Promoting optic nerve regeneration

Progress in Retinal and Eye Research 31 (2012) 688e701

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Promoting optic nerve regeneration Dietmar Fischer*,1, Marco Leibinger 1 Department of Neurology, Experimental Neurology, Heinrich Heine University Düsseldorf, Merowingerplatz 1a, 40225 Düsseldorf, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 7 July 2012

Vision is the most important sense for humans and it is irreversibly impaired by axonal damage of retinal ganglion cells (RGCs) in the optic nerve due to the lack of axonal regeneration. The failure of regeneration is partially attributable to factors located in the inhibitory environment of the forming glial scar and myelin as well as an insufficient intrinsic ability for axonal regrowth. Moreover, RGCs undergo apoptotic cell death after optic nerve injury, eliminating any chance for regeneration. In this review, we discuss the different aspects that cause regenerative failure in the optic nerve. Moreover, we describe discoveries of the last two decades demonstrating that under certain circumstances mature RGCs can be transformed into an active regenerative state allowing these neurons to survive axotomy and to regenerate axons in the injured optic nerve. In this context we focus on the role of the cytokines ciliary neutrophic factor (CNTF) and leukemia inhibitory factor (LIF), their receptors and the downstream signaling pathways. Furthermore, we discuss strategies to overcome inhibitory signaling induced by molecules associated with optic nerve myelin and the glial scar as well as the regenerative outcome after combinatorial treatments. These findings are encouraging and may open the possibility that clinically meaningful regeneration may become achievable one day in the future. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: CNTF LIF Taxol Inflammatory stimulation Optic nerve regeneration Lens injury

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688 Multiple causes for regenerative failure in the optic nerve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689 2.1. Axotomy induced cell death of mature RGCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689 2.1.1. Apoptotic cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689 2.1.2. Role of trophic factors in neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690 2.2. Insufficient intrinsic growth state of mature neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691 2.2.1. Activation of the intrinsic growth state of mature RGCs by lens injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691 2.3. Inhibition of axonal regeneration by the optic nerve extracellular environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696 2.3.1. Inhibitors of CNS myelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696 2.3.2. Glial scar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 697 2.3.3. Overcoming inhibitory signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 697 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698

1. Introduction

* Corresponding author. Tel.: þ49 (0)211 302039237; fax: þ49 (0)211 302039249. E-mail address: dietmar.fi[email protected] (D. Fischer). 1 Percentage of work contributed by each author in the production of the manuscript is as follows: Dietmar Fischer: 80%; Marco Leibnger: 20%. 1350-9462/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.preteyeres.2012.06.005

The retina and optic nerve are part of the central nervous system (CNS). Retinal ganglion cells (RGCs) are a population of neurons located in the innermost layer of the retina and convey visual signals from the retina along their axons to the brain. As with other mammalian CNS neurons, RGCs are normally unable to regenerate

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injured axons after optic nerve damage that can, in severe cases, cause complete visual loss with devastating consequences to the patient’s life (Cajal, 1991). Axonal damage can be induced by trauma, cancer, ischemia or hematoma and is also often associated with certain neurodegenerative/neuroinflammatory diseases such as multiple sclerosis (Compston, 2004; Plant, 2008). In addition, it can be induced by increased intraocular pressure at the optic nerve head which is considered one of the main reasons for the irreversible degeneration of RGCs in glaucomatous optic neuropathies, the second most common cause of blindness in developed countries (Buckingham et al., 2008; Soto et al., 2011; Vidal-Sanz et al., 2011). To date no therapeutic treatment is available to substantially stimulate axon regeneration and to functionally repair axonal connections in the visual pathway. However, research in the last two decades has 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 over long distances in the injured optic nerve. Important signaling pathways and molecules that are either limiting or facilitating axon regeneration have been identified and potentially provide novel therapeutic targets. It has become clear that combinatorial treatments overcoming the inhibitory environment of the glial scar and optic nerve myelin together with approaches activating the intrinsic growth program yield stronger regeneration than each single treatment alone. In this review, we aim to provide an updated overview of the cellular and molecular mechanisms limiting axon regeneration in the mammalian optic nerve and discuss recently developed strategies overcoming these limitations. 2. Multiple causes for regenerative failure in the optic nerve The reasons for the lack of axonal regeneration in the injured optic nerve of adult mammals are manifold. Regenerative failure is mainly attributable to: 1) delayed axotomy-induced apoptotic cell death of RGCs, 2) insufficient intrinsic ability of mature RGCs to regrow axons, 3) growth-inhibitory myelin in the optic nerve, and 4) formation of an inhibitory glial scar at the injury site (Fig. 1). In the following paragraphs, we will outline the molecular mechanisms underlying these different aspects in more detail. 2.1. Axotomy induced cell death of mature RGCs Axonal injury in the optic nerve triggers cell death and degeneration of mature RGCs. RGC death does not start instantly after axotomy but is rather delayed 5e6 days after intraorbital optic nerve injury in rats and slowly continues with less than 10% of the RGCs surviving after 14 days (Berkelaar et al., 1994; Fischer et al., 2004b). The onset and progeression of RGC death are dependent on the distance of the injury to the eyeball. Intracranial lesion 8e9 mm behind the rat eye delays the onset of cell death until day 8 and more than 80% of the RGCs survive 4 weeks after optic nerve injury (Berkelaar et al., 1994). Although much has been learned about the mechanisms underlying axotomy-induced RGC death, the signaling mechanisms leading to the initiation of cell death still remain to be unraveled. The delay and distance-dependent onset of RGC death point to a so far undefined retrogradely transported signal. As RGCs are disconnected from their central targets in the brain upon axotomy of the optic nerve, the RGC somata are cut off from their supply with neurotrophic factors that are retrogradely transported from the synaptic terminals in the brain. A shortage of target-derived neurotrophic factors is therefore believed to be involved in triggering RGC death observed upon optic nerve injury (Berkelaar et al., 1994; Rabacchi et al., 1994; Almasieh et al., 2012). Such a signal could be the axotomy-induced stop of the continuous

