Arthur Butt

Myelin-, reactive glia-, and scar-derived CNS Axon growth inhibitors: Expression, receptor signaling, and correlation with Axon regeneration

Axon regeneration is arrested in the injured central nervous system (CNS) by axon growth‐inhibitory ligands expressed in oligodendrocytes/myelin, NG2‐glia, and reactive astrocytes in the lesion and degenerating tracts, and by fibroblasts in scar tissue. Growth cone receptors (Rc) bind inhibitory ligands, activating a Rho‐family GTPase intracellular signaling pathway that disrupts the actin cytoskeleton inducing growth cone collapse/repulsion. The known inhibitory ligands include the chondroitin sulfate proteoglycans (CSPG) Neurocan, Brevican, Phosphacan, Tenascin, and NG2, as either membrane‐bound or secreted molecules; Ephrins expressed on astrocyte/fibroblast membranes; the myelin/oligodendrocyte‐derived growth inhibitors Nogo, MAG, and OMgp; and membrane‐bound semaphorins (Sema) produced by meningeal fibroblasts invading the scar. No definitive CSPG Rc have been identified, although intracellular signaling through the Rho family of G‐proteins is probably common to all the inhibitory ligands. Ephrins bind to signalling Ephs. The ligand‐binding Rc for all the myelin inhibitors is NgR and requires p75NTR for transmembrane signaling. The neuropilin (NP)/plexin (Plex) Rc complex binds Sema. Strategies for promoting axon growth after CNS injury are thwarted by the plethora of inhibitory ligands and the ligand promiscuity of some of their Rc. There is also paradoxical reciprocal expression of many of the inhibitory ligands/Rc in normal and damaged neurons, and NgR expression is restricted to a limited number of neuronal populations. All these factors, together with an incomplete understanding of the normal functions of many of these molecules in the intact CNS, presently confound interpretive acumen in regenerative studies.

Inwardly rectifying potassium channels (Kir) in central nervous system glia: a special role for Kir4.1 in glial functions

Glia in the central nervous system (CNS) express diverse inward rectifying potassium channels (Kir). The major function of Kir is in establishing the high potassium (K+) selectivity of the glial cell membrane and strongly negative resting membrane potential (RMP), which are characteristic physiological properties of glia. The classical property of Kir is that K+ flows inwards when the RMP is negative to the equilibrium potential for K+ (Ek), but at more positive potentials outward currents are inhibited. This provides the driving force for glial uptake of K+ released during neuronal activity, by the processes of “K+ spatial buffering” and “K+ siphoning”, considered a key function of astrocytes, the main glial cell type in the CNS. Glia express multiple Kir channel subtypes, which are likely to have distinct functional roles related to their differences in conductance, and sensitivity to intracellular and extracellular factors, including pH, ATP, G‐proteins, neurotransmitters and hormones. A feature of CNS glia is their specific expression of the Kir4.1 subtype, which is a major K+ conductance in glial cell membranes and has a key role in setting the glial RMP. It is proposed that Kir4.1 have a primary function in K+ regulation, both as homomeric channels and as heteromeric channels by co‐assembley with Kir5.1 and probably Kir2.0 subtypes. Significantly, Kir4.1 are also expressed by oligodendrocytes, the myelin‐forming cells of the CNS, and the genetic ablation of Kir4.1 are also expressed by Oligodendrocytes, the myelin‐forming cells of the CNS, and the genetic ablation of Kir4.1 results in severe hypomyelination. Hence, Kir, and in particular Kir4.1, are key regulators of glial functions, which in turn determine neuronal excitability and axonal conduction.

