Role of tumor necrosis factor receptor-1 in the death of retinal ganglion cells following optic nerve crush injury in mice

To assess the specific role of tumor necrosis factor (TNF) death receptor signaling in the induction of retinal ganglion cell (RGC) death, optic nerves of mice deficient for TNF receptor-1 (TNF-R1-/-) and control mice (C57BL/6J) were unilaterally subjected to crush injury. Counts of RGCs and their axons 6 weeks after the injury demonstrated that their loss was significantly less in TNF-R1-/- mice compared to controls. The most prominent decrease in neuronal loss detected in TNF-R1-/- mice was beyond the initial 2-week period after the injury. This time period was correlated with the period of glial activation and increased glial immunolabeling for TNF-alpha in these eyes. No further protection against neuronal loss was detectable in TNF-R1-/- mice treated with D-JNKI1, a specific inhibitor of c-Jun N-terminal protein kinase (JNK). However, anti-JNK treatment of control animals provided a significant protection against neuronal loss during the same secondary degeneration period. Phospho-JNK immunolabeling of RGCs in control mice subjected to optic nerve crush significantly decreased following their treatment with D-JNKI1, and anti-JNK treatment protected RGCs from degeneration in these animals, similar to the lack of TNF-R1. These findings provide evidence that TNF death receptor signaling is involved in the secondary degeneration of RGCs following optic nerve injury, and is associated with JNK signaling. Since secondarily degenerating neurons are viable targets for neuroprotection, inhibition of TNF death receptor signaling may be an effective strategy to protect RGCs in several neurodegenerative injuries.     

Tumor necrosis factor‐alpha mediates activation of NF‐κB and JNK signaling cascades in retinal ganglion cells and astrocytes in opposite ways

Tumor necrosis factor‐alpha (TNF) is an important mediator of the innate immune response in the retina. TNF can activate various signaling cascades, including NF‐κB, nuclear factor kappa B (NF‐κB) and c‐Jun N‐terminal kinase (JNK) pathways. The harmful role of these pathways, as well as of TNF, has previously been shown in several retinal neurodegenerative conditions including glaucoma and retinal ischemia. However, TNF and TNF‐regulated signaling cascades are capable not only of mediating neurotoxicity, but of being protective. We performed this study to delineate the beneficial and detrimental effects of TNF signaling in the retina. To this end, we used TNF‐treated primary retinal ganglion cell (RGC) and astrocyte cultures. Levels of expression of NF‐κB subunits in RGCs and astrocytes were evaluated by quantitative RT‐PCR (qRT‐PCR) and Western blot (WB) analysis. NF‐κB and JNK activity in TNF‐treated cells was determined in a time‐dependent manner using ELISA and WB. Gene expression in TNF‐treated astrocytes was measured by qRT‐PCR. We found that NF‐κB family members were present in RGCs and astrocytes at the mRNA and protein levels. RGCs failed to activate NF‐κB in the presence of TNF, a phenomenon that was associated with sustained JNK activation and RGC death. However, TNF initiated the activation of NF‐κB and mediated transient JNK activation in astrocytes. These events were associated with glial survival and increased expression of neurotoxic pro‐inflammatory factors. Our findings suggest that, in the presence of TNF, NF‐κB and JNK signaling cascades are activated in opposite ways in RGCs and astrocytes. These events can directly and indirectly facilitate RGC death.     

The optic nerve head is the site of axonal transport disruption,axonal cytoskeleton damage and putative axonal regeneration failure in a rat model of glaucoma