Fig. 1. Causes for regenerative failure and degenerative response of RGCs towards optic nerve injury. A) Schematic drawing of a rodent eye with RGCs projecting axons in the intact optic nerve. B) Axons are severed after optic nerve injury. Axons distal from the lesions site undergo degeneration (Wallerian degeneration), whereas proximal axons survive, but fail to regenerate beyond the injury site. Regenerative failure is attributable to different reasons as indicated: (1) Insufficiency to initiate a regenerative program that would allow mature RGCs to regrow axons, (2) inhibitory factors associated with the myelin of the optic nerve, and (3) inhibitory factors of the glial scar that functions as a barrier for regeneration (red). C) A few days after intraorbital injury in rodents RGCs start to undergo cell death (apoptosis) with less than 10% of RGCs surviving 2 weeks after injury.

supply with trophic factors. Possible other injury induced signals could include the uptake of extracellular particles at the injury site or the lesion induced de novo synthesis of proteins in the axoplasm. These putative molecules may be retrogradely transported to the RGC soma and then trigger cell death. 2.1.1. Apoptotic cell death Research of the last two decades strongly points towards an apoptotic mechanism underlying axotomy-induced death of mature RGCs, which is dependent on Ca2þ influx as well as Bax translocation on mitochondrial membranes (Pettmann and Henderson, 1998; Cellerino et al., 2000). As a consequence, further downstream, caspases are activated, which are the executers of cell death and cleave proteins that are essential for cellular integrity. Intrinsic as well as extrinsic pathways have been shown to activate initiator caspases (Fig. 2). The intrinsic pathway involves the activation of caspase-9 by the apoptosome, which is a complex formed by procaspase-9, apoptotic protease activating factor 1 (Apaf-1) and mitochondria-derived cytochrome C. Caspase-9 then

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Fig. 2. Intrinsic and extrinsic apoptotic pathways. Apoptosis can be initiated through two distinct pathways, the extrinsic pathway activated by receptors at the cell surface and the intrinsic pathway mediated by mitochondrial homeostasis. In the extrinsic pathway, an active death receptor recruits the intracellular adaptor protein fas-associated death domain (FADD) which then recruits procaspase-8 to form a death-inducing signaling complex leading to an activation of caspases-8. Caspase-8 subsequently activates other downstream caspases. The intrinsic pathway can be activated by the deprivation of neurotrophic factors leading to caspase activation via Bax-mediated cytochrome C release from mitochondria. Both, the intrinsic and extrinsic pathways engage a common set of executioner caspases which dismantle the cell by cleaving intracellular proteins, altering or negating protein function.

cleaves and thereby activates other caspases, such as caspase- 3. In the extrinsic pathway, ligand-activated death receptor recruits the intracellular adaptor protein fas-associated death domain (FADD), which forms a death-inducing signaling complex with procaspase 8 (Kischkel et al., 1995, 2000). Subsequently, caspase-8 is activated through autoproteolysis and in turn activates caspase-3 (Muzio et al., 1998). In addition, the activation of caspases-2, -3, -6, -8 and -9 following optic nerve injury has been documented (Fig. 2) (Chaudhary et al., 1999; Kermer et al., 1999; Weishaupt et al., 2003; Cheung et al., 2004; Grosskreutz et al., 2005; Ahmed et al., 2011; Monnier et al., 2011). Consistent with a role in axotomy-induced apoptosis, inhibition of specific caspases is reportedly neuroprotective on axotomized RGCs. For instance, intraocular application of inhibitors for caspase-3 or caspase-9 as well as the knockdown of Apaf-1 using a viral siRNA strategy modestly enhances RGC survival after optic nerve injury (Kermer et al., 1999; Cheung et al., 2004; Lingor et al., 2005). Inhibition of caspase-2 expression using chemically modified siRNA robustly protected axotomized RGCs from apoptosis in vivo, suggesting a key role of caspase-2 in injury-induced cell death (Ahmed et al., 2011). In addition to caspases, Bcl-2 family members, such as Bcl-2, BclXL, and Bax, have also been shown to contribute to axotomy induced RGC death. Messenger RNA levels of anti-apoptotic Bcl-2 and Bcl-XL decrease in the retina after optic nerve injury, whereas virusmediated overexpression of Bcl-XL in RGCs enhances the survival of injured neurons (Chaudhary et al., 1999; Malik et al., 2005). Similarly, mice overexpressing Bcl-2 exhibited almost no RGC death after axotomy (Chierzi et al., 1999; Chierzi and Fawcett, 2001; Inoue et al., 2002) and knockout of Bax, a pro-apoptotic factor, reduced the number of RGCs undergoing cell death (Li et al., 2000).

Apoptotic RGCs are eventually phagocytized by retinal microglia that become active, proliferate and switch from a ramified into a phagocytic state upon injury (Wohl et al., 2010). In addition to their role in removing apoptotic neurons, microglia may also be actively involved in initiating RGC death by releasing pro-apoptotic factors, such as tumor necrosis factor a (TNF a) or interleukin 1b (IL1 b) (Thanos et al., 1993; Rao et al., 2003; Sivakumar et al., 2011). 2.1.2. Role of trophic factors in neuroprotection Since the survival of RGCs is a prerequistite for axon regeneration, neuroprotection is important to achieve functional optic nerve repair. In this context, neuroprotective factors have received significant attention. The classical neurotrophins are a family of diffusible trophic proteins that mediate several cellular responses in the CNS, such as proliferation, differentiation, axon growth as well as dendrite and synapse formation (Ebadi et al., 1997). This family comprises four peptides, namely nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 4/5 (NT4/ 5), and neurotrophin 3 (NT3). Neurotrophins bind with high affinity to specific tyrosine kinase receptors (TrK) that are classified as Trk-A (NGF), Trk-B (BDNF, NT4/5), and Trk-C (NT3). NT-3 also binds Trk-A and -B with low affinity. Mature RGCs express all three Trk receptors, but significant neuroprotection of axotomized RGCs has been reported only for BDNF and NT4/5 (Mey and Thanos, 1993; Cohen et al., 1994; Mansour-Robaey et al., 1994). Besides the classical neurotrophins, cytokines of the IL-6 superfamily such as leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF), have been shown implicated in neuroprotection (Mey and Thanos, 1993; Zhang et al., 2005; Leaver et al., 2006b; Leibinger et al., 2009). Both cytokines mediate their effects through the

