Trying to understand axonal regeneration in the CNS of fish.

In contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish--such as the retinal ganglion cells--are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support--at least in vitro--axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growth-associated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tectum.     

Induction of α1-tubulin gene expression during development and regeneration of the fish central nervous system

The α1- and α2-tubulin encoding genes were cloned from a goldfish genomic DNA library. α1- and α2-tubulin RNA expression was examined in developing and adult retinas. These studies demonstrated increased α1-tubulin RNA in presumptive ganglion cells that grow axons early in retinal development and in adult retinal ganglion cells whose optic axons had been damaged. The α2-tubulin RNA was undetectable in developing retina and constitutively expressed in adult retinal ganglion cells regardless of optic nerve crush. To determine if these changes in α1-tubulin RNA reflected changes in α1-tubulin promoter activity, we introduced into zebrafish embryos and adult goldfish retinal explants expression vectors harboring the α1-tubulin gene's promoter. These studies showed that the α1-tubulin promoter confers a developmentally regulated, neuron-restricted pattern of reporter gene expression in vivo and its activity is increased in adult retinal neurons induced to regenerate their axons. Promoter deletions defined regions of α1-tubulin DNA necessary for this pattern of expression. These results suggest that DNA sequences necessary for α1-tubulin gene induction during central nervous system development and regeneration are contained within the α1-tubulin gene's 5′-flanking DNA and that this promoter will be useful for identifying these elements and their DNA binding proteins. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 429–440, 1998     

Trying to understand axonal regeneration in the CNS of fish

In contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish—such as the retinal ganglion cells—are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support—at least in vitro—axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growth associated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tectum. © 1992 John Wiley & Sons, Inc.     

Degenerative and regenerative responses of injured neurons in the central nervous system of adult mammals.

In adult mammals, the severing of the optic nerve near the eye is followed by a loss of retinal ganglion cells (RGCs) and a failure of axons to regrow into the brain. Experimental manipulations of the non-neuronal environment of injured RGCs enhance neuronal survival and make possible a lengthy axonal regeneration that restores functional connections with the superior colliculus. These effects suggest that injured nerve cells in the mature central nervous system (CNS) are strongly influenced by interactions with components of their immediate environment as well as their targets. Under these conditions, injured CNS neurons can express capacities for growth and differentiation that resemble those of normally developing neurons. An understanding of this regeneration in the context of the cellular and molecular events that influence the interactions of axonal growth cones with their non-neuronal substrates and neuronal targets should help in the further elucidation of the capacities of neuronal systems to recover from injury.     

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. I. Adult brain, retina, and peripheral nerve

Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and Muller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans.     

Neurolin, a cell surface glycoprotein on growing retinal axons in the goldfish visual system, is reexpressed during retinal axonal regeneration.

The mAb E 21 recognizes a cell surface glycoprotein selectively associated with fish retinal ganglion cell axons that are in a state of growth. All retinal axons and ganglion cells in goldfish embryos stained for E 21. In adult fish, however, E 21 immunoreactivity exhibited a patterned distribution in ganglion cells in the marginal growth zone of the continuously enlarging fish retina and the new axons emerging from these cells in the retina, optic nerve, and optic tract. The E 21 antigen was absent from older axons, except the terminal arbor layer in the tectum, the Stratum fibrosum et griseum superficiale where it was uniformly distributed. Upon optic nerve transection, the previously unlabeled axons reacquired E 21 positivity as they regenerated throughout their path to the tectum. Several months after ONS, however, E 21 staining disappeared from the regenerated axons over most of their lengths but reappeared as in normal fish in the terminal arbor layer. The immunoaffinity-purified E 21 antigen, called Neurolin, has an apparent molecular mass of 86 kD and contains the HNK1/L2 carbohydrate moiety, like several members of the class of cell adhesion molecules of the Ig superfamily. The NH2-terminal amino acid sequence has homologies to the cell adhesion molecule DM-Grasp recently described in the chicken. Thus, retinal ganglion cell axons express Neurolin during their development and are able to reexpress this candidate cell adhesion molecule during axonal regeneration, suggesting that Neurolin is functionally important for fish retinal axon growth.     

