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1.
J Neurovirol ; 14(5): 437-47, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18937121

ABSTRACT

Cytomegalovirus (CMV) is the leading transmittable cause of congenital brain abnormalities in children and infection results in fatal ventriculoencephalitis in advanced acquired immunodeficiency syndrome (AIDS) patients. Pathology associated with CMV brain infection is seen predominantly in the periventricular region, an area known to harbor neural stem cells (NSCs). In the present study, using an adult model of murine CMV brain infection, the authors demonstrated that nestin-positive NSCs in the subventricular zone are susceptible to murine CMV infection. Furthermore, primary NSC cultures supported productive murine CMV replication with a 1000-fold increase in viral titers by 5 days post infection (d.p.i). Previous studies from the authors' laboratory demonstrated that CD8 lymphocytes were essential in protecting the brain against murine CMV infection. In the present study, the authors found that interferon (IFN)-gamma treatment increased the expression of major histocompatibility complex (MHC) class I on NSCs. Viral infection, on the other hand, inhibited this IFN-gamma-induced MHC up-regulation. In addition to increasing MHC class I expression, IFN-gamma (but not tumor necrosis factor [TNF]-alpha, interleukin [IL]-1 beta, or IL-10) also suppressed NSC proliferation in vitro. This decrease in proliferation was not accompanied by apoptosis or extracellular release of cellular lactate dehydrogenase (LDH), suggesting that the effects were not due to direct cytotoxicity. These studies demonstrate that NSCs are susceptible to murine CMV infection and inflammatory mediators, such as IFN-gamma, alter cellular characteristics which may have an impact on their reparative functions.


Subject(s)
Cytomegalovirus Infections/immunology , Histocompatibility Antigens Class I/metabolism , Interferon-gamma/metabolism , Neurons/metabolism , Stem Cells/metabolism , Animals , Brain/immunology , Cytomegalovirus/immunology , Gene Expression Regulation/drug effects , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/pharmacology , L-Lactate Dehydrogenase/metabolism , Mice , Neurons/cytology , Neurons/immunology , Stem Cells/cytology , Stem Cells/immunology
2.
J Pharmacol Exp Ther ; 322(3): 957-63, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17538007

ABSTRACT

Neural stem cells (NSCs) play an important role in the developing as well as adult brain. NSCs have been shown to migrate toward sites of injury in the brain and to participate in the process of brain repair. Like NSCs, cultured human neural precursor cells (NPCs) are self-renewing, multipotent cells capable of differentiating into neurons, astrocytes, and oligodendrocytes and of migrating toward chemotactic stimuli. Cellular and environmental factors are important for NPC proliferation and migration. Expression of kappa-opioid receptors (KORs) and mu-opioid receptors (MORs) in murine embryonic stem cells and of MORs and delta-opioid receptors in rodent neuronal precursors, as well as hippocampal progenitors has been reported by other investigators. In this study, we demonstrated robust expression of KORs in highly enriched (>90% nestin-positive) human fetal brain-derived NPCs. We found that KOR ligands, dynorphin(1-17) and trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate (U50,488) but not dynorphin(2-17), stimulated proliferation and migration of NPCs in a concentration-dependent manner. NPC proliferation was maximally stimulated at 10(-14) M dynorphin(1-17) and 10(-12) M U50,488. The KOR selective antagonist, nor-binaltorphimine, partially blocked the migratory and proliferative effects of KOR agonists supporting, at least in part, the involvement of a KOR-related mechanism. As has been described for rodent P19 embryonal carcinoma stem cells, retinoic acid treatment markedly suppressed KOR mRNA expression in human NPCs. Taken together, the results of this study suggest that activation of KORs alters functional properties of NPCs/NSCs that are relevant to human brain development and repair.


