J Neuropathol Exp Neurol Vol. 69, No. 4 Copyright Ó 2010 by the American Association of Neuropathologists, Inc. April 2010 pp. 323Y334

REVIEW ARTICLE

Novel Therapeutic Targets for Axonal Degeneration in

Steven Petratos, PhD, Michael F. Azari, PhD, Ezgi Ozturk, BSc(Hons), Roula Papadopoulos, PhD, and Claude C.A. Bernard, DSc Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021

INTRODUCTION Abstract Multiple sclerosis (MS) is a major neurological disorder Multiple sclerosis (MS) is a devastating neurological condition in which there is inflammation, demyelination, and axonal that mainly affects young adults and is associated with long-standing damage in the brain and spinal cord. Approximately 2.5 million morbidity. The of MS is believed to involve people currently live with MS globally, and this causes a immune-mediated multifocal lesions in the CNS that are charac- significant health burden; more than 400,000 people in the terized by inflammation, demyelination, and axonal injury. Most United States alone are diagnosed with the . Women are research efforts to date have concentrated on the mechanisms of at least 2 to 3 times more likely to develop the disease than immune-mediated demyelination, whereas mechanisms of axonal men, and the disease onset is most often between the ages injury, the major determinant of neurological deficits in MS patients, of 20 and 50 years (1). Although MS occurs in most ethnic have been elusive beyond observational analyses. This review dis- groups (some more than others, particularly individuals from cusses current understanding of the pathology and novel clinical northern Europe or their direct descendants), strong genetic investigations of axonal injury in MS and the commonly used MS or environmental risk factors are still to be elucidated (2). animal model, experimental autoimmune encephalomyelitis. The The histopathologic patterns of MS have been well charac- review focuses on the etiology and the induction of axonal degen- terized, with much emphasis on the destructive immunologic eration through molecular signaling cascades downstream of - component of the disease in the brain and spinal cord (3). associated inhibitory factors. Defining and eventually elucidating the Currently, the mechanisms underlying axonal damage in MS signaling pathways elicited during the onset and progression of MS demonstrable in these histopathologic patterns are being de- may provide novel therapeutic strategies to limit axonal degeneration fined. Axonal damage can either develop secondary to white in the acute phase of the disease. Furthermore, blocking or poten- matter damage, or, as recently postulated, may be a primary tiating specific signaling pathways, particularly those that mediate or precipitating event in MS. A more plausible scenario would retraction and promote disassembly of the tubulin network, may be that axonal degeneration occurs concomitantly with demy- promote regrowth of damaged in CNS regions affected by elination (4). Trapp et al (5) reported the common appearance many acute and chronic disease processes. of newly transected axons in active and hypocellular chronic Key Words: Axonal degeneration, CRMP-2, Experimental auto- active MS lesions in brains obtained at autopsy, corroborat- immune encephalomyelitis, Multiple sclerosis, Myelin-associated ing such a hypothesis. Importantly, it is now recognized that inhibitory factors, Nogo-A, RhoA. permanent neurological impairment is a consequence of the level of axonal loss (4). Thus, understanding the molecular mechanisms leading to axonal degeneration is of critical im- portance for the development of novel therapeutic strategies From the Monash Immunology and Stem Cell Laboratories, Monash aimed at limiting the neurological decline during MS. University, Clayton Victoria (SP, MFA, EO, CCAB); and Molecular Experimental autoimmune encephalomyelitis (EAE) is Neuropathology and Experimental Neurology Laboratory, School of an animal model that mimics the clinical course as well as Medical Sciences and Health Innovations Research Institute, Royal Melbourne Institute of Technology University, Bundoora Victoria the pathophysiological hallmarks of MS. The EAE model (SP, RP), Australia. has thus been instrumental for the development of current Send correspondence and reprint requests to: Steven Petratos, PhD, Monash and novel MS therapeutics (6). The induction of EAE by Immunology and Stem Cell Laboratories, Monash University, Clayton immunization with the myelin glycoprotein Victoria 3800, Australia; E-mail: [email protected] (MOG) peptide spanning the 35 to 55 amino acid sequence Steven Petratos, Michael Azari, and Ezgi Ozturk contributed equally to this article. (MOG35Y55) is one of the most commonly used animal models Steven Petratos is supported by Australia Project Grant ID No. 384157 from to study MS (7). It has long been believed that axonal damage the National Health and Medical Research Council (NHMRC); Michael and loss is secondary to demyelination, commonly referred to Azari is supported by the NHMRC Health Professional Research Train- as Wallerian degeneration, and readily seen in the MOG Y ing Fellowship; Ezgi Ozturk is supported by the Faculty of Medicine 35 55 Postgraduate Award, Monash University Postgraduate Scholarship; Claude model of MS (8). On the other hand, it has recently been Bernard is supported by the NHMRC Project Grant ID No. 380832, the demonstrated that axonal degeneration can precede demyeli- National Multiple Sclerosis Society, and The Baker Foundation. nation in EAE (9). Therefore, the MOG35Y55 EAE model of

J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010 323

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. Petratos et al J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010 Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021

324 Ó 2010 American Association of Neuropathologists, Inc.

