Unconjugated Bilirubin Restricts Oligodendrocyte Differentiation and Axonal Myelination

High levels of serum unconjugated bilirubin (UCB) in newborns are associated with axonal damage and glial reactivity that may contribute to subsequent neurologic injury and encephalopathy (kernicterus). Impairments in myelination and white matter damage were observed at autopsy in kernicteric infants. We have recently reported that UCB reduces oligodendrocyte progenitor cell (OPC) survival in a pure OPC in vitro proliferative culture. Here, we hypothesized that neonatal hyperbilirubinemia may also impair oligodendrocyte (OL) maturation and myelination. We used an experimental model of hyperbilirubinemia that has been shown to mimic the pathophysiological conditions leading to brain dysfunction by unbound (free) UCB. Using primary cultures of OL, we demonstrated that UCB delays cell differentiation by increasing the OPC number and reducing the number of mature OL. This finding was combined with a downregulation of Olig1 mRNA levels and upregulation of Olig2 mRNA levels. Addition of UCB, prior to or during differentiation, impaired OL morphological maturation, extension of processes and cell diameter. Both conditions reduced active guanosine triphosphate (GTP)-bound Rac1 fraction. In myelinating co-cultures of dorsal root ganglia neurons and OL, UCB treatment prior to the onset of myelination decreased oligodendroglial differentiation and the number of myelinating OL, also observed when UCB was added after the onset of myelination. In both circumstances, UCB decreased the number of myelin internodes per OL, as well as the myelin internode length. Our studies demonstrate that increased concentrations of UCB compromise myelinogenesis, thereby elucidating a potential deleterious consequence of elevated UCB.     

Neuregulin and BDNF Induce a Switch to NMDA Receptor-Dependent Myelination by Oligodendrocytes

Myelination is essential for rapid impulse conduction in the CNS, but what determines whether an individual axon becomes myelinated remains unknown. Here we show, using a myelinating coculture system, that there are two distinct modes of myelination, one that is independent of neuronal activity and glutamate release and another that depends on neuronal action potentials releasing glutamate to activate NMDA receptors on oligodendrocyte lineage cells. Neuregulin switches oligodendrocytes from the activity-independent to the activity-dependent mode of myelination by increasing NMDA receptor currents in oligodendrocyte lineage cells 6-fold. With neuregulin present myelination is accelerated and increased, and NMDA receptor block reduces myelination to far below its level without neuregulin. Thus, a neuregulin-controlled switch enhances the myelination of active axons. In vivo, we demonstrate that remyelination after white matter damage is NMDA receptor-dependent. These data resolve controversies over the signalling regulating myelination and suggest novel roles for neuregulin in schizophrenia and in remyelination after white matter damage.     

Myelination of Neuronal Cell Bodies when Myelin Supply Exceeds Axonal Demand

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The UDP-Galactose:Ceramide Galactosyltransferase: Expression Pattern in Oligodendrocytes and Schwann Cells During Myelination and Substrate Preference for Hydroxyceramide

Abstract: Galactosylceramide ("galactocerebroside"; GalC) is a major glycolipid in the myelin sheath of the CNS and the PNS. The enzyme UDP-galactose:ceramide galactosyltransferase (CGalT) catalyzes the final step of the synthesis of GalC: the transfer of galactose to ceramide. By a differential screening approach, we have isolated a cDNA, the sequence of which is identical to the recently isolated cDNA clones for CGalT. By northern analysis and in situ hybridization we demonstrated that CGalT mRNA is expressed at birth in oligodendrocytes and Schwann cells, an expression pattern corresponding to the onset of myelination. In addition to the high expression levels of CGalT in oligodendrocytes and Schwann cells, in situ hybridization also showed expression in subtypes of neurons in spinal cord, cerebellum, and brainstem in the adult CNS, but at a much lower level than in oligodendrocytes. Expression of CGalT in COS cells demonstrated that CGalT has a preference for hydroxyceramide as a substrate. CGalT-expressing COS cells synthesize and transport GalC to their cell surface as shown by immunofluorescence and by lipid analysis of living cells. Our results suggested that the CGalT specifically uses hydroxyceramide for the synthesis of GalC and that separate (co)enzymes are not needed.     

