A role for ephrin-A5 in axonal sprouting, recovery, and activity-dependent plasticity after stroke.

Stroke causes loss of neurological function. Recovery after stroke is facilitated by forced use of the affected limb and is associated with sprouting of new connections, a process that is sharply confined in the adult brain. We show that ephrin-A5 is induced in reactive astrocytes in periinfarct cortex and is an inhibitor of axonal sprouting and motor recovery in stroke. Blockade of ephrin-A5 signaling using a unique tissue delivery system induces the formation of a new pattern of axonal projections in motor, premotor, and prefrontal circuits and mediates recovery after stroke in the mouse through these new projections. Combined blockade of ephrin-A5 and forced use of the affected limb promote new and surprisingly widespread axonal projections within the entire cortical hemisphere ipsilateral to the stroke. These data indicate that stroke activates a newly described membrane-bound astrocyte growth inhibitor to limit neuroplasticity, activity-dependent axonal sprouting, and recovery in the adult.

Glial scar borders are formed by newly proliferated, elongated astrocytes that interact to corral inflammatory and fibrotic cells via STAT3-dependent mechanisms after spinal cord injury.

Astroglial scars surround damaged tissue after trauma, stroke, infection, or autoimmune inflammation in the CNS. They are essential for wound repair, but also interfere with axonal regrowth. A better understanding of the cellular mechanisms, regulation, and functions of astroglial scar formation is fundamental to developing safe interventions for many CNS disorders. We used wild-type and transgenic mice to quantify and dissect these parameters. Adjacent to crush spinal cord injury (SCI), reactive astrocytes exhibited heterogeneous phenotypes as regards proliferation, morphology, and chemistry, which all varied with distance from lesions. Mature scar borders at 14 d after SCI consisted primarily of newly proliferated astroglia with elongated cell processes that surrounded large and small clusters of inflammatory, fibrotic, and other cells. During scar formation from 5 to 14 d after SCI, cell processes deriving from different astroglia associated into overlapping bundles that quantifiably reoriented and organized into dense mesh-like arrangements. Selective deletion of STAT3 from astroglia quantifiably disrupted the organization of elongated astroglia into scar borders, and caused a failure of astroglia to surround inflammatory cells, resulting in increased spread of these cells and neuronal loss. In cocultures, wild-type astroglia spontaneously corralled inflammatory or fibromeningeal cells into segregated clusters, whereas STAT3-deficient astroglia failed to do so. These findings demonstrate heterogeneity of reactive astroglia and show that scar borders are formed by newly proliferated, elongated astroglia, which organize via STAT3-dependent mechanisms to corral inflammatory and fibrotic cells into discrete areas separated from adjacent tissue that contains viable neurons.

A chemical screen identifies novel compounds that overcome glial-mediated inhibition of neuronal regeneration.

A major barrier to regeneration of CNS axons is the presence of growth-inhibitory proteins associated with myelin and the glial scar. To identify chemical compounds with the ability to overcome the inhibition of regeneration, we screened a novel triazine library, based on the ability of compounds to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. The screen produced four "hit compounds," which act with nanomolar potency on several different neuronal types and on several distinct substrates relevant to glial inhibition. Moreover, the compounds selectively overcome inhibition rather than promote growth in general. The compounds do not affect neuronal cAMP levels, PKC activity, or EGFR (epidermal growth factor receptor) activation. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions and potentiates regeneration of optic nerve axons after nerve crush in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury.

Conservation and divergence of vulnerability and responses to stressors between human and mouse astrocytes.

Astrocytes play important roles in neurological disorders such as stroke, injury, and neurodegeneration. Most knowledge on astrocyte biology is based on studies of mouse models and the similarities and differences between human and mouse astrocytes are insufficiently characterized, presenting a barrier in translational research. Based on analyses of acutely purified astrocytes, serum-free cultures of primary astrocytes, and xenografted chimeric mice, we find extensive conservation in astrocytic gene expression between human and mouse samples. However, the genes involved in defense response and metabolism show species-specific differences. Human astrocytes exhibit greater susceptibility to oxidative stress than mouse astrocytes, due to differences in mitochondrial physiology and detoxification pathways. In addition, we find that mouse but not human astrocytes activate a molecular program for neural repair under hypoxia, whereas human but not mouse astrocytes activate the antigen presentation pathway under inflammatory conditions. Here, we show species-dependent properties of astrocytes, which can be informative for improving translation from mouse models to humans.

