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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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Viral infection reduces the number of CD133(+) cells and alters nestin expression in the developing brain.A. Representative dotplots from mock-infected and MCMV-infected brains showing CD133(+) cells at 7 d p.i. Dissociated brain-derived cells were incubated with CD133(+) Mabs and analyzed by flow cytometry. Live cell gating was performed by using 7 AAD, the live cells were also excluded from immune cells that may infiltrate during the infection using CD45-PE-Cy5. B. The absolute numbers of CD133(+) cells obtained from infected brains versus control brains are shown. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus mock infected. The cells prepared from P7 brain were also stained for intracellular nestin expression. C. Representative histogram overlays from isotype (grey line, filled) uninfected (red line, tinge) and infected (blue line) brains. D. Mean fluorescent intensity for nestin expression was calculated and found to be significantly lower in the cells isolated from virus-infected brain. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus control new born mice.
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pone-0016211-g003: Viral infection reduces the number of CD133(+) cells and alters nestin expression in the developing brain.A. Representative dotplots from mock-infected and MCMV-infected brains showing CD133(+) cells at 7 d p.i. Dissociated brain-derived cells were incubated with CD133(+) Mabs and analyzed by flow cytometry. Live cell gating was performed by using 7 AAD, the live cells were also excluded from immune cells that may infiltrate during the infection using CD45-PE-Cy5. B. The absolute numbers of CD133(+) cells obtained from infected brains versus control brains are shown. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus mock infected. The cells prepared from P7 brain were also stained for intracellular nestin expression. C. Representative histogram overlays from isotype (grey line, filled) uninfected (red line, tinge) and infected (blue line) brains. D. Mean fluorescent intensity for nestin expression was calculated and found to be significantly lower in the cells isolated from virus-infected brain. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus control new born mice.

Mentions: Because NSCs were found to be infected with MCMV, we then assessed whether viral infection had any effect on their number. Brain tissues were harvested from MCMV-infected and non-infected control neonates at 7 d p.i. and were stained for CD133. Flow cytometric analysis showed that numbers of CD133(+) cells were significantly reduced in the infected brains (1.41±0.80%) when compared to controls (5.35±2.0%) (Fig 3A). Absolute numbers of CD133(+) cells in control and MCMV-infected mice were 3.09×105±4.1×104 and 3.45×104±2.1×104, p = 0.01 (Fig 3 B). Relatively low levels of nestin expression were detected in infected-brains by flow cytometry (Fig 3C). Mean fluorescence intensity of nestin expression was found to be significantly lower among cells purified from virus-infected neonates (57.00±2.2% versus 143.00±2.84%, respectively, p<0.01 Student's t test) (Fig. 3D).


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

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

Viral infection reduces the number of CD133(+) cells and alters nestin expression in the developing brain.A. Representative dotplots from mock-infected and MCMV-infected brains showing CD133(+) cells at 7 d p.i. Dissociated brain-derived cells were incubated with CD133(+) Mabs and analyzed by flow cytometry. Live cell gating was performed by using 7 AAD, the live cells were also excluded from immune cells that may infiltrate during the infection using CD45-PE-Cy5. B. The absolute numbers of CD133(+) cells obtained from infected brains versus control brains are shown. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus mock infected. The cells prepared from P7 brain were also stained for intracellular nestin expression. C. Representative histogram overlays from isotype (grey line, filled) uninfected (red line, tinge) and infected (blue line) brains. D. Mean fluorescent intensity for nestin expression was calculated and found to be significantly lower in the cells isolated from virus-infected brain. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus control new born mice.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020957&req=5

pone-0016211-g003: Viral infection reduces the number of CD133(+) cells and alters nestin expression in the developing brain.A. Representative dotplots from mock-infected and MCMV-infected brains showing CD133(+) cells at 7 d p.i. Dissociated brain-derived cells were incubated with CD133(+) Mabs and analyzed by flow cytometry. Live cell gating was performed by using 7 AAD, the live cells were also excluded from immune cells that may infiltrate during the infection using CD45-PE-Cy5. B. The absolute numbers of CD133(+) cells obtained from infected brains versus control brains are shown. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus mock infected. The cells prepared from P7 brain were also stained for intracellular nestin expression. C. Representative histogram overlays from isotype (grey line, filled) uninfected (red line, tinge) and infected (blue line) brains. D. Mean fluorescent intensity for nestin expression was calculated and found to be significantly lower in the cells isolated from virus-infected brain. Data were derived from 3 independent experiments, n = 3–5 neonates. *p<0.05 versus control new born mice.
Mentions: Because NSCs were found to be infected with MCMV, we then assessed whether viral infection had any effect on their number. Brain tissues were harvested from MCMV-infected and non-infected control neonates at 7 d p.i. and were stained for CD133. Flow cytometric analysis showed that numbers of CD133(+) cells were significantly reduced in the infected brains (1.41±0.80%) when compared to controls (5.35±2.0%) (Fig 3A). Absolute numbers of CD133(+) cells in control and MCMV-infected mice were 3.09×105±4.1×104 and 3.45×104±2.1×104, p = 0.01 (Fig 3 B). Relatively low levels of nestin expression were detected in infected-brains by flow cytometry (Fig 3C). Mean fluorescence intensity of nestin expression was found to be significantly lower among cells purified from virus-infected neonates (57.00±2.2% versus 143.00±2.84%, respectively, p<0.01 Student's t test) (Fig. 3D).

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