Hypoxia Alters Cell Cycle Regulatory Protein Expression and Induces Premature Maturation of Oligodendrocyte Precursor Cells

Periventricular white matter injury (PWMI) is a common form of brain injury sustained by preterm infants. A major factor that predisposes to PWMI is hypoxia. Because oligodendrocytes (OLs) are responsible for myelination of axons, abnormal OL development or function may affect brain myelination. At present our understanding of the influences of hypoxia on OL development is limited. To examine isolated effects of hypoxia on OLs, we examined the influences of hypoxia on OL development in vitro.Cultures of oligodendrocyte precursor cells (OPCs) were prepared from mixed glial cultures and were 99% pure. OPCs were maintained at 21% O(2) or hypoxia (1% or 4% O(2)) for up to 7 days. We observed that 1% O(2) lead to an increase in the proportion of myelin basic protein (MBP)-positive OLs after 1 week in culture, and a decrease in the proportion of platelet-derived growth factor receptor alpha (PDGFRalpha)-positive cells suggesting premature OL maturation. Increased expression of the cell cycle regulatory proteins p27(Kip1) and phospho-cdc2, which play a role in OL differentiation, was seen as well.These results show that hypoxia interferes with the normal process of OL differentiation by inducing premature OPC maturation

Hypoxia influences on cell viability.

<p>Images of calcein (live) and ethidium (dead) cells labeled OLs cultured in the presence or absence of GFs in 21%₂, 4% or 1% O₂ for 48 hrs, 96 hrs, and 7 days. Images shown are representative of 4 separate studies.</p

Hypoxia influences on VEGF A.

<p>OLs were cultured in the presence of GFs in 21%₂, 4% or 1% O₂. Cell culture media was collected at 48 hrs, 96 hrs, and 7 days and levels of secreted VEGF A were measured by ELISA (*<i>p</i><0.01; ANOVA vs. 21% O₂). Data shown are representative of three separate studies.</p

Changes in OL cell cycle proteins in 1% O₂.

<p>Densitometric analysis of data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004739#pone-0004739-g009" target="_blank">Figure 9</a> was done by normalizing protein levels to ribosomal S6 protein levels. The percent increase in the expression of cell cycle proteins is shown relative to levels in 21% O₂ (* <i>p</i><0.05, ** <i>p</i><0.01). Data shown are representative of three separate studies.</p

Hypoxia induces premature OL maturation.

<p>Percentages of cells that are positive for PDGFRα, GalC, or MBP cultured in 21% or 1% O₂ plus GFs at 48 hrs, 96 hrs or 7 days in culture. * <i>p</i><0.05 1% vs. 21% O₂ at each time. Data shown are representative of three separate studies. At least three separate coverslips were analyzed per study.</p

Demonstration of intracellular hypoxia.

<p>Intracellular hypoxia was assessed by incubating cells with pimonidazole (A, C and E), which forms adducts at pO₂ of <10 mm Hg (green). Cells are counterstained with DAPI (B, D, and F; blue). Note absent staining in 21% O₂ (A), moderate staining in 4% O₂ (C), and high level staining in 1% O₂ (E). Scale bars = 10 um. Data shown are representative of four separate studies.</p

Hypoxia influences on cell number.

<p>CyQuant analysis was performed at 48 hrs, 96 hrs, and at 7 days for OLs cultured in the presence or absence of GFs in 21%₂ or 1% O₂. Relative florescence units are shown (RFU). In GF-treated cultures in 1% O₂, levels was similar to the 21% O₂ group at 46 and 96 hours (p>0.05; ANOVA). At 7 days, levels were greatest in the cells cultures in 1% O₂ and GFs (*, p<0.05; ANOVA). Data are averages of three separate studies.</p

Changes in OL morphology in hypoxia.

<p>OLs were cultured in 21% O₂ in the presence of GFs (A, E), in the absence of GFs (B, F), or under with GFs in 4% O₂ (C, G) or 1% O₂ (D, H) for 1 week. Fluorescence images of cells stained with actin-specific phalloidin (A–D) and phase-contrast images (E–H) of OLs show that cultures deprived of GFs (B, F) or cultures exposed to hypoxia (C, D, G, H) undergo morphological changes consistent with enhanced maturation. Scale bars = 10 um. Data shown are representative of five separate studies. At least ten separate coverslips were analyzed per study.</p

Amyloid beta peptide (25-35) activates protein kinase C leading to cyclooxygenase-2 induction and prostaglandin E2 release in primary midbrain astrocytes

Prostaglandins (PGs) are generated by the enzymatic activity of cyclooxygenase-1 and -2 (COX-1/2) and modulate several functions in the CNS such as the generation of fever, the sleep/wake cycle, and the perception of pain. Moreover, the induction of COX-2 and the generation of PGs has been linked to neuroinflammatory aspects of Alzheimer's disease (AD). Non-steroidal anti-inflammatory drugs (NSAIDs) that block COX enzymatic activity have been shown to reduce the incidence of AD in various epidemiological studies. While several reports investigated the expression of COX-2 in neurons and microglia, expression of COX-2 in astroglial cells has not been investigated in detail. Here we show that amyloid beta peptide 25-35 (Abeta(25-35)) induces COX-2 mRNA and protein synthesis and a subsequent release of prostaglandin E(2) (PGE(2)) in primary midbrain astrocytes. We further demonstrate that protein kinase C (PKC) is involved in Abeta(25-35)-induced COX-2/PGE(2) synthesis. PKC-inhibitors prevent Abeta(25-35)-induced COX-2 and PGE(2) synthesis. Furthermore Abeta(25-35) rapidly induces the phosphorylation and enzymatic activation of PKC in primary rat midbrain glial cells and in primary human astrocytes from post mortem tissue. Our data suggest that the PKC isoforms alpha and/or beta are most probably involved in Abeta(25-35)-induced expression of COX-2 in midbrain astrocytes. The potential role of astroglial cells in the phagocytosis of amyloid and the involvement of PGs in this process suggests that a modulation of PGs synthesis may be a putative target in the prevention of amyloid deposition