Journal of Neuro-Ophthalmology, December 2010, Volume 30, Issue 4

Four Steps to Optic Nerve Regeneration

The failure of the optic nerve to regenerate after injury or in neurodegenerative disease remains a major clinical and scientific problem. Retinal ganglion cell (RGC) axons course through the optic nerve and carry all the visual information to the brain, but after injury, they fail to regrow through the optic nerve and RGC cell bodies typically die, leading to permanent loss of vision. There are at least 4 hurdles to overcome in preserving RGCs and regenerating their axons: 1) increase RGC survival, 2) overcome the inhibitory environment of the optic nerve, 3) enhance RGC intrinsic axon growth potential, and 4) optimize the mapping of RGC connections back into their targets in the brain.

Four Steps to Optic Nerve Regeneration Darcie L. Moore, PhD, Jeffrey L. Goldberg, MD, PhD Abstract: The failure of the optic nerve to regenerate after injury or in neurodegenerative disease remains a major clinical and scientific problem. Retinal ganglion cell (RGC) axons course through the optic nerve and carry all the visual information to the brain, but after injury, they fail to regrow through the optic nerve and RGC cell bodies typically die, leading to permanent loss of vision. There are at least 4 hurdles to overcome in preserving RGCs and regenerating their axons: 1) increase RGC survival, 2) overcome the inhibitory environment of the optic nerve, 3) enhance RGC intrinsic axon growth potential, and 4) optimize the mapping of RGC connections back into their targets in the brain. Journal of Neuro-Ophthalmology 2010;30:347-360 doi: 10.1097/WNO.0b013e3181e755af 2010 by North American Neuro-Ophthalmology Society Many disorders insult retinal ganglion cell (RGC) axons in the optic nerve (Fig. 1), including traumatic optic neuropathy (1), ischemic optic neuropathy (2), optic neuritis (3,4), and glaucoma (5). The underlying causes of these diverse disorders vary. In some diseases, like Leber hereditary optic neuropathy, the damage is thought to begin within the RGCs themselves; in others, such as in optic neuritis, damage to RGC axons is secondary to dysfunction or loss of the surrounding optic nerve glial cells. While there are multiple well-characterized animal models for these conditions (Table 1), much is still unknown about their initial causes and progression. In none of these diseases, however, can RGC axon fibers regenerate back to their targets, and in most of these, RGCs die (6). We review critical advances in the understanding of why regenerating optic nerves is such a daunting task. SURVIVAL OF RGCS AFTER OPTIC NERVE INJURY One of the effects following optic nerve axon injury is RGC death, which can be seen in histopathological samples from human optic neuropathies (7) and can be studied in greater detail in animal models. For example, in adult rats, 85%- 90% of RGCs die by 2 weeks after crushing or cutting the optic nerve (Fig. 2) (8). The more distant the injury from the eye, the less the severity of initial RGC death (8-10), possibly due to the support of optic nerve glial cells (see below, Fig. 1) or persistence of collateral axon branches to other supportive targets (11). The majority of dying RGCs undergo apoptosis, or programmed cell death (8,9,12). After RGC injury, there is an upregulation of proapoptotic proteins (8,13,14); conversely, overexpression of anti-apoptotic proteins, such as Bcl-2, results in an increased survival of injured RGCs (15,16). Alternatively, some RGCs undergo necrotic death or secondary degeneration after optic nerve injury but in minimal numbers (17). Depending on the location of the injury, at the cell body or along the axon, activation of distinct RGC death pathways occurs (18). What Causes RGCs to Die After Optic Nerve Injury? Axon injury disrupts the connections of RGCs to their target, resulting in a loss of target-derived neurotrophic support. Target-derived signals are retrogradely transported to the cell body and are hypothesized to be required for neuronal survival (19-23). Removal of this support leads to apoptosis, and addition of exogenous neurotrophic factors increases survival and regeneration (24). One of the neu-rotrophins shown to regulate RGC survival is brain-derived neurotrophic factor (BDNF), expressed in both the retina and the superior colliculus, which is targeted by Section Editors: Grant T. Liu, MD Randy H. Kardon, MD, PhD Institute of Cell Biology (DLM), ETH, Zurich, Switzerland; and Bascom Palmer Eye Institute (JLG), Miller School of Medicine, University of Miami, Miami, Florida. Funded by NEI (EY020913), NINDS (NS061348), The Glaucoma Foundation, the American Heart Association, the James and Esther King Foundation, an NEI P30 grant (EY014801), and an unrestricted grant from Research to Prevent Blindness to the University of Miami. D. L. Moore