Hypoxia dysregulates circadian rhythm and causes synapse elimination defects in astrocytes
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE255588 | GEO | 2025/02/12
REPOSITORIES: GEO
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Project description:Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered post-transcriptionally. We used ribosome profiling to examine post-transcriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5'UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells without altering the phase or period and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased PER2 protein levels and reduced total sleep time compared to that in wild-type mice. These results suggest that post-transcriptional processes shape translation of mRNA transcripts, which can impact physiological rhythms and sleep.
2023-09-13 | GSE231820 | GEO
Project description:Disruption of circadian rhythms is implicated in increasing the risk of cancer development. The Period2 (Per2) gene is one of the major components of mammalian circadian clock, which plays a key role in controlling the circadian rhythms in physiology and behavior. PER2 also has tumor suppression function, but its role in the regulation of susceptibility to chemotherapeutic drugs remains unclear. In this study, we identified up-regulation of Aldehyde dehydrogenase 3a1 (Aldh3a1) gene in oncogenic transformed mouse embryo fibroblasts prepared from Per2-mutant (Per2m/m) mice, which were associated with the resistance against chemotherapeutic drugs. Co-expression of H-rasV12 and SV40 large T antigen induced malignant transformation of both wild-type and Per2m/m cells, but the cytotoxic effects of methotrexate, gemcitabine, etoposide, vincristine, and oxaliplatin were significantly alleviated in oncogenic transformed Per2m/m cells. Although introduction of oncogenes increased the expression of Aldh3a1 in both wild-type and Per2m/m cells, the levels of ALDH3A1 protein in oncogenic transformed Per2m/m cells were 10-fold higher than those in wild-type cells. Elevated ALDH3A1 levels in oncogenic transformed Per2m/m cells were sufficient to prevent chemotherapeutic drug-induced production of reactive oxygen species. Consequently, suppression of Aldh3a1 expression by siRNA relived chemoresistance of Per2m/m cells. These results suggest an uncovered role for PER2 in the development of multiple drug resistance and offers new therapeutic strategies for treatment of cancers.
2018-10-10 | GSE113242 | GEO
Project description:Sirtuin 1 (SIRT1) is involved in both aging and circadian-clock regulation, yet the link between the two processes in relation to SIRT1 function is not clear. Using Sirt1-deficient mice, we found that Sirt1 and Period 2 (Per2) constitute a reciprocal negative regulation loop that plays important roles in modulating hepatic circadian rhythmicity and aging. Sirt1-deficient mice exhibited profound premature aging and enhanced acetylation of histone H4 on lysine16 (H4K16) in the promoter of Per2, the latter of which leads to its overexpression; in turn, Per2 suppresses Sirt1 transcription through binding to the Sirt1 promoter at the Clock/Bmal1 site. This negative reciprocal relationship between SIRT1 and PER2 was also observed in human hepatocytes. We further demonstrated that the absence of Sirt1 or the ectopic overexpression of Per2 in the liver resulted in a dysregulated pace of the circadian rhythm. The similar circadian rhythm was also observed in aged wild type mice. The interplay between Sirt1 and Per2 modulates aging gene expression and circadian-clock maintenance. To investigate hepatic SIRT1-dependent aging related genes, livers from wild type mice at 3 months (young), 12 months (middle age), and 19 months (old) of age, as well as Sirt1-deficient mice at 3 months of age were snap frozen and subject to RNA isolation and microarray analysis.
