Abrogation of ß-catenin signaling in oligodendrocyte precursor cells reduces glial scarring and promotes axon regeneration after CNS injury.

When the brain or spinal cord is injured, glial cells in the damaged area undergo complex morphological and physiological changes resulting in the formation of the glial scar. This scar contains reactive astrocytes, activated microglia, macrophages and other myeloid cells, meningeal cells, proliferating oligodendrocyte precursor cells (OPCs), and a dense extracellular matrix. Whether the scar is beneficial or detrimental to recovery remains controversial. In the acute phase of recovery, scar-forming astrocytes limit the invasion of leukocytes and macrophages, but in the subacute and chronic phases of injury the glial scar is a physical and biochemical barrier to axonal regrowth. The signals that initiate the formation of the glial scar are unknown. Both canonical and noncanonical signaling Wnts are increased after spinal cord injury (SCI). Because Wnts are important regulators of OPC and oligodendrocyte development, we examined the role of canonical Wnt signaling in the glial reactions to CNS injury. In adult female mice carrying an OPC-specific conditionally deleted ß-catenin gene, there is reduced proliferation of OPCs after SCI, reduced accumulation of activated microglia/macrophages, and reduced astrocyte hypertrophy. Using an infraorbital optic nerve crush injury, we show that reducing ß-catenin-dependent signaling in OPCs creates an environment that is permissive to axonal regeneration. Viral-induced expression of Wnt3a in the normal adult mouse spinal cord induces an injury-like response in glia. Thus canonical Wnt signaling is both necessary and sufficient to induce injury responses among glial cells. These data suggest that targeting Wnt expression after SCI may have therapeutic potential in promoting axon regeneration.

Entrapment via synaptic-like connections between NG2 proteoglycan+ cells and dystrophic axons in the lesion plays a role in regeneration failure after spinal cord injury.

NG2 is purportedly one of the most growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) produced after spinal cord injury. Nonetheless, once the severed axon tips dieback from the lesion core into the penumbra they closely associate with NG2+ cells. We asked if proteoglycans play a role in this tight cell-cell interaction and whether overadhesion upon these cells might participate in regeneration failure in rodents. Studies using varying ratios of CSPGs and adhesion molecules along with chondroitinase ABC, as well as purified adult cord-derived NG2 glia, demonstrate that CSPGs are involved in entrapping neurons. Once dystrophic axons become stabilized upon NG2+ cells, they form synaptic-like connections both in vitro and in vivo. In NG2 knock-out mice, sensory axons in the dorsal columns dieback further than their control counterparts. When axons are double conditioned to enhance their growth potential, some traverse the lesion core and express reduced amounts of synaptic proteins. Our studies suggest that proteoglycan-mediated entrapment upon NG2+ cells is an additional obstacle to CNS axon regeneration.

Atypical protein kinase C and Par3 are required for proteoglycan-induced axon growth inhibition.

When the CNS is injured, damaged axons do not regenerate. This failure is due in part to the growth-inhibitory environment that forms at the injury site. Myelin-associated molecules, repulsive axon guidance molecules, and extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs) found within the glial scar inhibit axon regeneration but the intracellular signaling mechanisms triggered by these diverse molecules remain largely unknown. Here we provide biochemical and functional evidence that atypical protein kinase C (PKCζ) and polarity (Par) complex proteins mediate axon growth inhibition. Treatment of postnatal rat neurons in vitro with the NG2 CSPG, a major component of the glial scar, activates PKCζ, and this activation is both necessary and sufficient to inhibit axonal growth. NG2 treatment also activates Cdc42, increases the association of Par6 with PKCζ, and leads to a Par3-dependent activation of Rac1. Transfection of neurons with kinase-dead forms of PKCζ, dominant-negative forms of Cdc42, or mutant forms of Par6 that do not bind to Cdc42 prevent NG2-induced growth inhibition. Similarly, transfection with either a phosphomutant Par3 (S824A) or dominant-negative Rac1 prevent inhibition, whereas expression of constitutively active Rac1 inhibits axon growth on control surfaces. These results suggest a model in which NG2 binding to neurons activates PKCζ and modifies Par complex function. They also identify the Par complex as a novel therapeutic target for promoting axon regeneration after CNS injury.