was a Lois Pope LIFE fellow and was supported by the National Institutes of Health Training Grants T32 NS07492 and T32 NS007459. The authors declare no conflict of interest. Address correspondence to Jeffrey L. Goldberg, MD, PhD, 1501 NW 10th Avenue, BRB 826, Miami, FL 33136; E-mail: jgoldberg@med. miami.edu Moore and Goldberg: J Neuro-Ophthalmol 2010; 30: 347-360 347 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. approximately 100% of RGC axons in the rodent and about 30% of RGC axons in humans. BDNF supports RGC survival in vitro (25,26) and in vivo (27) by binding to its receptor, tropomyosin receptor kinase B (trkB), resulting in activation of downstream effectors, including the Ras- MAPK and PI3K-Akt pathways. The increased survival after axotomy following BDNF application appears to be due to a combination of these 2 pathways (28). Would trophic factor injection alone be enough to save RGCs? After optic nerve injury, intraocular injection of BDNF neurotrophin 4/5, nerve growth factor, or insulin-like growth factor 1 leads to a temporary increase in the RGC survival (8,13,27,29-33). For example, with appli-cation of BDNF or ciliary neurotrophic factor (CNTF), 25% of RGCs are alive at 3 weeks, when nearly all RGCs without treatment would be dead, but by 7 weeks, 95% are dead with or without treatment (27). Sustained over-expression of trophic factors does not solve this problem. For example, transducing other retinal cells, such as Muller glial cells, to overexpress BDNF is neuroprotective for RGCs, but these effects do not persist (29). Neurotrophic factors, such as glial-derived neurotrophic factor (34-36) and CNTF (27,37,38), have also been found to increase RGC survival after injury. Why Is the Response to Trophic Factors So Limited? Receptors for these factors are expressed in RGCs (39-42), but after injury, these cells only transiently upregulate their neurotrophin receptors, followed by a long-term decrease in their expression (40,43). Interestingly, recent studies dem-onstrate that exogenous application of BDNF can lead to FIG. 1. The retina contains at least 6 major classes of neurons plus Muller glial cells and other astrocytes, but retinal ganglion cells (RGCs) are the only output neurons of the retina. Most of the processing of light begins with rod and cone photoreceptors in the outer nuclear layer (ONL), which transduce photons into a chemical code passed onto neurons of the inner nuclear layer (INL). The signal is modified and enhanced by these interneurons before providing input onto RGCs, which transmit both visual and non-image-forming information to the brain. In the optic nerve, RGC axons are myelinated by oligodendrocytes, increasing the speed and probably the fidelity of electrical signaling to the brain. Astrocytes also play critical roles throughout the optic nerve, communicating with both blood vessels and RGC axons in the spaces between the ensheathing myelin. The RGC axons in turn propagate an electrical signal down their length, arriving at their target structures in the brain. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium; ILM, inner limiting membrane; ELM, external limiting membrane. 348 Moore and Goldberg: J Neuro-Ophthalmol 2010; 30: 347-360 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. a decrease in regeneration (44), and application of neuro-trophic factors in general can lead to downregulation of the receptors, creating a longer-term reduced responsiveness to these factors (45), at least in animal models. It is not known whether this limited responsiveness would also be seen in humans. Neurotrophic factors for retinal neuroprotection are in the early phases of testing in humans (46,47). The failure of neurotrophins to support sustained RGC survival may also be due to a decreased ability of RGCs to respond to neurotrophins following optic nerve axon injury (48). After optic nerve injury in animal models, RGCs lose their trophic responsiveness, such that they are unable to respond to neurotrophic factors or activate their down-stream intracellular signaling components, even in the presence of BDNF, for example (26,49). This trophic responsiveness can be restored by increasing the number of trkB receptors present on the plasma membrane, either by directly overexpressing them (50) or by enhancing their recruitment to the surface. Surface recruitment can be eli-cited by elevating RGCs' intracellular cyclic adenosine monophosphate (cAMP) levels by pharmacologic treatment or depolarization (26,49). RGCs exhibit a decrease in cAMP levels after injury, possibly due to decreased electrical activity (49). These findings suggest a therapeutic approach, that is, to increase RGCs' cAMP levels after injury, although cAMP injections alone do not increase survival (51) and may need to be accompanied by neurotrophic factors. Trophic responsiveness and