Ferritin loss in astrocytes reduces spinal cord oxidative stress and demyelination in the experimental autoimmune encephalomyelitis (EAE) model.

Demyelinating diseases such as multiple sclerosis (MS) cause myelin degradation and oligodendrocyte death, resulting in the release of toxic iron and iron-induced oxidative stress. Astrocytes have a large capacity for iron transport and storage, however the role of astrocytic iron homeostasis in demyelinating disorders is not completely understood. Here we investigate whether astrocytic iron metabolism modulates neuroinflammation, oligodendrocyte survival, and oxidative stress following demyelination. To this aim, we conditionally knock out ferritin in astrocytes and induce experimental autoimmune encephalomyelitis (EAE), an autoimmune-mediated model of demyelination. Ferritin ablation in astrocytes reduced the severity of disease in both the acute and chronic phases. The day of onset, peak disease severity, and cumulative clinical score were all significantly reduced in ferritin KO animals. This corresponded to better performance on the rotarod and increased mobility in ferritin KO mice. Furthermore, the spinal cord of ferritin KO mice display decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes. Correspondingly, the size of demyelinated lesions, iron accumulation, and oxidative stress were attenuated in the CNS of ferritin KO subjects, particularly in white matter regions of the spinal cord. Thus, deleting ferritin in astrocytes reduced neuroinflammation, oxidative stress, and myelin deterioration in EAE animals. Collectively, these findings suggest that iron storage in astrocytes is a potential therapeutic target to lessen CNS inflammation and myelin loss in autoimmune demyelinating diseases.

Deletion of voltage-gated calcium channels in astrocytes decreases neuroinflammation and demyelination in a murine model of multiple sclerosis.

The experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis was used in combination with a Cav1.2 conditional knock-out mouse (Cav1.2KO) to study the role of astrocytic voltage-gated Ca++ channels in autoimmune CNS inflammation and demyelination. Cav1.2 channels were specifically ablated in Glast-1-positive astrocytes by means of the Cre-lox system before EAE induction. After immunization, motor activity was assessed daily, and a clinical score was given based on the severity of EAE symptoms. Cav1.2 deletion in astrocytes significantly reduced the severity of the disease. While no changes were found in the day of onset and peak disease severity, EAE mean clinical score was lower in Cav1.2KO animals during the chronic phase of the disease. This corresponded to better performance on the rotarod and increased motor activity in Cav1.2KO mice. Furthermore, decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes were found in the lumbar section of the spinal cord of Cav1.2KO mice 40 days after immunization. The degree of myelin protein loss and size of demyelinated lesions were also attenuated in Cav1.2KO spinal cords. Similar results were found in EAE animals treated with nimodipine, a Cav1.2 Ca++ channel inhibitor with high affinity to the CNS. Mice injected with nimodipine during the acute and chronic phases of the disease exhibited lower numbers of reactive astrocytes, activated microglial, and infiltrating immune cells, as well as fewer demyelinated lesions in the spinal cord. These changes were correlated with improved clinical scores and motor performance. In summary, these data suggest that antagonizing Cav1.2 channels in astrocytes during EAE alleviates neuroinflammation and protects the spinal cord from autoimmune demyelination.

The expression of ceruloplasmin in astrocytes is essential for postnatal myelination and myelin maintenance in the adult brain.

Ceruloplasmin (Cp) is a ferroxidase enzyme that is essential for cell iron efflux. The absence of this protein in humans and rodents produces progressive neurodegeneration with brain iron accumulation. Astrocytes express high levels of Cp and iron efflux from these cells has been shown to be central for oligodendrocyte maturation and myelination. To explore the role of astrocytic Cp in brain development and aging we generated a specific conditional KO mouse for Cp in astrocytes (Cp cKO). Deletion of Cp in astrocytes during the first postnatal week induced hypomyelination and a significant delay in oligodendrocyte maturation. This abnormal myelin synthesis was exacerbated throughout the first two postnatal months and accompanied by a reduction in oligodendrocyte iron content, as well as an increase in brain oxidative stress. In contrast to young animals, deletion of astrocytic Cp at 8 months of age engendered iron accumulation in several brain areas and neurodegeneration in cortical regions. Aged Cp cKO mice also showed myelin loss and oxidative stress in oligodendrocytes and neurons, and at 18 months of age, developed abnormal behavioral profiles, including deficits in locomotion and short-term memory. In summary, our results demonstrate that iron efflux-mediated by astrocytic Cp-is essential for both early oligodendrocyte maturation and myelin integrity in the mature brain. Additionally, our data suggest that astrocytic Cp activity is central to prevent iron accumulation and iron-induced oxidative stress in the aging CNS.