Regenerated retinal ganglion cell axons form normal numbers of boutons but fail to expand their arbors in the superior colliculus

Regenerated retinal ganglion cell (RGC) axons can re-form functional synapses with target neurons in the superior colliculus (SC). Because preterminal axon branching determines the size, shape and density of innervation fields, we investigated the branching patterns and bouton formation of individual RGC axons that had regrown along peripheral nerve (PN) grafts to the SC. Within the superficial layers of the SC, the regenerated axons formed terminal arbors with average numbers of terminal boutons that were similar to the controls. However, axonal branches were shorter than normal so that the mean area of the regenerated arbors was nearly one-tenth that of control arbors and the resulting fields of innervation contained greater than normal numbers of synapses concentrated in small areas of the target. Our results have delineated a critical defect in the reconstitution of retino-collicular circuitry in adult mammals: the failure of terminal RGC branches to expand appropriately. Because recent studies have documented that brain-derived neurotrophic factor (BDNF) can specifically lengthen RGC axonal branches not only during development in the SC but also within the adult retina after axotomy, the present quantitative studies should facilitate experimental attempts to correct this deficit of the regenerative response. © 1998 Chapman and Hall     

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments

Summary Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose severed axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.     

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.     

Extrinsic cellular and molecular mediators of peripheral axonal regeneration

The ability of injured peripheral nerves to regenerate and reinnervate their original targets is a characteristic feature of the peripheral nervous system (PNS). On the other hand, neurons of the central nervous system (CNS), including retinal ganglion cell (RGC) axons, are incapable of spontaneous regeneration. In the adult PNS, axonal regeneration after injury depends on well-orchestrated cellular and molecular processes that comprise a highly reproducible series of degenerative reactions distal to the site of injury. During this fine-tuned process, named Wallerian degeneration, a remodeling of the distal nerve fragment prepares a permissive microenvironment that permits successful axonal regrowth originating from the proximal nerve fragment. Therefore, a multitude of adjusted intrinsic and extrinsic factors are important for surviving neurons, Schwann cells, macrophages and fibroblasts as well as endothelial cells in order to achieve successful regeneration. The aim of this review is to summarize relevant extrinsic cellular and molecular determinants of successful axonal regeneration in rodents that contribute to the regenerative microenvironment of the PNS.     

Stimulating axonal regeneration of mature retinal ganglion cells and overcoming inhibitory signaling

Like other neurons of the central nervous system (CNS), retinal ganglion cells (RGCs) are normally unable to regenerate injured axons and instead undergo apoptotic cell death. This regenerative failure leads to lifelong visual deficits after optic nerve damage and is partially attributable to factors located in the inhibitory environment of the forming glial scar and myelin as well as to an insufficient intrinsic ability for axonal regrowth. In addition to its ophthalmological relevance, the optic nerve has long been used as a favorable paradigm for studying regenerative failure in the CNS as a whole. Findings over the last 15 years have shown that, under certain circumstances, mature RGCs can be transformed into an active regenerative state enabling these neurons to survive axotomy and to regenerate axons in the optic nerve. Moreover, combinatorial treatments overcoming the inhibitory environment of the glial scar and optic nerve myelin, together with approaches activating the intrinsic growth program, can further enhance the amount of regeneration in vivo. These findings are encouraging and open the possibility that clinically meaningful regenerationmay become achievable in the future.     

The survival of chick retinal ganglion cells in response to brain-derived neurotrophic factor depends on their embryonic age

