Astrocytes and axon regeneration in the central nervous system

The failure of axons to regenerate in the central nervous system is mainly due to inhibition by the environment, made up of astrocytes and oligodendrocytes, which surrounds regions of damage. Both cell types are inhibitory to axon regeneration, and it seems likely that each will have to be neutralised before significant axon regeneration is achieved. Axons regenerate over the surface of astrocytes grown in normal mononlayer culture but not through three-dimensional astrocyte cultures. Astrocyte cell lines have been created, some of which resemble embryonic astrocytes and form a loose tissue with extensive extracellular space which permits axon regeneration, and others which model astrocytes in the damaged brain having little extracellular space and much extracellular matrix material. There is no correlation between the inhibitory effect on axons and the expression of cell adhesion molecules, proteases, protease inhibitors, and a variety of extracellular matrix molecules. However, extracellular matrix produced by inhibitory cell lines is inhibitory to axon regeneration, while that produced by permissive cell lines is not. This difference depends on the production of a chondroitinase-sensitive proteoglycan which can block the neurite-inducing effects of laminin so that treatment of inhibitory extracellular matrix with chondroitinase renders it more permissive to axon regeneration.     

Immunocytochemical characterization of reactive optic nerve astrocytes and meningeal cells

Regeneration in the adult central nervous system (CNS) is thought to be hampered by the lesion-induced activation of astrocytes and meningeal cells and the consecutive formation of a glial scar. The substrate properties of reactive astrocytes differ significantly from their neonatal counterparts, which promote axon growth, but in spite of intensive studies the underlying molecular changes are still not fully understood. We have used two cell culture systems to compare the expression of certain surface molecules on neonatal astrocytes, reactive astrocytes and meningeal cells in vitro. Both, neonatal and reactive adult astrocytes exhibited a very similar expression of growth promoting molecules (NCAM, L1, laminin, fibronectin, DSD-1 proteoglycan) and potential inhibitors (tenascinC, chondroitin sulfate, and NG2-proteoglycan), whereas we could not detect the inhibitory keratan sulfate on either astrocyte population. In contrast, meningeal cells expressed considerable levels of keratan sulfate, but only minimal amounts of NCAM. In addition, the much higher expression of extracellular fibronectin around meningeal cells implies an excess formation of extracellular matrix (ECM). In coculture experiments, embryonic retinal ganglion cell (RGC) axons clearly avoided meningeal cells and instead preferred even reactive adult astrocytes. Our results suggest that the expression of inhibitory keratan sulfate proteoglycans together with a lack of NCAM and an excess production of ECM may be responsible for the non-permissiveness of meningeal cells. Compared to reactive astrocytes, meningeal cells are even worse a substrate for growing axons. None of the molecules investigated, however, seems to account for the different substrate properties of neonatal and reactive adult astrocytes.     

Neurite outgrowth over resting and reactive astrocytes

A classic problem in CNS fiber regeneration is that the glial scar, generated after a lesion, is not crossed by regenerating axons. We know that reactive astrocytes are important in the formation of this barrier and that the barrier is not mechanical. However, its precise nature remains unclear. To study interactions of normal and reactive astrocytes with central neurites, we have attempted to create an in vitro model of the glial scar. We found the following: (1) Cultured astrocytes, independently of their lineage, morphology, immunological type and treatment with differentiating agents, induced profuse neurite outgrowth from various kinds of embryonic CNS neurons. The outgrowth was comparable to that elicited by laminin. (2) Membranes from isomorphic gliotic tissue (induced by deafferentation or excitotoxic injury and containing a large number of reactive astrocytes), inhibited central neurite outgrowth as powerfully as myelin. Reactive astrocyte membranes from areas of anisomorphic gliosis (following penetrating trauma) were permissive for neurite outgrowth, but growth was more limited than on cultured astrocyte membranes. (3) When given a choice, growing neurites actively avoided membranes from isomorphic gliosis (similar to myelin), while they seemed to follow anisomorphic membrane boundaries and crossed unhindered into membranes of cultured astrocytes. In conclusion, reactive glia seem to contain both inhibitory and neurite promoting molecules, the proportion of which depends on the way gliosis has been generated. For isomorphic reactive astrocytes the balance is inhibitory for central neurite outgrowth, while anisomorphic reactive astrocytes probably express inhibitory components at lower levels and the growth promoting factors predominate. Overall, our observations suggest that reactive astrocytes are still the major problem for axonal regeneration in the CNS.     