2016-08-08 | E-GEOD-54436 | biostudies-arrayexpress
Project description:The peripheral circadian oscillator plays an essential role in synchronizing local physiology to operate in a circadian manner via regulation of the expression of clock-controlled genes. In the murine uterus, the endometrial stromal cells undergo proliferation and differentiation into decidual cells in response to ovarian steroids and blastocyst implantation at the early stage of pregnancy. The circadian clock genes are attenuated in the decidualizing cells only 2 days after implantation. The present study aimed to evaluate the circadian rhythms of clock genes and clock-controlled genes expressed in the rat uterus endometrial stromal cells (UESCs) during the stage of implantation. The real-time monitoring system of Per2 promoter activity was employed to precisely evaluate the generation of circadian rhythms in the UESCs prepared from transgenic rats constructed with mouse Per2 promoter-destabilized luciferase reporter gene (Per2-dLuc). During monitoring Per2-dLuc oscillation after synchronization with dexamethasone, total RNA was isolated from the cultured UESCs at four-time points (6-h interval) in the first to second phases and cDNA was synthesized. cRNA was synthesized from the double strand cDNA and hybridized on a DNA microarray. RT-qPCR was performed to confirm the expression of core clock genes revealed by DNA microarray analysis. Several clock genes such as Bmal1, Rev-erbα, and Per2 displayed significant rhythms. Of 12,252 genes showing significantly expression, 7,235 genes displayed significant alterations (p < 0.05). These genes were related to growth factors, transcription factors, receptors, channels, and enzymes. Some candidates as clock-controlled genes were evaluated by using RNA interference to Bmal1 mRNA. Down-regulation of Igf1 gene expression was observed by Bmal1 silencing, whereas the expression of Inhβa, Fas, and Caspase3 were significantly increased. These results indicate that clock-controlled genes are up- or down-regulated in rat UESCs during the stage of decidualization. DNA microarray analysis coupled with RNA interference will be helpful to understand the physiological roles of some oscillating genes in blastocyst implantation and placenta formation. The circadian clock positively or negatively regulates the expression of clock-controlled genes, including growth factors and apoptosis-related factors. To search the clock-controlled genes expressed during the period of implantation, we analyzed the clock genes and clock-controlled genes expressed in cultured uterus endometrial stromal cells prepared from pregnant rats at the stage of implantation using DNA microarray technology. We used transgenic rats constructed with mouse Per2 promoter-destabilized luciferase (Per2-dLuc) reporter gene to precisely adjust the time of gene expression. In addition, several genes of significantly expressed genes including growth factor genes and apoptosis-related genes were analyzed using RNA interference to Bmal1 mRNA whether these were controlled under circadian clockwork.
2013-07-23 | E-GEOD-49080 | biostudies-arrayexpress
Project description:This is a simple and reproducible protocol in which the hCOs generated are homogeneous and develop properly presenting proliferative ventricular zones (VZs) formed by neural and radial glia (RG) precursors that differentiate to give rise to mature neurons and glia cells. The hCOs at four weeks of maturation present additional cell types such as oligodendrocyte precursors, astrocytes, microglia and endothelial cells as shown by single‐cell RNA sequencing (scRNA‐seq) and immunohistochemistry (IHC).
2023-10-01 | GSE242329 | GEO
Project description:Circadian rhythms are a series of endogenous autonomous 24-hour oscillations generated by the circadian clock. At the molecular level, the circadian clock is generated by a transcription-translation feedback loop, where BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. In this project, we aimed at finding the interactome of PER2 protein in human U2OS osteosarcoma cell line using proximity-dependent biotin identification (BioID) technique. U2OS clones overexpressing PER2-BioID2 or BioID2 were treated with dexamethasone in order to reset the circadian rhythm, and cells were then incubated in biotin-containing media for 12 hours to label the proteins in close proximity of PER2-BioID2. Samples were collected after 36 and 48 hours of the resetting to identify the labeled proteins by mass spectrometry. In addition to known interactors such as CRY1 and CRY2, many novel interactors were identified. In summary, we obtained a network of PER2 interactome and confirmed some of the novel interactions using classical the co-immunoprecipitation method.
