Microglia-derived extracellular vesicles in homeostasis and demyelination/remyelination processes.

Microglia (MG) play a crucial role as the predominant myeloid cells in the central nervous system and are commonly activated in multiple sclerosis. They perform essential functions under normal conditions, such as actively surveying the surrounding parenchyma, facilitating synaptic remodeling, engulfing dead cells and debris, and protecting the brain against infectious pathogens and harmful self-proteins. Extracellular vesicles (EVs) are diverse structures enclosed by a lipid bilayer that originate from intracellular endocytic trafficking or the plasma membrane. They are released by cells into the extracellular space and can be found in various bodily fluids. EVs have recently emerged as a communication mechanism between cells, enabling the transfer of functional proteins, lipids, different RNA species, and even fragments of DNA from donor cells. MG act as both source and recipient of EVs. Consequently, MG-derived EVs are involved in regulating synapse development and maintaining homeostasis. These EVs also directly influence astrocytes, significantly increasing the release of inflammatory cytokines like IL-1ß, IL-6, and TNF-α, resulting in a robust inflammatory response. Furthermore, EVs derived from inflammatory MG have been found to inhibit remyelination, whereas Evs produced by pro-regenerative MG effectively promote myelin repair. This review aims to provide an overview of the current understanding of MG-derived Evs, their impact on neighboring cells, and the cellular microenvironment in normal conditions and pathological states, specifically focusing on demyelination and remyelination processes.

The Role of Galectin-3: From Oligodendroglial Differentiation and Myelination to Demyelination and Remyelination Processes in a Cuprizone-Induced Demyelination Model.

The aim of this work was to combine our previously published results with our new data to show how galectin-3 (Gal-3) controls myelin integrity and function, promotes oligodendroglial cell differentiation, and regulates microglial responses to limit cuprizone- (CPZ)-induced demyelination and foster remyelination. In this study, 8-week-old Gal-3-deficient (Lgals3 -/-) and wild type (WT) mice were fed a diet containing 0.2 % CPZ w/w for 6 weeks, after which CPZ was withdrawn in order to allow remyelination. Our results show that remyelination was less efficient in Lgals3 -/- than in WT mice. Electron microscopic images from remyelinated sections in Lgals3 -/- mice revealed collapsed axons with a defective myelin wrap, while remyelinated WT mice had normal axons without relevant myelin wrap disruption. MMP-3 expression increased during remyelination in WT but not in Lgals3 -/- mice. The number of CD45+, TNFα+ and TREM-2b+ cells decreased only in WT mice only, with no alterations in Lgals3 -/- mice during demyelination and remyelination. Therefore, Gal-3 influences remyelination by mechanisms involving the tuning of microglial cells, modulation of MMP activity, and changes in myelin architecture.

Combined effects of transferrin and thyroid hormone during oligodendrogenesis In vitro.

Thyroid hormones (THs) and transferrin (Tf) are factors capable of favoring myelination due to their positive effects on oligodendroglial cell (OLG) differentiation. The first notion of a combined effect of apotransferrin (aTf) and TH emerged from experiments conducted in young hyperthyroid animals, which showed a seven-fold increase in the expression of Tf mRNA and precocious myelination when compared with control animals. The mechanism underlying this phenomenon in young hyperthyroid rats could consist of an increase in Tf synthesis, which in the CNS is almost exclusively produced by OLG. Overall, our results show that, during the initial stages of OLG differentiation, Tf synthesis triggers thyroid hormone receptor alpha 1 (TRα1) expression in the subventricular zone (SVZ) and promotes proliferating cells to become responsive to this trophic factor. Exposure to TH could then regulate Tf expression through TRα1 and promote the induction of thyroid hormone receptor beta (TRß) expression, which mediates TH effects on myelination through the activation of final OLG differentiation. This regulation of the combined effects of Tf and THs implies that both factors are fundamental actors during oligodendrogenesis. GLIA 2016;64:1879-1891.

Inhalation of growth factors and apo-transferrin to protect and repair the hypoxic-ischemic brain.