P2Y and P2X purinoceptor mediated Ca2+ signalling in glial cell pathology in the central nervous system

Activation of purinoceptors by extracellular ATP is an important component of the glial response to injury in the central nervous system (CNS). ATP has been shown to evoke raised cytosolic [Ca(2+)] in astrocytes, oligodendrocytes, and microglia, the three major glial cell types in the CNS. Glial cells express a heterogenous collection of metabotropic P2Y and ionotropic P2X purinoceptors, which respectively mobilise Ca(2+) from intracellular stores and trigger Ca(2+) influx across the plasmalemma. It is likely that different receptors have distinct roles in glial cell physiology and pathology. Our studies on optic nerve glia in situ indicate that P2Y(1) and P2Y(2/4) receptors are activated at low ATP concentrations, suggesting they are the predominant purinoceptors mediating physiological Ca(2+) signalling. Glia also express P2X(1) and P2X(3) purinoceptors, which mediate fast, rapidly desensitising current and may also be important in signalling. At high concentrations, such as occur in CNS injury, ATP induces large and prolonged increases in glial [Ca(2+)](i) with a primary role for P2Y purinoceptors and inositol trisphosphate (IP(3))-dependent release of Ca(2+) from intracellular stores. In addition, we found that high concentrations of ATP activated a significant P2X component that did not desensitise or saturate and was dependent on extracellular Ca(2+). These are characteristic properties of the P2X(7) subtype, and we provide in situ evidence that application of the P2X(7) receptor agonist benzoyl-benzoyl ATP (BzATP) evokes raised [Ca(2+)](i) in optic nerve glia, and that the dye YO-PRO-1, which passes through pore-forming P2X(7) receptors, is taken up by astrocytes, oligodendrocytes and microglia. Glia also express P2X(2) and P2X(4) receptors that are also pore-forming in the presence of sustained high ATP concentrations and which may also be important in the glial injury response. There is evidence that activation of P2 purinoceptors is a key step in triggering reactive changes in glial cells, including expression of immediate early genes, induction of extracellular signal regulated kinase and cyclooxygenase-2, synthesis of phospholipase A(2), release of arachidonic acid, production of prostaglandins and release of interleukins. We show that the ATP-mediated increase in glial [Ca(2+)](i) is potentiated by arachidonic acid and reduced by the inhibition of phospholipase A(2) inhibition. Together, the results implicate ATP as a primary signalling molecule in glial cells and indicate specific roles for P2Y and P2X purinoceptors in glial cell pathology.

Cells expressing the NG2 antigen contact nodes of Ranvier in adult CNS white matter.

The NG2 antibody, which recognises an integral membrane chondroitin sulphate, labels a significant population of cells in adult CNS white matter tracts of the rat optic nerve and anterior medullary velum (AMV). Adult NG2+ cells are highly complex with multiple branching processes and we show by EM immunocytochemistry that they extend perinodal processes, which contact nodes of Ranvier. NG2+ cells do not react to conventional immunohistochemical markers for adult glia and so we reservedly term them NG2P cells. In vitro, NG2 labels oligodendrocyte‐type‐2 astrocyte (O‐2A) progenitors that can give rise to oligodendrocytes or type‐2 astrocytes, depending on the culture medium. Thus, it is possible that NG2P cells may be derived from the same stem cells as oligodendrocytes. Interestingly, NG2+ cells identified previously in adult CNS displayed phenotypic characteristics of O‐2Aadult progenitors and it is possible that, like them, NG2P cells might retain the capacity of generating oligodendrocytes in the adult CNS. This may be an important role of NG2P cells in demyelinating diseases such as multiple sclerosis. It is significant therefore that the perinodal processes of NG2P cells contact the only sites of exposed axolemma in myelinated axons, so that NG2P cells are ideally situated to detect and respond to changes in axonal function during demyelination. A further implication of our finding is that NG2P cells may perform functions at nodes of Ranvier previously attributed to perinodal astrocytes, including the clustering and maintenance of sodium channels in the axon membrane at nodes, during development and following demyelination. GLIA 26:84–91, 1999.

