Pathological Neuroinflammatory Conversion of Reactive Astrocytes Is Induced by Microglia and Involves Chromatin Remodeling

Following brain injury or in neurodegenerative diseases, astrocytes become reactive and may suffer pathological remodeling, features of which are the loss of their homeostatic functions and a pro-inflammatory gain of function that facilitates neurodegeneration. Pharmacological intervention to modulate this astroglial response and neuroinflammation is an interesting new therapeutic research strategy, but it still requires a deeper understanding of the underlying cellular and molecular mechanisms of the phenomenon. Based on the known microglial–astroglial interaction, the prominent role of the nuclear factor kappa B (NF-κB) pathway in mediating astroglial pathological pro-inflammatory gain of function, and its ability to recruit chromatin-remodeling enzymes, we first explored the microglial role in the initiation of astroglial pro-inflammatory conversion and then monitored the progression of epigenetic changes in the astrocytic chromatin. Different configurations of primary glial culture were used to modulate microglia–astrocyte crosstalk while inducing pro-inflammatory gain of function by lipopolysaccharide (LPS) exposure. In vivo, brain ischemia by cortical devascularization (pial disruption) was performed to verify the presence of epigenetic marks in reactive astrocytes. Our results showed that 1) microglia is required to initiate the pathological conversion of astrocytes by triggering the NF-κB signaling pathway; 2) this interaction is mediated by soluble factors and induces stable astroglial phenotypic changes; 3) the pathological conversion promotes chromatin remodeling with stable increase in H3K9K14ac, temporary increase in H3K27ac, and temporary reduction in heterochromatin mark H3K9me3; and 4) in vivo reactive astrocytes show increased H3K27ac mark in the neuroinflammatory milieu from the ischemic penumbra. Our findings indicate that astroglial pathological pro-inflammatory gain of function is associated with profound changes in the configuration of astrocytic chromatin, which in turn are initiated by microglia-derived cues. These results open a new avenue in the study of potential pharmacological interventions that modify the initiation and stabilization of astroglial pathological remodeling, which would be useful in acute and chronic CNS injury. Epigenetic changes represent a plausible pharmacological target to interfere with the stabilization of the pathological astroglial phenotype.

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Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01152.2007 ◽

2008 ◽

Vol 294 (2) ◽

pp. H699-H707 ◽

Cited By ~ 46

Author(s):

Ellen Steward Pentz ◽

Maria Luisa S. Sequeira Lopez ◽

Magali Cordaillat ◽

R. Ariel Gomez

Keyword(s):

Chromatin Remodeling ◽

In Vitro Model ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Histone H4 Acetylation ◽

Renin Gene ◽

Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

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Microvascular Mechanisms of Polyphosphate-Induced Neutrophil-Endothelial Cell Interactions in vivo

European Surgical Research ◽

10.1159/000497435 ◽

2019 ◽

Vol 60 (1-2) ◽

pp. 53-62

Author(s):

Feifei Du ◽

Yongzhi Wang ◽

Zhiyi Ding ◽

Matthias W. Laschke ◽

Henrik Thorlacius

Keyword(s):

Molecular Mechanisms ◽

Inflammatory Diseases ◽

Pharmacological Intervention ◽

Lymphocyte Function ◽

Microvascular Endothelium ◽

Recruitment Methods ◽

Leukocyte Accumulation ◽

Tnf Α

Background: Polyphosphates (PolyPs) have been reported to exert pro-inflammatory effects. However, the molecular mechanisms regulating PolyP-provoked tissue accumulation of leukocytes are not known. The aim of the present investigation was to determine the role of specific adhesion molecules in PolyP-mediated leukocyte recruitment. Methods: PolyPs and TNF-α were intrascrotally administered, and anti-P-selectin, anti-E-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1), and neutrophil depletion antibodies were injected intravenously or intraperitoneally. Intravital microscopy of the mouse cremaster microcirculation was used to examine leukocyte-endothelium interactions and recruitment in vivo. Results: Intrascrotal injection of PolyPs increased leukocyte accumulation. Depletion of neutrophils abolished PolyP-induced leukocyte-endothelium interactions, indicating that neutrophils were the main leukocyte subtype responding to PolyP challenge. Immunoneutralization of P-selectin and PSGL-1 abolished PolyP-provoked neutrophil rolling, adhesion, and emigration. Moreover, immunoneutralization of Mac-1 and LFA-1 had no impact on neutrophil rolling but markedly reduced neutrophil adhesion and emigration evoked by PolyPs. Conclusion: These results suggest that P-selectin and PSGL-1 exert important roles in PolyP-induced inflammatory cell recruitment by mediating neutrophil rolling. In addition, our data show that Mac-1 and LFA-1 are necessary for supporting PolyP-triggered firm adhesion of neutrophils to microvascular endothelium. These novel findings define specific molecules as potential targets for pharmacological intervention in PolyP-dependent inflammatory diseases.

