GPR68 Senses Flow and Is Essential for Vascular Physiology.

Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation of vascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology.

Mycobacterial toxin induces analgesia in buruli ulcer by targeting the angiotensin pathways.

Mycobacterium ulcerans, the etiological agent of Buruli ulcer, causes extensive skin lesions, which despite their severity are not accompanied by pain. It was previously thought that this remarkable analgesia is ensured by direct nerve cell destruction. We demonstrate here that M. ulcerans-induced hypoesthesia is instead achieved through a specific neurological pathway triggered by the secreted mycobacterial polyketide mycolactone. We decipher this pathway at the molecular level, showing that mycolactone elicits signaling through type 2 angiotensin II receptors (AT2Rs), leading to potassium-dependent hyperpolarization of neurons. We further validate the physiological relevance of this mechanism with in vivo studies of pain sensitivity in mice infected with M. ulcerans, following the disruption of the identified pathway. Our findings shed new light on molecular mechanisms evolved by natural systems for the induction of very effective analgesia, opening up the prospect of new families of analgesics derived from such systems.

Loss of function of the maternal membrane oestrogen receptor ERα alters expansion of trophoblast cells and impacts mouse fertility.

The binding of 17ß-oestradiol to oestrogen receptor alpha (ERα) plays a crucial role in the control of reproduction, acting through both nuclear and membrane-initiated signalling. To study the physiological role of membrane ERα in the reproductive system, we used the C451A-ERα mouse model with selective loss of function of membrane ERα. Despite C451A-ERα mice being described as sterile, daily weighing and ultrasound imaging revealed that homozygous females do become pregnant, allowing the investigation of the role of ERα during pregnancy for the first time. All neonatal deaths of the mutant offspring mice resulted from delayed parturition associated with failure in pre-term progesterone withdrawal. Moreover, pregnant C451A-ERα females exhibited partial intrauterine embryo arrest at about E9.5. The observed embryonic lethality resulted from altered expansion of Tpbpa-positive spiral artery-associated trophoblast giant cells into the utero-placental unit, which is associated with an imbalance in expression of angiogenic factors. Together, these processes control the trophoblast-mediated spiral arterial remodelling. Hence, loss of membrane ERα within maternal tissues clearly alters the activity of invasive trophoblast cells during placentogenesis. This previously unreported function of membrane ERα could open new avenues towards a better understanding of human pregnancy-associated pathologies.

Membrane and Nuclear Estrogen Receptor Alpha Actions: From Tissue Specificity to Medical Implications.

Estrogen receptor alpha (ERα) has been recognized now for several decades as playing a key role in reproduction and exerting functions in numerous nonreproductive tissues. In this review, we attempt to summarize the in vitro studies that are the basis of our current understanding of the mechanisms of action of ERα as a nuclear receptor and the key roles played by its two activation functions (AFs) in its transcriptional activities. We then depict the consequences of the selective inactivation of these AFs in mouse models, focusing on the prominent roles played by ERα in the reproductive tract and in the vascular system. Evidence has accumulated over the two last decades that ERα is also associated with the plasma membrane and activates non-nuclear signaling from this site. These rapid/nongenomic/membrane-initiated steroid signals (MISS) have been characterized in a variety of cell lines, and in particular in endothelial cells. The development of selective pharmacological tools that specifically activate MISS and the generation of mice expressing an ERα protein impeded for membrane localization have begun to unravel the physiological role of MISS in vivo. Finally, we discuss novel perspectives for the design of tissue-selective ER modulators based on the integration of the physiological and pathophysiological roles of MISS actions of estrogens.

Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.

Autophagy protein 5 controls flow-dependent endothelial functions.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Endothelin Receptor Blockade Improves Cerebral Blood Flow-Mediated Dilation in a Mouse Model of Alzheimer's Disease.