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responsiveness towards their ligands. Accordingly intravitreal application of FGF2, GDNF, HGF and GM-CSF reportedly increase the survival of mature RGCs upon optic nerve injury (Sievers et al., 1987; Bahr et al., 1989; Koeberle and Ball, 1998, 2002; Schmeer et al., 2002; Schallenberg et al., 2009; Tonges et al., 2011). For most neurotrophic factors it is, however, not clear whether their beneficial effects were mediated directly on RGCs or indirectly via activation of other retinal cells, which then release additional factors confering neuroprotection on RGCs. For instance, injection of high dosages of recombinant CNTF into the vitreous chamber activates retinal glia to express and release endogenous CNTF (Peterson et al., 2000; Müller et al., 2009). Accordingly, the beneficial effects of recombinant CNTF application are significantly reduced in CNTF knockout animals (Müller et al., 2009). BDNF is one of the most effective survival agents for axotomized RGCs (Mansour-Robaey et al., 1994; Pernet and Di Polo, 2006). However, the neuroprotective effects of BDNF and other factors even when continuously provided to RGCs, are transient and only delay the progress of neuronal degeneration rather than preventing it (Di Polo et al., 1998; Leaver et al., 2006a, 2006b). It is also important to note that increased survival, although essential for regeneration, does not per se enable RGCs to regrow injured axons. BDNF, for example, does not enhance axon regeneration, but rather blocks the effects of axon growth-promoting treatments (Cui et al., 1999; Pernet and Di Polo, 2006). Moreover, mice overexpressing the anti-apoptotic protein Bcl-2, show almost no RGC death after axotomy, but do not regenerate axons into the optic nerve (Chierzi et al., 1999; Chierzi and Fawcett, 2001; Inoue et al., 2002). Thus, neuroprotection and axon regeneration may be mediated by different signaling pathways in the mammalian visual system, although there seems to be a significant degree of overlap (Fischer, 2012). 2.2. Insufficient intrinsic growth state of mature neurons

Fig. 3. Mechanisms underlying the beneficial effects of inflammatory stimulation. A) Schematic drawing of eye, optic nerve and regenerating RGCs. Lens injury or intravitreal application of toll-like receptor agonists (zymosan, Pam3Cys) protect axotomized RGCs from cell death and enable these neurons to regenerate axons beyond the injury site of the optic nerve. B) Lens injury causes a release of crystallins of the b/gsuperfamily, which induce the expression of CNTF and LIF in retinal astrocytes/Müller cells and activate macrophages. Macrophage-derived factors may also influence the activation of retinal astrocytes and Müller cells and contribute to the induction of CNTF and LIF expression. Similar responses can be evoked by intravitreal injections of toll like receptor 2 agonists like zymosan and Pam3Cys. Glial-derived CNTF and LIF mediate the neuroprotective, axon growth promoting and disinhibitory effects of inflammatory stimulation. Additional glial or macrophage-derived factors may synergistically contribute or potentiate these effects.

signal transducing receptors glycoprotein 130 (gp130) and LIFreceptor. Whereas LIF directly interacts with the LIF receptor, which subsequently forms a complex with gp130, CNTF first binds to CNTF-receptor a, which then recruits the other signaling receptor subunits (Fig. 4) (Heinrich et al., 2003). All these receptor components are expressed by mature RGCs, and both cytokines have been demonstrated to be potent neuroprotective molecules for axotomized RGCs in vivo and in vitro (Mey and Thanos, 1993; Sarup et al., 2004; Leaver et al., 2006b; Müller et al., 2007; Leibinger et al., 2009). In addition, RGCs express several receptors of other trophic factors such as fibroblast growth factor receptor (FGFR1), GDNF family receptor a 1 (Ret/GFRa1), hepatocyte growth factor receptor (HGFR), and granulocyteemacrophage colony stimulating factor receptor (GM-CSF-a-R), suggesting a potential

During emryonic development, neurons extend axonal processes over long distances and form synapses with their appropriate targets in the brain. At this stage, RGCs are in an active growth mode, which is associated with the expression of specific “axonal growth associated genes” and the activation of specific signaling pathways (Fu and Gordon, 1997; Goldberg et al., 2002b; Moore et al., 2009). Thus, embryonic RGCs show high axonal growth rates in culture and in vivo. This growth rate decreases dramatically between embryonic day 21 and postnatal day 2 and even declines further for adult RGCs (Goldberg et al., 2002a). RGCs of adult mammals fail to switch into a robust regenerative state after axonal injury and only a small percentage of dissociated neurons extends axons in culture or can regrow into the growth permissive environment of sciatic nerve grafts transplanted to the proximal end of a transected optic nerve in vivo (Aguayo et al., 1981; So and Aguayo, 1985; Bray et al., 1987; Vidal-Sanz et al., 1987; 1988; Yin et al., 2003; Müller et al., 2007; Grozdanov et al., 2010). As pointed out later in more detail, removal or blockade of extracellular inhibitory activities alone is insufficient to yield significant regeneration in vivo (Fischer et al., 2004b; Sengottuvel and Fischer, 2011; Sengottuvel et al., 2011), underlining the importance of axon growth stimulation to achieve significant optic nerve repair. 2.2.1. Activation of the intrinsic growth state of mature RGCs by lens injury Puncture of the ocular lens induces a robust regenerative response in mature axotomized RGCs (Fischer et al., 2000; Leon et al., 2000). As a result, axotomy induced cell death is markedly delayed and most of these neurons regenerate lengthy axons into

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Fig. 4. Scheme of signaling pathway activation in RGCs following CNTF and LIF stimulation and the role of the JAK/STAT3 and PI3K/AKT/mTOR pathways regarding different aspects of axon regeneration. Binding of CNTF and LIF to their receptors (CNTFR, LIFR, and gp130) activates the Janus-kinases (JAK1, 2), which either phosphorylate the transcription factor STAT3 (JAK2) or activate SHP2 (JAK1). Phosphorylated STAT3 forms dimers, which translocate into the nucleus to initiate gene expression. STAT3 induced gene expression of suppressor of cytokine signaling 3 (SOCS3) serves as a negative feedback loop since SOCS3 inhibits JAK2 and thereby hinders STAT3 activation. SHP2 activates phosphatidylinositol 3-kinase (PI3K) which then converts phosphatidylinositol (4,5) bisphosphate (PIP2) into phosphatidylinositol (3,4,5) trisphosphate (PIP3). PIP3 stimulates phosphatidylinositoldependent kinase 1/2 (PDK1/2), which subsequently activates AKT via phosphorylation. Phosphatase and tensin homolog (PTEN) counteracts this activation by catalyzing the conversion of PIP3 to PIP2. One of the activated downstream signaling targets of AKT is the mammalian target of rapamycin (mTOR), whose activation can be specifically blocked by rapamycin. Activated mTOR phosphorylates and thereby inhibits the E4 binding protein1 (E4-BP1), a repressor of the eukaryotic translation initiation factor 4E (EIF-4E). Additionally, S6 Kinase 1 (S6K1) and its target, the ribosomal protein S6 are also controlled by mTOR. Cytokine mediated activation of mTOR is involved in maintaining RGCs in an active regenerative state and also conveys disinhibitory effects. Other unknown signaling pathways that are also activated by the CNTF- and LIF-receptors may also be involved in mediating the neuroprotective effects of IS and the initial transformation of RGCs into a regenerative state.

