Search

Sleep is crucial for healthy cognition, including memory. The two main phases of sleep, REM (rapid eye movement) and non-REM sleep, are associated with characteristic electrophysiological patterns that are recorded using surface and intracranial electrodes. These patterns include sharp-wave ripples, cortical slow oscillations, delta waves, and spindles during non-REM sleep and theta oscillations during REM sleep. They reflect the precisely timed activity of underlying neural circuits. Here, we review how these electrical signatures have been guiding our understanding of the circuits and processes sustaining memory consolidation during sleep, focusing on hippocampal theta oscillations and sharp-wave ripples and how they coordinate with cortical patterns. Finally, we highlight how these brain patterns could also sustain sleep-dependent homeostatic processes and evoke several potential future directions for research on the memory function of sleep., [Figure: see text].

International audience; Aspergillus fumigatus is a human-pathogenic mold that extracts nutrients from the environment or from host tissues by secreting hydrolytic enzymes. The ability of A. fumigatus to adjust secretion levels in proportion to demand relies on the assistance of the unfolded protein response (UPR), an adaptive stress response pathway that regulates the unique protein-folding environment of the endoplasmic reticulum (ER). The P5-type ATPase Spf1 has recently been implicated in a novel mechanism of ER homeostasis that involves correcting errors in ER-membrane protein targeting. However, the contribution of this protein to the biology of A. fumigatus is unknown. Here, we employed a gene knockout and RNA sequencing strategy to determine the functional role of the A. fumigatus gene coding for the orthologous P5 ATPase SpfA. The data reveal that the spfA gene is induced by ER stress in a UPR-dependent manner. In the absence of spfA, the A. fumigatus transcriptome shifts toward a profile of altered redox and lipid balance, in addition to a signature of ER stress that includes srcA, encoding a second P-type ATPase in the ER. A ΔspfA deletion mutant showed increased sensitivity to ER stress, oxidative stress, and antifungal drugs that target the cell wall or plasma membrane. The combined loss of spfA and srcA exacerbated these phenotypes and attenuated virulence in two animal infection models. These findings demonstrate that the ER-resident ATPases SpfA and SrcA act jointly to support diverse adaptive functions of the ER that are necessary for fitness in the host environment.IMPORTANCE The fungal UPR is an adaptive signaling pathway in the ER that buffers fluctuations in ER stress but also serves as a virulence regulatory hub in species of pathogenic fungi that rely on secretory pathway homeostasis for pathogenicity. This study demonstrates that the gene encoding the ER-localized P5-type ATPase SpfA is a downstream target of the UPR in the pathogenic mold A. fumigatus and that it works together with a second ER-localized P-type ATPase, SrcA, to support ER homeostasis, oxidative stress resistance, susceptibility to antifungal drugs, and virulence of A. fumigatus.

International audience; Pulmonary arterial hypertension (PAH) is a severe cardiovascular disease that is caused by the progressive occlusion of the distal pulmonary arteries, eventually leading to right heart failure and death. Almost 40% of patients with PAH are iron deficient. Although widely studied, the mechanisms linking between PAH and iron deficiency remain unclear. Here we review the mechanisms regulating iron homeostasis and the preclinical and clinical data available on iron deficiency in PAH. Then we discuss the potential implications of iron deficiency on the development and management of PAH.

International audience; With the aging of the general population, there is an increasing need for bone defect repair, prompting the development of reliable alternatives to autologous bone grafting, without the usually associated major drawbacks (i.e., limited volume and severe postoperative pain). Given the crucial role that miRNAs appear to have in bone tissue physiopathology, exploring their potential has recently garnered increased interest. In this review, we first describe the involvement of miRNAs in bone metabolism, and then focus on their potential therapeutic applications (as bone biomarkers and molecular targets). We also highlight the as yet unsolved biological (i.e., off-target effects) and technological (i.e., specific delivering) challenges associated with their use.

International audience; Iron is essential for retinal function but contributes to oxidative stress-mediated degeneration. Iron retinal homeostasis is highly regulated and transferrin (Tf), a potent iron chelator, is endogenously secreted by retinal cells. In this study, therapeutic potential of a local Tf delivery was evaluated in animal models of retinal degeneration. After intravitreal injection, Tf spread rapidly within the retina and accumulated in photoreceptors and retinal pigment epithelium, before reaching the blood circulation. Tf injected in the vitreous prior and, to a lesser extent, after light-induced retinal degeneration, efficiently protected the retina histology and function. We found an association between Tf treatment and the modulation of iron homeostasis resulting in a decrease of iron content and oxidative stress marker. The immunomodulation function of Tf could be seen through a reduction in macrophage/microglial activation as well as modulated inflammation responses. In a mouse model of hemochromatosis, Tf had the capacity to clear abnormal iron accumulation from retinas. And in the slow P23H rat model of retinal degeneration, a sustained release of Tf in the vitreous via non-viral gene therapy efficently slowed-down the photoreceptors death and preserved their function. These results clearly demonstrate the synergistic neuroprotective roles of Tf against retinal degeneration and allow identify Tf as an innovative and not toxic therapy for retinal diseases associated with oxidative stress.

International audience; MicroRNAs (miRNAs) are evolutionarily conserved small regulatory RNAs that participate in the adjustment of many, if not all, fundamental biological processes. Molecular mechanisms involved in miRNA biogenesis and mode of action have been elucidated in the past two decades. Similar to many cellular pathways, miRNA processing and function can be globally or specifically regulated at several levels and by numerous proteins and RNAs. Given their role as fine-tuning molecules, it is essential for miRNA expression to be tightly regulated in order to maintain cellular homeostasis. Here, we review our current knowledge of the first step of their maturation occurring in the nucleus and how it can be specifically and dynamically modulated.

International audience; Microglia are the resident macrophages of the central nervous system (CNS). Gene expression profiling has identified Sall1, which encodes a transcriptional regulator, as a microglial signature gene. We found that Sall1 was expressed by microglia but not by other members of the mononuclear phagocyte system or by other CNS-resident cells. Using Sall1 for microglia-specific gene targeting, we found that the cytokine receptor CSF1R was involved in the maintenance of adult microglia and that the receptor for the cytokine TGF-β suppressed activation of microglia. We then used the microglia-specific expression of Sall1 to inducibly inactivate the murine Sall1 locus in vivo, which resulted in the conversion of microglia from resting tissue macrophages into inflammatory phagocytes, leading to altered neurogenesis and disturbed tissue homeostasis. Collectively, our results show that transcriptional regulation by Sall1 maintains microglial identity and physiological properties in the CNS and allows microglia-specific manipulation in vivo.

