Tissue-specific neuro-glia interactions determine neurite differentiation in ganglion cells

Guided formation and extension of axons versus dendrites is considered crucial for structuring the nervous system. In the chick visual system, retinal ganglion cells (RGCs) extend their axons into the tectum opticum, but not into glial somata containing retina layers. We addressed the question whether the different glia of retina and tectum opticum differentially affect axon growth. Glial cells were purified from retina and tectum opticum by complement-mediated cytolysis of non-glial cells. RGCs were purified by enzymatic delayering from flat mounted retina. RGCs were seeded onto retinal versus tectal glia monolayers. Subsequent neuritic differentiation was analysed by immunofluorescence microscopy and scanning electron microscopy. Qualitative and quantitative evaluation revealed that retinal glia somata inhibited axons. Time-lapse video recording indicated that axonal inhibition was based on the collapse of lamellipodia- and filopodia-rich growth cones of axons. In contrast to retinal glia, tectal glia supported axonal extension. Notably, retinal glia were not inhibitory for neurons in general, because in control experiments axon extension of dorsal root ganglia was not hampered. Therefore, the axon inhibition by retinal glia was neuron type-specific. In summary, the data demonstrate that homotopic (retinal) glia somata inhibit axonal outgrowth of RGCs, whereas heterotopic (tectal) glia of the synaptic target area support RGC axon extension. The data underscore the pivotal role of glia in structuring the developing nervous system.     

Wnt/β‐catenin‐signaling and the formation of Müller glia‐derived progenitors in the chick retina

Müller glia can be stimulated to de‐differentiate, proliferate, and form Müller glia‐derived progenitor cells (MGPCs) that are capable of producing retinal neurons. The signaling pathways that influence the de‐differentiation of mature Müller glia and proliferation of MGPCs may include the Wnt‐pathway. The purpose of this study was to investigate how Wnt‐signaling influences the formation of MGPCs in the chick retina in vivo. In NMDA‐damaged retinas where MGPCs are known to form, we find dynamic changes in retinal levels of potential readouts of Wnt‐signaling, including dkk1, dkk3, axin2, c‐myc, tcf‐1, and cd44. We find accumulations of nuclear β‐catenin in MGPCs that peaks at 3 days and rapidly declines by 5 days after NMDA‐treatment. Inhibition of Wnt‐signaling with XAV939 in damaged retinas suppressed the formation of MGPCs, increased expression of ascl1a and decreased hes5, but had no effect upon the differentiation of progeny produced by MGPCs. Activation of Wnt‐signaling, with GSK3β‐inhibitors, in the absence of retinal damage, failed to stimulate the formation of MGPCs, whereas activation of Wnt‐signaling in damaged retinas stimulated the formation of MGPCs. In the absence of retinal damage, FGF2/MAPK‐signaling stimulated the formation of MGPCs by activating a signaling network that includes Wnt/β‐catenin. In FGF2‐treated retinas, inhibition of Wnt‐signaling reduced numbers of proliferating MGPCs, whereas activation of Wnt‐signaling failed to influence the formation of proliferating MGPCs. Our findings indicate that Wnt‐signaling is part of a network initiated by FGF2/MAPK or retinal damage, and activation of canonical Wnt‐signaling is required for the formation of proliferating MGPCs. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 983–1002, 2016     

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.     

Actions of neurotrophic factors and their signaling pathways in neuronal survival and axonal regeneration

Adult axons in the mammalian central nervous system do not elicit spontaneous regeneration after injury, although many affected neurons have survived the neurotrauma. However, axonal regeneration does occur under certain conditions. These conditions include: (a) modification of regrowth environment, such as supply of peripheral nerve bridges and transplantation of Schwann cells or olfactory ensheathing glia to the injury site; (b) application of neurotrophic factors at the cell soma and axon tips; (c) blockade of growth-inhibitory molecules such as Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein; (d) prevention of chondroitin-sulfate-proteoglycans-related scar tissue formation at the injury site using chondroitinase ABC; and (e) elevation of intrinsic growth potential of injured neurons via increasing intra-cellular cyclic adenosine monophosphate level. A large body of evidence suggests that these conditions achieve enhanced neuronal survival and axonal regeneration through sometimes over-lapping and sometimes distinct signal transduction mechanisms, depending on the targeted neuronal populations and intervention circumstances. This article reviews the available information on signal transduction pathways underlying neurotrophic-factor-mediated neuronal survival and neurite outgrowth/axonal regeneration. Better understanding of signaling transduction is important in helping us develop practical therapeutic approaches for encouraging neuronal survival and axonal regeneration after traumatic injury in clinical context.     

