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Murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain.

Mutnal MB, Cheeran MC, Hu S, Lokensgard JR - PLoS ONE (2011)

Bottom Line: In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain.In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin.This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development.

View Article: PubMed Central - PubMed

Affiliation: Neuroimmunology Laboratory, Department of Medicine, Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT

Background: Congenital cytomegalovirus (CMV) brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV) and the pattern of injury to the developing brain.

Methodology/principal findings: We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi) cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection.

Conclusions: MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons.

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Decreased expression of DCX in the infected brain.Cells isolated from MCMV-infected and control brains were analyzed for intracellular doublecortin (DCX) and glial fibrillary acidic protein (GFAP) at 7 d p.i. Histogram overlays from isotype (grey line, filled), infected (blue line, filled), and control (red line, tinge) are shown for: A. DCX, a marker for young/immature neurons and B. GFAP, a marker for glial precursors. Infected brains showed reduced expression levels of intracellular DCX compared to control brains while there was no difference in the expression levels of GFAP in MCMV infected versus control brains. C. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus mock infected.
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pone-0016211-g007: Decreased expression of DCX in the infected brain.Cells isolated from MCMV-infected and control brains were analyzed for intracellular doublecortin (DCX) and glial fibrillary acidic protein (GFAP) at 7 d p.i. Histogram overlays from isotype (grey line, filled), infected (blue line, filled), and control (red line, tinge) are shown for: A. DCX, a marker for young/immature neurons and B. GFAP, a marker for glial precursors. Infected brains showed reduced expression levels of intracellular DCX compared to control brains while there was no difference in the expression levels of GFAP in MCMV infected versus control brains. C. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus mock infected.

Mentions: We next examined if viral infection is associated with abnormal expression of structural proteins such as doublecortin (DCX) and GFAP. DCX is a microtubule associated protein expressed by neuroblasts and is accepted as an effective read-out for neurogenesis. GFAP is an intermediate filament protein that is thought to be specific for astrocytes. Using an intracellular staining technique and flow cytometry, we found that expression of DCX is altered in the virus-infected brain (Fig. 7A), while GFAP expression remained unaltered (Fig. 7B). Histogram overlays are shown for both DCX and GFAP expression and were prepared from isotype, control, and virus infected peaks. Mean fluorescence intensity of DCX and GFAP expression were also measured between the groups. Expression levels of DCX in virus-infected brains were found to be significantly lower compared to uninfected control animals (511±140% versus, 1178±161% respectively, p<0.05 Student's t test) and there was no significant difference in the MFI of GFAP expression among groups studied (Fig. 7C).


Murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain.

Mutnal MB, Cheeran MC, Hu S, Lokensgard JR - PLoS ONE (2011)

Decreased expression of DCX in the infected brain.Cells isolated from MCMV-infected and control brains were analyzed for intracellular doublecortin (DCX) and glial fibrillary acidic protein (GFAP) at 7 d p.i. Histogram overlays from isotype (grey line, filled), infected (blue line, filled), and control (red line, tinge) are shown for: A. DCX, a marker for young/immature neurons and B. GFAP, a marker for glial precursors. Infected brains showed reduced expression levels of intracellular DCX compared to control brains while there was no difference in the expression levels of GFAP in MCMV infected versus control brains. C. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus mock infected.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020957&req=5

pone-0016211-g007: Decreased expression of DCX in the infected brain.Cells isolated from MCMV-infected and control brains were analyzed for intracellular doublecortin (DCX) and glial fibrillary acidic protein (GFAP) at 7 d p.i. Histogram overlays from isotype (grey line, filled), infected (blue line, filled), and control (red line, tinge) are shown for: A. DCX, a marker for young/immature neurons and B. GFAP, a marker for glial precursors. Infected brains showed reduced expression levels of intracellular DCX compared to control brains while there was no difference in the expression levels of GFAP in MCMV infected versus control brains. C. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus mock infected.
Mentions: We next examined if viral infection is associated with abnormal expression of structural proteins such as doublecortin (DCX) and GFAP. DCX is a microtubule associated protein expressed by neuroblasts and is accepted as an effective read-out for neurogenesis. GFAP is an intermediate filament protein that is thought to be specific for astrocytes. Using an intracellular staining technique and flow cytometry, we found that expression of DCX is altered in the virus-infected brain (Fig. 7A), while GFAP expression remained unaltered (Fig. 7B). Histogram overlays are shown for both DCX and GFAP expression and were prepared from isotype, control, and virus infected peaks. Mean fluorescence intensity of DCX and GFAP expression were also measured between the groups. Expression levels of DCX in virus-infected brains were found to be significantly lower compared to uninfected control animals (511±140% versus, 1178±161% respectively, p<0.05 Student's t test) and there was no significant difference in the MFI of GFAP expression among groups studied (Fig. 7C).

Bottom Line: In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain.In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin.This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development.

View Article: PubMed Central - PubMed

Affiliation: Neuroimmunology Laboratory, Department of Medicine, Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT

Background: Congenital cytomegalovirus (CMV) brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV) and the pattern of injury to the developing brain.

Methodology/principal findings: We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi) cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection.

Conclusions: MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons.

Show MeSH
Related in: MedlinePlus