INTRODUCTION: Cerebral blood flow (CBF) is reduced in patients with Alzheimer's disease (AD). Flow-mediated dilation (FMD), which plays a key role in the regulation of blood flow, is attenuated by endothelin-1. We hypothesized that endothelin receptor blockade may improve CBF in AD. METHODS: We investigated cerebrovascular reactivity in a mouse model of AD (APP-PS1; 5-6-month-old male subjects). We assessed the in vivo response to normoxic hypercapnia and in vitro FMD in isolated cerebral and mesenteric resistance arteries before and after endothelin receptor blockade (bosentan). RESULTS: Normoxic hypercapnia increased basilar trunk blood flow velocity (+12.3 ± 2.4%; p = 0.006, n = 6) in wild-type (WT) mice but reduced blood flow in APP-PS1 mice (-11.4 ± 1.2%; p < 0.0001, n = 8). Bosentan (50 mg/kg, acute intraperitoneal injection) restored cerebrovascular reactivity in APP-PS1 mice (+10.2 ± 2.2%; p < 0.0001, n = 8) but had no effect in WT. FMD was reduced in the posterior cerebral artery of APP-PS1 compared to WT and was normalized by bosentan (1 µmol/L, 30 min, or 50 mg/kg/day for 28 days). FMD was similar in the mesenteric artery of APPS-PS1 and WT. CONCLUSION: APP-PS1 mice exhibited cerebrovascular endothelial dysfunction. Acute and chronic blockade of endothelin receptors restored endothelial vasomotor function, suggesting a promising therapeutic approach to restoring cerebral vasoreactivity in AD.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Evolutionary information helps understand distinctive features of the angiotensin II receptors AT1 and AT2 in amniota.

In vertebrates, the octopeptide angiotensin II (AngII) is an important in vivo regulator of the cardiovascular system. It acts mainly through two G protein-coupled receptors, AT1 and AT2. To better understand distinctive features of these receptors, we carried out a phylogenetic analysis that revealed a mirror evolution of AT1 and AT2, each one split into two clades, separating fish from terrestrial receptors. It also revealed that hallmark mutations occurred at, or near, the sodium binding site in both AT1 and AT2. Electrostatics computations and molecular dynamics simulations support maintained sodium binding to human AT1 with slow ingress from the extracellular side and an electrostatic component of the binding free energy around -3kT, to be compared to around -2kT for human AT2 and the δ opioid receptor. Comparison of the sodium binding modes in wild type and mutated AT1 and AT2 from humans and eels indicates that the allosteric control by sodium in both AT1 and AT2 evolved during the transition from fish to amniota. The unusual S7.46N mutation in AT1 is mirrored by a L3.36M mutation in AT2. In the presence of sodium, the N7.46 pattern in amniota AT1 stabilizes the inward orientation of N3.35 in the apo receptor, which should contribute to efficient N3.35 driven biased signaling. The M3.36 pattern in amniota AT2 favours the outward orientation of N3.35 and the receptor promiscuity. Both mutations have physiological consequences for the regulation of the renin-angiotensin system.

Tetrodotoxin Decreases the Contractility of Mesenteric Arteries, Revealing the Contribution of Voltage-Gated Na+ Channels in Vascular Tone Regulation.

Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na+ (NaV) channels. In arteries, NaV channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of NaV channels in vascular tone using TTX. We first characterized the expression of NaV channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which scn2a and scn1b were the most abundant transcripts, suggesting that murine vascular NaV channels consist of NaV1.2 channel subtype with NaVß1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks NaV channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications.

NTPDase1/CD39 Ectonucleotidase Is Necessary for Normal Arterial Diameter Adaptation to Flow.

NTPDase1/CD39, the major vascular ectonucleotidase, exerts thrombo-immunoregulatory function by controlling endothelial P2 receptor activation. Despite the well-described release of ATP from endothelial cells, few data are available regarding the potential role of CD39 as a regulator of arterial diameter. We thus investigated the contribution of CD39 in short-term diameter adaptation and long-term arterial remodeling in response to flow using Entpd1-/- male mice. Compared to wild-type littermates, endothelial-dependent relaxation was modified in Entpd1-/- mice. Specifically, the vasorelaxation in response to ATP was potentiated in both conductance (aorta) and small resistance (mesenteric and coronary) arteries. By contrast, the relaxing responses to acetylcholine were supra-normalized in thoracic aortas while decreased in resistance arteries from Entpd1-/- mice. Acute flow-mediated dilation, measured via pressure myography, was dramatically diminished and outward remodeling induced by in vivo chronic increased shear stress was altered in the mesenteric resistance arteries isolated from Entpd1-/- mice compared to wild-types. Finally, changes in vascular reactivity in Entpd1-/- mice were also evidenced by a decrease in the coronary output measured in isolated perfused hearts compared to the wild-type mice. Our results highlight a key regulatory role for purinergic signaling and CD39 in endothelium-dependent short- and long-term arterial diameter adaptation to increased flow.

