Cultured oligodendrocytes mimic in vivo phenotypic characteristics: Cell shape, expression of myelin-specific antigens, and membrane production

Primary cultures of neonatal mouse cerebra were maintained for up to 4 weeks in the absence of neurons. Oligodendrocytes in these cultures pass through a sequence of cytoarchitectural change and antigen expression which mimics the differentiation of oligodendrocytes in vivo. The cell bodies and processes of oligodendrocytes first express the myelin-specific antigen galactocerebroside (GC) by 2 days in vitro. Myelin basic protein (MBP) appears several days later. The majority of oligodendrocytes then proceed to elaborate large sheets of membranous material from the tips and lengths of cell processes. These membranous sheets, which contain GC and MBP, are reminiscent of unwrapped myelin profiles in vivo. As with the cell bodies and processes, GC is inserted into the sheets several days before MBP. Our results establish that oligodendrocytes cultured without neurons are able to produce extensive membranes containing myelin-specific antigens. They also suggest that oligodendrocyte shape and membrane production are, in part, regulated from within the oligodendrocyte itself.     

Emergence of three myelin proteins in oligodendrocytes cultured without neurons

Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat brain and optic nerve to allow us to analyze whether two transmembranous myelin proteins, myelin-associated glycoprotein (MAG) and proteolipid protein (PLP), were expressed together with myelin basic protein (MBP) in defined medium with low serum and in the absence of neurons. Using double label immunofluorescence, we investigated when and where these three myelin proteins appeared in cells expressing galactocerebroside (GC), a specific marker for the oligodendrocyte membrane. We found that a proportion of oligodendrocytes derived from brain and optic nerve invariably express MBP, MAG, and PLP about a week after the emergence of GC, which occurs around birth. In brain-derived oligodendrocytes, MBP and MAG first emerge between the fifth and the seventh day after birth, followed by PLP 1 to 2 d later. All three proteins were confined to the cell body at that time, although an extensive network of GC positive processes had already developed. Each protein shows a specific cytoplasmic localization: diffuse for MBP, mostly perinuclear for MAG, and particulate for PLP. Interestingly, MAG, which may be involved in glial-axon interactions, is the first myelin protein detected in the processes at approximately 10 d after birth. MBP and PLP are only seen in these locations after 15 d. All GC-positive cells express the three myelin proteins by day 19. Simultaneously, numerous membrane and myelin whorls accumulate along the oligodendrocyte surface. The sequential emergence, cytoplasmic location, and peak of expression of these three myelin proteins in vitro follow a pattern similar to that described in vivo and, therefore, are independent of continuous neuronal influences. Such cultures provide a convenient system to study factors regulating expression of myelin proteins.     

Dysmyelination, demyelination and reactive astrogliosis in the optic nerve of the taiep rat

Taiep is an autosomal recessive mutant rat that shows a highly hypomyelinated central nervous system (CNS). Oligodendrocytes accumulate microtubules (MTs) in association with endoplasmic reticulum (ER) membranes forming MT-ER complexes. The microtubular defect in oligodendrocytes, the abnormal formation of CNS myelin and the astrocytic reaction were characterized by immunocytochemical and ultrastructural methods during the first year of life. Optic nerves of both control and taiep rats were processed by the immunoperoxidase method using antibodies against tubulin, myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). Taiep oligodendrocytes are strongly immunoreactive against tubulin, indicative of a significant accumulation of microtubules. Early differentiated oligodendrocytes observed with electron microscopy show that MT-ER complexes are mainly present in the cell body. This defect increases during the first year of life; oligodendrocytes show large MT-ER complexes projected within oligodendrocyte processes. Using anti-MBP, there was a progressive reduction of immunolabeling in the myelin sheaths as taiep rats grew older. Ultrastructural analysis revealed severely dysmyelinated axons with a frequently collapsed periaxonal collar. However, through age the myelin sheath became gradually infiltrated by MTs, suggesting their contribution to premature loss of myelin in the taiep rat. Axons of one-year-old taiep rats were severely demyelinated. Modifications in astrocytes revealed by the GFAP antibody showed a strong hypertrophy with increased immunostaining in their processes. As demyelination of axons progressed, taiep rats developed a strong astrogliosis. The present findings suggest that in taiep rats the early abnormal myelination of axons affects the adequate maintenance of myelin, leading to a progressive loss of myelin components and severe astrogliosis, features that should be considered in the pathogenesis of dysmyelinating diseases.     