Synantocytes: New functions for novel NG2 expressing glia

In the adult CNS, antibodies to the NG2 chondroitin sulphate proteoglycan (CSPG) label a large population of glia that have the antigenic phenotype of oligodendrocyte progenitor cells (OPC). However, NG2 expressing glia have the morphological phenotype of astrocytes, not OPC. We propose adult NG2 expressing glia are a distinct mature glial type, which we have called syantocytes or synantoglia after the Greek ‘to contact’, because they specifically contact neurons and axons at synapses and nodes of Ranvier, respectively. Synantocytes are highly complex cells that elaborate multiple branching processes and are an equally significant population in both white and grey matter. We provide evidence that phenotypically distinct synantocytes develop postnatally and that neither postnatal nor adult synantocytes depend on axons for their survival, indicating they respond with markedly different behaviours to the environmental cues and axonal signals that control the differentiation of OPC into oligodendrocytes. The primary response of synantocytes to changes in the CNS environment is a rapid and localised reactive gliosis. Reactive synantocytes interact intimately with astrocytes and macrophages at lesion sites, consistent with them playing a key role in the orchestration of scar formation that protects the underlying neural tissue. It is our hypothesis that synantocytes are specialised to monitor and respond to changes in the integrity of the CNS, by way of their cellular contacts, repertoire of plasmalemmal receptors and the NG2 molecule itself. To paraphrase Del Rio Hortega, we propose that synantocytes are the fifth element in the CNS, in addition to neurons, astrocytes, oligodendrocytes and microglia.

Synantocytes: the fifth element

Classic studies have recognized neurons and three glial elements in the central nervous system (CNS) – astrocytes, oligodendrocytes and microglia. The identification of novel glia that specifically express the NG2 chondroitin sulphate proteoglycan (CSPG) raises the possibility of a fifth element. Until recently, all NG2‐expressing glia were considered to be oligodendrocyte precursor cells (OPCs) that persist in the adult CNS to generate oligodendrocytes throughout life. However, this narrow view of the function of ‘NG2‐glia’ is being challenged. The majority of NG2‐expressing glia in the adult CNS are a distinct class of cells that we have called ‘synantocytes’ (from the Greek synanto for contact). Synantocytes are stellate cells, with large process arborizations, and are exquisitely related to neurons. Individual cells traverse white and grey matter and form multiple contacts with neurons, astrocytes, oligodendrocytes and myelin. Synantocytes are an integral component of the ‘tetrapartite’ synapse, and provide a potential integrative neuron‐glial communications pathway. Neuronal activity, glutamate and adenosine triphosphate (ATP) act on synantocyte receptors and evoke raised intracellular calcium. It remains to be seen whether this serves a physiological function, but synantocytes may be specialized to monitor signals from neurons and glia, and to respond to changes in the integrity of the CNS via their specific contacts and ion channel and receptor profiles. The general consequences of synantocyte activation are proliferation and phenotypic changes, resulting in glial scar formation, or regeneration of oligodendrocytes, and possibly neurons.

Astrocytes and NG2-glia: what's in a name?

Classic studies recognize two functionally segregated macroglial cell types in the central nervous system (CNS), namely astrocytes and oligodendrocytes. A third macroglial cell type has now been identified by its specific expression of the NG2 chondroitin sulphate proteoglycan (NG2‐glia). These NG2‐glia exist abundantly in both grey and white matter of the mature CNS and are almost as numerous as astrocytes. It is well established that NG2‐glia give rise to oligodendrocytes. However, the majority of NG2‐glia in the adult CNS proliferate very slowly and are non‐motile. Both astrocytes and NG2‐glia display a stellate morphology and express ion channels and receptors to neurotransmitters used by neurons. Both types of glia make intimate contacts with neurons in grey and white matter, and their functional differences and similarities are only beginning to be unravelled. Recent observations emphasize the need to examine the relationship between astrocytes and NG2‐glia, and address the question of whether they represent overlapping or two distinct glial cell populations. To be of any relevance, this classification must relate to specific functions in the neural network. At present, the balance of evidence is that NG2‐glia and astrocytes are functionally segregated populations.

NG2 proteoglycan promotes angiogenesis-dependent tumor growth in CNS by sequestering angiostatin

During embryogenesis, the NG2 proteoglycan is expressed on immature capillary vessels, but as the vessels mature they lose this expression. NG2 is up‐regulated in high‐grade gliomas, but it is not clear to what extent it contributes to malignant progression. Using a combination of high spatial and temporal resolution functional magnetic resonance imaging and histopathological analyses, we show here that overexpression of NG2 increases tumor initiation and growth rates, neovascularization, and cellular proliferation, which predisposes to a poorer survival outcome. By confocal microscopy and cDNA gene array expression profiles, we also show that NG2 tumors express lower levels of hypoxia inducible factor‐1α, vascular endothelial growth factor, and endogenous angiostatin in vivo compared with wild‐type tumors. Moreover, we demonstrate that NG2‐positive cells bind, internalize, and coimmunoprecipitate with angiostatin. These results indicate a unique role for NG2 in regulating the transition from small, poorly vascularized tumors to large, highly vascular gliomas in situ by sequestering angiostatin.

