scholarly journals The Spike Glycoprotein of Murine Coronavirus MHV-JHM Mediates Receptor-Independent Infection and Spread in the Central Nervous Systems of Ceacam1a−/− Mice

2007 ◽  
Vol 82 (2) ◽  
pp. 755-763 ◽  
Author(s):  
Tanya A. Miura ◽  
Emily A. Travanty ◽  
Lauren Oko ◽  
Helle Bielefeldt-Ohmann ◽  
Susan R. Weiss ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Lethal Dose ◽  
Cell Types ◽  
Wild Type ◽  
Spike Glycoprotein ◽  
Susceptibility To Infection ◽  
Spread Of Infection ◽  
The Brain ◽  
Murine Coronavirus

ABSTRACT The MHV-JHM strain of the murine coronavirus mouse hepatitis virus is much more neurovirulent than the MHV-A59 strain, although both strains use murine CEACAM1a (mCEACAM1a) as the receptor to infect murine cells. We previously showed that Ceacam1a −/− mice are completely resistant to MHV-A59 infection (E. Hemmila et al., J. Virol. 78:10156-10165, 2004). In vitro, MHV-JHM, but not MHV-A59, can spread from infected murine cells to cells that lack mCEACAM1a, a phenomenon called receptor-independent spread. To determine whether MHV-JHM could infect and spread in the brain independent of mCEACAM1a, we inoculated Ceacam1a −/− mice. Although Ceacam1a −/− mice were completely resistant to i.c. inoculation with 106 PFU of recombinant wild-type MHV-A59 (RA59) virus, these mice were killed by recombinant MHV-JHM (RJHM) and a chimeric virus containing the spike of MHV-JHM in the MHV-A59 genome (SJHM/RA59). Immunohistochemistry showed that RJHM and SJHM/RA59 infected all neural cell types and induced severe microgliosis in both Ceacam1a −/− and wild-type mice. For RJHM, the 50% lethal dose (LD50) is <101.3 in wild-type mice and 103.1 in Ceacam1a −/− mice. For SJHM/RA59, the LD50 is <101.3 in wild-type mice and 103.6 in Ceacam1a −/− mice. This study shows that infection and spread of MHV-JHM in the brain are dependent upon the viral spike glycoprotein. RJHM can initiate infection in the brains of Ceacam1a −/− mice, but expression of mCEACAM1a increases susceptibility to infection. The spread of infection in the brain is mCEACAM1a independent. Thus, the ability of the MHV-JHM spike to mediate mCEACAM1a-independent spread in the brain is likely an important factor in the severe neurovirulence of MHV-JHM in wild-type mice.

scholarly journals A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology

2019 ◽  
Vol 294 (20) ◽  
pp. 8064-8087 ◽  
Author(s):  
Manmeet Singh ◽  
Abhinoy Kishore ◽  
Dibyajyoti Maity ◽  
Punnepalli Sunanda ◽  
Bankala Krishnarjuna ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Brain Parenchyma ◽  
Fusion Peptides ◽  
Spike Glycoprotein ◽  
Fusion Event ◽  
Viral Spread ◽  
Secondary Amino ◽  
The Brain

Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro. Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)–carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline's unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics and NMR experiments. We therefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis.

scholarly journals Microglia in Neurodegenerative Events—An Initiator or a Significant Other?

2021 ◽  
Vol 22 (11) ◽  
pp. 5818
Author(s):  
Gaylia Jean Harry
Keyword(s):  
Neurovascular Unit ◽  
Cell Types ◽  
In Vitro Assays ◽  
Significant Other ◽  
Neurodegenerative Process ◽  
Target Sites ◽  
Systemic Factors ◽  
Injury And Repair ◽  
The Brain

A change in microglia structure, signaling, or function is commonly associated with neurodegeneration. This is evident in the patient population, animal models, and targeted in vitro assays. While there is a clear association, it is not evident that microglia serve as an initiator of neurodegeneration. Rather, the dynamics imply a close interaction between the various cell types and structures in the brain that orchestrate the injury and repair responses. Communication between microglia and neurons contributes to the physiological phenotype of microglia maintaining cells in a surveillance state and allows the cells to respond to events occurring in their environment. Interactions between microglia and astrocytes is not as well characterized, nor are interactions with other members of the neurovascular unit; however, given the influence of systemic factors on neuroinflammation and disease progression, such interactions likely represent significant contributes to any neurodegenerative process. In addition, they offer multiple target sites/processes by which environmental exposures could contribute to neurodegenerative disease. Thus, microglia at least play a role as a significant other with an equal partnership; however, claiming a role as an initiator of neurodegeneration remains somewhat controversial.