Leptomeningeal cells modulate the neurite growth promoting properties of astrocytes in vitro

Leptomeningeal cells migrate into the lesion cavity after stab wounds to the adult mammalian central nervous system (CNS) and interact with astrocytes that form a new glia limitans. However, it is not known if leptomeningeal cells alter the ability of astrocytes near the lesion to support axon growth. In this study, we have used an in vitro approach to assess leptomeningeal cell-astrocyte interactions in a model that resembles the interactions of these cells in vivo. We cultured rat cortical astrocytes on top of monolayers of leptomeningeal cells or astrocytes. Differences in the morphology, neurite growth promoting properties, and expression of various extracellular matrix molecules and β1-integrin were assessed. Astrocytes acquired a long slender morphology when plated on leptomeningeal cells. Functionally, astrocytes cultured on top of leptomeningeal monolayers supported less neurite growth. Similar results were also obtained when astrocyte monolayers were treated with leptomeningeal cell-conditioned medium. Quantitative immunofluorescence labeling showed a reduction in cell surface bound laminin on astrocytes plated on leptomeningeal monolayers. Qualitative assessment of the immunofluorescence labeling showed an increase in matrix-like deposits of tenascin-C and chondroitin sulfate proteoglycan under similar culture conditions. This study provides the first direct evidence that leptomeningeal cells reduce the neurite growth promoting properties of astrocytes. These results suggest that interactions with leptomeningeal cells may 1) induce the formation of the slender astrocyte processes that form parallel to the lesion wall after penetrating injuries to the CNS; and 2) contribute along with other factors to alter astrocytes near the site of injury to a state that is less permissive for axon growth and regeneration. GLIA 19:47–57, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of plasminogen activators and type-1 plasminogen activator inhibitor by cyclic AMP and phorbol ester in rat astrocytes.

Two plasminogen activators (PAs): tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), as well as the type-1 plasminogen activator inhibitor (PAI-1) are synthesized and secreted by rat astrocytes. Preliminary studies suggest that PA activity plays a role in astrocyte development and differentiation. We have examined the regulation of the PA system by the cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in purified rat astrocyte cultures. PKA activity was increased by exposing cultured astrocytes to forskolin or dibutyryl cyclic AMP, whereas PKC activity was stimulated with phorbol-12-myristate 13-acetate (PMA). Activation of both second-messenger pathways produced a time- and dose-dependent increase in the total PA activity. However, based on SDS-PAGE/zymography we found that forskolin increased t-PA activity and reduced u-PA activity, whereas PMA treatment caused a significant increase in u-PA activity without altering t-PA activity. Reverse zymography analysis revealed that astrocyte PAI-1 activity is decreased by forskolin and increased by PMA. Together, these results demonstrate that the components of the PA system in rat astrocytes are independently and reciprocally regulated by PKA and PKC. Our findings raise the possibility that the plasminogen activator system could be involved in some of the actions of growth factors and/or neuromodulators that modulate PKC or PKA in astrocytes.     

The astrocyte/meningeal cell interface is a barrier to neurite outgrowth which can be overcome by manipulation of inhibitory molecules or axonal signalling pathways

Invading meningeal cells form a barrier to axon regeneration after damage to the spinal cord and other parts of the CNS, axons stopping at the interface between meningeal cells and astrocytes. Axon behavior was examined using an in vitro model of astrocyte/meningeal cell interfaces, created by plating aggregates of astrocytes and meningeal cells onto coverslips. At these interfaces growth of dorsal root ganglion axons attempting to grow from astrocytes to meningeal cells was blocked, but axons grew rapidly from meningeal cells onto astrocytes. Meningeal cells were examined for expression of axon growth inhibitory molecules, and found to express NG2, versican, and semaphorins 3A and 3C. Astrocytes express growth promoting molecules, including N-Cadherin, laminin, fibronectin, and tenascin-C. We treated cultures in various ways to attempt to promote axon growth across the inhibitory boundaries. Blockade of NG2 with antibody and blockade of neuropilin 2 but not neuropilin 1 both promoted axon growth from astrocytes to meningeal cells. Blockade of permissive molecules on astrocytes with N-Cadherin blocking peptide or anti beta-1 integrin had no effect. Manipulation of axonal signalling pathways also increased axon growth from astrocytes to meningeal cells. Increasing cAMP levels and inactivation of rho were both effective when the cultures were fixed in paraformaldehyde, demonstrating that their effect is on axons and not via effects on the glial cells.     

