Disruption of pathways regulated by Integrator complex in Galloway-Mowat syndrome due to WDR73 mutations.

Several studies have reported WDR73 mutations to be causative of Galloway-Mowat syndrome, a rare disorder characterised by the association of neurological defects and renal-glomerular disease. In this study, we demonstrate interaction of WDR73 with the INTS9 and INTS11 components of Integrator, a large multiprotein complex with various roles in RNA metabolism and transcriptional control. We implicate WDR73 in two Integrator-regulated cellular pathways; namely, the processing of uridylate-rich small nuclear RNAs (UsnRNA), and mediating the transcriptional response to epidermal growth factor stimulation. We also show that WDR73 suppression leads to altered expression of genes encoding cell cycle regulatory proteins. Altogether, our results suggest that a range of cellular pathways are perturbed by WDR73 loss-of-function, and support the consensus that proper regulation of UsnRNA maturation, transcription initiation and cell cycle control are all critical in maintaining the health of post-mitotic cells such as glomerular podocytes and neurons, and preventing degenerative disease.

Chondrodysplasia with multiple dislocations: comprehensive study of a series of 30 cases.

The group of chondrodysplasia with multiple dislocations includes several entities, characterized by short stature, dislocation of large joints, hand and/or vertebral anomalies. Other features, such as epiphyseal or metaphyseal changes, cleft palate, intellectual disability are also often part of the phenotype. In addition, several conditions with overlapping features are related to this group and broaden the spectrum. The majority of these disorders have been linked to pathogenic variants in genes encoding proteins implicated in the synthesis or sulfation of proteoglycans (PG). In a series of 30 patients with multiple dislocations, we have performed exome sequencing and subsequent targeted analysis of 15 genes, implicated in chondrodysplasia with multiple dislocations, and related conditions. We have identified causative pathogenic variants in 60% of patients (18/30); when a clinical diagnosis was suspected, this was molecularly confirmed in 53% of cases. Forty percent of patients remain without molecular etiology. Pathogenic variants in genes implicated in PG synthesis are of major importance in chondrodysplasia with multiple dislocations and related conditions. The combination of hand features, growth failure severity, radiological aspects of long bones and of vertebrae allowed discrimination among the different conditions. We propose key diagnostic clues to the clinician.

Bacterial ClpB heat-shock protein, an antigen-mimetic of the anorexigenic peptide α-MSH, at the origin of eating disorders.

The molecular mechanisms at the origin of eating disorders (EDs), including anorexia nervosa (AN), bulimia and binge-eating disorder (BED), are currently unknown. Previous data indicated that immunoglobulins (Igs) or autoantibodies (auto-Abs) reactive with α-melanocyte-stimulating hormone (α-MSH) are involved in regulation of feeding and emotion; however, the origin of such auto-Abs is unknown. Here, using proteomics, we identified ClpB heat-shock disaggregation chaperone protein of commensal gut bacteria Escherichia coli as a conformational antigen mimetic of α-MSH. We show that ClpB-immunized mice produce anti-ClpB IgG crossreactive with α-MSH, influencing food intake, body weight, anxiety and melanocortin receptor 4 signaling. Furthermore, chronic intragastric delivery of E. coli in mice decreased food intake and stimulated formation of ClpB- and α-MSH-reactive antibodies, while ClpB-deficient E. coli did not affect food intake or antibody levels. Finally, we show that plasma levels of anti-ClpB IgG crossreactive with α-MSH are increased in patients with AN, bulimia and BED, and that the ED Inventory-2 scores in ED patients correlate with anti-ClpB IgG and IgM, which is similar to our previous findings for α-MSH auto-Abs. In conclusion, this work shows that the bacterial ClpB protein, which is present in several commensal and pathogenic microorganisms, can be responsible for the production of auto-Abs crossreactive with α-MSH, associated with altered feeding and emotion in humans with ED. Our data suggest that ClpB-expressing gut microorganisms might be involved in the etiology of EDs.

A constant and similar assembly defect of mitochondrial respiratory chain complex I allows rapid identification of NDUFS4 mutations in patients with Leigh syndrome.