The RE1 binding protein REST regulates oligodendrocyte differentiation.

The RE1-silencing transcription factor (REST) represses the expression of neuronal-specific genes in non-neuronal cells by recruiting histone deacetylases (HDACs) and other histone modifying and chromatin remodeling proteins to the DNA. REST regulation of the expression of neuronal genes is required for the orderly developmental transition from a neuroepithelial stem cell to a functional neuron. Here, we examined the expression and function of REST in neonatal rat oligodendrocyte precursor cells (OPCs). OPCs develop from the same neuroepithelial stem cells as neurons, can be reprogrammed to act as neural stem-like cells in vitro, and require HDAC-mediated gene repression to develop into mature oligodendrocytes. We show that OPCs express functional REST protein and that REST interacts with several neuronal-specific genes whose expression is repressed in OPCs. REST transcript and protein expression increased fourfold during the first 48 h of oligodendrocyte differentiation. During this differentiation, the expression of RE1 containing neuronal genes further decreased as the transcription of oligodendrocyte-specific genes was activated. Expression of a dominant-negative form of REST in OPCs prevented the cells from developing into mature MBP-positive oligodendrocytes. Rather, the cells began to develop a neuronal phenotype characterized by increased expression of neuronal proteins, transcription factors, and cell-type-specific marker antigens. REST overexpression promoted the development of O4-positive pre-oligodendrocytes from OPCs. Together, these results show that REST function is required for the differentiation of OPCs into oligodendrocytes. By regulating the expression of neuronal genes, REST may also regulate the phenotypic plasticity of OPCs.

Role of chondroitin sulfate proteoglycans in axonal conduction in Mammalian spinal cord.

Chronic unilateral hemisection (HX) of the adult rat spinal cord diminishes conduction through intact fibers in the ventrolateral funiculus (VLF) contralateral to HX. This is associated with a partial loss of myelination from fibers in the VLF (Arvanian et al., 2009). Here, we again measured conduction through the VLF using electrical stimulation while recording the resulting volley and synaptic potentials in target motoneurons. We found that intraspinal injection of chondroitinase-ABC, known to digest chondroitin sulfate proteoglycans (CSPGs), prevented the decline of axonal conduction through intact VLF fibers across from chronic T10 HX. Chondroitinase treatment was also associated with behavior suggestive of an improvement of locomotor function after chronic HX. To further study the role of CSPGs in axonal conduction, we injected three purified CSPGs, NG2 and neurocan, which increase in the vicinity of a spinal injury, and aggrecan, which decreases, into the lateral column of the uninjured cord at T10 in separate experiments. Intraspinal injection of NG2 acutely depressed axonal conduction through the injected region in a dose-dependent manner. Similar injections of saline, aggrecan, or neurocan had no significant effect. Immunofluorescence staining experiments revealed the presence of endogenous and exogenous NG2 at some nodes of Ranvier. These results identify a novel acute action of CSPGs on axonal conduction in the spinal cord and suggest that antagonism of proteoglycans reverses or prevents the decline of axonal conduction, in addition to stimulating axonal growth.

Antibodies against the NG2 proteoglycan promote the regeneration of sensory axons within the dorsal columns of the spinal cord.