Subject(s)
Embryonic Stem Cells/chemistry , Embryonic Stem Cells/physiology , Neurons/cytology , Receptors, Opioid, kappa/analysis , Brain/growth & development , Cell Movement/drug effects , Cell Proliferation/drug effects , Fetus , Humans , Ligands , Multipotent Stem Cells , Regeneration
3.
J Pharmacol Exp Ther ; 318(3): 1280-6, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16766721

ABSTRACT

Maternal use of cocaine during pregnancy is associated with sustained morphological brain abnormalities and sustained cognitive deficits in the offspring. Here, we use a cell culture model of highly enriched human fetal brain-derived neural precursor cells (NPCs) to assess the effects of cocaine treatment on their proliferation, migration, and differentiation. Our data show that cocaine treatment markedly inhibited the proliferation of NPCs, a phenomenon that was associated with cell cycle arrest, possibly because of increased expression of the cyclin-dependent kinase inhibitor p21. In addition, treatment of NPCs with cocaine inhibited their migratory response to CXCL12 (stromal cell-derived factor-1alpha), a finding that correlated with cocaine-induced down-regulation of CXCR4 on NPCs. Finally, these data demonstrated that NPCs exposed to cocaine underwent differentiation into cells expressing neuronal markers that was associated with an inhibition of SOX2 (SRY-related HMG-box gene 2), a transcription factor that inhibits NPC differentiation. Taken together, these results point to several cellular mechanisms whereby exposure of human neural stem cells to cocaine in utero could contribute to subsequent neurodevelopmental and neurocognitive deficits.


Subject(s)
Brain/cytology , Cocaine/pharmacology , Neurons/cytology , Stem Cells/cytology , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , HMGB Proteins/analysis , Humans , Receptors, CXCR4/analysis , SOXB1 Transcription Factors , Transcription Factors/analysis
4.
J Neurosci Res ; 82(6): 839-50, 2005 Dec 15.
Article in English | MEDLINE | ID: mdl-16273540

ABSTRACT

Cytomegalovirus (CMV) is a major cause of congenital brain disease, and its neuropathogenesis may be related to viral infection of rapidly dividing, susceptible neural precursor cells (NPCs). In the present study, we evaluated the susceptibility of human fetal brain-derived NPCs (nestin(+), A2B5(+), CD133(+)) to infection with CMV. Data derived from these studies demonstrated that undifferentiated NPCs supported productive viral replication. After differentiation in the presence of serum, a treatment that promotes development of an astroglial cell phenotype (GFAP(+), nestin(-), A2B5(-)), viral expression was retained. However, differentiation of NPCs in medium containing platelet-derived growth factor and brain-derived neurotropic factor, conditions that support the development of neurons (Tuj-1(+), nestin(-), A2B5(-)), resulted in reduced viral expression, with corresponding decreased CMV major immediate-early promoter (MIEP) activity relative to undifferentiated cells. Further experiments showed that cellular differentiation into a neuronal phenotype was associated with elevated levels of various CCAAT/enhancer binding protein beta (C/EBP)-beta isoforms, which suppressed MIEP activity in cotransfected NPCs. Taken together, these data demonstrate that the susceptibility of primary human NPCs to CMV is retained concomitantly with differentiation into glial cells but is actively repressed following differentiation into neurons.


Subject(s)
Brain/cytology , Cell Differentiation/physiology , Cytomegalovirus/physiology , Neuroglia/virology , Neurons/virology , Stem Cells/virology , AC133 Antigen , Antigens, CD/metabolism , Blotting, Western/methods , Brain/embryology , Brain-Derived Neurotrophic Factor/pharmacology , Cell Proliferation , Fetus , Flow Cytometry/methods , Gene Expression Regulation/physiology , Glial Fibrillary Acidic Protein/metabolism , Glycoproteins/metabolism , Growth Substances/pharmacology , Humans , Immunohistochemistry/methods , Intermediate Filament Proteins/metabolism , L-Lactate Dehydrogenase/metabolism , Nerve Tissue Proteins/metabolism , Nestin , Neuroglia/cytology , Neuroglia/metabolism , Neurons/cytology , Neurons/metabolism , Peptides/metabolism , Platelet-Derived Growth Factor/pharmacology , Reverse Transcriptase Polymerase Chain Reaction/methods , Stem Cells/cytology , Stem Cells/metabolism , Tetrazolium Salts , Thiazoles , Time Factors , Transfection/methods , beta-Galactosidase/metabolism
5.
J Leukoc Biol ; 78(6): 1233-41, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16314440