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Demonstration of reduced in vivo surface expression of activating mutant thyrotrophin receptors in thyroid sections

Acta Endocrinologica ◽

10.1530/eje.0.1460163 ◽

2002 ◽

pp. 163-171 ◽

Cited By ~ 9

Author(s):

M Sequeira ◽

B Jasani ◽

D Fuhrer ◽

M Wheeler ◽

M Ludgate

Keyword(s):

Molecular Mechanisms ◽

Thyroid Tissue ◽

Surface Expression ◽

Germline Mutations ◽

Antigen Retrieval ◽

Graves Disease ◽

Paraffin Sections ◽

Gain Of Function

OBJECTIVE: Thyroid function and growth are controlled by TSH. Hyperthyroidism can be due to Graves' Disease (GD), in which thyroid-stimulating antibodies mimic TSH, or gain-of-function mutations in the TSH receptor (TSHR). These activating mutations have poor surface expression when assessed in non-thyroidal cells in vitro but nothing is known of their in vivo behaviour. Several TSHR antibodies have been produced but none has been applied to thyroid paraffin sections. This study aimed to develop a technique suitable for use on paraffin sections and apply it to investigate TSHR expression in thyroids harbouring activating TSHR germline mutations compared with normal and GD thyroids. DESIGN AND METHODS: Immunocytochemistry coupled with antigen retrieval, using a spectrum of antibodies to the TSHR, was applied to paraffin sections of GD thyroid tissue. Subsequently, TSHR immunoreactivity was examined in three normal thyroids, three patients with GD and three patients with familial hyperthyroidism, due to different gain-of-function TSHR germline mutations, using the optimised protocol. RESULTS: Two antibodies, A10 and T3-495, to the extracellular domain (ECD) and membrane spanning region (MSR) of the TSHR respectively, produced specific basolateral staining of thyroid follicular cells. In normal and GD thyroids, basolateral staining with T3-495 was generally more intense than with A10, suggesting a possible surfeit of MSR over ECD. Graves' Disease thyroids have more abundant TSHR than normal glands. In contrast, thyroids harbouring gain-of-function mutations have the lowest expression in vivo, mirroring in vitro findings. CONCLUSIONS: The development of an immunocytochemical method applicable to paraffin sections has demonstrated that different molecular mechanisms causing hyperthyroidism result in the lowest (mutation) and highest (autoimmunity) levels of receptor at the thyrocyte surface.

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The Intrinsic Virtues of EGCG, an Extremely Good Cell Guardian, on Prevention and Treatment of Diabesity Complications

Molecules ◽

10.3390/molecules25133061 ◽

2020 ◽

Vol 25 (13) ◽

pp. 3061

Author(s):

Maria Assunta Potenza ◽

Dominga Iacobazzi ◽

Luca Sgarra ◽

Monica Montagnani

Keyword(s):

Free Radicals ◽

Chromatin Remodeling ◽

Molecular Mechanisms ◽

Metabolic Diseases ◽

Cardiovascular Complications ◽

Epigenetic Changes ◽

Obesity And Diabetes ◽

Membrane Target ◽

Multiple Signaling ◽

Pathologic Processes

The pandemic proportion of diabesity—a combination of obesity and diabetes—sets a worldwide health issue. Experimental and clinical studies have progressively reinforced the pioneering epidemiological observation of an inverse relationship between consumption of polyphenol-rich nutraceutical agents and mortality from cardiovascular and metabolic diseases. With chemical identification of epigallocatechin-3-gallate (EGCG) as the most abundant catechin of green tea, a number of cellular and molecular mechanisms underlying the activities of this unique catechin have been proposed. Favorable effects of EGCG have been initially attributed to its scavenging effects on free radicals, inhibition of ROS-generating mechanisms and upregulation of antioxidant enzymes. Biologic actions of EGCG are concentration-dependent and under certain conditions EGCG may exert pro-oxidant activities, including generation of free radicals. The discovery of 67-kDa laminin as potential EGCG membrane target has broaden the likelihood that EGCG may function not only because of its highly reactive nature, but also via receptor-mediated activation of multiple signaling pathways involved in cell proliferation, angiogenesis and apoptosis. Finally, by acting as epigenetic modulator of DNA methylation and chromatin remodeling, EGCG may alter gene expression and modify miRNA activities. Despite unceasing research providing detailed insights, ECGC composite activities are still not completely understood. This review summarizes the most recent evidence on molecular mechanisms by which EGCG may activate signal transduction pathways, regulate transcription factors or promote epigenetic changes that may contribute to prevent pathologic processes involved in diabesity and its cardiovascular complications.

