The oligodendrocyte-type-2 astrocyte cell lineage is specialized for myelination

Astrocytes are one of the most numerous cell types in the vertebrate central nervous system (CNS) and yet their functions are largely unknown. In the rat optic nerve there are two distinct types of astrocyte: type-1 astrocytes develop from one type of precursor cell, and type-2 astrocytes develop from bipotential, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, that initially give rise to oligodendrocytes (which make myelin in the CNS), and then to type-2 astrocytes. Type-1 astrocytes form the glial limiting membrane at the periphery of the optic nerve and are probably responsible for glial scar formation following nerve transection. The functions of type-2 astrocytes, which, like oligodendrocytes, are found mainly in tracts of myelinated axons throughout the CNS, are unknown. In this report we provide evidence that processes from type-2 astrocytes contribute to the structure of nodes of Ranvier, suggesting that the O-2A cell lineage is specialized for constructing myelin sheaths and nodes in the mammalian CNS.     

Platelet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell

The mitogens which modulate cell-cell interactions during development of the central nervous system are unknown. One of the few interactions sufficiently well understood to allow identification of such molecules involves the two glial lineages which make up the rat optic nerve. One population of glial cells in this tissue, the type-1 astrocytes, secrete a soluble factor(s) which promotes division of a second population of bipotential oligodendrocyte/type-2 astrocyte (O-2A) progenitor cells; these progenitors give rise to oligodendrocytes, which myelinate large axons in the CNS, and type-2 astrocytes, which enwrap bare axons at nodes of Ranvier. Type-1 astrocytes also promote progenitor motility, and inhibit the premature differentiation of progenitors into oligodendrocytes which occur when these cells are grown in the absence of type-1 astrocytes. We have now found that platelet-derived growth factor mimics the effects of type-1 astrocytes on O-2A progenitor cells, and antibodies to PDGF block the effects of type-1 astrocytes.     

Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture

The various cell types in a multicellular animal differentiate on a predictable schedule but the mechanisms responsible for timing cell differentiation are largely unknown. We have studied a population of bipotential glial (O-2A) progenitor cells in the developing rat optic nerve that gives rise to oligodendrocytes beginning at birth and to type-2 astrocytes beginning in the second postnatal week. Whereas, in vivo, these O-2A progenitor cells proliferate and give rise to postimitotic oligodendrocytes over several weeks, in serum-free (or low-serum) culture they stop dividing prematurely and differentiate into oligodendrocytes within two or three days. The normal timing of oligodendrocyte development can be restored if embryonic optic-nerve cells are cultured in medium conditioned by type-1 astrocytes, the first glial cells to differentiate in the nerve: in this case the progenitor cells continue to proliferate, the first oligodendrocytes appear on the equivalent of the day of birth, and new oligodendrocytes continue to develop over several weeks, just as in vivo. Here we show that platelet-derived growth factor (PDGF) can replace type-1-astrocyte-conditioned medium in restoring the normal timing of oligodendrocyte differentiation in vitro and that anti-PDGF antibodies inhibit this property of the appropriately conditioned medium. We also show that PDGF is present in the developing optic nerve. These findings suggest that type-1-astrocyte-derived PDGF drives the clock that times oligodendrocyte development.     

Differentiation of a bipotential glial progenitor cell in a single cell microculture

Although it is known that most cells of the vertebrate central nervous system (CNS) are derived from the neuroepithelial cells of the neural tube, the factors determining whether an individual neuroepithelial cell develops into a particular type of neurone or glial cell remain unknown. A promising model for studying this problem is the bipotential glial progenitor cell in the developing rat optic nerve; this cell differentiates into a particular type of astrocyte (a type-2 astrocyte) if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serum-free medium. As the oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell can differentiate along either glial pathway in neurone-free cultures, living axons clearly are not required for its differentiation, at least in vitro. However, the studies on 0-2A progenitor cells were carried out in bulk cultures of optic nerve, and so it was possible that other cell-cell interactions were required for differentiation in culture. We show here that 0-2A progenitor cells can differentiate into type-2 astrocytes or oligodendrocytes when grown as isolated cells in microculture, indicating that differentiation along either glial pathway in vitro does not require signals from other CNS cells, apart from the signals provided by components of the culture medium. We also show that single 0-2A progenitor cells can differentiate along either pathway without dividing, supporting our previous studies using 3H-thymidine and suggesting that DNA replication is not required for these cells to choose between the two differentiation programmes.     