The neurodegenerative disease glaucoma is characterised by the progressive death of retinal ganglion cells (RGCs) and structural damage to the optic nerve (ON). New insights have been gained into the pathogenesis of glaucoma through the use of rodent models; however, a coherent picture of the early pathology remains elusive. Here, we use a validated, experimentally induced rat glaucoma model to address fundamental issues relating to the spatio-temporal pattern of RGC injury. The earliest indication of RGC damage was accumulation of proteins, transported by orthograde fast axonal transport within axons in the optic nerve head (ONH), which occurred as soon as 8 h after induction of glaucoma and was maximal by 24 h. Axonal cytoskeletal abnormalities were first observed in the ONH at 24 h. In contrast to the ONH, no axonal cytoskeletal damage was detected in the entire myelinated ON and tract until 3 days, with progressively greater damage at later time points. Likewise, down-regulation of RGC-specific mRNAs, which are sensitive indicators of RGC viability, occurred subsequent to axonal changes at the ONH and later than in retinas subjected to NMDA-induced somatic excitotoxicity. After 1 week, surviving, but injured, RGCs had initiated a regenerative-like response, as delineated by Gap43 immunolabelling, in a response similar to that seen after ON crush. The data presented here provide robust support for the hypothesis that the ONH is the pivotal site of RGC injury following moderate elevation of IOP, with the resulting anterograde degeneration of axons and retrograde injury and death of somas.     

The WldS gene delays axonal but not somatic degeneration in a rat glaucoma model

Glaucoma is a leading cause of blindness caused by progressive degeneration of retinal ganglion cells (RGCs) and their axons. The pathogenesis of glaucoma remains incompletely understood, but optic nerve (ON) axonal injury appears to be an important trigger of RGC axonal and cell body degeneration. Rat models are widely used in glaucoma research to explore pathogenic mechanisms and to test novel neuroprotective approaches. Here we investigated the mechanism of axon loss in glaucoma, studying axon degeneration in slow Wallerian degeneration (Wld(S)) rats after increasing intraocular pressure. Wld(S) delays degeneration of experimentally transected axons for several weeks, so it can provide genetic evidence for Wallerian-like degeneration in disease. As apoptosis is unaffected, Wld(S) also provides information on whether cell death results from axon degeneration or arises independently, an important question yet to be resolved in glaucoma. Having confirmed expression of Wld(S) protein, we found that Wld(S) delayed ON axonal degeneration in experimental rat glaucoma for at least 2 weeks, especially in proximal ON where wild-type axons are most severely affected. The duration of axonal protection is similar to that after ON transection and crush, suggesting that axonal degeneration in glaucoma follows a Wallerian-like mechanism. Axonal degeneration must be prevented for RGCs to remain functional, so pharmacologically mimicking and enhancing the protective mechanism of Wld(S) could offer an important route towards therapy. However, Wld(S) did not protect RGC bodies in glaucoma or after ON lesion, suggesting that combination treatments protecting both axons and cell bodies offer the best therapeutic prospects.     

Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury

Basic fibroblast growth factor (or FGF-2) has been shown to be a potent stimulator of retinal ganglion cell (RGC) axonal growth during development. Here we investigated if FGF-2 upregulation in adult RGCs promoted axon regrowth in vivo after acute optic nerve injury. Recombinant adeno-associated virus (AAV) was used to deliver the FGF-2 gene to adult RGCs providing a sustained source of this neurotrophic factor. FGF-2 gene transfer led to a 10-fold increase in the number of axons that extended past 0.5 mm from the lesion site compared to control nerves. Detection of AAV-mediated FGF-2 protein in injured RGC axons correlated with growth into the distal optic nerve. The response to FGF-2 upregulation was supported by our finding that FGF receptor-1 (FGFR-1) and heparan sulfate (HS), known to be essential for FGF-2 signaling, were expressed by adult rat RGCs. FGF-2 transgene expression led to only transient protection of injured RGCs. Thus the effect of this neurotrophic factor on axon extension could not be solely attributed to an increase in neuronal survival. Our data indicate that selective upregulation of FGF-2 in adult RGCs stimulates axon regrowth within the optic nerve, an environment that is highly inhibitory for regeneration. These results support the hypothesis that key factors involved in axon outgrowth during neural development may promote regeneration of adult injured neurons.     

