Biology subjects

Leon, S.

Publications and source records attributed to Leon, S..

13 recordsLinked to original sources

bioRxiv2026

CD1b-specific T cells are transcriptionally closer to conventional CD4 T cells than to innate-like NKT and MAIT cells

Unconventional T cells recognize non-peptide antigens presented by molecules other than the major histocompatibility complex (MHC) proteins. Among unconventional T cells, natural killer T (NKT) cells, which recognize CD1d-lipid complexes, mucosal-associated invariant T (MAIT) cells, which recognize MR1-metabolite complexes, and {gamma}{delta} T cells, are thoroughly studied. CD1b presents self- and mycobacterial lipids to relatively understudied T cell subsets. Like MAIT cells and type I NKT cells, CD1b-specific cells include subpopulations with conserved TCRs. Consequent to their recognition of a nearly monomorphic antigen-presenting molecule, CD1b-specific T cells might share innate-like features with MAIT, type I NKT, and {gamma}{delta} T cells. Due to their low frequency in the peripheral blood, CD1a-, CD1b-, and CD1c-specific T cells have been studied predominantly as in vitro-expanded clones, so even basic information about their native ex vivo immunophenotypes is lacking. Here, we sort and transcriptionally profile ex vivo two T cell populations that recognize CD1b presenting mycobacterial mycolipids and compare them with conventional CD4 and CD8 T cells, {gamma}{delta} T cells, NK cells, MAIT cells, and NKT cells. We show that both the invariant TCR-expressing CD4+, CD1b-GMM-specific germline encoded mycolyl-reactive (GEM) T cells, as well as the diverse TCR-expressing CD1b-GMM-specific T cells, are transcriptionally closer to conventional T cells than to the innate-like T cell populations {gamma}{delta}, MAIT and type I NKT cells. Thus, despite their recognition of non-polymorphic antigen presenting molecules, CD1b-specific T cells show adaptive rather than innate-like transcriptional features.

immunology↗

bioRxiv2024

Direct observation of fluorescent proteins in gels: a rapid, cost-efficient and quantitative alternative to immunoblotting

The discovery of Green Fluorescent Protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation. In this study, we report that various green, orange and red FPs can be maintained in a native, fluorescent state during the entire process of protein sample extraction, incubation with sample buffer, loading and migration on SDS-PAGE with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent in immunoblotting such as transfer onto a membrane and antibody incubations. Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable or better to antibody-based detection, a better quantification and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.

biochemistry↗

bioRxiv2024

POMC neurons control fertility through differential signaling of MC4R in Kisspeptin neurons in the female mouse

Inactivating mutations in the melanocortin 4 receptor (MC4R) gene cause monogenic obesity. Interestingly, female patients also display various degrees of reproductive disorders, in line with the subfertile phenotype of MC4RKO female mice. However, the cellular mechanisms by which MC4R regulates reproduction are unknown. Kiss1 neurons directly stimulate gonadotropin-releasing hormone (GnRH) release through two distinct populations; the Kiss1ARH neurons, controlling GnRH pulses, and the sexually dimorphic Kiss1AVPV/PeN neurons controlling the preovulatory LH surge. Here, we show that Mc4r expressed in Kiss1 neurons regulates fertility in females. In vivo, deletion of Mc4r from Kiss1 neurons in female mice replicates the reproductive impairments of MC4RKO mice without inducing obesity. Conversely, reinsertion of Mc4r in Kiss1 neurons of MC4R null mice restores estrous cyclicity and LH pulsatility without reducing their obese phenotype. In vitro, we dissect the specific action of MC4R on Kiss1ARH vs Kiss1AVPV/PeN neurons and show that MC4R activation excites Kiss1ARH neurons through direct synaptic actions. In contrast, Kiss1AVPV/PeN neurons are normally inhibited by MC4R activation except under elevated estradiol levels, thus facilitating the activation of Kiss1AVPV/PeN neurons to induce the LH surge driving ovulation in females. Our findings demonstrate that POMCARH neurons acting through MC4R, directly regulate reproductive function in females by stimulating the "pulse generator" activity of Kiss1ARH neurons and restricting the activation of Kiss1AVPV/PeN neurons to the time of the estradiol-dependent LH surge, and thus unveil a novel pathway of the metabolic regulation of fertility by the melanocortin system.

neuroscience↗

bioRxiv2024

Efficacy of Topical Strategies to Prevent the Onset of Root Caries Lesions, In vitro.