Evidence for Cooperative Selection of Axons for Myelination by Adjacent Oligodendrocytes in the Optic Nerve

The cellular mechanisms that regulate the topographic arrangement of myelin internodes along axons remain largely uncharacterized. Recent clonal analysis of oligodendrocyte morphologies in the mouse optic nerve revealed that adjacent oligodendrocytes frequently formed adjacent internodes on one or more axons in common, whereas oligodendrocytes in the optic nerve were never observed to myelinate the same axon more than once. By modelling the process of axonal selection at the single cell level, we demonstrate that internode length and primary process length constrain the capacity of oligodendrocytes to myelinate the same axon more than once. On the other hand, probabilistic analysis reveals that the observed juxtaposition of myelin internodes among common sets of axons by adjacent oligodendrocytes is highly unlikely to occur by chance. Our analysis may reveal a hitherto unknown level of communication between adjacent oligodendrocytes in the selection of axons for myelination. Together, our analyses provide novel insights into the mechanisms that define the spatial organization of myelin internodes within white matter at the single cell level.     

Oligodendrocytes in the Mouse Corpus Callosum Maintain Axonal Function by Delivery of Glucose

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Clemastine Enhances Myelination,Delays Axonal Loss and Promotes Functional Recovery in Spinal Cord Injury

Recent evidence has shown that demyelination occurs along with axonal degeneration in spinal cord injury (SCI) during the secondary injury phase. Oligodendrocyte precursor cells (OPC) are present in the lesions but fail to differentiate into mature oligodendrocytes and form new myelin. Given the limited recovery of neuronal functions after SCI in adults without effective treatment available so far, it remains unknown whether enhancing OPC differentiation and myelination could benefit the recovery of SCI. To show the significance of myelin regeneration after SCI, the injury was treated with clemastine in the rat model. Clemastine is an FDA-approved drug that is potent in promoting oligodendrocyte differentiation and myelination in vivo, for four weeks following SCI. Motor function was assessed using sloping boards and grid walking tests and scored according to the Basso, Beattie, and Bresnahan protocol. The myelin integrity and protein expression were evaluated using transmission electron microscopy and immunofluorescence, respectively. The results indicated that clemastine treatment preserves myelin integrity, decreases loss of axons and improves functional recovery in the rat SCI model. The presented data suggest that myelination-enhancing strategies may serve as a potential therapeutic approach for the functional recovery in SCI.

     

Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination

Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti-large basic protein serum was shown by the immunoblot technique to cross-react with all five forms of basic protein present in the myelin of 8-day-old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.     

HERC1 Ubiquitin Ligase Is Required for Normal Axonal Myelination in the Peripheral Nervous System

A missense mutation in HERC1 provokes loss of cerebellar Purkinje cells, tremor, and unstable gait in tambaleante (tbl) mice. Recently, we have shown that before cerebellar degeneration takes place, the tbl mouse suffers from a reduction in the number of vesicles available for release at the neuromuscular junction (NMJ). The aim of the present work was to study to which extent the alteration in HERC1 may affect other cells in the nervous system and how this may influence the motor dysfunction observed in these mice. The functional analysis showed a consistent delay in the propagation of the action potential in mutant mice in comparison with control littermates. Morphological analyses of glial cells in motor axons revealed signs of compact myelin damage as tomacula and local hypermyelination foci. Moreover, we observed an alteration in non-myelinated terminal Schwann cells at the level of the NMJ. Additionally, we found a significant increment of phosphorylated Akt-2 in the sciatic nerve. Based on these findings, we propose a molecular model that could explain how mutated HERC1 in tbl mice affects the myelination process in the peripheral nervous system. Finally, since the myelin abnormalities found in tbl mice are histological hallmarks of neuropathic periphery diseases, tbl mutant mice could be considered as a new mouse model for this type of diseases.     

Myelination: some receptors required.

Feltri et al. (2001)(this issue) succeed in disrupting beta 1 integrin specifically in Schwann cells, and in so doing, demonstrate that it is required for normal myelination. Their results reveal that signaling by an extracellular matrix receptor plays a key role in the differentiation of myelinating Schwann cells.     