Increased axon growth through astrocyte cell lines transfected with urokinase

The ability of cells to migrate through tissues depends on their production of a variety of proteases, and the same may be true of growth cones. Urokinase (plasminogen activator) regulates much of the extracellular proteolytic activity, by activating other proteases and as a result of its own proteolytic activity. In order to evaluate the potential role of urokinase as a promoter of axon growth, we have used a plasmid expressing urokinase under a cytomegalovirus promoter to transfect an astrocyte cell line, Neu7, which we have previously shown to provide a poor environment for axon regeneration. Five transfected lines all showed greatly increased ability to promote axon regeneration in both monolayer and three-dimensional cultures. The critical change in the transfected cells was largely within the extracellular matrix, since extracellular matrix laid down by urokinase-secreting cells was more permissive to axon growth than matrix from the parent Neu7 line. The effect was due to urokinase since treatment of the transfected cells with the urokinase inhibitors B623 and B428 rendered both the cells and their matrix much less permissive to axon growth, but did not require plasminogen, since it was blocked neither by serum-free medium nor by plasmin inhibitors. GLIA 23:24–34, 1998.© 1998 Wiley-Liss, Inc.     

Immune function of astrocytes

Astrocytes are the major glial cell within the central nervous system (CNS) and have a number of important physiological properties related to CNS homeostasis. The aspect of astrocyte biology addressed in this review article is the astrocyte as an immunocompetent cell within the brain. The capacity of astrocytes to express class II major histocompatibility complex (MHC) antigens and costimulatory molecules (B7 and CD40) that are critical for antigen presentation and T-cell activation are discussed. The functional role of astrocytes as immune effector cells and how this may influence aspects of inflammation and immune reactivity within the brain follows, emphasizing the involvement of astrocytes in promoting Th2 responses. The ability of astrocytes to produce a wide array of chemokines and cytokines is discussed, with an emphasis on the immunological properties of these mediators. The significance of astrocytic antigen presentation and chemokine/cytokine production to neurological diseases with an immunological component is described.     

Neurite Outgrowth on Non-permissive Substrates In Vitro is Enhanced by Ectopic Expression of the Neural Adhesion Molecule L1 by Mouse Astrocytes

Axonal regrowth in the lesioned central nervous system (CNS) of adult mammals is, in part, prevented by non-permissive properties of glial cells and myelin. To test if ectopic expression of the neurite outgrowth promoting recognition molecule L1 will overcome these non-permissive influences and promote neurite outgrowth, L1 was expressed in astrocytes of transgenic mice using regulatory sequences of the glial fibrillary acidic protein (GFAP) gene. Northern blot analysis of different transgenic lines revealed different levels of transgenically expressed L1. Cultured astrocytes derived from transgenic animals displayed L1 immunoreactivity at the cell surface and in situ hybridization and immunocytochemical analysis of optic nerves from adult transgenic mice localized L1 expression to astrocytes. Expression of L1 protein by transgenic astrocytes was significantly upregulated in lesioned optic nerves. When mouse small cerebellar neurons or chick dorsal root ganglion neurons were cultured on cryosections of lesioned optic nerves or astrocyte monolayers from transgenic mice, respectively, neurite outgrowth was increased up to 400% on tissue sections and 50% on astrocytes compared with similar preparations from non-transgenic mice. The increase in neurite outgrowth on tissue sections or astrocyte monolayers from different transgenic lines was proportional to the different levels of L1 expression. Moreover, increased neurite outgrowth on these substrates was specifically inhibited by polyclonal L1 antibodies. In vivo , rescue of severed axons was enhanced in transgenic versus wild type animals, while regrowth of axons was slightly, but not significantly, increased. Together, our observations demonstrate that L1 promotes neurite outgrowth when expressed ectopically by astrocytes and that L1 is able to overcome, at least partially, the non-permissive substrate properties of differentiated CNS glial cells in vitro.     