Three-dimensional morphology of astrocytes and oligodendrocytes in the intact mouse optic nerve

SummaryThe three-dimensional morphology of astrocytes and oligodendrocytes was analysed in the isolated intact mature mouse optic nerve, by correlating laser scanning confocal microscopy and camera lucida drawings of single cells, dye-filled with lysinated rhodamine dextran or horseradish peroxidase, respectively. These techniques enabled the entire process field of single dye-filled cells to be visualized in all planes and resolved the fine details of glial morphology. Morphometric analysis showed that the processes of all astrocytes had branches ending at the pial surface, on blood vessels, and freely in the nerve; branches ending in the nerve were described to end at nodes of Ranvier in the accompanying paper. Astrocytes were classified into a single morphological population in which each cell subserved multiple functions. The results of this study do not support the contention that astrocytes can be subdivided into two morphological and functional subtypes, namely type-1 and type-2, which have processes ending either at the glia limitans or at nodes, respectively. Three-dimensional analysis of oligodendrocyte units, defined as the oligodendrocyte, its processes and the axons it ensheaths, showed the provision of single myelin segments for an average of 19 nearby axons (range 12–35) with a mean internodal length of 138 μm (range 50–350 μm). Mouse optic nerve oligodendrocytes were a homogeneous population and were markedly similar to those in the rat optic nerve. The results of our analysis of oligodendrocyte morphology are consistent with the view that the number and internodal length of myelin sheaths supported by a single oligodendrocyte are related to the diameter of the ensheathed axons.

In vivo actions of fibroblast growth factor-2 and insulin-like growth factor-I on oligodendrocyte development and myelination in the central nervous system

The in vivo effects of fibroblast growth factor‐2 (FGF‐2) and insulin‐like growth factor‐I (IGF‐I) on oligodendrocytes and CNS myelination were determined in the postnatal rat anterior medullary velum (AMV) following injection of both cytokines into the cerebrospinal fluid. Either FGF‐2, IGF‐I, or saline were administered via the lateral ventricle, twice daily commencing at postnatal day (P) 6. At P9, AMV were immunohistochemically labeled with the Rip antibody, to enable analysis of the numbers of myelin sheaths and of promyelinating and myelinating oligodendrocytes; promyelinating oligodendrocytes are a recognisable immature phenotype which express myelin‐related proteins prior to forming myelin sheaths. In parallel experiments, AMV were treated for Western blot analysis to determine relative changes in expression of the myelin proteins 2′, 3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) and myelin oligodendrocyte glycoprotein (MOG), which, respectively, characterise early and late stages of myelin maturation. In FGF‐2–treated AMV, the number of promyelinating oligodendrocytes increased by 87% compared to saline‐injected controls. The numbers of myelinating oligodendrocytes and myelin sheaths were not decreased, but conspicuous unmyelinated gaps within fibre tracts were indications of retarded myelination following FGF‐2 treatment. Western blot analysis demonstrated decreased expression of CNP and a near‐total loss of MOG, confirming that FGF‐2 decreased myelin maturation. In contrast, IGF‐I had no effect on the number of promyelinating oligodendrocytes, but increased the numbers of myelinating oligodendrocytes and myelin sheaths by 100% and 93%, respectively. Western blot analysis showed that the amount of CNP was increased following IGF‐I treatment, correlating with the greater number of oligodendrocytes, but that MOG expression was lower than in controls, suggesting that the increased number of myelin sheaths in IGF‐I was not matched by increased myelin maturation. The results provide in vivo evidence that FGF‐2 and IGF‐I control the numbers of oligodendrocytes in the brain and, respectively, retard and promote myelination. J. Neurosci. Res. 57:74–85, 1999.