Reproductive disorders can result from a defective action of the neuropeptide gonadotropin-releasing hormone (GnRH), the master regulator of reproduction. We have previously shown that selenoprotein T (SELENOT), a newly described thioredoxin-like selenoprotein highly expressed in endocrine and neuroendocrine cells, plays a role in hormone secretion and neuroprotection. However, whether SELENOT is involved in neuroendocrine regulation in vivo is totally unknown. We found that SELENOT deficiency in the brain impaired sexual behavior, leading to a decline in fertility in both male and female mice. Biochemical and histological analyses of the gonadotrope axis of these mice revealed a higher expression of GnRH, which is associated with circulating luteinizing hormone (LH) excess, and elevated steroid hormones in males and a polycystic ovary syndrome-like phenotype in females. In addition, SELENOT deficiency impaired LH pulse secretion in both male and female mice. These changes were reverted after administration of a GnRH antagonist. Together, our data demonstrate for the first time to our knowledge the role of a selenoprotein in the central control of sexual behavior and reproduction, and identify a redox effector of GnRH neuron activity impacting both male and female reproductive function.

Non-small cell lung cancers (NSCLCs) treated with tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) almost invariably relapse in the long term, due to the emergence of subpopulations of resistant cells. Through a DNA barcoding approach, we show that the clinically approved drug sorafenib specifically abolishes the selective advantage of EGFR-TKI-resistant cells, while preserving the response of EGFR-TKI-sensitive cells. Sorafenib is active against multiple mechanisms of resistance/tolerance to EGFR-TKIs and its effects depend on early inhibition of MAPK-interacting kinase (MKNK) activity and signal transducer and activator of transcription 3 (STAT3) phosphorylation, and later down-regulation of MCL1 and EGFR. Using different xenograft and allograft models, we show that the sorafenib-EGFR-TKI combination can delay tumor growth and promote the recruitment of inflammatory cells. Together, our findings indicate that sorafenib can prolong the response to EGFR-TKIs by targeting NSCLC capacity to adapt to treatment through the emergence of resistant cells.

Graphical abstract: Abstract:The present study aims to investigate the impact of orexin deficiency on the regulation of energy and glucose metabolism using a mouse model depleted of the prepro-orexin gene. Our data reveal that, despite a decrease in food consumption (at least in males), orexin deficiency induces a significant increase in body weight that is associated with an alteration in the body composition, as male and female orexin-deficient mice display increased fat mass compared to wild-type littermates. Nevertheless, no significant differences of global energy expenditure and locomotor activity were observed in the mutant mice relative to the control. Glucose homeostasis is also impaired in the absence of orexins, since glucose tolerance and insulin secretion are diminished, and insulin sensitivity is slightly reduced. In addition, the livers of male orexin-KO mice are significantly larger and heavier with more adipose tissue than wild-type mice. Interestingly, orexin-deficient mice present an upregulation of liver enzymes involved in gluconeogenesis and a downregulation of GCK, an enzyme that promotes glycogen storage, which may participate to the altered glucose metabolism of orexin mutant mice. To conclude, the present study indicates that orexin deficiency induces profound alterations in the regulation of energy and glucose metabolism, which is more pronounced in males than in females. These findings support the idea that dysfunction of this orexin system may promote obesity and diabetes, and could represent an interesting therapeutic target in the context of 'diabesity'.

