scholarly journals Neuron-Glia Crosstalk Plays a Major Role in the Neurotoxic Effects of Ketamine via Extracellular Vesicles

2021 ◽  
Vol 9 ◽  
Author(s):  
Donald H. Penning ◽  
Simona Cazacu ◽  
Aharon Brodie ◽  
Vesna Jevtovic-Todorovic ◽  
Steve N. Kalkanis ◽  
...  
Keyword(s):  
Cell Death ◽  
Extracellular Vesicles ◽  
Neuronal Cells ◽  
Neural Cells ◽  
General Anesthetics ◽  
Rt Pcr ◽  
Neurotoxic Effects ◽  
The Impact ◽  
Bdnf Pathway

Background: There is a compelling evidence from animal models that early exposure to clinically relevant general anesthetics (GAs) interferes with brain development, resulting in long-lasting cognitive impairments. Human studies have been inconclusive and are challenging due to numerous confounding factors. Here, we employed primary human neural cells to analyze ketamine neurotoxic effects focusing on the role of glial cells and their activation state. We also explored the roles of astrocyte-derived extracellular vesicles (EVs) and different components of the brain-derived neurotrophic factor (BDNF) pathway.Methods: Ketamine effects on cell death were analyzed using live/dead assay, caspase 3 activity and PARP-1 cleavage. Astrocytic and microglial cell differentiation was determined using RT-PCR, ELISA and phagocytosis assay. The impact of the neuron-glial cell interactions in the neurotoxic effects of ketamine was analyzed using transwell cultures. In addition, the role of isolated and secreted EVs in this cross-talk were studied. The expression and function of different components of the BDNF pathway were analyzed using ELISA, RT-PCR and gene silencing.Results: Ketamine induced neuronal and oligodendrocytic cell apoptosis and promoted pro-inflammatory astrocyte (A1) and microglia (M1) phenotypes. Astrocytes and microglia enhanced the neurotoxic effects of ketamine on neuronal cells, whereas neurons increased oligodendrocyte cell death. Ketamine modulated different components in the BDNF pathway: decreasing BDNF secretion in neurons and astrocytes while increasing the expression of p75 in neurons and that of BDNF-AS and pro-BDNF secretion in both neurons and astrocytes. We demonstrated an important role of EVs secreted by ketamine-treated astrocytes in neuronal cell death and a role for EV-associated BDNF-AS in this effect.Conclusions: Ketamine exerted a neurotoxic effect on neural cells by impacting both neuronal and non-neuronal cells. The BDNF pathway and astrocyte-derived EVs represent important mediators of ketamine effects. These results contribute to a better understanding of ketamine neurotoxic effects in humans and to the development of potential approaches to decrease its neurodevelopmental impact.

scholarly journals Autophagy Function and Regulation in Kidney Disease

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 100 ◽  
Author(s):  
Gur P. Kaushal ◽  
Kiran Chandrashekar ◽  
Luis A. Juncos ◽  
Sudhir V. Shah
Keyword(s):  
Cell Death ◽  
Renal Injury ◽  
Mammalian Target Of Rapamycin ◽  
Kidney Injury ◽  
Therapeutic Approaches ◽  
Regulated Necrosis ◽  
Atg Genes ◽  
Cellular Components ◽  
The Impact

Autophagy is a dynamic process by which intracellular damaged macromolecules and organelles are degraded and recycled for the synthesis of new cellular components. Basal autophagy in the kidney acts as a quality control system and is vital for cellular metabolic and organelle homeostasis. Under pathological conditions, autophagy facilitates cellular adaptation; however, activation of autophagy in response to renal injury may be insufficient to provide protection, especially under dysregulated conditions. Kidney-specific deletion of Atg genes in mice has consistently demonstrated worsened acute kidney injury (AKI) outcomes supporting the notion of a pro-survival role of autophagy. Recent studies have also begun to unfold the role of autophagy in progressive renal disease and subsequent fibrosis. Autophagy also influences tubular cell death in renal injury. In this review, we reported the current understanding of autophagy regulation and its role in the pathogenesis of renal injury. In particular, the classic mammalian target of rapamycin (mTOR)-dependent signaling pathway and other mTOR-independent alternative signaling pathways of autophagy regulation were described. Finally, we summarized the impact of autophagy activation on different forms of cell death, including apoptosis and regulated necrosis, associated with the pathophysiology of renal injury. Understanding the regulatory mechanisms of autophagy would identify important targets for therapeutic approaches.

Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae

2015 ◽  
Vol 25 (2-3) ◽  
pp. 120-128 ◽  
Author(s):  
Irfan Manzoor ◽  
Sulman Shafeeq ◽  
Muhammad Afzal ◽  
Oscar P. Kuipers
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transcriptional Activation ◽  
Promoter Region ◽  
Transcriptional Regulator ◽  
Transcriptional Activator ◽  
Regulatory Site ◽  
Rt Pcr ◽  
The Impact

In this study, we explore the impact of fucose on the transcriptome of <i>S. pneumoniae</i> D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (<i>fcs</i> operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the <i>fcs</i> operon, as a transcriptional activator of the <i>fcs</i> operon. We also predict a 19-bp putative FcsR regulatory site (5′-ATTTGAACATTATTCAAGT-3′) in the promoter region of the <i>fcs</i> operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the <i>fcs</i> operon.

Thrombopoietin Has Neural Protective Effect in a Neonatal Rat Model of Hypoxic-Ischemic Brain Damage

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5059-5059 ◽  
Author(s):  
Mo Yang ◽  
Lei Liu ◽  
Enyu Liang ◽  
Beng H Chong ◽  
Chunfu Li
Keyword(s):  
Brain Damage ◽  
Rat Model ◽  
Akt Signaling ◽  
Cerebral Hemispheres ◽  
Neonatal Rat ◽  
Neural Cells ◽  
Rt Pcr ◽  
Hematopoietic Growth Factors ◽  
Neonatal Rat Model

Abstract Objective: The infusion of bone marrow cells into the damaged brain has been proposed as a new clinical practice for this disorder. Alternatively, hematopoietic growth factors may have a direct role on neural protection or have a mobilizing effect on bone marrow stem/progenitor cells to circulation for brain repair. Based on our previous findings, there are many similarities between megakaryocytes and neurons on functions and antigen expression such as neural marker MAP2, GFAP and Tau, 5-HT2A, 2B and 2C receptors (Stem Cells, 2014). Thrombopoietin (TPO) is a growth factor for megakaryocytic lineage. We postulate that TPO may play a role on neural protection or regeneration. The effect of TPO on nervous system has not been well investigated. Methods: To validate this hypothesis, we investigated the expression and role of TPO/TPO receptors in neural cells and a neonatal rat model of hypoxic-ischemic (HIE) brain damage. Results: To investigate the effect of TPO on in-vivo neural protection, a neonatal rat model of HIE brain damage was established. Brain injury was measured by the percentage weight reduction of the ipsilateral cerebral hemisphere as compared to the contralateral hemisphere. There was significantly less brain atrophy in TPO treated animals (12.0±1.2% and 11.5±1.0%) when compared with the saline control (21.0±1.6% and 24.4±2.2%) at 7 and 28 days post-operation (P<0.05, n=12). The percentage of NSE (Neuron-specific enolase) positive cells in the forelimb area of the cortex in the right hemisphere was significantly higher in the TPO group than that of the saline group (P<0.05, n=12). An improvement in sensory motor functions was also demonstrated after TPO treatment. TPO mRNA was also identified in human cerebral hemispheres, cerebellum, and mouse neural stem cell line C17.2 by RT-PCR methods. TPO protein was detected in human cerebrospinal fluids (n=10) by ELISA. Moreover, TPO receptor (c-mpl) mRNA was identified in human cerebral hemispheres and cerebellum, and C17.2 cells using RT-PCR. The expression of c-mpl protein was also confirmed on neurons in the human cerebral hemispheres, hippocampus, cerebellum, brainstem and spinal cord using immune-cytochemical staining. TPO also showed a stimulating effect on the in-vitro growth of C17.2 cells by the MTT assay. TPO activated the phosphoinositide 3-kinase(PI3K)/Akt signaling pathway which was demonstrated by Western blot. The Akt activation by TPO was inhibited by the PI3-kinase inhibitor LY294002. Conclusions: Our study provided the evidences showing the expression of TPO and TPO receptor (c-mpl) in neural cells and this effect may be mediated by c-mpl and Akt signaling. More importantly, our observation further demonstrated the functional role of TPO on neural protection in a rat model. These findings point to the possibility of a new strategy for treating brain damage by hematopoietic growth factors. Disclosures Yang: National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

scholarly journals Age-related Attenuation of Isoflurane Preconditioning in Human Atrial Cardiomyocytes

2008 ◽  
Vol 108 (4) ◽  
pp. 612-620 ◽  
Author(s):  
Yasushi Mio ◽  
Martin W. Bienengraeber ◽  
Jasna Marinovic ◽  
David D. Gutterman ◽  
Mladen Rakic ◽  
...  
Keyword(s):  
Cell Death ◽  
Respiratory Function ◽  
Right Atrial ◽  
Channel Activity ◽  
Age Related ◽  
Isolated Myocardium ◽  
The Impact ◽  
Mitochondrial Respiratory

