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 cells (OPCs) 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 knockdown 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 OPCs maturation as well as the role played by these Ca++ channels during the early phases of myelination.

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We have found a significant upregulation of L-type voltage-operated Ca++ channels (VOCCs) in reactive astrocytes. To test if VOCCs are centrally involved in triggering astrocyte reactivity, we used in vitro models of astrocyte activation in combination with pharmacological inhibitors, siRNAs and the Cre/lox system to reduce the activity of L-type VOCCs in primary cortical astrocytes. The endotoxin lipopolysaccharide (LPS) as well as high extracellular K+, glutamate and ATP promote astrogliosis in vitro. L-type VOCC inhibitors drastically reduce the number of reactive cells, astrocyte hypertrophy and cell proliferation after these treatments. Astrocytes transfected with siRNAs for the Cav1.2 subunit that conducts L-type Ca++ currents as well as Cav1.2 knockout astrocytes showed reduce Ca++ influx by ~80% after plasma membrane depolarization. Importantly, Cav1.2 knock-down/out prevents astrocyte activation and proliferation induced by LPS. Similar results were found using the scratch wound assay. After injuring the astrocyte monolayer, cells extend processes toward the cell-free scratch region and subsequently migrate and populate the scratch. We found a significant increase in the activity of L-type VOCCs in reactive astrocytes located in the growing line in comparison to quiescent astrocytes situated away from the scratch. Moreover, the migration of astrocytes from the scratching line as well as the number of proliferating astrocytes was reduced in Cav1.2 knock-down/out cultures. In summary, our results suggest that Cav1.2 L-type VOCCs play a fundamental role in the induction and/or proliferation of reactive astrocytes, and indicate that the inhibition of these Ca++ channels may be an effective way to prevent astrocyte activation.

The divalent metal transporter 1 (DMT1) is a multimetal transporter with a primary role in iron transport. Although DMT1 has been described previously in the CNS, nothing was known about the role of this metal transporter in oligodendrocyte maturation and myelination. To determine whether DMT1 is required for oligodendrocyte progenitor cell (OPC) maturation, we used siRNAs and the system to knock down/knock out DMT1 expression as well as Blocking DMT1 synthesis in primary cultures of OPCs reduced oligodendrocyte iron uptake and significantly delayed OPC development., a significant hypomyelination was found in DMT1 conditional knock-out mice in which DMT1 was postnatally deleted in NG2- or Sox10-positive OPCs. The brain of DMT1 knock-out animals presented a decrease in the expression levels of myelin proteins and a substantial reduction in the percentage of myelinated axons. This reduced postnatal myelination was accompanied by a decrease in the number of myelinating oligodendrocytes and a rise in proliferating OPCs. Furthermore, using the cuprizone model of demyelination, we established that DMT1 deletion in NG2-positive OPCs lead to less efficient remyelination of the adult brain. These results indicate that DMT1 is vital for OPC maturation and for the normal myelination of the mouse brain. To determine whether divalent metal transporter 1 (DMT1), a multimetal transporter with a primary role in iron transport, is essential for oligodendrocyte development, we created two conditional knock-out mice in which DMT1 was postnatally deleted in NG2- or Sox10-positive oligodendrocyte progenitor cells (OPCs). We have established that DMT1 is necessary for normal OPC maturation and is required for an efficient remyelination of the adult brain. Since iron accumulation by OPCs is indispensable for myelination, understanding the iron incorporation mechanism as well as the molecules involved is critical to design new therapeutic approaches to intervene in diseases in which the myelin sheath is damaged or lost.