scholarly journals SUN-LB56 Steroid and Sex Specific Responses of Neural Stem Cells to Prenatal Dexamethasone versus Betamethasone Administration

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Neeru Silswal ◽  
Suban Burale ◽  
Joe Bean ◽  
Fatma Talib ◽  
Herschel Gupta ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Developmental Stages ◽  
Inositol Phosphate ◽  
Biological Response ◽  
Fold Change ◽  
Developmental Trajectory ◽  
Developing Brain ◽  
Oligodendrocyte Differentiation ◽  
Increased Risk

Abstract Synthetic glucocorticoids (sGCs) are widely administered to pregnant women for their anti-inflammatory, immunosuppressive and organ maturation properties. Worldwide, Dexamethasone (Dex) and Betamethasone (Beta) are the two most commonly administered prenatal sGCs to reduce morbidity and mortality associated with respiratory distress, intraventricular hemorrhage and necrotizing enterocolitis. Preterm administration of sGCs is associated with reduced birthweight and increased risk for hypertension, cardiovascular, metabolic, and neurological problems later in life. Adverse neurological outcome has been shown to depend on the type of sGCs used, the dose, timing of sGCs administration and sex. We have previously shown that the glucocorticoid receptor (GR) is expressed in the developing brain in stem and progenitor cells, neurons and glia from early developmental stages, and that prenatal Dex alters neural stem cell (NSC) biology and the developmental trajectory of the cerebral cortex, hypothalamus and adult behavior. To identify the molecular and cellular basis of the sex and steroid specific responses in the developing brain, we compared the consequence of Dex versus Beta exposure on embryonic cerebral cortical NSC biology. Murine NSC were isolated from the E14.5 cerebral cortex and exposed to 10-7 M Dex, 10-7 M Beta, or Vehicle for 4 or 24 hours and the immediate and long-term impact on transcription, proliferation and neuronal, glial and oligodendrocyte differentiation examined. Affymetrix complete genome transcriptional analyses reveal sex specific responses to Dex versus Beta within 4 hours. At >+/-1.5-fold change 548 genes were differentially regulated by Dex, 452 by Beta and 256 were altered by both Dex and Beta (P < 0.05). Distinct sex specific responses to Dex versus Beta were observed. At >+/-2-fold change 126 genes were significantly different in the Dex versus Beta female transcriptome, 146 in the male transcriptome with 18 genes unique to both male and female transcriptome. Ingenuity Pathway Analysis revealed that the most significantly altered pathway altered (Z score >2) with both sGCs is Inositol Phosphate metabolism. Cardiac hypertrophy, Tec kinase, and Th1 pathways were unique to Beta stimulation, whereas Melatonin, Neuropathic Pain and IL6 signaling pathways were specific to Dex stimulation. Both Dex and Beta significantly alter genes implicated in proliferation and differentiation as also described in other studies, therefore the biological response of NSC to sGCs stimulation was compared. Only Dex significantly decreased the rate of proliferation over a 72 hour. In-vitro differentiation studies reveal that both Dex and Beta reduced oligodendrocyte differentiation without altering neuronal differentiation when cells were exposed to sGCs as progenitors. However, when cells were exposed to sGCs during differentiation, Dex increased oligodendrocyte and neuronal maturation while Beta only increased oligodendrocyte differentiation. These results reveal gene targets, cellular pathways and processes that are differentially altered by prenatal Dex versus Beta exposure. Prenatal sGCs administration provides clear benefits for neonatal outcome, however, a detailed understanding of their targets in the brain is required to identify alternative sGCs drug regimens to reduce adverse neurological effects. Our finds may provide insights into the sex specific neurological outcomes observed in children exposed to sGCs in-utero.

scholarly journals Betamethasone Induces a Unique Transcriptome in Neural Stem Cells

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A802-A802
Author(s):  
Neerupma Silswal ◽  
Joe Bean ◽  
Herschel Gupta ◽  
Fatma Talib ◽  
Suban Burale ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Serotonin Receptor ◽  
Target Genes ◽  
Biological Response ◽  
The United States ◽  
Oligodendrocyte Differentiation

