Deciphering proteins and their functions in the regenerating retina

Neurons of the mammalian CNS, including retinal ganglion cells, lack, in contrast to the PNS, the ability to regenerate axons spontaneously after injury. Regeneration of the CNS is extremely complex and involves various molecular factors and cells. Therewith the regenerative process remains an enormous scientific and clinical challenge. This article provides an overview of proteins that play a crucial role in axon regeneration of retinal ganglion cells and their underlying signaling pathways. In this context, we elucidate the role of 2D gel electrophoresis and highlight some additional proteins, altered upon regeneration by using this highly sensitive method.     

周围神经或其组织成分移植修复成年哺乳动物视网膜节细胞的损伤

本综述重点阐述了移植周围神经或其组织成分雪旺细胞、成纤维细胞和神经营养因子,改善成年哺乳动物中枢神经系统抑制神经再生的微环境、增强受损神经元的内在再生潜力,以促进细胞损伤后的存活和轴突再生。     

A novel biological function for CD44 in axon growth of retinal ganglion cells identified by a bioinformatics approach

The failure of CNS regeneration and subsequent motor and sensory loss remain major unsolved questions despite massive accumulation of experimental observations and results. The sheer volume of data and the variety of resources from which these data are generated make it difficult to integrate prior work to build new hypotheses. To address these challenges we developed a prototypic suite of computer programs to extract protein names from relevant publications and databases and associated each of them with several general categories of biological functions in nerve regeneration. To illustrate the usefulness of our data mining approach, we utilized the program output to generate a hypothesis for a biological function of CD44 interaction with osteopontin (OPN) and laminin in axon outgrowth of CNS neurons. We identified CD44 expression in retinal ganglion cells and when these neurons were plated on poly- l -lysine 3% of them initiated axon growth, on OPN 15%, on laminin-111 (1×) 41%, on laminin-111 (0.5×) 56%, and on a mixture of OPN and laminin (1×) 67% of neurons generated axon growth. With the aid of a deoxyribozyme (DNA enzyme) to CD44 that digests the target mRNA, we demonstrated that a reduction of CD44 expression led to reduced axon initiation of retinal ganglion cells on all substrates. We suggest that such an integrative, applied systems biology approach to CNS trauma will be critical to understand and ultimately overcome the failure of CNS regeneration.     

Electrophoretic Analysis of Axonally Transported Proteins in Toad Retinal Ganglion Cells

As a preliminary step to studying changes in axonal transport in regenerating neurons, we have analyzed the composition and organization of polypeptides normally axonally transported in a neuronal system capable of regeneration, i.e., the retinal ganglion cells of the toad, Bufo marinus. We labeled proteins synthesized in the retina with 35S-methionine and subsequently used one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to analyze labeled, transported proteins in tissues containing segments of the axons (the optic nerve, optic tract, and optic tecta) of the retinal ganglion cells. The transported polypeptides could be divided into five groups according to their apparent transport velocities. Many of the polypeptides of each group were electrophoretically similar to polypeptides of corresponding groups previously described in rabbit and guinea pig retinal ganglion cells, and in some cases, additional properties of the polypeptides indicated that the transported materials of the two vertebrate classes were homologous. These results serve two purposes. First they establish the retinal ganglion cells of the toad Bufo marinus as a model system in which changes in gene expression related to regeneration may be studied. Second they show that the organization and many aspects of the composition of axonal transport in retinal ganglion cells have been conserved in animals as unrelated as amphibians, and mammals.     

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.     

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.     

Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo

While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma.     

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.     

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.     

Astrocytes as gate-keepers in optic nerve regeneration--a mini-review

Animals that develop without extra-embryonic membranes (anamniotes--fish, amphibians) have impressive regenerative capacity, even to the extent of replacing entire limbs. In contrast, animals that develop within extra-embryonic membranes (amniotes--reptiles, birds, mammals) have limited capacity for regeneration as adults, particularly in the central nervous system (CNS). Much is known about the process of nerve development in fish and mammals and about regeneration after lesions in the CNS in fish and mammals. Because the retina of the eye and optic nerve are functionally part of the brain and are accessible in fish, frogs, and mice, optic nerve lesion and regeneration (ONR) has been extensively used as a model system for study of CNS nerve regeneration. When the optic nerve of a mouse is severed, the axons leading into the brain degenerate. Initially, the cut end of the axons on the proximal, eye-side of the injury sprout neurites which begin to grow into the lesion. Simultaneously, astrocytes of the optic nerve become activated to initiate wound repair as a first step in reestablishing the structural integrity of the optic nerve. This activation appears to initiate a cascade of molecular signals resulting in apoptotic cell death of the retinal ganglion cells axons of which make up the neural component of the optic nerve; regeneration fails and the injury is permanent. Evidence specifically implicating astrocytes comes from studies showing selective poisoning of astrocytes at the optic nerve lesion, along with activation of a gene whose product blocks apoptosis in retinal ganglion cells, creates conditions favorable to neurites sprouting from the cut proximal stump, growing through the lesion and into the distal portion of the injured nerve, eventually reaching appropriate targets in the brain. In anamniotes, astrocytes ostensibly present no such obstacle since optic nerve regeneration occurs without intervention; however, no systematic study of glial involvement has been done. In fish, vigorously growing neurites sprout from the cut axons and within a few days begin to re-enervate the brain. This review offers a new perspective on the role of glia, particularly astrocytes, as "gate-keepers;" i.e., as being permissive or inhibitory, by comparison between fish and mammals of glial function during ONR.     