Retinal ganglion cell and nonneuronal cell responses to a microcrush lesion of adult rat optic nerve

Injury of the optic nerve has served as an important model for the study of cell death and axon regeneration in the CNS. Analysis of axon sprouting and regeneration after injury by anatomical tracing are aided by lesion models that produce a well-defined injury site. We report here the characterization of a microcrush lesion of the optic nerve made with 10-0 sutures to completely transect RGC axons. Following microcrush lesion, 62% of RGCs remained alive 1 week later, and 28% of RGCs, at 2 weeks. Optic nerve sections stained by hematoxylin-based methods showed a thin line of intensely stained cells that invaded the lesion site at 24 h after microcrush lesion. The lesion site became increasingly disorganized by 2 weeks after injury, and both macrophages and blood vessels invaded the lesion site. The microcrush lesion was immunoreactive for chondroitin sulfate proteoglycans (CSPG), and an adjacent GFAP-negative zone developed early after the lesion, disappearing by 1 week. Luxol fast blue staining showed a myelin-free zone at the lesion site, and myelin remained distal to the lesion at 8 weeks. To study the axonal response to microcrush lesion, anterograde tracing was used. Within 6 h after injury all RGC axons retracted back from the site of lesion. By 1 week after injury, axons regrew toward the lesion, but most stopped abruptly at the injury scar. The few axons that were able to cross the injury site did not extend further in the optic nerve white matter by 8 weeks postlesion. Our observations suggest that both the CSPG-positive scar and the myelin-derived growth inhibitory proteins contribute to the failure of RGC regeneration after injury.     

Regenerating optic axons restore topography after incomplete optic nerve injury

Following complete optic nerve injury in a lizard, Ctenophorus ornatus, retinal ganglion cell (RGC) axons regenerate but fail to restore retinotectal topography unless animals are trained on a visual task (Beazley et al. [ 1997] J Comp Neurol 370:105-120, [2003] J Neurotrauma 20:1263-1270). Here we show that incomplete injury, which leaves some RGC axons intact, restores normal topography. Strict RGC axon topography allowed us to preserve RGC axons on one side of the nerve (projecting to medial tectum) while lesioning those on the other side (projecting to lateral tectum). Topography and response properties for both RGC axon populations were assessed electrophysiologically. The majority of intact RGC axons retained appropriate topography in medial tectum and had normal, consistently brisk, reliable responses. Regenerate RGC axons fell into two classes: those that projected topographically to lateral tectum with responses that tended to habituate and those that lacked topography, responded weakly, and habituated rapidly. Axon tracing by localized retinal application of carbocyanine dyes supported the electrophysiological data. RGC soma counts were normal in both intact and axotomized RGC populations, contrasting with the 30% RGC loss after complete injury. Unlike incomplete optic nerve injury in mammals, where RGC axon regeneration fails and secondary cell death removes many intact RGC somata, lizards experience a "win-win" situation: intact RGC axons favorably influence the functional outcome for regenerating ones and RGCs do not succumb to either primary or secondary cell death.     

Precrushed sciatic nerve grafts enhance the survival and axonal regrowth of retinal ganglion cells in adult rats.

We have examined the influence of normal and precrushed ("conditioned") sciatic nerve grafts on the survival and axonal growth of retinal ganglion cells (RGCs) in adult rats. Normal sciatic nerves (group A) or sciatic nerves which had been crushed 1 week before transplantation (group B, conditioned grafts) were used as grafts. The nerves were removed and sutured to the proximal stump of intraorbitally axotomized optic nerves. Neuronal survival and axon growth were determined by counting the numbers of surviving, DiI-prelabled RGCs, cresyl violet-stained RGCs and the numbers of axons which had grown into the grafts 3 and 6 months after transplantation. Counting of axons was performed by combined use of light and electron microscopy. We observed that the use of conditioned grafts (group B) significantly enhanced RGC survival and axonal regrowth as compared to normal grafts 3 months after transplantation. Six months after grafting, RGC survival (as determined in DiI-stained retinae) and axonal growth were not significantly different in both groups. These results suggest that the functional status of a peripheral nerve used for grafting in the CNS influences neuronal viability and axonal reelongation especially during the first 3 months after grafting. Very long-term RGC survival, however, may be determined by functional reconnection of regenerating RGC axons rather than by the graft itself.     

