May anomalous X chromosome methylation be responsible for the spontaneous abortion of a male foetus?

Pregnancy loss is an important reproductive problem which appears to be highly associated with genetic factors. A spontaneous abortion occurred before prenatal diagnosis could be performed, in a woman who carried a fragile X full mutation. DNA extracted from formalin-fixed para ffin-embedded chorionic villi preparations indicated that it was a male embryo with an apparently methylated X chromosome. The previous analysis of the family showed that her daughter, who also carried a full mutation, exhibited an extremely skewed X inactivation of the normal allele (100%) and a severe fragile X phenotype. Thus, we speculate that the aberrant pattern of X chromosome methylation in this family may provoke the spontaneous miscarriage of this pregnancy that could be explained by at least partial inactivation of the unique X chromosome in a male foetus. Spontaneous abortion occurs quite frequently in humans, and recurrent pregnancy loss is a significant problem in women’s health (Christiansen 2006). Many cases of spontaneous abortion defy diagnosis and genetic factors have been proposed as a major contribution (Lanasa and Hogge 2000; Sierra and Stephenson 2006). The X chromosome inactiva tion (XCI) is the process in which one of the two X chromosomes present in each cell of female mammals is inactivated during early embryogenesis, to achieve dosage compensation with males (Avner and Heard 2001; Heard 2004). Initial steps of XCI involve a ‘counting process’, which senses the X chromosome/autosome ratio that restricts XCI to female embryos and, thereafter, the choice of which chromosome is inactivated (Morey et al . 2004).Skewed XCI leads to an expression of X-linked recessive disorders in females (Plenge et al. 2002; Mart´ınez et al. 2005; Renault et al. 2007). Another form of X-inactivation called meiotic sex chromosome inactivation (MSCI), takes place in males, during spermatogenesis and is a manifestation of the general meiotic-silencing mechanism (Turner 2007). In this study, we report the spontaneous abortion of a male foetus with an apparently methylated X chromosome, and propose that anomalous inactivation of its uniqueX chromosome could explain nonviability of the embryo and pregnancy loss. Therefore, we strongly suggest, as a follow up, that the methylation status of the X chromosome be tested in early spontaneous abortion of males in the absence of any other known genetic or nongenetic cause

Deletion of the von Hippel-Lindau gene causes sympathoadrenal cell death and impairs chemoreceptor-mediated adaptation to hypoxia

Mutations of the von Hippel–Lindau (VHL) gene are associated with pheochromocytomas and paragangliomas, but the role of VHL in sympathoadrenal homeostasis is unknown. We generated mice lacking Vhl in catecholaminergic cells. They exhibited atrophy of the carotid body (CB), adrenal medulla, and sympathetic ganglia. Vhl‐null animals had an increased number of adult CB stem cells, although the survival of newly generated neuron‐like glomus cells was severely compromised. The effects of Vhl deficiency were neither prevented by pharmacological inhibition of prolyl hydroxylases or selective genetic down‐regulation of prolyl hydroxylase‐3, nor phenocopied by hypoxia inducible factor overexpression. Vhl‐deficient animals appeared normal in normoxia but survived for only a few days in hypoxia, presenting with pronounced erythrocytosis, pulmonary edema, and right cardiac hypertrophy. Therefore, in the normal sympathoadrenal setting, Vhl deletion does not give rise to tumors but impairs development and plasticity of the peripheral O2‐sensing system required for survival in hypoxic conditions