Hypoxic-ischemic brain damage is a major contributor to chronic neurological dysfunction and acute mortality in infants as well as in adults. In this review, we summarize recent publications demonstrating that the intranasal administration (INA) of apo-transferrin (aTf) and different growth factors provides neuroprotection to the mouse and rat brain after a hypoxic-ischemic event. The intranasal delivery of growth factors such as insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) has been found to improve neurological function and reduce infarct size in adult rats after a hypoxic-ischemic event. On the other hand, INA of aTf and epidermal growth factor (EGF) were effective in reducing white matter damage and inflammation and in promoting the proliferation and survival of oligodendroglial progenitor cells (OPCs) in a model of hypoxic-ischemic encephalopathy. Therefore, data summarized in this review suggest that INA of growth factors and aTf can be used in combination in clinical treatment in order to protect and repair the hypoxic-ischemic brain.

Transferrin and thyroid hormone converge in the control of myelinogenesis.

Myelination is a concerted mechanism tightly regulated in the brain. Although several factors are known to participate during this process, the complete sequence of events is far from being fully elucidated. Separate effects of apotransferrin (aTf) and thyroid hormone (TH) are well documented on rat myelin formation. TH promotes the maturation of oligodendrocyte progenitors (OPCs) into myelinating oligodendrocytes (OLGs), while aTf is able to induce the commitment of neural stem cells (NSCs) toward the oligodendroglial linage and favors OLG maturation. We have also demonstrated that Tf mRNA exhibited a seven-fold increase in hyperthyroid animals. These observations have led us to hypothesize that both factors may interplay during oligodendrogenesis. To assess the combined effects of aTf and TH on proper myelination in the rat brain, Tf expression and oligodendroglial maturation were evaluated at postnatal days 10 (P10) and 20 (P20) in several experimental groups. At P10, an up-regulation of both Tf mRNA and protein, as well as myelination, was found in hyperthyroid animals, while a decrease in Tf mRNA levels and myelin formation was detected in the hypothyroid group. At P20, no differences were found either in Tf mRNA or protein levels between hyperthyroid and control (Ctrol) rats, although differences in OLG differentiation remained. Also at P20, hypothyroid animals showed decreased Tf mRNA and protein levels accompanied with a less mature myelinating phenotype. Moreover, TH and aTf differentially regulate the expression of KLF9 transcription factor as well as TRα and TRß at P10 and P20. Our results suggest that TH is necessary early in OLG development for aTf action, as exogenous aTf administration was unable to counteract the effect of low TH levels in the hypothyroid state in all the time points analyzed. Furthermore, the fact that hyperthyroidism induced an increase in Tf expression and aTf-dependent regulation of TRα strongly suggests that Tf could be involved in some of TH later effects on OLG maturation. Here we describe the possible relationship between TH and aTf and its implication in oligodendrogenesis.

Glycan-dependent binding of galectin-1 to neuropilin-1 promotes axonal regeneration after spinal cord injury.

Following spinal cord injury (SCI), semaphorin 3A (Sema3A) prevents axonal regeneration through binding to the neuropilin-1 (NRP-1)/PlexinA4 receptor complex. Here, we show that galectin-1 (Gal-1), an endogenous glycan-binding protein, selectively bound to the NRP-1/PlexinA4 receptor complex in injured neurons through a glycan-dependent mechanism, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. Although both Gal-1 and its monomeric variant contribute to de-activation of microglia, only high concentrations of wild-type Gal-1 (which co-exists in a monomer-dimer equilibrium) bind to the NRP-1/PlexinA4 receptor complex and promote axonal regeneration. Our results show that Gal-1, mainly in its dimeric form, promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A binding to NRP-1/PlexinA4 complex, supporting the use of this lectin for the treatment of SCI patients.

Galectin-3 controls the response of microglial cells to limit cuprizone-induced demyelination.