neuroprotection of RGCs after axon injury can also be enhanced by electrical stim-ulation (52). RGCs are less electrically active after optic FIG. 2. Retinal ganglion cell (RGC) survival is dramatically decreased following optic nerve crush in adult mice. A. RGCs can be labeled by injections of a fluorescent dye into the superior colliculus, the primary target for RGC axons in the rodent. The dye (in this example, Fluorogold, shown in red) is retrogradely transported along the length of the axons back to the cell bodies in the retina, allowing for specific labeling of RGCs. The dissected retina can be viewed en face in flatmount as above and can also be immunostained for RGC markers like beta-III tubulin (shown in green), which label RGC bodies as well as their bundled axons in the nerve fiber layer (NFL). B. When one optic nerve is crushed after such retrograde labeling, by 2 weeks, a severe loss of retrogradely labeled or tubulin-positive RGCs is noted, along with atrophy of the axon bundles in the NFL. Scale bar, 50 mm. TABLE 1. Animal models of optic nerve disease Disease Animal Model References Glaucoma DBA/2J mouse (187-190) Decreasing aqueous outflow (191,192); for review, see (193) Ischemic optic neuropathy Photochemically induced ischemic optic neuropathy (194,195) Optic neuritis 2D2 myelin oligodendrocyte glycoprotein-specific T-cell receptor mice (196,197) Traumatic optic neuropathy Optic nerve crush (198) Optic nerve transection (199) Leber hereditary optic neuropathy Mutated human ND4 gene targeted to mitochondria (200) For an additional review see Levkovitch-Verbin (201). Moore and Goldberg: J Neuro-Ophthalmol 2010; 30: 347-360 349 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. nerve injury (53). Increasing activity through electrical stimulation increases cAMP levels in RGCs (49) and greatly potentiates the neuroprotective effects of neurotrophic factor treatment (54), possibly by increasing the number of neurotrophin receptors on the neuronal surface (26). In the peripheral nervous system (PNS), electrical stimulation after injury results in an accelerated expression of regeneration-associated genes (55,56), suggesting that electrical activity may also positively influence axon growth. Recent studies have shown that transcorneal stimulation after optic nerve crush increases not only the number of surviving RGCs but also the number of axons projecting past the lesion (57,58). This experimental result suggests that electrical stimulation may be used as a therapeutic strategy to increase RGC survival and growth in future studies. In addition to the loss of positive signals such as trophic support and electrical activity, there is an increase in neg-ative prodeath signals after optic nerve injury. For example, there is an increase in superoxide levels in RGCs from the mitochondrial electron transport chain; blocking this increase leads to a reduction in RGC death (59). Providing extra neurotrophins cannot block this increase in super-oxides (59), suggesting that the cell death pathway for trophic factor withdrawal is separate from that of free radical-induced cell death. Treating RGCs with novel reducing agents to block this rise in superoxides is neuro-protective in RGCs at low doses (60). These studies suggest that there are multiple strategies for increasing RGC survival after injury, including elevating cAMP, providing multiple exogenous trophic factors, elec-trically stimulating RGCs to increase their activity, and re-ducing superoxide levels. It is likely that combinations of these strategies will be required to optimally increase RGC survival and regeneration. Some of the same signals that increase survival also increase the RGC ability to regenerate (48). THE INHIBITORY ENVIRONMENT OF THE OPTIC NERVE Why Do RGC Axons Fail to Regenerate in the Injured or Diseased Optic Nerve? After injury anywhere in the adult central nervous system (CNS), the ability of axons to regenerate is actively inhibited by the mature CNS environment and the cellular response to injury (Fig. 3). The response by meningeal cells, microglia, oligodendrocytes, and astrocytes can include migration to the site of injury, proliferation, and changes in cellular morphology and protein expression. The expression and secretion of inhibitory molecules and proteins, and the presence of myelin debris, create an unfavorable environ-ment for axon regeneration in the CNS. These inhibitory phenomena do not occur in the PNS, where axons regen-erate after injury. One difference between the CNS and PNS is the makeup of the glial cells. Whereas peripheral nerve Schwann cells are supportive of axon growth due to their secretion of neurotrophins and lack of associated in-hibitory factors, optic nerve oligodendrocytes and reactive astrocytes are inhibitory to axon growth, expressing many inhibitory proteins, as has also been