Nogo‐A does not inhibit retinal axon regeneration in the lizard Gallotia galloti

The myelin‐associated protein Nogo‐A contributes to the failure of axon regeneration in the mammalian central nervous system (CNS). Inhibition of axon growth by Nogo‐A is mediated by the Nogo‐66 receptor (NgR). Nonmammalian vertebrates, however, are capable of spontaneous CNS axon regeneration, and we have shown that retinal ganglion cell (RGC) axons regenerate in the lizard Gallotia galloti. Using immunohistochemistry, we observed spatiotemporal regulation of Nogo‐A and NgR in cell bodies and axons of RGCs during ontogeny. In the adult lizard, expression of Nogo‐A was associated with myelinated axon tracts and upregulated in oligodendrocytes during RGC axon regeneration. NgR became upregulated in RGCs following optic nerve injury. In in vitro studies, Nogo‐A‐Fc failed to inhibit growth of lizard RGC axons. The inhibitor of protein kinase A (pkA) activity KT5720 blocked growth of lizard RGC axons on substrates of Nogo‐A‐Fc, but not laminin. On patterned substrates of Nogo‐A‐Fc, KT5720 caused restriction of axon growth to areas devoid of Nogo‐A‐Fc. Levels of cyclic adenosine monophosphate (cAMP) were elevated over sustained periods in lizard RGCs following optic nerve lesion. We conclude that Nogo‐A and NgR are expressed in a mammalian‐like pattern and are upregulated following optic nerve injury, but the presence of Nogo‐A does not inhibit RGC axon regeneration in the lizard visual pathway. The results of outgrowth assays suggest that outgrowth‐promoting substrates and activation of the cAMP/pkA signaling pathway play a key role in spontaneous lizard retinal axon regeneration in the presence of Nogo‐A. Restriction of axon growth by patterned Nogo‐A‐Fc substrates suggests that Nogo‐A may contribute to axon guidance in the lizard visual system. J. Comp. Neurol. 525:936–954, 2017. © 2016 Wiley Periodicals, Inc.     

Receptor protein tyrosine phosphatase sigma inhibits axon regrowth in the adult injured CNS

Recently, receptor protein tyrosine phosphatase-sigma (RPTPsigma) has been shown to inhibit axon regeneration in injured peripheral nerves. Unlike the peripheral nervous system (PNS), central nervous system (CNS) neurons fail to regenerate their axons after injury or in disease. In order to assess the role of RPTPsigma in CNS regeneration, we used the retinocollicular system of adult mice lacking RPTPsigma to evaluate retinal ganglion cell (RGC) axon regrowth after optic nerve lesion. Quantitative analysis demonstrated a significant increase in the number of RGC axons that crossed the glial scar and extended distally in optic nerves from RPTPsigma (-/-) mice compared to wild-type littermate controls. Although we found that RPTPsigma is expressed by adult RGCs in wild-type mice, the retinas and optic nerves of adult RPTPsigma (-/-) mice showed no histological defects. Furthermore, the time-course of RGC death after nerve lesion was not different between knockout and wild-type animals. Thus, enhanced axon regrowth in the absence of RPTPsigma could not be attributed to developmental defects or increased neuronal survival. Finally, we show constitutively elevated activity of mitogen-activated protein kinase (MAPK) and Akt kinase in adult RPTPsigma (-/-) mice retinas, suggesting that these signaling pathways may contribute to promoting RGC axon regrowth following traumatic nerve injury. Our results support a model in which RPTPsigma inhibits axon regeneration in the adult injured CNS.     