Isolated complex I deficiency is a frequent cause of respiratory chain defects in childhood. In this study, we report our systematic approach with blue native PAGE (BN-PAGE) to study mitochondrial respiratory chain assembly in skin fibroblasts from patients with Leigh syndrome and CI deficiency. We describe five new NDUFS4 patients with a similar and constant abnormal BN-PAGE profile and present a meta-analysis of the literature. All NDUFS4 mutations that have been tested with BN-PAGE result in a constant and similar abnormal assembly profile with a complete loss of the fully assembled complex I usually due to a truncated protein and the loss of its canonical cAMP dependent protein kinase phosphorylation consensus site. We also report the association of abnormal brain MRI images with this characteristic BN-PAGE profile as the hallmarks of NDUFS4 mutations and the first founder NDUFS4 mutations in the North-African population.

Identification of novel striatal genes by expression profiling in adult mouse brain.

Large-scale transcriptome analysis in the brain is a powerful approach to identify novel genes of potential interest toward understanding cerebral organization and function. We utilized the microarray technology to measure expression levels of about 24,000 genes and expressed sequence tags in mouse hippocampus, frontal cortex and striatum. Using expression profile obtained from whole brain as a reference, we categorized the genes into groups of genes either enriched in, or restricted to, one of the three areas of interest. We found enriched genes for each target area. Further, we identified 14 genes in the category of genes restricted to the striatum, among which were the orphan G protein-coupled receptor GPR88 and retinoic acid receptor-beta. These two genes were already reported to be selectively expressed in the striatum, thus validating our experimental approach. We selected 6 striatal-restricted genes, as well as 10 striatal-enriched candidates, that were previously undescribed. We analyzed their expression by in situ hybridization analysis in the brain, and quantitative RT-PCR in both brain and peripheral organs. Two of these unknown genes displayed a notable expression pattern. The striatal-restricted gene H3076B11 shows uniform expression throughout and uniquely in the striatum, representing a genuine striatal marker. The striatal-enriched gene 4833421E05Rik is preferentially expressed in the rostral striatum, and is also abundant in kidney, liver and lung. These two genes may contribute to some of the many striatal-controlled behaviors, including initiation of movement, habit formation, or reward and motivation.

Analysis of prolactin-modulated gene expression profiles during the Nb2 cell cycle using differential screening techniques.

BACKGROUND: Rat Nb2-11C lymphoma cells are dependent on prolactin for proliferation and are widely used to study prolactin signaling pathways. To investigate the role of this hormone in the transcriptional mechanisms that underlie prolactin-stimulated mitogenesis, five different techniques were used to isolate differentially expressed transcripts: mRNA differential display, representational difference analysis (RDA), subtractive suppressive hybridization (SSH), analysis of weakly expressed candidate genes, and differential screening of an organized library. RESULTS: About 70 transcripts were found to be modulated in Nb2 cells following prolactin treatment. Of these, approximately 20 represent unknown genes. All cDNAs were characterized by northern blot analysis and categorized on the basis of their expression profiles and the functions of the known genes. We compared our data with other cell-cycle-regulated transcripts and found several new potential signaling molecules that may be involved in Nb2 cell growth. In addition, abnormalities in the expression patterns of several transcripts were detected in Nb2 cells, including the constitutive expression of the immediate-early gene EGR-1. Finally, we compared the differential screening techniques in terms of sensitivity, efficiency and occurrence of false positives. CONCLUSIONS: Using these techniques to determine which genes are differentially expressed in Nb2 lymphoma cells, we have obtained valuable insight into the potential functions of some of these genes in the cell cycle. Although this information is preliminary, comparison with other eukaryotic models of cell-cycle progression enables identification of expression abnormalities and proteins potentially involved in signal transduction, which could indicate new directions for research.

From the molecular biology of prolactin and its receptor to the lessons learned from knockout mice models.

Prolactin (PRL), a polypeptide hormone secreted mainly by the pituitary and, to a lesser extent, by peripheral tissues, affects more physiological processes than all other pituitary hormones combined since it is involved in > 300 separate functions in vertebrates. Its main actions are related to lactation and reproduction. The initial step of PRL action is the binding to a specific membrane receptor, the PRLR, which belongs to the class 1 cytokine receptor superfamily. PRL-binding sites have been identified in a number of tissues and cell types in adult animals. Signal transduction by this receptor is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases and signal transducers and activators of transcription (STATs). Disruption of the PRLR gene has provided a new mouse model with which to identify actions directly associated with PRL or any other PRLR ligands, such as placental lactogens. To date, several different phenotypes have been analyzed and are briefly described in this review. Coupled with the SAGE technique, this PRLR knockout model is being used to qualitatively and quantitatively evaluate the expression pattern of hepatic genes in two physiological situations: transcriptomes corresponding to livers from both wild type and PRLR KO mice are being compared, and following statistical analyses, candidate genes presenting a differential profile will be further characterized. Such a new approach will undoubtedly open future avenues of research for PRL targets. To date, no pathology linked to any mutation in the genes encoding PRL or its receptor have been identified. The development of genetic models provides new opportunities to understand how PRL can participate to the development of pathologies throughout life, as for example the initiation and progression of breast cancer.

Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.

PRL is an anterior pituitary hormone that, along with GH and PLs, forms a family of hormones that probably resulted from the duplication of an ancestral gene. The PRLR is also a member of a larger family, known as the cytokine class-1 receptor superfamily, which currently has more than 20 different members. PRLRs or binding sites are widely distributed throughout the body. In fact, it is difficult to find a tissue that does not express any PRLR mRNA or protein. In agreement with this wide distribution of receptors is the fact that now more than 300 separate actions of PRL have been reported in various vertebrates, including effects on water and salt balance, growth and development, endocrinology and metabolism, brain and behavior, reproduction, and immune regulation and protection. Clearly, a large proportion of these actions are directly or indirectly associated with the process of reproduction, including many behavioral effects. PRL is also becoming well known as an important regulator of immune function. A number of disease states, including the growth of different forms of cancer as well as various autoimmune diseases, appear to be related to an overproduction of PRL, which may act in an endocrine, autocrine, or paracrine manner, or via an increased sensitivity to the hormone. The first step in the mechanism of action of PRL is the binding to a cell surface receptor. The ligand binds in a two-step process in which site 1 on PRL binds to one receptor molecule, after which a second receptor molecule binds to site 2 on the hormone, forming a homodimer consisting of one molecule of PRL and two molecules of receptor. The PRLR contains no intrinsic tyrosine kinase cytoplasmic domain but associates with a cytoplasmic tyrosine kinase, JAK2. Dimerization of the receptor induces tyrosine phosphorylation and activation of the JAK kinase followed by phosphorylation of the receptor. Other receptor-associated kinases of the Src family have also been shown to be activated by PRL. One major pathway of signaling involves phosphorylation of cytoplasmic State proteins, which themselves dimerize and translocate to nucleus and bind to specific promoter elements on PRL-responsive genes. In addition, the Ras/Raf/MAP kinase pathway is also activated by PRL and may be involved in the proliferative effects of the hormone. Finally, a number of other potential mediators have been identified, including IRS-1, PI-3 kinase, SHP-2, PLC gamma, PKC, and intracellular Ca2+. The technique of gene targeting in mice has been used to develop the first experimental model in which the effect of the complete absence of any lactogen or PRL-mediated effects can be studied. Heterozygous (+/-) females show almost complete failure to lactate after the first, but not subsequent, pregnancies. Homozygous (-/-) females are infertile due to multiple reproductive abnormalities, including ovulation of premeiotic oocytes, reduced fertilization of oocytes, reduced preimplantation oocyte development, lack of embryo implantation, and the absence of pseudopregnancy. Twenty per cent of the homozygous males showed delayed fertility. Other phenotypes, including effects on the immune system and bone, are currently being examined. It is clear that there are multiple actions associated with PRL. It will be important to correlate known effects with local production of PRL to differentiate classic endocrine from autocrine/paracrine effects. The fact that extrapituitary PRL can, under some circumstances, compensate for pituitary PRL raises the interesting possibility that there may be effects of PRL other than those originally observed in hypophysectomized rats. The PRLR knockout mouse model should be an interesting system by which to look for effects activated only by PRL or other lactogenic hormones. On the other hand, many of the effects reported in this review may be shared with other hormones, cytokines, or growth factors and thus will be more difficult to study. (ABSTRACT TRUNCATED)

Prolactin: a hormone at the crossroads of neuroimmunoendocrinology.