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010 Mechanisms of Axonal Degeneration in MS

MS can be used to help define the pathogenetic mechanisms of ing components of CNS myelin (14). This notion is attested axonal degeneration. to by the extensive axonal regeneration that is observed when The normal neurobiological response to axonal injury mice are immunized with purified CNS myelin before spinal is an initial attempt at regrowing the distal end of the axon. cord injury (SCI) (15). To date, 5 MAIFs have been identi- Local surrounding the lesion also attempt to compen- fied: myelin-associated glycoprotein (MAG) (16, 17); Nogo- sate for the injury by Bsprouting[ or extending new neurites. A (18Y20); oligodendrocyte myelin glycoprotein (OMgp) However, there are factors within the CNS environment that (21); semaphorin-4D/CD100 (22); and ephrin-B3 (23). Of inhibit these compensatory responses and cause failure of these, MAG and Nogo-A have been most intensively studied. regeneration/sprouting. Chief among these factors are com- Biochemical and structural biological reviews of these ponents of myelin or the myelin-associated inhibitory factors molecules have been previously described and will not be (MAIFs), such as the potent inhibitor of neurite outgrowth, discussed here (24). Nogo-A (Fig. 1). Expression of Nogo-A in Myelin-associated glycoprotein (17), Nogo-A (18, 20), has been reported to be upregulated in chronic active demye- and OMgp (25) are all normally localized on the innermost Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021 linating lesions of MS (10). In addition, immunization against lamella of the myelin sheath in direct contact with axons, Nogo-A or deletion of the nogo has been shown to preventing aberrant sprouting (26). Of these 3 inhibitors, ameliorate the pathophysiological hallmarks of EAE in Nogo-A (1,192 amino acids), a Reticulon family member C57Bl/6 mice (11). Strong evidence supporting the Nogo-A (Reticulon 4A), has the most interesting structure because it signaling pathway regulating axonal injury in EAE derives has at least 2 domains that can individually inhibit axonal from recent studies showing that deletion of the coreceptor growth (24). The binding of Nogo-A with its cognate re- for Nogo-A, LINGO-1 (which can signal through the down- ceptor, NgR1 (Fig. 1), is a trivalent interaction involving the stream cytoskeletal regulator, RhoA-GTP), improves axonal Nogo-66, Nogo-A-24, and Nogo-C39 domains, with the core integrity and protects against neurological decline in MOG- of the binding domain centered in the middle of the concave induced EAE mice (12). This raises the question of whether surface of NgR1 leucine-rich repeat domain and surrounded Nogo-A can signal axonal degeneration in EAE and, by im- by differentially used residues (27). plication, in MS. The nogo gene is differentially spliced to generate Current treatment of MS mostly targets the inflamma- 3 isoforms, Nogo-A, Nogo-B, and Nogo-C. Nogo-A is the tory nature of the disease to prevent the presumed autoimmune longest; it is CNS specific and contains a unique N-terminal attack on the CNS myelin and hence limit the devastating sequence (Amino-Nogo). The C-terminal portion of the neurological complications that ensue. It is possible, however, Nogo-A contains 2 transmembrane domains that are that by limiting Nogo receptor 1 (NgR1)Ydependent signal- separated by a short extracellular domain (Nogo-66) (Fig. 1). ing, axonal degeneration and hence neurological impairment Nogo-66 acts via the Nogo-66 receptor (NgR1) (28) in during EAE might be diminished. The precise dissection of conjunction with 2 coreceptors: the leucine-rich repeat and the NgR1 signaling pathways eliciting axonal degeneration immunoglobulin (Ig) domainYcontaining Nogo-receptor in- during neuroinflammatory such as MS may open teracting protein 1 (LINGO-1) receptor, and either the low- novel avenues for therapeutic intervention to limit the neuro- affinity neurotrophin receptor (p75NTR) or a receptor named logical decline that accompanies the disease. TROY (29). Activation of this receptor complex mediates inhibition of neurite growth by regulation of intracellular signaling through RhoA GTPase (30), a regulator of MAIFs AND THEIR ROLE IN MS/EAE cytoskeletal changes in neurons (31). The receptor through Although lesioned CNS axons can form growth cones which Amino-Nogo acts is not yet characterized, but a recent in response to injury, this regenerative attempt is transitory, report suggests that Amino-Nogo can bind and inhibit and no significant regrowth occurs over long distances (13). signaling by >5 and >v integrins and inhibit activation of The reason for the aborted axonal growth in the CNS is not focal adhesion kinase, thereby modulating fibronectin- caused by the absence of growth-promoting molecules, but dependent adhesion of the growth cone to the extracellular rather by the presence of axon outgrowth inhibitors, includ- matrix (32). Amino-Nogo also increases the affinity of the

