Connexins-Mediated Glia Networking Impacts Myelination and Remyelination in the Central Nervous System

In the central nervous system (CNS), the glial gap junctions are established among astrocytes (ASTs), oligodendrocytes (OLs), and/or between ASTs and OLs due to the expression of membrane proteins called connexins (Cxs). Together, the glial cells form a network of communicating cells that is important for the homeostasis of brain function for its involvement in the intercellular calcium wave propagation, exchange of metabolic substrates, cell proliferation, migration, and differentiation. Alternatively, Cxs are also involved in hemichannel function and thus participate in gliotransmission. In recent years, pathologic changes of oligodendroglia or demyelination found in transgenic mice with different subsets of Cxs or pharmacological insults suggest that glial Cxs may participate in the regulation of the myelination or remyelination processes. However, little is known about the underlying mechanisms. In this review, we will mainly focus on the functions of Cx-mediated gap junction channels, as well as hemichannels, in brain glial cells and discuss the way by which they impact myelination and remyelination. These aspects will be considered at the light of recent genetic and non-genetic studies related to demyelination and remyelination.     

Generation of demyelination models by targeted ablation of oligodendrocytes in the zebrafish CNS

Demyelination is the pathological process by which myelin sheaths are lost from around axons, and is usually caused by a direct insult targeted at the oligodendrocytes in the vertebrate central nervous system (CNS). A demyelinated CNS is usually remyelinated by a population of oligodendrocyte progenitor cells, which are widely distributed throughout the adult CNS. However, myelin disruption and remyelination failure affect the normal function of the nervous system, causing human diseases such as multiple sclerosis. In spite of numerous studies aimed at understanding the remyelination process, many questions still remain unanswered. Therefore, to study remyelination mechanisms in vivo, a demyelination animal model was generated using a transgenic zebrafish system in which oligodendrocytes are conditionally ablated in the larval and adult CNS. In this transgenic system, bacterial nitroreductase enzyme (NTR), which converts the prodrug metronidazole (Mtz) into a cytotoxic DNA cross-linking agent, is expressed in oligodendrocyte lineage cells under the control of the mbp and sox10 promoter. Exposure of transgenic zebrafish to Mtz-containing media resulted in rapid ablation of oligodendrocytes and CNS demyelination within 48 h, but removal of Mtz medium led to efficient remyelination of the demyelinated CNS within 7 days. In addition, the demyelination and remyelination processes could be easily observed in living transgenic zebrafish by detecting the fluorescent protein, mCherry, indicating that this transgenic system can be used as a valuable animal model to study the remyelination process in vivo, and to conduct high-throughput primary screens for new drugs that facilitate remyelination.     

Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury

Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages.     

In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

A demyelinating disease induced in C57B1/6N mice by intracranial injection of a coronavirus (murine hepatitis virus strain A59) is followed by functional recovery and efficient CNS myelin repair. To study the biological properties of the cells involved in this repair process, glial cells were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. Using three-color immunofluorescence combined with [3H]thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. We identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells with the O4 antibody. Cultures from demyelinated tissue differed in several ways from those of age-matched controls: first, the total number of O-2A lineage cells was strikingly increased; second, the O-2A population consisted of a higher proportion of O4-positive astrocytes and cells of mixed oligodendrocyte-astrocyte phenotype; and third, all the cell types within the O-2A lineage showed enhanced proliferation. This proliferation was not further enhanced by adding PDGF, basic fibroblast growth factor (bFGF), or insulin-like growth factor I (IGF-I) to the defined medium. However, bFGF and IGF-I seemed to influence the fate of O-2A lineage cells in cultures of demyelinated tissue. Basic FGF decreased the percentage of cells expressing galactocerebroside. In contrast, IGF-I increased the relative proportion of oligodendrocytes. Thus, O-2A lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. These properties may be linked to the efficient remyelination achieved in this demyelinating disease.     

Antibody-mediated CNS demyelination II. Focal spinal cord lesions induced by implantation of an IgM antisulfatide-secreting hybridoma

We showed previously that spinal cord implants of hybridoma cells (O1) that secrete an IgM antigalactocerebroside cause focal multiple-sclerosis-like plaques of demyelination followed by remyelination to form “shadow plaques” (Rosenbluth et al., 1999). The antibody in that case was directed against a glycolipid present in mature oligodendrocytes and myelin but not in precursor cells. We now report the effects of implanting a different hybridoma (O4) that secretes IgM antibodies directed against sulfatide, a constituent not only of mature myelin and oligodendrocytes but also of late precursor cells, in order to determine whether this hybridoma too would generate focal demyelination and would, in addition, block remyelination. Our results show that focal plaques of demyelination indeed appear after O4 implantation, and that remyelination does occur, but only in cases where the hybridoma cells have degenerated, probably through host rejection. The occurrence of remyelination suggests that oligodendrocyte precursor cells are capable of migrating in rapidly from adjacent areas or that early precursors, not yet expressing sulfatide, remain undamaged within the lesions. In cases where intact hybridoma cells persist at lesion sites, remyelination does not occur. Failure of remyelination in this model thus appears to result from the continuing presence of antimyelin antibodies rather than from depletion of oligodendrocyte precursors.     