Proliferating bipotential glial progenitor cells in adult rat optic nerve

We have shown previously that the rat optic nerve contains three types of macroglial cells--oligodendrocytes and two types of astrocytes--which develop as two distinct lineages. Type-1 astrocytes develop from one type of precursor cell beginning at embryonic day 16 (E16), while oligodendrocytes and then type-2 astrocytes develop from a common, bipotential progenitor cell beginning at birth (E21) and postnatal days 7-10 (P7-10), respectively. Here we report that proliferating bipotential oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells are present in the adult rat optic nerve, raising the possibility that these cells are produced continually from self-renewing stem cells throughout life.     

A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium

We have identified a cell type in 7-day-old rat optic nerve that differentiates into a fibrous astrocyte if cultured in the presence of fetal calf serum and into an oligodendrocyte if cultured in the absence of serum. In certain culture conditions some of these cells acquire a mixed phenotype, displaying properties of both astrocytes and oligodendrocytes. These observations suggest that fibrous astrocytes and oligodendrocytes develop from a common progenitor cell and provide a striking example of developmental plasticity and environmental influence in the differentiation of CNS glial cells.     

Facilitation of voltage-gated ion channels in frog neuroglia by nerve impulses

The functions of glial cells in the nervous system are not well defined, with the exception of myelin production by oligodendrocytes, uptake of amino-acid synaptic transmitters, and a contribution to extracellular potassium homeostasis. Neuroglia have receptors for neurotransmitters which may be involved in neuron-glia interactions. Recent studies have demonstrated voltage-gated ion channels in glial membranes. In a study of the optic nerve of the frog, small areas of the surface were examined with the loose patch-clamp method, and voltage-gated Na+ and K+ channels, presumably located in the membranes of the astrocytes forming the glia limitans, were identified. We now report that nerve impulses in the axons of the frog optic nerve transiently alter the properties of the voltage-dependent membrane channels of the surface glial cells (astrocytes), a demonstration of a new form of neuron-glia interaction.     

Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids

L-GLUTAMATE and L-aspartate are thought to have a widespread function as synaptic transmitters in the mammalian central nervous system and there are at least three types of neuronal glutamate receptors, which can be activated by the selective agonists N-methyl-D-aspartate (NMDA), quisqualate and kainate. Recent experiments indicate that glutamate receptors also occur in astrocytes. We have used patch-clamp methods to determine whether one type of macroglial cell, the type-2 astrocyte, possesses glutamate receptors, as previously proposed from neurochemical studies. We find that glutamate and related amino acids can evoke whole-cell and single-channel currents in type-2 astrocytes from rat cerebellum. Although these cells are found mainly in white matter, where neurotransmission does not occur, their processes are closely associated with axons at nodes of Ranvier, suggesting that such receptors are involved in neuronal-glial signalling at the node. Our experiments show that glial cells possess quisqualate- and kainate-receptor channels but lack receptors for NMDA. Interestingly, these glutamate channels exhibit multiple conductance levels that are similar in amplitude to the neuronal glutamate channels.     

Effect of ciliary neurotrophic factor on the reactive astrogliosis of cultured astrocytes from newborn rat brain

The effect of ciliary neurotrophic factor (CNTF) on reactive astrogliosis was studied on a mechanical scratch model of the confluent astrocytic cultures from newborn rat brain. Following injury, the astrocytes at the edge of the injured area displayed a typical process of the reactive astrogliosis. This process included apparently hyperplastic change and significantly increased GFAP expression of the flat astrocytes, and migration to the injured area of the O-2A progenitor cells and their differentiation into process-bearing astrocytes. Exogenous CNTF applied to the cell cultures significantly promoted the hyperplasia and GFAP expression of the flat astrocytes. The results suggest that CNTF can enhance the reactive astrogliosis in the injured area.     

