of the P2Y1 receptor prevented restoration of platelet aggregation by the combination of epinephrine and ADP, as well as intracellular Ca(2+) mobilisation. In combination with ADP, epinephrine induced platelet aggregation of ticagrelor-treated platelets through inhibition of the cAMP pathway and activation of the PI3K pathway, thus enabling the P2Y1 receptor signalling and subsequent Ca(2+) mobilisation. This proof-of-concept study needs to be challenged in vivo for the management of bleeding in ticagrelor-treated patients.

Abstract:
INTRODUCTION: Clinical benefit-risk balance of direct oral anticoagulants (DOAC) in atherothrombosis prevention differs between anti-Xa and anti-IIa drugs and their specific effect on platelet functions remains controversial. We hence investigated rivaroxaban and dabigatran effect on platelets in identical experimental conditions. MATERIALS AND METHODS: Blood of fifteen healthy volunteers was spiked with DOAC which plasma concentrations were measured by specific anti-Xa or anti-IIa assays. Light transmission aggregometry measured in platelet-rich plasma used low doses of agonists: 0.5mM arachidonic acid, 2.5muM ADP, 0.5muM epinephrine, 0.8mug/ml collagen, 7.5muM TRAP-6 and 0.5 pM tissue factor in the presence of H-Gly-Pro-Arg-Pro-OH to prevent fibrin polymerization. Platelet adhesion on collagen fibres was evaluated in whole blood under flow. Same experiments were reproduced in the presence of aspirin. RESULTS: Median [95% CI] plasma concentrations were of 28 [23-36], 128 [119-144] and 321 [293-361] ng/ml for rivaroxaban and 39 [34-45], 171 [166-193] and 353 [349-382] ng/ml for dabigatran. DOAC did not modify platelet aggregation or adhesion on collagen fibres at any tested concentrations. However, they delayed platelet aggregation secondary to coagulation activation with a more potent effect with dabigatran (p<0.001). Aspirin did not modify DOAC effect. CONCLUSION: Efficacy of combining DOAC and aspirin in atherothrombosis prevention would not stem from a direct antiplatelet effect of the formers but to their additive inhibitory effect on platelet aggregation secondary to coagulation activation. This effect differs according to DOAC molecules and may also result from the pleiotropic roles of the different coagulation factors targeted by DOAC.