Abstract 925: Rit GTPase Protects Cells from Oxidative Damage

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Jennifer L Rudolph ◽  
Geng-Xian Shi ◽  
Susan M Harrison ◽  
Douglas A Harrison ◽  
Douglas A Andres
Keyword(s):  
Cortical Neurons ◽  
Map Kinases ◽  
Critical Role ◽  
Cell Types ◽  
Signaling Cascades ◽  
Neuronal Cultures ◽  
Wild Type ◽  
Developmental Abnormalities ◽  
Cellular Survival ◽  
Basic Biology

Signaling cascades that contribute to the regulation of cellular survival influence many cardiovascular diseases. Elucidating the mechanisms of these cascades is thus important for understanding basic biology and for therapeutic intervention. We have identified an evolution-arily conserved group of Ras-related GTPases, including two mammalian genes (Rit and Rin) and a single Drosophila ortholog (Ric), that we hypothesize plays a critical role in trophic factor-mediated anti-apoptotic signaling. Expression of activated Rit in PC6 cells induces potent activation of the MAP kinases ERK1/2 and p38, and promotes neurite outgrowth and cell survival. Furthermore, RNAi-mediated Rit silencing sensitized PC6 cells to a wide variety of stresses, in part by inhibiting NGF-mediated activation of ERK and p38 MAP kinases and CREB. Here we present studies from two genetic models designed to further assess our hypothesis. First, we generated a transgenic mouse over-expressing constitutively activated Rit (RitL79). To begin to assess the contribution of Rit signaling to cellular survival, we examined the ability of cortical neuronal cultures isolated from Rit-TG versus wild-type littermates to survive reactive-oxygen-species (ROS)-mediated cell death. Cell viability for wild-type cortical neurons following a 4 h exposure to H2O2 (60 μM) was reduced approximately 90%, while cortical neurons isolated from Rit-TG mice were largely unaffected by this treatment. Thus, RitL79-mediated signaling protects cortical neurons against ROS-mediated apoptosis. As further confirmation of the important role this protein family plays in survival from stress, we generated a Drosophila strain null for D-Ric. Mutants are homozygous viable and show no obvious developmental abnormalities. However, D-Ric mutants are susceptible to environmental stresses, including heat stress and dry starvation. Taken together, these studies suggest a conserved role for Rit/Ric in promoting cellular survival and suggest that Rit signaling is a potential target for pharmaceutical intervention. We look forward to assessing Rit’s role in other systems including cardiac and vascular cell types. This work was supported by a Predoctoral Fellowship from the AHA.

oko meduzy mutations affect neuronal patterning in the zebrafish retina and reveal cell-cell interactions of the retinal neuroepithelial sheet

Development ◽  
1999 ◽  
Vol 126 (6) ◽  
pp. 1235-1246 ◽  
Author(s):  
J. Malicki ◽  
W. Driever
Keyword(s):  
Critical Role ◽  
Cell Types ◽  
Cell Interactions ◽  
Retinal Cell ◽  
Wild Type ◽  
Vertebrate Retina ◽  
Neuroepithelial Cells ◽  
Patterning Defect ◽  
Cell Cell

Mutations of the oko meduzy (ome) locus cause drastic neuronal patterning defect in the zebrafish retina. The precise, stratified appearance of the wild-type retina is absent in the mutants. Despite the lack of lamination, at least seven retinal cell types differentiate in oko meduzy. The ome phenotype is already expressed in the retinal neuroepithelium affecting morphology of the neuroepithelial cells. Our experiments indicate that previously unknown cell-cell interactions are involved in development of the retinal neuroepithelial sheet. In genetically mosaic animals, cell-cell interactions are sufficient to rescue the phenotype of oko meduzy retinal neuroepithelial cells. These cell-cell interactions may play a critical role in the patterning events that lead to differentiation of distinct neuronal laminae in the vertebrate retina.