Excitability differs among muscle fibers and undergoes continuous changes during development and growth, yet the neuromuscular synapse maintains a remarkable fidelity of execution. Here we show in two evolutionarily distant vertebrates (Xenopus laevis cell culture and mouse nerve-muscle ex-vivo) that the skeletal muscle cell constantly senses, through two identified calcium signals, synaptic events and their efficacy in eliciting spikes. These sensors trigger retrograde signal(s) that control presynaptic neurotransmitter release, resulting in synaptic potentiation or depression. In the absence of spikes, synaptic events trigger potentiation. Once the synapse is sufficiently strong to initiate spiking, the occurrence of these spikes activates a negative retrograde feedback. These opposing signals dynamically balance the synapse in order to continuously adjust neurotransmitter release to a level matching current muscle cell excitability. DOI: http://dx.doi.org/10.7554/eLife.12190.001, eLife digest Nerve cells communicate with each other, and with targets such as muscle cells, at junctions called synapses. The nerve cell before the synapses releases a chemical called a neurotransmitter, which binds to receptors on the cell after the synapses. However, the first cell cannot determine by itself whether it is releasing the correct amount of neurotransmitter to activate its partner. For this, it requires feedback from the second cell. This feedback is particularly important at synapses between nerve cells and muscle cells, which are known as neuromuscular junctions. The likelihood that a given amount of transmitter will activate a muscle cell can vary with age and after exercise. Muscle cells must therefore be able to instruct their nerve cell partners to increase or decrease neurotransmitter release to accommodate these changes. Ouanounou et al. have now identified the mechanism by which muscle cells determine whether nerve cells are releasing an appropriate amount of neurotransmitter. Experiments in two distantly related animals – mice and embryos from a frog called Xenopus – revealed that muscle cells use two calcium-based signals. The first is the flow of calcium ions into the muscle cell in response to binding of neurotransmitter to receptors at the synapses: this tells the muscle cell how active the nerve cell is. The second is the release of calcium ions from internal stores inside the muscle cell: this occurs whenever neurotransmitter release is sufficient to activate the muscle cell. In response to the first calcium signal, the muscle cell sends positive feedback to the neuron, telling it to increase neurotransmitter release further. In response to the second signal, the muscle cell sends negative feedback to reduce neurotransmitter release. Thus, when neurotransmitter release is not enough to activate the muscle, positive feedback dominates and neurotransmitter release increases. However, when the muscle is activated, the two types of feedback act in balance to maintain efficient communication across the synapse. The next steps are to identify the cell signaling cascades that are mobilized by the two calcium signals, including the specific molecule (or molecules) that regulate neurotransmitter release. DOI: http://dx.doi.org/10.7554/eLife.12190.002

Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer’s diseases. Here, we identify tryptophan 2,3-dioxygenase ( tdo-2 ), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra l -tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases.

Development of Myeloid Immune Cells As leukocytes develop to maturity, they proceed through an array of phenotypically distinct intermediates. For T and B lymphocyte populations, the different developmental stages, anatomical locations, and cell signals required for progression are well established. However, until recently, much less has been known about how development proceeds in the myeloid lineage, which includes monocytes, macrophages, and dendritic cells. Geissmann et al. (p. 656 ) review our current understanding of myeloid lineage development and describe the developmental pathways and cues that drive differentiation.

Objective— Bexarotene (Targretin) is a clinically used antitumoral agent which exerts its action through binding to and activation of the retinoid-X-receptor (RXR). The most frequent side-effect of bexarotene administration is an increase in plasma triglycerides, an independent risk factor of cardiovascular disease. The molecular mechanism behind this hypertriglyceridemia remains poorly understood. Methods and Results— Using wild-type and LXRα/β-deficient mice, we show here that bexarotene induces hypertriglyceridemia and activates hepatic LXR-target genes of lipogenesis in an LXR-dependent manner, hence exerting a permissive effect on RXR/LXR heterodimers. Interestingly, RNA analysis and Chromatin Immunoprecipitation assays performed in the liver reveal that the in vivo permissive effect of bexarotene on the RXR/LXR heterodimer is restricted to lipogenic genes without modulation of genes controlling cholesterol homeostasis. Conclusion— These findings demonstrate that the hypertriglyceridemic action of bexarotene occurs via the RXR/LXR heterodimer and show that RXR heterodimers can act with a selective permissivity on target genes of specific metabolic pathways in the liver.

International audience; Disruption of iron homeostasis at the levels of intestinal absorption or erythropoiesis contributes to cadmium toxicity. Cellular iron homeostasis in metazoans is maintained by the iron regulatory proteins (IRPs) that regulate the synthesis of proteins involved in the transport, use, and storage of iron. The effect of cadmium intoxication on this regulatory system has been investigated in a cellular model of human epithelium. Cadmium exposure of HeLa cells did not activate the IRPs; rather, the amount of these proteins relative to that of housekeeping proteins decreased. Accordingly, the transferrin receptor mRNA level decreased upon cadmium insult. In a more integrated investigation, separate groups of mice had free access to different doses of cadmium in drinking water for 3 weeks. Cadmium accumulated in all analyzed organs, but its concentration in mouse tissues did not correlate with changes of the activity of the IRPs. The intoxicated mice did not show any sign of anemia, indicating that iron homeostasis was not immediately disrupted after the onset of cadmium accumulation. These data establish that cadmium destabilizes IRPs in mammalian cells, but that iron imbalance is not an early event of cadmium intoxication.

International audience; The maturation of cytosolic iron-sulfur (Fe/S) proteins in mammalian cells requires components of the mitochondrial iron-sulfur cluster assembly and export machineries. Little is known about the cytosolic components that may facilitate the assembly process. Here, we identified the cytosolic soluble P-loop NTPase termed huNbp35 (also known as Nubp1) as an Fe/S protein, and we defined its role in the maturation of Fe/S proteins in HeLa cells. Depletion of huNbp35 by RNA interference decreased cell growth considerably, indicating its essential function. The deficiency in huNbp35 was associated with an impaired maturation of the cytosolic Fe/S proteins glutamine phosphoribosylpyrophosphate amidotransferase and iron regulatory protein 1 (IRP1), while mitochondrial Fe/S proteins remained intact. Consequently, huNbp35 is specifically involved in the formation of extramitochondrial Fe/S proteins. The impaired maturation of IRP1 upon huNbp35 depletion had profound consequences for cellular iron metabolism, leading to decreased cellular H-ferritin, increased transferrin receptor levels, and higher transferrin uptake. These properties clearly distinguished huNbp35 from its yeast counterpart Nbp35, which is essential for cytosolic-nuclear Fe/S protein assembly but plays no role in iron regulation. huNbp35 formed a complex with its close homologue huCfd1 (also known as Nubp2) in vivo, suggesting the existence of a heteromeric P-loop NTPase complex that is required for both cytosolic Fe/S protein assembly and cellular iron homeostasis.