Background Clinical trials suggest that anesthetic-induced preconditioning (APC) produces cardioprotection in humans, but the mechanisms of APC and significance of aging for APC in humans are not well understood. Here, the impact of age on the role of two major effectors of APC, mitochondria and sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels, in preconditioning of the human atrial myocardium were investigated. Methods Right atrial appendages were obtained from adult patients undergoing cardiac surgery and assigned to mid-aged (MA) and old-aged (OA) groups. APC was induced by isoflurane in isolated myocardium and isolated cardiomyocytes. Mitochondrial oxygen consumption measurements, myocyte survival testing, and patch clamp techniques were used to investigate mitochondrial respiratory function and sarcKATP channel activity. Results After in vitro APC with isoflurane, the respiratory function of isolated mitochondria was better preserved after hypoxia-reoxygenation stress in MA than in OA. In isolated intact myocytes, APC significantly decreased oxidative stress-induced cell death in MA but not in OA, and isoflurane protection from cell death was attenuated by the sarcKATP channel inhibitor HMR-1098. Further, the properties of single sarcKATP channels were similar in MA and OA, and isoflurane sensitivity of pinacidil-activated whole cell KATP current was no different between MA and OA myocytes. Conclusion Anesthetic-induced preconditioning with isoflurane decreases stress-induced cell death and preserves mitochondrial respiratory function to a greater degree in MA than in OA myocytes; however, sarcKATP channel activity is not differentially affected by isoflurane. Therefore, effectiveness of APC in humans may decrease with advancing age partly because of altered mitochondrial function of myocardial cells.

scholarly journals Actin dynamics regulation by TTC7A/PI4KIIIα axis limits DNA damage and cell death during leukocyte migration

2021 ◽  
Author(s):  
Tania Gajardo ◽  
Marie Lo ◽  
Mathilde Bernard ◽  
Claire Leveau ◽  
Marie-Therese El-Daher ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Cell Migration ◽  
Actin Cytoskeleton ◽  
Leukocyte Migration ◽  
Actin Dynamics ◽  
Immune Homeostasis ◽  
Cellular Functions ◽  
The Impact

The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cell processes, including cell migration. Mutations in the TTC7A gene have been described as the cause of a primary immunodeficiency associated to different degrees of gut involvement and alterations in the actin cytoskeleton dynamics. Although several cellular functions have been associated with TTC7A, the role of the protein in the maintenance of the immune homeostasis is still poorly understood. Here we leverage microfabricated devices to investigate the impact of TTC7A deficiency in leukocytes migration at the single cell level. We show that TTC7A-deficient leukocytes exhibit an altered cell migration and reduced capacity to deform through narrow gaps. Mechanistically, TTC7A-deficient phenotype resulted from impaired phosphoinositides signaling, leading to the downregulation of the PI3K/AKT/RHOA regulatory axis and imbalanced actin cytoskeleton dynamic. This resulted in impaired cell motility, accumulation of DNA damage and increased cell death during chemotaxis in dense 3D gels. Our results highlight a novel role of TTC7A as a critical regulator of leukocyte migration. Impairment of this cellular function is likely to contribute to pathophysiology underlying progressive immunodeficiency in patients.

scholarly journals Analysis of the use of photosensitization in human glioblastoma multiforme to induce cell death

2019 ◽  
Vol 98 (Suppl) ◽  
pp. 25-25
Author(s):  
Mario Minor Murakami Junior ◽  
Yollanda E. Moreira Franco ◽  
Maurício Da Silva Baptista ◽  
Suely Kazue Nagahashi Marie
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Methylene Blue ◽  
Cell Proliferation ◽  
Cell Death ◽  
Acridine Orange ◽  
Light Irradiation ◽  
Rt Pcr ◽  
Low Concentration ◽  
The Impact