Bios2cor: an R package integrating dynamic and evolutionary correlations to identify functionally important residues in proteins.

SUMMARY: Both dynamic correlations in protein sidechain motions during molecular dynamics (MD) simulations and evolutionary correlations in multiple sequence alignments (MSAs) of homologous proteins may reveal functionally important residues. We developed the R package Bios2cor that provides a unique framework to investigate and, possibly, integrate both analyses. Bios2cor starts with an MSA or an MD trajectory and computes correlation/covariation scores between positions in the MSA or between sidechain dihedral angles or rotamers in the MD trajectory. In addition, Bios2cor provides a variety of tools for the analysis, the visualization and the interpretation of the data. AVAILABILITY AND IMPLEMENTATION: The R package Bios2cor is available from the Comprehensive R Archive Network, at https://CRAN.R-project.org/package=Bios2cor.

Protective role of the mitochondrial fusion protein OPA1 in hypertension.

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.

Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle Cells.

RATIONALE: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an ER (estrogen receptor) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17ß-estradiol in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear. OBJECTIVE: Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms. METHODS AND RESULTS: Using 3 complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with 17ß-estradiol and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα subfunctions (membrane/extranuclear versus genomic/transcriptional) demonstrated that 17ß-estradiol-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells. CONCLUSIONS: Whereas tamoxifen acts as an antiestrogen and ERα antagonist in breast cancer but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα.

Sildenafil-Induced Revascularization of Rat Hindlimb Involves Arteriogenesis through PI3K/AKT and eNOS Activation.

Hypoxia and inflammation play a major role in revascularization following ischemia. Sildenafil inhibits phosphodiesterase-5, increases intracellular cGMP and induces revascularization through a pathway which remains incompletely understood. Thus, we investigated the effect of sildenafil on post-ischemic revascularization. The left femoral artery was ligated in control and sildenafil-treated (25 mg/kg per day) rats. Vascular density was evaluated and expressed as the left/right leg (L/R) ratio. In control rats, L/R ratio was 33 ± 2% and 54 ± 9%, at 7- and 21-days post-ligation, respectively, and was significantly increased in sildenafil-treated rats to 47 ± 4% and 128 ± 11%, respectively. A neutralizing anti-VEGF antibody significantly decreased vascular density (by 0.48-fold) in control without effect in sildenafil-treated animals. Blood flow and arteriolar density followed the same pattern. In the ischemic leg, HIF-1α and VEGF expression levels increased in control, but not in sildenafil-treated rats, suggesting that sildenafil did not induce angiogenesis. PI3-kinase, Akt and eNOS increased after 7 days, with down-regulation after 21 days. Sildenafil induced outward remodeling or arteriogenesis in mesenteric resistance arteries in association with eNOS protein activation. We conclude that sildenafil treatment increased tissue blood flow and arteriogenesis independently of VEGF, but in association with PI3-kinase, Akt and eNOS activation.

Early Inactivation of Membrane Estrogen Receptor Alpha (ERα) Recapitulates the Endothelial Dysfunction of Aged Mouse Resistance Arteries.

Flow-mediated dilation (FMD) of resistance arteries is essential for tissue perfusion but it decreases with ageing. As estrogen receptor alpha (Erα encoded by Esr1), and more precisely membrane ERα, plays an important role in FMD in young mice in a ligand-independent fashion, we evaluated its influence on this arteriolar function in ageing. We first confirmed that in young (6-month-old) mice, FMD of mesenteric resistance arteries was reduced in Esr1-/- (lacking ERα) and C451A-ERα (lacking membrane ERα). In old (24-month-old) mice, FMD was reduced in WT mice compared to young mice, whereas it was not further decreased in Esr1-/- and C451A-ERα mice. Markers of oxidative stress were similarly increased in old WT and C451A-ERα mice. Reduction in oxidative stress with superoxide dismutase plus catalase or Mito-tempo, which reduces mitochondrial superoxide restored FMD to a normal control level in young C451A-ERα mice as well as in old WT mice and old C451A-ERα mice. Estradiol-mediated dilation was absent in old WT mice. We conclude that oxidative stress is a key event in the decline of FMD, and that an early defect in membrane ERα recapitulates phenotypically and functionally ageing of these resistance arteries. The loss of this function could take part in vascular ageing.