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BAF60a Mediates Critical Interactions between Nuclear Receptors and the BRG1 Chromatin-Remodeling Complex for Transactivation

Molecular and Cellular Biology ◽

10.1128/mcb.23.17.6210-6220.2003 ◽

2003 ◽

Vol 23 (17) ◽

pp. 6210-6220 ◽

Cited By ~ 134

Author(s):

Pei-Wen Hsiao ◽

Christy J. Fryer ◽

Kevin W. Trotter ◽

Weidong Wang ◽

Trevor K. Archer

Keyword(s):

Nuclear Receptors ◽

Chromatin Remodeling ◽

Molecular Mechanisms ◽

Hormone Receptors ◽

Nuclear Receptor Coactivators ◽

Chromatin Remodeling Complex ◽

Truncation Mutant ◽

Promoter Recruitment

ABSTRACT Nuclear hormone receptors are ligand-dependent transcriptional regulators that modulate chromatin structure. However, the precise molecular mechanisms by which receptors recruit chromatin-remodeling activity are not fully elucidated. We show that in the absence of its ligand-binding domain, the glucocorticoid receptor (GR) is able to interact with both nuclear receptor coactivators and the BRG1 chromatin-remodeling complex in vivo. Individually, the GR makes direct interactions with BRG1-associated factor 60a (BAF60a) and BAF57, but not with BRG1, BAF155, or BAF170. Further, BAF60a possesses at least two interaction surfaces, one for GR and BRG1 and a second for BAF155 and BAF170. A GR mutant, GR(R488Q), that fails to interact with BAF60a in vitro has reduced chromatin-remodeling activity and reduced transcriptional activity from the promoter assembled as chromatin in vivo. Stable expression of a BAF60a truncation mutant, BAF60a4-140, caused chromatin-specific loss of GR functions in vivo. In the presence of the BAF60a mutant, the GR fails to interact with the BRG1 complex and consequently is also deficient in its ability to activate transcription from chromatin. Thus, in addition to previously identified BAF250, BAF60a may provide another critical and direct link between nuclear receptors and the BRG1 complex that is required for promoter recruitment and subsequent chromatin remodeling.

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miR-155 accelerates proliferation of mouse hepatocytes during liver regeneration by directly targeting SOCS1

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00072.2018 ◽

2018 ◽

Vol 315 (4) ◽

pp. G443-G453 ◽

Cited By ~ 7

Author(s):

Xia Lin ◽

Li Chen ◽

Haiyan Li ◽

Yu Liu ◽

Yanhong Guan ◽

...

Keyword(s):

Cell Cycle ◽

Liver Regeneration ◽

Partial Hepatectomy ◽

Molecular Mechanisms ◽

Pharmacological Intervention ◽

Cytokine Signaling ◽

Suppressor Of Cytokine Signaling ◽

Proliferation Of Hepatocytes

Liver regeneration after two-thirds partial hepatectomy (PH) is a clinically significant repair process for restoring proper liver architecture. Although microRNA-155 (miR-155) has been found to serve as a crucial microRNA regulator that controls liver cell function and proliferation, little is known about its specific role in the regenerating liver. Using a mouse model with miR-155 overexpression or miR-155 knockout, we investigated the molecular mechanisms of miR-155 in liver regeneration. We found a marked induction of miR-155 in C57BL/6 mice after PH. Furthermore, RL-m155 mice showed enhanced liver regeneration as a result of accelerated progression of hepatocytes into the cell cycle, mainly through an increase in cyclin levels. However, proliferation of hepatocytes was delayed in miR-155-deficient livers. Expression of suppressor of cytokine signaling 1 (SOCS1) was dramatically downregulated in the process of liver regeneration, and enhancement of SOCS1 contributed to impaired proliferation of hepatocytes. Additionally, in vitro and in vivo experiments showed that adenovirus- or adeno-associated virus-mediated overexpression of SOCS1 attenuated improved liver regeneration induced by miR-155 overexpression. Our study shows that miR-155 is a pro-proliferative regulator in liver regeneration by facilitating the cell cycle and directly targeting SOCS1. NEW & NOTEWORTHY Our findings suggest a microRNA-155 (miR-155)-mediated positive regulation pattern in liver regeneration. A series of in vivo and in vitro studies showed that miR-155 upregulation enhanced partial hepatectomy-induced proliferation of hepatocytes by promoting the cell cycle without inducing DNA damage or apoptosis. Suppressor of cytokine signaling 1, a target gene of miR-155, antagonized the proliferation-promoting effect of miR-155. Therefore, pharmacological intervention targeting miR-155 may be therapeutically beneficial in various liver diseases.