Use of a rat Y chromosome probe to determine the long-term survival of glial cells transplanted into areas of CNS demyelination

A lack of suitable markers for cells which undergo division following transplantation has hindered studies assessing the long-term survival of glial cell grafts in the CNS. A probe specific to the rat Y chromosome was used to identify male glial cells grafted into an area of ethidium bromide-induced demyelination in syngeneic adult female rat spinal cord 4 weeks, 6 months and 12 months post-transplantation. At all time points there was extensive oligodendrocyte remyelination of transplanted lesions, and graft-derived cells were present within the lesion up to 12 months post-transplantation. In order to demonstrate graft-derived oligodendrocytes in the remyelinated region at 6 and 12 months, double-labelling studies were performed using the oligodendrocyte-specific antibodies carbonic anhydrase II or phosphatidyl ethanolamine-binding protein in combination with the Y chromosome probe. It was found that the majority of oligodendrocytes in the transplanted region were graft-derived. Graft-mediated remyelination was associated with a reduction in myelin sheath thickness and increase in nodal frequency similar to that observed in spontaneous remyelination, suggesting that, like axons remyelinated spontaneously, axons remyelinated by grafted cells will be capable of secure conduction. An alteration in the immunoreactivity of oligodendrocytes from carbonic anhydrase II-negative in the unlesioned dorsal funiculus to carbonic anhydrase II-positive in the remyelinated dorsal funiculus was considered to reflect a reduction in the amount of myelin supported by each oligodendrocyte, leading to the proposal that carbonic anhydrase II immunoreactivity may provide a means of identifying areas of remyelination in normally carbonic anhydrase II-negative white matter tracts.     

Glia Disease and Repair—Remyelination

The inability of the mammalian central nervous system (CNS) to undergo spontaneous regeneration has long been regarded as a central tenet of neurobiology. However, although this is largely true of the neuronal elements of the adult mammalian CNS, save for discrete populations of granular neurons, the same is not true of its glial elements. In particular, the loss of oligodendrocytes, which results in demyelination, triggers a spontaneous and often highly efficient regenerative response, remyelination, in which new oligodendrocytes are generated and myelin sheaths are restored to denuded axons. Yet, remyelination in humans is not without limitation, and a variety of demyelinating conditions are associated with sustained and disabling myelin loss. In this review, we will review the biology of remyelination, including the cells and signals involved; describe when remyelination occurs and when and why it fails and the consequences of its failure; and discuss approaches for therapeutically enhancing remyelination in demyelinating diseases of both children and adults, both by stimulating endogenous oligodendrocyte progenitor cells and by transplanting these cells into demyelinated brain.     

Remyelination properties of human embryonic nerve cells in the course of long-term cultivation

We studied the mitogenic and differentiation potential and the remyelination properties of human embryonic nerve cells in culture. After 1 month of cultivation without differentiation agents, a 3.6-fold increase in the number of CNP-positive cells (the mitotically active precursors of oligodendrocytes) was observed. At the same time, the number of GalC-positive cells (mature oligodendrocytes) remained low. The remyelination properties of embryonic nerve cells can thus be explained by a high density of oligodendrocyte precursors. After cultivation, the population of cells maintained and enhanced their remyelination potential by increasing the number of CNP-positive cells under conditions of experimental demyelination.     

Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination

Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal role for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis.     

Geissoschizine Methyl Ether, an Alkaloid from the Uncaria Hook, Improves Remyelination After Cuprizone-Induced Demyelination in Medial Prefrontal Cortex of Adult Mice

