scholarly journals Human lactoferrin enhances the expression of transcription factor c-Fos in neuronal cultures under stimulated conditions

2021 ◽  
pp. 74-78
Author(s):  
М.Ю. Копаева ◽  
А.М. Азиева ◽  
А.Б. Черепов ◽  
М.В. Нестеренко ◽  
И.Ю. Зарайская
Keyword(s):  
Transcription Factor ◽  
Cellular Localization ◽  
Culture Medium ◽  
Human Lactoferrin ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Fos Expression ◽  
Factor C ◽  
Number Of Cells

Целью настоящей работы стало исследование влияния лактоферрина (Лф) человека на экспрессию транскрипционного фактора c-Fos в первичных нейрональных культурах после физиологической стимуляции, определение клеточной локализации Лф человека и возможной колокализации экзогенного белка с индуцированной экспрессией c-Fos. Методы. Первичные диссоциированные клеточные культуры получали из гиппокампа головного мозга новорожденных мышей (Р0-Р1) линии С57Вl/6. Индукцию экспрессии белка c-Fos в клетках осуществляли путем трехкратного добавления 50 мМ KСl в культуральную среду на 8-й день культивирования in vitro. Анализ содержания c-Fos проводили иммунофлюоресцентным методом через 2 часа после стимуляции. Результаты. Лф детектировался как в цитоплазме, так и в ядрах отдельных клеток культуры после стимуляции KСl. В ядрах некоторых клеток была выявлена колокализация включения Лф и экспрессии c-Fos. Было обнаружено, что предварительное введение Лф в культуральную среду увеличивало количество клеток, экспрессирующих c-Fos после добавления 50 мМ KСl. The aims of this research were 1) to study the effect of human lactoferrin (Lf) on the expression of the c-Fos transcription factor in primary neuronal cultures after physiological stimulation; 2) to determine the cellular localization of human Lf and possible colocalization of an exogenous protein with induced c-Fos expression. Methods. Primary dissociated cell cultures were obtained from the hippocampus of newborn C57Bl/6 mice (P0-P1). The expression of c-Fos was induced by addition of 50 mM KCl to the culture medium at 8 day in vitro. c-Fos content was analyzed by immunofluorescence 2 hrs after stimulation. Results. Lf was detected in cytoplasm and in nuclei after stimulation KCl. Lf inclusion and c-Fos expression were colocalized in the nuclei of some cells. Thus, results showed that pretreatment with Lf led to increase in the number of cells expressing c-Fos after exposure to 50 mM KCl.

scholarly journals Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

2009 ◽  
Vol 29 (9) ◽  
pp. 2431-2442 ◽  
Author(s):  
Zhongmin Yuan ◽  
Shoufang Gong ◽  
Jingyan Luo ◽  
Zhihao Zheng ◽  
Bin Song ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neuronal Apoptosis ◽  
Target Genes ◽  
Dominant Negative ◽  
Bimolecular Fluorescence Complementation ◽  
Activator Protein 1 ◽  
Fos Expression ◽  
Activating Transcription Factor ◽  
Living Neurons ◽  
Factor C

ABSTRACT The activator protein 1 (AP-1) transcription factor c-Jun is crucial for neuronal apoptosis. However, c-Jun dimerization partners and the regulation of these proteins in neuronal apoptosis remain unknown. Here we report that c-Jun-mediated neuronal apoptosis requires the concomitant activation of activating transcription factor-2 (ATF2) and downregulation of c-Fos. Furthermore, we have observed that c-Jun predominantly heterodimerizes with ATF2 and that the c-Jun/ATF2 complex promotes apoptosis by triggering ATF activity. Inhibition of c-Jun/ATF2 heterodimerization using dominant negative mutants, small hairpin RNAs, or decoy oligonucleotides was able to rescue neurons from apoptosis, whereas constitutively active ATF2 and c-Jun mutants were found to synergistically stimulate apoptosis. Bimolecular fluorescence complementation analysis confirmed that, in living neurons, c-Fos downregulation facilitates c-Jun/ATF2 heterodimerization. A chromatin immunoprecipitation assay also revealed that c-Fos expression prevents the binding of c-Jun/ATF2 heterodimers to conserved ATF sites. Moreover, the presence of c-Fos is able to suppress the expression of c-Jun/ATF2-mediated target genes and, therefore, apoptosis. Taken together, our findings provide evidence that potassium deprivation-induced neuronal apoptosis is mediated by concurrent upregulation of c-Jun/ATF2 heterodimerization and downregulation of c-Fos expression. This paradigm demonstrates opposing roles for ATF2 and c-Fos in c-Jun-mediated neuronal apoptosis.