A new in vitro model of the glial scar inhibits axon growth.

Astrocytes respond to central nervous system (CNS) injury with reactive astrogliosis and participate in the formation of the glial scar, an inhibitory barrier for axonal regeneration. Little is known about the injury-induced mechanisms underlying astrocyte reactivity and subsequent development of an axon-inhibitory scar. We combined two key aspects of CNS injury, mechanical trauma and co-culture with meningeal cells, to produce an in vitro model of the scar from cultures of highly differentiated astrocytes. Our model displayed widespread morphological signs of astrocyte reactivity, increases in expression of glial fibrillary acidic protein (GFAP), and accumulation of GFAP in astrocytic processes. Expression levels of scar-associated markers, phosphacan, neurocan, and tenascins, were also increased. Importantly, neurite growth from various CNS neuronal populations was significantly reduced when neurons were seeded on the scar-like cultures, compared with growth on cultures of mature astrocytes. Quantification of neurite growth parameters on the scar model demonstrated significant reductions in neuronal adhesion and neurite lengths. Interestingly, neurite outgrowth of postnatal neurons was reduced to a greater extent than that of embryonic neurons, and outgrowth inhibition varied among neuronal populations. Scar-like reactive sites and neurite-inhibitory patches were found throughout these cultures, creating a patchwork of growth-inhibitory areas mimicking a CNS injury site. Thus, our model showed relevant aspects of scar formation and produced widespread inhibition of axonal regeneration; it should be useful both for examining mechanisms underlying scar formation and to assess various treatments for their potential to improve regeneration after CNS injury. (c) 2008 Wiley-Liss, Inc.

Purinergic receptor signaling regulates N-cadherin expression in primary astrocyte cultures.

Extracellular ATP exerts both short-term and long-term effects in the CNS by stimulating cell-surface purinergic receptors. Here we have examined the effect of purinergic receptor activation on N-cadherin expression, a calcium-dependent cell adhesion molecule involved in many processes, including glia-glia and axon-glia interactions. When primary cultures of rat cortical astrocytes were treated with ATP, N-cadherin protein expression increased in a time- and concentration-dependent manner. In addition, ATP treatment caused an increase in N-cadherin immunoreactivity in both the cytoplasm and on the cell surface membrane. Interestingly, experiments with cycloheximide revealed that relocalization of N-cadherin to the cell surface membrane were independent of protein synthesis. The ATP-induced increase in N-cadherin protein expression was blocked by reactive blue 2 and 8-(p-sulfophenyl)-theophylline, suggesting involvement of both P2 and P1 purinergic receptors, respectively. In addition, N-cadherin expression was partially blocked when signaling from purinergic receptors to extracellular signal regulated protein kinase or Akt was inhibited by 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene or wortmannin, respectively. By using an in vitro model of traumatic CNS injury, we found that N-cadherin expression was increased when astrocytes were subjected to rapid and reversible mechanical strain. The findings presented here demonstrate a role for extracellular ATP, purinergic receptors and protein kinase signaling in regulating N-cadherin expression and suggest a role for this mechanism in cell-cell interactions.

Inhibition of N-cadherin and beta-catenin function reduces axon-induced Schwann cell proliferation.

N-cadherin and beta-catenin are involved in cell adhesion and cell cycle in tumor cells and neural crest. Both are expressed at key stages of Schwann cell (SC) development, but little is known about their function in the SC lineage. We studied the role of these molecules in adult rat derived SC-embryonic dorsal root ganglion cocultures by using low-Ca(2+) conditions and specific blocking antibodies to interfere with N-cadherin function and by using small interfering RNA (siRNA) to decrease beta-catenin expression in both SC-neuron cocultures and adult rat-derived SC monocultures. N-cadherin blocking conditions decreased SC-axon association and reduced axon-induced SC proliferation. In SC monocultures, beta-catenin reduction diminished the proliferative response of SCs to the mitogen beta1-heregulin, and, in SC-DRG cocultures, beta-catenin reduction inhibited axon-contact-dependent SC proliferation. Stimulation of SC cultures with beta1-heregulin increased total beta-catenin protein amount, phosphorylation of GSK-3beta and beta-catenin presence in nuclear extracts. In conclusion, our findings suggest a previously unrecognized contribution of beta-catenin and N-cadherin to axon-induced SC proliferation.