Preventive fluoride-based topical products, such as silver diamine fluoride (SDF) and others with chlorhexidine have been widely used as a strategy for preventing dental caries lesions, at all ages. Whether they are effective against root caries is less clear. AimTo evaluate the efficacy of various dental products in preventing root caries lesions, using a biological model of root caries. MethodologyAn experimental in vitro study was devised using a validated model with biofilms of Streptococcus mutans formed on bovine root dentin slabs. Slabs were divided into 5 experimental groups: (1) 1% chlorhexidine gel, (2) 22,600 ppm fluoride varnish, (3) 5000 ppm fluoride toothpaste, (4) 38% SDF and (5) negative control (0.9% NaCl). After the experimental phases, biofilms were detached from the dentin slabs to assess biomass and the number of viable cells (CFU). Demineralization of dentin slabs was evaluated by laser fluorescence and surface Knoop microhardness loss. ResultsDentin slabs treated with SDF did not significantly differ in surface microhardness loss compared to those treated with 1% chlorhexidine gel (p>0.05). However, both materials showed significantly lower surface demineralization than the rest of the groups. SDF had a higher impact on the properties of Streptococcus mutans biofilms. ConclusionSDF seems more effective than other materials in preventing root caries lesions in a highly cariogenic environment.

microbiology↗

bioRxiv2022

Fusion-negative Rhabdomyosarcoma 3D-organoids as an innovative model to predict resistance to cell death inducers

Rhabdomyosarcoma (RMS) is the main form of soft-tissue sarcoma in children and adolescents. For 20 years, and despite international clinical trials, its cure rate has not really improved, and remains stuck at 20% in case of relapse. The definition of new effective therapeutic combinations is hampered by the lack of reliable models, which complicate the transposition of promising results obtained in pre-clinical studies into efficient solutions for young patients. Inter-patient heterogeneity, particularly in the so-called fusion-negative group (FNRMS), adds an additional level of difficulty in optimizing the clinical management of children and adolescents with RMS. Here, we describe an original 3D-organoid model derived from relapsed FNRMS and show that it finely mimics the characteristics of the original tumor, including inter- and intra-tumoral heterogeneity. Moreover, we have established the proof-of-concept of their preclinical potential by re-evaluating the therapeutic opportunities of targeting apoptosis in FNRMS from a streamlined approach based on the exploitation of bulk and single-cell omics data.

cancer biology↗

bioRxiv2022

Dissecting the KNDy hypothesis: KNDy neuron-derived kisspeptins are dispensable for puberty but essential for preserved female fertility and gonadotropin pulsatility