scholarly journals DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

2019 ◽  
Vol 116 (50) ◽  
pp. 25322-25328 ◽  
Author(s):  
Yi Liu ◽  
Xiaopin Ma ◽  
Hisashi Fujioka ◽  
Jun Liu ◽  
Shengdi Chen ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Cellular Mechanism ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Efflux ◽  
And Function ◽  
The Brain ◽  
Early Onset Parkinson’S Disease

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson’s disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

scholarly journals Virus-Neutralizing Monoclonal Antibody Expressed in Milk of Transgenic Mice Provides Full Protection against Virus-Induced Encephalitis

2001 ◽  
Vol 75 (6) ◽  
pp. 2803-2809 ◽  
Author(s):  
Andreas F. Kolb ◽  
Lecia Pewe ◽  
John Webster ◽  
Stanley Perlman ◽  
C. Bruce A. Whitelaw ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Neutralizing Antibodies ◽  
Defense Mechanism ◽  
Viral Infections ◽  
Hepatitis Virus ◽  
Recombinant Antibody ◽  
Lethal Dose ◽  
Neutralizing Antibody ◽  
Murine Hepatitis Virus

ABSTRACT Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine β-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.

scholarly journals Toll-Like Receptor 2 Controls the Gamma Interferon Response to Francisella tularensis by Mouse Liver Lymphocytes

2007 ◽  
Vol 75 (11) ◽  
pp. 5338-5345 ◽  
Author(s):  
Kee-Jong Hong ◽  
Jason R. Wickstrum ◽  
Hung-Wen Yeh ◽  
Michael J. Parmely
Keyword(s):  
Francisella Tularensis ◽  
Cell Types ◽  
Gamma Interferon ◽  
Interferon Response ◽  
Interleukin 12 ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Ifn Γ ◽  
Deficient Mice

ABSTRACT The production of gamma interferon (IFN-γ) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-γ expression. The present study was undertaken to determine whether or not macrophages (Mφ) or dendritic cells (DC) provide coactivating signals for the liver IFN-γ response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mφ and DC significantly augmented the IFN-γ response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-γ production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-γ at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-γ responses to F. tularensis by regulating DC IL-12 production. While Mφ also coinduced hepatic IFN-γ production in response to F. tularensis, they did so in a fashion less dependent on TLR2.

scholarly journals SUN-260 Dual Role of Carboxypeptidase E in Prohormone Processing and a Novel Neurotrophic Factor Mediating Neuroprotection and Cognitive Functions in Hippocampal CA3 Neurons in Mice

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Lan Xiao ◽  
Vinay Sharma ◽  
Leila Toulabi ◽  
Xuyu Yang ◽  
Cheol Lee ◽  
...  
Keyword(s):  
Neurotrophic Factor ◽  
Neuronal Degeneration ◽  
Tyrosine Kinase Receptor ◽  
Wild Type ◽  
Prohormone Processing ◽  
Hippocampal Ca3 ◽  
Ca3 Neurons ◽  
The Brain