P2 receptor signalling, proliferation of astrocytes, and expression of molecules involved in cell-cell interactions.

Growing evidence indicates that trophic actions of extracellular nucleotides are involved in CNS development, injury and repair. For example, upon CNS injury, ATP is released and contributes to the formation of reactive astrocytes, cells that produce molecules that can impede or promote axonal regeneration. Proliferation is one of the features of reactive astrogliosis, particularly in traumatic injury. Fibroblast growth factor (FGF)2 is also increased after injury and can stimulate astrocyte proliferation. Extracellular ATP enhances FGF2-induced proliferation in a process mediated by P2Y receptors and increased cyclin expression. However, when P2X receptors are activated, FGF2-induced proliferation is inhibited. P2 receptors are coupled to extracellular signal regulated protein kinase (ERK), and differences in the extent and duration of ERK activation by P2Y and P2X receptors may mediate the opposing effects of these receptors on FGF2-induced mitogenesis. Trauma also activates P2 receptor/ERK signalling, and stimulation of this and other protein kinase pathways by extracellular ATP increases expression of cell adhesion and extracellular matrix molecules involved in migration, glial contact formation, neuronal guidance and synapse formation. These findings support the hypothesis that purinergic signalling via protein kinase cascades plays a key role in astrocyte proliferation, glia-glia connections, and neuron-glia interactions in both normal and pathological conditions.

N-cadherin mediates axon-aligned process growth and cell-cell interaction in rat Schwann cells.

The molecular mechanisms underlying the contact behavior of Schwann cells (SCs) and SC-axon association are poorly understood. SC-SC and SC-axon interactions were studied using purified adult rat SCs and cocultures of SCs with embryonic dorsal root ganglion neurons. After contact of SCs with axons, SCs start to extend processes in alignment with axons. This unique alignment was quantitated using a new assay. SC-axon alignment and SC-SC band formation were disrupted in medium containing low extracellular calcium, indicating the involvement of calcium-dependent adhesion molecules. N-cadherin expression was strong in developing rat sciatic nerves but weak in adult sciatic nerves. In purified adult-derived rat SCs, N-cadherin expression was increased by mitogens (neuregulins) and decreased by high cell density. High-resolution confocal images show intense N-cadherin signals in SC process tips. Subcellular N-cadherin was accumulated in bands at intercellular junctions between SCs and was clustered at axon-SC contact sites. Blocking antibodies (rabbit and guinea pig IgG directed against the first extracellular domain of N-cadherin) and cyclic pentapeptides (including the HAV motif) were used to perturb N-cadherin function. All blocking agents reduced the number of N-cadherin-positive SC-SC junctions and perturbed axon-aligned growth of SC processes. Averaging over all N-cadherin-perturbation experiments, in controls 67-86% of SCs exhibited axon-aligned process growth, whereas in treated cultures only 41% of the SCs aligned with axons. These results are evidence that in mammals N-cadherin is important for formation of SC-SC junctions and SC process growth in alignment with axons.

Olfactory ensheathing cell-neurite alignment enhances neurite outgrowth in scar-like cultures.