Abstract Twelve percent of pregnant women receive glucocorticoids (sGCs) to reduce the risks to reduce morbidity and mortality associated with preterm birth in infants. The two most commonly administered sGC are Dexamethasone (Dex) and Betamethasone (Beta) and they serve to decrease the severity of respiratory distress, intraventricular hemorrhage and necrotizing enterocolitis. However, repeated administration of sGC has been shown to be associated with adverse neurological outcome and depends on the type of sGCs used, dose, timing of sGCs administration and sex. We have previously shown that prenatal exposure to Dex in a murine model lead to sex specific changes in the transcription response and in the biological function of neural stem cells and to long-term changes in brain architecture and behavior. Beta is the predominant sGC used prenatally in the United States, therefore these studies investigated the cellular and molecular responses to beta exposure on the neural stem cells in-vitro and anatomical organization of the brain in-vivo. Murine NSCs were isolated from the E14.5 cerebral cortex and exposed to 10-7 M Dex, 10-7 M Beta, or Vehicle for 4 or 24 hours and the immediate and long-term impact on transcription, proliferation and neuronal, glial and oligodendrocyte differentiation examined. Affymetrix genome transcriptional analyses reveal sex specific responses to Dex vs Beta in 4 hours. In females 682 genes were differentially regulated by Dex compared to 576 by Beta. In contrast, 875 were altered by Dex and 576 by Beta in males (Fold change > +/- 1.5, P< 0.05). Select target genes were independently validated by QPCR. Ingenuity Pathway Analysis was used to identify unique and overlapping pathways that were altered by Dex vs Beta. In males, Dex uniquely altered 34 pathways including, Thyroid Hormone Metabolism, ERK5 Signaling and Opioid Signaling while Bata altered 33 pathways including, Phagasome formation, IL-7 Signaling and JAK STAT signaling. In Females, Dex altered 45 pathways including Calcium Signaling, Serotonin Receptor Signaling and Xenobiotic Signaling, while Beta altered 46 pathways including, FXR/RXR Activation, Tec Kinase Signaling and D-myo-Inositol-5-Phosphate Metabolism. Another 35 pathways were altered by both Dex and Beta but they showed differences in genes activated or repressed. Dex and Beta, both significantly altered genes involved in proliferation and differentiation therefore the biological response of NSC to sGCs stimulation in vitro and the long term consequences of sGC exposure in-vivo was compared. Distinct differences in cell proliferation, glial and oligodendrocyte differentiation were observed. These results reveal gene targets, cellular pathways and processes that are differentially altered by prenatal Dex vs Beta exposure. Our finds may provide insights into the sex specific neurological outcomes observed in children exposed to sGCs in-utero.

scholarly journals SUN-LB53 Role of the Glucocorticoid Receptor Phosphorylation in Neural Development

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Neeru Silswal ◽  
Herschel Gupta ◽  
Joe Bean ◽  
Suban Burale ◽  
Alexis Franks ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Glucocorticoid Receptor ◽  
Neural Development ◽  
Transcriptional Activation ◽  
Biological Response ◽  
Neuronal Morphology ◽  
Receptor Phosphorylation ◽  
Increased Risk ◽  
Z Scores

Abstract Synthetic glucocorticoid (sGC) administration in pregnancy has greatly reduced the risk of respiratory distress, intraventricular hemorrhage and necrotizing enterocolitis in premature infants. Significant evidence has accumulated in human and animal models that prenatal exposure to sGCs can lead to adverse side effects such as reduced birthweight, increased risk for hypertension, cardiovascular, metabolic, and neurological problems later in life. Phosphorylation of the glucocorticoid receptor (GR) has been shown to play a significant role in a cells response to sGC administration, altering target gene activation versus repression, the magnitude and duration of the response. The GR receptor is phosphorylated on three sites (S203, S211, S226) in the N-terminal. An increased in the ratio of phosphorylation on S211 to S226 is associated with enhanced transcriptional activation. Furthermore, changes in S221/S226 ratio are associated with distinct neurological disorders in humans. We have previously shown that in-utero exposure to a single dose of dexamethasone (Dex) reduces proliferation in cerebral cortical and hypothalamic neural stem cells (NSCs), alters neuronal differentiation, neuronal morphology and adult behavior. To investigate the role of receptor phosphorylation on NSCs biology and brain development, mice with a serine (S211) to alanine (S211A) knockin were generated. NSCs were isolated from the mouse E14.5 cerebral cortex and the transcriptional and biological response of cells were examined in response to sGC or vehicle stimulation. Affymetrix complete genome arrays were used to identify changes in global gene expression in response to 4 hours of 10-7 M Dex exposure. Basally, 2651 genes were >1.5 fold (p < 0.05) differentially regulated in S211A versus wildtype, with 929 distinct upregulated and 1722 downregulated. Sex specific differences were observed basally, with 382 upregulated and 824 down regulated in females compared to 1191 upregulated and 1353 downregulated in males. Ingenuity pathway analysis (IPA) revealed that the only significant pathways that were altered basally in S211A versus wildtype were valine and isoleucine degradation, fatty acid beta oxidation and glutathione redox reaction I, all with negative Z scores (Z scores -2.1 to -3.16, P < 1.3E-01 to 1.3E-06). In response to a 4-hour Dex stimulation, 473 and 657 genes were upregulated and 782 and 996 genes were downregulated in females versus male respectively in S211A compared to wildtype. IPA analysis revealed that only one significant pathway with a Z score >2 that was altered in S211A versus wildtype in response to dex was of activation LPS/IL1 mediated inhibition of RXR function (Z = 2.82, p <3.08E-03). Some of the most significant genes changed basally in S211A versus wildtype include genes involved in the cell cycle. To determine if these transcriptional changes led to a distinct biological response, proliferation and differentiation studies were performed. Basally, S211A cells exhibit enhanced proliferation compared to wildtype cells in vitro. These findings were validated by in-vivo findings demonstrated by increased expression of TBR2, an immediate progenitor cell marker in the cerebral cortex at E17.5. These studies identify distinct pathways and developmental neurological processes that are sensitive to phosphorylation of GR on S211 basally and in response to sGC exposure.