Differential addressing of 5-HT1A and 5-HT1B receptors in epithelial cells and neurons

1999 ◽  
Vol 112 (6) ◽  
pp. 967-976
Author(s):  
A. Ghavami ◽  
K.L. Stark ◽  
M. Jareb ◽  
S. Ramboz ◽  
L. Segu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neuronal Cultures ◽  
Striatal Neurons ◽  
Primary Neuronal Cultures ◽  
Axon Terminals ◽  
In Vivo Experiments ◽  
In Vitro Systems ◽  
The Central Nervous System

The 5-HT1A and 5-HT1B serotonin receptors are expressed in a variety of neurons in the central nervous system. While the 5-HT1A receptor is found on somas and dendrites, the 5-HT1B receptor has been suggested to be localized predominantly on axon terminals. To study the intracellular addressing of these receptors, we have used in vitro systems including Madin-Darby canine kidney (MDCK II) epithelial cells and primary neuronal cultures. Furthermore, we have extended these studies to examine addressing in vivo in transgenic mice. In epithelial cells, 5-HT1A receptors are found on both apical and basolateral membranes while 5-HT1B receptors are found exclusively in intracellular vesicles. In hippocampal neuronal cultures, 5-HT1A receptors are expressed on somatodendritic membranes but are absent from axons. In contrast, 5-HT1B receptors are found on both dendritic and axonal membranes, including growth cones where they accumulate. Using 5-HT1A and 5-HT1B knockout mice and the binary tTA/tetO system, we generated mice expressing these receptors in striatal neurons. These in vivo experiments demonstrate that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. Therefore, in all systems we have examined, there is a differential sorting of the 5-HT1A and 5-HT1B receptors. Furthermore, we conclude that our in vivo transgenic system is the only model that reconstitutes proper sorting of these receptors.

scholarly journals Nuclear Import of c-Jun Is Mediated by Multiple Transport Receptors

2007 ◽  
Vol 282 (38) ◽  
pp. 27685-27692 ◽  
Author(s):  
Inga Waldmann ◽  
Sarah Wälde ◽  
Ralph H. Kehlenbach
Keyword(s):  
Transcription Factor ◽  
Nuclear Import ◽  
Leucine Zipper ◽  
Dependent Manner ◽  
Reporter Protein ◽  
Permeabilized Cells ◽  
Import Receptors ◽  
Factor C

c-Jun and c-Fos are major components of the transcriptional complex AP-1. Here, we investigate the nuclear import pathway(s) of the transcription factor c-Jun. c-Jun bound specifically to the nuclear import receptors importin β, transportin, importin 5, importin 7, importin 9, and importin 13. In digitonin-permeabilized cells, importin β, transportin, importin 7, and importin 9 promoted efficient import of c-Jun into the nucleus. Importin α, by contrast, inhibited nuclear import of c-Jun in vitro. A single basic region preceding the leucine zipper of c-Jun functions as a nuclear localization signal (NLS) and was required for interaction with all tested import receptors. In vivo, nuclear import of a c-Jun reporter protein lacking the leucine zipper strictly depended on this NLS. In a leucine zipper-dependent manner, c-Jun with mutations in its NLS was still imported into the nucleus in a complex with endogenous leucine zipper proteins or, for example, with cotransfected c-Fos. Together, these results explain the highly efficient nuclear import of the transcription factor c-Jun.

scholarly journals Quantifying Amyloid Beta (Aβ)–Mediated Changes in Neuronal Morphology in Primary Cultures

2012 ◽  
Vol 17 (6) ◽  
pp. 835-842 ◽  
Author(s):  
Lan Nguyen ◽  
Sarah Wright ◽  
Mike Lee ◽  
Zhao Ren ◽  
John-Michael Sauer ◽  
...  
Keyword(s):  
Amyloid Beta ◽  
Cell Injury ◽  
Primary Cultures ◽  
Neuronal Morphology ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Blocking Antibody ◽  
Cortical Cultures ◽  
Hippocampal Cultures

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) in the brain. Herein, the authors quantify Aβ-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aβ caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aβ-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aβ-mediated cytotoxicity. Finally, we showed that Aβ challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aβ-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.