FIGURE 1. Oligodendrocyte myelin membrane localized Nogo-A and its interaction with its cognate receptors. (A) Alternatively spliced isoforms of Nogo (Reticulon 4). Nogo A is 200 kD, Nogo B is 55 kD, and Nogo C is 25 kD. Despite the evolutionary conservation of the C-terminus among these isoforms, the N-terminus is longer in Nogo A than the other 2 isoforms, with limited conservation. (B) Full-length Nogo A is 1,192 amino acids (AA) in length and consists of 2 transmembrane domains, a C-terminus extracellular Nogo-66 domain (ligand binding domain), and 2 intracellular tails (one at the N-terminus, one at the C-terminus in the cis configuration). Nogo A in the trans configuration has its N-terminus in the extracellular compartment, which is able to interact with integrin receptors, commonly referred to as Amino-Nogo. (C) The Nogo-66 domain interaction with the leucine rich repeat ectodomain of Nogo receptor 1 (NgR1), is a high affinity binding, at a nanomolar range. This produces conformational changes of the NgR1 coreceptors to signal downstream, modifying the axonal cytoskeleton and favoring retraction of the distal growth end of the injured axon. Microfilament and microtubule dynamics are modified through the activation of RhoA (RhoA-GTP) and its effector kinase, Rho kinase (ROCKII). GDI, guanine dissociation inhibitor; LINGO-1, LRR and immunoglobulin domainYcontaining Nogo receptorYinteracting protein 1 receptor; p75, low-affinity neurotrophin receptor (p75NTR).

Ó 2010 American Association of Neuropathologists, Inc. 325

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. Petratos et al J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010