The Role of Vesicle Trafficking and Release in Oligodendrocyte Biology

Oligodendrocytes are a subtype of glial cells found within the central nervous system (CNS), responsible for the formation and maintenance of specialized myelin membranes which wrap neuronal axons. The development of myelin requires tight coordination for the cell to deliver lipid and protein building blocks to specific myelin segments at the right time. Both internal and external cues control myelination, thus the reception of these signals also requires precise regulation. In late years, a growing body of evidence indicates that oligodendrocytes, like many other cell types, may use extracellular vesicles (EVs) as a medium for transferring information. The field of EV research has expanded rapidly over the past decade, with new contributions that suggest EVs might have direct involvement in communications with neurons and other glial cells to fine tune oligodendroglial function. This functional role of EVs might also be maladaptive, as it has likewise been implicated in the spreading of toxic molecules within the brain during disease. In this review we will discuss the field’s current understanding of extracellular vesicle biology within oligodendrocytes, and their contribution to physiologic and pathologic conditions.

     

The cellular and molecular events of central nervous system remyelination

Central nervous system (CNS)* regeneration is a subject of great interest, particularly in diseases causing a dramatic loss of neurons. However, some CNS diseases do not affect neurons but damage other cells, such as the myelin-forming cells--called oligodendrocytes--which are also crucial to the harmonious function of the nervous system. Diseases in which oligodendrocytes and myelin are attacked can cause devastating neurological dysfunction which is sometimes followed by recovery and myelin repair or remyelination. The question of the regeneration potential of oligodendrocytes in experimental and human demyelinating diseases such as multiple sclerosis has been debated for a long time. Present evidence suggests that oligodendrocyte precursor cells persist in the adult CNS and that oligodendrocyte regeneration can occur but may be limited by ongoing disease processes. Here we will briefly review recent advances which have broadened our understanding of the cellular and molecular events of CNS remyelination.     

Oligodendrocyte ablation affects the coordinated interaction between granule and Purkinje neurons during cerebellum development

Oligodendrocytes (OLs) are the glial cells of the central nervous system (CNS) classically known to be devoted to the formation of myelin sheaths around most axons of the vertebrate brain. We have addressed the role of these cells during cerebellar development, by ablating OLs in vivo. Previous analyses had indicated that OL ablation during the first six postnatal days results into a striking cerebellar phenotype, whose major features are a strong reduction of granule neurons and aberrant Purkinje cells development. These two cell types are highly interconnected during cerebellar development through the production of molecules that help their proliferation, differentiation and maintenance. In this article, we present data showing that OL ablation has major effects on the physiology of Purkinje (PC) and granule cells (GC). In particular, OL ablation results into a reduction of sonic hedgehog (Shh), Brain Derived Neurotrophic Factor (BDNF), and Reelin (Rln) expression. These results indicate that absence of OLs profoundly alters the normal cerebellar developmental program.     

Glioblastoma infiltration into central nervous system tissue in vitro: involvement of a metalloprotease

Differentiated oligodendrocytes and central nervous system (CNS) myelin are nonpermissive substrates for neurite growth and for cell attachment and spreading. This property is due to the presence of membrane-bound inhibitory proteins of 35 and 250 kD and is specifically neutralized by monoclonal antibody IN-1 (Caroni, P., and M. E. Schwab. 1988. Neuron. 1:85-96). Using rat optic nerve explants, CNS frozen sections, cultured oligodendrocytes or CNS myelin, we show here that highly invasive CNS tumor line (C6 glioblastoma) was not inhibited by these myelin- associated inhibitory components. Lack of inhibition was due to a specific mechanism as the metalloenzyme blocker 1,10-phenanthroline and two synthetic dipeptides containing metalloprotease-blocking sequences (gly-phe, tyr-tyr) specifically impaired C6 cell spreading on CNS myelin. In the presence of these inhibitors, C6 cells were affected by the IN-1-sensitive inhibitors in the same manner as control cells, e.g., 3T3 fibroblasts or B16 melanomas. Specific blockers of the serine, cysteine, and aspartyl protease classes had no effect. C6 cell spreading on inhibitor-free substrates such as CNS gray matter, peripheral nervous system myelin, glass, or poly-D-lysine was not sensitive to 1,10-phenanthroline. The nonpermissive substrate properties of CNS myelin were strongly reduced by incubation with a plasma membrane fraction prepared from C6 cells. This reduction was sensitive to the same inhibitors of metalloproteases. In our in vitro model for CNS white matter invasion, cell infiltration of optic nerve explants, which occurred with C6 cells but not with 3T3 fibroblasts or B16 melanomas, was impaired by the presence of the metalloprotease blockers. These results suggest that C6 cell infiltrative behavior in CNS white matter in vitro occurs by means of a metalloproteolytic activity, which probably acts on the myelin-associated inhibitory substrates.     