Melanopsin,ganglion-cell photoreceptors,and mammalian photoentrainment

An understanding of the retinal mechanisms in mammalian photoentrainment will greatly facilitate optimization of the wavelength, intensity, and duration of phototherapeutic treatments designed to phase shift endogenous biological rhythms. A small population of widely dispersed retinal ganglion cells projecting to the suprachiasmatic nucleus in the hypothalamus is the source of the critical photic input. Recent evidence has shown that many of these ganglion cells are directly photosensitive and serve as photoreceptors. Melanopsin, a presumptive photopigment, is an essential component in the phototransduction cascade within these intrinsically photosensitive ganglion cells and plays an important role in the retinal photoentrainment pathway. This review summarizes recent findings related to melanopsin and melanopsin ganglion cells and lists other retinal proteins that might serve as photopigments in the mammalian photoentrainment input pathway.     

CALEB binds via its acidic stretch to the fibrinogen-like domain of tenascin-C or tenascin-R and its expression is dynamically regulated after optic nerve lesion

Recently, we described a novel chick neural transmembrane glycoprotein, which interacts with the extracellular matrix proteins tenascin-C and tenascin-R. This protein, termed CALEB, contains an epidermal growth factor-like domain and appears to be a novel member of the epidermal growth factor family of growth and differentiation factors. Here we analyze the interaction between CALEB and tenascin-C as well as tenascin-R in more detail, and we demonstrate that the central acidic peptide segment of CALEB is necessary to mediate this binding. The fibrinogen-like globe within tenascin-C or -R enables both proteins to bind to CALEB. We show that two isoforms of CALEB in chick and rodents exist that differed in their cytoplasmic segments. To begin to understand the in vivo function of CALEB and since in vitro antibody perturbation experiments indicated that CALEB might be important for neurite formation, we analyzed the expression pattern of the rat homolog of CALEB during development of retinal ganglion cells, after optic nerve lesion and during graft-assisted retinal ganglion cell axon regeneration by in situ hybridization. These investigations demonstrate that CALEB mRNA is dynamically regulated after optic nerve lesion and that this mRNA is expressed in most developing and in one-third of the few regenerating (GAP-43 expressing) retinal ganglion cells.     

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.     

损伤视神经后斑马鱼视网膜神经节细胞数量和分布的变化（简报）

Changes in number and distribution of retinal ganglion cells were studied after optic nerve crush in zebrafish (Brachydanio rerio) with retinal wholemount. There were approximately 40,000 to 56,000 cells in the retinal ganglion cell layer. The density of ganglion cells was divided into six classes and the area of highest cell density (central area) was located at the temporal area to the optic disc in normal fish. At the early regeneration stages after optic nerve crush, the percentage of lost cells increased gradually. Cell density had fallen first in the central area. At the late regeneration stages, there was an approximately 20% loss of ganglion cells during optic nerve regeneration. The results suggest that the loss of cells may undergo apoptosis rather than necrosis. A wave of cell loss started in the central area and spread progressively further into periphery. The reason caused these changes may be due to temporal interruption of optic nerve function, recovery from crush and the ability to quickly regenerate in optic nerve of the fish.     

损伤视神经后斑马鱼视网膜神经节细胞数量和分布的变化

中枢神经系统的再生是神经科学领域的一个重要课题。鱼类和两栖类的视神经作为中枢神经系统的一部分,具有再生的能力。已知在损伤视神经后,对与视神经纤维直接相连的视网膜神经节细胞的形态结构,数量和分布等产生一系列的影响。视神经再生过程中细胞学研究在很大程度上依赖于示踪方法和其它技术的发展,结合光镜和电镜,它们仅对神经细胞末梢的精细结构和神经细胞间突触连接构筑等研究较准确详实,但对视网膜神经节细     

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.     

