Murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain - PubMed (original) (raw)
Murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain
Manohar B Mutnal et al. PLoS One. 2011.
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.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. CD133(+) cells are infected with MCMV in the developing brain.
A. Day-old neonates from timed breeders were infected intra-cerebrally (i.c.) either with 200 TCID50 of virulent, salivary gland passed recombinant MCMV expressing green fluorescent protein (GFP) or with mock inoculum, representing controls. A single cell suspension was prepared from the harvested brains by papain enzyme digestion at 7 d p.i. One million cells from both infected and control brains were stained for CD133, and analyzed by flow cytometry. B. Representative histogram showing MCMV infected CD133(+) cells, the black line represents control and the green line was obtained from MCMV-infected brain. Data shown are representative of three independent experiments using cells prepared from P7 brains obtained from 4, 3 and 5 neonates, respectively.
Figure 2. Neural stem/progentior cells (NSPC) expressing nestin are targets for MCMV.
Representative dotplot showing nestin(+)/nestin(−) cells from P7 brain. Day-old neonates were infected through intra-cerebral injections with 200 TCID50 of recombinant virus expressing GFP and control neonates received similar dose of mock inoculum. Brain tissues from infected and control animals were harvested at 7 d p.i., and dissociated into single cells using TrypLE enzyme. The cells were stained for intracellular nestin and analyzed for nestin(−)GFP(+) as well as nestin(+)GFP(+) cells. Gates in the histograms were drawn based on mock-infected controls. Data shown are representative of three independent experiments using cells prepared from P7 brains (n = 12 neonates).
Figure 3. 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.
Figure 4. CD24(hi), neuronal precursors cells are infected with the virus.
We first established a surface biomarker code based on published literature to identify the types of cells that were infected with MCMV. Brain-derived cells prepared from either infected or mock-infected animals were incubated with different Mabs for surface markers including CD15, CD24 and CD29. Representative contour plot prepared from control mice, gated on the CD45(−)CD15(−) population showing the classification of cells (lower panel) and the ratio of MCMV-GFP(+) among the subtypes are shown (upper panel). CD24 expression in the developing brain has been attributed to transit amplifying cells and is also required for terminal differentiation of neuronal progenitors. High CD29 expression is associated with neural crest like progenitor cells. Three different subtypes of cells were identified using CD24 and CD29 surface markers as shown in the representative contour plot. CD24(hi)CD29(−), CD24(hi)CD29(+) cell types were found to be infected.
Figure 5. Reduced BrdU uptake by high CD24 expressing neuronal precursor cells in virus-infected brains.
Mice were treated with BrdU on 7 d p.i. and 24 h later the brain samples were collected and processed for intranuclear BrdU staining as described in the methods. Cells were then analyzed by flow cytometry. A. Representative contour plot, gated on the CD45(−)CD15(−) population showing the classification of cells (lower panel) and the ratio of BrdU+ cells among the respective cell types is shown (upper panel). B. The absolute numbers of BrdU+ cells within each subtype, 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.
Figure 6. 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.
Figure 7. 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.
Figure 8. MCMV brain infection of the neonates down-regulates neurotrphin expression levels.
BDNF and NT3 mRNA expression was assessed using quantitative real-time RT-PCR on total RNA extracted from whole brain homogenates of control and virus- infected neonates at 7 d p.i. mRNA levels were normalized to HPRT and are presented as mean ± SD normalized expression from pooled data obtained using 3–5 animals per group from 2 independent experiments.
References
- Bale JF., Jr Human cytomegalovirus infection and disorders of the nervous system. Arch Neurol. 1984;41:310–320. - PubMed
- Becroft DM. Prenatal cytomegalovirus infection: epidemiology, pathology and pathogenesis. Perspect Pediatr Pathol. 1981;6:203–241. - PubMed
- Stagno S, Pass RF, Cloud G, Britt WJ, Henderson RE, et al. Primary cytomegalovirus infection in pregnancy. Incidence, transmission to fetus, and clinical outcome. Jama. 1986;256:1904–1908. - PubMed
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