International audience; Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal-intact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium- and potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium- and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum- and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium- and potassium-dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. Conclusion: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium- and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium- and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids. (HEPATOLOGY 2007;46:173–179.)

International audience; In bacteria-host interactions, competition for iron is critical for the outcome of the infection. As a result of its redox properties, this metal is essential for the growth and proliferation of most living organisms, including pathogenic bacteria. This metal is also potentially toxic, making the precise maintenance of iron homeostasis necessary for survival. Iron acquisition and storage control is mediated in most bacteria by the global ferric uptake regulator (Fur) and iron-responsive small regulatory non-coding RNAs (RyhB in the model organism Escherichia coli). While the role of these regulators in iron homeostasis is well documented in both pathogenic and non-pathogenic bacteria, many recent studies also demonstrate that these regulators are involved in the virulence of pathogenic bacteria. By sensing iron availability in the environment, Fur and RyhB are able to regulate, either directly or indirectly via other transcriptional regulators or modulation of intracellular iron concentration, many virulence determinants of pathogenic bacteria. Iron is thus both a nutritional and regulatory element, allowing bacteria to adapt to various host environments by adjusting expression of virulence factors. In this review, we present evidences that Fur and RyhB are the major regulators of this adaptation, as they are involved in diverse functions ranging from iron homeostasis to regulation of virulence by mediating key pathogen responses such as invasion of eukaryotic cells, toxin production, motility, quorum sensing, stress resistance or biofilm formation. Therefore, Fur and RyhB play a major role in regulating an adaptative response during bacterial infections, making them important targets in the fight against pathogenic bacteria.

International audience; Kupffer cells, the phagocytes of fetal origin that line the liver sinusoids, are key contributors of host defense against enteroinvasive bacteria. Here, we found that infection by Listeria monocytogenes induced the early necroptotic death of Kupffer cells, which was followed by monocyte recruitment and an anti-bacterial type 1 inflammatory response. Kupffer cell death also triggered a type 2 response that involved the hepatocyte-derived alarmin interleukin-33 (IL-33) and basophil-derived interleukin-4 (IL-4). This led to the alternative activation of the monocyte-derived macrophages recruited to the liver, which thereby replaced ablated Kupffer cells and restored liver homeostasis. Kupffer cell death is therefore a key signal orchestrating type 1 microbicidal inflammation and type-2-mediated liver repair upon infection. This indicates that beyond the classical dichotomy of type 1 and type 2 responses, these responses can develop sequentially in the context of a bacterial infection and act interdependently, orchestrating liver immune responses and return to homeostasis, respectively.

We previously showed that pancreatic β-cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now provide evidence that the endogenous protein inhibitor of nNOS (PIN) is expressed in rat pancreatic islets and INS-1 cells. Double-immunofluorescence studies showed a colocalization of PIN with both nNOS and myosin Va in insulin-secreting β-cells. Electron microscopy studies confirmed that PIN is mainly associated with insulin secretory granules and colocated with nNOS in the latter. In addition, PIN overexpression in INS-1 cells enhanced glucose-induced insulin secretion, which is only partly reversed by addition of an NO donor, sodium nitroprusside (SNP), and unaffected by the inhibitor of cytochrome c reductase activity, miconazole. In contrast, the pharmacological inhibitor of nNOS, Nω-nitro-l-arginine methyl ester, amplified glucose-induced insulin secretion, an effect insensitive to SNP but completely normalized by the addition of miconazole. Thus, PIN insulinotropic effect could be related to its colocalization with the actin-based molecular motor myosin Va and as such be implicated in the physiological regulation of glucose-induced insulin secretion at the level of the exocytotic machinery.

International audience; Bile acids (BAs), a group of structurally diverse molecules that are primarily synthesized in the liver from cholesterol, are the chief components of bile. Besides their well-established roles in dietary lipid absorption and cholesterol homeostasis, it has recently emerged that BAs are also signaling molecules, with systemic endocrine functions. BAs activate mitogen-activated protein kinase pathways, are ligands for the G-protein-coupled receptor TGR5, and activate nuclear hormone receptors such as farnesoid X receptor alpha. Through activation of these diverse signaling pathways, BAs can regulate their own enterohepatic circulation, but also triglyceride, cholesterol, energy, and glucose homeostasis. Thus, BA-controlled signaling pathways are promising novel drug targets to treat common metabolic diseases, such as obesity, type II diabetes, hyperlipidemia, and atherosclerosis.

International audience; With the aim of finding important mediators of muscle atrophy, we cloned SMHS1, a novel gene that was found to be upregulated in rat soleus muscle atrophied by restriction of activity. The SMHS1 amino acid sequence shares 65% similarity with RTP801-which is a cellular stress response protein regulated by HIF-1-but SMHS1 expression was demonstrated to be independent of HIF-1. SMHS1 was found to be mainly expressed in skeletal muscle, and comparisons of its expression in atrophied versus hypertrophied muscles and in oxidative versus glycolytic muscles suggested that SMHS1 contributes to the muscle energy metabolism phenotypes.

NCB5OR is a highly conserved NAD(P)H reductase that contains a cytochrome b5-like domain at the N terminus and a cytochrome b5 reductase-like domain at the C terminus. The enzyme is located in the endoplasmic reticulum (ER) and is widely expressed in organs and tissues. Targeted inactivation of this gene in mice has no impact on embryonic or fetal viability. At 4 weeks of age, Ncb5or -/- mice have normal blood glucose levels but impaired glucose tolerance. Isolated Ncb5or -/- islets have markedly impaired glucose- or arginine-stimulated insulin secretion. By 7 weeks of age, these mice develop severe hyperglycemia with markedly decreased serum insulin levels and nearly normal insulin tolerance. As the animals age, there is a progressive loss of beta cells in pancreatic islets, but there is no loss of alpha, delta, or PP cells. Electron microscopy reveals degranulation of beta cells and hypertrophic and hyperplastic mitochondria, some of which contain electron dense inclusions. Four-week-old Ncb5or -/- mice have enhanced sensitivity to the diabetogenic agent streptozotocin. NCB5OR appears to play a critical role in protecting pancreatic beta cells against oxidant stress.