Dissection of astrocyte-mediated cues in neuronal guidance and process extension

Neurites are believed to be guided by astrocyte boundaries during development. We have previously shown that in vitro astrocyte boundaries can be generated by combining two different astrocyte cell lines, one which is inhibitory to neurite outgrowth (Neu7) with one that is permissive (A7). The extracellular matrix molecules tenascin-C, chondroitin sulfate proteoglycans (CSPG) and keratan sulfate proteoglycans (KSPG) were implicated in boundary formation. We have now further addressed the roles of these molecules using additional astrocyte cell lines that differ in their potential to permit neurite extension and in their expression of extracellular matrix molecules. T34-2 and 27A1 cells are permissive to neurite extension. T34-2 cells express high amounts of tenascin-C, but very low levels of proteoglycans, while 27A1 cells express CSPG and KSPG, but very little tenascin-C. T34-2 cells formed boundaries to neurites, and these boundaries are greatly reduced in the presence of blocking antitenascin-C antiserum. The addition of the antiserum did not affect neurite extension. 27A1 cells also formed boundaries without affecting neurite extension. Chondroitinase ABC, but not keratanase, treatment reduced the boundary, suggesting that CSPG is a major boundary component. These results demonstrate that astrocyte tenascin-C and proteoglycans are distinct components of astrocyte boundaries. More importantly, these results suggest that growing neurites can be directed to their targets by astrocyte-derived guidance molecules independent of effects on process extension.     

Concentration-dependent actions of glial chondroitin sulfate on the neuritic growth of midbrain neurons

Astrocytes located in two distinct regions of midbrain differ in their neuritic growth support abilities. Midbrain neurons cultured onto astrocyte monolayers from the lateral (L) region develop long and branched neurites while neurons cultured onto astrocyte monolayers from the medial (M) region develop short or no neurites. The extracellular matrix of these astrocytes has an important role in promoting or inhibiting the growth of these neurons. Differences on the compartmental distribution, as well as on the concentration of GAGs of L and M astrocytes, may be related to their differential capacity of supporting neuritic growth. Indeed, enzymatic digestion of heparan sulfate (HS) and chondroitin sulfate (CS) chains also pointed to an important function for GAGs on axon navigation. In order to better characterize the role of CS on the growth of midbrain neurites, we treated L and M astrocyte monolayers with 1 mM of beta-D-xyloside. Under these conditions, astrocytes oversynthesized and secreted CS protein-free chains to the culture medium. M astrocytes had a significant reduction in their neuritic growth-inhibiting ability after xyloside treatment, suggesting a promoting role for soluble CS in neuritic growth. Chondroitin 4-sulfate (CS-4) added in different concentrations to M astrocyte cultures turned this glia into a permissive substrate, acting in a linear way as far as the largest neurite was concerned. However, a U-shaped dose-effect curve on neurite growth resulted from the similar treatment of L astrocytes. These results suggest that glial CS-4 could be involved in the neurite growth modulating properties of midbrain neurons in a complex concentration-dependent way.     

Central nervous system-infiltrated immune cells induce calcium increase in astrocytes via astroglial purinergic signaling

Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naïve astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell–astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca²⁺ increase in astrocytes by promoting glial release of ATP. ATP propagated Ca²⁺ elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca²⁺ increase is specifically triggered by the autoreactive CD4⁺ T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca²⁺ signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell–astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.     

Retinal neurite growth on astrocytes is not modified by extracellular matrix, anti-L1 antibody, or oligodendrocytes.

Two factors that may influence the course of axonal regeneration in the central nervous system (CNS) are extracellular matrix (ECM) and cell surface molecules that may enhance or inhibit neurite outgrowth. Whereas cultured astrocytes have been reported to be a good substratum for neurite outgrowth, there is recent evidence that cultured oligodendrocytes are inhibitory. To test the influences of 1) ECM components, 2) the L1 adhesion molecule, and 3) the inhibitory potential of mature oligodendrocytes in the astrocytic environment, we have utilized a culture system in which neurites from embryonic rat retina grow vigorously on astrocyte monolayers. The major ECM components were assembled in neonatal rat cortical astrocyte-retina co-cultures only when the medium contained serum. In electron microscopic studies of serum containing cultures, retinal neurites were seen to be related to astrocyte surfaces but rarely were found in contact with ECM; in serum-free medium the association between neurites and astrocytes was similar. In addition, the growth of neurites was vigorous whether ECM was present or absent. Presence of antibodies against the cell surface adhesion molecule L1 did not inhibit retinal neurite elongation on glial fibrillary acidic protein-positive astrocytes. When oligodendrocytes from adult rat spinal cord were combined with the astrocytes, retinal neurites grew as well on the mixed glial population as on astrocytes alone. Immunostaining for galactocerebroside showed many oligodendrocyte processes to be aligned in the direction of neurite growth, suggesting association between the two cell types. This association was verified by electron microscopy. Furthermore, retinal explants extended neurites among myelin basic protein-positive oligodendrocytes cultured without astrocytes. Thus, the astrocyte surface is a strong promoter of neurite growth from embryonic rat retina. This growth did not depend upon either ECM or the L1 adhesion molecule. Because neurites grew on astrocytes in the presence of mature oligodendrocytes or among oligodendrocytes alone, we conclude that oligodendrocytes do not inhibit neurite growth under certain conditions.     