demonstrated throughout the brain and spinal cord (61). Damaged oligodendrocytes degenerate after injury, leav-ing myelin debris containing inhibitory proteins such as Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (Omgp) at the site of injury. Astrocytes, however, respond to injury by becoming hypertrophic and proliferating, forming a ‘‘glial scar'' at the optic nerve injury site. Astrocytes release inhibitory extra-cellular matrix molecules, such as chondroitin sulfate pro-teoglycans (CSPGs), creating a molecular barrier to regeneration (61). In the spinal cord, treatment with a bac-terial enzyme chondroitinase ABC degrades the sulfated glycosaminoglycan side chains and can partially interfere with this inhibition (62). In addition to actively inhibiting axon growth, CSPGs may also mask growth-promoting proteins such as laminin (63) or may change normally growth-attracting proteins like semaphorin 5A into repulsive cues (64). Other semaphorins secreted from infiltrating meningeal cells and expressed by oligodendrocytes and neuroepithelial cells also inhibit axon regeneration (65,66). Some of these inhibitory molecules are used as guidance cues during development and may be reexpressed after injury. For example, semaphorin 3A acts as a repulsive guidance cue during development (67) but is also upregulated after injury, inhibiting axon regeneration (68). The expression of netrin-1 at the optic nerve head, which normally attracts RGC axons to exit the retina and grow into the optic nerve in early development, becomes an inhibitory signal in the later life due to the low levels of cAMP in adult RGCs (69). The complexity with which these inhibitory proteins interact with RGCs needs to be further defined. Can We Simply Turn Off the RGCs' Response to Such Negative Cues? Semaphorin receptors such as neuropilin-1 are upregulated following injury (70). Nogo-A, MAG, and Omgp activate neuronal Nogo receptor (NgR) protein complexes that may be constitutively expressed. Downstream signaling mole-cules such as Rho and Rho kinase (ROCK) are normally found in RGC axons. Research involving these proteins has led to varied results, limiting the ability to draw complete conclusions as to their exact mechanisms. After optic nerve crush in the chick, inactivation of MAG increased axon regeneration (71); however, optic nerve crush in MAG-knockout mice did not lead to RGC regeneration (72). CNS injuries performed on the multiple different Nogo and NgR knockouts have revealed varied results in regeneration, from none to modest regeneration and sprouting, although more positive results have been seen using dominant-negative and pharmacoinhibition strategies against these 350 Moore and Goldberg: J Neuro-Ophthalmol 2010; 30: 347-360 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. same molecules (73). Treatment with the IN-1 antibody, for example, which neutralizes Nogo-A, resulted in greater regeneration than Nogo-A gene knockouts (74,75). Treat-ment of spinal cord injury with anti-Nogo antibodies has entered clinical trials in Europe and, if successful, is almost certain to be followed by optic nerve regeneration clinical trials (http://www.axregen.eu/team/university-of-zurich/). Pretreating neurons with neurotrophins prior to expo-sure to MAG and myelin decreases their ability to inhibit axon growth. Neurotrophin pretreatment increases levels of intracellular cAMP (76), and a developmental decrease in cAMP correlates with a developmental increase in the negative response to MAG/myelin (77). These phenomena identify cAMP as an important modulator of axon growth after injury. Many of these inhibitory environmental signals con-verge on a downstream target called Rho, a small GTPase (78). Targeting a convergent downstream signal relieves the need to block each glial-associated inhibitor separately. Rho signaling leads to actin cytoskeleton remodeling (79) and growth cone collapse (80). Experiments inactivating Rho with C3 transferase, which ribosylates Rho proteins, showed increased regeneration of CNS fibers (81-84) when Rho was inactivated soon after injury (85). Multiple injections of this inhibitor after injury also increased RGC survival (85). The combination of Rho inactivation, overexpression of CNTF, cAMP treatment, and peripheral nerve grafting after axotomy enhanced viability as well as increased regeneration of those surviving fibers (86). Recently, collapsin response mediator protein 4b (CRMP4) was identified as interacting with Rho to carry out its inhibitory functions. Knockdown of CRMP4 or blocking of CRMP4 and Rho interaction resulted in attenuation of inhibition from myelin substrates, identifying an even FIG. 3. Optic nerve crush experiments allow researchers to test approaches to increase optic nerve regeneration. Rodent optic nerves can be crushed a short distance behind the eyeball using fine forceps