We show that the lymphoid hyperplasia observed in IL-2Rα- and IL-2-deficient mice is due to the lack of a population of regulatory cells essential for CD4 T cell homeostasis. In chimeras reconstituted with bone marrow cells from IL-2Rα-deficient donors, restitution of a population of CD25+CD4+ T cells prevents the chaotic accumulation of lymphoid cells, and rescues the mice from autoimmune disease and death. The reintroduction of IL-2-producing cells in IL-2-deficient chimeras establishes a population of CD25+CD4+ T cells, and restores the peripheral lymphoid compartments to normal. The CD25+CD4+ T cells regulated selectively the number of naive CD4+ T cells transferred into T cell-deficient hosts. The CD25+CD4+/naive CD4 T cell ratio and the sequence of cell transfer determines the homeostatic plateau of CD4+ T cells. Overall, our findings demonstrate that IL-2Rα is an absolute requirement for the development of the regulatory CD25+CD4+ T cells that control peripheral CD4 T cell homeostasis, while IL-2 is required for establishing a sizeable population of these cells in the peripheral pools.

International audience; It is well known that the physiological impact imposed by events or behaviors displayed during the waking period determines the way organisms sleep. Among the situations known to affect sleep both in its duration and quality, stress has been widely studied and it is now admitted that its effects on sleep architecture depend on several factors specific to the stressor or the individual itself. Although numerous reports have highlighted the prominent role of the circadian cycle in the physiological, endocrine and behavioral consequences of restraint stress, a possible circadian influence in the effects of stress on the sleep-wake cycle has never been studied. Thus the present study was designed to compare the effects on sleep of a 1 h-lasting restraint stress applied at light onset to those observed after the same stressor was applied at light offset. We report that in both conditions stress induced a marked paradoxical sleep increase, whereas wakefulness displayed a moderate decrease and slow wave sleep a moderate augmentation. Although the effects of stress at lights on were of similar magnitude than those of stress at lights off, important differences in the sleep rebound latencies were observed: whatever the time of day the stress was applied, its effects on sleep always occurred during the dark period. This result thus shows that restraint stress could be efficiently used to study the interaction between the circadian and homeostatic components of sleep regulation.

International audience; The B cell-activating factor of the tumor-necrosis factor family (BAFF) plays a dominant role in the B cell homeostasis. By rescuing autoreactive B cells, excessive BAFF favors the development of autoimmune diseases. Given the numbers of variants of this B cell-specific cytokine, caution must be exercized when determining its serum level. Alternate splice isoforms, such as Δ3 BAFF and Δ4 BAFF, have been identified. They raise the possibility that their overproduction impact the synthesis of full-length BAFF.

NK cell differentiation mainly occurs in the bone marrow (BM) where a critical role in the regulation of developing lymphocyte distribution is played by members of the chemokine receptor family. In mouse, the chemokine receptor CX3CR1 identifies a late stage of NK cell development characterized by decreased effector functions and expression of the inhibitory receptor KLRG1. The role of CX3CR1 in the regulation of differentiation and positioning of NK cell subsets in the BM is not known. In this study, we found that CX3CR1 deficiency leads to accumulation of KLRG1+ NK cells in BM during steady-state conditions. The NK cell subset that expresses the receptor in wild-type mice was expanded in several tissues of CX3CR1-deficient mice, and NK cell degranulation in response to sensitive target cell stimulation was enhanced, suggesting a regulatory role of CX3CR1 in NK cell positioning and differentiation in BM. Indeed, the observed NK cell expansion was not due to altered turnover rate, whereas it was associated with preferential accumulation in the BM parenchyma. In addition, a role of CX3CR1 in NK cell trafficking from BM and spleen was evidenced also during inflammation, as CX3CR1-deficient NK cells were more prompt to exit the BM and did not decrease in spleen in response to polyinosinic-polycytidylic acid–promoted hepatitis. Overall, our results evidenced a relevant role of CX3CR1 in the regulation of NK cell subset exit from BM during homeostasis, and suggest that defect in the CX3CR1/CX3CL1 axis alters NK cell trafficking and functional response during inflammatory conditions.

International audience; Prenatal stress impairs activity of the hypothalamo-pituitary-adrenal (HPA) axis in response to stress in adult offspring. So far, very few data are available on the effects of prenatal stress on circadian functioning of the HPA axis. Here, we studied the effects of prenatal stress on the circadian rhythm of corticosterone secretion in male and female adult rats. To evaluate the effects of prenatal stress on various regulatory components of corticosterone secretion, we also assessed the diurnal fluctuation of adrenocorticotropin, total and free corticosterone levels, and hippocampal corticosteroid receptors. Finally, in the search of possible maternal factors, we studied the effects of repeated restraint stress on the pattern of corticosterone secretion in pregnant female rats. Results demonstrate that prenatal stress induced higher levels of total and free corticosterone secretion at the end of the light period in both males and females, and hypercorticism over the entire diurnal cycle in females. No diurnal fluctuation of adrenocorticotropin was observed in any group studied. The effects of prenatal stress on corticosterone secretion could be mediated, at least in part, by a reduction in corticosteroid receptors at specific times of day. Results also show that prepartal stress alters the pattern of corticosterone secretion in pregnant females. Those data indicate that prenatally stressed rats exhibit an altered temporal functioning of the HPA axis, which, taken together with their abnormal response to stress, reinforces the idea of a general homeostatic dysfunction in those animals.

Although earlier, seminal studies demonstrated that the gut per se has the intrinsic ability to regulate the rates of iron absorption, the spotlight in the past decade has been placed on the systemic regulation of iron homeostasis by the hepatic hormone hepcidin and the molecular mechanisms that regulate its expression. Recently, however, attention has returned to the gut based on the finding that hypoxia inducible factor-2 (HIF-2α) regulates the expression of key genes that contribute to iron absorption. Here we review the current understanding of the molecular mechanisms that regulate iron homeostasis in the gut by focusing on the role of HIF-2 under physiological steady-state conditions and in the pathogenesis of iron-related diseases. We also discuss implications for adapting HIF-2–based therapeutic strategies in iron-related pathological conditions.

Objective— Endosomal signature phospholipid bis(monoacylglycero)phosphate (BMP) has been involved in the regulation of cellular cholesterol homeostasis. Accumulation of BMP is a hallmark of lipid storage disorders and was recently reported as a noticeable feature of oxidized low-density lipoprotein–laden macrophages. This study was designed to delineate the consequences of macrophage BMP accumulation on intracellular cholesterol distribution, metabolism, and efflux and to unravel the underlying molecular mechanisms. Approach and Results— We have developed an experimental design to specifically increase BMP content in RAW 264.7 macrophages. After BMP accumulation, cell cholesterol distribution was markedly altered, despite no change in low-density lipoprotein uptake and hydrolysis, cholesterol esterification, or total cell cholesterol content. The expression of cholesterol-regulated genes sterol regulatory element–binding protein 2 and hydroxymethylglutaryl-coenzyme A reductase was decreased by 40%, indicative of an increase of endoplasmic reticulum–associated cholesterol. Cholesterol delivery to plasma membrane was reduced as evidenced by the 20% decrease of efflux by cyclodextrin. Functionally, BMP accumulation reduced cholesterol efflux to both apolipoprotein A1 and high-density lipoprotein by 40% and correlated with a 40% decrease in mRNA contents of ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, and liver-X receptor α and β. Foam cell formation induced by oxidized low-density lipoprotein exposure was exacerbated in BMP-enriched cells. Conclusions— The present work shows for the first time a strong functional link between BMP and cholesterol-regulating genes involved in both intracellular metabolism and efflux. We propose that accumulation of cellular BMP might contribute to the deregulation of cholesterol homeostasis in atheromatous macrophages.