Introduction: The most frequent primary tumor of the central nervous system is the malignant glioma, being the glioblastoma (GBM), grade IV astrocytoma, the most aggressive and lethal glioma. Malignant astrocytomas are responsive for therapy targeting autophagy as temozolomide, the standard adjuvant treatment which induces autophagic cell death. Autophagy is a homeostatic intracellular process that eliminate old proteins and recycle cellular components. Mitophagy is a subtype of autophagy that regulates the removal of damaged, dysfunctional or redundant mitochondria. Parallel damage against lysosomes and mitochondria membranes using photosensitized oxidations and strong redox stress leads to activation of mitophagy and malfunction of autophagy. This mechanism of photosensitization, ultimately, causes cell death. Challenging cells with a low concentration of a photosensitizer as 1,9-dimethyl methylene blue (DMMB) combined with light- irradiation of 12 joules/cm2 have induced mitochondrial damage with activation of mitophagy and concomitant lysosome damage, in skin-derived cell lines. This experimental design was applied to U87MG GBM cells to verify if tumor cell death may be obtained with combined mitochondrial and lysosomal damages to open new therapeutic strategies for GBM and to better understand the mechanisms of mitophagy.Objectives: Our primary objective is to analyze the impact of challenging GBM cells with a low concentration of 1,9-dimethyl methylene blue (DMMB) with combined light- irradiation of 12 joules/cm2.Methodology: U87MG a human GBM cell line was used. The photodamage was performed using DMMB photosensitized by a LED with maximum emission wavelength at 630 nm providing 12 J/cm2. Cell proliferation and viability assays were performed using MTT to assess whether there was proliferation inhibition and/or alteration of cell viability after photosensitization. Quantification of cells in different stages of apoptosis, and in the various phases of the cell cycle were analyzed using flow cytometry after photosensitization. Acridine orange assay was used to assess lysosome damage. RT-PCR and Western Blotting were performed to evaluate the expression levels of the main autophagy and mitophagy genes and proteins.Partial Results: Cell proliferation and viability assays demonstrated that the concentration of DMMB to cause 50% inhibition of biological activity of cells (IC50) was 10 nM after 48h. The apoptosis and cell cycle experiments were performed in this concentration. Increase in apoptosis was observed after 24hs of photosensitization. Currently, the cell cycle flow cytometry assay has been performed, followed by the quantification of lysosomes damage by Acridine Orange assay. The genes and proteins involved in the mechanisms of autophagy and mitophagy will be determined by expression analysis through RT-PCR and Western blot assays.Discussion and Conclusion: For a future perspective, if this prove of concept is achieved, i.e. death of tumor cells by the combined approach of photosensitizer with irradiation, a new therapeutic strategy of light-activated drugs may be offered to cancer patients.

scholarly journals Adverse Effects of Methylene Blue on the Central Nervous System

2008 ◽  
Vol 108 (4) ◽  
pp. 684-692 ◽  
Author(s):  
Laszlo Vutskits ◽  
Adrian Briner ◽  
Paul Klauser ◽  
Eduardo Gascon ◽  
Alexandre G. Dayer ◽  
...  
Keyword(s):  
Methylene Blue ◽  
Cell Death ◽  
Neuronal Cultures ◽  
Dendritic Arbor ◽  
Field Potentials ◽  
Neurotoxic Effects ◽  
The Central Nervous System ◽  
The Impact

Background An increasing number of clinical observations suggest adverse neurologic outcome after methylene blue (MB) infusion in the setting of parathyroid surgery. Hence, the aim of the current study was to investigate the potentially neurotoxic effects of MB using a combination of in vivo and in vitro experimental approaches. Methods Isoflurane-anesthetized adult rats were used to evaluate the impact of a single bolus intravascular administration of MB on systemic hemodynamic responses and on the minimum alveolar concentration (MAC) of isoflurane using the tail clamp test. In vivo, MB-induced cell death was evaluated 24 h after MB administration using Fluoro-Jade B staining and activated caspase-3 immunohistochemistry. In vitro, neurotoxic effects of MB were examined in hippocampal slice cultures by measuring excitatory field potentials as well as propidium iodide incorporation after MB exposure. The impact of MB on dendritic arbor was evaluated in differentiated single cell neuronal cultures. Results Bolus injections of MB significantly reduced isoflurane MAC and initiated widespread neuronal apoptosis. Electrophysiologic recordings in hippocampal slices revealed a rapid suppression of evoked excitatory field potentials by MB, and this was associated with a dose-dependent effect of this drug on cell death. Dose-response experiments in single cell neuronal cultures revealed that a 2-h-long exposure to MB at non-cell-death-inducing concentrations could still induce significant retraction of dendritic arbor. Conclusions These results suggest that MB exerts neurotoxic effects on the central nervous system and raise questions regarding the safety of using this drug at high doses during parathyroid gland surgery.

scholarly journals Immune Suppression Induced by Gallibacterium anatis GtxA During Interaction with Chicken Macrophage-Like HD11 Cells

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 536
Author(s):  
Bo Tang ◽  
Anders M. Bojesen
Keyword(s):  
Cell Death ◽  
Bacterial Strains ◽  
Rt Pcr ◽  
Spleen Tissue ◽  
Host Cell Response ◽  
Tnf Α ◽  
Chicken Macrophage ◽  
Adhesion And Invasion ◽  
Invasion Assays