the axon growth supportive environment of a peripheral nerve graft (Fischer et al., 2000; Yin et al., 2003). Moreover, lens injury also enables RGCs to regenerate axons into the inhibitory environment of either a transected/resutured or crushed optic nerve (Figs. 3A and 7D, E) (Fischer et al., 2000, 2001; Leon et al., 2000; Yin et al., 2003). Thus, lens injury exerts neuroprotection, axon growth promotion and to some degree disinhibitory effects on RGCs. The lens-injury induced ability to regrow axons is not a simple consequence of its neuroprotective effect on RGCs. This is because RGCs that have been pretreated in vivo by lens injury show, in contrast to untreated controls, spontaneous axon growth with a 2.3 times higher growth rate on a growth-permissive substrate (Fischer et al.,

2004b; Müller et al., 2007). As shown by a microarray study on isolated and purified RGCs, these neurons also alter their gene expression pattern when entering the regenerative state 3e4 days after an optic nerve crush/lens injury (Fischer et al., 2004b). These changes include genes such as Galanin, SPRR1A, and GAP43 whose expression is also upregulated in regenerating neurons of the peripheral nervous system and indicate a switch of RGCs into an active regenerative state upon lens injury (Bonilla et al., 2002; Fischer et al., 2004b). 2.2.1.1. Mechanisms underlying the effects of lens injury. In order to identify the lenticular components mediating the beneficial effects

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Fig. 5. Inhibitory signaling of myelin and glial scar derived factors. Schematic drawing depicting myelin and glial scar derived inhibitory factors, their receptors and the converging downstream signaling pathway causing a destabilization of the actin cytoskeleton in the growth cones. Myelin derived inhibitory proteins NogoA (with two inhibitory domains, Nogo66 and Amino-Nogo), myelin associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) bind to the paired immunoglobulin-like receptor B (PirB) or the Nogo receptor 1 (NgR1). MAG also binds to NgR2. Binding of myelin derived inhibitory ligands to NgR initiates the formation of a functional ternary complex consisting of NgR, leucine rich repeat and Ig domain-containing NgR interacting protein 1 LINGO-1 and either p75NTR or alternatively TROY. Further downstream, the ras homolog gene A (RhoA) and its target the rho-associated protein kinase (ROCK) are activated and induce depolymerization of actin filaments (f-actin) via activation of LIM kinase (LIMK) and cofilin. Additionally, oligodendrocyte derived sulfatide and other inhibitors, such as ephrins and netrins, activate RhoA via their respective receptors receptors. Chondroitin sulfate proteoglycans (CSPGs) released by reactive astrocytes also activate RhoA after binding to either NgR1, NgR3 or the protein tyrosine phosphatase s (PTPs) followed by actin depolymerization. Since most inhibitors activate RhoA/ROCK signaling, blocking this pathway by the RhoA antagonist ADP-ribosyltransferase C3 or by ROCK inhibitors like Y27632 can be used to overcome inhibition of axonal growth.

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Fig. 6. Taxol overcomes the inhibitory effects of myelin and CSPGs. A) Schematic drawing of an intact neuronal growth cone showing the distribution of the cytoskeleton elements. Bundled actin filaments (F-actin bundles, red) form the filopodia, while mesh-like branched F-actin networks give structure to lammelopodia-like veils. Individual dynamic ‘pioneer’ microtubules (MTs, green) explore this region, usually along F-actin bundles. The central part of the growth cone contains stable, bundled MTs that enter the growth cone from the axon shaft. B) Scheme of the Taxol effect on microtubules, which are subjected to the equilibrium between polymerization and depolymerization of tubulin dimers at plus ends. Low concentrations of Taxol lead to a shift of this equilibrium towards the polymerization site, resulting in enhanced binding of the a-tubulin subunit to the plus ends of the microtubules and therefore promotion of axonal growth. C) Growth cones of RGCs stained with phalloidin (red) and antibodies against bIII-tubulin (green) demonstrating that Taxol stabilizes axonal growth cones against myelin-induced collapse. RGCs were cultured for 3 days, then some of the cells were treated with Taxol for 3 h, and afterwards half of the cultures were exposed to CNS myelin extract for 10 min as indicated. Myelin-treated growth cones are also depicted at higher exposure times, indicating that microtubules (green) are also located in the extensions (arrows) of the growth cones that were exposed to Taxol. Scale bar, 2 mm. D) Quantification of neurite outgrowth of RGCs in cultures 3 days after exposure to vehicle () or Taxol (3 nM). In addition, half of the cultures were also exposed to CNS myelin extract (Myelin). Treatment effects: ***p < 0.001. E) Quantification of neurite outgrowth of RGCs in cultures 3 d after exposure to vehicle () or Taxol (3 nM). In addition, half of the cultures were exposed to the proteoglycan neurocan (NC; 5 mg/ml). Treatment effects: ***p ¼ 0.001. Data presented in D and E demonstrate that Taxol treatment overcomes myelin and neurocan (CSPG) induced inhibition of neurite growth.

of lens injury, Fischer et al. (2008) fractionized and purified lens homogenate by gel filtration chromatography and applied each purified fraction into the vitreous body after optic nerve injury. Crystallins are the most abundant proteins in the lens (Wistow, 1993) and intravitreal applications of b- and g-crystallins, which are members of a common superfamily, fully mimicked the beneficial effects of lens injury. Moreover, exposure of retinal explants to b- and g-crystallins enhanced axon regeneration in culture, suggesting that these proteins directly interact with retinal cells

(Liedtke et al., 2007; Fischer et al., 2008). However, it was shown later, that the effects of crystallins are mainly indirectly mediated via the activation of retinal astrocytes and Müller cells and the subsequent release of CNTF and LIF (Fischer et al., 2008; Leibinger et al., 2009). Alternatively, lens injury effects can be induced by implanting pieces of peripheral nerve into the vitreous body or by intravitreal injection of toll-like receptor 2 agonists, such as the yeast wall extract zymosan or the small molecule Pam3Cys (Berry et al., 1996; Leon et al., 2000; Yin et al., 2003; Hauk et al., 2009;