International audience; Patrolling Ly6C(-) monocytes are blood-circulating cells that play a role in inflammation and in the defense against pathogens. Here, we show that similar to natural killer (NK) cells, patrolling monocytes express high levels of S1PR5, a G-coupled receptor for sphingosine-1 phosphate. We found that S1pr5(-/-) mice lack peripheral Ly6C(-) monocytes but have a normal number of these cells in the bone marrow (BM). Various lines of evidence exclude a direct contribution of S1PR5 in the survival of Ly6C(-) monocytes at the periphery. Rather, our data support a role for S1PR5 in the egress of Ly6C(-) monocytes from the BM. In particular, we observed a reduced frequency of patrolling monocytes in BM sinusoids of S1PR5 KO mice. Unexpectedly, S1P was not a chemoattractant for patrolling monocytes and had no significant effect on their viability in vitro. Moreover, the disruption of S1P gradients in vivo did not alter Ly6C(-) monocyte trafficking and viability. These data suggest that S1PR5 regulates the trafficking of monocytes via a mechanism independent of S1P gradients.

International audience; Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross-linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3(ECD) cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3(ECD) in the brain vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could contribute to mutant Notch3(ECD) toxicity by impairing extracellular matrix homeostasis in small vessels.

Lymphopenia induces T cells to undergo cell divisions as part of a homeostatic response mechanism. The clonal response to lymphopenia is extremely diverse, and it is unknown whether this heterogeneity represents distinct mechanisms of cell-cycle control or whether a common mechanism can account for the diversity. We addressed this question by combining in vivo and mathematical modeling of lymphopenia-induced proliferation (LIP) of two distinct T cell clonotypes. OT-I T cells undergo rapid LIP accompanied by differentiation that superficially resembles Ag-induced proliferation, whereas F5 T cells divide slowly and remain naive. Both F5 and OT-I LIP responses were most accurately described by a single stochastic division model where the rate of cell division was exponentially decreased with increasing cell numbers. The model successfully identified key biological parameters of the response and accurately predicted the homeostatic set point of each clone. Significantly, the model was successful in predicting interclonal competition between OT-I and F5 T cells, consistent with competition for the same resource(s) required for homeostatic proliferation. Our results show that diverse and heterogenous clonal T cell responses can be accounted for by a single common model of homeostasis.

International audience; Proteinase-activated receptor 2 (PAR2) is a G protein-coupled membrane receptor that is activated upon cleavage of its extracellular N-terminal domain by trypsin and related proteases. PAR2 is expressed in kidney collecting ducts, a main site of control of Na(+) and K(+) homeostasis, but its function remains unknown. We evaluated whether and how PAR2 might control electrolyte transport in collecting ducts, and thereby participate in the regulation of blood pressure and plasma K(+) concentration. PAR2 is expressed at the basolateral border of principal and intercalated cells of the collecting duct where it inhibits K(+) secretion and stimulates Na(+) reabsorption, respectively. Invalidation of PAR2 gene impairs the ability of the kidney to control Na(+) and K(+) balance and promotes hypotension and hypokalemia in response to Na(+) and K(+) depletion, respectively. This study not only reveals a new role of proteases in the control of blood pressure and plasma potassium level, but it also identifies a second membrane receptor, after angiotensin 2 receptor, that differentially controls sodium reabsorption and potassium secretion in the late distal tubule. Conversely to angiotensin 2 receptor, PAR2 is involved in the regulation of sodium and potassium balance in the context of either stimulation or nonstimulation of the renin/angiotensin/aldosterone system. Therefore PAR2 appears not only as a new actor of the aldosterone paradox, but also as an aldosterone-independent modulator of blood pressure and plasma potassium.

International audience; CLIPR-59 is a new member of the cytoplasmic linker proteins (CLIP) family mainly localized to the trans-Golgi network. We show here that Clipr-59 expression in mice is restricted to specific pools of neurons, in particular motoneurons (MNs), and progressively increases from embryonic day 12.5 (E12.5) until the first postnatal days. We generated a Clipr-59 knockout mouse model that presents perinatal lethality due to respiratory defects. Physiological experiments revealed that this altered innervation prevents the normal nerve-elicited contraction of the mutant diaphragm that is reduced both in amplitude and fatigue-resistance at E18.5, despite unaffected functional muscular contractility. Innervation of the mutant diaphragm is not altered until E15.5, but is then partially lost in the most distal parts of the muscle. Ultrastructural observations of neuromuscular junctions (NMJs) in the distal region of the diaphragm reveal a normal organization, but a lower density of nerve terminals capped by terminal Schwann cells in E18.5 mutant when compared with control embryos. Similar defects in NMJ stability, with a hierarchy of severity along the caudo-rostral axis, are also observed in other muscles innervated by facial and spinal MNs in Clipr-59 mutant mice. Clipr-59 deficiency therefore affects axon maintenance but not axon guidance toward muscle targets. Thus, CLIPR-59 is involved in the stabilization of specific motor axons at the NMJ during mouse late embryogenesis and its role is crucial for mouse perinatal development.

Hematopoietic stem cells self-renew for life to guarantee the continuous supply of all blood cell lineages. Here we show that Poly(ADP-ribose) polymerase-2 (Parp-2) plays an essential role in hematopoietic stem/progenitor cells (HSPC) survival under steady-state conditions and in response to stress. Increased levels of cell death were observed in HSPC from untreated Parp-2-/- mice, but this deficit was compensated by increased rates of self-renewal, associated with impaired reconstitution of hematopoiesis upon serial bone marrow transplantation. Cell death after γ-irradiation correlated with an impaired capacity to repair DNA damage in the absence of Parp-2. Upon exposure to sublethal doses of γ-irradiation, Parp-2-/- mice exhibited bone marrow failure that correlated with reduced long-term repopulation potential of irradiated Parp-2-/- HSPC under competitive conditions. In line with a protective role of Parp-2 against irradiation-induced apoptosis, loss of p53 or the pro-apoptotic BH3-only protein Puma restored survival of irradiated Parp-2-/- mice, whereas loss of Noxa had no such effect. Our results show that Parp-2 plays essential roles in the surveillance of genome integrity of HSPC by orchestrating DNA repair and restraining p53-induced and Puma-mediated apoptosis. The data may affect the design of drugs targeting Parp proteins and the improvement of radiotherapy-based therapeutic strategies.