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside⁺ (GalC) and O4⁺ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.     

Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter

CNS white matter from higher vertebrates and cultured differentiated oligodendrocytes are nonpermissive substrates for neurite growth and fibroblast spreading. Membrane proteins of 35 kd and 250 kd with highly nonpermissive substrate properties could be extracted from CNS myelin fractions. Monoclonal antibodies were raised against these proteins: IN-1 and IN-2 bound both to the 35 kd and 250 kd inhibitors and to the surface to differentiated cultured oligodendrocytes. Adsorption of nonpermissive CNS myelin or nonpermissive oligodendrocytes with either antibody markedly improved their substrate properties. Optic nerve explants injected with IN-1 or IN-2 allowed axon ingrowth of cocultured sensory and sympathetic neurons. We conclude that the nonpermissive substrate properties of CNS white matter are due to these membrane proteins on the surface of differentiated oligodendrocytes and to their in vivo product, myelin.     

Extrinsic Factors Driving Oligodendrocyte Lineage Cell Progression in CNS Development and Injury

Neurochemical Research - Oligodendrocytes (OLs) generate myelin membranes for the rapid propagation of electrical signals along axons in the central nervous system (CNS) and provide metabolites to...     

Characterization of mature rat oligodendrocytes: a proteomic approach

Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.     

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.     

The Polarity Protein Scribble Regulates Myelination and Remyelination in the Central Nervous System

The development and regeneration of myelin by oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), requires profound changes in cell shape that lead to myelin sheath initiation and formation. Here, we demonstrate a requirement for the basal polarity complex protein Scribble in CNS myelination and remyelination. Scribble is expressed throughout oligodendroglial development and is up-regulated in mature oligodendrocytes where it is localised to both developing and mature CNS myelin sheaths. Knockdown of Scribble expression in cultured oligodendroglia results in disrupted morphology and myelination initiation. When Scribble expression is conditionally eliminated in the myelinating glia of transgenic mice, myelin initiation in CNS is disrupted, both during development and following focal demyelination, and longitudinal extension of the myelin sheath is disrupted. At later stages of myelination, Scribble acts to negatively regulate myelin thickness whilst suppressing the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAP) kinase pathway, and localises to non-compact myelin flanking the node of Ranvier where it is required for paranodal axo-glial adhesion. These findings demonstrate an essential role for the evolutionarily-conserved regulators of intracellular polarity in myelination and remyelination.     

The cfy mutation disrupts cell divisions in a stage-dependent manner in zebrafish embryos

The zebrafish curly fry (cfy) mutation leads to embryonic lethality and abnormal cell divisions starting at 12-14 h postfertilization (hpf) during neural tube formation. The mitotic defect is seen in a variety of tissues including the central nervous system (CNS). In homozygous mutant embryos, mitoses are disorganized with an increase in mitotic figures throughout the developing neural tube. One consequence of aberrant mitoses in cfy embryos is an increase in cell death. Despite this, patterning of the early CNS is relatively unperturbed with distribution of the early, primary neurons indistinguishable from that of wild-type embryos. At later stages, however, the number of neurons was dramatically decreased throughout the CNS. The effect on neurons in older cfy embryos but not young ones correlates with the time of birth of neurons: primary neurons are born before the action of the cfy gene and later neurons after. Presumably, death of neuronal progenitors that divide beginning at the neural keel stage or death of their neuronal progeny accounts for the diminution of neurons in older mutant embryos. In addition, oligodendrocytes, which also develop late in the CNS, are greatly reduced in number in cfy embryos due to an apparent decrease in oligodendrocyte precursors. Genetic mosaic analysis demonstrates that the mutant phenotype is cell-autonomous. Furthermore, there are no obvious defects in apical/basal polarity within the neuroepithelium, suggesting that the cfy gene is not critical for epithelial polarity and that polarity defects are unlikely to account for the increased mitotic figures in mutants. These results suggest that the cfy gene regulates mitosis perhaps in a stage-dependent manner in vertebrate embryos.     