Arabidopsis gynoecium structure in the wild and in ettin mutants

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1519-1532 ◽  
Author(s):  
R.A. Sessions ◽  
P.C. Zambryski
Keyword(s):  
Cell Types ◽  
Flowering Plants ◽  
Wild Type ◽  
Reproductive Structure ◽  
Developmental Abnormalities ◽  
Transmitting Tract ◽  
Type Pattern ◽  
In The Wild ◽  
Gynoecium Development ◽  
Scanning Electron

The gynoecium is the female reproductive structure of flowering plants. Here we present a description of the Arabidopsis thaliana gynoecium at anthesis. The cylindrical organ can be broken down into three longitudinal regions arranged in an apical-basal order: stigma, style, and ovary. Each region can be distinguished histologically and morphologically. The transmitting (pollination) tract is axially positioned along the center of the gynoecium and spans stigma, style and ovary. Histochemistry, scanning electron microscopy and a style-specific reporter gene are used to compare the wild-type pattern of gynoecium cell types and regions, with patterns formed in gynoecia of individuals homozygous for a series of allelic mutations at the ETTIN locus. ettin gynoecia show morphological and histological alterations that appear to result from the merging of apical and basal regions and the development of abaxial into adaxial tissues. These developmental abnormalities result in a reduction of the ovary region, an expansion of the stylar and stigmatic regions, and the abaxial (outward) proliferation of transmitting tract tissue. The alterations in the mutants can be interpreted as resulting from misspecifications of position along the longitudinal and transverse axes during gynoecium development. The results suggest that early patterning events underlie wild-type gynoecium development, and that ETT functions during this early programming.

scholarly journals Development of a High-Throughput AlphaScreen Assay for Modulators of Synapsin I Phosphorylation in Primary Neurons

2013 ◽  
Vol 19 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Betty Chan ◽  
Jeffrey R. Cottrell ◽  
Bing Li ◽  
Kelley C. Larson ◽  
Crystle J. Ashford ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
High Throughput ◽  
Cortical Neurons ◽  
High Throughput Screening ◽  
Critical Role ◽  
Neuronal Cultures ◽  
Synapsin I ◽  
Primary Neurons ◽  
Primary Neuronal Cultures ◽  
Small Molecule Modulators

Alterations in synaptic transmission have been implicated in a number of psychiatric and neurological disorders. The discovery of small-molecule modulators of proteins that regulate neurotransmission represents a novel therapeutic strategy for these diseases. However, high-throughput screening (HTS) approaches in primary neurons have been limited by challenges in preparing and applying primary neuronal cultures under conditions required for generating sufficiently robust and sensitive HTS assays. Synapsin I is an abundant presynaptic protein that plays a critical role in neurotransmission through tethering synaptic vesicles to the actin cytoskeleton. It has several phosphorylation sites that regulate its modulation of synaptic vesicle trafficking and, therefore, the efficacy of synaptic transmission. Here, we describe the development of a rapid, sensitive, and homogeneous assay to detect phospho-synapsin I (pSYN1) in primary cortical neurons in 384-well plates using AlphaScreen technology. From results of a pilot screening campaign, we show that the assay can identify compounds that modulate synapsin I phosphorylation via multiple signaling pathways. The implementation of the AlphaScreen pSYN1 assay and future development of additional primary neuronal HTS assays provides an attractive approach for discovery of novel classes of therapeutic candidates for a variety of CNS disorders.

scholarly journals Inducible alpha-synuclein overexpression affects human Neural Stem Cells behavior

2018 ◽  
Author(s):  
J Zasso ◽  
M Ahmed ◽  
A Cutarelli ◽  
L Conti
Keyword(s):  
Cell Model ◽  
Critical Role ◽  
Alpha Synuclein ◽  
Cellular Systems ◽  
Neuronal Cultures ◽  
Wild Type ◽  
Gene Encoding ◽  
Human Neural Stem Cells ◽  
Cell Model System