Carotid body chemosensory responses in mice deficient of TASK channels

Background K+ channels of the TASK family are believed to participate in sensory transduction by chemoreceptor (glomus) cells of the carotid body (CB). However, studies on the systemic CB-mediated ventilatory response to hypoxia and hypercapnia in TASK1- and/or TASK3-deficient mice have yielded conflicting results. We have characterized the glomus cell phenotype of TASK-null mice and studied the responses of individual cells to hypoxia and other chemical stimuli. CB morphology and glomus cell size were normal in wild-type as well as in TASK1−/− or double TASK1/3−/− mice. Patch-clamped TASK1/3-null glomus cells had significantly higher membrane resistance and less hyperpolarized resting potential than their wild-type counterpart. These electrical parameters were practically normal in TASK1−/− cells. Sensitivity of background currents to changes of extracellular pH was drastically diminished in TASK1/3-null cells. In contrast with these observations, responsiveness to hypoxia or hypercapnia of either TASK1−/− or double TASK1/3−/− cells, as estimated by the amperometric measurement of catecholamine release, was apparently normal. TASK1/3 knockout cells showed an enhanced secretory rate in basal (normoxic) conditions compatible with their increased excitability. Responsiveness to hypoxia of TASK1/3-null cells was maintained after pharmacological blockade of maxi-K+ channels. These data in the TASK-null mouse model indicate that TASK3 channels contribute to the background K+ current in glomus cells and to their sensitivity to external pH. They also suggest that, although TASK1 channels might be dispensable for O2/CO2 sensing in mouse CB cells, TASK3 channels (or TASK1/3 heteromers) could mediate hypoxic depolarization of normal glomus cells. The ability of TASK1/3−/− glomus cells to maintain a powerful response to hypoxia even after blockade of maxi-K+ channels, suggests the existence of multiple sensor and/or effector mechanisms, which could confer upon the cells a high adaptability to maintain their chemosensory function

Metilación del ADN: relevancia en el sistema inmune y el síndrome X frágil

El objetivo general de este proyecto ha sido el estudio de la regulación de la metilación del ADN en células del sistema inmune y en células de pacientes con el síndrome X frágil. Para el estudio de la regulación de la metilación del ADN en el sistema inmune los objetivos específicos fueron: 1) Estudiar la implicación de diferentes vías de activación en la regulación de la metilación del ADN en linfocitios T. 2) Determinar si la regulación de la metilación del ADN se correlaciona con la expresión de genes susceptibles del sistema inmune. 3) Estudiar el efecto de activadores de la PKC e inhibidores de fosfatasas sobre la metilación del ADN y la expresión del IFN-γ. 4) Estudiar el efecto de agentes bloqueantes de grupos sulfidrilos, los cuales bloquean potentemente a grupos SH presentes en las ADN metiltransferasas, sobre la metilación y la expresión del IFN-γ. 5) Determinar el estado de metilación de los genes que contienen secuencias CpG en el promotor inducidos por las rutas anteriormente mencionadas. Para el estudio de la regulación de la expresión del gen FMR1 y su estado de metilación en células de pacientes con el síndrome X frágil los objetivos específicos fueron: 1) Inmortalizar células con el virus de Epstein Barr para la obtención de líneas celulares de sujetos control y de pacientes X frágil. 2) Establecer nuevas estrategias para la reversión del gen FMR1 en las células inmortalizadas de pacientes X frágil utilizando plásmidos de expresión de factores de transcripción implicados en la regulación de dicho gen de forma aislada o conjunta. 3) Estudiar la regulación de la expresión del gen FMR1 en células inmortalizadas de pacientes X frágil tratadas con un oligo antisentido de la ADN metiltransferasa 1 (DNMT1). 4) Comparar diferentes tratamientos para conocer cuales pueden ser los factores necesarios para la reversión de la expresión del gen FMR1

Gene expression analyses reveal metabolic specifications in acute O2 -sensing chemoreceptor cells.

Glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM) are essential for reflex cardiorespiratory adaptation to hypoxia. However, the mechanisms whereby these cells detect changes in O2 tension are poorly understood. The metabolic properties of acute O2 -sensing cells have been investigated by comparing the transcriptomes of CB and AM cells, which are O2 -sensitive, with superior cervical ganglion neurons, which are practically O2 -insensitive. In O2 -sensitive cells, we found a characteristic prolyl hydroxylase 3 down-regulation and hypoxia inducible factor 2α up-regulation, as well as overexpression of genes coding for three atypical mitochondrial electron transport subunits and pyruvate carboxylase, an enzyme that replenishes tricarboxylic acid cycle intermediates. In agreement with this observation, the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. The responsiveness of peripheral chemoreceptor cells to acute hypoxia depends on a 'signature metabolic profile'. Acute O2 sensing is a fundamental property of cells in the peripheral chemoreceptors, e.g. glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM), and is necessary for adaptation to hypoxia. These cells contain O2 -sensitive ion channels, which mediate membrane depolarization and transmitter release upon exposure to hypoxia. However, the mechanisms underlying the detection of changes in O2 tension by cells are still poorly understood. Recently, we suggested that CB glomus cells have specific metabolic features that favour the accumulation of reduced quinone and the production of mitochondrial NADH and reactive oxygen species during hypoxia. These signals alter membrane ion channel activity. To investigate the metabolic profile characteristic of acute O2 -sensing cells, we used adult mice to compare the transcriptomes of three cell types derived from common sympathoadrenal progenitors, but exhibiting variable responsiveness to acute hypoxia: CB and AM cells, which are O2 -sensitive (glomus cells > chromaffin cells), and superior cervical ganglion neurons, which are practically O2 -insensitive. In the O2 -sensitive cells, we found a characteristic mRNA expression pattern of prolyl hydroxylase 3/hypoxia inducible factor 2α and up-regulation of several genes, in particular three atypical mitochondrial electron transport subunits and some ion channels. In addition, we found that pyruvate carboxylase, an enzyme fundamental to tricarboxylic acid cycle anaplerosis, is overexpressed in CB glomus cells. We also observed that the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. Our data suggest that responsiveness to acute hypoxia depends on a 'signature metabolic profile' in chemoreceptor cells

Oxygen regulation of breathing is abolished in mitochondrial complex III-deficient arterial chemoreceptors.

Acute oxygen (O2) sensing is essential for adaptation of organisms to hypoxic environments or medical conditions with restricted exchange of gases in the lung. The main acute O2-sensing organ is the carotid body (CB), which contains neurosecretory chemoreceptor (glomus) cells innervated by sensory fibers whose activation by hypoxia elicits hyperventilation and increased cardiac output. Glomus cells have mitochondria with specialized metabolic and electron transport chain (ETC) properties. Reduced mitochondrial complex (MC) IV activity by hypoxia leads to production of signaling molecules (NADH and reactive O2 species) in MCI and MCIII that modulate membrane ion channel activity. We studied mice with conditional genetic ablation of MCIII that disrupts the ETC in the CB and other catecholaminergic tissues. Glomus cells survived MCIII dysfunction but showed selective abolition of responsiveness to hypoxia (increased [Ca2+] and transmitter release) with normal responses to other stimuli. Mitochondrial hypoxic NADH and reactive O2 species signals were also suppressed. MCIII-deficient mice exhibited strong inhibition of the hypoxic ventilatory response and altered acclimatization to sustained hypoxia. These data indicate that a functional ETC, with coupling between MCI and MCIV, is required for acute O2 sensing. O2 regulation of breathing results from the integrated action of mitochondrial ETC complexes in arterial chemoreceptors

Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion.

Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin-deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. Biotin is a water-soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin-deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin-deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin-deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin-deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells

Acute O2 Sensing: Role of Coenzyme QH2/Q Ratio and Mitochondrial ROS Compartmentalization.

Acute O2 sensing by peripheral chemoreceptors is essential for mammalian homeostasis. Carotid body glomus cells contain O2-sensitive ion channels, which trigger fast adaptive cardiorespiratory reflexes in response to hypoxia. O2-sensitive cells have unique metabolic characteristics that favor the hypoxic generation of mitochondrial complex I (MCI) signaling molecules, NADH and reactive oxygen species (ROS), which modulate membrane ion channels. We show that responsiveness to hypoxia progressively disappears after inducible deletion of the Ndufs2 gene, which encodes the 49 kDa subunit forming the coenzyme Q binding site in MCI, even in the presence of MCII substrates and chemical NAD+ regeneration. We also show contrasting effects of physiological hypoxia on mitochondrial ROS production (increased in the intermembrane space and decreased in the matrix) and a marked effect of succinate dehydrogenase activity on acute O2 sensing. Our results suggest that acute responsiveness to hypoxia depends on coenzyme QH2/Q ratio-controlled ROS production in MCI