Local generation of glia is a major astrocyte source in postnatal cortex

Glial cells constitute nearly 50% of the cells in the human brain. Astrocytes, which make up the largest glial population, are crucial to the regulation of synaptic connectivity during postnatal development. Because defects in astrocyte generation are associated with severe neurological disorders such as brain tumours, it is important to understand how astrocytes are produced. Astrocytes reportedly arise from two sources: radial glia in the ventricular zone and progenitors in the subventricular zone, with the contribution from each region shifting with time. During the first three weeks of postnatal development, the glial cell population, which contains predominantly astrocytes, expands 6-8-fold in the rodent brain. Little is known about the mechanisms underlying this expansion. Here we show that a major source of glia in the postnatal cortex in mice is the local proliferation of differentiated astrocytes. Unlike glial progenitors in the subventricular zone, differentiated astrocytes undergo symmetric division, and their progeny integrate functionally into the existing glial network as mature astrocytes that form endfeet with blood vessels, couple electrically to neighbouring astrocytes, and take up glutamate after neuronal activity.     

Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro

Neuropeptides may have functions in the central nervous system (CNS) other than altering neuronal excitability. For example, they may act as regulators of brain metabolism by affecting glycogenolysis. Since it has been suggested that glial cells might provide metabolic support for neuronal activity, they may well be one of the targets for neuropeptide regulation of metabolism. Consistent with this view are reports that peptide-containing nerve terminals have been seen apposed to astrocytes, but it is also quite possible that peptides could act at sites lacking morphological specialization. Primary cultures containing CNS glial cells have been shown to respond to beta-adrenergic agonists with an increase in cyclic AMP and, as a result, with an increase in glycogenolysis and have also been shown to respond to a variety of peptides with changes in cyclic AMP. In the study reported here, we have examined the effects of several peptides on relatively pure cultures of rat astrocytes. We demonstrate that the increase in intracellular cyclic AMP induced by noradrenaline is markedly enhanced by somatostatin and substance P and is inhibited by enkephalin, even though these peptides on their own have little or no effect on the basal levels of cyclic AMP. Vasoactive intestinal peptide (VIP) on the other hand increases cyclic AMP in the absence of noradrenaline. These results suggest that neuropeptides influence glial cells as well as neurones in the CNS and, in the case of somatostatin and substance P, provide further examples of neuropeptides modulating the response to another chemical signal without having a detectable action on their own.     

Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity

Oligodendrocytes, the myelin-forming glial cells of the central nervous system, maintain long-term axonal integrity. However, the underlying support mechanisms are not understood. Here we identify a metabolic component of axon-glia interactions by generating conditional Cox10 (protoheme IX farnesyltransferase) mutant mice, in which oligodendrocytes and Schwann cells fail to assemble stable mitochondrial cytochrome c oxidase (COX, also known as mitochondrial complex IV). In the peripheral nervous system, Cox10 conditional mutants exhibit severe neuropathy with dysmyelination, abnormal Remak bundles, muscle atrophy and paralysis. Notably, perturbing mitochondrial respiration did not cause glial cell death. In the adult central nervous system, we found no signs of demyelination, axonal degeneration or secondary inflammation. Unlike cultured oligodendrocytes, which are sensitive to COX inhibitors, post-myelination oligodendrocytes survive well in the absence of COX activity. More importantly, by in vivo magnetic resonance spectroscopy, brain lactate concentrations in mutants were increased compared with controls, but were detectable only in mice exposed to volatile anaesthetics. This indicates that aerobic glycolysis products derived from oligodendrocytes are rapidly metabolized within white matter tracts. Because myelinated axons can use lactate when energy-deprived, our findings suggest a model in which axon-glia metabolic coupling serves a physiological function.     