Nogo-A/NgR1 interaction (33) and can act through the Rho which Nogo-A contributes to the pathophysiology of EAE GTPases (30, 34). A third segment of Nogo-A, a C-terminal are still unclear, however. domain, can also interact specifically with NgR1 (35). Despite the ambiguity of the role of Nogo-A in the The action of the MAIFs on severed axons attempting induction of EAE (either immune or neurobiological or both), regeneration after injury or disease has been determined to be Mi et al (12) defined a neurobiological role for LINGO-1, the one of the most critical inhibitory events that limits CNS signaling coreceptor for the MAIFs (including Nogo-A) repair. The molecular interaction of the MAIFs with NgR1 is during the course of EAE. These investigators showed that a high-affinity ligand-receptor interaction that initiates in- Lingo1-knockout mice or mice treated with an antiYLINGO-1 tracellular signaling in affected neurons, converging on the functionYblocking antibody had a reduced locomotor deficit Rho-GTPases to inhibit axonal regrowth (Fig. 1). The down- throughout the course of the disease. These results were inde- stream effectors of Nogo-A/NgR1 signaling are RhoA-GTP pendent of the activity of immune cell infiltrates, suggesting and the collapsin response mediator protein 2 (CRMP-2) that a neurobiologically specific role for LINGO-1 in the induc- regulate actin and tubulin dynamics after injury (36). We hy- tion of the neurological complications of EAE. They demon- Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021 pothesize that by reducing the capacity of the myelin-derived strated this specifically by testing the proliferative indices deposits to signal through their cognate receptor in EAE or in of knockout versus wild-type littermateYisolated T-cells after MS, it may be possible to limit the activation of the RhoA/ MOG35Y55 immunization and showing that their MOG re- phosphorylated CRMP-2 pathway in the axonal cytoskeleton, sponsiveness was almost identical (12). Along the same lines, thereby preventing degeneration. the cytokine profiles of isolated T-cells from wild-type litter- On the other hand, MAG can exert its inhibitory effects mates with EAE were not different from those of Lingo1- on neurite outgrowth through either the NgR or gangliosides, knockout mice. Moreover, EAE could be transferred to naive depending on the neuronal type. Using primary rat neuronal wild-type littermates with encephalitogenic T-cells isolated cultures, the inhibitory effects of MAG were shown to be from Lingo1-knockout mice. By contrast, encephalitogenic mediated predominantly by NgRs in dorsal root ganglion T-cells from wild-type mice could not adoptively transfer EAE (DRG) neurons, mainly by gangliosides in hippocampal neu- into knockout littermates, clearly indicating an endogenous rons and exclusively by gangliosides (GD1a and GT1b) in role for LINGO-1 in the CNS during the neurological decline cerebellar granule neurons (37). associated with EAE. Receptor systems other than through NgR may also The neurobiological role of LINGO1 during EAE was contribute to the inhibitory effects of the MAIFs on neurites. eventually attributed to the function of the receptor in remye- For example, paired Ig-like receptor B (PirB), which has been linating oligodendrocyte precursor cells (12). Lower EAE previously implicated in visual system plasticity (38), now scores of locomotor deficit in the Lingo1-knockout group seems to be important in MAIF signaling for neurite out- were correlated with a lower extent of oligodendrogliopathy growth inhibition. Atwal et al (39) reported that MAG, OMgp, compared with EAE induced in wild-type littermates, as and Nogo-66 bind with high affinity to PirB. Moreover, demonstrated by electron microscopy. Oligodendrogliopathy blocking this high-affinity interaction with the use of a His- is characteristic of an Binside-out[ degeneration by which tagged fusion PirB ectodomain peptide, an anti-PirB anti- the loss of the axon initiates oligodendrocyte and myelin body, or the PirB genetic mutant, produces a restoration of degeneration. Furthermore, when EAE was induced in the maximal neurite length of cerebellar granule neurons in the Lingo1-deficient mice, there was an abundance of remyelin- presence of the MAIFs. These investigators did show, how- ated axons and an increased number of myelinated axons ever, that there was a requirement for both NgR and PirB compared with those in the wild-type littermates. Likewise, to potentiate MAIF-mediated growth cone collapse of DRG treatment of rats with EAE with an antagonistic antiYLINGO- neurons, suggesting complexity to the axonal degeneration 1 antibody led to more myelinated and remyelinated fibers system mediated through the myelin (39). It may be in the spinal cord compared with those in rats treated with worthwhile to investigate the effect of combinatorial treat- control IgG (12). The contention that LINGO-1 plays an ments for inhibition of the NgR and PirB ligand interactions important role in is further supported by the in the context of axonal degeneration in MS. demonstration that LINGO-1 plays a dominant role in the Given the apparent redundancy in signaling axonal inhibition of oligodendrocyte differentiation and myelination growth inhibition and the tendency of the organism to com- when it is expressed either on the oligodendrocyte precursor pensate for genetic deletion of any of these specific ligands, cell (44) or on the axon that the preYmyelinating oligoden- it is not surprising that studies of axonal regeneration in 3 drocyte eventually myelinates (45). These data suggest that different strains of nogo-deficient mice have shown variable therapeutic targeting of LINGO-1 may be a viable mecha- degrees of regeneration (40Y42). An additional reason for this nism for repair in demyelinating diseases. variability may be strain-specific differences between the mice used in these studies (43). Recently, the relevance of Nogo-A in neuroinflammation was demonstrated by its direct role MODELS OF AXONAL INJURY AND THE in the pathogenesis of EAE (11). Furthermore, immunization GENETIC EVIDENCE FOR NgR1-MEDIATED against Nogo-A ameliorated EAE in mice and prevented AXONAL DEGENERATION axonal injury (11). It is therefore plausible that abrogation of Nogo-AYknockout mice, including nogo-abtrap/trap (40) Nogo-A signaling by deletion of the cognate receptor for and nogo-aEIII/EIII (41, 43) mice, have been reported to show Nogo-A may provide resistance to EAE. The mechanisms by enhanced growth of corticospinal tract axons after SCI,

326 Ó 2010 American Association of Neuropathologists, Inc.