2022-04-22 | PXD025179 | Pride
Project description:Disrupted sleep-wake and molecular circadian rhythms are a feature of aging associated with metabolic disease and reduced levels of NAD+, yet whether changes in nucleotide metabolism control circadian behavioral and genomic rhythms remains unknown. Here we reveal that supplementation with the NAD+ precursor nicotinamide riboside (NR) markedly reprograms metabolic and stress-response pathways that decline with aging through inhibition of the clock repressor PER2. NR enhances BMAL1 chromatin binding genome-wide through PER2K680 deacetylation, which in turn primes PER2 phosphorylation within a domain that controls nuclear transport and stability and which is mutated in human advanced sleep phase syndrome. In old mice, dampened BMAL1 chromatin binding, transcriptional oscillations, mitochondrial respiration rhythms, and late evening activity are restored by NAD+ repletion to youthful levels with NR. These results reveal effects of NAD+ on metabolism and the circadian system with aging through the spatiotemporal control of the molecular clock.
2020-05-04 | GSE133989 | GEO
Project description:Oxygen is vital for the development and survival of mammals. In response to hypoxia, the brain initiates numerous adaptive responses at the organ level as well as at the molecular and cellular levels, including the alteration of gene expression. Astrocytes play critical roles in the proper functioning of the brain; thus the manner in which astrocytes respond to hypoxia is likely important in determining the outcome of brain hypoxia. Here, we used microarray gene expression profiling and data-analysis algorithms to identify and analyze hypoxia-responsive genes in primary human astrocytes. We also compared gene expression patterns in astrocytes with those in human HeLa cells and pulmonary artery endothelial cells (ECs). Remarkably, in astrocytes, five times as many genes were induced as suppressed, whereas in HeLa and pulmonary ECs, as many as or more genes were suppressed than induced. More genes encoding hypoxia-inducible functions, such as glycolytic enzymes and angiogenic growth factors, were strongly induced in astrocytes compared with HeLa cells. Furthermore, gene ontology and computational algorithms revealed that many target genes of the EGF and insulin signaling pathways and the transcriptional regulators Myc, Jun, and p53 were selectively altered by hypoxia in astrocytes. Indeed, Western blot analysis confirmed that two major signal transducers mediating insulin and EGF action, Akt and MEK1/2, were activated by hypoxia in astrocytes. These results provide a global view of the signaling and regulatory network mediating oxygen regulation in human astrocytes.. This SuperSeries is composed of the following subset Series:; GSE3045: Astrocytic response to hypoxia; GSE3051: HeLa response to hypoxia Experiment
2007-10-26 | E-GEOD-4483 | biostudies-arrayexpress
Project description:Many mammalian proteins have circadian cycles of production and degradation, but de novo transcription is only responsible for a small fraction of this rhythmicity. We used ribosome profiling to quantify RNA translation in liver from circadian-entrained mice transferred to constant darkness conditions over a 24-h period and compared translation levels to absolute protein production for 16 circadian proteins. We observed a delay between translation and peak protein levels for several circadian genes covering a wide range of translation efficiencies. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). Increased uORFs in 5' UTRs was associated with decreased ribosome binding in downstream ORFs and reduced expression of synthetic reporter constructs in vitro and in single cells. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells without altering phase or period. Genomic Per2 uORF mutation using CRISPR/Cas9 increased PER2 expression in PER2:Luc MEFs and reduced sleep in Per2 uORF mutant mice. These results suggest that post-transcriptional processes shape translation of mRNA transcripts, which can impact physiological rhythms and sleep.
2023-05-05 | GSE201732 | GEO
Project description:Astrocytes were purified from mouse cortices and human cortex tissue by immunopanning with HepaCAM. All treatments were proceeded on 3DIV in serum-free medium. Astrocytes were treated by TNFα for 48hr, by Poly I:C for 72hr, or by hypoxia for 72hr. Acutely purified atrocytes RNA was harvest immediately after HepaCAM immunopanning. Serum selected astrocytes RNA was collected after 4-6 days culturing in serum medium. Serum-free cultured astrocytes RNA was collected on 5-6 DIV. Serum-selection purified human astrocytes were transplanted into P2-11 Rag2 immunodeficient mouse brains, and after about 8 months, transplanted human and host mouse astocytes RNA was collected acutely on HepaCAM immunopanning dish.
2021-04-21 | GSE147870 | GEO