scholarly journals Non-proliferative neurogenesis in human periodontal ligament stem cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carlos Bueno ◽  
Marta Martínez-Morga ◽  
Salvador Martínez
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Periodontal Ligament ◽  
Cell Biology ◽  
Basic Knowledge ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Periodontal Ligament Stem Cells ◽  
Sequence Of Events

AbstractUnderstanding the sequence of events from undifferentiated stem cells to neuron is not only important for the basic knowledge of stem cell biology, but also for therapeutic applications. In this study we examined the sequence of biological events during neural differentiation of human periodontal ligament stem cells (hPDLSCs). Here, we show that hPDLSCs-derived neural-like cells display a sequence of morphologic development highly similar to those reported before in primary neuronal cultures derived from rodent brains. We observed that cell proliferation is not present through neurogenesis from hPDLSCs. Futhermore, we may have discovered micronuclei movement and transient cell nuclei lobulation coincident to in vitro neurogenesis. Morphological analysis also reveals that neurogenic niches in the adult mouse brain contain cells with nuclear shapes highly similar to those observed during in vitro neurogenesis from hPDLSCs. Our results provide additional evidence that it is possible to differentiate hPDLSCs to neuron-like cells and suggest the possibility that the sequence of events from stem cell to neuron does not necessarily requires cell division from stem cell.

scholarly journals The ingestion and digestion of human lactoferrin by mouse peritoneal macrophages and the transfer of its iron into ferritin.

1977 ◽  
Vol 146 (3) ◽  
pp. 817-827 ◽  
Author(s):  
J L van Snick ◽  
B Markowetz ◽  
P L Masson
Keyword(s):  
Iron Content ◽  
Culture Medium ◽  
Reticuloendothelial System ◽  
Peritoneal Macrophages ◽  
Human Lactoferrin ◽  
Saturation Point ◽  
Intracellular Digestion ◽  
Additional Support ◽  
Mouse Peritoneal Macrophages

Human lactoferrin (Lf) labeled with 125I and/or 59Fe was found to be ingested in vitro by mouse peritoneal macrophages (MPM). The uptake measured after 15 h incubation reached a saturation point at a concentration of 200 microgram/ml in the culture medium, whatever was the iron content of Lf. In such conditions, the uptake of transferrin (Tf) used as a control was 10 times lower. At a concentration of 80 microgram/ml in the medium, one cell picked up about 0.7 X 10(6) molecules of Lf per hour, and 0.13 X 10(6) molecules of Tf per hour. Iron-saturated Lf disappeared from MPM with a half life of 14.5 h, whereas the halflife of iron-free Lf was 4.2 h. Concomitant with the intracellular digestion of Lf, the iron was transmitted to ferritin. These data provide additional support for the hypothesis that Lf plays a key role in iron turnover, especially at the level of the reticuloendothelial system where iron is recovered from the catabolism of erythrocytes.

scholarly journals Isoflurane but not Fentanyl Causes Apoptosis in Immature Primary Neuronal Cells

2017 ◽  
Vol 11 (1) ◽  
pp. 39-47
Author(s):  
Monika Berns ◽  
Anna Christine Wolter ◽  
Christoph Bührer ◽  
Stefanie Endesfelder ◽  
Thoralf Kerner
Keyword(s):  
Cell Viability ◽  
Neuronal Cells ◽  
Neuronal Cultures ◽  
Primary Neurons ◽  
Primary Neuronal Cultures ◽  
Amino Butyric Acid ◽  
Wide Range ◽  
Primary Neuronal Cells ◽  
The Impact

Background: Anaesthetics are widely used in new-borns and preterm infants, although it is known that they may adversely affect the developing brain. Objective: We assessed the impact of the volatile anaesthetic, isoflurane, and the intravenous analgesic, fentanyl, on immature and mature embryonic neuronal cells. Methods: Primary neuronal cultures from embryonic rats (E18) cultured for 5 (immature) or 15 days (mature) in vitro (DIV), respectively, were exposed to isoflurane (1.5 Vol.%) or fentanyl (0.8 - 200 ng/ml) for 24 hours. Experiments were repeated in the presence of the γ-amino butyric acid-A (GABAA) receptor antagonists, bicuculline or picrotoxin (0.1 mmol/l), or the pancaspase inhibitor zVAD-fmk (20 nmol/l). Cell viability was assessed by methyltetrazolium (MTT) metabolism or lactate dehydrogenase (LDH) release. Results: Isoflurane reduced cell viability significantly in primary neuronal cells cultured for 5 DIV (Δ MTT -28 ±13%, Δ LDH +143 ±15%). Incubation with bicuculline, picrotoxin or zVAD-fmk protected the cells mostly from isoflurane toxicity. After 15 DIV, cell viability was not reduced by isoflurane. Viability of primary neurons cultured for 5 DIV did not change with fentanyl over the wide range of concentrations tested. Conclusion: Immature primary neurons may undergo apoptosis following exposure to isoflurane but are unaffected by fentanyl. Mature primary neurons were not affected by isoflurane exposure.

