Axonal Signals and Oligodendrocyte Differentiation

Axons produce signals that regulate oligodendrocyte proliferation, survival, terminal differentiation, and myelinogenesis. We review here recent in vitro and in vivo experimental approaches that aim to characterize axonal signals to oligodendroglia and to identify molecular mediators that regulate differentiation of oligodendendrocytes. We propose that the promoters of myelin genes, whose activation during terminal differentiation is modulated by axonal signals, can provide a means to identify molecular mediators of axo-oligodendroglial signals.     

Protein Tyrosine Phosphatase Receptor Type Z Negatively Regulates Oligodendrocyte Differentiation and Myelination

Background

Fyn tyrosine kinase-mediated down-regulation of Rho activity through activation of p190RhoGAP is crucial for oligodendrocyte differentiation and myelination. Therefore, the loss of function of its counterpart protein tyrosine phosphatase (PTP) may enhance myelination during development and remyelination in demyelinating diseases. To test this hypothesis, we investigated whether Ptprz, a receptor-like PTP (RPTP) expressed abuntantly in oligodendrocyte lineage cells, is involved in this process, because we recently revealed that p190RhoGAP is a physiological substrate for Ptprz.

Methodology/Principal Findings

We found an early onset of the expression of myelin basic protein (MBP), a major protein of the myelin sheath, and early initiation of myelination in vivo during development of the Ptprz-deficient mouse, as compared with the wild-type. In addition, oligodendrocytes appeared earlier in primary cultures from Ptprz-deficient mice than wild-type mice. Furthermore, adult Ptprz-deficient mice were less susceptible to experimental autoimmune encephalomyelitis (EAE) induced by active immunization with myelin/oligodendrocyte glycoprotein (MOG) peptide than were wild-type mice. After EAE was induced, the tyrosine phosphorylation of p190RhoGAP increased significantly, and the EAE-induced loss of MBP was markedly suppressed in the white matter of the spinal cord in Ptprz-deficient mice. Here, the number of T-cells and macrophages/microglia infiltrating into the spinal cord did not differ between the two genotypes after MOG immunization. All these findings strongly support the validity of our hypothesis.

Conclusions/Significance

Ptprz plays a negative role in oligodendrocyte differentiation in early central nervous system (CNS) development and remyelination in demyelinating CNS diseases, through the dephosphorylation of substrates such as p190RhoGAP.     

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dst^dt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dst^dt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dst^dt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dst^dt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dst^dt-27J optic nerves was normal, as was myelin basic protein expression in both cerebral cortex and spinal cord. Together these data suggest that, unlike Schwann cells, oligodendrocytes do not have an intrinsic requirement for neuronal dystonin for differentiation and myelination.     