Modulating astrogliosis after neurotrauma

Traumatic injury to the adult central nervous system (CNS) results in a rapid response from resident astrocytes, a process often referred to as reactive astrogliosis or glial scarring. The robust formation of the glial scar and its associated extracellular matrix (ECM) molecules have been suggested to interfere with any subsequent neural repair or CNS axonal regeneration. A series of recent in vivo experiments has demonstrated a distinct inhibitory influence of the glial scar on axonal regeneration. Here we review several experimental strategies designed to elucidate the roles of astrocytes and their associated ECM molecules after CNS damage, including astrocyte ablation techniques, transgenic approaches, and alterations in the deposition of the ECM. In the short term, mediators that modulate the inflammatory mechanisms responsible for eliciting astrogliotic scarring hold strong potential for establishing a favorable environment for neuronal repair. In the future, the conditional (inducible) genetic manipulation of astrocytes holds promise for further increasing our understanding of the functional biology of astrocytes as well as opening new therapeutic windows. Nevertheless, it is most likely that, to obtain long distance axonal regeneration within the injured adult CNS, a combinatorial approach involving different repair strategies, including but not limited to astrogliosis modulation, will be required.     

Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors

In utero alcohol exposure can lead to fetal alcohol spectrum disorders, characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. We have recently shown that stimulation of M₃ muscarinic receptors in astrocytes increases the synthesis and release of fibronectin, laminin, and plasminogen activator inhibitor‐1, causing neurite outgrowth in hippocampal neurons. As M₃ muscarinic receptor signaling in astroglial cells is strongly inhibited by ethanol, we hypothesized that ethanol may also inhibit neuritogenesis in hippocampal neurons induced by carbachol‐stimulated astrocytes. In the present study, we report that the effect of carbachol‐stimulated astrocytes on hippocampal neuron neurite outgrowth was inhibited in a concentration‐dependent manner (25–100 mM) by ethanol. This effect was because of the inhibition of the release of fibronectin, laminin, and plasminogen activator inhibitor‐1. Similar effects on neuritogenesis and on the release of astrocyte extracellular proteins were observed after the incubation of astrocytes with carbachol in the presence of 1‐butanol, another short‐chain alcohol, which like ethanol is a competitive substrate for phospholipase D, but not by tert‐butanol, its analog that is not a substrate for this enzyme. This study identifies a potential novel mechanism involved in the developmental effects of ethanol mediated by the interaction of ethanol with cell signaling in astrocytes, leading to an impairment in neuron–astrocyte communication. © 2010 Wiley‐Liss, Inc.     

Endothelin B receptors are expressed by astrocytes and regulate astrocyte hypertrophy in the normal and injured CNS

The ability of mammalian central nervous system (CNS) neurons to survive and/or regenerate following injury is influenced by surrounding glial cells. To identify the factors that control glial cell function following CNS injury, we have focused on the endothelin B receptor (ET(B)R), which we show is expressed by the majority of astrocytes that are immunoreactive for glial acid fibrillary protein (GFAP) in both the normal and crushed rabbit optic nerve. Optic nerve crush induces a marked increase in ET(B)R and GFAP immunoreactivity (IR) without inducing a significant increase in the number of GFAP-IR astrocytes, suggesting that the crush-induced astrogliosis is due primarily to astrocyte hypertrophy. To define the role that endothelins play in driving this astrogliosis, artificial cerebrospinal fluid (CSF), ET-1 (an ET(A)R and ET(B)R agonist), or Bosentan (a mixed ET(A)R and ET(B)R antagonist) were infused via osmotic minipumps into noninjured and crushed optic nerves for 14 days. Infusion of ET-1 induced a hypertrophy of ET(B)R/GFAP-IR astrocytes in the normal optic nerve, with no additional hypertrophy in the crushed nerve, whereas infusion of Bosentan induced a significant decrease in the hypertrophy of ET(B)R/GFAP-IR astrocytes in the crushed but not in the normal optic nerve. These data suggest that pharmacological blockade of astrocyte ET(B)R receptors following CNS injury modulates glial scar formation and may provide a more permissive substrate for neuronal survival and regeneration.     