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010 Mechanisms of Axonal Degeneration in MS whereas one of the mutants, termed nogo-abatg/atg, failed to clonal antibody IN-1 (52). Whether blocking antibodies such show enhanced axonal regrowth (42). These conflicting ob- as IN-1 produce similar blockade of neurite outgrowth inhibi- servations after injury have been mimicked in NgR1 mutant tion in vivo, particularly in models of MS, has yet to be mice after dorsal hemisection (46, 47), but the ngr1j/j mice defined. Interestingly, expression of Nogo-A in oligodendro- did recover hindlimb function after SCI and showed regrowth cytes is upregulated in chronic active demyelinating lesions of rubrospinal and raphespinal axons (46). In a selective py- of MS (10). Fry et al (53) recently demonstrated that NgR ramidotomy paradigm, there is pronounced collateral sprout- induces efflux of macrophages from the injured peripheral ing of intact corticospinal tract fibers across the midline and nerve to terminate inflammation at the end of Wallerian de- into injured spinal cord, with a return of preferred forelimb generation. This effect is postulated to contribute to the re- usage in both ngr1j/j and nogo-abatg/atg mice (48). These data generative capacity of peripheral nerves. Given that depletion confirm a role for Nogo-A and NgR1 in limiting functional of peripheral macrophages reduces the severity of EAE (54), recovery through axonal growth within the injured CNS, but it is possible that NgR may also play a role in the efflux these in vivo models do not provide molecular downstream of macrophages from the CNS after injury or inflammatory Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021 signaling evidence of axonal regrowth. In view of the grow- insult. It is intriguing that when SJL mice were injected with ing body of pharmacological studies that show enhanced peptides from different regions of Nogo-66, T- and B-cell regrowth of axons after injury and disease in the CNS by immune responses were stimulated and EAE was either blocking the binding of the MAIFs with the ectodomain of induced or suppressed depending on the specific epitope of NgR1 (49), it is imperative to identify the signaling cascade Nogo-66 to which the peptide corresponded (50). The in- that promotes axonal degeneration to provide therapeutic vestigators further showed that Nogo-66Yspecific T-cell lines targets for CNS repair. The role of NgR1 in axonal regrowth could ameliorate EAE in this mouse model through a TH2- related to a neuroinflammatory-induced disease such as EAE dependent mechanism. Despite these data, they showed in- is now awaiting investigation. creased spreading of the antibody response against other myelin antigens after immunization with the Nogo-45Y66 peptide and enhanced demyelination and paralysis in the THE ROLE OF NOGO-A SIGNALING IN THE mice. Recent data using DNA vaccination for the generation INDUCTION OF EAE of antibodies against the full-length Nogo-A protein did not It has recently been suggested that Nogo-A is involved induce demyelination or inflammatory disease in naive mice in autoimmune-mediated demyelination (50). Accordingly, nor did it exacerbate the EAE in mice immunized with it is important to determine whether the role of Nogo-A in myelin proteolipid protein (55). Interpretation of these data EAE is related to axonal signaling through NgR1 initiating may not advocate Nogo-A as a potent encephalitogen. degeneration or through initiating the autoimmune response. Recently, the complexity of the NgR1 signaling system Karnezis et al (11) demonstrated that blocking Nogo-A inter- in vivo has been established with the identification of the action with its cognate receptor limits axonal degeneration in expression of the receptor in human T-cells and monocytes EAE. Interestingly, in that study, nogo-aj/j mice or mice that from both healthy individuals and MS patients (56). More- were vaccinated with the Nogo-623Y640 peptide exhibited over, Zhang et al (57) demonstrated that a tumor necrosis reduced CNS inflammatory infiltrates, increased anti- and factor family protein involved during B-cell development, immunomodulatory cytokines such as transforming growth known as B-lymphocyte stimulator, can act as a high-affinity factor-beta and interleukin-19 (IL-10), and enhanced proin- ligand for NgR1 signaling independent of the MAIFs. It flammatory cytokines. These data indicate modulation of would, therefore, be imperative to dissect out the effects of inflammatory mechanisms. It is plausible that this dampening pharmacological antagonists to NgR1 in the context of height- of the neuroinflammatory response in these mice may be ened neuroinflammation for the future application of ther- caused by the reduced axonal pathology also manifested apeutic manipulation of this pathway in patients at different in these mice, but whether the reduction in axonal damage stages of MS. occurs before the appearance of the inflammatory component of the disease or is a consequence of the reduced immune response remains unresolved. NgR1 SIGNALING AND CYTOSKELETAL ORGANIZATION: TRANSDUCTION OF THE EXTRACELLULAR SIGNAL INTO THE AXOPLASM THE ROLE OF NOGO-A AND NgR IN THE RhoA-GTP (active RhoA) has been implicated in neu- IMMUNE RESPONSE IN MS AND EAE rological disease paradigms as an intracellular effector of AntiYNogo-A autoantibodies have been reported in the neurite retraction, primarily involving the MAIFs (58, 59). serum and cerebrospinal fluid of MS patients (51). Notably, Inhibition of neurite outgrowth by the MAIFs occurs by using these antibodies seem to be more frequently present in the receptor cluster composed of NgR1, the low-affinity neu- relapsing-remitting, rather than in the chronic progressive rotrophin receptor, p75NTR, LINGO-1, and TROY to activate forms of MS, as well as being more frequent in younger endogenously bound RhoA (Fig. 1) (29). Downstream, RhoA- patients (51). It is plausible that this antibody response may GTP can mediate the reorganization of the microfilament and play a role in clinical remissions of the disease. Neutralizing microtubule networks forming the neuronal cytoskeleton (59). the potent MAIF effect on neurite outgrowth inhibition was In the , this can include contraction between myosin first demonstrated against Nogo-A in culture using the mono- head groups on actin filaments at filopodia pulling actin

Ó 2010 American Association of Neuropathologists, Inc. 327

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. Petratos et al J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010 Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021

328 Ó 2010 American Association of Neuropathologists, Inc.