and left for 2 or more weeks to assess for any regeneration to occur. One day before collecting the nerves for processing, a fluorescent anterograde tracer is injected into the vitreous and is taken up by retinal cells including retinal ganglion cells (RGCs), which transport the dye down the length of their axons. A. In animals with no therapeutic treatment (control, top), the axons typically stop at the crush site (asterisk) and very few will regenerate any further into the distal optic nerve. B. In contrast, animals treated with multiple injections of ciliary neurotrophic factor plus cyclic adenosine monophosphate (cAMP) demonstrate increased RGC axon regeneration past the crush site (bottom). Scale bar, 200 mm. Moore and Goldberg: J Neuro-Ophthalmol 2010; 30: 347-360 351 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. more specific therapeutic target (87). Inhibition of ROCK, a downstream effector of Rho, has also shown promising results in overcoming environmental inhibition and promoting neurite outgrowth both in vitro and in vivo (38,88-93). Besides activation of RhoA by these inhibitory envi-ronmental signals, there is an increase in intracellular cal-cium that may be involved in the downstream activation of the epidermal growth factor receptor (EGFR) and protein kinase C (PKC), although whether these 2 are interrelated has yet to be determined. Inhibition of PKC activity by CSPG and myelin-based activation increases regeneration in dorsal column axons (94). Inhibiting EGFR pharma-cologically after optic nerve crush blocks myelin and CSPG inhibition on neurite growth and promotes regeneration of RGCs (95), although this EGFR inhibitor may be acting through other mechanisms (96,97). A number of drugs that block the responses of RGCs and other CNS neurons to inhibition are now in clinical trials for spinal cord injury, and identification of further downstream targets of in-hibitory signaling will create more specific therapeutic targeting strategies for future studies (98). Not all the cellular responses to injury or disease are negative or inhibitory. For example, macrophages associated with inflammation can potentially be neuroprotective and induce axon outgrowth (99). Macrophages are recruited with lens injury and elicit an 8-fold increase in RGC sur-vival and a 100-fold increase in regeneration of RGC axons past the site of an optic nerve crush (100-102). Similar macrophage activation can be elicited by injection of zymosan, a yeast cell wall preparation that activates mac-rophages (101,103,104). How does lens injury create these effects? Macrophages migrating into the retina express oncomodulin, which causes extensive outgrowth of RGCs (with concurrent elevation of cAMP) after optic nerve crush in the adult optic nerve (105). However, activated macro-phages and oncomodulin may not be the primary effectors of increased regeneration after injury (106). Combinatorial approaches may further enhance regenerative response, as by using macrophage-derived factors to ‘‘sensitize'' neurons prior to dominant-negative suppression of the activity of NgR (107) or in combination with Rho inactivation (108). The immune system has recently been targeted for clinical trials in optic nerve neuropathies and spinal cord injuries. Immunization with a peptide derived from Nogo-A, an inhibitory protein present on myelin, can increase recovery after spinal cord injury (109). In addition, vaccination with copolymer 1 (Cop-1), a synthetic chain of amino acids that cross-reacts with myelin basic protein, could activate the im-mune system and decrease secondary degeneration of surviving fibers after optic nerve injury. Cop-1 is a drug presently used to treat multiple sclerosis and is not known to create any addi-tional immunogenic effects, making it a good candidate for clinical testing (110). Clinical trials were started for Cop-1 treatment in progressive optic nerve degeneration and started but suspended for transplantation of autologous activated macrophages into the injured spinal cord (111). Further studies to find additional proteins released following lens in-jury or macrophage activation may reveal potential candidates involved in increasing CNS survival and regeneration. Bypassing the inhibitory optic nerve environment en-tirely is the oldest approach. As early as 1911, Tello (112) used peripheral nerve grafts attached to cut optic nerve to demonstrate that RGCs could regenerate a short distance if given a permissive substrate. Such experiments were rejuvenated by Aguayo et al (113) in the 1980s, using sciatic nerve transplants to connect the retina to the superior colliculus. Although the majority of RGCs died as a result of the optic nerve injury, replacement of a portion of the injured optic nerve with a piece of peripheral nerve enabled about 20% of the surviving RGCs to regrow long axons back to