International audience; The control of fat cell lipolysis by the catecholamines involves at least four different adrenoceptor subtypes; three beta (beta 1-, beta 2-, and beta 3-ARs) and one alpha 2-adrenoceptor (alpha 2-AR). The physiological importance of the beta- and alpha 2A-ARs varies according to the species, the sex, the age, the anatomical location of fat deposits and the degree of obesity in humans and animals. The physiological amines operate through differential recruitment of these sites on the basis of their relative affinities. This point has been assessed by in vitro studies and has partly been confirmed in in vivo experiments using selected alpha/beta-AR antagonists and in situ microdialysis. The affinity of the beta 3-AR for catecholamines is less than that of the classical beta 1- and beta 2-ARs in the various species investigated. Conversely, it is the alpha 2-AR which exhibit the highest affinity for the physiological amines in all fat cells. The relative order of affinity of the various fat cell ARs for the physiological amines defined in binding studies and in vitro assays is alpha 2 > beta 1 > or = beta 2 > beta 3 for norepinephrine and alpha 2 > beta 2 > beta 1 > beta 3 for epinephrine. When considering differential beta-AR recruitment by catecholamines, it is the beta 1-AR which is always activated at the lowest norepinephrine levels, whatever the species, while the activation of the beta 3-AR requires higher norepinephrine levels. In addition to the differential recruitment, differential regulation by hormones could also occur for each fat cell AR subtype. The alpha 2-and beta 3-ARs are less prone to desensitization and down-regulation by comparison with the beta 1- and beta 2-AR.

Vertebrates need to maintain extracellular chloride (Cl−) concentrations to ensure the normal operation of physiological processes; the transition from aquatic to terrestrial environments necessitated the development of sophisticated mechanisms to ensure Cl−homeostasis in the face of fluctuating Cl−levels. Zebrafish calcitonin gene-related peptide (CGRP), unlike its splice variant calcitonin, does not respond to environmental Ca2+levels. This study aimed to test the hypothesis that CGRP is involved in the control of body fluid Cl−homeostasis. Acclimation to high-Cl−artificial water stimulated the mRNA expression ofcgrpand the receptor (crlr1) when compared with low-Cl−. CGRP knockdown induced upregulation of the Na+-Cl−co-transporter (ncc2b), while overexpression of CGRP resulted in the downregulation ofncc2bmRNA synthesis and a simultaneous decrease in Cl−uptake in embryos. Consistent with these findings, knockdown of eithercgrporcrlr1was found to increase the density of NCC2b-expressing cells in embryos. This is the first demonstration that CGRP acts as a hypochloremic hormone through suppressing NCC2b expression and the differentiation of NCC-expressing ionocytes. Elucidation of this novel function of CGRP in fish body fluid Cl−homeostasis promises to enhance our understanding of the related physiology in vertebrates.

International audience; Mutations of clock genes can lead to diabetes and obesity. REV-ERB alpha, a nuclear receptor involved in the circadian clockwork, has been shown to control lipid metabolism. To gain insight into the role of REV-ERB alpha in energy homeostasis in vivo, we explored daily metabolism of carbohydrates and lipids in chow-fed, unfed, or high-fat-fed Rev-erb alpha(-/-) mice and their wild-type littermates. Chow-fed Rev-erb alpha(-/-) mice displayed increased adiposity (2.5-fold) and mild hyperglycemia (similar to 10%) without insulin resistance. Indirect calorimetry indicates that chow-fed Rev-erb alpha(-/-) mice utilize more fatty acids during daytime. A 24-h nonfeeding period in Rev-erb alpha(-/-) animals favors further fatty acid mobilization at the expense of glycogen utilization and gluconeogenesis, without triggering hypoglycemia and hypothermia. High-fat feeding in Rev-erb alpha(-/-) mice amplified metabolic disturbances, including expression of lipogenic factors. Lipoprotein lipase (Lpl) gene, critical in lipid utilization/storage, is triggered in liver at night and constitutively up-regulated (similar to 2-fold) in muscle and adipose tissue of Rev-erb alpha(-/-) mice. We show that CLOCK, up-regulated (2-fold) at night in Rev-erb alpha(-/-) mice, can transactivate Lpl. Thus, overexpression of Lpl facilitates muscle fatty acid utilization and contributes to fat overload. This study demonstrates the importance of clock-driven Lpl expression in energy balance and highlights circadian disruption as a potential cause for the metabolic syndrome.

Mitochondria are dynamic organelles that play a key role in energy conversion. Optimal mitochondrial function is ensured by a quality-control system tightly coupled to fusion and fission. In this connection, mitofusin 2 (Mfn2) participates in mitochondrial fusion and undergoes repression in muscle from obese or type 2 diabetic patients. Here, we provide in vivo evidence that Mfn2 plays an essential role in metabolic homeostasis. Liver-specific ablation of Mfn2 in mice led to numerous metabolic abnormalities, characterized by glucose intolerance and enhanced hepatic gluconeogenesis. Mfn2 deficiency impaired insulin signaling in liver and muscle. Furthermore, Mfn2 deficiency was associated with endoplasmic reticulum stress, enhanced hydrogen peroxide concentration, altered reactive oxygen species handling, and active JNK. Chemical chaperones or the antioxidant N -acetylcysteine ameliorated glucose tolerance and insulin signaling in liver-specific Mfn2 KO mice. This study provides an important description of a unique unexpected role of Mfn2 coordinating mitochondria and endoplasmic reticulum function, leading to modulation of insulin signaling and glucose homeostasis in vivo.

International audience; Millions of people suffer from inflammatory diseases of the intestine, some of them potentiating gastrointestinal cancer. These gut-associated pathologies arise from imbalanced interactions between the host gut epithelia and resident or ingested microbes, interactions that are still poorly understood at the molecular level. Drosophila has been a very powerful model to study development and diseases. Its relatively simple tissue organization and sophisticated genetics are some of the advantages of using it as an experimental model to dissect gut-microbe interactions. Recent progress made in various research fields such as Drosophila microbiota composition, gut epithelium structure or gut immune reactions led us to believe that Drosophila is becoming an ad hoc model system to dissect the mechanisms that cooperate to maintain intestinal homeostasis in higher eukaryotes. It further may help us understand how an alteration of these finely tuned processes precipitates the inflammatory processes found in some inflammatory bowel diseases.