The NG2 chondroitin sulfate proteoglycan inhibits axon growth in vitro. Levels of NG2 increase rapidly in the glial scars that form at sites of CNS injury, suggesting that NG2 may inhibit axon regeneration. To determine the functions of NG2, we infused mixtures of neutralizing or non-neutralizing anti-NG2 monoclonal antibodies into the dorsally transected adult rat spinal cord and analyzed the regeneration of ascending mechanosensory axons anatomically. At 1 week after injury, ascending sensory axons in control animals terminated caudal to the lesion within an area containing dense deposits of NG2 immunoreactivity. In animals treated with the neutralizing anti-NG2 antibodies, labeled axons penetrated the caudal border of the lesion and grew into and beyond the lesion center. The low intrinsic growth capacity of adult neurons may also limit the ability of damaged axons to regenerate. To enhance growth, we combined antibody treatment with a peripheral nerve conditioning lesion. After a conditioning lesion and treatment with control, non-neutralizing antibodies, many sensory axons grew into the lesion core. These axons did not grow past the rostral border of the lesion; rather, they grew along the dorsal surface of the spinal cord and within any remaining pieces of the dorsal roots. In contrast, combining a peripheral nerve conditioning lesion with neutralizing anti-NG2 antibodies resulted in sensory axon regeneration past the glial scar and into the white matter rostral to the injury site. The combinatorial approach used here that neutralizes extrinsic inhibition and increases intrinsic growth results in anatomically correct axon regeneration, a prerequisite for functional recovery.

Multiple regions of the NG2 proteoglycan inhibit neurite growth and induce growth cone collapse.

The NG2 chondroitin sulfate proteoglycan, an integral membrane proteoglycan, inhibits axon growth from cerebellar granule neurons and dorsal root ganglia (DRG) neurons in vitro. The extracellular domain of the NG2 core protein contains three subdomains: an N-terminal globular domain (domain 1), a central extended domain that has the sites for glycosaminoglycan (GAG) attachment (domain 2), and a juxtamembrane domain (domain 3). Here, we used domain-specific fusion proteins and antibodies to map the inhibitory activity within the NG2 core protein. Fusion proteins encoding domain 1 (D1-Fc) or domain 3 (D3-Fc) of NG2 inhibited axon growth from cerebellar granule neurons when the proteins were substrate-bound. These proteins also induced growth cone collapse from newborn DRG neurons when added to the culture medium. Domain 2 only inhibited axon growth when the GAG chains were present. Neutralizing antibodies directed against domain 1 or 3 blocked completely the inhibition from substrates coated with D1-Fc or D3-Fc. When the entire extracellular domain of NG2 was used as a substrate, however, both neutralizing antibodies were needed to reverse completely the inhibition. When NG2 was expressed on the surface of HEK293 cells, the neutralizing anti-D1 antibody was sufficient to block the inhibition, whereas the anti-D3 antibody had no effect. These results suggest that domains 1 and 3 of NG2 can inhibit neurite growth independently. These inhibitory domains may be differentially exposed depending on whether NG2 is presented as an integral membrane protein or as a secreted protein associated with the extracellular matrix.

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-1a, 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.

tPA-mediated generation of plasmin is catalyzed by the proteoglycan NG2.

Paralysis resulting from spinal cord injury is devastating and persistent. One major reason for the inability of the body to heal this type of injury ensues from the local increase of glial cells leading to the formation of a glial scar, and the upregulation of chondroitin sulfate proteoglycans (CSPGs) at the site of injury through which axons are unable to regenerate. Experimental approaches to overcome this problem have accordingly focused on reducing the inhibitory properties of CSPGs, for example by using chondroitinase to remove the sugar chains and reduce the CSPGs to their core protein constituents, although this step alone does not provide dramatic benefits as a monotherapy. Using in vitro and in vivo approaches, we describe here a potentially synergistic therapeutic opportunity based on tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen (plg) into the active protease plasmin. We show that tPA and plg both bind to the CSPG protein NG2, which functions as a scaffold to accelerate the tPA-driven conversion of plg to plasmin. The binding occurs via the tPA and plg kringle domains to domain 2 of the NG2 CSPG core protein, and is enhanced in some settings after chondroitinase-mediated removal of the NG2 proteoglycan side chains. Once generated, plasmin then degrades NG2, both in an in vitro setting using recombinant protein, and in vivo models of spinal cord injury. Our finding that the tPA and plg binding is in some instances more efficient after exposure of the NG2 proteoglycan to chondroitinase treatment suggests that a combined therapeutic approach employing both chondroitinase and the tPA/plasmin proteolytic system could be of significant benefit in promoting axonal regeneration through glial scars after spinal cord injury.