Tenascin-R and axon growth-promoting molecules are up-regulated in the regenerating visual pathway of the lizard (Gallotia galloti)

It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down-regulation of axon growth-inhibitory proteins, and to what extent outgrowth-promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth-inhibiting extracellular matrix molecules tenascin-R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth-promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin-R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin-R and CSPG were expressed in an extracellular matrix-, oligodendrocyte/myelin- and neuron-associated pattern and up-regulated in the regenerating optic pathway. The expression pattern of tenascin-R was not indicative of a role in channeling or restriction of re-growing RGC axons. Up-regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin-R expression. Moreover, we analyzed the influence of substrates containing tenascin-R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin-R, and further improved on mixed substrates containing tenascin-R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down-regulation of tenascin-R or CSPG. Presence of tenascin-R is insufficient to prevent RGC axon growth, and concomitant up-regulation of axon growth-promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up-regulation of contactin in RGCs suggests that tenascin-R may have an instructive function during axon regeneration in the lizard optic pathway.     

Permissive role of Cdc2 activity induced from astrocytes in neurite outgrowth

Following spinal cord injury, glial cells are recognized as major environmental factors hampering axon's regenerative responses. However, recent studies suggested that, in certain circumstances, reactive astrocytes may have a permissive role for axonal regeneration and functional recovery. Here, we report that Cdc2 activation in astrocytes is positively linked to axon growth. Cdc2 was strongly, but transiently, induced from reactive astrocytes within and around the injury cavity. Cdc2 levels in primary, non‐neuronal cells prepared from injured spinal cord were up‐regulated by extending the pre‐injury period. Cdc2‐mediated vimentin phosphorylation was strongly induced in astrocytes after long‐term culture (7 days, LTC) as compared with short‐term culture (3 days, STC). Induction levels of phospho‐vimentin in LTC astrocytes were positively associated with increased neurite outgrowth in co‐cultured dorsal root ganglion neurons. β3 integrin mRNA was induced in LTC astrocytes and activation of β3 integrin was regulated by Cdc2 activity. Furthermore, genetic depletion and pharmacological blockade experiments demonstrate that activation of Cdc2 and β3 integrin in LTC astrocytes is required for neurite outgrowth. Our data suggest that the Cdc2 pathway may play an important role in determining phenotypic expression of astrocytes such that astrocytes provide permissive environments for axonal regeneration following spinal cord injury.     

Retinal regeneration is facilitated by the presence of surviving neurons

Teleost fish regenerate their retinas after damage, in contrast to mammals. In zebrafish subjected to an extensive ouabain‐induced lesion that destroys all neurons and spares Müller glia, functional recovery and restoration of normal optic nerve head (ONH) diameter take place at 100 days postinjury. Subsequently, regenerated retinas overproduce cells in the retinal ganglion cell (RGC) layer, and the ONH becomes enlarged. Here, we test the hypothesis that a selective injury, which spares photoreceptors and Müller glia, results in faster functional recovery and fewer long‐term histological abnormalities. Following this selective retinal damage, recovery of visual function required 60 days, consistent with this hypothesis. In contrast to extensively damaged retinas, selectively damaged retinas showed fewer histological errors and did not overproduce neurons. Extensively damaged retinas had RGC axons that were delayed in pathfinding to the ONH, and showed misrouted axons within the ONH, suggesting that delayed functional recovery following an extensive lesion is related to defects in RGC axons exiting the eye and/or reaching their central targets. The atoh7, fgf8a, Sonic hedgehog (shha), and netrin‐1 genes were differentially expressed, and the distribution of hedgehog protein was disrupted after extensive damage as compared with selective damage. Confirming a role for Shh signaling in supporting rapid regeneration, shha^t4+/‐ zebrafish showed delayed functional recovery after selective damage. We suggest that surviving retinal neurons provide structural/molecular information to regenerating neurons, and that this patterning mechanism regulates factors such as Shh. These factors in turn control neuronal number, retinal lamination, and RGC axon pathfinding during retinal regeneration. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 851–876, 2014     