Galectin-3 (Gal-3) is a ß-galactoside-binding lectin that plays an important role in inflammatory and neurodegenerative diseases. Cuprizone (CPZ)-induced demyelination is characterized by the loss of mature oligodendrocytes (OLG) by apoptosis, myelin sheath degeneration and recruitment of microglia and astrocytes to the lesioned area. We compared CPZ-induced demyelination of 8-week-old Lgals3(-/-) vs WT mice. Lgals3(-/-) mice displayed a similar susceptibility to CPZ-induced demyelination up to the fifth week, as evaluated by MBP immunostaining and electronic microscopy. However, OLG progenitors (OPC) generated in CPZ-treated Lgals3(-/-) mice showed diminished arborization, suggesting decreased ability of these cells to differentiate. Surprisingly, while WT mice experienced spontaneous remyelination in the fifth week of CPZ treatment-even though the CPZ diet was maintained up to sixth week-Lgals3(-/-) mice lacked this capacity and suffered continuous demyelination up to the sixth week, accompanied by pronounced astroglial activation. Moreover, after 2weeks of CPZ treatment, WT and Lgals3(-/-) mice showed lower innate anxiety as compared with respective naive mice, but only CPZ-treated Lgals3(-/-) mice showed decreased locomotor activity and exhibited spatial working memory impairment. Expression of Gal-3 increased during CPZ-induced demyelination in microglia but not in astrocytes. While CPZ-treated WT mice displayed heightened microglial activation associated with ED1 expression and pronounced upregulation of the phagocytic receptor TREM-2b, this effect was not observed in CPZ-treated Lgals3(-/-) mice which, in spite of showing an increased number of microglia, these cells evidenced caspase-3 activation. Our results indicate that Gal-3 is expressed in microglial cells to modulate their phenotype, facilitating the onset of remyelination and OLG differentiation.

Glatiramer promotes oligodendroglial cell maturation in a cuprizone-induced demyelination model.

The therapeutic potential of glatiramer acetate (GA) in Multiple Sclerosis has been apparent for many years and has been proven effective in experimental allergic encephalomyelitis, one of its animal models. The cuprizone (CPZ) model for the CNS de/remyelination has gained a renewed interest during the past decade. CPZ-induced demyelination is considered to be primarily an oligodendrocyte loss with participation of the inflammatory response. As the blood brain barrier remains intact, we found this model advantageous for studying GA effects on CNS remyelination with minimum influence of the peripheral immune cellular component. Our results show that GA, given one week before the CPZ treatment, had a maturational effect functional to remyelination. However, myelin was unorganized as compared to controls. When GA was concomitantly injected with CPZ, oligodendroglial precursor proliferation diminished in favor of maturation and myelin recovered an organized disposition. GA-treated animals also show microglial cell (MG) activation. In vitro assays demonstrated that GA-primed MG cultures had a significant increase in IL-10 and IL-4 secretion. GA-challenged MG-conditioned media induced oligodendrocyte proliferation and subsequent differentiation. Our results suggest that, in addition to its well-recognized immunoregulatory properties, GA also has an effect on resident immuno-response, which leads mature oligodendrocytes towards CPZ-induced demyelination repair.

Iron and holotransferrin induce cAMP-dependent differentiation of Schwann cells.

The differentiation of myelin-forming Schwann cells (SC) is completed with the appearance of myelin proteins MBP and P(0) and a concomitant downregulation of markers GFAP and p75NTR, which are expressed by immature and adult non-myelin-forming SC. We have previously demonstrated that holotransferrin (hTf) can prevent SC dedifferentiation in culture (Salis et al., 2002), while apotransferrin (aTf) cannot. As a consequence, we used pure cultured SC and submitted them to serum deprivation in order to promote dedifferentiation and evaluate the prodifferentiating ability of ferric ammonium citrate (FAC) through the expression of MBP, P(0), p75NTR and c-myc. The levels of cAMP, CREB and p-CREB were also measured. Results show that Fe(3+), either in its free form or as hTf, can prevent the dedifferentiation promoted by serum withdrawal. Both FAC and hTf were proven to promote differentiation, probably through the increase in cAMP levels and CREB phosphorylation, as well as levels of reactive oxygen species. This effect was inhibited by deferroxamine (Dfx, an iron chelator), H9 (a cAMP-PKA antagonist) and N-acetylcysteine (NAC, a powerful antioxidant).

Galectin-3 drives oligodendrocyte differentiation to control myelin integrity and function.