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manner (Jo et al., 1999; Yin et al., 2003; Müller et al., 2007, 2009; Ahmed et al., 2009; Leibinger et al., 2009; Sengottuvel et al., 2011), but do not necessarily require the presence of compounds elevating intracellular levels of cyclic adenosine monophosphate (cAMP) as reported for other factors (Yin et al., 2003). Nevertheless, elevation of cAMP potentiates the beneficial effects of CNTF and IS (Cui et al., 2003; Yin et al., 2003; Müller et al., 2007, 2009; Park et al., 2009). The neuroprotective and axon growth promoting effects of intravitreally applied recombinant CNTF are less pronounced than those seen after IS (Müller et al., 2007, 2009; Lingor et al., 2008; Smith et al., 2009). This discrepancy may be explained by the short halflife of exogenous CNTF in the vitreous body whereas astrocytederived CNTF and LIF are constantly released over at least several days and are directly accessible for RGCs after IS (Müller et al., 2007; Leibinger et al., 2009). Accordingly, Harvey and colleagues demonstrated that a constant supply of releasable CNTF via viral expression resulted in significantly stronger neuroprotection and regeneration, with axons reaching the optic chiasm 5 weeks after an intraorbital optic nerve crush (Leaver et al., 2006a, 2006b; Hellstrom et al., 2011b). Therefore, glial-derived CNTF and LIF are the principal factors mediating the beneficial effects of IS, but additional glial- or macrophage-derived factors may synergistically contribute or potentiate their effects (Fig. 3B). For instance, expression of Apolipoprotein E (ApoE) potentiates the biological activity of CNTF (Gutman et al., 1997) and it is reportedly upregulated in Müller cells upon IS. IS triggered neuroprotective and axon growth promoting effects are significantly reduced in ApoE deficient mice (Lorber et al., 2009). Fig. 7. Regeneration of axons in the injured optic nerve after activating the intrinsic growth state and Taxol treatment. A) Schematic drawing of the local application of PBS or Taxol at the lesion site and inflammatory stimulation (IS). Gel foam soaked with the solutions is shown in light blue. IS was induced by lens injury. BeE) Longitudinal sections through the rat optic nerve were stained with an antibody against GAP-43 2 weeks after an optic nerve crush (ONC). IS induces axon growth beyond the lesion site of the optic nerve (D), whereas an ONC alone shows almost no regeneration (B). Local treatment of the optic nerve with Taxol allows some axons to regenerate in the optic nerve (C), whereas the amount of regeneration after a combinatorial treatment of Taxol and IS is markedly enhanced (E). Asterisks indicate the lesion sites. Arrows point to the longest axons. Scale bar ¼ 100 mm.

Pernet et al., 2011). Intravitreal application of lens proteins, zymosan or Pam3Cys activates retinal astrocytes/Müller cells and also induces infiltration of activated macrophages into the eye (Fig. 3). Thus, the term “inflammatory stimulation” (IS) can be used as a more general phrase to describe all these treatments. To identify the mechanisms underlying the IS induced effects, research has been initially focused on the role of macrophages (Leon et al., 2000; Yin et al., 2003). However, intravitreal injections of activated primary macrophages do not improve axon regeneration or increase GAP-43 expression in RGCs (Leon et al., 2000). Furthermore, macrophage depletion in the inner eye does not significantly affect the beneficial effects of IS (Hauk et al., 2008; Fischer, 2010), suggesting that macrophage independent factors are the principal mediators of IS. In contrast to macrophages, the activation of retinal glia is closely correlated with the transformation of RGCs into a regenerative state (Müller et al., 2007; Lorber et al., 2008, 2009; Fischer, 2010). Moreover, IS induces the expression of neuroprotective CNTF and LIF in retinal astrocytes (Müller et al., 2007; Leibinger et al., 2009). Intravitreal injections of neutralizing antibodies against CNTF or the genetic knockout of this cytokine significantly reduce IS effects. All neuroprotective and axon growth promoting effects of IS are absent in mice deficient for CNTF and LIF, underlining the key role of both cytokines in this context (Leibinger et al., 2009). In vitro, CNTF and LIF potently enhance neurite outgrowth of RGCs in a concentration dependent

2.2.1.2. Signaling pathways involved in cytokine induced axon regeneration of mature RGCs. As mentioned before, CNTF and LIF belong to the family of interleukin-6 (IL-6)-type cytokines, which comprises IL-6, interleukin-11 (IL-11), oncostatin M (OSM), cardiotrophin-1 and cardiotrophin-like cytokine (Heinrich et al., 1998). Receptors mediating the effects of IL-6-type cytokines can are classified in 1) non-signaling a-receptors (IL-6Ra, IL-11Ra, and CNTFRa, where R refers to receptor) and 2) signal transducing receptors (gp130, LIFR, and OSMR). The latter receptors become tyrosine phosphorylated and associate with Janus-Kinases (JAKs) in response to cytokine stimulation. Each of the IL-6-type cytokines is characterized by a certain profile of receptor recruitment that in all cases involves at least one molecule of gp130. CNTF first binds specifically to the CNTFa-receptor subunit. The complex of cytokine and a-receptor efficiently recruits the signaling receptor subunits (LIFR and g130) and forms a ternary receptor complex. In contrast, LIF directly engages its signaling receptor subunits and signals through a heterodimeric receptor without requirement for an additional a-receptor subunit (Fig. 4) (Heinrich et al., 2003). As pointed out previously, mature uninjured and axotomized RGCs express CNTFRa and LIFR as well as the ubiquitously expressed gp130, suggesting that CNTF and LIF mediate their beneficial effects directly on RGCs through these receptors (Sarup et al., 2004). Following CNTF or LIF binding, the gp130-associated JAKs (JAK1, JAK2, and TYK2) phosphorylate tyrosine residues in the cytoplasmic tail of the receptors, which serves as docking sites for signal transducers and activators of transcription (STAT; mainly STAT3) and the protein tyrosine phosphatase SHP-2 (Rane and Reddy, 2000; Müller et al., 2007). Recruitment of STAT3 monomers to gp130 results in their phosphorylation, dimerization and translocation to the nucleus, where activated STAT3 binds to specific DNA response elements in the promoters of target genes (Stahl et al., 1994; Hemmann et al., 1996). Recruitment and phosphorylation of SHP-2 results in activation of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) and phosphatidyl inositol-3-phosphatekinase (PI3K/Akt) signaling