Ephrin‐B2 elicits differential growth cone collapse and axon retraction in retinal ganglion cells from distinct retinal regions

The circuit for binocular vision and stereopsis is established at the optic chiasm, where retinal ganglion cell (RGC) axons diverge into the ipsilateral and contralateral optic tracts. In the mouse retina, ventrotemporal (VT) RGCs express the guidance receptor EphB1, which interacts with the repulsive guidance cue ephrin‐B2 on radial glia at the optic chiasm to direct VT RGC axons ipsilaterally. RGCs in the ventral retina also express EphB2, which interacts with ephrin‐B2, whereas dorsal RGCs express low levels of EphB receptors. To investigate how growth cones of RGCs from different retinal regions respond upon initial contact with ephrin‐B2, we utilized time‐lapse imaging to characterize the effects of ephrin‐B2 on growth cone collapse and axon retraction in real time. We demonstrate that bath application of ephrin‐B2 induces rapid and sustained growth cone collapse and axon retraction in VT RGC axons, whereas contralaterally‐projecting dorsotemporal RGCs display moderate growth cone collapse and little axon retraction. Dose response curves reveal that contralaterally‐projecting ventronasal axons are less sensitive to ephrin‐B2 treatment compared to VT axons. Additionally, we uncovered a specific role for Rho kinase signaling in the retraction of VT RGC axons but not in growth cone collapse. The detailed characterization of growth cone behavior in this study comprises an assay for the study of Eph signaling in RGCs, and provides insight into the phenomena of growth cone collapse and axon retraction in general. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 781–794, 2010     

A Proteomics Approach to Identify Candidate Proteins Secreted by Müller Glia that Protect Ganglion Cells in the Retina

The retinal Müller glial cells, can enhance the survival and activity of neurons, especially of retinal ganglion cells (RGCs), which are the neurons affected in diseases such as glaucoma, diabetes, and retinal ischemia. It has been demonstrated that Müller glia release neurotrophic factors that support RGC survival, yet many of these factors remain to be elucidated. To define these neurotrophic factors, a quantitative proteomic approach was adopted aiming at identifying neuroprotective proteins. First, the conditioned medium from porcine Müller cells cultured in vitro under three different conditions were isolated and these conditioned media were tested for their capacity to promote survival of primary adult RGCs in culture. Mass spectrometry was used to identify and quantify proteins in the conditioned medium, and osteopontin (SPP1), clusterin (CLU), and basigin (BSG) were selected as candidate neuroprotective factors. SPP1 and BSG significantly enhance RGC survival in vitro, indicating that the survival‐promoting activity of the Müller cell secretome is multifactorial, and that SPP1 and BSG contribute to this activity. Thus, the quantitative proteomics strategy identify proteins secreted by Müller glia that are potentially novel neuroprotectants, and it may also serve to identify other bioactive proteins or molecular markers.     

Attenuation of Axonal Degeneration by Calcium Channel Inhibitors Improves Retinal Ganglion Cell Survival and Regeneration After Optic Nerve Crush

Axonal degeneration is one of the initial steps in many traumatic and neurodegenerative central nervous system (CNS) disorders and thus a promising therapeutic target. A focal axonal lesion is followed by acute axonal degeneration (AAD) of both adjacent axon parts, before proximal and distal parts follow different degenerative fates at later time points. Blocking calcium influx by calcium channel inhibitors was previously shown to attenuate AAD after optic nerve crush (ONC). However, it remains unclear whether the attenuation of AAD also promotes consecutive axonal regeneration. Here, we used a rat ONC model to study the effects of calcium channel inhibitors on axonal degeneration, retinal ganglion cell (RGC) survival, and axonal regeneration, as well as the molecular mechanisms involved. Application of calcium channel inhibitors attenuated AAD after ONC and preserved axonal integrity as visualized by live imaging of optic nerve axons. Consecutively, this resulted in improved survival of RGCs and improved axonal regeneration at 28 days after ONC. We show further that calcium channel inhibition attenuated lesion-induced calpain activation in the proximity of the crush and inhibited the activation of the c-Jun N-terminal kinase pathway. Pro-survival signaling via Akt in the retina was also increased. Our data thus show that attenuation of AAD improves consecutive neuronal survival and axonal regeneration and that calcium channel inhibitors could be valuable tools for therapeutic interventions in traumatic and degenerative CNS disorders.     