Kiss1 neurons in the hypothalamic arcuate-nucleus (ARC) play key roles in the control of GnRH pulsatility and fertility. A fraction of ARC Kiss1 neurons, termed KNDy, co-express neurokinin B (NKB; encoded by Tac2). Yet, NKB- and Kiss1-only neurons are also found in the ARC, while a second major Kiss1-neuronal population is present in the rostral hypothalamus. The specific contribution of different Kiss1 neuron sub-sets to reproductive control remains unfolded. To tease apart the physiological roles of KNDy-born kisspeptins, conditional ablation of Kiss1 in Tac2-expressing cells was implemented in vivo. Mice with Tac2 cell-specific Kiss1 KO (TaKKO) displayed reduced ARC kisspeptin content and Kiss1 expression, with greater suppression in females, which was detectable at infantile-pubertal age. In contrast, Tac2/NKB levels were fully preserved. Despite the drop of ARC Kiss1/kisspeptin, pubertal timing was normal in TaKKO mice of both sexes. However, young-adult TaKKO females displayed disturbed LH pulsatility and sex steroid levels, with suppressed basal LH and pre-ovulatory LH surges, early-onset subfertility and premature ovarian insufficiency. Conversely, testicular histology and fertility were grossly conserved in TaKKO males. Ablation of Kiss1 in Tac2-cells led also to sex-dependent alterations in body composition, glucose homeostasis and locomotor activity. Our data document that KNDy-born kisspeptins are dispensable/compensable for puberty in both sexes, but required for maintenance of female gonadotropin pulsatility and fertility, as well as adult metabolic homeostasis. Significance StatementNeurons in the hypothalamic arcuate nucleus (ARC) co-expressing kisspeptins and NKB, named KNDy, have been recently suggested to play a key role in pulsatile secretion of gonadotropins, and hence reproduction. However, the relative contribution of this Kiss1 neuronal-subset, vs. ARC Kiss1-only and NKB-only neurons, as well as other Kiss1 neuronal populations, has not been assessed in physiological settings. We report here findings in a novel mouse-model with elimination of KNDy-born kisspeptins, without altering other kisspeptin compartments. Our data highlights the heterogeneity of ARC Kiss1 populations and document that, while dispensable/compensable for puberty, KNDy-born kisspeptins are required for proper gonadotropin pulsatility and fertility, specifically in females. Characterization of this functional diversity is especially relevant, considering the potential of kisspeptin-based therapies for management of human reproductive disorders. Disclosure StatementThe authors have nothing to disclose in relation to the contents of this work.

physiology↗

bioRxiv2022

2-deoxyglucose inhibits yeast AMPK signaling and triggers glucose transporter endocytosis, potentiating the drug toxicity

2-deoxyglucose is a glucose analog that impacts many aspects of cellular physiology. After its uptake and its phosphorylation into 2-deoxyglucose-6-phosphate (2DG6P), it interferes with several metabolic pathways including glycolysis and protein N-glycosylation. Despite this systemic effect, resistance can arise through strategies that are only partially understood. In yeast, 2DG resistance is often associated with mutations causing increased activity of the yeast 5-AMP activated protein kinase (AMPK), Snf1. Here we focus on the contribution of a Snf1 substrate in 2DG resistance, namely the alpha-arrestin Rod1 involved in nutrient transporter endocytosis. We report that 2DG triggers the endocytosis of many plasma membrane proteins, mostly in a Rod1-dependent manner. Rod1 participates in 2DG-induced endocytosis because 2DG, following its phosphorylation by hexokinase Hxk2, triggers changes in Rod1 post-translational modifications and promotes its function in endocytosis. Mechanistically, this is explained by a transient, 2DG-induced inactivation of Snf1/AMPK by protein phosphatase 1 (PP1). We show that 2DG-induced endocytosis is detrimental to cells, and the lack of Rod1 counteracts this process by stabilizing glucose transporters at the plasma membrane. This facilitates glucose uptake, which may help override the metabolic blockade caused by 2DG, and 2DG export - thus terminating the process of 2DG detoxification. Altogether, these results shed a new light on the regulation of AMPK signaling in yeast and highlight a remarkable strategy to bypass 2DG toxicity involving glucose transporter regulation. Author summaryIn this article, we study the resistance to a drug named 2-deoxyglucose (2DG). 2DG is an efficient inhibitor of several metabolic pathways, particularly glycolysis which is of prime importance for tumor cell proliferation. Cancer cells but also cells from other organisms can develop various resistance strategies that only being unraveled. Here, using bakers yeast as a model organism, we document the resistance mechanism of a mutant lacking the arrestin gene ROD1, known to regulate nutrient transporter endocytosis. First, we show that 2DG triggers the endocytosis of many nutrient transporters. Mechanistically, 2DG leads to a transient inhibition of the AMPK signaling pathway, which in turn promotes the activation of Rod1, a pre-requisite for 2DG-induced endocytosis. Consequently, cells lacking Rod1 maintain various proteins at the plasma membrane including hexose transporters, the latter being essential for 2DG resistance. Hexose transporter stabilization at the cell surface may sustain glucose import and help cells cope with the 2DG-induced metabolic blockade, while also allowing 2DG detoxication.