The RTX toxin GtxA expressed by Gallibacterium anatis biovar haemolytica has been proposed a major virulence factor during disease manifestations in the natural host, the chicken. To better understand the role of GtxA in the pathogenesis of G. anatis, we compared the GtxA expressing wildtype strain with its isogenic ∆gtxA mutant that was unable to express GtxA during exposure to chicken macrophage-like HD11 cells. From adhesion and invasion assays, we showed that GtxA appears to promote adhesion and invasion of HD11 cells. By using quantitative RT-PCR, we also demonstrated that the G. anatis expressing GtxA induced a mainly anti-inflammatory (IL-10) host cell response as opposed to the pro-inflammatory (IL-1β, IL-6 and TNF-α) response induced by the GtxA deletion mutant. Interestingly, these results, at least partly, resemble recent responses observed from spleen tissue of chickens infected with the same two bacterial strains. The effect of the GtxA toxin on the type of cell death was less clear. While GtxA clearly induced cell death, our efforts to characterize whether this was due to primarily necrosis or apoptosis through expression analysis of a broad range of apoptosis genes did not reveal clear answers.

scholarly journals Calpains mediate acute renal cell death: role of autolysis and translocation

2001 ◽  
Vol 281 (4) ◽  
pp. F728-F738 ◽  
Author(s):  
Xiuli Liu ◽  
Juanita J. Rainey ◽  
Jay F. Harriman ◽  
Rick G. Schnellmann
Keyword(s):  
Cell Death ◽  
Mitochondrial Function ◽  
Immunoblot Analysis ◽  
Calpain Inhibitor ◽  
Antimycin A ◽  
Rt Pcr ◽  
Plasma Membrane Permeability ◽  
Injury Death ◽  
Casein Zymography

The goals of this study were to determine 1) the expression of calpain isoforms in rabbit renal proximal tubules (RPT); 2) calpain autolysis and translocation, and calpastatin levels during RPT injury; and 3) the effect of a calpain inhibitor (PD-150606) on calpain levels, mitochondrial function, and ion transport during RPT injury. RT-PCR, immunoblot analysis, and FITC-casein zymography demonstrated the presence of only μ- and m-calpains in rabbit RPT. The mitochondrial inhibitor antimycin A decreased RPT μ- and m-calpain and calpastatin levels in conjunction with cell death and increased plasma membrane permeability. No increases in either μ- or m-calpain were observed in the membrane nor were increases observed in autolytic forms of either μ- or m-calpain in antimycin A-exposed RPT. PD-150606 blocked antimycin A-induced cell death, preserved calpain levels in antimycin A-exposed RPT, and promoted the recovery of mitochondrial function and active Na+ transport in RPT after hypoxia and reoxygenation. The present study suggests that calpains mediate RPT injury without undergoing autolysis or translocation, and ultimately they leak from cells subsequent to RPT injury/death. Furthermore, PD-150606 allows functional recovery after injury.

scholarly journals Biology and Role of Extracellular Vesicles (EVs) in the Pathogenesis of Thrombosis

2019 ◽  
Vol 20 (11) ◽  
pp. 2840 ◽  
Author(s):  
Marta Zarà ◽  
Gianni Francesco Guidetti ◽  
Marina Camera ◽  
Ilaria Canobbio ◽  
Patrizia Amadio ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Cell Communication ◽  
Parental Cell ◽  
Biochemical Properties ◽  
Protein Markers ◽  
Surface Receptors ◽  
The Impact ◽  
Physiological And Biochemical ◽  
Physiological And Biochemical Properties

Extracellular vesicles (EVs) are well-established mediators of cell-to-cell communication. EVs can be released by every cell type and they can be classified into three major groups according to their biogenesis, dimension, density, and predominant protein markers: exosomes, microvesicles, and apoptotic bodies. During their formation, EVs associate with specific cargo from their parental cell that can include RNAs, free fatty acids, surface receptors, and proteins. The biological function of EVs is to maintain cellular and tissue homeostasis by transferring critical biological cargos to distal or neighboring recipient cells. On the other hand, their role in intercellular communication may also contribute to the pathogenesis of several diseases, including thrombosis. More recently, their physiological and biochemical properties have suggested their use as a therapeutic tool in tissue regeneration as well as a novel option for drug delivery. In this review, we will summarize the impact of EVs released from blood and vascular cells in arterial and venous thrombosis, describing the mechanisms by which EVs affect thrombosis and their potential clinical applications.

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