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pathways (Fukada et al., 1996; Kim and Baumann, 1999; Nishida et al., 1999; Ernst and Jenkins, 2004; Leibinger et al., 2012). Activated SHP-2 can also act as a negative regulator of IL-6 type cytokine signaling by inhibiting JAK mediated activation of STAT3 (Fig. 4) (Lehmann et al., 2003). CNTF and LIF treatment or IS activate the JAK/STAT3-, PI3K/AKT- and MAPK/ERK-signaling pathways and intravitreal application of specific inhibitors of all three signaling pathways significantly compromises CNTF-stimulated neuroprotection and axon regeneration in vivo (Park et al., 2004; Müller et al., 2009). However, only inhibition of the JAK/STAT3 and the PI3K/Akt, but not the MAPK/ERK-signaling pathway reduced CNTFinduced neurite outgrowth in cell culture, suggesting that the activation of the MAPK/ERK pathway is not directly involved in stimulating axon growth (Müller et al., 2009). Consistent with this notion, intravitreal application of recombinant CNTF activates the MAPK/ERK pathway mainly in retinal Müller cells, which is essential for the upregulation of endogenous CNTF expression in these cells. Since endogenous glial derived CNTF has been shown to contribute to the beneficial effects of CNTF injection it has to be concluded that MAPK/ERK inhibition compromises the beneficial effects of IS rather indirectly (Müller et al., 2009). 2.2.1.2.1. PI3K/AKT/mTOR pathway. CNTF and LIF also activate via JAK 1 and SHP2 phosphatidylinositol 3-kinase (PI3K), which then converts phosphatidylinositol (4,5) biphosphate (PIP2) into phosphatidylinositol (3,4,5) triphosphate (PIP3). PIP3 in turn activates phosphatidylinositol-dependent kinase 1/2 (PKD1/2), which subsequently phosphorylates AKT. Phosphatase and tensin homolog (PTEN) counteracts this activation of AKT by catalyzing the conversion of PIP3 to PIP2 and thereby functions as a negative regulator of the PI3K/AKT pathway. A downstream signaling target of AKT is the mammalian target of rapamycin (mTOR), whose activity can be specifically blocked by rapamycin. mTOR controls protein translation via E4 binding protein1 (E4-BP1), a repressor of the eukaryotic translation initiation factor 4E (EIF-4E) and S6 Kinase 1 (S6K1; Fig. 4). The genetic deletion of PTEN is reportedly neuroprotective and potently promotes RGC axon regeneration to an extent similar to IS (Park et al., 2008; Sun et al., 2011). Consistently, the axon growth stimulatory effects of PTEN deletion are blocked by mTOR inhibition. Moreover, deletion of tuberous sclerosis complex 1 (TSC1), a negative regulator of mTOR signaling, leads to a constitutive activation of mTOR and, for the most part, mimics the effects of PTEN deletion. Activation of the mTOR pathway therefore seems to potently enhance neuroprotection and axon regeneration (Park et al., 2008). However, the role of mTOR in CNTF and IS mediated axon regeneration of RGCs proved to be more complex. Although CNTF treatment and IS prevent the downregulation of mTOR activity in axotomized RGCs in a PI3K dependent manner, inhibition of mTOR activity by rapamycin does neither prevent CNTF-induced neurite outgrowth stimulation in culture nor the initial transformation of RGCs into a regenerative state or the neuroprotective effects of IS in vivo. Rapamycin treatment compromises mainly long distance, but not short distance axon regeneration in the optic nerve following IS, indicating that mTOR activity is important for sustaining RGCs in the regenerative state (Leibinger et al., 2012). Consistently, PTEN depletion in floxed mice in combination with IS allowed some axons to regenerate over about 5 mm through the whole optic nerve and to reach their central targets in the brain (Kurimoto et al. 2011). Although the reported reinnervation of the target area was still very limited and although this treatment caused cataracts and intraocular inflammation, this amount of regeneration was allegedly sufficient to partially recover some visually guided behaviors (de Lima et al., 2012). Moreover, mTOR activity seems to also be involved in mediating disinhibitory effects, as has been shown before for the PI3K/AKT

axis (Perdigoto et al., 2011). Neurite outgrowth of cultured RGCs on a growth-permissive substrate is not reduced by rapamycin, but mTOR inhibition markedly reduced growth on myelin and CSPGs containing inhibitory substrates, suggesting that the effect of mTOR is downstream of specific inhibitory receptors and may affect a common converging point (Leibinger et al., 2012). 2.2.1.2.2. JAK/STAT3 pathway. Pharmacological inhibition of JAK2 by AG490 blocks CNTF-induced neurite outgrowth of mature RGCs in culture and significantly reduces the regenerative response of RGCs to IS in vivo, suggesting that JAK- and subsequent STAT3 activation is important in initiating axonal growth (Müller et al., 2007, 2009). One of the genes, which is induced upon JAK/STAT3 activation is suppressor of cytokine signaling 3 (SOCS3; Fig. 4). SOCS3 acts as feedback inhibitor of the JAK/STAT3 pathway and avoids excessive STAT3 phosphorylation (Nicholson et al., 2000). Thus, elevated expression of SOCS3 blocks the JAK/STAT3 pathway and thereby limits some of the physiological consequences of STAT3-mediated signaling (Shouda et al., 2001; Jo et al., 2005). Consistently, viral overexpression of SOCS3 in RGCs markedly compromises the axon growth promoting effects of intravitreally applied recombinant CNTF (Hellstrom and Harvey, 2011; Hellstrom et al., 2011a). Upregulation of SOCS3 expression is also detected in mature axotomized RGCs after IS (Fischer et al., 2004b) and may influence JAK/STAT3 signaling. In fact, pSTAT3 levels reach a maximum about 2.5 days after IS and decrease afterwards (Müller et al., 2007). However, pSTAT3 levels remain significantly increased even 14 days after IS compared with untreated controls, demonstrating that endogenous SOCS3 expression prevents excessive JAK/ STAT3 signaling, but does not completely prevent its activity when cytokines are continuously supplied to RGCs (Müller et al., 2007). Accordingly, conditional knockout of SOCS3 in RGCs allows RGCs to regenerate axons beyond the lesion site of a crushed optic nerve and enhances the effects of intravitreally applied recombinant CNTF, suggesting that SOCS3 functions as an intrinsic brake for CNTF-mediated regeneration (Smith et al., 2009; Sun et al., 2011). SOCS3 expression has been shown to be suppressed by cAMP elevation (Park et al., 2009), which might explain the enhancing effects of cAMP elevating drugs on CNTF and IS induced axon regeneration (Cui et al., 2003; Müller et al., 2007, 2009; Kurimoto et al., 2011). 2.3. Inhibition of axonal regeneration by the optic nerve extracellular environment Beside the intrinsic insufficiency of RGCs to regrow axons, the extracellular environment of the optic nerve is inhibitory to axonal growth, particularly at the injury site to which the axonal tips are exposed, representing another obstacle for axonal regeneration. The significant contribution of the inhibitory environment in counteracting axon regeneration in vivo becomes evident by the fact that RGC axons, which normally do not regenerate in the injured optic nerve, are able to grow into a peripheral nerve graft that is transplanted at the stump after optic nerve transection (Aguayo et al., 1987; Vidal-Sanz et al., 1987). Using this peripheral nerve bridge, Aguayo and colleagues guided injured axons back to their original targets resulting in synapse formation in the superior colliculus (Vidal-Sanz et al., 1987; Keirstead et al., 1989). These studies demonstrate, despite a generally weak regenerative response, that mature RGCs are not completely incapable of regrowing axons and initiated the search for axon growth inhibitory factors, their receptors and downstream signaling pathways. 2.3.1. Inhibitors of CNS myelin Myelin that normally wraps around axons in the optic nerve, gets fragmented after injury. Thus, damage to the optic nerve