Metabolomic Profiling of Angiotensin-II-Induced Abdominal Aortic Aneurysm in Ldlr-/- Mice Points to Alteration of Nitric Oxide, Lipid, and Energy Metabolisms.

Aneurysm is the second-most common disease affecting the aorta worldwide after atherosclerosis. While several clinical metabolomic studies have been reported, no study has reported deep metabolomic phenotyping in experimental animal models of aortic aneurysm. We performed a targeted metabolomics study on the blood and aortas of an experimental mice model of aortic aneurysm generated by high-cholesterol diet and angiotensin II in Ldlr-/- mice. The mice model showed a significant increase in media/lumen ratio and wall area, which is associated with lipid deposition within the adventitia, describing a hypertrophic remodeling with an aneurysm profile of the abdominal aorta. Altered aortas showed increased collagen remodeling, disruption of lipid metabolism, decreased glucose, nitric oxide and lysine metabolisms, and increased polyamines and asymmetric dimethylarginine (ADMA) production. In blood, a major hyperlipidemia was observed with decreased concentrations of glutamine, glycine, taurine, and carnitine, and increased concentrations of the branched amino acids (BCAA). The BCAA/glycine and BCAA/glutamine ratios discriminated with very good sensitivity and specificity between aneurysmatic and non-aneurysmatic mice. To conclude, our results reveal that experimental induction of aortic aneurysms causes a profound alteration in the metabolic profile in aortas and blood, mainly centered on an alteration of NO, lipid, and energetic metabolisms.

Pharmacological Dissection of the Crosstalk between NaV and CaV Channels in GH3b6 Cells.

Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the ß-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.

Screening an In-House Isoquinoline Alkaloids Library for New Blockers of Voltage-Gated Na+ Channels Using Voltage Sensor Fluorescent Probes: Hits and Biases.

Voltage-gated Na+ (NaV) channels are significant therapeutic targets for the treatment of cardiac and neurological disorders, thus promoting the search for novel NaV channel ligands. With the objective of discovering new blockers of NaV channel ligands, we screened an In-House vegetal alkaloid library using fluorescence cell-based assays. We screened 62 isoquinoline alkaloids (IA) for their ability to decrease the FRET signal of voltage sensor probes (VSP), which were induced by the activation of NaV channels with batrachotoxin (BTX) in GH3b6 cells. This led to the selection of five IA: liriodenine, oxostephanine, thalmiculine, protopine, and bebeerine, inhibiting the BTX-induced VSP signal with micromolar IC50. These five alkaloids were then assayed using the Na+ fluorescent probe ANG-2 and the patch-clamp technique. Only oxostephanine and liriodenine were able to inhibit the BTX-induced ANG-2 signal in HEK293-hNaV1.3 cells. Indeed, liriodenine and oxostephanine decreased the effects of BTX on Na+ currents elicited by the hNaV1.3 channel, suggesting that conformation change induced by BTX binding could induce a bias in fluorescent assays. However, among the five IA selected in the VSP assay, only bebeerine exhibited strong inhibitory effects against Na+ currents elicited by the hNav1.2 and hNav1.6 channels, with IC50 values below 10 µM. So far, bebeerine is the first BBIQ to have been reported to block NaV channels, with promising therapeutical applications.

Metabolomic Sexual Dimorphism of the Mouse Brain is Predominantly Abolished by Gonadectomy with a Higher Impact on Females.

The importance of sexual dimorphism of the mouse brain metabolome was recently highlighted, in addition to a high regional specificity found between the frontal cortex, the cerebellum, and the brain stem. To address the origin of this dimorphism, we performed gonadectomy on both sexes, followed by a metabolomic study targeting 188 metabolites in the three brain regions. While sham controls, which underwent the same surgical procedure without gonadectomy, reproduced the regional sexual dimorphism of the metabolome previously identified, no sex difference was identifiable after gonadectomy, through both univariate and multivariate analyses. These experiments also made it possible to identify which sex was responsible for the dimorphism for 35 metabolites. The female sex contributed to the difference for more than 80% of them. Our results show that gonads are the main contributors to the brain sexual dimorphism previously observed, especially in females.