Neuronal STAT3 activation is essential for CNTF- and inflammatory stimulation-induced CNS axon regeneration

CNS neurons, such as retinal ganglion cells (RGCs), do not normally regenerate injured axons, but instead undergo apoptotic cell death. Regenerative failure is due to inhibitory factors in the myelin and forming glial scar as well as due to an insufficient intrinsic capability of mature neurons to regrow axons. Nevertheless, RGCs can be transformed into an active regenerative state upon inflammatory stimulation (IS) in the inner eye, for instance by lens injury, enabling these RGCs to survive axotomy and to regenerate axons into the lesioned optic nerve. The beneficial effects of IS are mediated by various factors, including CNTF, LIF and IL-6. Consistently, IS activates various signaling pathways, such as JAK/STAT3 and PI3K/AKT/mTOR, in several retinal cell types. Using a conditional knockdown approach to specifically delete STAT3 in adult RGCs, we investigated the role of STAT3 in IS-induced neuroprotection and axon regeneration. Conditional STAT3 knockdown in RGCs did not affect the survival of RGCs after optic nerve injury compared with controls, but significantly reduced the neuroprotective effects of IS. STAT3 depletion significantly compromised CNTF-stimulated neurite growth in culture and IS-induced transformation of RGCs into an active regenerative state in vivo. As a consequence, IS-mediated axonal regeneration into the injured optic nerve was almost completely abolished in mice with STAT3 depleted in RGCs. In conclusion, STAT3 activation in RGCs is involved in neuroprotection and is a necessary prerequisite for optic nerve regeneration upon IS.     

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.     

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish ( Carassius auratus ). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo . None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.     

Suppression of the p75 receptor signal attenuates the effect of ephrin-B3 and promotes axonal regeneration of the injured optic nerve

The p75 neurotrophin receptor (p75NTR) is known to transduce the signal from some myelin-associated axon growth inhibitors, including Nogo and myelin-associated glycoprotein. As ephrin-B3, a member of the ephrin family, is also expressed in myelin and inhibits axon growth, the purpose of this study was to assess the possible involvement of p75NTR in ephrin-B3 signaling. Here, we report that p75NTR is required for the inhibitory effect of ephrin-B3 on neurite growth in vitro. While ephrin-B3 inhibited neurite elongation of embryonic cortical neurons, the neurons with p75NTR knockdown or with EphA4 knockdown were less sensitive to ephrin-B3. Although no direct interaction of p75NTR with ephrin-B3 was observed, Pep5, a peptide that specifically inhibits RhoA activation mediated by p75NTR, reduced the effect of ephrin-B3. Therefore, p75NTR functions as a signal transducer for ephrin-B3. Moreover, axonal regeneration in vivo was induced by Pep5 application after optic nerve crush injury in mice. Thus, Pep5 is a promising agent that contributes to axonal regeneration in the central nervous system.     

AAV-mediated inhibition of ULK1 promotes axonal regeneration in the central nervous system in vitro and in vivo

Axonal damage is an early step in traumatic and neurodegenerative disorders of the central nervous system (CNS). Damaged axons are not able to regenerate sufficiently in the adult mammalian CNS, leading to permanent neurological deficits. Recently, we showed that inhibition of the autophagic protein ULK1 promotes neuroprotection in different models of neurodegeneration. Moreover, we demonstrated previously that axonal protection improves regeneration of lesioned axons. However, whether axonal protection mediated by ULK1 inhibition could also improve axonal regeneration is unknown. Here, we used an adeno-associated viral (AAV) vector to express a dominant-negative form of ULK1 (AAV.ULK1.DN) and investigated its effects on axonal regeneration in the CNS. We show that AAV.ULK1.DN fosters axonal regeneration and enhances neurite outgrowth in vitro. In addition, AAV.ULK1.DN increases neuronal survival and enhances axonal regeneration after optic nerve lesion, and promotes long-term axonal protection after spinal cord injury (SCI) in vivo. Interestingly, AAV.ULK1.DN also increases serotonergic and dopaminergic axon sprouting after SCI. Mechanistically, AAV.ULK1.DN leads to increased ERK1 activation and reduced expression of RhoA and ROCK2. Our findings outline ULK1 as a key regulator of axonal degeneration and regeneration, and define ULK1 as a promising target to promote neuroprotection and regeneration in the CNS.Subject terms: Cell death in the nervous system, Neurodegeneration, Spinal cord injury     

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.     

Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.     