Sensory afferents regenerated into dorsal columns after spinal cord injury remain in a chronic pathophysiological state.

Axon regeneration after experimental spinal cord injury (SCI) can be promoted by combinatorial treatments that increase the intrinsic growth capacity of the damaged neurons and reduce environmental factors that inhibit axon growth. A prior peripheral nerve conditioning lesion is a well-established means of increasing the intrinsic growth state of sensory neurons whose axons project within the dorsal columns of the spinal cord. Combining such a prior peripheral nerve conditioning lesion with the infusion of antibodies that neutralize the growth inhibitory effects of the NG2 chondroitin sulfate proteoglycan promotes sensory axon growth through the glial scar and into the white matter of the dorsal columns. The physiological properties of these regenerated axons, particularly in the chronic SCI phase, have not been established. Here we examined the functional status of regenerated sensory afferents in the dorsal columns after SCI. Six months post-injury, we located and electrically mapped functional sensory axons that had regenerated beyond the injury site. The regenerated axons had reduced conduction velocity, decreased frequency-following ability, and increasing latency to repetitive stimuli. Many of the axons that had regenerated into the dorsal columns rostral to the injury site were chronically demyelinated. These results demonstrate that regenerated sensory axons remain in a chronic pathophysiological state and emphasize the need to restore normal conduction properties to regenerated axons after spinal cord injury.

NG2: a component of the glial scar that inhibits axon growth.

NG2 is a high-molecular-weight chondroitin sulphate proteoglycan found on the surfaces of oligodendrocyte precursor cells (OPCs). Here we review the history and biology of OPCs with an emphasis on their functions after experimentally induced CNS injury. Injury to brain or spinal cord results in the rapid accumulation of NG2-expressing OPCs in the glial scar that forms at the injury site. The glial scar is considered a biochemical and physical barrier to successful axon regeneration and the functional properties of NG2 suggest that it, along with other macromolecules, participates in the creation of this growth-inhibitory environment. NG2 is an important target for therapies designed to promote successful axon regrowth.

Two separate metalloproteinase activities are responsible for the shedding and processing of the NG2 proteoglycan in vitro.

A high proportion of NG2 in the adult rat spinal cord is saline-soluble and migrates slightly faster than intact NG2 on SDS-PAGE, suggesting that it represents the shed ectodomain of NG2. In the injured cerebral cortex, much of the overall increase in NG2 is due to the saline-soluble (shed), rather than the detergent-soluble (intact), form. Hydroxamic acid metalloproteinase inhibitors, but not TIMPs, were able to prevent NG2 shedding in oligodendrocyte precursor cells (OPCs) in vitro. The generation of another truncated form of NG2 was, however, sensitive to TIMP-2 and TIMP-3. Two observations suggest that NG2 is involved in PDGF signaling in OPCs: the rate of NG2 shedding increased with cell density and NG2 expression was increased in the absence of PDGF. Ectodomain shedding converts NG2 into a diffusible entity able to interact with the growth cone, and we suggest that this release is likely to enhance its axon growth-inhibitory activity.

Inhibition of axon growth by oligodendrocyte precursor cells.

The glial scar that forms at the site of injury is thought to be a biochemical and physical barrier to successful regeneration, although the molecules responsible for this barrier function are not well understood. Glia scars contain large numbers of oligodendrocyte precursor cells (OPCs) and these cells can produce several different growth-inhibitory chondroitin sulfate proteoglycans (CSPGs), including NG2, neurocan, and phosphacan. Here, we used membrane-based assays to show that the surface of OPCs is both nonpermissive and inhibitory for neurite outgrowth. Inhibition of growth by OPC is reversed by treatment with antibodies against the NG2 CSPG and the expression of NG2 is sufficient to change a growth-permissive cell surface to a nonpermissive surface. These result suggest that the OPCs that accumulate rapidly at sites of CNS injury can contribute to the creation of an environment that inhibits nerve regeneration and that NG2 is a necessary feature of that environment.

NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.

Glial progenitor cells of the developing CNS committed to the oligodendrocyte lineage (OPCs) express the chondroitin sulfate proteoglycan, NG2. A proportion of OPCs fail to differentiate past the stage at which they express NG2 and the lipid antigen O4 and persist in the adult CNS in a phenotypically immature form. However, the physiological function of NG2(+) cells in the adult CNS is unknown. Using antibodies against NG2 we show that NG2 is expressed by a distinct cell population in the mature CNS with the homogeneous antigenic phenotype of oligodendrocyte progenitors. The morphology of NG2(+) OPCs varies from region to region, reflecting the different structural environments, but they appear to represent a homogeneous population within any one gray or white matter region. A study of nine CNS regions showed that NG2(+) OPCs are numerous throughout the CNS and numbers in the white matter are only 1.5 times that in the gray. Whereas the ratio of OPCs to myelinating oligodendrocytes in the spinal cord gray and white matter approximates 1:4, gray matter regions of the forebrain have a 1:1 ratio, a phenomenon that will have consequences for oligodendrocyte replacement following demyelination. BrdU incorporation experiments showed that NG2(+) cells are the major dividing cell population of the adult rat CNS. Since very little apoptosis was detected and BrdU became increasingly present in oligodendrocytes after a 10-day pulse chase, with a concomitant decrease in NG2(+) BrdU incorporating cells, we suggest that the size of the oligodendrocyte population may actually increase during adult life.

Oligodendrocyte precursor cells: reactive cells that inhibit axon growth and regeneration.

Oligodendrocyte precursor cells (OPCs) are a newly recognized glial component of the adult central nervous system of unknown function. Antibodies against the NG2 chondroitin sulfate proteoglycan have been useful tools to identify these cells in intact tissue. Here we review studies that show that OPCs react to several types of experimentally induced brain injury. Injury stimulates OPCs to re-enter the cell cycle, divide, and accumulate at the site of damage. OPCs, together with microglia and astrocytes, form the glial scar. Glial scars are thought to inhibit or prevent axonal regeneration and reactive OPCs contribute to this inhibition by producing growth-inhibiting chondroitin sulfate proteoglycans, particularly NG2. In developing animals, NG2 is found in areas, such as the perinotochordal mesenchyme, that are avoided by growing motor and sensory axons. Within the developing CNS, NG2-expressing cells surround the developing optic chiasm and tract and separate it from the overlying diencephalon. Thus, NG2-expressing cells are well positioned to inhibit axonal growth from developing as well as regenerating neurons.

Expression and glycanation of the NG2 proteoglycan in developing, adult, and damaged peripheral nerve.

We have investigated expression of the axon growth-inhibitory proteoglycan NG2 in peripheral nerve. In the adult, NG2 was present on endoneurial and perineurial fibroblasts, but not on Schwann cells. At birth, peripheral nerve NG2 was heavily glycanated, but was much less so in the adult. In vitro, sciatic nerve fibroblasts also produced heavily glycanated NG2. After peripheral nerve injury in rats and humans, an accumulation of NG2-positive cells was observed at the injury site. In the rat, there was an increase in NG2 glycanation for at least 2 weeks following injury. In mixed cultures of Schwann cells and peripheral nerve fibroblasts, the axons preferred to grow on the Schwann cells and seldom crossed onto the fibroblasts. Three-dimensional cultures of sciatic nerve fibroblasts were inhibitory to the growth of dorsal root ganglion axons. Inhibition of proteoglycan synthesis made the cells more permissive. NG2 may play a part in blocking axon regeneration through scar tissue in injured human peripheral nerve.

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.