The survival effects of brain-derived neurotrophic factor (BDNF) on the ganglion cells of the chick retina were studied in vitro at different embryonic ages. We found these effects to be strongly age-dependent: at E5, when the first ganglion cell axons have crossed the optic chiasm, but not yet reached the tectum, ganglion cells survived on a laminin substrate irrespective of the presence or absence of BDNF. At E6, when the axons of the first-generated ganglion cells reached the rostral pole of the tectum, the ganglion cells began to show a dependency on BDNF for survival, but the majority of them were alive after 2 days in vitro in the absence of BDNF. With increasing age, the BDNF dependency for survival increased, and at E11, the majority of the ganglion cells plated were dependent on BDNF for survival. It is at this age that the maximal number of axons can be found in vivo in the optic nerve, the subsequent elimination of ganglion cells and their axons resulting in the loss of hundreds of thousands of them over the next few days. Taken together, these data indicate that retinal ganglion cells depend on BDNF for survival only when their axons have reached their target in vivo. This situation is reminiscent of that described in the peripheral nervous system for the nerve growth factor responsiveness of mouse trigeminal sensory neurons during the period of innervation of their target.     

RSK1 promotes mammalian axon regeneration by inducing the synthesis of regeneration-related proteins

In contrast to the adult mammalian central nervous system (CNS), the neurons in the peripheral nervous system (PNS) can regenerate their axons. However, the underlying mechanism dictating the regeneration program after PNS injuries remains poorly understood. Combining chemical inhibitor screening with gain- and loss-of-function analyses, we identified p90 ribosomal S6 kinase 1 (RSK1) as a crucial regulator of axon regeneration in dorsal root ganglion (DRG) neurons after sciatic nerve injury (SNI). Mechanistically, RSK1 was found to preferentially regulate the synthesis of regeneration-related proteins using ribosomal profiling. Interestingly, RSK1 expression was up-regulated in injured DRG neurons, but not retinal ganglion cells (RGCs). Additionally, RSK1 overexpression enhanced phosphatase and tensin homolog (PTEN) deletion-induced axon regeneration in RGCs in the adult CNS. Our findings reveal a critical mechanism in inducing protein synthesis that promotes axon regeneration and further suggest RSK1 as a possible therapeutic target for neuronal injury repair.

This study shows that p90 ribosomal S6 kinase 1 (RSK1) responds differentially to nerve injury in the peripheral and central nervous systems, and identifies it as a crucial regulator of axonal regeneration; mechanistically, RSK1 preferentially induces the synthesis of regeneration-related proteins via the RSK1-eEF2K-eEF2 axis.     

Target-Dependent Regulation of Retinal Nicotinic Acetylcholine Receptor and Tubulin RNAs During Optic Nerve Regeneration in Goldfish

A fundamental issue in central nervous system development regards the effect of target tissue on the differentiation of innervating neurons. We address this issue by characterizing the role the retinal ganglion cell target, i.e., the optic tectum, plays in regulating expression of tubulin and nicotinic acetylcholine receptor genes in regenerating retinal ganglion cells. Tubulins are involved in axonal growth, whereas nicotinic acetylcholine receptors mediate communication across synapses. Retinal ganglion cell axons were induced to regenerate by crushing the optic nerve. Following crush, there was a rapid increase in alpha-tubulin RNAs (3 days), which preceded the increase in nicotinic acetylcholine receptor RNAs (10-15 days). Both classes of RNAs approached control levels by the time retinotectal synapses and functional recovery were restored (4-6 weeks). If the optic nerve was repeatedly crushed or its target ablated, tubulin RNAs remained elevated, and the increase in receptor RNAs that would otherwise be seen 2 weeks after a single nerve crush did not occur. The interaction of retinal ganglion cell axons with their targets in the optic tectum appears, then, to exert a suppressive effect on the RNA encoding a cytoskeletal protein, tubulin, and an inductive effect on RNAs encoding nicotinic acetylcholine receptors involved in synaptic communication.     

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.     

NgR RNA interference, combined with zymosan intravitreal injection, enhances optic nerve regeneration

Mature retinal ganglion cells like other CNS neurons are unable to regenerate their axons after injury. Regenerative failure has been attributed, in part, to two factors: the existence of myelin-derived inhibitors that bind to the Nogo receptor (NgR) and a deficiency of trophic support factors. We investigated the regrowth of injured axons both by inhibiting NgR by RNA interference and by recruiting exogenous trophic support by zymosan intravitreal injection. Our results showed that either approach can stimulate optic nerve axon regrowth but regenerated axons can grow longer and extend further when both methods are combined. We conclude that endogenous NgR inhibition and exogenous trophic support both play independent, important roles in enhancing optic nerve axon regrowth and that the regenerative effect can be augmented when the two are combined. This may provide a therapeutic strategy for promoting axon regeneration in the CNS as well.     