RANK and its ligand RANKL play important roles in the development and regulation of the immune system. We show that mice transgenic for Rank in hair follicles display massive postnatal growth of skin-draining lymph nodes. The proportions of hematopoietic and nonhematopoietic stromal cells and their organization are maintained, with the exception of an increase in B cell follicles. The hematopoietic cells are not activated and respond to immunization by foreign Ag and adjuvant. We demonstrate that soluble RANKL is overproduced from the transgenic hair follicles and that its neutralization normalizes lymph node size, inclusive area, and numbers of B cell follicles. Reticular fibroblastic and vascular stromal cells, important for secondary lymphoid organ formation and organization, express RANK and undergo hyperproliferation, which is abrogated by RANKL neutralization. In addition, they express higher levels of CXCL13 and CCL19 chemokines, as well as MAdCAM-1 and VCAM-1 cell-adhesion molecules. These findings highlight the importance of tissue-derived cues for secondary lymphoid organ homeostasis and identify RANKL as a key molecule for controlling the plasticity of the immune system.

International audience; Iron disorders of genetic origin are mainly composed of iron overload diseases, the most frequent being HFE-related hemochromatosis. Hepcidin deficiency underlies iron overload in HFE-hemochromatosis as well as in several other genetic iron excess disorders, such as hemojuvelin or hepcidin-related hemochromatosis and transferrin receptor 2-related hemochromatosis. Deficiency of ferroportin, the only known cellular protein iron exporter, produces iron overload in the typical form of ferroportin disease. By contrast, genetically enhanced hepcidin production, as observed in matriptase-2 deficiency, generates iron-refractory iron deficiency anemia. Diagnosis of these iron storage disorders is usually established noninvasively through combined biochemical, imaging and genetic approaches. Moreover, improved knowledge of the molecular mechanisms accounting for the variations of iron stores opens the way of novel therapeutic approaches aiming to restore normal iron homeostasis. In this review, we will summarize recent findings about these various genetic entities that have been identified owing to an exemplary interplay between clinicians and basic scientists.

International audience; OBJECTIVES: Molecular mechanisms controlling cell fate decision making in self-renewing cells are poorly understood. A previous transcriptomic study, carried out in primary avian erythroid progenitor cells (T2ECs), revealed that the gene encoding oxidosqualene cyclase (OSC/LSS), an enzyme involved in cholesterol biosynthesis, is significantly up-regulated in self-renewing cells. The aim of the present work is to understand whether this up-regulation is required for self-renewal maintenance and what are the mechanisms involved. MATERIALS AND METHODS: To investigate OSC function, we studied effects of its enzymatic activity inhibition using Ro48-8071, a specific OSC inhibitor. In addition, we completed this pharmacological approach by RNAi-mediated OSC/LSS knockdown. The study of OSC inhibition was carried out on both self-renewing and differentiating cells to observe any state-dependent effect. Results: Our data show that OSC acts both by protecting self-renewing T2EC cells from apoptosis and by blocking their differentiation program, as OSC inhibition is sufficient to trigger spontaneous commitment of self-renewing cells towards an early differentiation state. This is self-renewal specific, as OSC inhibition has no effect on erythroid progenitors that have already differentiated. CONCLUSIONS: Taken together, our results suggest that OSC/LSS expression and activity are required to maintain cell self-renewal and may be involved in the self-renewal versus differentiation/apoptosis decision making, by keeping cells in a self-renewal state.

http://www.sciencedirect.com/; International audience; Free cholesterol is the predominant form of cholesterol in the neural retina. The vertebrate neural retina exhibits its own capacity to synthesize cholesterol and meets its demand also by taking it from the circulation. Defects in cholesterol synthesis and trafficking in the neural retina has detrimental consequences on its structure and function, highlighting the crucial importance of maintaining cholesterol homeostasis in the retina. Our purpose was to give a review on the functioning of the retina, the role of cholesterol and cholesterol metabolism therein, with special emphasis on cholesterol-24S-hydroxylase (CYP46A1). Similar to the brain, the retina expresses cholesterol-24S-hydroxylase (CYP46A1) and is enriched in its metabolic product, 24S-hydroxycholesterol. We recently published that one single nucleotide polymorphism in CYP46A1 gene, designated as rs754203, was a risk factor for glaucoma. Glaucoma is the second leading cause of blindness worldwide, affecting more than 60 million people. Glaucoma is characterized by the loss of retinal ganglion cells, which show high CYP46A1 expression. These data suggest the potential involvement of CYP46A1 and 24S-hydroxycholesterol in the pathophysiology of glaucoma.

International audience; Cadmium (Cd) is a toxic metal and can induce and/or promote diseases in humans (cancer, aging diseases, kidney and bone diseases, etc.). Its toxicity involves many mechanisms including the alteration of copper (Cu) and zinc (Zn) homeostasis leading to reactive oxygen species (ROS) production, either directly or through the inhibition of antioxidant activities. Importantly, ROS can induce oxidative damages in cells. Cadmium, Cu and Zn are also able to induce glutathione (GSH) and metallothioneins (MT) synthesis in a cell-type-dependent manner. As a consequence, the effects induced by these three metals result simultaneously from the inhibition of antioxidant activities and the induction of other factors such as GSH and MT synthesis. MT levels are regulated not only by the p53 protein in a cell-type-dependent manner, or by transcription factors such as metal-responsive transcription factor 1 (MTF-1) and cellular Zn levels but also by cellular GSH level. As described in the literature, DNA damage, GSH and MT levels are sensitive biomarkers used to identify Cd-induced toxicity alone or together with Cu and Zn homeostasis alteration.