scholarly journals Effects of Lipopolysaccharide on Oligodendrocyte Differentiation at Different Developmental Stages: an In Vitro Study

2021 ◽  
Vol 36 (49) ◽  
Author(s):  
Ja-Hye Ahn ◽  
Hyun Ju Lee ◽  
Kyeongmi Lee ◽  
Jean Lim ◽  
Jae Kyoon Hwang ◽  
...  
Keyword(s):  
Developmental Stages ◽  
In Vitro Study ◽  
Vitro Study ◽  
Oligodendrocyte Differentiation

327 Developmental Trajectories of Insomnia and Risk of Internalizing Disorders in Young Adulthood

SLEEP ◽  
2021 ◽  
Vol 44 (Supplement_2) ◽  
pp. A131-A131
Author(s):  
Julio Fernandez-Mendoza ◽  
Kristina Puzino ◽  
Myra Qureshi ◽  
Susan Calhoun ◽  
Savannah Toth ◽  
...  
Keyword(s):  
Young Adulthood ◽  
Developmental Stages ◽  
Cox Regression ◽  
Developmental Trajectories ◽  
Medication Use ◽  
Internalizing Disorders ◽  
Developmental Trajectory ◽  
Self Report ◽  
Increased Risk ◽  
Insomnia Symptoms

Abstract Introduction Internalizing disorders (ID) are the most common form of psychopathology and a large proportion of individuals experience their first onset after the age of 18. Childhood insomnia symptoms, i.e., difficulties initiating or maintaining sleep (DIMS), have been shown to be associated with ID. However, little is known about the developmental trajectories of insomnia symptoms and their associated risk of ID as the child transitions into adulthood. The present study examined the risk of ID in young adulthood associated with the longitudinal trajectories of insomnia symptoms across three developmental stages. Methods The Penn State Child Cohort is a population-based sample of 700 children (Mdn=9y), who were followed-up 8 years later as adolescents (N=421, Mdn=16y) and 15 years later as young adults (N=492, Mdn=24y). Insomnia symptoms were defined as parent-reported (childhood) or self-reported (adolescence and young adulthood) moderate-to-severe DIMS. The developmental trajectories of insomnia symptoms across the three time-points were identified as never, remitted, waxing-and-waning, persistent and incident. The presence of ID was defined as a self-report of a diagnosis or treatment for mood and/or anxiety disorders. Cox regression models were adjusted for sex, race/ethnicity, age and childhood/adolescent history of ID or psychoactive medication use. Results A persistent developmental trajectory was associated with a 2.8-fold increased risk of adult ID (HR=2.83, 95%CI=1.79–4.49) and an incident trajectory with a 1.9-fold risk (HR=1.88, 95%CI=1.10–3.20), while a waxing-and-waning trajectory was marginally associated with adult ID (HR=1.70, 95%CI=0.99–2.91). A remitting trajectory was not associated with an increased risk of adult ID (HR=0.92, 95%CI=0.38–2.24). Conclusion This 15-year longitudinal study with three developmental stages shows that childhood-onset insomnia symptoms that persist across the life-course are strong determinants of ID in young adulthood, independent of past diagnosis or medication use. In contrast, childhood insomnia symptoms that remit in the transition to adolescence do not confer increased risk of ID in young adulthood. Given that insomnia symptoms may precipitate and/or maintain ID, these data further reinforce the need for early sleep interventions to prevent mental health disorders. Support (if any) NIH Awards Number R01HL136587, R01MH118308, R01HL97165, R01HL63772, UL1TR000127

scholarly journals Coordination of Neuron Production in Mouse and Human Cerebral Cortex by the Homolog of Drosophila Mastermind Protein