AbstractConverging evidence suggest that levels of alpha-Synuclein (aSyn) expression play a critical role in Parkinson’s disease (PD). Several mutations of the SNCA gene, encoding for aSyn have been associated to either the familial or the sporadic forms of PD. Nonetheless, the mechanism underlying wild type aSyn-mediated neurotoxicity in neuronal cells as well as its specific driving role in PD pathogenesis has yet to be fully clarified. In this view, the development of proper in vitro cellular systems is a crucial step.Here we present a novel human Tet-on hNSC cell line, in which aSyn timing and level of expression can be tightly experimentally tuned. Induction of aSyn in self-renewing hNSCs leads to progressive formation of aSyn aggregates and impairs their proliferation and cell survival. Furthermore, aSyn induction during the neuronal differentiation process results in impaired neurogenic potential due to enhanced refractoriness to exit self-renewal and to increase of gliogenic vs neurogenic competence. Finally, acute aSyn induction in hNSC-derived dopaminergic neuronal cultures results in cell toxicity.This novel conditional in vitro cell model system may be a valuable tool for dissecting of aSyn pathogenic effects in hNSCs and neurons and in developing new potential therapeutic strategies.

scholarly journals Increasing the TRPM2 Channel Expression in Human Neuroblastoma SH-SY5Y Cells Augments the Susceptibility to ROS-Induced Cell Death

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 28 ◽  
Author(s):  
Xinfang An ◽  
Zixing Fu ◽  
Chendi Mai ◽  
Weiming Wang ◽  
Linyu Wei ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Model ◽  
Critical Role ◽  
Neuronal Cell ◽  
Cell Types ◽  
Wild Type ◽  
Human Neuroblastoma ◽  
Channel Activation ◽  
Trpm2 Channel

Human neuroblastoma SH-SY5Y cells are a widely-used human neuronal cell model in the study of neurodegeneration. A recent study shows that, 1-methyl-4-phenylpyridine ion (MPP), which selectively causes dopaminergic neuronal death leading to Parkinson’s disease-like symptoms, can reduce SH-SY5Y cell viability by inducing H2O2 generation and subsequent TRPM2 channel activation. MPP-induced cell death is enhanced by increasing the TRPM2 expression. By contrast, increasing the TRPM2 expression has also been reported to support SH-SY5Y cell survival after exposure to H2O2, leading to the suggestion of a protective role for the TRPM2 channel. To clarify the role of reactive oxygen species (ROS)-induced TRPM2 channel activation in SH-SY5Y cells, we generated a stable SH-SY5Y cell line overexpressing the human TRPM2 channel and examined cell death and cell viability after exposure to H2O2 in the wild-type and TRPM2-overexpressing SH-SY5Y cells. Exposure to H2O2 resulted in concentration-dependent cell death and reduction in cell viability in both cell types. TRPM2 overexpression remarkably augmented H2O2-induced cell death and reduction in cell viability. Furthermore, H2O2-induced cell death in both the wild-type and TRPM2-overexpressing cells was prevented by 2-APB, a TRPM2 inhibitor, and also by PJ34 and DPQ, poly(ADP-ribose) polymerase (PARP) inhibitors. Collectively, our results show that increasing the TRPM2 expression renders SH-SY5Y cells to be more susceptible to ROS-induced cell death and reinforce the notion that the TRPM2 channel plays a critical role in conferring ROS-induced cell death. It is anticipated that SH-SY5Y cells can be useful for better understanding the molecular and signaling mechanisms for ROS-induced TRPM2-mediated neurodegeneration in the pathogenesis of neurodegenerative diseases.

scholarly journals Inflammatory Monocytes but Not Neutrophils Are Necessary To Control Infection with Toxoplasma gondii in Mice

2010 ◽  
Vol 78 (4) ◽  
pp. 1564-1570 ◽  
Author(s):  
Ildiko R. Dunay ◽  
Anja Fuchs ◽  
L. David Sibley
Keyword(s):  
Acute Infection ◽  
Critical Role ◽  
Cell Types ◽  
Wild Type ◽  
Inflammatory Monocytes ◽  
Intestinal Pathology ◽  
Parasite Replication ◽  
Acute Toxoplasmosis ◽  
Acute Ileitis

