Corina I. García

Oligodendrocytes and myelination: The role of iron

Iron is an essential trophic factor that is required for oxygen consumption and ATP production. Thus it plays a key role in vital cell functions. Although the brain has a relatively high rate of oxygen consumption compared to other organs, oligodendrocytes are the principal cells in the CNS that stain for iron under normal conditions. The importance of iron in myelin production has been demonstrated by studies showing that decreased availability of iron in the diet is associated with hypomyelination. The timing of iron delivery to oligodendrocytes during development is also important because hypomyelination and the associated neurological sequelae persist long after the systemic iron deficiency has been corrected. Therefore, identifying the molecular roles of iron in oligodendrocyte development and myelin production, and the mechanisms and timing of iron acquisitions are important prerequisites to developing effective therapies for dysmyelinating disorders. It is the purpose of this review to give a comprehensive overview of the existing literature on role of iron in oligodendrocytes and the mechanisms of iron acquisition and intracellular handling.

Multidrug Resistance Protein 4 (MRP4/ABCC4) Regulates cAMP Cellular Levels and Controls Human Leukemia Cell Proliferation and Differentiation

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Apotransferrin promotes the differentiation of two oligodendroglial cell lines

We have previously shown that addition of apotransferrin (aTf) accelerates maturation of oligodendroglial cells (OLGcs) in primary cultures. In this work, we examined the effect of aTf on two conditionally immortalized cell lines: N19 and N20.1. These cells proliferate at 34°C and differentiate into mature OLGcs at 39°C. In vitro addition of aTf to both cell lines at the differentiation temperature for 7 days showed increased expression of galactocerebroside, O4, and myelin basic protein (MBP) and a drop in the percentage of BrdU+ cells. The effect on MBP expression was particularly interesting in the less mature N19 cells. These cells do not express either MBP mRNAs or proteins, so aTf induced, rather than modulated, MBP expression in this cell line. In addition, even though MBP mRNAs for all four isoforms were induced, only the 17 and 21.5 kDa appeared to be translated. OLGc differentiation has been shown to be stimulated by the cAMP‐CREB pathway. In N19 cells, following a pulse of aTf, there was a 10‐fold increase in cAMP levels accompanied by elevated levels of pCREB. In the more mature N20.1 cells, there were no changes in cAMP levels. We conclude that addition of aTf to immature OLGc lines can enhance their expression of differentiated markers, such as MBP. The action of aTf on MBP gene expression in the least mature line is likely to be mediated by the cAMP pathway. In the N20.1 cells, it appears that different signals and/or mechanisms are involved in modulating myelin lipid and MBP expression. The results suggest that aTf can influence OLGc gene expression and differentiation through multiple mechanisms depending on the maturation of the cell.

Apotransferrin decreases the response of oligodendrocyte progenitors to PDGF and inhibits the progression of the cell cycle

In the CNS, transferrin (Tf) is expressed by the oligodendroglial cells (OLGcs) and is essential for their development. We have previously shown that apotransferrin (aTf) accelerates maturation of OLGcs in vivo as well as in vitro. The mechanisms involved in this action appear to be complex and have not been completely elucidated. The aim of this study was to investigate if Tf participates in the regulation of the cell cycle of oligodendroglial progenitor cells (OPcs). Primary cultures of OPcs were treated with aTf and/or with different combinations of mitogenic factors. Cell cycle progression was studied by BrdU incorporation, flow cytometry and by the expression of cell cycle regulatory proteins. Apotransferrin decreased the number of BrdU+ cells, increasing the cell cycle time and decreasing the number of cells in S phase. The cell cycle inhibitors p27kip1, p21cip1 and p53 were increased, and in agreement with these results, the activity of the complexes involved in G1-S progression (cyclin D/CDK4, cyclin E/CDK2), was dramatically decreased. Apotransferrin also inhibited the mitogenic effects of PDGF and PDGF/IGF on OPcs, but did not affect their proliferation rate in the presence of bFGF, bFGF/PDGF or bFGF/IGF. Our results indicate that inhibition of the progression of the cell cycle of OPcs by aTf, even in the presence of PDGF, leads to an early beginning of the differentiation program, evaluated by different maturation markers (O4, GC and MBP) and by morphological criteria. The modulation by aTf of the response of OPcs to PDGF supports the idea that this glycoprotein might act as a key regulator of the OLGc lineage progression.