2019 ◽  
Vol 93 (2-3) ◽  
pp. 152-165
Author(s):  
Albert E. Ayoub ◽  
Martin H. Dominguez ◽  
Jaime Benoit ◽  
Juan Alberto Ortega ◽  
Nevena Radonjic ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Signaling Pathways ◽  
Neuronal Migration ◽  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Ectopic Expression ◽  
Genetic Network ◽  
Dominant Negative ◽  
Beta Catenin

The coordination of progenitor self-renewal, neuronal production, and migration is essential to the normal development and evolution of the cerebral cortex. Numerous studies have shown that the Notch, Wnt/beta-catenin, and Neurogenin pathways contribute separately to progenitor expansion, neurogenesis, and neuronal migration, but it is unknown how these signals are coordinated. In vitro studies suggested that the mastermind-like 1 (MAML1) gene, homologue of the Drosophila mastermind, plays a role in coordinating the aforementioned signaling pathways, yet its role during cortical development remains largely unknown. Here we show that ectopic expression of dominant-negative MAML (dnMAML) causes exuberant neuronal production in the mouse cortex without disrupting neuronal migration. Comparing the transcriptional consequences of dnMAML and Neurog2 ectopic expression revealed a complex genetic network controlling the balance of progenitor expansion versus neuronal production. Manipulation of MAML and Neurog2 in cultured human cerebral stem cells exposed interactions with the same set of signaling pathways. Thus, our data suggest that evolutionary changes that affect the timing, tempo, and density of successive neuronal layers of the small lissencephalic rodent and large convoluted primate cerebral cortex depend on similar molecular mechanisms that act from the earliest developmental stages.

Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

1974 ◽  
Vol 32 ◽  
pp. 320-321
Author(s):  
J. P. Revel
Keyword(s):  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Movement ◽  
Cellular Interactions ◽  
Serial Sections ◽  
Cell Sheets ◽  
Freeze Etching ◽  
Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

Thrombin Generation Measured Ex Vivo Following Microvasculor Injury Is Increased in SLE Patients with Antiphospholipid-protein Antibodies

1997 ◽  
Vol 78 (04) ◽  
pp. 1173-1177 ◽  
Author(s):  
Jacek Musiał ◽  
Jakub Swadźba ◽  
Miłosz Jankowski ◽  
Marek Grzywacz ◽  
Stanisława Bazan-Socha ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Skin Incision ◽  
Anticardiolipin Antibodies ◽  
Small Blood Vessel ◽  
Anionic Phospholipids ◽  
Increased Risk ◽  
High Concentrations

SummaryAntiphospholipid-protein antibodies (APA) include lupus-type anticoagulant (LA) and antibodies recognizing complexes of anionic phospholipids (e.g. cardiolipin) and proteins (e.g. prothrombin and (β2-glycoprotein I). The presence of APA is associated with an increased risk of both arterial and venous thrombosis. However, the pathogenic mechanism leading to thrombosis in patients with APA remains unclear. We studied 32 patients with systemic lupus erythematosus (SLE) who were divided into two groups depending on the presence (n = 19) or absence (n = 13) of APA. Healthy volunteers (n = 12) matched by age and sex served as controls. In all subjects LA and IgG class anticardiolipin antibodies (ACA) were determined. Thrombin generation was monitored ex vivo measuring fibrinopeptide A (FPA) and prothrombin fragment F1 + 2 (F1 + 2) in blood emerging from a skin microvasculature injury, collected at 30 second intervals. In subjects with antiphospholipid antibodies mean FPA and F1 + 2 concentrations were signiF1cantly higher at most blood sampling times than in controls. In some SLE patients with APA the process of thrombin generation was clearly disturbed and very high concentrations of F1brinopeptide A were detected already in the F1rst samples collected. Two minutes after skin incision SLE patients without APA produced slightly more FPA, but not F1 + 2, as compared to healthy subjects. Mathematical model applied to analyze the thrombin generation kinetics revealed that APA patients generated signiF1cantly greater amounts of thrombin than healthy controls (p = 0.02 for either marker). In contrast, in the same patients generation of thrombin in recalciF1ed plasma in vitro was delayed pointing to the role of endothelium in the phenomenon studied. In summary, these data show for the F1rst time that in SLE patients with antiphospholipid-protein antibodies thrombin generation after small blood vessel injury is markedly increased. Enhanced thrombin generation might explain thrombotic tendency observed in these patients.

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