genetics↗

bioRxiv2021

Dual TCR-alpha expression on MAIT cells as a potential confounder of TCR interpretation

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant T cell receptor (TCR) chain that uses TRAV1-2 joined to TRAJ33/20/12 and recognize metabolites from bacterial riboflavin synthesis bound to the antigen-presenting molecule, MR1. Recently, our attempts to identify alternative MR1-presented antigens led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered antigen specificity is likely to lead to altered affinity for the most potent known antigen, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), we performed bulk TCR and {beta} chain sequencing, and single cell-based paired TCR sequencing, on T cells that bound the MR1-5-OP-RU tetramer, but with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Whereas we initially interpreted these as diverse MR1-restricted TCRs, single cell TCR sequencing revealed that cells expressing atypical TCR chains also co-expressed an invariant MAIT TCR chain. Transfection of each non-TRAV1-2 TCR chain with the TCR{beta} chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCR chain expression in human T cells and competition for the endogenous {beta} chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and non-canonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of antigen specificity.

immunology↗

bioRxiv2020

POMC neurons functional heterogeneity relies on mTORC1 signaling

Hypothalamic Pro-opiomelanocortin (POMC) neurons are classically known to trigger satiety. However, they encompass heterogeneous subpopulations whose functions are unknown. Here we show that POMC neurons releasing GABA, glutamate or both neurotransmitters possess distinct spatial distribution, molecular signatures and functions. Functional specificity of these subpopulations relies on the energy sensor mechanistic Target of Rapamycin Complex 1 (mTORC1), since pharmacological blockade of mTORC1, by mimicking a cellular negative energy state, simultaneously inhibited POMC/glutamatergic and activated POMC/GABAergic neurons. Chemogenetics and conditional deletion of mTORC1 then demonstrated that mTORC1 blockade in POMC neurons causes hyperphagia. This is due to decreased POMC-derived anorexigenic -melanocyte-stimulating hormone and the recruitment of POMC/GABAergic neurotransmission, which is restrained by cannabinoid type 1 receptor signaling. Genetic inhibition of glutamate release from POMC neurons also produced hyperphagia, recapitulating the phenotype caused by mTORC1 blockade. Altogether, these findings pinpoint the molecular mechanisms engaged by POMC neurons to oppositely control feeding, thereby challenging conventional views about their functions.

neuroscience↗

bioRxiv2018

Multiple signaling pathways converge onto the regulation of HAD-like phosphatases to modulate cellular resistance to the metabolic inhibitor 2-deoxyglucose

Cancer cells display an altered metabolism with an increased glycolysis and glucose uptake. Anti-cancer strategies targeting glycolysis through metabolic inhibitors have been considered. Particularly, the glucose analogue 2-deoxyglucose (2DG) is imported into cells and phosphorylated into 2DG-6-phosphate, a toxic by-product that inhibits glycolysis. Recent data suggest that 2DG has additional effects in the cell, and resistance to 2DG has also been observed. Using yeast as a model, we engaged an unbiased, mass-spectrometry-based approach to probe the cellular effects of 2DG on the proteome and study resistance mechanisms. This revealed that two 2DG-6-phosphate phosphatases, Dog1 and Dog2, are induced upon exposure to 2DG and participate in 2DG detoxication. 2DG induces Dog2 by upregulating several signaling pathways, such as the MAPK (Hog1/p38)-based stress-responsive pathway, the Unfolded Protein Response (UPR) triggered by 2DG-induced ER stress, and the MAPK (Slt2)-based Cell Wall Integrity pathway. Thus, 2DG-induced interference with cellular signaling rewires the expression of these endogenous phosphatases to promote 2DG resistance. Consequently, loss of the UPR or CWI pathways leads to hypersensitivity to 2DG. In contrast, DOG2 is transcriptionally repressed by glucose availability in a Snf1/AMPK-dependent manner, and mutants impaired in this pathway are 2DG-resistant. The characterization and genome resequencing of spontaneous 2DG-resistant mutants revealed that DOG2 overexpression is a common strategy to achieve 2DG resistance. The human Dog2 orthologue, HDHD1, also displays 2DG-6-phosphate phosphatase activity in vitro, and its overexpression confers 2DG resistance in HeLa cells, which has important implications for potential future chemotherapies involving 2DG.