The regenerative capacity of adult CNS neurons after injury is strongly inhibited by the spinal cord lesion site environment that is composed primarily of the reactive astroglial scar and invading meningeal fibroblasts. Olfactory ensheathing cell (OEC) transplantation facilitates neuronal survival and functional recovery after a complete spinal cord transection, yet the mechanisms by which this recovery occurs remain unclear. We used a unique multicellular scar-like culture model to test if OECs promote neurite outgrowth in growth-inhibitory areas. Astrocytes were mechanically injured and challenged by meningeal fibroblasts to produce key inhibitory elements of a spinal cord lesion. Neurite outgrowth of postnatal cerebral cortical neurons was assessed on three substrates: quiescent astrocyte control cultures, reactive astrocyte scar-like cultures, and scar-like cultures with OECs. Initial results showed that OECs enhanced total neurite outgrowth of cortical neurons in a scar-like environment by 60%. We then asked if the neurite growth-promoting properties of OECs depended on direct alignment between neuronal and OEC processes. Neurites that aligned with OECs were nearly three times longer when they grew on inhibitory meningeal fibroblast areas and twice as long on reactive astrocyte zones compared to neurites not associated with OECs. Our results show that OECs can independently enhance neurite elongation and that direct OEC-neurite cell contact can provide a permissive substrate that overcomes the inhibitory nature of the reactive astrocyte scar border and the fibroblast-rich spinal cord lesion core.

Invariant mantling of growth cones by Schwann cell precursors characterize growing peripheral nerve fronts.

Little is known about the cytoarchitecture of growth fronts in developing mammalian nerves. We report here the first quantitative, ultrastructural analysis of growth cones (GCs) and their immediate cellular and tissue environment at tips of growing nerves that are nearing their targets in fore limbs of E14 rat embryos. Schwann cell precursor (SCP) marker, p75 neurotrophin receptor, and growth cone marker, SCG10, were used to identify nerve fronts, respectively. Using confocal 3D reconstructions and immunoelectron microscopy, we found that growth cone and Schwann cell precursor migrate together at the nerve front, where growth cone contact adjacent growth cone and Schwann cell precursor with similar frequency. Schwann cell precursor are extensively connected by adherens junctions and form elaborate scaffolds that enmantle growth cone at nerve fronts, so that 80% of the nerve front surface is covered by Schwann cell precursor. Although they interdigitate in complex ways among growth cone, the total contact area between growth cone and glial membranes is remarkably constant among the 100 growth fronts analyzed. In contrast to this consistency, other growth cone contacts varied markedly from front to front such that the frequencies of GC-GC contacts are increasing proportional to their decreasing contacts with mesenchymal tissue. Thus, at the nerve front, it is the Schwann cell precursor that are most exposed to extracellular environment while forming a surprisingly invariant substrate for advancing growth cone. This study shows for the first time that Schwann cell precursor are close and consistent cellular companions of growth cone in their approach to their final targets in the developing limb and suggests a previously unappreciated role for Schwann cell precursor in growth cone advance through the limb mesenchyme.

Role of N-cadherin in Schwann cell precursors of growing nerves.

In the present paper, we determine the localization and developmental regulation of N-cadherin in embryonic rat nerves and examine the role of N-cadherin in this system. We also identify a major transition in the architecture of embryonic nerves and relating it to N-cadherin expression. We find that in early embryonic nerves, N-cadherin is primarily expressed in Schwann cell precursors. Pronounced expression is seen at distal nerve fronts where these cells associate with growth cones, and the proximal nerve ends, in boundary cap cells. Unexpectedly, N-cadherin is downregulated as precursors generate Schwann cells, coinciding with the time at which most axons make target connections. Therefore, glial N-cadherin expression is essentially restricted to the period of axon outgrowth. We also provide evidence that N-cadherin supports the formation of contacts between Schwann cell precursors and show that these cells are a favorable substrate for axon growth, unlike N-cadherin-negative Schwann cells. Induction of N-cadherin expression in Schwann cells by neuregulin-1 restores their ability to form contacts and support axon growth. Finally, we show that the loss of glial N-cadherin during embryonic nerve development is accompanied by a transformation of nerve architecture, involving the appearance of endoneurial connective tissue space, fibroblasts, Schwann cell basal lamina, and blood vessels. Because N-cadherin is likely to promote the extensive glial contacts typical of the compact embryonic nerve, we suggest that N-cadherin loss at the time of Schwann cell generation allows endoneurial space to appear between the glial cells, a development that eventually permits the extensive interactions between connective tissue and individual axon-Schwann cell units necessary for myelination.