Prolactin (PRL), secreted by the pituitary, decidua, and lymphoid cells, has been shown to have a regulatory role in reproduction, immune function, and cell growth in mammals. The effects of PRL are mediated by a membrane-bound receptor that is a member of the superfamily of cytokine receptors. Formation of a trimer, consisting of one molecule of ligand and two molecules of receptor, appears to be a necessary prerequisite for biological activity. The function of these receptors is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). To study these receptors, we have used two approaches: mutational analysis of their cytoplasmic domains coupled with functional tests and inactivation (knockout) of the receptor gene by homologous recombination in mice. We have produced mice by gene targeting in embryonic stem cells carrying a germline null mutation of the prolactin receptor gene. Heterozygous (+/-) females show almost complete failure to lactate, following their first, but not subsequent pregnancies. Homozygous (-/-) females are infertile as a result of multiple reproductive abnormalities, including ovulation of premiotic oocytes, reduced fertilization of oocytes, reduced preimplantation oocyte development, lack of embryo implantation, and the absence of pseudopregnancy. Half of the homozygous males are infertile or show reduced fertility. In view of the wide-spread distribution of PRL receptors, other phenotypes including those on the immune system, are currently being evaluated in -/- animals. This study establishes the prolactin receptor as a key regulator of mammalian reproduction and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.

Suppression subtractive hybridization (SSH) identifies prolactin stimulation of p38 MAP kinase gene expression in Nb2 T lymphoma cells: molecular cloning of rat p38 MAP kinase.

Suppression Subtractive Hybridization (SSH) has been used to compare rat Nb2 cells treated with prolactin for 1 hour with untreated cells. This new method for identifying differentially expressed genes showed that the mRNAs for at least three genes were elevated by such treatment, including a p38 mitogen activated protein (MAP) kinase. The p38 MAP kinase was cloned and the full length cDNA sequence was determined.

In vivo study of prolactin (PRL) intracellular signalling during lactogenesis in the rat: JAK/STAT pathway is activated by PRL in the mammary gland but not in the liver.

The rat prolactin receptor (PRL-R) exists in two forms, which differ in the length of the cytoplasmic domains, tissue distribution, and biological activity. The short form predominates in liver while the long form is prevalent in mammary gland. We have compared activation by PRL of the JAK2-STAT pathway (protein tyrosine phosphorylation and STAT5 activation) in mammary gland and liver in an in vivo rat model of induction of lactogenesis by PRL injections, and we have studied the relative proportion of both forms of the receptor in these tissues by reverse transcription-polymerase chain reaction. Rats were ovario-hysterectomized on Day 19 of pregnancy, treated with bromocriptine, subsequently injected with 250 micrograms ovine PRL i.p. on Day 20, and killed 0-12 h after. Western blots of solubilized mammary gland and liver membranes immunoprecipitated with anti-PRL-R or anti-JAK2 antibodies showed that the PRL-R is constitutively associated with JAK2 and that the long form of the PRL-R is present in both tissues, while the short form was detected only in liver. Phosphorylated proteins corresponding to the long form of PRL-R and JAK2 appeared 15-60 min after ovine PRL injection in mammary extracts but not in liver. At these same times, an electrophoretic mobility shift assay, using a rat beta-casein probe specific for STAT5 binding, showed activated STAT5 in mammary gland cytosol and nuclear extracts. In the liver, low levels of activated STAT5 were detected in non-treated animals, which were not modified by PRL. Quantitative RT-PCR of liver and mammary PRL-R mRNA showed that the amount of the long form of PRL-R mRNA is roughly comparable in both tissues, while the short form is predominant in liver and in a minority in mammary tissue. Both forms were down-regulated by PRL only in mammary glands. Thus, during lactogenesis, mammary tissue responds to PRL by activation of JAK2 and STAT5, while the liver does not respond to PRL in spite of the presence of PRL-R associated with JAK2 and pre-existing activated STAT5. Thus, liver tissue may lack a critical component for activation of the PRL pathway, or the large quantities of the short form of the PRL-R may associate with the long form to constitute inactive heterodimers.

Regulation of prolactin receptor mRNA expression in peripheral lymphocytes in rats in response to changes in serum concentrations of prolactin.

In the present study we have evaluated the absolute number of the two forms of prolactin (PRL) receptor mRNA in rat peripheral blood lymphocytes and the modulation of receptor mRNA induced by changes in serum levels of endogenous PRL or by administration of ovine PRL. Lymphocytes expressed low levels of both forms of PRL receptor transcripts. Repeated treatments with ovine PRL significantly reduced levels of mRNA encoding the long form PRL receptor, whereas expression was markedly increased by repeated doses of bromocriptine. In contrast, the mRNA level of short form PRL receptor was unchanged by both treatments. The expression of long form transcripts was also markedly decreased in lymphocytes from pituitary-grafted rats. Therefore it appears that in rat peripheral lymphocytes PRL has a negative effect on the expression of its own receptor.