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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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Differential expression of the transcription factor Oct4 in infected brains.Brain-derived cells prepared from either infected or mock-infected animals were incubated with different MAbs for surface markers including CD15, CD24, and CD29. The cellular subsets were then analyzed for expression of the transcription factor Oct4 and compared with mock-infected brains in the histogram overlays. Histogram overlays show a blue line representing control neonates, an orange line MCMV infected, and a black line representing isotype for Oct4 staining. Data were derived from 3 independent experiments, n = 3–5 neonates.
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pone-0016211-g006: Differential expression of the transcription factor Oct4 in infected brains.Brain-derived cells prepared from either infected or mock-infected animals were incubated with different MAbs for surface markers including CD15, CD24, and CD29. The cellular subsets were then analyzed for expression of the transcription factor Oct4 and compared with mock-infected brains in the histogram overlays. Histogram overlays show a blue line representing control neonates, an orange line MCMV infected, and a black line representing isotype for Oct4 staining. Data were derived from 3 independent experiments, n = 3–5 neonates.

Mentions: Oct4 is critically involved in self-renewal of embryonic stem cells, so it is frequently used as a marker for undifferentiated cells. Oct4 expression must be closely regulated; too much or too little will actually induce differentiation [26]. The transcription factors Oct4, Sox2, and Nanog are capable of inducing the expression of each other, and are essential for maintaining the self-renewing undifferentiated state of the inner cell mass of the blastocyst, as well as in embryonic stem cells [27]. It has been previously shown that CD24 cells express Sox2 indicating they still retain stemness [28]. To determine if CD24(hi) cells from the developing brain expressed Oct4, we performed intracellular staining for Oct4 as well as surface stained for CD15, CD24 and CD29 and analyzed these cell populations using flow cytometry. In this study, we demonstrated that CD24(hi)CD29(−) and CD24(hi)CD29(+) cells, isolated from uninfected control brains, expressed Oct4 (Fig. 6, upper panel, histogram with blue line). Although CD24(hi)CD29(−) negative cells were found to express Oct4, it appeared that once CD29 expression occurred on these CD24(hi) cells the number of Oct4-expressing cells was reduced (Fig. 6, upper panel, histogram on the right). We then went on to determine if MCMV infection had any effect on Oct4 expression among this defined subset of cells. The upper panel shows histogram overlays for Oct4 staining from brain cells isolated from control and infected brains, as well as isotype control staining. Data obtained from these experiments demonstrated that Oct4 expression was reduced in cells obtained from virus-infected brains compared to the cells from the brains of uninfected animals (upper panel, histogram overlays).


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

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

Differential expression of the transcription factor Oct4 in infected brains.Brain-derived cells prepared from either infected or mock-infected animals were incubated with different MAbs for surface markers including CD15, CD24, and CD29. The cellular subsets were then analyzed for expression of the transcription factor Oct4 and compared with mock-infected brains in the histogram overlays. Histogram overlays show a blue line representing control neonates, an orange line MCMV infected, and a black line representing isotype for Oct4 staining. Data were derived from 3 independent experiments, n = 3–5 neonates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020957&req=5

pone-0016211-g006: Differential expression of the transcription factor Oct4 in infected brains.Brain-derived cells prepared from either infected or mock-infected animals were incubated with different MAbs for surface markers including CD15, CD24, and CD29. The cellular subsets were then analyzed for expression of the transcription factor Oct4 and compared with mock-infected brains in the histogram overlays. Histogram overlays show a blue line representing control neonates, an orange line MCMV infected, and a black line representing isotype for Oct4 staining. Data were derived from 3 independent experiments, n = 3–5 neonates.
Mentions: Oct4 is critically involved in self-renewal of embryonic stem cells, so it is frequently used as a marker for undifferentiated cells. Oct4 expression must be closely regulated; too much or too little will actually induce differentiation [26]. The transcription factors Oct4, Sox2, and Nanog are capable of inducing the expression of each other, and are essential for maintaining the self-renewing undifferentiated state of the inner cell mass of the blastocyst, as well as in embryonic stem cells [27]. It has been previously shown that CD24 cells express Sox2 indicating they still retain stemness [28]. To determine if CD24(hi) cells from the developing brain expressed Oct4, we performed intracellular staining for Oct4 as well as surface stained for CD15, CD24 and CD29 and analyzed these cell populations using flow cytometry. In this study, we demonstrated that CD24(hi)CD29(−) and CD24(hi)CD29(+) cells, isolated from uninfected control brains, expressed Oct4 (Fig. 6, upper panel, histogram with blue line). Although CD24(hi)CD29(−) negative cells were found to express Oct4, it appeared that once CD29 expression occurred on these CD24(hi) cells the number of Oct4-expressing cells was reduced (Fig. 6, upper panel, histogram on the right). We then went on to determine if MCMV infection had any effect on Oct4 expression among this defined subset of cells. The upper panel shows histogram overlays for Oct4 staining from brain cells isolated from control and infected brains, as well as isotype control staining. Data obtained from these experiments demonstrated that Oct4 expression was reduced in cells obtained from virus-infected brains compared to the cells from the brains of uninfected animals (upper panel, histogram overlays).

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