Ceruloplasmin deletion in myelinating glial cells induces myelin disruption and oxidative stress in the central and peripheral nervous systems.

Ceruloplasmin (Cp) is a ferroxidase enzyme that is essential for cell iron efflux and has been postulated to have a neuroprotective role. During the myelination process, oligodendrocytes (OLs) and Schwann cells (SCs) express high levels of Cp, but the role of this enzyme in glial cell development and function is completely unknown. To define the function of Cp in the myelination of the central and peripheral nervous systems, we have conditionally knocked-out Cp specifically in OLs and SCs during early postnatal development as well as in aged mice. Cp ablation in early OLs (postnatal day 2, P2) significantly affects the differentiation of these cells and the synthesis of myelin through the first four postnatal weeks. The total number of mature myelinating OLs was reduced, and the density of apoptotic OLs was increased. These changes were accompanied with reductions in the percentage of myelinated axons and increases in the g-ratio of myelinated fibers. Cp ablation in young myelinating OLs (P30 or P60) did not affect myelin synthesis and/or OL numbers, however, Cp loss in aged OLs (8 months) induced cell iron overload, apoptotic cell death, brain oxidative stress, neurodegeneration and myelin disruption. Furthermore, Cp deletion in SCs affected postnatal SC development and myelination and produced motor coordination deficits as well as oxidative stress in young and aged peripheral nerves. Together, our data indicate that Cp ferroxidase activity is essential for OLs and SCs maturation during early postnatal development and iron homeostasis in matured myelinating cells. Additionally, our results suggest that Cp expression in myelinating glial cells is crucial to prevent oxidative stress and neurodegeneration in the central and peripheral nervous systems.

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.

Golli Myelin Basic Proteins Modulate Voltage-Operated Ca(++) Influx and Development in Cortical and Hippocampal Neurons.

The golli proteins, products of the myelin basic protein gene, are widely expressed in oligodendrocyte progenitor cells and neurons during the postnatal development of the brain. While golli appears to be important for oligodendrocyte migration and differentiation, its function in neuronal development is completely unknown. We have found that golli proteins function as new and novel modulators of voltage-operated Ca(++) channels (VOCCs) in neurons. In vitro, golli knock-out (KO) neurons exhibit decreased Ca(++) influx after plasma membrane depolarization and a substantial maturational delay. Increased expression of golli proteins enhances L-type Ca(++) entry and processes outgrowth in cortical neurons, and pharmacological activation of L-type Ca(++) channels stimulates maturation and prevents cell death in golli-KO neurons. In situ, Ca(++) influx mediated by L-type VOCCs was significantly decreased in cortical and hippocampal neurons of the golli-KO brain. These Ca(++) alterations affect cortical and hippocampal development and the proliferation and survival of neural progenitor cells during the postnatal development of the golli-KO brain. The CA1/3 sections and the dentate gyrus of the hippocampus were reduced in the golli-KO mice as well as the density of dendrites in the somatosensory cortex. Furthermore, the golli-KO mice display abnormal behavior including deficits in episodic memory and reduced anxiety. Because of the expression of the golli proteins within neurons in learning and memory centers of the brain, this work has profound implication in neurodegenerative diseases and neurological disorders.

Voltage-gated Ca2+ entry promotes oligodendrocyte progenitor cell maturation and myelination in vitro.