ABSTRACT Previous studies have suggested that both inflammatory monocytes and neutrophils are important for controlling acute toxoplasmosis in the mouse model. To test the role of these cell types, we used monoclonal antibody (MAb) RB6-8C5 to deplete both subsets of cells or MAb 1A8 to selectively remove neutrophils. RB6-8C5 MAb-treated mice succumbed to oral infection with T oxoplasma gondii, similar to Ccr2−/− mice, which are deficient in monocyte recruitment but have normal neutrophils. In contrast, mice treated with MAb 1A8 controlled parasite replication and survived acute infection. Ccr2−/− mice suffered from acute ileitis and inflammation in the spleen that was associated with a lack of inflammatory monocytes and elevated numbers of neutrophils. RB6-8C5 MAb-treated C57BL/6 mice also suffered from intestinal pathology and splenic damage, although this was less extensive due to the reduced numbers of neutrophils. Neutrophil-depleted infected wild-type mice displayed no pathological changes, compared to untreated infected controls. Collectively, these observations demonstrate the critical role of inflammatory monocytes during the acute infection with the parasite T. gondii and reveal that neutrophils are not protective but rather contribute to the pathology.

scholarly journals Neural Stem Cells: What Happens When They Go Viral?

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1468
Author(s):  
Yashika S. Kamte ◽  
Manisha N. Chandwani ◽  
Alexa C. Michaels ◽  
Lauren A. O’Donnell
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Viral Infections ◽  
Wide Spectrum ◽  
Cell Types ◽  
Developmental Abnormalities ◽  
Multipotent Progenitor Cells ◽  
The Central Nervous System ◽  
Simplex Virus ◽  
The Brain

Viruses that infect the central nervous system (CNS) are associated with developmental abnormalities as well as neuropsychiatric and degenerative conditions. Many of these viruses such as Zika virus (ZIKV), cytomegalovirus (CMV), and herpes simplex virus (HSV) demonstrate tropism for neural stem cells (NSCs). NSCs are the multipotent progenitor cells of the brain that have the ability to form neurons, astrocytes, and oligodendrocytes. Viral infections often alter the function of NSCs, with profound impacts on the growth and repair of the brain. There are a wide spectrum of effects on NSCs, which differ by the type of virus, the model system, the cell types studied, and the age of the host. Thus, it is a challenge to predict and define the consequences of interactions between viruses and NSCs. The purpose of this review is to dissect the mechanisms by which viruses can affect survival, proliferation, and differentiation of NSCs. This review also sheds light on the contribution of key antiviral cytokines in the impairment of NSC activity during a viral infection, revealing a complex interplay between NSCs, viruses, and the immune system.

scholarly journals Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

2016 ◽  
Vol 113 (34) ◽  
pp. E4995-E5004 ◽  
Author(s):  
Wen Lu ◽  
Michael Winding ◽  
Margot Lakonishok ◽  
Jill Wildonger ◽  
Vladimir I. Gelfand
Keyword(s):  
Cytoplasmic Streaming ◽  
Cell Types ◽  
Nurse Cells ◽  
Wild Type ◽  
Actin Cortex ◽  
Microtubule Motor ◽  
Multiple Cell ◽  
Cytoplasmic Microtubules ◽  
Two Populations

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

scholarly journals Recent Progress in the Study of Peroxiredoxin in the Harmful Algal Bloom Species Chattonella marina

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 162
Author(s):  
Yohei Shimasaki ◽  
Koki Mukai ◽  
Yuki Takai ◽  
Xuchun Qiu ◽  
Yuji Oshima
Keyword(s):  
Oxidative Stress ◽  
Growth Phase ◽  
Critical Role ◽  
High Irradiance ◽  
Growth Potential ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Chattonella Marina ◽  
Strong Light ◽  
Expression Strain