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010 Mechanisms of Axonal Degeneration in MS

filaments away from the area of directional growth (Fig. 1). the intracellular cytoskeleton. They are ubiquitously expressed Furthermore, RhoA-GTP activates the serine-threonine kinase, and are involved in diverse cellular processes including cell Rho kinase (ROKI/ROCKII), which then proceeds to phos- division and morphogenesis (66) and migration (67). Of par- phorylate Lin-11, Isl-1, and Mec-3 kinase (LIMK). The LIMK ticular significance is that they represent the point of conver- can itself then impede (by phosphorylation) the ability of gence of multiple signaling pathways involved in inhibition the actin-depolymerizing factor/cofilin by phosphorylation, to of neurite outgrowth. These include signals mediated by provide actin monomers for the extension of microfilaments MAIFs, Ephrins, Semaphorin 3A, and chondroitin sulfate to ensue, thus initiating growth cone collapse (59). proteoglycans. Cytoskeletal rearrangement initiated from the growth Stimulation of the Nogo receptor signaling complex cone eventually becomes transferred retrogradely to the larger dissociates RhoA guanine diphosphate dissociation inhibitor neuronal microtubule network at the core of the axon/neurite. > from the small GTPase RhoA, resulting in RhoA activation This larger cytoskeletal network is assembled from a 100-kD (21, 59) (Fig. 1). Activated RhoA integrates with the plasma

> and A tubulin heterodimer and transported to the plus ends membrane through isoprenylation, allowing ROCKII to dock Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021 of microtubules to potentiate neurite outgrowth (60). Rapid with it, resulting in activation of its kinase domain (59). microtubule assembly from the core (C-domain) of the neurite Active ROCKII can phosphorylate CRMP-2 at its threonine toward the actively growing peripheral domain (P-domain) oc- 555 site to confer decreased association with tubulin hetero- curs where there is substantial actin rearrangement (60). There- dimers, thereby decreasing microtubule polymerization and fore, dynamic growth cone regions provide an ideal platform in turn reducing neurite growth (68). The ROCKII can also for microtubule assembly and neurite outgrowth. This is an im- phosphorylate LIM kinase-1 (LIMK1) (69) and the regula- portant event that regulates the synaptic connection of various tory light chain of myosin II (MLC) (70). These cumulative neurons with each other through long-range and short-range phosphorylation events that are instigated by the activation of cues during CNS development (61). Assembly/polymerization RhoA and ROCKII promote the contraction of microfila- of this intricate neuronal cytoskeletal network, which during ments and destabilization and disassembly of microtubules, development provides growth, can also be accompanied by thereby resulting in neurite retraction from the growth cone, negative chemical and contact cues that prevent aberrant or that is, growth cone collapse. Both Amino-Nogo and Nogo- inappropriate growth to specific CNS regions (62). This is 66 have been reported to be inhibitory in vitro. achieved through transduction of inhibitory extracellular cues to intracellular signals that promote contraction and disas- THE ROLE OF CRMP-2 IN AXONAL sembly of the neuronal cytoskeleton in an opposing direction to the signal. In the adult CNS, this important signaling mech- DEGENERATION AND AXONAL TRANSPORT More than a decade ago, a 62-kD cytoplasmic protein anism prevents aberrant growth of axons and dendrites and Y the formation of pathophysiological connections. The MAIFs, was first discovered to mediate the growth cone inhibitory such as Nogo-A, are major extracellular inhibitory molecular effects of collapsin (semaphorin-3A) (71), which was later cues in the adult CNS. After injury, deposits of these myelin termed CRMP-2. The gene for CRMP-2 was found to be proteins at lesion sites prevent endogenous axonal regrowth expressed during neuronal differentiation in the rat and to after degeneration of axons; they may also initiate axonal de- be related to unc-33, a Caenorhabditis elegans gene that is generation in their own right (63, 64). Because myelin debris also involved in axonal growth (72). Mutations of unc-33 in is deposited after neuroinflammatory-mediated demyelination C elegans produce axonal abnormalities and uncoordinated in EAE and MS (65), induction of axonal degeneration in bodily movements (73). The CRMPs are a family of neu- these conditions might be potentiated by these MAIFs, ronal phosphoproteins with some sequence homology to thereby prohibiting any endogenous regrowth capacity. dihydropyrimidinase (74). The CRMPs regulate microtubule assembly as well as anterograde vesicular transport of impor- tant growth-related molecular cargo along neuronal micro- RHO-GTPases AND THEIR ROLE IN tubules (75). The CRMPs, particularly CRMP-2, are involved AXONAL DAMAGE IN EAE in the formation, outgrowth, and guidance of neurites. The Rho-GTPases are key molecular switches that trans- CRMP-2 is phosphorylated by cyclin-dependent kinase 5 at duce MAIF-inhibitory signals from the axonal membrane to serine 522 (76, 77), glycogen synthase kinase 3A at threonine