Pathfinding, target recognition, and synapse formation of single regenerating fibers in the adult grasshopper Schistocerca gregaria

After lesion of the peripheral tympanal nerve of the adult locust (Schistocerca gregaria), sensory axons regenerate into their original target areas. We examined the individual behavior of single regenerating auditory afferents during pathway and target selection by intracellularly recording and labeling them at different times postlesion. During axotomy, spontaneous activity is not increased in either the distal or proximal part of the cells. Stimulus response properties of lesioned cells with or without regenerating axons are not influenced. Surprisingly, only 55% of sensory neurons regenerate through the lesion site and often give rise to more than one axonal fiber. Within the central nervous system, 70% of regenerated axons consistently follow an incorrect pathway to reach the correct target region. Often, one of two processes formed by a cell chooses the correct pathway, and the other the incorrect one. In the target region, regenerated axons reconstitute somatotopically ordered projections and form synapses that resemble those of intact fibers in number and structure. The regeneration process does not induce a detectable expression of antigens that are known to be expressed during neural development in these neurons. Our study clearly demonstrates that precise synaptic regeneration is possible in adult animals within a completely differentiated central nervous system, although pathfinding and formation of arborizations are disturbed in a particular and probably system-related manner. The results strongly suggest that accurate pathfinding is unlikely to be a decisive factor in target area recognition and synaptogenesis.     

The role of myelin in Theiler's virus persistence in the central nervous system

Theiler's virus, a picornavirus, persists for life in the central nervous system of mouse and causes a demyelinating disease that is a model for multiple sclerosis. The virus infects neurons first but persists in white matter glial cells, mainly oligodendrocytes and macrophages. The mechanism, by which the virus traffics from neurons to glial cells, and the respective roles of oligodendrocytes and macrophages in persistence are poorly understood. We took advantage of our previous finding that the shiverer mouse, a mutant with a deletion in the myelin basic protein gene (Mbp), is resistant to persistent infection to examine the role of myelin in persistence. Using immune chimeras, we show that resistance is not mediated by immune responses or by an efficient recruitment of inflammatory cells into the central nervous system. With both in vivo and in vitro experiments, we show that the mutation does not impair the permissiveness of neurons, oligodendrocytes, and macrophages to the virus. We demonstrate that viral antigens are present in cytoplasmic channels of myelin during persistent infection of wild-type mice. Using the optic nerve as a model, we show that the virus traffics from the axons of retinal ganglion cells to the cytoplasmic channels of myelin, and that this traffic is impaired by the shiverer mutation. These results uncover an unsuspected axon to myelin traffic of Theiler's virus and the essential role played by the infection of myelin/oligodendrocyte in persistence.     

Slow transport of the cytoskeleton after axonal injury

The delivery of cytoskeletal proteins to the axon occurs by slow axonal transport. We examined how the rate of slow transport was altered after axonal injury. When retinal ganglion cell (RGC) axons regenerated through peripheral nerve grafts, an increase in the rate of slow transport occurred during regrowth of the injured axons. We compared these results to axonal injury in the optic nerve where no substantial regrowth occurs and found a completely different response. Slow transport was decreased approximately tenfold in rate in the proximal segment of crushed optic nerves. This decreased rate of slow transport was not induced immediately, but occurred about 1 week after injury. To explore whether a decrease in the rate of slow transport was induced when the regeneration of peripheral nerves was physically blocked, we examined slow transport in motor neurons after the sciatic nerve was transected and ligated. In this case, no change in the rate of the comigrating tubulin and neurofilament (NF) radioactive peaks were observed. We discuss how the changes in the rate of slow transport may reflect different neuronal responses to injury and speculate about the possible molecular changes in the expression of tubulin which may contribute to the observed changes. © 1992 John Wiley & Sons, Inc.