their targets. This process took approximately 2 months (114-116). Why Are Peripheral Nerve Grafts Able to Support CNS Regeneration? The less inhibitory environment of the peripheral nerve, as well as the trophic environment secreted by Schwann cells, creates a very permissive substrate for growth. In addition, the peripheral nerve graft may act to change the role of cells already present in the injured optic nerve. Whereas as-trocytes typically respond to CNS injury by hypertrophy, proliferation, expression of inhibitory proteins such as CSPGs, and creation of a glial scar in peripheral nerve grafts, astrocytes were found to encircle axonal bundles and act in conjunction with the Schwann cells of the peripheral nerve to guide those axons, which regenerated through the peripheral graft, ultimately changing the environmental response at the injury site (117). In addition to peripheral nerve grafts, other substrates have been grafted in experiments to increase RGC out-growth and regeneration. Transplanted perinatal optic nerves (118), RGC target tissue from fetal brain (119-122), cell transplants (123-125), various bridge matrices con-taining Schwann cells (126-130), exogenously delivered neurotrophic factors (131-134), olfactory ensheathing cells (135,136), and peripheral nerve transplants into the vitre-ous (137,138) have shown varied results. The graft and transplantation studies have shown that, at a minimum, some CNS neurons can regenerate to their targets. It is now important to determine if these techniques can be translated into therapeutic treatments for patients. THE DEVELOPMENTAL LOSS OF RGC INTRINSIC AXON GROWTH ABILITY In nearly all the experiments described above, only a small percentage of RGCs have regenerated and typically very slowly, even when some of the inhibitory signals have been neutralized. Could it be that the adult neurons have lost 352 Moore and Goldberg: J Neuro-Ophthalmol 2010; 30: 347-360 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. their capacity to regrow axons? In spinal cord injury studies in cats, the neonatal nervous system retains its ability to regenerate, but this ability is lost as the animals develop (139). This finding was confirmed in rats whose spinal cords were injured at birth or in adulthood, demonstrating that the neonatal CNS retains the ability to regenerate, whereas the adult CNS does not (140). Is this simply because the adult glia turned on their expression of inhibitory mole-cules? This question has been addressed by using ‘‘hetero-chronic cultures,'' in which tissues from younger and older animals are cocultured. For example, embryonic retinal explants can extend axons into embryonic or adult brain explants, but adult retinal explants cannot extend axons into either, suggesting that the problem is with the retina or RGCs (141). Similarly, coculturing mature postnatal explants with young environmentally permissive explants have demonstrated a differential effect of tissue age on regenerative ability in hippocampal (142), cerebellar (143,144), and hindbrain tissues (145). Such studies have suggested that intrinsic changes within the neurons them-selves limit their regenerative ability. Pure RGC cultures were finally used to demonstrate definitively that RGCs turn off their intrinsic capacity for rapid axon growth during early development. To remove all influence of any potential extrinsic inhibitory environment, RGCs from embryonic or postnatal ages were purified away from all other cell types, allowing the study of their intrinsic axon growth capacity. Whether cultured in a strongly tro-phic environment or even transplanted back in vivo, pu-rified embryonic RGCs extended their axons up to 10-fold faster than postnatal or adult RGCs (Fig. 4) (146). Altering the extrinsic environment did not change this fundamental observation, pointing to intrinsic limitations in RGC axon growth ability. Additionally, it was found that this developmental decrease can be initiated by a membrane-associated signal on presynaptic amacrine cells (Fig. 1) (146). These findings confirm the in vivo and in situ ex-periments, suggesting that a developmental program in RGCs is involved in their inability to regenerate. Can the Intrinsic Capacity of RGCs to Regenerate in Adulthood Be Increased to Embryonic Levels? One method of increasing intrinsic axon growth ability may be to alter the expression of specific genes that are upregulated or downregulated during development or after injury or in disease. For example, overexpression of Bcl-2, an anti-apoptotic gene whose expression is decreased de-velopmentally, increases RGC survival and slightly increases the regenerative capacity of RGCs in tissue explants (147). In vivo, Bcl-2 overexpression increases regeneration of RGC axons after injury in early postnatal rodents but not in later postnatal rodents, even when the