Oligodendrocytes: Myelination and Axonal Support

Myelinated nerve fibers have evolved to enable fast and efficient transduction of electrical signals in the nervous system. To act as an electric insulator, the myelin sheath is formed as a multilamellar membrane structure by the spiral wrapping and subsequent compaction of the oligodendroglial plasma membrane around central nervous system (CNS) axons. Current evidence indicates that the myelin sheath is more than an inert insulating membrane structure. Oligodendrocytes are metabolically active and functionally connected to the subjacent axon via cytoplasmic-rich myelinic channels for movement of macromolecules to and from the internodal periaxonal space under the myelin sheath. This review summarizes our current understanding of how myelin is generated and also the role of oligodendrocytes in supporting the long-term integrity of myelinated axons.When Virchow analyzed the fine structure of the brain in the 1850s, he recognized that there were more cells within the “Nervenkitt” than astrocytes, but because of the imperfect staining methods, they remained obscure and were only named the “third element” (reviewed in Somjen 1988; Rosenbluth 1999). It was decades later that Pío del Río-Hortega (1921) applied a staining method involving silver carbonate, thereby shedding new light on the rest of the interstitial cells. These cells were found to contain numerous short processes and were named oligodendroglia and microglia. Defining features of oligodendrocytes were their small cell bodies filled with nuclei containing large amounts of chromatin, and their cellular extensions that lacked fibers but were filled with cytoplasmic granules (Fig. 1). When the tissue was optimally preserved, the silver impregnation uncovered a tremendous complexity of extensions (Fig. 1). del Río-Hortega was able to distinguish four types of oligodendrocytes (Fig. 1): Type I cells generate many different myelin segments on small diameter axons in diverse orientations; type II cells are similar to type I in size and number, but myelin segments run in parallel to each other; type III oligodendrocytes ensheath fewer axons of larger diameter; and type IV oligodendrocytes have a cell body closely apposed to a single very large axon similar to Schwann cells. From the staining, it became clear that some of the processes run in parallel to axons and appeared to cover the axons with “myelin,” a term that was introduced by Virchow already in 1858, long before its origin and function were known.Open in a separate windowFigure 1.Morphology of oligodendroglia in the cerebellum of a cat. (Top right) Cellular processes and branches follow the orientation of the nerve fibers and form complex wraps as shown in del Río-Hortega (1921). (Top left) White matter of a newborn human brain: A, Elongated interfascicular cells filled with spherical granules of variable size; B and C, round granular cells; D, astrocytes with long processes; and E, nucleus of a microglia as shown in del Río-Hortega (1921). (Bottom left) The four types of oligodendrocytes recognized by del Río-Hortega. (Bottom right) Oligodendrocytes expressing proteolipid protein (PLP)–enhanced green fluorescent protein (EGFP) in transgenic mice. Because of its bulky EGFP tag, most of it is found within the cytoplasmic-rich spaces or myelin, including the myelinic channels.Penfield (1924) reinforced the view that the formation of myelin is likely the main role of oligodendrocytes. However, myelination was not considered their only function. Within the gray matter, a fraction of oligodendrocytes were termed “perineuronal” satellite cells, which did not form myelin, but were in close contact with the cell body of neurons, suggesting an interdependent relation. Some oligodendroglia were found in close association with small vessels and were, therefore, subclassified as “perivascular satellites.”The major findings that supported the role of oligodendrocytes in myelin generation were their high number in white matter tracts, their appearance in development at the time of myelination, and their position close to the myelin sheaths. Curiously, the large number of granules in the cytosol of oligodendrocytes, reminiscent of cells of secretory glands, was also taken as evidence for a function in myelin formation. At that time, myelin was not considered to be an extension of the oligodendroglial plasma membrane, however, but rather a fatty axonal substance secreted into the extracellular space. The importance of the “axon cylinder” for saltatory impulse propagation was well recognized, but it was the seminal work of Betty Ben Geren (1954), using electron microscopy (EM) in the chick peripheral nervous system, which showed that myelin is not axon derived, but rather a continuous membranous extension of Schwann cells. A principally similar relationship, with spiral wrapping of the glial plasma membrane around the axon, was confirmed later for oligodendrocytes in the central nervous system (CNS) (for an excellent early review, see Bunge 1968; Hildebrand et al. 1993). Together, these studies have turned oligodendrocytes and myelin from a putty-amorphous “Nervenkitt” into a fascinating study object of cell biology.     

Induction of Oligodendrocyte Differentiation and In Vitro Myelination by Inhibition of Rho-Associated Kinase

In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies.     

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.     

Unconjugated Bilirubin Exposure Impairs Hippocampal Long-Term Synaptic Plasticity

Background

Jaundice is one of the most common problems encountered in newborn infants, due to immaturity of hepatic conjugation and transport processes for bilirubin. Although the majority of neonatal jaundice is benign, some neonates with severe hyperbilirubinemia develop bilirubin encephalopathy or kernicterus. Accumulation of unconjugated bilirubin (UCB) in selected brain regions may result in temporary or permanent impairments of auditory, motor, or cognitive function; however, the molecular mechanisms by which UCB elicits such neurotoxicity are still poorly understood. The present study is undertaken to investigate whether prolonged exposure of rat organotypic hippocampal slice cultures to UCB alters the induction of long-term synaptic plasticity.

Methodology/Principal Findings

Using electrophysiological recording techniques, we find that exposure of hippocampal slice cultures to clinically relevant concentrations of UCB for 24 or 48 h results in an impairment of CA1 long-term potentiation (LTP) and long-term depression (LTD) induction in a time- and concentration-dependent manner. Hippocampal slice cultures stimulated with UCB show no changes in the secretion profiles of the pro-inflammatory cytokines, interleukin-1β and tumor necrosis factor-α, or the propidium ioide uptake. UCB treatment produced a significant decrease in the levels of NR1, NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptors through a calpain-mediated proteolytic cleavage mechanism. Pretreatment of the hippocampal slice cultures with NMDA receptor antagonist or calpain inhibitors effectively prevented the UCB-induced impairment of LTP and LTD.

Conclusion/Significance

Our results indicate that the proteolytic cleavage of NMDA receptor subunits by calpain may play a critical role in mediating the UCB-induced impairment of long-term synaptic plasticity in the hippocampus. These observations provide new insights into the molecular mechanisms underlying UCB-induced impairment of hippocampal synaptic plasticity which, in turn, might provide opportunities for the development of novel therapeutic strategies that targets these pathways for treatment.     