Peroxiredoxin (Prx) is a relatively recently discovered antioxidant enzyme family that scavenges peroxides and is known to be present in organisms from biological taxa ranging from bacteria to multicellular eukaryotes, including photosynthetic organisms. Although there have been many studies of the Prx family in higher plants, green algae, and cyanobacteria, few studies have concerned raphidophytes and dinoflagellates, which are among the eukaryotic algae that cause harmful algal blooms (HABs). In our proteomic study using 2-D electrophoresis, we found a highly expressed 2-Cys peroxiredoxin (2-CysPrx) in the raphidophyte Chattonella marina var. antiqua, a species that induces mass mortality of aquacultured fish. The abundance of the C. marina 2-CysPrx enzyme was highest in the exponential growth phase, during which photosynthetic activity was high, and it then decreased by about a factor of two during the late stationary growth phase. This pattern suggested that 2-CysPrx is a key enzyme involved in the maintenance of high photosynthesis activity. In addition, the fact that the depression of photosynthesis by excessively high irradiance was more severe in the 2-CysPrx low-expression strain (wild type) than in the normal-expression strain (wild type) of C. marina suggested that 2-CysPrx played a critical role in protecting the cell from oxidative stress caused by exposure to excessively high irradiance. In the field of HAB research, estimates of growth potential have been desired to predict the population dynamics of HABs for mitigating damage to fisheries. Therefore, omics approaches have recently begun to be applied to elucidate the physiology of the growth of HAB species. In this review, we describe the progress we have made using a molecular physiological approach to identify the roles of 2-CysPrx and other antioxidant enzymes in mitigating environmental stress associated with strong light and high temperatures and resultant oxidative stress. We also describe results of a survey of expressed Prx genes and their growth-phase-dependent behavior in C. marina using RNA-seq analysis. Finally, we speculate about the function of these genes and the ecological significance of 2-CysPrx, such as its involvement in circadian rhythms and the toxicity of C. marina to fish.

scholarly journals Differential Effects of Toll-Like Receptor Activation and Differential Mediation by MAP Kinases of Immune Responses in Microglial Cells

2021 ◽  
Author(s):  
Jaedeok Kwon ◽  
Christos Arsenis ◽  
Maria Suessmilch ◽  
Alison McColl ◽  
Jonathan Cavanagh ◽  
...  
Keyword(s):  
Map Kinase ◽  
Microglial Activation ◽  
Map Kinases ◽  
Mrna Level ◽  
Cell Types ◽  
Receptor Activation ◽  
Microglial Cells ◽  
Toll Like Receptor ◽  
Disease States ◽  
Nitrite Production

AbstractMicroglial activation is believed to play a role in many psychiatric and neurodegenerative diseases. Based largely on evidence from other cell types, it is widely thought that MAP kinase (ERK, JNK and p38) signalling pathways contribute strongly to microglial activation following immune stimuli acting on toll-like receptor (TLR) 3 or TLR4. We report here that exposure of SimA9 mouse microglial cell line to immune mimetics stimulating TLR4 (lipopolysaccharide—LPS) or TLR7/8 (resiquimod/R848), results in marked MAP kinase activation, followed by induction of nitric oxide synthase, and various cytokines/chemokines. However, in contrast to TLR4 or TLR7/8 stimulation, very few effects of TLR3 stimulation by poly-inosine/cytidine (polyI:C) were detected. Induction of chemokines/cytokines at the mRNA level by LPS and resiquimod were, in general, only marginally affected by MAP kinase inhibition, and expression of TNF, Ccl2 and Ccl5 mRNAs, along with nitrite production, were enhanced by p38 inhibition in a stimulus-specific manner. Selective JNK inhibition enhanced Ccl2 and Ccl5 release. Many distinct responses to stimulation of TLR4 and TLR7 were observed, with JNK mediating TNF protein induction by the latter but not the former, and suppressing Ccl5 release by the former but not the latter. These data reveal complex modulation by MAP kinases of microglial responses to immune challenge, including a dampening of some responses. They demonstrate that abnormal levels of JNK or p38 signalling in microglial cells will perturb their profile of cytokine and chemokine release, potentially contributing to abnormal inflammatory patterns in CNS disease states.

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