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environment leads to the exposure of transected axon tips to inhibitory myelin molecules. These inhibitors bind to their specific receptors on the axon, which susequently leads to destabilization of the actin cytoskeleton in filopodia and lamellipodia of the growth cone and thereby counteracting axonal regeneration (Yiu and He, 2006; Berry et al., 2008). Inhibitory proteins of the CNS myelin comprise molecules such as Nogo, myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMgp; Fig. 5) (McKerracher et al., 1994; Mukhopadhyay et al., 1994; Chen et al., 2000; GrandPre et al., 2000; Wang et al., 2002b). The transmembrane protein MAG is a member of the immunoglobulin (Ig) superfamily and is expressed in myelin of both the CNS as well as the peripheral nervous system (PNS) (McKerracher et al., 1994). Nogo belongs to the reticulon family of transmembrane proteins usually located in the endoplasmatic reticulum, although it has also been found on the oligodendrocyte surface and the innermost loop of the myelin membrane. It exists in three known isoforms; i.e., NogoA, NogoB and NogoC. While NogoB and NogoC are widely distributed outside the CNS, the expression of NogoA occurs mainly in oligodendrocytes. NogoA possesses two distinct inhibitory domains: 1) Nogo66 (a 66-amino acid loop) that is present in all three isoforms and 2) Amino-Nogo, a unique amino terminal region found only in Nogo A at the N-terminus of the molecule (Chen et al., 2000; GrandPre et al., 2000; Prinjha et al., 2000; Fournier et al., 2001; Oertle et al., 2003). The glycosylphosphatidylinositol-anchored glycoprotein (GPI) OMgp is also expressed by oligodendrocytes and is reportedly as potent as Nogo66 in mediating axon growth inhibition (Kottis et al., 2002; Wang et al., 2002b). Even though these three myelin-associated proteins are structurally heterogeneous, they can all bind with similar affinity to the Nogo receptor (NgR), a GPI-linked (Domeniconi et al., 2002; Liu et al., 2002; Wang et al., 2002b). Different homologues of the NgR exist in neurons; i.e., NgR1, NgR2, and NgR3 (Lauren et al., 2003; Pignot et al., 2003). Because the NgR lacks an intracellular domain downstream signaling depends on coreceptors. NgR1 forms a functional ternary complex with members of the tumor necrosis factor (TNF) receptor family, either p75NTR or alternatively TROY, and with leucine rich repeat (LRR) and Ig domain-containing NgR interacting protein 1 LINGO-1; Fig. 5) (Wang et al., 2002a; Wong et al., 2002; Yamashita et al., 2002; Mi et al., 2004). More recently, the paired immunoglobulin-like receptor B (PirB), which is expressed in the optic nerve and retina was identified as another functional receptor for Nogo, MAG, and OMgp (Atwal et al., 2008; Cai et al., 2012). Knockdown of NgR or overexpression of a dominant negative form of the receptor lacking the binding site to its co-receptors markedly enhanced axonal regeneration in the optic nerve in vivo when RGCs were transformed into an active regenerative state by a lens injury (Fischer et al., 2004a; Chen et al., 2009; Su et al., 2009). In contrast, expression of the dominant negative form alone showed almost no improvement of axonal regeneration, implying that overcoming myelin inhibition alone is not sufficient to yield significant regeneration in the optic nerve. However, other myelin associated proteins may also contribute to axon growth inhibition in the injured optic nerve. For example, repellents that prevent axons from targeting inappropriate areas during development are upregulated in the adult CNS. Among these factors are Ephrin-B3 (Benson et al., 2005; Duffy et al., 2012), semaphorins 5A and 4D (Moreau-Fauvarque et al., 2003; Goldberg et al., 2004), and netrin-1 (Ellezam et al., 2001; Manitt et al., 2001), which are expressed by oligodendrocytes. More recently, sulfatide, a major constituent of CNS myelin has been proposed as another myelin-associated inhibitor of neurite outgrowth. Mice that are unable to make sulfatide exhibit a small but significant enhancement in the extent of IS-induced regeneration in the optic nerve (Winzeler et al., 2011).