Upregulation of Gem relates to retinal ganglion cells apoptosis after optic nerve crush in adult rats

GTP-binding protein Gem, a member protein of the Ras superfamily, can regulate actin cytoskeleton reorganization mediated by Rho-associated coiled-coil-containing protein kinase (ROCK). One attractive activity of the ROCK is playing a potential role in physiological and pathological process in retinal ganglion cells (RGCs) apoptosis. However, the function of Gem in retina is still with limited understanding. To investigate whether Gem is involved in optic nerve injury, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis indicated that Gem was significantly increased in the retina at the 3rd day after ONC. Meanwhile, double-immunofluorescent staining showed that Gem expression was mainly up-regulated in ganglion cell layer and co-localized with NeuN (a marker of RGCs). Additionally, the co-localizations of Gem/active-caspase-3 and Gem/TUNEL-positive cells were detected in RGCs. Furthermore, the expression of active-caspase-3 and TUNEL-positive cells was parallel with that of Gem. Finally, expression pattern of ROCK family (only ROCK2 but not ROCK1) was increased in the differentiated process, which was collected with the expression of GEM and active-caspase-3. Based on the present results, it is suggested that Gem might play a crucial role in RGCs apoptosis after ONC, which might be involved in ROCK pathway.     

Ginkgolide B promotes axonal growth of retina ganglion cells by anti-apoptosis in vitro

One common feature of glaucoma, optic neuritis and some other optic nerve diseases is sustained and irreversible apoptosis of retinal ganglion cells (RGCs). Ginkgolide B is believed to protect neurons in brain and contribute to neurite outgrowth and synapse formation. The aim of the present study was to explore the effects of Ginkgo biloba extract (EGB761) and ginkgolide B on axonal growth of RCGs. Retina explants were cultured in three-dimensional tissue culture system, and the number and length of neurites were analyzed. Immunohistochemistry staining was performed to confirm that the neurite observed was axon of RGCs. TUNEL and activated caspase-3 staining were also applied to observe RGCs apoptosis. The result shows that neurites of RGCs treated with EGB761 or ginkgolide B were more and longer than those in control. The neurite is proved to be the axon of RGCs by immunostaining. Furthermore, compared with control group, RGCs treated with ginkgolide B showed decreased cellular apoptosis and inhibited caspase-3 activation. These results suggest ginkgolide B can promote RGCs axon growth by protecting RGCs against apoptosis.     

The RNA Binding Protein Igf2bp1 Is Required for Zebrafish RGC Axon Outgrowth In Vivo

Attractive growth cone turning requires Igf2bp1-dependent local translation of β-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the β-actin 3’UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.     

Regeneration of optic nerve fibers of adult mammals

The pathway from the retina to the brain in mammals provides a well-defined model system for investigation of not only surviving axotomy but also axonal regeneration of injured neurons. Here I introduce our recent works on axonal regeneration in the optic nerve (OpN) of adult cats. Fibers of retinal ganglion cells (RGCs) extend beyond the crush site of OpN with injections of a macrophage stimulator (oxidized galectin-1) or a Rho kinase (ROCK) inhibitor (Y-39983 or Y-27632) while axonal extension is blocked with injection of saline. Elongation of crushed optic fibers, however, is slowed after 2 weeks. Transplantation of peripheral nerve makes RGCs regenerate their transected axons into a graft but regenerated fibers extend only a few mm in the brain. Effectiveness of combination of the drugs and treatments has to be verified in future.     

Comparative Evaluation of Methods for Estimating Retinal Ganglion Cell Loss in Retinal Sections and Wholemounts

To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a⁺, βIII-tubulin⁺ and Islet-1⁺ RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG⁺ and Brn3a⁺ RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin⁺ RGCs was greater than Brn3a⁺ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.     

Early cellular signaling responses to axonal injury

Background

We have used optic nerve injury as a model to study early signaling events in neuronal tissue following axonal injury. Optic nerve injury results in the selective death of retinal ganglion cells (RGCs). The time course of cell death takes place over a period of days with the earliest detection of RGC death at about 48 hr post injury. We hypothesized that in the period immediately following axonal injury, there are changes in the soma that signal surrounding glia and neurons and that start programmed cell death. In the current study, we investigated early changes in cellular signaling and gene expression that occur within the first 6 hrs post optic nerve injury.

Results

We found evidence of cell to cell signaling within 30 min of axonal injury. We detected differences in phosphoproteins and gene expression within the 6 hrs time period. Activation of TNFα and glutamate receptors, two pathways that can initiate cell death, begins in RGCs within 6 hrs following axonal injury. Differential gene expression at 6 hrs post injury included genes involved in cytokine, neurotrophic factor signaling (Socs3) and apoptosis (Bax).

Conclusion

We interpret our studies to indicate that both neurons and glia in the retina have been signaled within 30 min after optic nerve injury. The signals are probably initiated by the RGC soma. In addition, signals activating cellular death pathways occur within 6 hrs of injury, which likely lead to RGC degeneration.     

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.     

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.