Abstract Stress causes release of glucocorticoids from the adrenals which then circulate to the brain. High concentrations glucocorticoid from chronic severe stress results in pathophysiology in the brain, including neuronal degeneration, cell death and cognitive dysfunction, leading to diseases such as Alzheimer Disease and Major Depressive Disorders. Neurotrophic/growth factors such as BDNF, NGF and NT3 have been linked to these pathological conditions. Carboxypeptidase E (CPE), a proneuropeptide/prohormone processing enzyme, also named neurotrophic factor-α1(NFα1) is highly expressed in the stress-vulnerable hippocampal CA3 neurons, and was shown to have neuroprotective activity from in vitro studies. Here we investigated if CPE-NFα1 functions in vivo, independent of its enzymatic activity, and the mechanism underlying its action. We generated knock-in mice expressing a non-enzymatic form of CPE, CPE-E342Q, but not wild-type CPE. The CPE-E342Q mice showed significantly decreased neuropeptide content and exhibited obesity, diabetes and infertility due to lack of prohormone processing activity, similar to CPE-KO mice. However, they showed no hippocampal CA3 degeneration, exhibited neurogenesis in the dentate gyrus, and displayed normal spatial learning and memory, similar to CPE wild-type mice, after weaning stress; unlike CPE-KO mice which showed hippocampal CA3 neuronal degeneration and cognitive deficits. Binding studies showed that radiolabeled CPE bound hippocampal cell membrane specifically, in a saturable manner. Binding of CPE and CPE-E342Q to hippocampal neurons activated Erk signaling and pre-treatment with either of these proteins protected neurons against H2O2- or glutamate-induced neurotoxcity by increasing BCL2 expression. In vitro and in vivo inhibitor studies demonstrated that this neuroprotective effect was independent of tyrosine kinase receptor signaling. Taken together, the data provide evidence that CPE-NFα1 is a unique neurotrophic factor which acts through a non-tyrosine kinase receptor to activate Erk-BCL2 signaling to protect hippocampal CA3 neurons against stress-induced neurodegeneration and maintaining normal cognitive functions in mice.

scholarly journals Absence of the Macrophage Mannose Receptor in Mice Does Not Increase Susceptibility to Pneumocystis carinii Infection In Vivo

2003 ◽  
Vol 71 (11) ◽  
pp. 6213-6221 ◽  
Author(s):  
Steve D. Swain ◽  
Sena J. Lee ◽  
Michel C. Nussenzweig ◽  
Allen G. Harmsen
Keyword(s):  
Pneumocystis Carinii ◽  
Hydrolytic Enzymes ◽  
Mannose Receptor ◽  
Opportunistic Pathogen ◽  
Cell Types ◽  
Surface Expression ◽  
Wild Type ◽  
Macrophage Mannose Receptor

ABSTRACT Host defense against the opportunistic pathogen Pneumocystis carinii requires functional interactions of many cell types. Alveolar macrophages are presumed to be a vital host cell in the clearance of P. carinii, and the mechanisms of this interaction have come under scrutiny. The macrophage mannose receptor is believed to play an important role as a receptor involved in the binding and phagocytosis of P. carinii. Although there is in vitro evidence for this interaction, the in vivo role of this receptor in P. carinii clearance in unclear. Using a mouse model in which the mannose receptor has been deleted, we found that the absence of this receptor is not sufficient to allow infection by P. carinii in otherwise immunocompetent mice. Furthermore, when mice were rendered susceptible to P. carinii by CD4+ depletion, mannose receptor knockout mice (MR-KO) had pathogen loads equal to those of wild-type mice. However, the MR-KO mice exhibited a greater influx of phagocytes into the alveoli during infection. This was accompanied by increased pulmonary pathology in the MR-KO mice, as well as greater accumulation of glycoproteins in the alveoli (glycoproteins, including harmful hydrolytic enzymes, are normally cleared by the mannose receptor). We also found that the surface expression of the mannose receptor is not downregulated during P. carinii infection in wild-type mice. Our findings suggest that while the macrophage mannose receptor may be important in the recognition of P. carinii, in vivo, this mechanism may be redundant, and the absence of this receptor may be compensated for.

scholarly journals Loss of a Protein Phosphatase 2A Regulatory Subunit (Cdc55p) Elicits Improper Regulation of Swe1p Degradation

2000 ◽  
Vol 20 (21) ◽  
pp. 8143-8156 ◽  
Author(s):  
Haifeng Yang ◽  
Wei Jiang ◽  
Matthew Gentry ◽  
Richard L. Hallberg
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cell Types ◽  
Regulatory Subunit ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Phosphatase 2A