International audience; Despite the tremendous amount of data over the last 40years, lack of gap junctional intercellular communication (GJIC) or altered expression of gap junction proteins is still a lesser known 'hallmark' of cancer. Expression of astrocytic gap junction protein, connexin43 (Cx43), is often reduced in astrocytomas, the most common neoplasia of the central nervous system (CNS) in adults. Supported by a number of evidences, the global decrease of Cx43 expression appears to be advantageous for the growth of glioma cells. Although the mechanisms by which Cx43 regulates the expression levels of proteins involved in cell growth is unclear, there are evidences to suggest that it might be independent of their channel forming properties. In this regard, the carboxyl tail of Cx43 may have the ability to control the translocation of transcription factor regulators into the nucleus. However, this putative tumor suppressor effect of Cx43 is counterbalanced by its capacity to enhance the migration of glioma cells out of the tumor core through mechanisms that seems to implicate its carboxyl tail, possibly by interacting with the actin cytoskeleton. This ambivalence between the tumor suppressor effect and promotion of cell migration may partly be explained by the heterogeneous expression of Cx43 in the glioma core especially at the malignant glioblastoma stage; some tumor cells would be expected to migrate (Cx43 expressing cells) and others to proliferate (non-expressing Cx43 cells). Moreover, the involvement of Cx43 in glioma progression seems to be more complex since, in addition, GJIC may increase their resistance to apoptosis and Cx43 may also affect cell homeostasis in a paracrine fashion via hemichannel action. In conclusion, Cx43 appears to be involved at different levels of the glioma progression by acting on cell growth regulation, promotion of cell migration and resistance to apoptosis. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

OBJECTIVE AMP-activated protein kinase (AMPK) signaling acts as a sensor of nutrients and hormones in the hypothalamus, thereby regulating whole-body energy homeostasis. Deletion of Ampkα2 in pro-opiomelanocortin (POMC) neurons causes obesity and defective neuronal glucose sensing. LKB1, the Peutz-Jeghers syndrome gene product, and Ca2+-calmodulin–dependent protein kinase kinase β (CaMKKβ) are key upstream activators of AMPK. This study aimed to determine their role in POMC neurons upon energy and glucose homeostasis regulation. RESEARCH DESIGN AND METHODS Mice lacking either Camkkβ or Lkb1 in POMC neurons were generated, and physiological, electrophysiological, and molecular biology studies were performed. RESULTS Deletion of Camkkβ in POMC neurons does not alter energy homeostasis or glucose metabolism. In contrast, female mice lacking Lkb1 in POMC neurons (PomcLkb1KO) display glucose intolerance, insulin resistance, impaired suppression of hepatic glucose production, and altered expression of hepatic metabolic genes. The underlying cellular defect in PomcLkb1KO mice involves a reduction in melanocortin tone caused by decreased α-melanocyte–stimulating hormone secretion. However, Lkb1-deficient POMC neurons showed normal glucose sensing, and body weight was unchanged in PomcLkb1KO mice. CONCLUSIONS Our findings demonstrate that LKB1 in hypothalamic POMC neurons plays a key role in the central regulation of peripheral glucose metabolism but not body-weight control. This phenotype contrasts with that seen in mice lacking AMPK in POMC neurons with defects in body-weight regulation but not glucose homeostasis, which suggests that LKB1 plays additional functions distinct from activating AMPK in POMC neurons.

International audience; Dominant tolerance to self-antigen requires the presence of sufficient numbers of CD4(+) Foxp3(+) Treg cells with matching antigen specificity. However, the size and role of TCR repertoire diversity for antigen-specific immuno-regulation through Treg cells is not clear. Here, we developed and applied a novel high-throughput (HT) TCR sequencing approach to analyze the TCR repertoire of Treg cells and revealed the importance of high diversity for Treg-cell homeostasis and function. We found that highly polyclonal Treg cells from WT mice vigorously expanded after adoptive transfer into non-lymphopenic TCR-transgenic recipients with low Treg-cell diversity. In that system, we identified specific Treg-cell TCR preferences in distinct anatomic locations such as the mesenteric LN indicating that Treg cells continuously compete for MHC class-II-presented self-, food-, or flora-antigen. Functionally, we showed that high TCR diversity was required for optimal suppressive function of Treg cells in experimental acute graft versus host disease (GvHD). In conclusion, we suggest that efficient immuno-regulation by Treg cells requires high TCR diversity. Thereby, continuous competition of peripheral Treg cells for limited self-antigen shapes an organ-optimized, yet highly diverse, local TCR repertoire.

Rationale: Macrophages cannot limit the uptake of lipids and rely on cholesterol efflux mechanisms for maintaining cellular cholesterol homeostasis. Important mediators of macrophage cholesterol efflux are ATP-binding cassette transporter 1 (ABCA1), which mediates the efflux of cholesterol to lipid-poor apolipoprotein AI, and scavenger receptor class B type I (SR-BI), which promotes efflux to mature high-density lipoprotein. Objective: The aim of the present study was to increase the insight into the putative synergistic roles of ABCA1 and SR-BI in foam cell formation and atherosclerosis. Methods and Results: Low-density lipoprotein receptor knockout (LDLr KO) mice were transplanted with bone marrow from ABCA1/SR-BI double knockout mice, the respective single knockouts, or wild-type littermates. Serum cholesterol levels were lower in ABCA1/SR-BI double knockout transplanted animals, as compared to the single knockout and wild-type transplanted animals on Western-type diet. Despite the lower serum cholesterol levels, massive foam cell formation was found in macrophages from spleen and the peritoneal cavity. Interestingly, ABCA1/SR-BI double knockout transplanted animals also showed a major increase in proinflammatory KC (murine interleukin-8) and interleukin-12p40 levels in the circulation. Furthermore, after 10 weeks of Western-type diet feeding, atherosclerotic lesion development in the aortic root was more extensive in the LDLr KO mice reconstituted with ABCA1/SR-BI double knockout bone marrow. Conclusions: Deletion of ABCA1 and SR-BI in bone marrow–derived cells enhances in vivo macrophage foam cell formation and atherosclerotic lesion development in LDLr KO mice on Western diet, indicating that under high dietary lipid conditions, both macrophage ABCA1 and SR-BI contribute significantly to cholesterol homeostasis in the macrophage in vivo and are essential for reducing the risk for atherosclerosis.

International audience; Beta-aminoisobutyric acid (BAIBA) is a catabolite of thymine and antiretroviral thymine analogues AZT and d4T. We recently discovered that this beta-amino acid is able to enhance fatty acid oxidation and reduce body weight in mice through an increased production of leptin by the white adipose tissue (WAT). Furthermore, BAIBA could have favourable effects on nonalcoholic steatohepatitis in a leptin-independent manner. In the present review, we shall recall the circumstances that led us to discover the effects of BAIBA on body fat mass and lipid homeostasis. In addition, we put forward several hypothetical mechanisms whereby BAIBA could enhance leptin secretion by WAT and present some anti-inflammatory effects in the liver. We also discuss in this review (i) the deleterious impacts caused by the absence of, or low leptin expression on lipid homeostasis and body weight in humans and animals and (ii) recent data from other investigators suggesting that increasing leptin levels and/or responsiveness may be indeed an attractive pharmacological strategy in order to prevent (and/or treat) obesity, at least in some individuals.

International audience; Host cells secrete antimicrobial proteins en masse to counter extracellular pathogens, placing a strain on the endoplasmic reticulum. The interplay between defence and cellular homeostasis has now been dissected genetically in Caenorhabditis elegans.