Martin Berry

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.

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.

Hippocampal FGF-2 and FGFR1 mRNA expression in major depression, schizophrenia and bipolar disorder

INTRODUCTIONnFGF-2 is important for stem cell proliferation, neocortical development and adult neuronal survival and growth. Reduced frontal cortical FGF-2 expression is described in major depression and is attenuated by antidepressants. We determined the distribution of hippocampal FGF-2 and its receptor (FGFR1) mRNA in post-mortem brains of people who suffered from major depression, bipolar disorder and schizophrenia and those of controls.nnnMETHODSnFGF-2 and FGFR1 mRNA were measured within hippocampal CA1, CA4 regions and the dentate gyrus (DG), using in situ hybridization. Within hippocampal regions, cellular staining was compared between diagnostic groups, using repeated measures analysis of variance.nnnRESULTSnThe density of FGF-2 mRNA+ cells in CA4 was reduced in depression compared to controls. The percentage of FGFR1 mRNA+ cells was higher in depression (CA1 and CA4) and schizophrenia (CA4) than in controls. FGFR1 mRNA expression was higher in depression than in the other groups in CA1, CA4 and DG. Overall FGF-2 mRNA expression was higher in DG than in CA1 and CA4.nnnCONCLUSIONSnWe found raised measures of FGFR1 mRNA+ in major depression and, less so, in schizophrenia, along with reduced FGF-2 mRNA density in depression. Perturbations of FGF regulation could be relevant to the pathogenesis of both disorders as FGF-2 and FGFR1 are implicated in normal hippocampal synaptology, stem cell recruitment, and connectivity, and are modulated by corticosteroids.

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.

Oligodendrocytes and the control of myelination in vivo: new insights from the rat anterior medullary velum.

The rat anterior medullary velum (AMV) is representative of the brain and spinal cord, overall, and provides an almost two‐dimensional preparation for investigating axon‐glial interactions in vivo. Here, we review some of our findings on axon‐oligodendrocyte unit relations in our adult, development, and injury paradigms: (1) adult oligodendrocytes are phenotypically heterogeneous, conforming to Del Rio Hortegas types I–IV, whereby differences in oligodendrocyte morphology, metabolism, myelin sheath radial and longitudinal dimensions, and biochemistry correlate with the diameters of axons in the unit; (2) oligodendrocytes derive from a common premyelinating oligodendrocyte phenotype, and divergence of types I–IV is related to the age they emerge and the presumptive diameter of axons in the unit; (3) during myelination, axon‐oligodendrocyte units progress through a sequence of maturation phases, related to axon contact, ensheathment, establishment of internodal myelin sheaths, and finally the radial growth and compaction of the myelin sheath; (4) we provide direct in vivo evidence that platelet‐derived growth factor‐AA (PDGF‐AA), fibroblast growth factor (FGF‐2), and insulin‐like growth factor‐I (IGF‐I) differentially regulate these events, by injecting the growth factors into the cerebrospinal fluid of neonatal rat pups; (5) in lesioned adult AMV, transected central nervous system (CNS) axons regenerate through the putatively inhibitory environment of the glial scar, but remyelination by oligodendrocytes is incomplete, indicating that axon‐oligodendrocyte interactions are defective; and (6) in the adult AMV, cells expressing the NG2 chondroitin sulphate have a presumptive adult oligodendrocyte progenitor antigenic phenotype, but are highly complex cells and send processes to contact axolemma at nodes of Ranvier, suggesting they subserve a specific perinodal function. Thus, axons and oligodendrocyte lineage cells form interdependent functional units, but oligodendrocyte numbers, differentiation, phenotype divergence, and myelinogenesis are governed by axons in the units, mediated by growth factors and contact‐dependent signals. J. Neurosci. Res. 59:477–488, 2000

Astrocyte associations with nodes of Ranvier: ultrastructural analysis of HRP-filled astrocytes in the mouse optic nerve