Hepatocyte growth factor protects retinal ganglion cells by increasing neuronal survival and axonal regeneration in vitro and in vivo

Hepatocyte growth factor (HGF) is known to promote the survival and foster neuritic outgrowth of different subpopulations of CNS neurons during development. Together with its corresponding receptor c-mesenchymal-epithelial transition factor (Met), it is expressed in the developing and the adult murine, rat and human CNS. We have studied the role of HGF in paradigms of retinal ganglion cell (RGC) regeneration and cell death in vitro and in vivo. After application of recombinant HGF in vitro, survival of serum-deprived RGC-5 cells and of growth factor-deprived primary RGC was significantly increased. This was shown to be correlated to the phosphorylation of c-Met and subsequent activation of serine/threonine protein kinase Akt and MAPK downstream signalling pathways involved in neuronal survival. Furthermore, neurite outgrowth of primary RGC was stimulated by HGF. In vivo, c-Met expression in RGC was up-regulated after optic nerve axotomy lesion. Here, treatment with HGF significantly improved survival of axotomized RGC and enhanced axonal regeneration after optic nerve crush. Our data demonstrates that exogenously applied HGF has a neuroprotective and regeneration-promoting function for lesioned CNS neurons. We provide strong evidence that HGF may represent a trophic factor for adult CNS neurons, which may play a role as therapeutic target in the treatment of neurotraumatic and neurodegenerative CNS disorders.     

Retinal Glia Promote Dorsal Root Ganglion Axon Regeneration

Axon regeneration in the adult central nervous system (CNS) is limited by several factors including a lack of neurotrophic support. Recent studies have shown that glia from the adult rat CNS, specifically retinal astrocytes and Müller glia, can promote regeneration of retinal ganglion cell axons. In the present study we investigated whether retinal glia also exert a growth promoting effect outside the visual system. We found that retinal glial conditioned medium significantly enhanced neurite growth and branching of adult rat dorsal root ganglion neurons (DRG) in culture. Furthermore, transplantation of retinal glia significantly enhanced regeneration of DRG axons past the dorsal root entry zone after root crush in adult rats. To identify the factors that mediate the growth promoting effects of retinal glia, mass spectrometric analysis of retinal glial conditioned medium was performed. Apolipoprotein E and secreted protein acidic and rich in cysteine (SPARC) were found to be present in high abundance, a finding further confirmed by western blotting. Inhibition of Apolipoprotein E and SPARC significantly reduced the neuritogenic effects of retinal glial conditioned medium on DRG in culture, suggesting that Apolipoprotein E and SPARC are the major mediators of this regenerative response.     

Schwann Cell Exosomes Mediate Neuron–Glia Communication and Enhance Axonal Regeneration

The functional and structural integrity of the nervous system depends on the coordinated action of neurons and glial cells. Phenomena like synaptic activity, conduction of action potentials, and neuronal growth and regeneration, to name a few, are fine tuned by glial cells. Furthermore, the active role of glial cells in the regulation of neuronal functions is underscored by several conditions in which specific mutation affecting the glia results in axonal dysfunction. We have shown that Schwann cells (SCs), the peripheral nervous system glia, supply axons with ribosomes, and since proteins underlie cellular programs or functions, this dependence of axons from glial cells provides a new and unexplored dimension to our understanding of the nervous system. Recent evidence has now established a new modality of intercellular communication through extracellular vesicles. We have already shown that SC-derived extracellular vesicles known as exosomes enhance axonal regeneration, and increase neuronal survival after pro-degenerative stimuli. Therefore, the biology nervous system will have to be reformulated to include that the phenotype of a nerve cell results from the contribution of two nuclei, with enormous significance for the understanding of the nervous system in health and disease.     

The Insulin-Like Growth Factor 1 Receptor Is Essential for Axonal Regeneration in Adult Central Nervous System Neurons

Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the βgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both βgc and the β subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, βgc was localised to distal regions and leading growth cones in RGC. IGF-1R-βgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, all significantly reduced axon regeneration from adult RGC in vitro (∼40% RGC possessed axons in controls vs 2–8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma.     

Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.     

Neuronal Injury External to the Retina Rapidly Activates Retinal Glia,Followed by Elevation of Markers for Cell Cycle Re-Entry and Death in Retinal Ganglion Cells

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.