Novel axon projection after stress and degeneration in the Dscam mutant retina

The Down syndrome cell adhesion molecule gene (Dscam) is required for normal dendrite patterning and promotes developmental cell death in the mouse retina. Loss-of-function studies indicate that Dscam is required for refinement of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus, and in this study we report and describe a requirement for Dscam in the maintenance of RGC axon projections within the retina. Mouse Dscam loss of function phenotypes related to retinal ganglion cell axon outgrowth and targeting have not been previously reported, despite the abundance of axon phenotypes reported in Drosophila Dscam1 loss and gain of function models. Analysis of the Dscam deficient retina was performed by immunohistochemistry and Western blot analysis during postnatal development of the retina. Conditional targeting of Dscam and Jun was performed to identify factors underlying axon-remodeling phenotypes. A subset of RGC axons were observed to project and branch extensively within the Dscam mutant retina after eye opening. Axon remodeling was preceded by histological signs of RGC stress. These included neurofilament accumulation, axon swelling, axon blebbing and activation of JUN, JNK and AKT. Novel and extensive projection of RGC axons within the retina was observed after upregulation of these markers, and novel axon projections were maintained to at least one year of age. Further analysis of retinas in which Dscam was conditionally targeted with Brn3b or Pax6α Cre indicated that axon stress and remodeling could occur in the absence of hydrocephalus, which frequently occurs in Dscam mutant mice. Analysis of mice mutant for the cell death gene Bax, which executes much of Dscam dependent cell death, identified a similar axon misprojection phenotype. Deleting Jun and Dscam resulted in increased axon remodeling compared to Dscam or Bax mutants. Retinal ganglion cells have a very limited capacity to regenerate after damage in the adult retina, compared to the extensive projections made in the embryo. In this study we find that DSCAM and JUN limit ectopic growth of RGC axons, thereby identifying these proteins as targets for promoting axon regeneration and reconnection.     

EphB regulates L1 phosphorylation during retinocollicular mapping

Interaction of the cell adhesion molecule L1 with the cytoskeletal adaptor ankyrin is essential for topographic mapping of retinal ganglion cell (RGC) axons to synaptic targets in the superior colliculus (SC). Mice mutated in the L1 ankyrin-binding motif (FIGQY(1229)H) display abnormal mapping of RGC axons along the mediolateral axis of the SC, resembling mouse mutant phenotypes in EphB receptor tyrosine kinases. To investigate whether L1 functionally interacts with EphBs, we investigated the role of EphB kinases in phosphorylating L1 using a phospho-specific antibody to the tyrosine phosphorylated FIGQY(1229) motif. EphB2, but not an EphB2 kinase dead mutant, induced tyrosine phosphorylation of L1 at FIGQY(1229) and perturbed ankyrin recruitment to the membrane in L1-transfected HEK293 cells. Src family kinases mediated L1 phosphorylation at FIGQY(1229) by EphB2. Other EphB receptors that regulate medial-lateral retinocollicular mapping, EphB1 and EphB3, also mediated phosphorylation of L1 at FIGQY(1229). Tyrosine(1176) in the cytoplasmic domain of L1, which regulates AP2/clathrin-mediated endocytosis and axonal trafficking, was not phosphorylated by EphB2. Accordingly mutation of Tyr(1176) to Ala in L1-Y(1176)A knock-in mice resulted in normal retinocollicular mapping of ventral RGC axons. Immunostaining of the mouse SC during retinotopic mapping showed that L1 colocalized with phospho-FIGQY in RGC axons in retinorecipient layers. Immunoblotting of SC lysates confirmed that L1 was phosphorylated at FIGQY(1229) in wild type but not L1-FIGQY(1229)H (L1Y(1229)H) mutant SC, and that L1 phosphorylation was decreased in the EphB2/B3 mutant SC. Inhibition of ankyrin binding in L1Y(1229)H mutant RGCs resulted in increased neurite outgrowth compared to WT RGCs in retinal explant cultures, suggesting that L1-ankyrin binding serves to constrain RGC axon growth. These findings are consistent with a model in which EphB kinases phosphorylate L1 at FIGQY(1229) in retinal axons to modulate L1-ankyrin binding important for mediolateral retinocollicular topography.     