Abstract Cardiac lymphatic alterations and insufficient lymphatic drainage have been found in cardiovascular diseases (CVDs). To unravel the mechanisms underlying lymphatic dysfunction, we applied single-cell (sc) analyses in murine heart failure (HF) models. Transaortic constriction (TAC) in C57BL/6J and BALB/c mice modeled chronic pressure -overload-induced cardiac hypertrophy and HF, respectively. Cardiac lymphatic (LEC) and blood vascular endothelial cells (BEC) were analyzed by scRNAseq, and targets validated by immunohistochemistry and human LEC cultures. While LEC profiles were comparable between strains in healthy mice, we found expansion of lymphatic capillaries and loss of valves post-TAC only in BALB/c. Differentially expressed gene (DEG) analysis revealed a reduction post-TAC only in BALB/c of lymphatic junctional components. Conversely, LEC expression of immune cell cross-talk mediators was mostly preserved post-TAC. Interestingly, around 35% of DEGs identified in cardiac LECs post-TAC were similarly altered in interleukin (IL)1β-stimulated human LECs. In conclusion, loss of lymphatic valves and dysregulated lymphatic barrier may underlie poor drainage capacity during pressure-overload-induced HF, despite potent lymphangiogenesis and preserved LEC immune attraction. Our work provides tractable targets to restore lymphatic health in CVDs.

26RFa is a regulatory peptide initially isolated from the brain. 26RFa was found to be involved in the regulation of vital functions such as the regulation of energy and glucose metabolism. However, the whole distribution of 26RFa in the organs/tissues of the organism remains fragmentary although it represents a crucial step to discover novel physiological functions for this regulatory peptide. To this aim, we have generated a mouse line that expresses the fluorescent protein tdTomato in 26RFa-expressing cells, and visualization of tdTomato immunostaining in toto was performed using the tridimensional imaging approach. Our observations reveal that 26RFa is largely distributed among the organism of male and female mice. 26RFa-expressing cells were notably found in numerous regions of the central nervous system including the olfactory bulbs, the cortex, the hippocampus, the hypothalamus, the cerebellum, the brainstem and the spinal cord. At the periphery, 26RFa-expressing structures were detected all along the gastrointestinal tract, in the liver, the white adipose tissue, the kidney, the adrenal gland, the lungs, the male and female reproductive tracts, the striated muscles, the thymus and a number of exocrine glands such as the salivary glands, the prostate, the seminal vesicles. In conclusion, the present anatomical observations are in agreement with the main physiological functions previously reported for 26RFa such the regulation of energy and glucose metabolism or the control of the hypothalamo-pituitary-gonadal or adrenal axis. However, 26RFa is present in other organs/tissues in which its physiological relevance is totally unknown opening therefore a new field of research for this regulatory peptide.

During corticogenesis, projection neurons migrate along the radial glial axis to form cortical layers, the alteration of which is associated with functional deficits in adulthood. As byproducts of cell metabolism, reactive oxygen species act as second messengers to contribute to neurodevelopment; however, free radical excess may impede this process. Selenoprotein T (SELENOT) is a newly identified thioredoxin-like enzyme of the endoplasmic reticulum abundantly expressed during embryogenesis whose gene disruption in the brain leads to neuroblast cell demise and neuromorphological alterations due to increased free radical levels. To determine the potential contribution of SELENOT to the establishment of cortical networks, we first analyzed its expression profile in the neocortex at different stages of development using RNA scope in situ hybridization. These studies revealed the expression of SELENOT in different cortical layers, and its localization in glutamatergic and GABAergic neurons. Targeted SELENOT gene knockout in the cortex using in utero electroporation-mediated gene disruption or Nes-Cre/loxP transgenesis system resulted in an alteration of neuroblast migration polarity, at the level of radial scaffolding, and projection neuron positioning. These results indicate that SELENOT which is highly expressed in the cortex during neurodevelopment plays a crucial role in corticogenesis by promoting projection neuron migration.

The inflammatory response to acute kidney injury (AKI) likely dictates future renal health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Due to the relative sparsity of lymphatic endothelial cells (LECs) in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. Here we characterized murine renal LEC subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI. We validated our findings by qPCR in LECs isolated from both cisplatin-injured and ischemia reperfusion injury, by immunofluorescence, and confirmation in in vitro human LECs. We have identified renal LECs and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin injured conditions. Following AKI, renal LECs alter genes involved endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models are also identified with renal LECs further demonstrating changed gene expression between cisplatin and ischemia reperfusion injury models, indicating the renal LEC response is both specific to where they lie in the lymphatic vasculature and the renal injury type. How LECs respond to AKI may therefore be key in regulating future kidney disease progression.