Development of oligodendrocytes. Studies of rat glial cells cultured in chemically-defined medium

Oligodendrocytes are macroglial cells that synthesize and maintain myelin in the central nervous system. Oligodendrocytes in rodent brain are formed postnatally from glial progenitor cells. These progenitors cells are bipotential and differentiate in a later stage of development into type-2 astrocytes. Recent studies with cultured cells indicate that growth factors such as platelet-derived growth factor and ciliary neurotrophic factor are instrumental in the control of these events. This paper discusses various methods for the isolation of oligodendrocytes and for their maintenance in culture. We use cerebra or spinal cords from one-week old rat pups to prepare glial cultures that are enriched in oligodendrocytes (60-80% or greater than or equal to 90%, respectively). After one day in serum-containing medium the cells are kept in chemically-defined medium, supplemented with the hormones insulin, T3 and hydrocortisone. The activities of astrocyte-and oligodendrocyte-specific marker enzymes were measured to evaluate the influence of these hormones on the differentiation of the oligodendrocytes. Finally, glial energy metabolism and the utilization of ketone bodies and of fatty acids are discussed briefly.     

Testosterone metabolism in brain cells and membranes

The central nervous system (CNS) is considered a target structure for the action of all the classes of hormonal steroids produced by the organism. Well-characterized genomic and less well-understood membrane mechanisms of action are probably involved in the steroid modulation of brain activities. Moreover, some classes of steroids need to be converted into “active” metabolites before interacting with their effector systems. In particular, testosterone (T) exerts many of its effects after conversion to 5-dihydrotestosterone (DHT) and estrogens. The CNS possesses both the 5-reductase, the enzyme which produces DHT and the aromatase which transforms T into estrogens; however, the relative role and distribution of these enzymes in the various structural components of the CNS has not been clarified so far. The 5-reductase has been found to be present in high concentrations in brain white matter structures because these are particularly rich in myelin membranes, to which the enzymatic activity appears to be associated. This membrane localization might suggest a possible involvement of steroidal 5-reduced metabolites in membrane-mediated events in the CNS. Moreover, the distribution of 5-reductase was studied in neurons, astrocytes and oligodendrocytes isolated from the brain of male rats by density gradient ultracentrifugation, as well as in neurons and glial cells grown in culture. The aromatase activity was also evaluated in neurons and glial cells grown in culture and in isolated oligodendrocytes. Among the three cell types isolated, neurons appear to be more active than oligodendrocytes and astrocytes, respectively, in converting T into DHT. Also, in cell culture experiments, neurons are more active in forming DHT than glial cells. Only neurons possess aromatase activity, while glial cells are apparently unable to aromatize T.     

Contact with central nervous system myelin inhibits oligodendrocyte progenitor maturation

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated during development through the proliferation and differentiation of a distinct progenitor population. Not all oligodendrocyte progenitors generated during development differentiate, however, and large numbers of oligodendrocyte progenitors are present in the adult CNS, particularly in white matter. These "adult progenitors" can be identified through expression of the NG2 proteoglycan. Adult oligodendrocyte progenitors are thought to develop from the original pool of progenitors and in vitro are capable of differentiating into oligodendrocytes. Why these cells fail to differentiate in the intact CNS is currently unclear. Here we show that contact with CNS myelin inhibits the maturation of immature oligodendrocyte progenitors. The inhibition of oligodendrocyte progenitor maturation is a characteristic of CNS myelin that is not shared by several other membrane preparations including adult and neonatal neural membrane fractions, PNS myelin, or liver. This inhibition is concentration dependent, is reversible, and appears not to be mediated by either myelin basic protein or basic fibroblast growth factor. Myelin-induced inhibition of oligodendrocyte progenitor maturation provides a mechanism to explain the generation of a residual pool of immature oligodendrocyte progenitors in the mature CNS.     

Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.     

Microglia and neuronal cell death

Microglia, the brain's innate immune cell type, are cells of mesodermal origin that populate the central nervous system (CNS) during development. Undifferentiated microglia, also called ameboid microglia, have the ability to proliferate, phagocytose apoptotic cells and migrate long distances toward their final destinations throughout all CNS regions, where they acquire a mature ramified morphological phenotype. Recent studies indicate that ameboid microglial cells not only have a scavenger role during development but can also promote the death of some neuronal populations. In the mature CNS, adult microglia have highly motile processes to scan their territorial domains, and they display a panoply of effects on neurons that range from sustaining their survival and differentiation contributing to their elimination. Hence, the fine tuning of these effects results in protection of the nervous tissue, whereas perturbations in the microglial response, such as the exacerbation of microglial activation or lack of microglial response, generate adverse situations for the organization and function of the CNS. This review discusses some aspects of the relationship between microglial cells and neuronal death/survival both during normal development and during the response to injury in adulthood.