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2.3.2. Glial scar Apart from myelin-derived inhibitors, the glial scar formation induced by inflammatory events at the injury site represents another significant barrier for axonal regeneration in the CNS (Silver and Miller, 2004). The damaged tissue around the epicenter of the lesion reacts with proliferation and activation of resident microglia and recruitment of macrophages from the bloodstream. In addition, astrocytes proliferate and together with the production and release of specific proteins form a glial scar. Inhibitory molecules identified in the glial scar are Semaphorin 3A, Slit-1, TenascinR, and, of most importance, chondroitin sulfate proteoglycans (CSPGs) (McKeon et al., 1991; Niederost et al., 1999; Becker et al., 2000; Hagino et al., 2003; Tang, 2003; Kaneko et al., 2006). The release of CSPGs is triggered by the signaling of various cytokines and growth factors associated with the process of scaring (Logan and Berry, 2002). For instance, transforming growth factor b (TGFb), which is released into the traumatic CNS wounds by platelets and macrophages induces fibrotic scarring and the deposition of CSPGs from reactive astrocytes (Logan et al., 1994, 1999; Rimaniol et al., 1995; Lagord et al., 2002). Recently, the transmembrane protein tyrosine phosphatase (PTPs) was discovered as a functional receptor for CSPGs. Accordingly, neurons from PTPs knockout mice displayed reduced sensitivity towards CSPGs in culture (Shen et al., 2009) and enhanced axonal regeneration beyond the glial scar in the optic nerve (Sapieha et al., 2005). More recently, NgR1 and NgR3 have been proposed as additional receptors for CSPGs (Fig. 5) (Dickendesher et al., 2012). 2.3.3. Overcoming inhibitory signaling 2.3.3.1. Inhibition of the RhoA/ROCK pathway. Intracellular signaling pathways of myelin inhibitors as well as molecules associated with the inhibitory glial scar converge on the ras homolog gene A/rho-associated protein kinase (RhoA/ROCK)pathway activation, leading to actin depolymerisation via LIM kinase and cofilin stimulation (Fig. 5). Ultimately, actin filament degradation induces immobility and/or collapse of the growth cone pathways (Mueller, 1999; Wong et al., 2002; Hsieh et al., 2006; Ji et al., 2006; Lingor et al., 2007). It might therefore be more efficient to target the axon growth inhibitory signaling cascade at the level of RhoA and/or ROCK rather than to directly neuralize molecules or receptors. ADP ribosyltransferase C3 is a bacterial protein that efficiently and irreversibly inactivates RhoA. Treatment of primary retinal neurons with C3-protein reverses the inhibitory effects of myelin and MAG on these cells (Lehmann et al., 1999). Application of C3 at the injury site of the optic nerve or intravitreal injection of a cell-permeable version of C3 leads to RGC axons crossing the lesion site and growing into the distal nerve segment (Lehmann et al., 1999; Bertrand et al., 2005). Furthermore, viral expression of C3 in RGCs enables axons to regenerate in the distal part of the injured optic nerve and further enhances the extent of axon regeneration after lens injury (Fischer et al., 2004b). Similarly treatment of RGCs with specific inhibitors for ROCK overcomes myelin and CSPG inhibition in vitro and allows axons to regenerate beyond the lesion site of the optic nerve in vivo (Lingor et al., 2007, 2008; Ahmed et al., 2009). 2.3.3.2. Stabilization of microtubules. Microtubules are noncovalent cytoskeletal polymers with a polarized structure composed of a- and b-tubulin heterodimer subunits (Fig. 6 A, B). Axonal microtubules are characterized by a uniform orientation, with their plus ends facing the axon tip and the minus ends facing the cell body. Extension of microtubules at the plus ends in growth cones is an essential event for axonal growth and depends on the equilibrium between polymerization and depolymerization of tubulin dimers. Because microtubule dynamics in the growth cones

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are affected by the state of polymerization of actin filaments it is also indirectly modulated by RhoA/ROCK signaling (Mimura et al., 2006; Conde and Caceres, 2009; Geraldo and Gordon-Weeks, 2009). The clinically established anti-cancer drug Paclitaxel (Taxol) directly modulates microtubule dynamics. At high concentrations, as used for cancer therapy, Taxol evokes a hyperstabilization of microtubules and thereby abrogates the extension of the spindle apparatus during mitosis resulting in cell cycle arrest. However, at low concentrations, Taxol favors polymerization and therefore promotes axonal growth (Fig. 6CeE) (Sengottuvel et al., 2011). Because of the disentanglement of the coupled interaction between actin and microtubules in the growth cone, microtubule polymerization at the plus ends becomes independent of the state of actin polymerization. This improves the motility of the growth cones and decreases the sensitivity towards inhibitory signaling (Derry et al., 1995, 1997; Geraldo and Gordon-Weeks, 2009; Sengottuvel et al., 2011). Consistently, low concentrations of Taxol enhance axon growth of mature primary RGCs per se. Taxol treatment overcomes growth inhibition of RGC axons by myelin and CSPG in vitro (Sengottuvel et al., 2011; Witte et al., 2008). In vivo, treatment by enwrapping the lesioned optic nerve with a piece of Taxol-soaked gel foam allows RGC axons to grow across the injury site of the optic nerve (Fig. 7AeE). Importantly, Taxol treatment markedly increases the amount of regeneration when combined with activation of the neuronal intrinsic growth state by IS ((Sengottuvel et al., 2011) (Fig. 7E)), demonstrating that combinatorial approaches yield stronger regeneration than each treatment alone. Besides the direct effects on the microtubules in growth cones, Taxol application also delays glial scar formation and suppresses the expression of CSPGs at the injury site, possibly exerting its beneficial effects in vivo via several mechanisms (Hellal et al., 2011; Sengottuvel and Fischer, 2011). 3. Concluding remarks Damage of axons in the mammalian optic nerve causes irreversible loss of vision due to the lack of regeneration and to date no clinical treatment is available for patients. As summarized in this review, regenerative failure in the injured optic nerve is attributable to several factors. These include apoptotic cell death of RGCs, intrinsic insufficiency for axonal growth and inhibitory signaling in the axonal growth cone induced by molecules associated with CNS myelin and glial scar. However, findings of the last two decades demonstrate that axotomy-induced cell death can be significantly delayed and inhibitory signaling overcome. Moreover, the intrinsic growth state can be stimulated by selected cytokines and modulation of specific intrinsic signaling pathways. Moreover, combinatorial treatments have been shown to yield stronger regeneration than each treatment alone. Although these findings are encouraging, several milestones have to be reached before functional restoration of the visual pathway may become achievable for patients suffering from optic nerve damage. Next to further optimization of treatments enhancing the number and length of regenerating axons, strategies have to be developed to guide and reconnect axons to their former targets in a topographically correct order. Moreover, regenerated axons need to be appropriately remyelinated and, eventually, treatments developed in animals have to be assessed for their transferability towards therapeutic approaches in human patients. Acknowledgments We want to thank Dr. Heike Diekmann, Philipp Gobrecht and Dr. Vetrivel Sengottuvel for critically reading of the manuscript and for their suggestions. We also want to apologize to all colleagues

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