FIGURE 2. The collapsin response mediator protein 2 (CRMP-2) and its role in the axonal cytoskeleton. (A) The alternatively spliced variants of CRMP-2 are CRMP-2A (75 kD) and CRMP-2B (62 kD). Full-length CRMP-2 is 571 amino acids in length with a dihydropyrimidinase domain and a tubulin-binding domain 323 to 381 amino acids. The CRMP-2 can be phosphorylated by specific kinases that regulate microtubule dynamics within the growing axon. The priming kinase for CRMP-2 is cyclin-dependent kinase 5 at Ser522 followed by glycogen synthase kinase 3A (GSK-3A) at Thr509/514 and experimentally at Ser518. The CRMP-2 can also be phosphorylated independently by Rho kinase (ROCKII) at Thr555. The CRMP-2 activity can be modulated by calpain cleavage of the C-terminus domain (calpain cleavage sites denoted by red arrows). (B) ROCKII phosphorylation of CRMP-2 at Thr555 may also affect the binding of CRMP-2 to kinesin-1, the primary anterograde transport protein along the axonal microtubule system. Phosphorylation of CRMP-2 produces dissociation of kinesin-1 and of the >- and A-tubulin heterodimers, preventing their transport to the plus ends (growth ends) of microtubules. (C) This blockade of molecular cargo transported down the axonal microtubules limits the growth of the axon.

Ó 2010 American Association of Neuropathologists, Inc. 329

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. Petratos et al J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010