Soluble adenylyl cyclase activity is necessary for retinal ganglion cell survival and axon growth

cAMP is a critical second messenger mediating activity-dependent neuronal survival and neurite growth. We investigated the expression and function of the soluble adenylyl cyclase (sAC, ADCY10) in CNS retinal ganglion cells (RGCs). We found sAC protein expressed in multiple RGC compartments including the nucleus, cytoplasm and axons. sAC activation increased cAMP above the level seen with transmembrane adenylate cyclase (tmAC) activation. Electrical activity and bicarbonate, both physiologic sAC activators, significantly increased survival and axon growth, whereas pharmacologic or siRNA-mediated sAC inhibition dramatically decreased RGC survival and axon growth in vitro, and survival in vivo. Conversely, RGC survival and axon growth were unaltered in RGCs from AC1/AC8 double knock-out mice or after specifically inhibiting tmACs. These data identify a novel sAC-mediated cAMP signaling pathway regulating RGC survival and axon growth, and suggest new neuroprotective or regenerative strategies based on sAC modulation.     

Selective impairment of slow axonal transport after optic nerve injury in adult rats.

To investigate cellular responses of injured mammalian CNS neurons, we examined the slow transport of cytoskeletal proteins in rat retinal ganglion cell (RGC) axons within the ocular stump of optic nerves that were crushed intracranially. RGC proteins were labeled by an intravitreal injection of 35S-methionine, and optic nerves were examined by SDS PAGE at different times after injury. In one group of rats, the RGC proteins were labeled 1 week after crushing. From 14 to 67 d after axotomy, the labeling of tubulin and neurofilaments was reduced in relation to other labeled proteins and to the labeling of tubulin and neurofilaments in the intact optic nerve of controls. To determine whether this reduction in labeling was due to an alteration in axonal transport after axotomy, we prelabeled RGC proteins 1 week before crushing. In such experiments, the rate of slow axonal transport of tubulin and neurofilaments decreased approximately 10-fold from 6 to 60 d after injury. Our results cannot be due only to the retrograde degeneration of RGCs and injured axons caused by axotomy in the optic nerve, because fast axonal protein transport and the fluorescent labeling of many axons were preserved in the ocular stumps of these optic nerves. This selective failure of the slow axonal transport of tubulin and neurofilaments may affect the renewal of the cytoskeleton and contribute to the gradual degeneration of RGCs that is observed after axotomy. The alterations in slow transport we document here differ from the enhanced rates we previously reported when injured RGC axons regenerated along peripheral nerve segments grafted to the ocular stump of transected optic nerves (McKerracher et al., 1990).     

Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons

In vitro, cyclic AMP (cAMP) elevation alters neuronal responsiveness to diffusible growth factors and myelin-associated inhibitory molecules. Here we used an established in vivo model of adult central nervous system injury to investigate the effects of elevated cAMP on neuronal survival and axonal regeneration. We studied the effects of intraocular injections of neurotrophic factors and/or a cAMP analogue (CPT-cAMP) on the regeneration of axotomized rat retinal ganglion cell (RGC) axons into peripheral nerve autografts. Elevation of cAMP alone did not significantly increase RGC survival or the number of regenerating RGCs. Ciliary neurotrophic factor increased RGC viability and axonal regrowth, the latter effect substantially enhanced by coapplication with CPT-cAMP. Under these conditions over 60% of surviving RGCs regenerated their axons. Neurotrophin-4/5 injections also increased RGC viability, but there was reduced long-distance axonal regrowth into grafts, an effect partially ameliorated by cAMP elevation. Thus, cAMP can act cooperatively with appropriate neurotrophic factors to promote axonal regeneration in the injured adult mammalian central nervous system.     