In addition to brain disorders, which constitute a devastating consequence of prenatal alcohol exposure (PAE), eye development is also significantly affected. Given that the retina is a readily accessible part of the central nervous system, a better understanding of the impact of ethanol on retinal development might provide ophthalmological landmarks helpful for early diagnosis of fetal alcohol syndrome. This study aimed to provide a fine morphometric and cellular characterization of the development of retinal microvasculature and neurovascular interactions in a mouse model of fetal alcohol spectrum disorder (FASD). The data revealed that PAE impaired superficial vascular plexus development. In particular, progression of the vascular migration front was significantly decreased in PAE retinas, supporting a delay in plexus progression. Moreover, a significant decrease in the vessel density and number of perforating vessels was quantified in PAE mice, supporting less angiogenesis. The present study provides also the first evidence of a close interaction between migrating calretinin-positive interneurons and perforating microvessels in the inner nuclear layer of the developing retina. This neurovascular association was significantly impaired by PAE. Moreover, projections of amacrine cells were abnormally distributed and densified in stratum S1 and S2. In humans, comparison of a five-month-old control infant with a three-month-old alcohol-exposed case revealed a similar mispositioning of calretinin-positive interneurons. This opens new research avenues regarding a neurovascular contribution in the deleterious effects of alcohol in the developing retina and support that ophthalmological examination could become a promising approach for early detection of alcohol-exposed infants presenting with neurovascular brain defects.

ABSTRACT Non-small cell lung cancers (NSCLCs) treated with tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) almost invariably relapse in the long term, due to the emergence of subpopulations of resistant cells. Here we show that the lack of sensitivity of these cells to EGFR-TKIs constitutes a vulnerability that can be potentially targeted. Through a DNA barcoding approach, we demonstrate that the clinically approved drug sorafenib specifically abolishes the selective advantage of EGFR-TKI-resistant cells, while preserving the response of EGFR-TKI-sensitive cells, thus resulting in overall inhibition of clonal evolution within the tumor cell mass population. Sorafenib is active against multiple mechanisms of resistance/tolerance to EGFR-TKIs and its effects depend on early inhibition of MAPK interacting kinase (MNK) activity and signal transducer and activator of transcription 3 (STAT3) phosphorylation, and later down-regulation of MCL1 and EGFR. Using several xenograft and allograft models to recapitulate different mechanisms and kinetics of acquired resistance, we show that the sorafenib-EGFR-TKI combination can substantially delay tumor growth and promote the recruitment of inflammatory cells. Together, our findings indicate that sorafenib can substantially prolong the response to EGFR-TKIs by targeting NSCLC capacity to adapt to treatment through the emergence of resistant cells.

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

Rationale Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphatics in non-ischemic cardiomyopathy induced by pressure-overload remains to be determined. Objective Investigate cardiac lymphangiogenesis following transverse aortic constriction (TAC) in adult male or female C57Bl/6J or Balb/c mice, and in patients with end-stage HF. Methods & Result Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, edema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis, respectively. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. The gender- and strain-dependent mouse cardiac hypertrophic response to TAC, especially increased ventricular wall stress, led to lymphatic expansion in the heart. Our experimental findings that ventricular dilation triggered cardiac lymphangiogenesis was mirrored by observations in clinical HF samples, with increased lymphatic density found in patients with dilated cardiomyopathy. Surprisingly, the striking lymphangiogenesis observed post-TAC in Balb/c mice, linked to increased cardiac Vegfc, did not suffice to resolve myocardial edema, and animals progressed to dilated cardiomyopathy and HF. Conversely, selective inhibition of the essentially Vegfd-driven capillary lymphangiogenesis observed post-TAC in male C57Bl/6J mice did not significantly aggravate cardiac edema. However, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, while the TAC-triggered development of interstitial cardiac fibrosis was unaffected by anti-VEGFR3, inhibition of lymphangiogenesis increased perivascular fibrosis and accelerated the development of left ventricular dilation and cardiac dysfunction. Conclusions We demonstrate for the first time that endogenous cardiac lymphangiogenesis limits pressure-overload-induced cardiac inflammation and perivascular fibrosis, thus delaying HF development. While these findings remain to be confirmed in a larger study of HF patients, we propose that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