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Mesocorticolimbic circuit mechanisms underlying the effects of ketamine on dopamine: a translational imaging study

10.1101/748665 ◽

2019 ◽

Author(s):

Michelle Kokkinou ◽

Elaine E. Irvine ◽

David R. Bonsall ◽

Sridhar Natesan ◽

Lisa A. Wells ◽

...

Keyword(s):

Locomotor Activity ◽

Molecular Mechanisms ◽

Imaging Study ◽

Dopamine Neurons ◽

Striatal Dopamine ◽

Pharmacological Intervention ◽

Dopamine Synthesis ◽

Midbrain Dopamine ◽

Midbrain Dopamine Neurons

ABSTRACTPatients with schizophrenia show increased striatal dopamine synthesis capacity in imaging studies. However, the mechanism underlying this is unclear but may be due to N-methyl-D-aspartate receptor (NMDAR) hypofunction and parvalbumin (PV) neuronal dysfunction leading to disinhibition of mesostriatal dopamine neurons. Here, we test this in a translational mouse imaging study using a ketamine model. Mice were treated with sub-chronic ketamine (30mg/kg) or saline followed by in-vivo positron emission tomography of striatal dopamine synthesis capacity, analogous to measures used in patients. Locomotor activity was measured using the open field test. In-vivo cell-type-specific chemogenetic approaches and pharmacological interventions were used to manipulate neuronal excitability. Immunohistochemistry and RNA sequencing were used to investigate molecular mechanisms. Sub-chronic ketamine increased striatal dopamine synthesis capacity (Cohen’s d=2.5, P<0.001) and locomotor activity. These effects were countered by inhibition of midbrain dopamine neurons, and by activation of cortical and ventral subiculum PV interneurons. Sub-chronic ketamine reduced PV expression in these neurons. Pharmacological intervention with SEP-363856, a novel psychotropic agent with agonism at trace amine receptor 1 (TAAR1), significantly reduced the ketamine-induced increase in dopamine synthesis capacity. These results show that sub-chronic ketamine treatment in mice mimics the dopaminergic alterations in patients with psychosis, and suggest an underlying neurocircuit involving PV interneuron hypofunction in frontal cortex and hippocampus as well as activation of midbrain dopamine neurons. A novel TAAR1 agonist reversed the dopaminergic alterations suggesting a therapeutic mechanism for targeting presynaptic dopamine dysfunction in patients.

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SAT-144 Cooperative Mechanism of SREBP-Dependent Cholesterol Synthesis Pathway and P53 on Malignant Formation in Breast Cancer

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1670 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Akitoshi Nakayama ◽

Masataka Yokoyama ◽

Hidekazu Nagano ◽

Ikki Sakuma ◽

Naoko Hashimoto ◽

...

Keyword(s):

Breast Cancer ◽

Cholesterol Synthesis ◽

Molecular Mechanisms ◽

Three Dimensional ◽

Breast Cancer Cell Lines ◽

Mutant P53 ◽

Dependent Manner ◽

Gain Of Function ◽

Necessary And Sufficient

Abstract p53 is mutated more than half of human cancers, and mutant p53, a gain of function, can actively have functional consequences with tumorigenesis. However, its action of molecular mechanisms, particularly in vivo conditions, has not been fully are clarified. Here, we generated KO and KI (R280K) breast cancer cell lines for p53 using CRISPR/Cas9 system, and then performed a three-dimensional culture model. We found that the introduction of mutant p53 was solely able to mediate the transformation to poor architectural structure. Interestingly, our findings in statin-effect along with cholesterol synthesis pathway, especially isoprenoid dependency, revealed that this pathway is necessary and sufficient for the regulation of malignant architecture in SREBP2-dependent manner with cooperatively being controlled by mutant p53 on 3D-cultured breast cancer. Furthermore, in accordance with the malignancy progresses, SREBP2 was accumulated in nuclear and nuclear membrane portion with enhancement in malignant formation. In addition, we found that mutant p53 interacts with SREBP2, and consistently mutant p53 was associated with DHCR7 promoter in parallel with binding pattern of SREBP2. Thus, our results provide the novel insight into the mutant p53, a gain of function, and its linkage to poor architectural structure in 3D-cultured breast cancer cells via SREBP2-dependent isoprenoids regulation as potential therapeutic targets.