Restricted expression of the neuronal intermediate filament protein plasticin during zebrafish development

In the adult goldfish visual pathway, expression of the neuronal intermediate filament (nIF) protein plasticin is restricted to differentiating retinal ganglion cells (RGCs) at the margin of the retina. Following optic nerve injury, plasticin expression is elevated transiently in all RGCs coincident with the early stages of axon regeneration. These results suggest that plasticin may be expressed throughout the nervous system during the early stages of axonogenesis. To test this hypothesis, we analyzed plasticin expression during zebrafish (Danio rerio) neuronal development. By using immunocytochemistry and in situ hybridization, we found that plasticin is expressed in restricted subsets of early zebrafish neurons. Expression coincides with axon outgrowth in projection neurons that pioneer distinct axon tracts in the embryo. Plasticin is expressed first in trigeminal, Rohon-Beard, and posterior lateral line ganglia neurons, which are among the earliest neurons to initiate axonogenesis in zebrafish. Plasticin is expressed also in reticulospinal neurons and in caudal primary motoneurons. Together, these neurons establish the first behavioral responses in the embryo. Plasticin expression also coincides with initial RGC axonogenesis and progressively decreases after RGC axons reach the tectum. At later developmental stages, plasticin is expressed in a subset of the cranial nerves. The majority of plasticin-positive neurons are within or project axons to the peripheral nervous system. Our results suggest that plasticin subserves the changing requirements for plasticity and stability during axonal outgrowth in neurons that project long axons. J. Comp. Neurol. 399:561–572, 1998. © 1998 Wiley-Liss, Inc.     

Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve

Axon regenerative failure in the mature CNS contributes to functional deficits following many traumatic injuries, ischemic injuries, and neurodegenerative diseases. The complement cascade of the innate immune system responds to pathogen threat through inflammatory cell activation, pathogen opsonization, and pathogen lysis, and complement is also involved in CNS development, neuroplasticity, injury, and disease. Here, we investigated the involvement of the classical complement cascade and microglia/monocytes in CNS repair using the mouse optic nerve injury (ONI) model, in which axons arising from retinal ganglion cells (RGCs) are disrupted. We report that central complement C3 protein and mRNA, classical complement C1q protein and mRNA, and microglia/monocyte phagocytic complement receptor CR3 all increase in response to ONI, especially within the optic nerve itself. Importantly, genetic deletion of C1q, C3, or CR3 attenuates RGC axon regeneration induced by several distinct methods, with minimal effects on RGC survival. Local injections of C1q function-blocking antibody revealed that complement acts primarily within the optic nerve, not retina, to support regeneration. Moreover, C1q opsonizes and CR3⁺ microglia/monocytes phagocytose growth-inhibitory myelin debris after ONI, a likely mechanism through which complement and myeloid cells support axon regeneration. Collectively, these results indicate that local optic nerve complement-myeloid phagocytic signaling is required for CNS axon regrowth, emphasizing the axonal compartment and highlighting a beneficial neuroimmune role for complement and microglia/monocytes in CNS repair.SIGNIFICANCE STATEMENT Despite the importance of achieving axon regeneration after CNS injury and the inevitability of inflammation after such injury, the contributions of complement and microglia to CNS axon regeneration are largely unknown. Whereas inflammation is commonly thought to exacerbate the effects of CNS injury, we find that complement proteins C1q and C3 and microglia/monocyte phagocytic complement receptor CR3 are each required for retinal ganglion cell axon regeneration through the injured mouse optic nerve. Also, whereas studies of optic nerve regeneration generally focus on the retina, we show that the regeneration-relevant role of complement and microglia/monocytes likely involves myelin phagocytosis within the optic nerve. Thus, our results point to the importance of the innate immune response for CNS repair.     