ABSTRACT CDC55 encodes a Saccharomyces cerevisiaeprotein phosphatase 2A (PP2A) regulatory subunit.cdc55-null cells growing at low temperature exhibit a failure of cytokinesis and produce abnormally elongated buds, butcdc55-null cells producing the cyclin-dependent kinase Cdc28-Y19F, which is unable to be inhibited by Y19 phosphorylation, show a loss of the abnormal morphology. Furthermore,cdc55-null cells exhibit a hyperphosphorylation of Y19. For these reasons, we have examined in wild-type and cdc55-null cells the levels and activities of the kinase (Swe1p) and phosphatase (Mih1p) that normally regulate the extent of Cdc28 Y19 phosphorylation. We find that Mih1p levels are comparable in the two strains, and an estimate of the in vivo and in vitro phosphatase activity of this enzyme in the two cell types indicates no marked differences. By contrast, while Swe1p levels are similar in unsynchronized and S-phase-arrested wild-type and cdc55-null cells, Swe1 kinase is found at elevated levels in mitosis-arrestedcdc55-null cells. This excess Swe1p incdc55-null cells is the result of ectopic stabilization of this protein during G2 and M, thereby accounting for the accumulation of Swe1p in mitosis-arrested cells. We also present evidence indicating that, in cdc55-null cells, misregulated PP2A phosphatase activity is the cause of both the ectopic stabilization of Swe1p and the production of the morphologically abnormal phenotype.

scholarly journals Cleavage between Replicase Proteins p28 and p65 of Mouse Hepatitis Virus Is Not Required for Virus Replication

2004 ◽  
Vol 78 (11) ◽  
pp. 5957-5965 ◽  
Author(s):  
Mark R. Denison ◽  
Boyd Yount ◽  
Sarah M. Brockway ◽  
Rachel L. Graham ◽  
Amy C. Sims ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Wild Type ◽  
Virus Growth ◽  
Amino Terminal ◽  
Normal Expression ◽  
Infected Cells ◽  
Critical Residues

ABSTRACT The p28 and p65 proteins of mouse hepatitis virus (MHV) are the most amino-terminal protein domains of the replicase polyprotein. Cleavage between p28 and p65 has been shown to occur in vitro at cleavage site 1 (CS1), 247Gly↓Val248, in the polyprotein. Although critical residues for CS1 cleavage have been mapped in vitro, the requirements for cleavage have not been studied in infected cells. To define the determinants of CS1 cleavage and the role of processing at this site during MHV replication, mutations and deletions were engineered in the replicase polyprotein at CS1. Mutations predicted to allow cleavage at CS1 yielded viable virus that grew to wild-type MHV titers and showed normal expression and processing of p28 and p65. Mutant viruses containing predicted noncleaving mutations or a CS1 deletion were also viable but demonstrated delayed growth kinetics, reduced peak titers, decreased RNA synthesis, and small plaques compared to wild-type controls. No p28 or p65 was detected in cells infected with predicted noncleaving CS1 mutants or the CS1 deletion mutant; however, a new protein of 93 kDa was detected. All introduced mutations and the deletion were retained during repeated virus passages in culture, and no phenotypic reversion was observed. The results of this study demonstrate that cleavage between p28 and p65 at CS1 is not required for MHV replication. However, proteolytic separation of p28 from p65 is necessary for optimal RNA synthesis and virus growth, suggesting important roles for these proteins in the formation or function of viral replication complexes.

scholarly journals Glucocorticoid receptor action in metabolic and neuronal function

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1208 ◽  
Author(s):  
Michael J. Garabedian ◽  
Charles A. Harris ◽  
Freddy Jeanneteau
Keyword(s):  
Gene Expression ◽  
Glucocorticoid Receptor ◽  
Metabolic Diseases ◽  
Cell Types ◽  
Health And Disease ◽  
And Behavior ◽  
External Signals ◽  
The Brain

Glucocorticoids via the glucocorticoid receptor (GR) have effects on a variety of cell types, eliciting important physiological responses via changes in gene expression and signaling. Although decades of research have illuminated the mechanism of how this important steroid receptor controls gene expression using in vitro and cell culture–based approaches, how GR responds to changes in external signals in vivo under normal and pathological conditions remains elusive. The goal of this review is to highlight recent work on GR action in fat cells and liver to affect metabolism in vivo and the role GR ligands and receptor phosphorylation play in calibrating signaling outputs by GR in the brain in health and disease. We also suggest that both the brain and fat tissue communicate to affect physiology and behavior and that understanding this “brain-fat axis” will enable a more complete understanding of metabolic diseases and inform new ways to target them.

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