negative influence of as-trocytes is minimized (148). Using lithium to induce Bcl-2 expression and removing astrocytes at the injury site by means of an astrocyte toxin resulted in an increase in optic nerve regenerations but did not increase RGC survival (149). Bcl-2 may yet be a promising target to increase RGC survival and possibly regeneration in vivo despite these mixed results. The levels of cAMP within neurons also appear to be developmentally regulated such that embryonic neurons have high cAMP levels that drop sharply postnatally and remain low throughout adulthood (77). The response of these neurons to MAG/myelin is also dependent on the neuron's stage of development and the neuron's intrinsic level of cAMP, as described previously (77). Embryonic axons, possessing endogenously high cAMP levels, are promoted by MAG/myelin, and this effect requires the activation of the transcription factor cAMP response ele-ment- binding protein (CREB) (150). CREB's upregulation of arginase I (Arg I), an enzyme that synthesizes polyamines, may be a part of the molecular pathway involved in over-coming myelin inhibitors (150-152). The expression pro-file of Arg I parallels that of cAMP levels during development, and its overexpression is sufficient to block the switch from promotion to inhibition by MAG/myelin (150,151). Therefore, the change in cAMP levels and downstream effectors during development is a switch that allows neurons to respond differently to extrinsic inhibitory environments (77,151). Other developmentally regulated signaling pathways could be important in axon regeneration. For example, the FIG. 4. Retinal ganglion cells (RGCs) lose their intrinsic axon growth ability during development. Purified RGCs from embryonic day 20 (E20) and postnatal day 8 (P8) were cultured and immunostained against beta-III tubulin to visualize neurites. A. E20 RGCs (top) extended longer axons than P8 RGCs (bottom) after 18 hours in culture. B. At 3 days in vitro (DIV), the percentage of neurons that extended an axon were quantified at each age, demon-strating that there was no difference in the ability to extend an axon between E20 and P8 RGCs. C. The growth of E20 and P8 RGCs were measured over 3 days, revealing that E20 RGCs grew their axons approximately 10-fold faster than P8 RGCs. Taken together, these results suggest that postnatal RGCs lose their ability to rapidly extend their axons during development. Reprinted with permission from Goldberg et al (146). Moore and Goldberg: J Neuro-Ophthalmol 2010; 30: 347-360 353 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. activation of the MAP kinase pathway was recently found to be important in the regeneration of motor neurons in Caenorhabditis elegans (153). The Dlk-1/MKK-4/PMK-3 MAP kinase pathway was found to be required for axon regeneration and also for normal growth cone formation and morphology. This pathway was not necessary during development, suggesting that there are specific signaling pathways activated during regeneration that are separate from those activated during development (153). Dlk has also been shown to be important for the degeneration of severed axons, a necessary component in successful regen-erative systems, clearing the way for regeneration following injury (154). Thus, the effect of Dlk seen in regeneration could be due, at least in part, to its effect on degeneration. Another signaling pathway that is developmentally regulated centers on the mammalian target of rapamycin (mTOR) protein. Deleting the phosphatase and tensin homolog protein or the tuberous sclerosis complex 1, both of which normally inhibit mTOR, leads to a dramatic in-crease in the number of regenerating axons after optic nerve injury, as well as to an increase in RGC survival (155). Could the Activation or Inactivation of Transcriptional Programs Be Important in the Loss of Intrinsic Axon Growth Ability? In the cerebellar granule neurons (another type of CNS neuron), the ubiquitin ligase Cdh1-anaphase promoting complex (Cdh1-APC) and its downstream targets have been identified as important players in the intrinsic regenerative ability of CNS neurons. Cdh1-APC was first identified to be a cell cycle ubiquitin ligase; however, a new role for this complex has been discovered in regulating axonal growth, in particular for axon growth inhibition (156-160). Cdh1- APC targets the inhibitor of differentiation 2 (Id2) for degradation, releasing a basic helix-loop-helix transcription factor (E47) to upregulate genes involved in axon growth inhibition, including NgR (157). Cdh1 also targets the transcription factor SnoN, whose degradation results in reduced axonal growth (158). These results suggest that the loss of intrinsic axon growth ability could be induced by critical transcriptional changes. It is not known, however, if these pathways function similarly in RGCs. Our lab has