SummaryAstrocytes are implicated in the function of nodes of Ranvier because their perinodal processes form contacts with the axonal membrane at nodes. We have filled astrocytes iontophoretically with horseradish peroxidase in the intact mouse optic nerve to resolve the precise relationship between perinodal processes and astrocyte three dimensional structure. We confirm that nodal contacts were formed either by single processes which almost completely enveloped nodes, or by delicate, finger-like projections from larger processes which made discrete nodal contacts. A single perinodal process can form multiple contacts with a node and nodes were contacted by processes from more than one astrocyte. Perinodal processes emanated from larger processes, which terminated as end-feet on blood vessels and at the pia, as well as collateral branches which subsequently ended at nodes; these latter may specifically subserve nodes. Perinodal contacts were also formed directly by the soma and cytoplasmic expansions of the cell body. Both primary processes and collateral branches formed multiple associations with nodes which often appeared in clusters. Thus, all astrocytes formed multiple contacts with nodes, blood vessels and the subpial glia limitans. We conclude that perinodal processes are not formed by a specialized astrocyte in the mouse optic nerve.

Cytology and lineage of NG2-positive glia.

We present evidence that NG2+ glia are an integral part of an oligodendrocyte/synantocyte (OS) lineage stream the progenitors of which begin to produce both glial phenotypes at about birth. The NG2 CSPG is differentially distributed within the OS lineage, being expressed in progenitors and synantocytes but not in oligodendrocytes. All cells in the OS lineage, except the primordial stem cells, express O4. The oligodendrocyte line reacts with CD9, but synantocytes are CD9−. Nonetheless, synantocytes are morphologically complex and specialised glia which contact axolemma in myelinated fibres at nodes of Ranvier and synaptic terminals, and form >99% of all NG2+ glia in the adult CNS. Thus, the other NG2+ phenotype, the adult oligodendrocyte progenitor cell (AOPC), constitutes a small population of <1% of all NG2+ glia in the mature CNS. AOPC are a heterogeneous set of cells probably originating from multiple sources which, by definition, produce oligodendrocytes in the adult to replace loss after trauma, demyelination and normal ‘wear and tear’. The definitive functions of synantocytes remain undefined.

Effective gene delivery to adult neurons by a modified form of electroporation

Non-viral methods of transfection of cDNAs into adult neurons and other post-mitotic cells are generally very inefficient. However, the recent development of Nucleofector technology developed by Amaxa Biosystems allows direct delivery of cDNAs into the nucleus, enabling transfection of non-dividing cells. In this study, we describe a reliable method for culturing large numbers of retinal cells from adult rats and using Nucleofection, we were able to transfect cDNA-encoding GFP (jellyfish green fluorescent protein) into retinal ganglion cells (RGCs) with relatively high efficiency (up to 28%). Neuronal GFP expression was observed within 18 h and continued for up to 14 days. This compares with values up to 60% of RGCs expressing GFP following infection with an HSV-1 vector. Adult rat dorsal root ganglion (DRG) neurons were also successfully transfected. Thus, in summary, Nucleofection provides the possibility for a fast and efficient method for cDNA delivery and study of gene function in adult mammalian neurons.

Fibroblast growth factor-2 inhibits myelin production by oligodendrocytes in vivo

Fibroblast growth factor-2 (FGF-2) controls in part the timely differentiation of oligodendrocytes into the myelin-producing cells of the CNS. However, although differentiated oligodendrocytes express FGF receptors (R), the effect of FGF-2 on myelin-producing oligodendrocytes in vivo was unknown. In the present study, we show that delivery of FGF-2 into the cerebrospinal fluid of anaesthetized rat pups, aged postnatal day (P) 6 to 9, induced a severe loss of myelin in the caudal anterior medullary velum (AMV). Furthermore, we show that the caudal AMV was myelinated at the time of treatment, so the effects of FGF-2 represent a loss of myelin and not delayed differentiation. This was confirmed by injection of platelet-derived growth factor-AA (PDGF-AA), a factor known to affect the differentiation of PDGF-alphaR expressing oligodendrocyte progenitors, but which did not induce myelin loss in the caudal AMV and did not affect differentiated oligodendrocytes, which do not express PDGF-alphaR. Compared to controls treated with saline or PDGF-AA, FGF-2 induced an accumulation of PLP protein and MBP mRNA within the somata of myelin-producing oligodendrocytes. The results indicate that FGF receptor signalling disrupts myelin production in differentiated oligodendrocytes in vivo and interrupted the transport of myelin-related gene products from the oligodendrocyte cell body to their myelin sheaths.