Galectins control critical pathophysiological processes, including the progression and resolution of central nervous system (CNS) inflammation. In spite of considerable progress in dissecting their role within lymphoid organs, their functions within the inflamed CNS remain elusive. Here, we investigated the role of galectin-glycan interactions in the control of oligodendrocyte (OLG) differentiation, myelin integrity and function. Both galectin-1 and -3 were abundant in astrocytes and microglia. Although galectin-1 was abundant in immature but not in differentiated OLGs, galectin-3 was upregulated during OLG differentiation. Biochemical analysis revealed increased activity of metalloproteinases responsible for cleaving galectin-3 during OLG differentiation and modulating its biological activity. Exposure to galectin-3 promoted OLG differentiation in a dose- and carbohydrate-dependent fashion consistent with the 'glycosylation signature' of immature versus differentiated OLG. Accordingly, conditioned media from galectin-3-expressing, but not galectin-3-deficient (Lgals3(-/-)) microglia, successfully promoted OLG differentiation. Supporting these findings, morphometric analysis showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio in the corpus callosum and striatum of Lgals3(-/-) compared with wild-type (WT) mice. Moreover, the myelin structure was loosely wrapped around the axons and less smooth in Lgals3(-/-) mice versus WT mice. Behavior analysis revealed decreased anxiety in Lgals3(-/-) mice similar to that observed during early demyelination induced by cuprizone intoxication. Finally, commitment toward the oligodendroglial fate was favored in neurospheres isolated from WT but not Lgals3(-/-) mice. Hence, glial-derived galectin-3, but not galectin-1, promotes OLG differentiation, thus contributing to myelin integrity and function with critical implications in the recovery of inflammatory demyelinating disorders.

Oligodendrogenesis in iron-deficient rats: effect of apotransferrin.

In rats, iron deficiency produces an alteration in myelin formation. However, there is limited information on the effects of this condition on oligodendroglial cell (OLGc) proliferation and maturation. In the present study, we further analyzed the hypomyelination associated with iron deficiency by studying the dynamics of oligodendrogenesis. Rats were fed control (40 mg Fe/kg) or iron-deficient (4 mg Fe/kg) diets from gestation day 5 until postnatal day 3 (P3) or 11 (P11). OLGc proliferation, migration and differentiation were investigated before and after an intracranial injection of apotransferrin at 3 days of age (P3). The proliferating cell population was evaluated at P3. Iron-deficient (ID) animals showed an increase in the oligodendrocyte precursors cell (OPC) population in comparison with controls. The overall pattern of migration of cells labeled with BrdU was investigated at P11. Iron deficiency increased the amount of BrdU(+) cells in the corpus callosum (CC) and decreased OLGc maturation and myelin formation. Changes in nerve conduction were analyzed by measuring visual evoked potentials. Latency and amplitude were significantly disturbed in ID rats compared with controls. Both parameters were substantially normalized when animals were treated with a single intracranial injection of 350 ng apotransferrin (aTf). The current results give support to the idea that iron deficiency increases the number of proliferating and undifferentiated cells in the CC compared with the control. Treatment with aTf almost completely reverted the effects of iron deficiency, both changing the migration pattern and increasing the number of mature cells in the CC and myelin formation.

Partial inhibition of proteasome activity enhances remyelination after cuprizone-induced demyelination.

We have previously demonstrated that addition of low concentrations of lactacystin (a specific inhibitor of the proteasome) to oligodendroglial cell cultures containing a high percentage of precursor cells induces their exit from the cell cycle and their differentiation. On the other hand, we have recently shown that the mechanism of cuprizone toxicity on oligodendroglial cells involves the recruitment of microglia and their secretion of pro-inflammatory cytokines and in the increased production of oxidant species, which results in a decrease in the activities of the mitochondrial respiratory chain. In the present paper we investigated the effect of a decrease in proteasome activity induced by the injection of lactacystin in the corpus callosum in the remyelination process that normally occurs after cuprizone-induced demyelination. This treatment markedly improves the remyelination process that normally occurs in cuprizone-induced demyelination. It also attenuates the activation of NFkappaB and the recruitment of microglia and astrocytes, thus helping in the recovery of the mitochondrial respiratory chain activities that are affected by cuprizone treatment.

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 p21(Cip1), p27(Kip1) 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 p27(Kip1) and Sp1.