Regulation of caspase activation in axotomized retinal ganglion cells

Transection of the optic nerve initiates massive death of retinal ganglion cells (RGCs). Interestingly, despite the severity of the injury, RGC loss was not observed until several days after axotomy. The mechanisms responsible for this initial lack of RGC death remained unknown. In the current study, immunohistochemical analysis revealed that caspases-3 and -9 activation in the RGCs were not detected until day 3 post-axotomy, coinciding with the onset of axotomy-induced RGC loss. Interestingly, elevated Akt phosphorylation was observed in axotomized retinas during the absence of caspase activation. Inhibiting the increase in Akt phosphorylation by intravitreal injection of wortmannin and LY294002, inhibitors of PI3K, resulted in premature nuclear fragmentation, caspases-3 and -9 activation in the ganglion cell layer. Our findings thus indicate that the PI3K/Akt pathway may serve as an endogenous regulator of caspase activation in axotomized RGCs, thereby, contributing to the late onset of RGC death following axotomy.     

Axon injury induced endoplasmic reticulum stress and neurodegeneration

Injury to central nervous system axons is a common early characteristic of neurodegenerative diseases. Depending on its location and the type of neuron, axon injury often leads to axon degeneration, retrograde neuronal cell death and progressive permanent loss of vital neuronal functions. Although these sequential events are clearly connected, ample evidence indicates that neuronal soma and axon degenerations are active autonomous processes with distinct molecular mechanisms. By exploiting the anatomical and technical advantages of the retinal ganglion cell(RGC)/optic nerve(ON) system, we demonstrated that inhibition of the PERK-e IF2α-CHOP pathway and activation of the X-box binding protein 1 pathway synergistically protect RGC soma and axon, and preserve visual function, in both acute ON traumatic injury and chronic glaucomatous neuropathy. The autonomous endoplasmic reticulum(ER) stress pathway in neurons has been implicated in several other neurodegenerative diseases. In addition to the emerging role of ER morphology in axon maintenance, we propose that ER stress is a common upstream signal for disturbances in axon integrity, and that it leads to a retrograde signal that can subsequently induce neuronal soma death. Therefore manipulation of the ER stress pathway may be a key step toward developing the effective neuroprotectants that are greatly needed in the clinic.     

Lack of Jun-N-terminal kinase 3 (JNK3) does not protect against neurodegeneration induced by 3-nitropropionic acid

F. Junyent, L. de Lemos, E. Verdaguer, M. Pallàs, J. Folch, C. Beas‐Zárate, A. Camins and C. Auladell (2012) Neuropathology and Applied Neurobiology 38, 311–321 Lack of Jun‐N‐terminal kinase 3 (JNK3) does not protect against neurodegeneration induced by 3‐nitropropionic acid Aims: 3‐Nitropropionic acid (3‐NP) is a toxin that replicates most of the clinical and pathophysiological symptoms of Huntington's disease, inducing neurodegeneration in the striatum due to the inhibition of mitochondrial succinate dehydrogenase. Different pathways have been implicated in the cell death induced by 3‐NP in rodents. One of them is the Jun‐N‐terminal kinase (JNK) pathway, which may play a role in the neurodegenerative process in different diseases. Moreover, the lack of one isoform of JNK (JNK3) has been associated with neuroprotection in different experimental models of neurodegeneration. Therefore, in the present study the role of JNK3 in the experimental Huntington's model induced by 3‐NP administration was evaluated. Methods: 3‐NP was intraperitoneally administered once a day for 3 days to wild‐type and Jnk3‐null mice. Coronal brain sections were used to determine cell death and astrogliosis in striatum. Western blots were performed to determine the involvement of different pathways in both wild‐type and Jnk3‐null mice. Results: Although JNK activation was observed following 3‐NP administration, the results indicate that the lack of JNK3 does not confer neuroprotection against 3‐NP toxicity. Thus, other pathways must be involved in the neurodegeneration induced in this model. One of the possible pathways towards 3‐NP‐induced apoptosis could involve the calpains, as their activity was increased in wild‐type and Jnk3‐null mice. Conclusion: Although JNK3 is a key protein involved in cell death in different neurodegenerative diseases, the present study demonstrates that the lack of JNK3 does not confer neuroprotection against 3‐NP‐induced neuronal death.