We have previously shown that the expression of voltage-operated Ca(++) channels (VOCCs) is highly regulated in the oligodendroglial lineage and is essential for proper oligodendrocyte progenitor cell (OPC) migration. Here we assessed the role of VOCCs, in particular the L-type, in oligodendrocyte maturation. We used pharmacological treatments to activate or block voltage-gated Ca(++) uptake and siRNAs to specifically knock down the L-type VOCC in primary cultures of mouse OPCs. Activation of VOCCs by plasma membrane depolarization increased OPC morphological differentiation as well as the expression of mature oligodendrocyte markers. On the contrary, inhibition of L-type Ca(++) channels significantly delayed OPC development. OPCs transfected with siRNAs for the Cav1.2 subunit that conducts L-type Ca(++) currents showed reduce Ca(++) influx by ~75% after plasma membrane depolarization, indicating that Cav1.2 is heavily involved in mediating voltage-operated Ca(++) entry in OPCs. Cav1.2 knockdown induced a decrease in the proportion of oligodendrocytes that expressed myelin proteins, and an increase in cells that retained immature oligodendrocyte markers. Moreover, OPC proliferation, but not cell viability, was negatively affected after L-type Ca(++) channel knockdown. Additionally, we have tested the ability of L-type VOCCs to facilitate axon-glial interaction during the first steps of myelin formation using an in vitro co-culture system of OPCs with cortical neurons. Unlike control OPCs, Cav1.2 deficient oligodendrocytes displayed a simple morphology, low levels of myelin proteins expression and appeared to be less capable of establishing contacts with neurites and axons. Together, this set of in vitro experiments characterizes the involvement of L-type VOCCs on OPC maturation as well as the role played by these Ca(++) channels during the early phases of myelination.

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.

Voltage-operated Ca(2+) and Na(+) channels in the oligodendrocyte lineage.

It is becoming increasingly clear that expression of Ca(2+) and Na(+) channels in the OL lineage is highly regulated and may be functionally related to different stages of development and myelination. Characterization of the mechanisms of voltage-dependent Ca(2+) and Na(+) entry are important because changes in intracellular Ca(2+) and Na(+) are central to practically all cellular activities. In nonexcitable cells, voltage-dependent Ca(2+) influx plays a key role in several important processes, including proliferation, apoptosis, and cell migration. It has been demonstrated that Ca(2+) signaling is essential in the development and functioning of OLs. For example, Ca(2+) uptake is required for the initiation of myelination, and perturbation of Ca(2+) homeostasis, e.g., overwhelming influxes of Ca(2+), leads to demyelination. Although OL progenitor cell Na(+) channels are present at a much lower density, their physiological properties appear to be indistinguishable from those recorded in neurons. Interestingly, recent data indicate that, as with neurons, some white matter OPCs possess the ability to generate Na(+)-dependent action potentials. This Mini-Review focuses on the mechanisms of Ca(2+) and Na(+) signaling in cells within the OL lineage mediated by voltage-operated ion channels, with a particular focus on the relevance of these voltage-dependent currents to oligodendroglial development, myelination, and demyelination. Overall, it is clear that cells in the OL lineage exhibit remarkable plasticity with regard to the expression of voltage-gated Ca(2+) and Na(+) channels and that perturbation of Ca(2+) and Na(+) homeostasis likely plays an important role in the pathogenesis underlying demyelinating diseases.

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.

Remyelination after cuprizone-induced demyelination in the rat is stimulated by apotransferrin.

Twenty-one-day-old Wistar rats were fed a diet containing 0.6% cuprizone for 2 weeks. Studies carried out after withdrawal of cuprizone showed histological evidences of marked demyelination in the corpus callosum. Biochemical studies of isolated myelin showed a marked decrease in myelin proteins, phospholipids, and galactocerebrosides as well as a marked decrease in myelin yield. Treatment of these animals with a single intracranial injection of 350 ng of apotransferrin at the time of withdrawal of cuprizone induced a marked increase in myelin deposition resulting in a significantly improved remyelination, evaluated by histological, immunocytochemical, and biochemical parameters, in comparison to what was observed in spontaneous recovery. Immunocytochemical studies of cryotome sections to analyze developmental parameters of the oligodendroglial cell population at the time of termination of cuprizone and at different times thereafter showed that in the untreated animals, there was a marked increase in the number of NG2-BrdU-positive precursor cells together with a marked decrease in MBP expression at the peak of cuprizone-induced demyelination. As expected, the amount of precursor cells decreased markedly during spontaneous remyelination and was accompanied by an increase in MBP reactivity. In the apotransferrin-treated animals, these phenomena occurred much faster, and remyelination was much more efficient than in the untreated controls. The results of this study suggest that apotransferrin is a very active promyelinating agent which could be important for the treatment of certain demyelinating conditions.