Fyn kinase is involved in oligodendroglial cell differentiation induced by apotransferrin.

Mechanisms that regulate oligodendroglial cell (OLGc) differentiation are the focus of intensive research in the field of cellular and molecular neurobiology. We have previously shown that the addition of apotransferrin (aTf) to primary OLGc cultures accelerates their differentiation and induces an increase in the expression of different components of the myelin cytoskeleton (CSK) such as actin, tubulin, and some of the microtubule-associated proteins, particularly the stable tubulin only peptide (STOP). Fyn protein-tyrosine kinase (Fyn kinase), a member of the Src family, participates in signalling pathways that regulate OLGs/myelin cytoskeletal reorganization. It is essential for myelin development in the central nervous system (CNS), and its absence results in hypomyelination. In the present study, we used both primary cell and N19 cell line cultures to investigate further the mechanisms of action involved in the accelerated differentiation of OLGcs induced by aTf. In particular, we were interested in studying the participation of Fyn kinase in the different pathways involved in the reorganization of the OLGc/myelin cytoskeleton. In agreement with results already published, we found that in OLGcs, Fyn kinase is associated with Tau and tubulin. Using a dominant-negative of Tau in which the Fyn-Tau-microtubules (MTs) interaction is blocked, we found that aTf was unable to induce OLGc morphological differentiation. It was also observed that aTf decreases the activated RhoA content in coincidence with a redistribution of actin immunoreactivity. These results give support to our hypothesis that Fyn kinase plays a key role in the differentiation process of OLGcs promoted by aTf.

Thyroid hormones promote differentiation of oligodendrocyte progenitor cells and improve remyelination after cuprizone-induced demyelination.

In the present work we analyzed the capacity of thyroid hormones (THs) to improve remyelination using a rat model of cuprizone-induced demyelination previously described in our laboratories. Twenty one days old Wistar rats were fed a diet containing 0.6% cuprizone for two weeks to induce demyelination. After cuprizone withdrawal, rats were injected with triiodothyronine (T3). Histological studies carried out in these animals revealed that remyelination in the corpus callosum (CC) of T3-treated rats improved markedly when compared to saline treated animals. The cellular events occurring in the CC and in the subventricular zone (SVZ) during the first week of remyelination were analyzed using specific oligodendroglial cell (OLGc) markers. In the CC of saline treated demyelinated animals, mature OLGcs decreased and oligodendroglial precursor cells (OPCs) increased after one week of spontaneous remyelination. Furthermore, the SVZ of these animals showed an increase in early progenitor cell numbers, dispersion of OPCs and inhibition of Olig and Shh expression compared to non-demyelinated animals. The changes triggered by demyelination were reverted after T3 administration, suggesting that THs could be regulating the emergence of remyelinating oligodendrocytes from the pool of proliferating cells residing in the SVZ. Our results also suggest that THs receptor beta mediates T3 effects on remyelination. These results support a potential role for THs in the remyelination process that could be used to develop new therapeutic approaches for demyelinating diseases.

Effect of transferrin on hypomyelination induced by iron deficiency.

We have used a model of iron deficiency in the rat to analyze the effects of a disruption in iron availability on oligodendroglial cell (OLGc) maturation and myelinogenesis and to explore the possible beneficial influence of an intracranial injection (ICI) of apotransferrin (aTf) at 3 days of age on this process. Studies carried out on postnatal days 17 and 24 showed that iron deficiency produced a decrease in myelin proteins and lipids at 24 days of age. Immunohistochemistry showed that in untreated iron-deficient (ID) rats, the immunoreactivity of anti-adenomatous polyposis coli (APC) and anti-MBP antibodies decreased markedly with reference to normal controls, whereas in ID rats treated with an ICI of aTf, the immunoreactivity of these markers increased. A similar situation occurred with the immunoreactivity of H-ferritin. In primary OLGc cultures from ID rats, there was a high number of cells positive to the antibody against the polysialylated form of the cell surface glycoprotein NCAM (PSA-NCAM) compared with in OLGc cultures prepared from normal controls or from ID animals treated with aTf. The number of MBP+ cells in cultures from ID rats increased after treatment with aTf. The presence of lipid rafts evaluated with a specific anti-protein prion cellular (PrPc) antibody showed a smaller number of PrPc-positive structures in ID rat cultures. Treatment of the ID animals with a single ICI of aTf stimulated myelination, producing a significant correction in the different biochemical parameters affected by ID.