Glial cells express N-CAM/D2-CAM-like polypeptides in vitro

The joining together of neurites to form fascicles and the growth of axons along glial surfaces during early development suggest that neurone-neurone and neurone-glial adhesion interactions are of considerable importance for defining nerve tracts. In vitro studies have indicated that adhesion between neurones involves a glycoprotein that has been independently studied under the names of N-CAM (for neural cell adhesion molecule), D2-CAM and BSP-2 (refs 10, 11). As N-CAM/D2-CAM appears to be a homophilic ligand that binds to N-CAM/D2-CAM polypeptide on adjacent cells, this glycoprotein is potentially important in adhesion interactions between any two N-CAM/D2-CAM-expressing cells. While it has been suggested that neurone-glial adhesion involves molecules other than N-CAM/D2-CAM, it is known that N-CAM/D2-CAM antigenic determinants are expressed by glial cells in vivo and that injection of anti-N-CAM antibodies into the eye-cup of chick embryos disrupts normal patterns of neuritic apposition to glial endfeet in the developing optic stalk. Do the molecules expressed by glia share restricted antigenic determinants, or binding domains, with N-CAM/D2-CAM, or are N-CAM/D2-CAM polypeptides expressed by glia? Here we present immunocytochemical evidence which suggests that all classes of macroglia express N-CAM/D2-CAM antigenic determinants on their surfaces and immunochemical analyses which indicate that the molecules expressed by purified astrocytes are closely similar, or identical, to at least some forms of N-CAM/D2-CAM obtained from whole brain or purified neurones. However, our results also suggest that different N-CAM/D2-CAM polypeptides may be separately expressed by neurones and astrocytes.     

Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1

The capacity of the adult brain and spinal cord to repair lesions by axonal regeneration or compensatory fibre growth is extremely limited. A monoclonal antibody (IN-1) raised against NI-220/250, a myelin protein that is a potent inhibitor of neurite growth, promoted axonal regeneration and compensatory plasticity following lesions of the central nervous system (CNS) in adult rats. Here we report the cloning of nogo A, the rat complementary DNA encoding NI-220/250. The nogo gene encodes at least three major protein products (Nogo-A, -B and -C). Recombinant Nogo-A is recognized by monoclonal antibody IN-1, and it inhibits neurite outgrowth from dorsal root ganglia and spreading of 3T3 fibroblasts in an IN-1-sensitive manner. Antibodies against Nogo-A stain CNS myelin and oligodendrocytes and allow dorsal root ganglion neurites to grow on CNS myelin and into optic nerve explants. These data show that Nogo-A is a potent inhibitor of neurite growth and an IN-1 antigen produced by oligodendrocytes, and may allow the generation of new reagents to enhance CNS regeneration and plasticity.     

Ia-restricted encephalitogenic T lymphocytes mediating EAE lyse autoantigen-presenting astrocytes

T lymphocytes specific for myelin basic protein (MBP) are responsible for the cellular events leading to autoimmune disease within the central (CNS) and peripheral (PNS) nervous systems. Both in actively induced and T-cell transfer versions of experimental autoimmune encephalomyelitis (EAE) and neuritis (EAN), the autoaggressive T cells are activated outside the nervous system and reach their target tissue via the blood circulation. The target specificity of the autoaggressive T cells is impressive; T-cell lines specific for MBP predominantly home to and affect the white matter of the CNS whereas T cells specific for PNS myelin protein P2 exclusively infiltrate peripheral nerves. Having penetrated the tight blood tissue barriers, the lymphocytes seem to interact with local cells expressing the relevant autoantigen in an immunogenic form. Although the exact mechanism of target finding and destruction is unknown, studies from our laboratory have shown that astrocytes, a main component of the normal CNS glia, can actively present antigen to specific T cells. This observation suggests that astrocytes are involved in natural immune reactivity within the CNS, and that they may be involved in pathological aberrations, such as in the development of autoimmune lesions. Having studied astrocyte/T-cell interactions in more detail, we discovered that encephalitogenic T-cell lines recognizing MBP on astrocytes will subsequently proceed to kill the presenting cells. Here we report that astrocyte killing follows the rules governing 'classical' T-cell-mediated cytolysis; it is antigen-specific, restricted by antigens of the major histocompatibility complex (MHC) and apparently contact-dependent. Our data suggest that the nature of the recognized antigenic epitope determines whether or not antigen recognition is followed by killing; moreover, killing of antigen-presenting astrocytes seems to be correlated with the capacity to transfer encephalomyelitis to normal syngeneic rats.     