Accumulating evidence indicates that the medial prefrontal cortex (mPFC) is a site of myelin and oligodendrocyte abnormalities that contribute to psychotic symptoms of schizophrenia. The development of therapeutic approaches to enhance remyelination, a regenerative process in which new myelin sheaths are formed on demyelinated axons, may be an attractive remedial strategy. Geissoschizine methyl ether (GM) in the Uncaria hook, a galenical constituent of the traditional Japanese medicine yokukansan (Yi-gan san), is one of the active components responsible for the psychotropic effects of yokukansan, though little is known about the mechanisms underlying the effects of either that medicine or GM itself. In the present study, we employed a cuprizone (CPZ)-induced demyelination model and examined the cellular changes in response to GM administration during the remyelination phase in the mPFC of adult mice. Using the mitotic marker 5-bromo-2′-deoxyuridine (BrdU), we demonstrated that CPZ treatment significantly increased the number of BrdU-positive NG2 cells, as well as microglia and mature oligodendrocytes in the mPFC. Newly formed oligodendrocytes were increased by GM administration after CPZ exposure. In addition, GM attenuated a decrease in myelin basic protein immunoreactivity caused by CPZ administration. Taken together, our findings suggest that GM administration ameliorated the myelin deficit by mature oligodendrocyte formation and remyelination in the mPFC of CPZ-fed mice. The present findings provide experimental evidence supporting the role for GM and its possible use as a remedy for schizophrenia symptoms by promoting the differentiation of progenitor cells to and myelination by oligodendrocytes.     

Characterization of Glial Populations in the Aging and Remyelinating Mouse Corpus Callosum

Cells in the white matter of the adult brain have a characteristic distribution pattern in which several cells are contiguously connected to each other, making a linear array (LA) resembling pearls-on-a-string parallel to the axon axis. We have been interested in how this pattern of cell distribution changes during aging and remyelination after demyelination. In the present study, with a multiplex staining method, semi-quantitative analysis of the localization of oligodendrocyte lineage cells (oligodendrocyte progenitors, premyelinating oligodendrocytes, and mature oligodendrocytes), astrocytes, and microglia in 8-week-old (young adult) and 32-week-old (aged) corpus callosum showed that young adult cells still include immature oligodendrocytes and that LAs contain a higher proportion of microglia than isolated cells. In aged mice, premyelinating oligodendrocytes were decreased, but microglia continued to be present in the LAs. These results suggest that the presence of microglia is important for the characteristic cell localization pattern of LAs. In a cuprizone-induced demyelination model, we observed re-formation of LAs after completion of cuprizone treatment, concurrent with remyelination. These re-formed LAs again contained more microglia than the isolated cells. This finding supports the hypothesis that microglia contribute to the formation and maintenance of LAs. In addition, regardless of the distribution of cells (LAs or isolated cells), astrocytes were found to be more abundant than in the normal corpus callosum at 24 weeks after cuprizone treatment when remyelination is completed. This suggests that astrocytes are involved in maintaining the functions of remyelinated white matter.

     

The demonstration by transplantation of the very restricted remyelinating potential of post-mitotic oligodendrocytes

To examine the remyelinating ability of post-mitotic oligodendrocytes, we subjected cell preparations derived from neonatal and adult rats to 40 Grays of X-irradiation to remove mitotically active cells and injected them into areas of demyelination in which the inherent ability to generate remyelinating cells had been inhibited. The extensive remyelination seen following implantation of non-irradiated neonatal and adult cells was almost completely abolished when the transplanted cell suspension was exposed to 40 Grays of X-irradiation, demonstrating that effective remyelination requires the generation of cells by mitosis. Radiation-resistant and therefore non-dividing oligodendrocytes were detected in areas of demyelination following transplantation of neonatal cultures and oligodendrocyte preparations derived from the adult nervous system. However, the pattern of myelin formation associated with the radiation-resistant oligodendrocytes from the two sources was different. Following implantation of X-irradiated neonatal cultures, a small number of oligodendrocytes could be found within the area of demyelination, and although these cells formed sheets of myelin membrane, they did not form myelin sheaths. After implantation of X-irradiated adult cells, in addition to the aberrant myelin formation seen with the neonatal cells, some myelin sheaths were observed. Our findings confirm that effective remyelination requires cell division and suggest that there may be diverse populations of radiation-resistant oligodendrocytes in the adult nervous system, some of which can form myelin sheaths and others of which can only make myelin sheets. Important for the interpretation of our previous studies is the demonstration here that 40 Grays of X-irradiation per se does not inhibit oligodendrocytes from remyelinating axons.     

Erythropoietin promotes oligodendrogenesis and myelin repair following lysolecithin-induced injury in spinal cord slice culture

Here, we sought to delineate the effect of EPO on the remyelination processes using an in vitro model of demyelination. We report that lysolecithin-induced demyelination elevated EPO receptor (EpoR) expression in oligodendrocyte progenitor cells (OPCs), facilitating the beneficial effect of EPO on the formation of oligodendrocytes (oligodendrogenesis). In the absence of EPO, the resultant remyelination was insufficient, possibly due to a limiting number of oligodendrocytes rather than their progenitors, which proliferate in response to lysolecithin-induced injury. By EPO treatment, lysolecithin-induced proliferation of OPCs was accelerated and the number of myelinating oligodendrocytes and myelin recovery was increased. EPO also enhanced the differentiation of neural progenitor cells expressing EpoR at high level toward the oligodendrocyte-lineage cells through activation of cyclin E and Janus kinase 2 pathways. Induction of myelin-forming oligodendrocytes by high dose of EPO implies that EPO might be the key factor influencing the final differentiation of OPCs. Taken together, our data suggest that EPO treatment could be an effective way to enhance remyelination by promoting oligodendrogenesis in association with elevated EpoR expression in spinal cord slice culture after lysolecithin-induced demyelination.     