Overexpression of human transferrin in two oligodendroglial cell lines enhances their differentiation.

We have previously demonstrated that the addition of apotransferrin (aTf) to oligodendroglial cell (OLGc) primary cultures accelerates their maturation. Cells treated with aTf developed a multipolar morphology and displayed increased expression of mature OLGc markers. In this work, we studied the effect of Tf overexpression in two OLGc lines, N19 and N20.1. The former cells exhibit characteristics of OLGc precursors (O2A), while N20.1 cells express markers of more mature OLGcs. Using the complete cDNA of the human Tf gene, we obtained clones overexpressing Tf in both cell lines. These clones were evaluated for the expression of OLGc differentiation markers. In agreement with our previous results, we found that in the cells overexpressing Tf, there was an increased O4, GC, and MBP immunoreactivity. To study the myelinogenic potential of these cells, we co‐cultured N19 and N20.1 Tf‐transfected cells together with cortical neurons. There was a dramatic increase in the morphological differentiation of the OLGcs accompanied by enhanced GC and MBP expression. The OLGcs appeared to establish contact with neurites and extend their processes along them. Only two MBP isoforms were detected in Tf‐overexpressing clones, while all the isoforms were present in the co‐cultures, suggesting that there was a modulation of MBP expression by neurons. Concomitantly, we found an increase in several proteins involved in axon–glia interaction, such as MAG, N‐CAM, and F3/Contactin. This co‐culture system represents a potentially powerful tool to study neuron–glia interactions that occur during myelinogenesis and the role of Tf in this process.

Apotransferrin induces cAMP/CREB pathway and cell cycle exit in immature oligodendroglial cells.

We have demonstrated previously that a single intracranial injection of apotransferrin (aTf) in neonatal rats increases myelination and accelerates differentiation of oligodendroglial cells (OLGc). In addition, we have shown through in vitro experiments that OLGc isolated from 4‐day‐old rats (OLGc‐4) treated with aTf were more differentiated than were controls although aTf had no effect upon OLGc isolated from 10‐day‐old animals (OLGc‐10). In the present work, we analyzed the role of second messengers in the effect of aTf upon the maturation of OLGc at different stages of development. We isolated OLGc‐4 and OLGc‐10 from rat brain using a Percoll density gradient and briefly treated the cells with a pulse of aTf or kept them in culture during 2 days in the presence or absence of aTf. In OLGc‐4, after a short pulse of aTf, there was an increase in the levels of cyclic AMP (cAMP), in the phosphorylation of cAMP response element‐binding protein (CREB) and in the DNA‐binding capacity of cAMP‐responsive transcription factors. Treatment of OLGc‐4 with aTf diminished bromodeoxyuridine (BrdU) incorporation and changed levels of p27 and cyclin D1. This glycoprotein seemed to act on OLGc through the cAMP pathway only at early stages of development and on a certain sensitive cell population, accelerating their differentiation, probably as a consequence of premature withdrawal from the cell cycle.

Differential effects of apotransferrin on two populations of oligodendroglial cells.

In the central nervous system (CNS), apotransferrin (aTf) is produced by oligodendroglial cells (OLGcs), and aTf is essential for cell survival. We previously demonstrated that a single intracranial injection of aTf in 3‐day‐old rats accelerates differentiation of OLGc and that aTf acts at early stages of development on certain populations of OLGcs, promoting accelerated maturation, with no effect on late markers of cell differentiation. The objective of the present study was to analyze OLGc maturation at two different stages of rat development, 4 and 10 days of age, in OLGcs isolated from the brain after intracranial injection of aTf at 3 days of age, and to explore the in vitro effect of aTf added to cultures of OLGc isolated from aTf‐injected and control brains. The maturational cell stages were identified by immunocytochemistry with different OLGc markers and by analysis of their morphological complexity. The OLGcs isolated from 4‐ and 10‐day‐old animals intracranially injected with aTf were more differentiated than control cells. Treatment with aTf of the cultures of OLGcs that were isolated from 4‐day‐old saline‐injected control animals induced their differentiation, while a similar treatment of the cultures of OLGcs that were isolated from 10‐day‐old animals did not induce further maturation of the cells. The results presented in the present report demonstrate that the in vivo effects of aTf on OLGc maturation can be reproduced in cultures and that the effects of aTf occur early in development during a narrow, transient “temporal window” within which OLGcs are sensitive to its action. GLIA 42:406–416, 2003.