The mRNA of transferrin is expressed in Schwann cells during their maturation and after nerve injury.

Transferrin, the iron carrier protein, has been shown to be involved in oligodendroglial cell differentiation in the central nervous system but little is known about its role in the peripheral nervous system. In the present work, we have studied the presence of transferrin and of its mRNA in rat sciatic nerves and in Schwann cells isolated at embryonic and adult ages as well as during the regeneration process that follows nerve crush. We have also studied the correlation between the expression of the mRNAs of transferrin and the expression of mature myelin markers in the PNS. We show that transferrin is present in whole sciatic nerves at late stages of embryonic life as well as at postnatal day 4 and in adult rats. We demonstrate for the first time, that in normal conditions, the transferrin mRNA is expressed in Schwann cells isolated from sciatic nerves between embryonic days 14 and 18, being absent at later stages of development and in adult animals. In adult rats, 3 days after sciatic nerve crushing, the mRNA of transferrin is expressed in the injured nerve, but 7 days after injury its expression disappears. Transferrin protein in the sciatic nerve closely follows the expression of its mRNA indicating that under these circumstances, it appears to be locally synthesized. Transferrin in the PNS could have a dual role. During late embryonic ages it could be locally synthesized by differentiating Schwann cells, acting as a pro-differentiating factor. A similar situation would occur during the regeneration that follows Wallerian degeneration. In the adult animals on the other hand, Schwann cells could pick up transferrin from the circulation or/and from the axons, sub serving possible trophic actions closely related to myelin maintenance.

The neurotoxic effect of cuprizone on oligodendrocytes depends on the presence of pro-inflammatory cytokines secreted by microglia.

In order to further characterize the still unknown mechanism of cuprizone-induced demyelination, we investigated its effect on rat primary oligodendroglial cell cultures. Cell viability was not significantly affected by this treatment. However, when concentrations of IFNgamma and/or TNFalpha having no deleterious effects per se on cell viability were added together with cuprizone, cell viability decreased significantly. In mitochondria isolated from cuprizone-treated glial cells, we observed a marked decrease in the activities of the various complexes of the respiratory chain, indicating a disruption of mitochondrial function. An enhancement in oxidant production was also observed in cuprizone and/or TNFalpha-treated oligodendroglial cells. In in vivo experiments, inhibition of microglial activation with minocycline prevented cuprizone-induced demyelination. Based on the above-mentioned results we suggest that these microglial cells appear to have a very active role in cuprizone-induced oligodendroglial cell death and demyelination, through the production and secretion of pro-inflammatory cytokines.

Presence of alpha-globin mRNA and migration of bone marrow cells after sciatic nerve injury suggests their participation in the degeneration/regeneration process.

We have previously reported that in the distal stump of ligated sciatic nerves, there is a change in the distribution of myelin basic protein (MBP) and P0 protein immunoreactivities. These results agreed with the studies of myelin isolated from the distal stump of animals submitted to ligation of the sciatic nerve, showing a gradual increase in a 14 kDa band with an electrophoretic mobility similar to that of an MBP isoform, among other changes. This band, which was resolved into two bands of 14 and 15 kDa using a 16% gel, was found to contain a mixture of MBP fragments and peptides with great homology with alpha- and beta-globins. In agreement with these results, we have demonstrated that the mRNA of alpha-globin is present in the proximal and distal stumps of the ligated nerve. It is also detected at very low levels in Schwann cells isolated from normal nerves. These results could be due to the presence of alpha- and/or beta-globin arising from immature cells of the erythroid series. Also, they could be present in macrophages, which spontaneously migrate to the injured nerve to promote the degradation of myelin proteins. Cells isolated from normal adult rat bone marrow which were injected intraortically were found to migrate to the injured area. These cells could contribute to the remyelination of the damaged area participating in the removal of myelin debris, through their transdifferentiation into Schwann cells or through their fusion with preexisting Schwann cells in the distal stump of the injured sciatic nerve.

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.