Identification of a novel cell-penetrating peptide from human phosphatidate phosphatase LPIN3

Biomolecules such as proteins, DNA, and RNA are macromolecules and can not cross the cell membrane. However, cell-penetrating peptide (CPP) has been shown to deliver therapeutic biomolecules successfully into cells. The various and widely used CPPs including TAT, VP22, and Antp are mostly non-human originated CPPs, and are limited by their potential toxicity and immunogenicity. We report here on a newly identified novel cell-penetrating sequence (LPIN; RRKRRRRRK) from the nuclear localization sequence (NLS) of human nuclear phosphatase, LPIN3. LPIN-EGFP recombinant protein was concentration- and time-dependently delivered into cells and localized to the nucleus as well as the cytoplasm. It penetrated the cell membrane by lipid raft-mediated endocytosis by binding to heparan sulfate proteoglycan. LPIN-EGFP was successfully delivered into primary mouse splenocytes in vitro and it could be delivered into various tissues including liver, kidney, and intestine in mice after intra-peritoneal injection. This research suggests that LPIN-CPP could be used in a drug delivery system to deliver therapeutic biomolecules including peptides, proteins, DNA, and RNA and without the limitations of non-human originated CPPs such as TAT-CPP.     

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.     

In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Expression of carbonic anhydrase II mRNA and protein in oligodendrocytes during toxic demyelination in the young adult mouse

The aim of this study was to identify events that might take place in oligodendrocytes early in the process of demyelination, i.e., before the occurrence of massive loss of myelin. It was considered important to focus on demyelination and remyelination in young adults, in whose brains there would be relatively few juvenile glial precursor cells. CAII mRNA and protein were used to monitor changes in oligodendrocytes during cuprizone intoxication in the mice. After four or eight weeks of cuprizone feeding CAII message became less plentiful in oligodendrocyte processes. Two days after removal of cuprizone CAII message had appeared in those cell processes. Four or eight weeks after beginning cuprizone feeding CAII protein had decreased∼25% in forebrain homogenates. The loss of CAII protein was reversible after four weeks on cuprizone, but not after eight weeks. After four weeks of cuprizone feeding the numbers of CAII mRNA-prositive oligodendrocytes had decreased by ∼50%m and after eight weeks, by ∼80%. By 12 weeks, however, the number of oligodendrocytes expressing CAII mRNA had spontaneously returned to normal levels. Before eight weeks of cuprizone feeding, loss of myelinated tracts in the corpus striatum was reversible. Demyelination appreared to become irreversible after nine weeks of intoxication, although expression of CAII mRNA remained reversible. The results suggest that in the brain of the young adult, oligodendrocytes expressing message for CAII can be generated spontaneously shortly before demyelination becomes irreversible, and can survive and continue to express CAII mRNA but not CAII protein. Special issue dedicated to Dr. Marion E. Smith.     

Therapeutic strategies in multiple sclerosis. II. Long-term repair

Spontaneous myelin repair in multiple sclerosis (MS) provides a striking example of the brain's inherent capacity for sustained and stable regenerative tissue repair--but also clearly emphasizes the limitations of this capacity; remyelination ultimately fails widely in many patients, and disability and handicap accumulate. The observation of endogenous partial myelin repair has raised the possibility that therapeutic interventions designed to supplement or promote remyelination might have a useful and significant impact both in the short term, in restoring conduction, and in the long term, in safeguarding axons. Therapeutic remyelination interventions must involve manipulations to either the molecular or the cellular environment within lesions; both depend crucially on a detailed understanding of the biology of the repair process and of those glia implicated in spontaneous repair, or capable of contributing to exogenous repair. Here we explore the biology of myelin repair in MS, examining the glia responsible for successful remyelination, oligodendrocytes and Schwann cells, their 'target' cells, neurons and the roles of astrocytes. Options for therapeutic remyelinating strategies are reviewed, including glial cell transplantation and treatment with growth factors or other soluble molecules. Clinical aspects of remyelination therapies are considered--which patients, which lesions, which stage of the disease, and how to monitor an intervention--and the remaining obstacles and hazards to these approaches are discussed.