Type 1 astrocytes and oligodendrocyte-type 2 astrocyte glial progenitors migrate toward distinct molecules

During central nervous system (CNS) development, glial precursors proliferate in subventricular zones and then migrate throughout the CNS to adopt their final destinations and differentiate into various types of mature glial cells. Although several growth factors promoting the proliferation and/or differentiation of glial precursors have been identified, very little is known about the nature of signals that guide glial cell migration in the CNS. Therefore, we have investigated whether polypeptide growth factors and/or extracellular matrix molecules may mediate the migration of two major glial cell types, type 1 astrocytes and oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells. We show that, in a microchemotaxis chamber assay, type 1 astrocytes move toward laminin and complement-derived C5a. Astrocyte migration toward laminin is inhibited by a laminin-specific pentapeptide, YIGSR-NH2. In contrast, O-2A progenitors migrate toward platelet-derived growth factor (PDGF), which also functions as a mitogen for these cells. Using a new method to simultaneously assay migration and DNA synthesis, we also demonstrate that O-2A progenitors can migrate toward PDGF even when DNA replication is inhibited with an antimitotic agent. Thus, migration of different types of glial cells can be induced in vitro by specific signaling molecules, which are present in the developing brain and may stimulate migration of glial cells prior to CNS myelination.     

Protein kinase C mediates neurite guidance at an astrocyte boundary

During development, astrocytes play an active role in directing axons to their final targets. This guidance has been attributed in part to the increased expression of guidance molecules, such as tenascin-C and chondroitin sulfate proteoglycans, by boundary-forming astrocytes. We have previously used a culture model of astrocyte boundaries to demonstrate that neurites growing on permissive astrocytes alter their trajectory as they encounter less-permissive astrocytes. The present study investigated the role of the protein kinase C (PKC) family of signal transduction molecules in this form of axonal guidance. Neurons were plated onto mixed astrocyte monolayers in the presence of agents that either downregulate the phorbol ester-sensitive PKC isoforms or inhibit PKC. Both downregulation and inhibition of PKC increased the percentage of neurons that crossed onto the nonpermissive astrocytes. On astrocyte monolayers, phorbol ester modulation of PKC but not PKC inhibitors resulted in a decrease in overall neurite extension. PKC inhibitors also caused a similar alteration in the neuronal response to cell-free boundaries, at concentrations that did not inhibit neurite extension. Thus, phorbol-ester-sensitive PKC isoforms direct the guidance of neurites by astrocyte-derived matrix molecules.     

Regeneration of axons in the visual system

This review will describe the unique advantages that are offered by the visual system of mammals and other vertebrates for studying the regenerative responses of the central nervous system (CNS) to injury, and recent insights provided by such studies. In the mouse and rat visual system a variety of experimental paradigms promote survival of retinal ganglion cells (RGC) and optic nerve regeneration, probably through stimulation by neurotrophic factors (NTF) either directly, or indirectly through retinal astrocyte/Müller cell intermediary activation. NTF induce disinhibition of axon growth through regulated intramembranous proteolysis of p75NTR, and the inactivation of RhoA and EGFR signalling. The concomitant release of metalloproteinases (MMP) and plasminogen activators from RGC axons, and tissue inhibitors of metalloproteinases from optic nerve glia repress scarring and thereby reduce titres of scar-derived inhibitory ligands expressed in the wound. MMP also degrade myelin-derived inhibitory ligands along regenerating axon trajectories after regulated release from glia at the growing front of regenerating RGC axons. Optic nerve transection induces apoptosis of RGC which is blocked by anti-apoptotic regimes and thus, in combination with blockers of axon-growth inhibitory signalling and promoters of axon growth may be a therapeutic formula for promoting sustained axon regeneration. All these findings in the visual system are translatable to the CNS as a whole and thus strategies that successfully promote visual axon regeneration will be equally effective elsewhere in the CNS. Future developments likely to advance the field of regenerative research include a greater understanding of phylogenetic differences in the response of the CNS to injury, the role of NTF, cAMP, EGFR, glia/neuron interactions in disinhibiting and promoting axon growth, the control of neuron death, and effective drug delivery.