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Adhesive Mechanisms of Histone-Induced Neutrophil-Endothelium Interactions in the Muscle Microcirculation

European Surgical Research ◽

10.1159/000441778 ◽

2015 ◽

Vol 56 (1-2) ◽

pp. 19-31 ◽

Cited By ~ 2

Author(s):

Johanna Puegge ◽

Yongzhi Wang ◽

Jonas Roller ◽

Su Zhang ◽

Lingtao Luo ◽

...

Keyword(s):

Molecular Mechanisms ◽

Tissue Injury ◽

Cell Damage ◽

Leukocyte Recruitment ◽

Pharmacological Intervention ◽

Lymphocyte Function ◽

Leukocyte Accumulation ◽

Histone 3 ◽

Neutrophil Depletion

Background: Extracellular histones released during cell damage have the capacity to cause tissue injury associated with increased leukocyte accumulation. However, the molecular mechanisms regulating histone-induced leukocyte recruitment remain elusive. The objective of this study was to examine the role of adhesion molecules in histone-dependent leukocyte accumulation by use of intravital microscopy of the mouse cremaster microcirculation. Methods: Histone 3 and TNF-α were intrascrotally administered, and anti-P-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1) antibody and neutrophil depletion antibody were injected intravenously or intraperitoneally. Results: Intrascrotal injection of histone 3 dose-dependently increased leukocyte recruitment. Neutrophil depletion abolished intravascular and extravascular leukocytes after histone 3 challenge, suggesting that neutrophils were the dominating leukocyte subtype responding to histone stimulation. Pretreatment with an anti-P-selectin and an anti-PSGL-1 antibody abolished histone-stimulated neutrophil rolling, adhesion and emigration. When the anti-P-selectin or the anti-PSGL-1 antibody was administrated after histone 3 stimulation, neutrophil rolling was reduced, whereas the number of firmly adherent and emigrated neutrophils were unchanged, suggesting that the inhibitory effect of blocking P-selectin and PSGL-1 on neutrophil adhesion and recruitment was due to the reduction in neutrophil rolling. Moreover, pretreatment with antibodies against Mac-1 and LFA-1 had no effect of neutrophil rolling but abolished adhesion and emigration evoked by histone 3. Thus, our data demonstrate that P-selectin and PSGL-1 play an important role in histone-induced inflammatory cell recruitment by mediating neutrophil rolling as a precondition for histone-provoked firm adhesion and emigration in vivo. Moreover, we conclude that both Mac-1 and LFA-1 are critical in supporting histone-provoked firm adhesion of neutrophils to endothelial cells. Conclusion: These novel findings define specific selectins and integrins as potential targets for pharmacological intervention in histone-dependent inflammatory diseases.

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Aberrant Calcium Signals in Reactive Astrocytes: A Key Process in Neurological Disorders

International Journal of Molecular Sciences ◽

10.3390/ijms20040996 ◽

2019 ◽

Vol 20 (4) ◽

pp. 996 ◽

Cited By ~ 17

Author(s):

Eiji Shigetomi ◽

Kozo Saito ◽

Fumikazu Sano ◽

Schuichi Koizumi

Keyword(s):

Neurological Disorders ◽

Molecular Mechanisms ◽

Brain Disease ◽

Reactive Astrocytes ◽

Brain Functions ◽

Progression Of Disease ◽

Brain Insults ◽

The Brain

Astrocytes are abundant cells in the brain that regulate multiple aspects of neural tissue homeostasis by providing structural and metabolic support to neurons, maintaining synaptic environments and regulating blood flow. Recent evidence indicates that astrocytes also actively participate in brain functions and play a key role in brain disease by responding to neuronal activities and brain insults. Astrocytes become reactive in response to injury and inflammation, which is typically described as hypertrophy with increased expression of glial fibrillary acidic protein (GFAP). Reactive astrocytes are frequently found in many neurological disorders and are a hallmark of brain disease. Furthermore, reactive astrocytes may drive the initiation and progression of disease processes. Recent improvements in the methods to visualize the activity of reactive astrocytes in situ and in vivo have helped elucidate their functions. Ca2+ signals in reactive astrocytes are closely related to multiple aspects of disease and can be a good indicator of disease severity/state. In this review, we summarize recent findings concerning reactive astrocyte Ca2+ signals. We discuss the molecular mechanisms underlying aberrant Ca2+ signals in reactive astrocytes and the functional significance of aberrant Ca2+ signals in neurological disorders.

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