Glutamate Transporters: Expression and Function in Oligodendrocytes

Glutamate, the main excitatory neurotransmitter of the vertebrate central nervous system (CNS), is well known as a regulator of neuronal plasticity and neurodevelopment. Such glutamate function is thought to be mediated primarily by signaling through glutamate receptors. Thus, it requires a tight regulation of extracellular glutamate levels and a fine-tuned homeostasis that, when dysregulated, has been associated with a wide range of central pathologies including neuropsychiatric, neurodevelopmental, and neurodegenerative disorders. In the mammalian CNS, extracellular glutamate levels are controlled by a family of sodium-dependent glutamate transporters belonging to the solute carrier family 1 (SLC1) that are also referred to as excitatory amino acid transporters (EAATs). The presumed main function of EAATs has been best described in the context of synaptic transmission where EAATs expressed by astrocytes and neurons effectively regulate extracellular glutamate levels so that synapses can function independently. There is, however, increasing evidence that EAATs are expressed by cells other than astrocytes and neurons, and that they exhibit functions beyond glutamate clearance. In this review, we will focus on the expression and functions of EAATs in the myelinating cells of the CNS, oligodendrocytes. More specifically, we will discuss potential roles of oligodendrocyte-expressed EAATs in contributing to extracellular glutamate homeostasis, and in regulating oligodendrocyte maturation and CNS myelination by exerting signaling functions that have traditionally been associated with glutamate receptors. In addition, we will provide some examples for how dysregulation of oligodendrocyte-expressed EAATs may be involved in the pathophysiology of neurologic diseases.

     

Myelination: all about Rac 'n' roll

During the development of the peripheral nervous system, Schwann cells select individual axons from a nerve bundle and establish a one-to-one relationship through a process termed "radial sorting". Recent findings identify the Rho family GTPase Rac1 as the downstream effector molecule responsible for process extension and lamellipodia formation in Schwann cells, allowing for proper radial sorting and myelination. These findings begin to shed light on our understanding of the distinct and yet essential molecular mechanisms involved in developmental processes preceding myelination.     

Poster Sessions BP02: Oligodendrocytes and Schwann Cells

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro , the NF1-derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1-derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1-derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1-derived Schwann cells have increased RAS activity and a three-fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1-derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP-related domain of neurofibromin into the NF1-derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1-derived Schwann cells the Schwann cells expressing the GAP-related domain expressed about one-half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1-drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.     

Oligodendrocytes in neurodegenerative diseases

Oligodendrocyte is a highly specialized glial cell type in the vertebrate central nervous system, which guarantees the long-distance transmission of action potential by producing myelin sheath wrapping adjacent axons. Disrupted myelin and oligodendrocytes are hallmarks of some devastating neurological diseases, such as multiple sclerosis, although their contribution to neurodegeneration in a given disease is still controversial. However, accumulating evidence from clinical studies and genetic animal models implicates oligodendrocyte dysfunction as one of major events in the processes of initiation and progression of neurodegeneration. In this article, we will review recent progress in understanding non-traditional function of oligodendrocytes in neuronal support and protection independent of myelin sheath and its possible contribution to neurodegeneration. Oligodendrocytes play a pivotal role in neurodegenerative diseases among which special emphasis is given to multiple system atrophy and Alzheimer’s disease in this review.     

Lysophosphatidic Acid can Support the Formation of Membranous Structures and an Increase in MBP mRNA Levels in Differentiating Oligodendrocytes

During development, differentiating oligodendrocytes progress in distinct maturation steps from premyelinating to myelinating cells. Such maturing oligodendrocytes express both the receptors mediating signaling via extracellular lysophosphatidic acid (LPA) and the major enzyme generating extracellular LPA, namely phosphodiesterase-Iα/autotaxin (PD-Iα/ATX). However, the biological role of extracellular LPA during the maturation of differentiating oligodendrocytes is currently unclear. Here, we demonstrate that application of exogenous LPA induced an increase in the area occupied by the oligodendrocytes’ process network, but only when PD-Iα/ATX expression was down-regulated. This increase in network area was caused primarily by the formation of membranous structures. In addition, LPA increased the number of cells positive for myelin basic protein (MBP). This effect was associated by an increase in the mRNA levels coding for MBP but not myelin oligodendrocyte glycoprotein (MOG). Taken together, these data suggest that LPA may play a crucial role in regulating the later stages of oligodendrocyte maturation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Special issue article in honor of Dr. George DeVries. Luciana Nogaroli and Larra M. Yuelling contributed equally to this work.