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

During corticogenesis, projection neurons migrate along the radial glial axis to form cortical layers, the alteration of which is associated with functional deficits in adulthood. As byproducts of cell metabolism, reactive oxygen species act as second messengers to contribute to neurodevelopment; however, free radical excess may impede this process. SELENOT is a thioredoxin-like enzyme of the endoplasmic reticulum abundantly expressed during embryogenesis whose gene disruption in the brain leads to neuroblast cell demise due to increased free radical levels. To determine the potential contribution of SELENOT to the establishment of cortical networks, we analyzed first its expression profile in the neocortex at different stages of development. These studies revealed the widespread expression of SELENOT in all cortical layers, and its continous increase throughout mouse lifespan. In addition, we disrupted the SELENOT gene in the cortex using in utero electroporation and Nes-Cre/lox knockout. SELENOT deficiency altered neuroblast migration polarity, at the level of radial scaffolding, and projection neuron positionning. These results indicate that SELENOT plays a crucial role during neurodevelopment by sustaining projection neuron migration.

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

Metabolic syndrome (MetS) is associated with Heart Failure with Preserved Ejection Fraction (HFpEF), characterized by diastolic dysfunction, notably affecting elderly menopausal women. To improve treatment strategies, deepened pathophysiological understanding of potential gender-specific mechanisms contributing to ventricular stiffening is needed. We hypothesized that lymphatic alterations in the heart may contribute to cardiac inflammation and edema promoting cardiac fibrosis and dysfunction in a gender-specific manner. We investigated cardiovascular function by echocardiography, hemodynamic analyses, MRI, ex vivo coronary arteriography, and cardiac remodeling and inflammation by immunohistochemistry and flow cytometry in male and ovariectomized (ovx.) female obese Zucker (fa/fa) rats and their lean controls up to 24-weeks of age. Whereas body weight gain, insulin resistance, and diastolic dysfunction were similar in 24-weeks-old obese animals of both genders, only male obese rats displayed coronary endothelial dysfunction and reduced cardiac perfusion as compared to lean animals. In contrast, only female ovx. obese rats displayed significant cardiac hypertrophy as well as remodeling and rarefaction of cardiac lymphatics accompanied by persistent low-grade cardiac inflammation at both 14- and 24-weeks of age. The cardiac infiltration was characterized by increased levels of B lymphocytes, and both pro-inflammatory (M1) and non-classical (M2-like) monocytes, as well as increased frequency of M1 type macrophages. The monocytosis was linked to increased cardiac expression of CCL2. Studies are ongoing to determine chemokine expression changes in coronary endothelial cells. In conclusion, our data show that gender-specific mechanisms involving cardiac lymphatics may underlie development of MetS-related diastolic dysfunction, suggesting novel therapeutic targets to prevent HFpEF in patients with MetS.

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

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

Working on catecholamine systems for years, the neuropharmacologist Arvid Carlsson has made a number of important and pioneering discoveries, which have highlighted the key role of these neuronal and peripheral neurotransmitters in brain functions and adrenal regulations. Since then, major advances have been made concerning the distribution of the catecholaminergic systems in particular by studying their rate-limiting enzyme, tyrosine hydroxylase (TH). Recently new methods of tissue transparency coupled with in toto immununostaining and three-dimensional (3D) imaging technologies allow to precisely map TH immunoreactive pathways in the mouse brain and adrenal glands. High magnification images and movies obtained with combined technologies (iDISCO+ and light-sheet microscopy) are presented in this review dedicated to the pioneer work of Arvid Carlsson and his collaborators.