BDNF production by olfactory ensheathing cells contributes to axonal regeneration of cultured adult CNS neurons

Olfactory ensheathing cells (OECs) are the main glial cell type that populates mammalian olfactory nerves. These cells have a great capacity to promote the regeneration of axons when transplanted into the injured adult mammalian CNS. However, little is still known about the molecular mechanisms they employ in mediating such a task. Brain-derived neurotrophic factor (BDNF) was identified as a candidate molecule in a genomic study that compared three functionally different OEC populations: Early passage OECs (OEC Ep), Late passage OECs (OEC Lp) and the OEC cell line TEG3 [Pastrana, E., Moreno-Flores, M.T., Gurzov, E.N., Avila, J., Wandosell, F., Diaz-Nido, J., 2006. Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2. J. Neurosci. 26, 5347-5359]. We have here set out to determine the role played by BDNF in the stimulation of axon outgrowth by OECs. We compared the extracellular BDNF levels in the three OEC populations and show that it is produced in significant amounts by the OECs that can stimulate axon regeneration in adult retinal neurons (OEC Ep and TEG3) but it is absent from the extracellular medium of OEC Lp cells which lack this capacity. Blocking BDNF signalling impaired axonal regeneration of adult retinal neurons co-cultured with TEG3 cells and adding BDNF increased the proportion of adult neurons that regenerate their axons on OEC Lp monolayers. Combining BDNF with other extracellular proteins such as Matrix Metalloproteinase 2 (MMP2) further augmented this effect. This study shows that BDNF production by OECs plays a direct role in the promotion of axon regeneration of adult CNS neurons.     

In Vitro Protein Synthesis in the Goldfish Retinotectal Pathway During Regeneration: Evidence for Specific Axonal Proteins of Retinal Origin in the Optic Nerve

Four proteins with molecular weights of 58,000 can be separated as a linear array by two-dimensional gel electrophoresis. They are highly concentrated in the goldfish optic nerve and are designated as ON1, ON2, ON3, and ON4. Proteins ON1 and ON2 are undetectable in the optic nerve after disconnection and their concentration is gradually restored during regeneration. In vitro incubations of retinas, optic nerves, or tecta in the presence of [35S]methionine indicate that proteins ON1 and ON2 are of retinal origin. The labeling rate of these proteins in the retina increases fourfold after optic nerve crush whereas the overall labeling rate in the retina remains largely constant. Their synthesis cannot be detected in tissues devoid of retinal ganglion cells. This is consistent with the view that ON1 and ON2 are synthesized by retinal ganglion cells and are consequently of neuronal origin in the optic nerve. In contrast, similar experiments indicate that ON3 and ON4 are of nonneuronal origin. They are synthesized in the optic nerve in the absence of retinal ganglion cells.     

Temporary disruption of the retinal basal lamina and its effect on retinal histogenesis.

An experimental paradigm was devised to remove the retinal basal lamina for defined periods of development: the basal lamina was dissolved by injecting collagenase into the vitreous of embryonic chick eyes, and its regeneration was induced by a chase with mouse laminin-1 and alpha2-macroglobulin. The laminin-1 was essential in reconstituting a new basal lamina and could not be replaced by laminin-2 or collagen IV, whereas the macroglobulin served as a collagenase inhibitor that did not directly contribute to basal lamina regeneration. The regeneration occurred within 6 h after the laminin-1 chase by forming a morphologically complete basal lamina that included all known basal lamina proteins from chick embryos, such as laminin-1, nidogen-1, collagens IV and XVIII, perlecan, and agrin. The temporary absence of the basal lamina had dramatic effects on retinal histogenesis, such as an irreversible retraction of the endfeet of the neuroepithelial cells from the vitreal surface of the retina, the formation of a disorganized ganglion cell layer with an increase in ganglion cells by 30%, and the appearance of multiple retinal ectopias. Finally, basal lamina regeneration was associated with aberrant axons failing to correctly enter the optic nerve. The present data demonstrate that a transient disruption of the basal lamina leads to dramatic and probably irreversible aberrations in the histogenesis in the developing central nervous system.     

Changes in goldfish retinal ganglion cells during axonal regeneration

Recent work suggests that mammalian retinal ganglion cells may become more like developing ganglion cells in form while regenerating through a peripheral nerve graft. We have injected Lucifer Yellow into regenerating ganglion cells of goldfish to look for similar changes. Within three weeks of injury, we saw dye-coupling to nearby cells, which is a common developmental feature in many species. Dendrites and axons, which in most mature ganglion cells are smooth, became varicose and hairy, like those examined in mammalian development. Secondary axons arose later, not only as side-branches of the primary axon but also from the soma, as in mammalian development and regeneration. Since, in fish, these responses are clearly an intrinsic part of functional regeneration, their equivalence in fish and mammals strengthens the view that a similar regenerative competence may exist in the retinal ganglion cells of all vertebrates.