genetics↗

bioRxiv2018

Redundancy in the central tachykinin systems safeguards puberty onset and fertility

The tachykinin neurokinin B (NKB, Tac2) is critical for GnRH release. NKB signaling deficiency leads to infertility in humans. However, some patients reverse this hypogonadism resembling the fertile phenotype of Tac2KO and Tacr3KO (encoding NKB receptor, NK3R) mice despite the absence of NKB signaling. Here, we demonstrate that in the absence of NKB signaling, other tachykinins (substance P and neurokinin A [NKA], encoded by Tac1) may take over to preserve fertility. The complete absence of tachykinins in Tac1/Tac2KO mice leads to delayed puberty onset in both sexes and infertility in 80% of females (but not males), in contrast to the 100% fertile phenotype of Tac1KO and Tac2KO mice separately. Furthermore, we demonstrate that NKA controls puberty onset and LH release through NKB-independent mechanisms in the presence of sex steroids and NKB-dependent mechanisms in their absence. In summary, tachykinins interact in a coordinated manner to ensure reproductive success in female mice.

physiology↗

bioRxiv2018

NKB Signaling in the Medial Amygdala Stimulates Gonadotropin Release in a Kisspeptin-Independent Manner in Female Mice

Neurokinin B (NKB) signaling is critical for reproduction in all studied species. The existing consensus is that NKB induces GnRH release via kisspeptin (Kiss1) stimulation in the arcuate nucleus. However, the stimulatory action of NKB is dependent on circulating estrogen (E2) levels, without which, NKB inhibits LH release. Importantly, the evidence supporting the kisspeptin-dependent role of NKB, derives from models of persistent hypogonadal state [e.g. Kiss1r knockout (KO) mice], with reduced E2 levels. Here, we demonstrate that in the presence of E2, NKB signaling induces LH release in a kisspeptin-independent manner. Moreover, senktide (NKB receptor agonist) delivery to the medial amygdala (MeA) increases LH in E2-treated Kiss1 KO females (but not males or sham-treated females) similar to controls, and thus, this increase is independent of Kiss1 neurons. These results document a novel kisspeptin-independent regulatory pathway of reproductive function in females mediated by NKB-responsive neurons in the MeA.

neuroscience↗

bioRxiv2018

PACAP neurons in the ventral premammillary nucleus regulate reproductive function in the female mouse

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuromodulator implicated in anxiety, metabolism and reproductive behavior. PACAP global knockout mice have decreased fertility and PACAP modulates LH release. However, its source and role at the hypothalamic level remain unknown. We demonstrate that PACAP-expressing neurons of the ventral premamillary nucleus of the hypothalamus (PMVPACAP) project to, and make direct contact with, kisspeptin neurons in the arcuate and AVPV/PeN nuclei and a subset of these neurons respond to PACAP exposure. Targeted deletion of PACAP from the PMV through stereotaxic virally mediated cre- injection or genetic cross to LepR-i-cre mice with PACAPfl/fl mice led to delayed puberty onset and impaired reproductive function in female, but not male, mice. We propose a new, sex-specific role for PACAP-expressing neurons in the PMV in the relay of nutritional state information to regulate GnRH release by modulating the activity of kisspeptin neurons, thereby regulating reproduction.

neuroscience↗