Reformation of long axon pathways in adult rat central nervous system by human forebrain neuroblasts.

The failure of lesioned axons to regenerate over long distances in the mammalian central nervous system (CNS) is not due to an inability of central neurons to regenerate, but rather to the non-permissive nature of the CNS tissue environment. Regenerating CNS axons, which grow well within a peripheral nerve, for example, fail to penetrate mature CNS tissue by more than about 1 mm. Recent evidence indicates that this may be due to inhibitory membrane proteins associated with CNS oligodendrocytes and myelin. We report here that human telencephalic neuroblasts implanted into the excitotoxically lesioned striatum of adult rats can escape or neutralize this inhibitory influence of the adult CNS environment and extend axons along major myelinated fibre tracts for distances of up to approximately 20 mm. The axons were seen to elongate along the paths of the striato-nigral and cortico-spinal tracts to reach the substantia nigra, the pontine nuclei and the cervical spinal cord, which are the normal targets for the striatal and cortical projection neurons likely to be present in these implants.     

神经干细胞及其临床应用潜能

近年来神经干细胞已在成年哺乳动物中的中枢神经系统中分离成功。神经干细胞的最基本特征是具有分化为神经元、星状胶质细胞和少突胶质细胞的潜能，具有自我更新能力，并足以维持整个大脑所需。神经干细胞在修复受伤神经组织及治疗神经系统退行性疾病，如帕金森病、阿尔茨海默病、和亨庭顿病等方面有很好的应用前景。但在达到临床实际应用之前仍有一系列问题需要解决，最首要的是搞清神经干细胞的分化机制。     

Structure, expression and function of a schwannoma-derived growth factor

During the development of the nervous system, cells require growth factors that regulate their division and survival. To identify new growth factors, serum-free growth-conditioned media from many clonal cell lines were screened for the presence of mitogens for central nervous system glial cells. A cell line secreting a potent glial mitogen was established from a tumour (or 'schwannoma') derived from the sheath of the sciatic nerve. The cells of the tumour, named JS1 cells, were adapted to clonal culture and identified as Schwann cells. Schwann cells secrete an autocrine mitogen and human schwannoma extracts have mitogenic activity on glial cells. Until now, neither mitogen has been purified. Here we report the purification and characterization of a mitogenic molecule, designated schwannoma-derived growth factor (SDGF), from the growth-conditioned medium of the JS1 Schwann cell line. SDGF belongs to the epidermal growth factor family, and is an autocrine growth factor as well as a mitogen for astrocytes, Schwann cells and fibroblasts.     

Guidance of optic nerve fibres by N-cadherin adhesion molecules

The dendritic branches (neurites) of developing neurons migrate along specific pathways to reach their targets. It has been suggested that this migration is guided by factors present on the surface of other neurons or glial cells. The molecular nature of such factors, however, remains to be elucidated. N-cadherin is a cell-surface glycoprotein which belongs to the cadherin family of cell-cell adhesion molecules. This adhesion molecule is expressed in various neuronal cells as well as in glial cells of the central and peripheral nervous systems in vertebrate embryos and recent immunological studies suggested that N-cadherin may play a role in guiding the migration of neurites on myotubes or astrocytes. To further examine this possibility, we used a molecular-genetic approach; that is, we examined the outgrowth of chicken embryonic optic axons on monolayer cultures of Neuro 2a or L cells transfected with the complementary DNA encoding chicken N-cadherin. The data indicate that N-cadherin is used as a guide molecule for the migration of optic axons on cell surfaces.