recently found that a family of transcription factors, called Kru¨ppel-like factors (KLFs), may affect axon growth ability during development and regeneration. There are 17 members of the KLF family; 15 are expressed in RGCs, and many of these are developmentally regulated (161). For example, the expression of KLF4 and KLF9 increases during postnatal development. When overex-pressed in RGCs, these KLFs decrease neurite growth sig-nificantly. Using a gene knockout of KLF4 in RGCs, we have found that removal of KLF4 increases neurite growth of RGCs in vitro, and more importantly, increases axon regeneration after optic nerve injury. Taken together, these results suggest that the KLF family of transcription factors may be involved in the loss of intrinsic axon growth ability seen during development in RGCs. The presence of such a large number of signaling pathways, proteins, and transcription factors suggests that not one single target will be adequate to increase optic nerve regeneration. Coaxing RGC axons through an injured or degenerating optic nerve may take a multifactorial approach, addressing RGC survival, glial inhibitors, and RGCs' intrinsic capacity for rapid axon regeneration. TARGET REINNERVATION AFTER OPTIC NERVE INJURY Once We Are Able to Enhance RGC Survival After Injury, Overcome the Inhibitory Molecules Present at the Lesion, and Re-establish an Embryonic Growth Phenotype, Will the Axons Be Guided Back to Their Developmental Targets and Create Functional Maps of Visual Space? Work on regeneration is in too early a stage to permit pre-dictions on what will happen ‘‘postregeneration.'' However, much is known about developmental mapping from which assumptions can be drawn. For example, during embryonic development, RGC axons are attracted to the optic nerve head by glial cells expressing netrin-1 (162). Half of these axons are guided by ephrin B2 ligands to remain ipsilateral at the optic chiasm (163) and are funneled within the optic nerve by semaphorin 5A, expressed by neuroepithelial cells (164), and Slit (165-167). To keep spatial orientation intact, RGC axons must topographically map onto the superior colliculus, their target in the midbrain. This mapping occurs through gradients of ephrin ligands, which create the spec-ificity of the visual map, allowing RGCs to communicate their positional relevance (168). Are Any of These Molecules Available for Regenerating Axons Finding Their Way to Their Target Brain Regions in the Adult? Experiments have shown that after deafferentation of the superior colliculus (the major target for RGCs in rodents), there is reexpression of some of the same guidance cues, creating a crude topographic map (169-172). After optic nerve injury, ephrin A2 expression in the superior colliculus is upregulated (173-175). If RGC survival is enhanced, RGCs express the appropriate Eph A5 receptors in a gradient that mimics development (176). Other developmental guidance molecules such as ephrin B1, however, are only minimally expressed in the adult and deafferented superior colliculus (175). In experiments in which a small percentage of regenerating axons were able to reach their target through peripheral nerve grafts, functional synapses were identified at 354 Moore and Goldberg: J Neuro-Ophthalmol 2010; 30: 347-360 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. the target tissue (177-183), although the axonal arbors made by these neurons were much smaller than normal (184). The fibers that reinnervated the superior colliculus created a rough topographic map, suggesting that developmental cues may be reexpressed in the target tissue to allow for appropriate mapping (172). Additional grafting studies connecting a severed optic nerve with the pretectum (important for pupillary reflexes) revealed a restoration of a functional pupillary reflex after 2-4 months (182,185,186), supporting the notion that regenerating axons can functionally reinnervate their target tissues. Taken together, these findings suggest that if RGCs can be promoted to regenerate, there are some developmental cues available to guide axons back to their targets and possibly re-create topographic maps of visual space. CONCLUSIONS Promoting optic nerve regeneration may seem an insur-mountable task. Research in the field of CNS regeneration suggests that we must simultaneously address RGC death after optic nerve injury, the inhibitory glial environment, and changes in the RGCs' intrinsic potential for axon regen-eration. We must ultimately think about how regenerating axonsmay innervate their targets in the brain. With a number of these approaches entering into human clinical trials in the optic nerve or the spinal cord, and with many new tech-nologies and strategies, we are getting closer to offering real hope to those with optic nerve disease. 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