Expression of myelin basic protein in two oligodendroglial cell lines is modulated by apotransferrin through different transcription factors

We have shown that apotransferrin (aTf) promotes the differentiation of two oligodendroglial cell (OLGc) lines, N19 and N20.1, representing different stages of OLGc maturation. Although in both cell lines aTf promoted myelin basic protein (MBP) expression, an increase in cAMP levels and CREB phosphorylation was observed only in the less mature cells (N19), suggesting that the maturation induced by aTf is achieved probably through different signaling pathways. We transfected both cell lines with the proximal region of the human MBP promoter fused to the lacZ reporter gene. In both transfected cell lines, addition of aTf produced an activation of the promoter. To elucidate the mechanisms involved in this action, Western blot analysis, EMSAs, and RT‐PCR were performed for different transcription factors involved in mbp regulation. In the N20.1 line, treatment with aTf increased the expression and the DNA‐binding capacity of thyroid hormone (TH) receptors, Sp1, and nuclear factor‐κB (NFκB). For these cells we found that an inductor of NFκB (tumor necrosis factor‐α) promoted MBP messenger synthesis, whereas mithramycin, a specific inibitor of Sp1, and a cAMP analog (db‐cAMP) inhibited its transcription. In the N19 cell line, aTf stimulated NF‐I and NFκB activation, but, aside from aTf, only db‐cAMP induced mbp transcription. These data suggest that, depending on the OLGc maturational stage, aTf modulates MBP expression and OLGc differentiation through different signaling pathways and different transcription factors.

Differential Gene Expression during Development in Two Oligodendroglial Cell Lines Overexpressing Transferrin: A cDNA Array Analysis

In the central nervous system, transferrin (Tf) is produced by oligodendroglial cells (OLGcs) and is essential for their development. Recently, using the complete cDNA of the human Tf gene, we obtained clones overexpressing Tf in two OLGc lines, N19 and N20.1, which represent different stages of differentiation. We showed that the overexpression of this glycoprotein promotes the maturation and myelinogenic capacity of both cell lines. In this work, using cDNA array technology, we examined changes induced by Tf in 1,176 genes. We found 41 genes differentially expressed in both cell lines, all of them involved in OLGc development. In the less mature cells (N19) overexpressing Tf, there was a significant increase in key enzymes of neurosteroid metabolism, such as cholesterol side chain cleavage cytochrome P450, 3β-hydroxysteroid dehydrogenase and 5α-reductase type 1. In the more mature cell line (N20.1), Tf overexpression produced an induction of several mRNAs of the GABAA receptor subunits, of thyroid hormone receptors and of proteins involved in axon-glia interactions such as F3/contactin. In addition, in both cell lines, Tf overexpression induced an increase in the expression of different isoforms of transforming growth factor β receptors and in several genes related to mitochondrial function and to complex lipid metabolism, crucial steps in myelin synthesis. Differentiation produced by Tf in both cell lines seems to occur by modulation of different genes depending on the maturational stage of the cells. Our findings provide new insights into the molecular basis of OLGc differentiation and on the role played by Tf in this process.

Partial Inhibition of the Proteasome Enhances the Activity of the Myelin Basic Protein Promoter

We have previously shown that low concentrations of a specific proteasome inhibitor accelerate exit from the cell cycle and enhance oligodendroglial cell (OLGc) differentiation. To elucidate the mechanisms involved in this process, OLGcs of the N20.1 cell line, transfected with a reporter gene driven by the MBP promoter, were treated with proteasome inhibitors and/or inhibitors of different signaling pathways. Partial proteasome inhibition resulted in enhanced activation of the MBP promoter which involved the tyrosine kinase, PI3-Akt and PKC pathways, accompanied by an increase in the levels of p21Cip1, p27Kip1 and Sp1 and by a decrease in Nkx2.2. Binding of Sp1 to DNA was also increased. These results were not observed when the Sp1 binding site was mutated. We conclude that the enhanced activation of the MBP promoter induced by partial inhibition of the proteasome could be due, at least in part, to the stabilization of p27Kip1 and Sp1.