Myelination of Neuronal Cell Bodies when Myelin Supply Exceeds Axonal Demand

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SSeCKS is a Suppressor in Schwann Cell Differentiation and Myelination

Src-suppressed protein kinase C substrate (SSeCKS) plays an important role in the differentiation process. In regeneration of sciatic nerve injury, expression of SSeCKS decreases, mainly in Schwann cells. However, the function of SSeCKS in Schwann cells differentiation remains unclear. We observed that SSeCKS was decreased in differentiated Schwann cells. In long-term SSeCKS-reduced Schwann cells, cell morphology changed and myelin gene expression induced by cAMP was accelerated. Myelination was also enhanced in SSeCKS-suppressed Schwann cells co-culture with dorsal root ganglion (DRG). In addition, we found suppression of SSeCKS expression promoted Akt serine 473 phosphorylation in cAMP-treated Schwann cells. In summary, our data indicated that SSeCKS was a negative regulator of myelinating glia differentiation.     

Role of Sonic Hedgehog Signaling in Oligodendrocyte Differentiation

During development, the secreted molecule Sonic Hedgehog (Shh) is required for lineage specification and proliferation of oligodendrocyte progenitors (OLPs), which are the glia cells responsible for the myelination of axons in the central nervous system (CNS). Shh signaling has been implicated in controlling both the generation of oligodendrocytes (OLGs) during embryonic development and their production in adulthood. Although, some evidence points to a role of Shh signaling in OLG development, its involvement in OLG differentiation remains to be fully determined. The objective of this study was to assess whether Shh signaling is involved in OLG differentiation after neural stem cell commitment to the OLG lineage. To address these questions, we manipulated Shh signaling using cyclopamine, a potent inhibitor of Shh signaling activator Smoothened (Smo), alone or combined with the agonist SAG in OLG primary cultures and assessed expression of myelin-specific markers. We found that inactivation of Shh signaling caused a dose-dependent decrease in myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in differentiating OLGs. Co-treatment of the cells with SAG reversed the inhibitory effect of cyclopamine on both myelin-specific protein levels and morphological changes associated with it. Further experiments are required to elucidate the molecular mechanism by which Shh signaling regulates OLG differentiation.     

Use of Transgenic Systems to Investigate Oligodendrocyte Differentiation and Function

Transgenic techniques are generating new strains of animals that are of great importance for many neurological research projects. This includes new animal models of human diseases that should allow analysis of disease etiology and treatment. The insertion of new genetic material into the mouse genome enables the investigator to study the effects of overexpression of normal or mutated genes under a variety of experimental conditions. The use of cell-specific and/or developmentally regulated promoters permits studies on the expression of the specific DNA in selected cells within the nervous system at important developmental stages. This article focuses on the techniques for generating transgenic mice, noting specific advantages or problems that should be considered when designing a transgenic project. The use of reporter genes such as the LacZ gene is discussed, using the particular example of the myelin proteolipid protein promoter directing expression of the LacZ gene in differentiating oligodendrocytes.     

二甲双胍(Metformin)在少突胶质前体细胞分化中的作用和机制*

目的：研究二甲双胍(metformin)在少突胶质前体细胞(oligodendrocyte precursor cell, OPC)分化过程中的作用,并对其分子机制进行初步探讨。方法：使用免疫吸附法直接分离纯化OPC后诱导培养,通过免疫荧光染色对细胞进行鉴定。在不同浓度二甲双胍处理OPC后,使用CCK8检测细胞活性;通过免疫荧光染色、流式细胞分析、实时荧光定量PCR和蛋白质印迹检测二甲双胍对OPC分化中细胞数量、mRNA和蛋白质水平的影响。结果：使用免疫吸附法可分离出高纯度OPC;CCK8检测结果显示在100 μmol/L浓度以内,二甲双胍对细胞无毒性;免疫荧光染色结果显示,二甲双胍处理OPC后,PDGFRα ⁺ OLIG2⁺阳性细胞数明显增加,且MBP⁺细胞数显著增加;流式细胞分析结果显示,PDGFRα⁺细胞数显著增加;实时荧光定量PCR结果显示,OPC分化相关基因Mag、Mbp等的mRNA水平显著增加;蛋白质印迹结果显示,分化相关蛋白OLIG2和MBP表达增加。机制上,少突胶质细胞系Oli-neu、OPC分别经二甲双胍处理5 min后,RAS、p-MEK、p-ERK蛋白量显著增加。结论：二甲双胍通过RAS-MEK-ERK信号通路促进少突胶质前体细胞的分化。     

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.