ABSTRACT

Recent studies have shown that proinflammatory cytokines damage rodent neural precursor cells (NPCs), a source of self-renewing, multipotent cells that play an important role in the developing as well as adult brain. In this study, the effects of tumor necrosis factor alpha (TNF-alpha) on cytokine and chemokine production by human NPCs (>98% nestin- and >90% A2B5-positive), obtained from 6- to 8-week-old fetal brain specimens, were evaluated. NPCs stimulated with this proinflammatory cytokine were found to produce abundant amounts of the chemokines monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL2) and interferon-inducible protein 10 (IP-10)/CXC chemokine ligand 10 (CXCL10) in a time- and concentration-dependent manner. TNF-alpha treatment also induced NPC apoptosis. Receptors for TNF [TNFRI (p55) and TNFRII (p75)] mRNA were constitutively expressed on NPCs. However, only TNFRI was involved in TNF-alpha-induced chemokine production and apoptosis by NPCs, as anti-TNFRI but not anti-TNFRII antibodies blocked the stimulatory effect. TNF-alpha treatment induced p38 mitogen-activated protein kinase (MAPK) phosphorylation in NPCs, and SB202190, an inhibitor of p38 MAPK, blocked TNF-alpha-induced chemokine production. Thus, this study demonstrated that NPCs constitutively express receptors for TNF-alpha, which when activated, trigger via a p38 MAPK signaling pathway production of two chemokines, MCP-1/CCL2 and IP-10/CXCL10, which are involved in infectious and inflammatory diseases of the brain.


Subject(s)
Apoptosis/immunology , Brain/immunology , Chemokines/immunology , Neurons/immunology , Stem Cells/immunology , Tumor Necrosis Factor-alpha/immunology , Apoptosis/drug effects , Brain/cytology , Brain/physiopathology , Chemokine CCL2/immunology , Chemokine CCL2/metabolism , Chemokine CXCL10 , Chemokines/metabolism , Chemokines, CXC/immunology , Chemokines, CXC/metabolism , Cytokines/immunology , Cytokines/metabolism , Encephalitis/chemically induced , Encephalitis/immunology , Encephalitis/metabolism , Humans , Intermediate Filament Proteins/metabolism , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , MAP Kinase Signaling System/immunology , Nerve Degeneration/chemically induced , Nerve Degeneration/immunology , Nerve Degeneration/metabolism , Nerve Tissue Proteins/metabolism , Nestin , Neurons/drug effects , Neurons/metabolism , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/immunology , Receptors, Tumor Necrosis Factor, Type I/metabolism , Stem Cells/drug effects , Stem Cells/metabolism , Tumor Necrosis Factor-alpha/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/immunology , p38 Mitogen-Activated Protein Kinases/metabolism
6.
Brain Res Dev Brain Res ; 152(2): 159-69, 2004 Sep 17.
Article in English | MEDLINE | ID: mdl-15351504

ABSTRACT

Neural precursor cells (NPCs) are self-renewing, multipotent progenitors that give rise to neurons, astrocytes and oligodendrocytes in the central nervous system (CNS). Fetal NPCs have attracted attention for their potential use in studying normal CNS development. Several studies of rodent neural progenitors have suggested that chemokines and their receptors are involved in directing NPC migration during CNS development. In this study, we established a consistent system to culture human NPCs and examined the expression of chemokine receptors on these cells. NPCs were found to express the markers nestin and CD133 and to differentiate into neurons, astrocytes and oligodendrocytes at the clonal level. Flow cytometry and RNase protection assay (RPA) indicated that NPCs express high levels of CXCR4 and low levels of several other chemokine receptors. When examined using a chemotaxis assay, NPCs were able to respond to CXCL12/SDF-1alpha, a ligand of CXCR4. Treatment with anti-CXCR4 antibody or HIV-1 gp120 abolished the migratory response of NPCs towards CXCL12/SDF-1alpha. These findings suggest that CXCR4 may play a significant role in directing NPC migration during CNS development.


Subject(s)
Chemokines/metabolism , Neurons/metabolism , Receptors, CXCR4/metabolism , Stem Cells/metabolism , Telencephalon/embryology , Telencephalon/metabolism , AC133 Antigen , Antigens, CD/metabolism , Astrocytes/cytology , Astrocytes/drug effects , Astrocytes/metabolism , Biomarkers , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Lineage/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Chemokine CXCL12 , Chemokines, CXC/metabolism , Chemokines, CXC/pharmacology , Glycoproteins/metabolism , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp120/pharmacology , Humans , Intermediate Filament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Nestin , Neurons/cytology , Neurons/drug effects , Oligodendroglia/cytology , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Peptides/metabolism , Stem Cells/cytology , Stem Cells/drug effects , Telencephalon/cytology , Up-Regulation/drug effects , Up-Regulation/physiology
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