Platelet-derived growth factor C signaling is a potential therapeutic target for radiation proctopathy

2021 ◽  
Vol 13 (582) ◽  
pp. eabc2344
Author(s):  
Weisi Lu ◽  
Yunling Xie ◽  
Binjie Huang ◽  
Tenghui Ma ◽  
Huaiming Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Growth Factor ◽  
Platelet Derived Growth Factor ◽  
In Vitro Assays ◽  
Tissue Samples ◽  
Pelvic Malignancies ◽  
Genome Wide ◽  
Factor C ◽  
Chemokine Receptor 4

Radiation proctopathy (RP) is characterized by inflammation of colorectal tissue and is a common complication of radiation therapy for pelvic malignancies with high incidence but lacking effective treatment. Here, we found that platelet-derived growth factor C (PDGF-C) and fibrosis markers were up-regulated in tissue samples from patients with RP and in rectal tissues after irradiation in a mouse model of RP. Genetic deletion of Pdgf-c in mice ameliorated RP-induced injuries. Genome-wide gene expression profiling and in vitro assays revealed that the promotive effect of PDGF-C in RP development was mediated by activation of PDGF receptors (PDGFRs) and C-X-C motif chemokine receptor 4, a proinflammatory chemokine regulated by transcription factor ETS variant transcription factor 1. Treatment with crenolanib, a selective inhibitor of PDGFRs, prevented or reduced RP in mice after irradiation. These results reveal that inhibition of PDGF-C signaling may have therapeutic value for the treatment of RP.

scholarly journals Endotoxin Preconditioning Protects against the Cytotoxic Effects of TNFα after Stroke: A Novel Role for TNFα in LPS-Ischemic Tolerance

2007 ◽  
Vol 27 (10) ◽  
pp. 1663-1674 ◽  
Author(s):  
Holly L Rosenzweig ◽  
Manabu Minami ◽  
Nikola S Lessov ◽  
Sarah C Coste ◽  
Susan L Stevens ◽  
...  
Keyword(s):  
Ischemic Injury ◽  
Artery Occlusion ◽  
Ischemic Tolerance ◽  
Neuronal Cultures ◽  
Death Domain ◽  
Primary Neuronal Cultures ◽  
Cytotoxic Effects ◽  
Intracerebral Administration ◽  
Lps Preconditioning

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury. Tumor necrosis factor-α (TNFα) is protective in LPS-induced preconditioning yet exacerbates neuronal injury in ischemia. Here, we define dual roles of TNFα in LPS-induced ischemic tolerance in a murine model of stroke and in primary neuronal cultures in vitro, and show that the cytotoxic effects of TNFα are attenuated by LPS preconditioning. We show that LPS preconditioning significantly increases circulating levels of TNFα before middle cerebral artery occlusion in mice and show that TNFα is required to establish subsequent neuroprotection against ischemia, as mice lacking TNFα are not protected from ischemic injury by LPS preconditioning. After stroke, LPS preconditioned mice have a significant reduction in the levels of TNFα (~ threefold) and the proximal TNFα signaling molecules, neuronal TNF-receptor 1 (TNFR1), and TNFR-associated death domain (TRADD). Soluble TNFR1 (s-TNFR1) levels were significantly increased after stroke in LPS-preconditioned mice (~ 2.5-fold), which may neutralize the effect of TNFα and reduce TNFα-mediated injury in ischemia. Importantly, LPS-preconditioned mice show marked resistance to brain injury caused by intracerebral administration of exogenous TNFα after stroke. We establish an in vitro model of LPS preconditioning in primary cortical neuronal cultures and show that LPS preconditioning causes significant protection against injurious TNFα in the setting of ischemia. Our studies suggest that TNFα is a twin-edged sword in the setting of stroke: TNFα upregulation is needed to establish LPS-induced tolerance before ischemia, whereas suppression of TNFα signaling during ischemia confers neuroprotection after LPS preconditioning.

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