509/514, serine 518 (78, 79), and also Rho kinase at threo- tubule assembly and therefore axonal regrowth. The role of nine 555 (68, 80); all of these can mediate neurite retraction. MAIF signaling in axonal injury has been shown to be linked Such phosphorylation disrupts the association of CRMP-2 with axonal RhoA activation in white matter tracts surround- with tubulin heterodimers so that tubulin cannot be trans- ing a transection SCI lesion in the rat (36). Importantly, these ported to the plus ends of microtubules for assembly, thereby findings also correlated with an increase in phosphorylated impeding directional growth of the neurite (75). Importantly, CRMP-2 in axons with depolymerizing tubulin (36). Treat- CRMP-2 phosphorylation also reduces its binding to the ment of these rats with the ROCK inhibitor Y-27632 imme- microtubule-related motor protein kinesin-1, which is involved diately after spinal cord thoracic transection confirmed that in anterograde vesicular axonal transport of molecules that phosphorylation of CRMP-2 was dependent on ROCK (36). regulate synaptic integrity and plasticity (e.g. brain-derived Inhibition of ROCK rescued levels of polymerized tubulin, neurotrophic factor) to the distal ends of axons (81) (Fig. 2). and phosphorylated CRMP-2 levels returned to baseline in The overexpression of CRMP-2 in rat cerebellar neurons axons. Collectively, these data raise the important question results in neurite outgrowth even in the presence of MAG or of whether interference with the Nogo-A signal transduction Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021 Nogo-66 (36). Using purified tubulin and CRMP-2 purified pathway in neurons would promote axonal integrity in EAE. from bovine brain, Fukata et al (75) found that CRMP-2 directly binds tubulin heterodimers and that CRMP-2 has greater affinity for tubulin heterodimers than microtubules CURRENT TREATMENT OF MS by a factor of 10. Indeed, coincubation with video micros- Currently, 4 disease-modifying agents have been ap- copy experiments shows that CRMP-2 dramatically promotes proved by the US Federal Drug Administration for the treat- microtubule assembly (75). The ROCKII inactivation of ment of MS. They are interferon-A1a, glatiramer acetate, CRMP-2 by phosphorylation at the Thr555 residue was dem- mitoxantrone, and natalizumab (82). All of these compounds onstrated in rat cerebellar neuron transfection experiments are targeted toward modulating the immune response in an using a mutant form of CRMP-2 with a single point muta- attempt to limit damage that occurs as a consequence of neu- tion at T555A that rendered the molecule incapable of being roinflammation. Novel experimental therapies that focus on phosphorylated by ROCKII (36). Furthermore, DRG neurons the neurobiological aspects of the disease are now emerging. transfected with the same construct showed an increase in There has been significant success in blunting neurological axonal length similar to DRG neurons transfected with wild- complications in EAE models. type CRMP-2 (68). Inhibition of neurite outgrowth is also observed with siRNA knockdown of CRMP-2 in cerebellar granule cells or by transfection of these cells with the dom- NOVEL THERAPEUTIC TARGETS IN MS inant negative form of CRMP-2, which lacks the microtubule- A number of promising therapeutic strategies tested in binding domain (CRMP-2$C), thereby demonstrating the EAE have involved inhibition of the Nogo-A/NgR/LINGO-1/ central role of CRMP-2 in neurite outgrowth (36). Immuno- RhoA/ROCKII/CRMP-2 signaling pathways. These strategies histochemical studies using antibodies against CRMP-2 and include blockade of either Nogo-A/NgR or LINGO-1/NgR/ phosphorylated Thr555YCRMP-2 on DRG neurons revealed p75(or TROY) interactions, the use of statins or geranylger- that CRMP-2 is localized to the growth cone and colocalizes anyl transferase inhibitor to block RhoA-GTP isoprenylation, with microtubules and actin at distal parts of the axon (68). inhibition of ROCKII by the use of fasudil, Y27632 to bind Phosphorylated CRMP-2, however, was localized diffusely in to and inactivate the coiled-coiled region of the phosphoryla- the growth cone; it was present as punctate structures and was ting enzyme, and antagonizing the Rho kinase through the also localized in the filopodia. Unlike CRMP-2, phosphory- use of a therapeutic dose of the Clostridium botulinum pro- lated CRMP-2 was not localized with microtubule bundles. tein C3 exoenzyme or its recently developed pharmaceutical Electron microscopy and freeze-etching replica method using agent, Cethrin, a recombinant fusion protein of C3 and a mem- DRG neurons revealed CRMP-2 to be localized on filamen- brane transport sequence (BioAxone Therapeutics, Montreal, tous microtubules, on clathrin-coated pits, and actin filaments Canada). The latter is currently in clinical-phase trials for its at the plasma membrane of the growth cone. However, phos- use in SCI patients (83). An alternative mechanism for limiting phorylated CRMP-2 was only localized with actin filaments axonal degeneration in EAE has also involved the inhibition at the growth cone (68). of calpain, a calcium-dependent cysteine protease highly ex- Considering the physiological relevance of CRMP-2 in pressed during neurological disease and injury. the regulation of axonal growth, it has recently been shown that there is an increase in the levels of phosphorylated thre- INHIBITION OF THE NgR COMPLEX onine 555 CRMP-2 after axonal transection in a model of SCI (64). These increases correlated with increased levels of Inhibition of Nogo-A Nogo-A deposits at and around the site of injury. When the As previously indicated, passive or active immuniza- level of Nogo-A deposits was reduced by daily administra- tion against Nogo-A decreases the clinical and histopatho- tion of the neuropoietic cytokine, leukemia inhibitory factor, logic manifestations of EAE (11). This may have been caused a corresponding reduction in the levels of phospho-Thr555 by either increased clearance of Nogo-A deposits in CNS CRMP-2 also ensued (64). These data may suggest that an lesions, thereby decreasing the interaction of Nogo-A with increase in extracellular MAIF deposits after CNS injury can its cognate receptor on injured axons, or reduction in pro- signal CRMP-2 phosphorylation, possibly limiting micro- inflammatory cytokines. Clinical trials are currently being

330 Ó 2010 American Association of Neuropathologists, Inc.

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010 Mechanisms of Axonal Degeneration in MS performed by Schwab et al in collaboration with Novartis ROCK INHIBITION: PREVENTION OF Europharm Ltd, using a recombinant human monoclonal anti- AND AXONAL body (IgG4K) directed against the human Nogo-A protein to DEGENERATION enhance axonal regrowth after SCI (84). Current data show a Rho kinase (ROCK) inhibitors have already reached significant enhancement of locomotor performance in a rat the clinic; more than 30,000 patients are receiving the first model of SCI after the intrathecal administration of a ro- generation of ROCK inhibitors, fasudil (89). Fasudil is a dent version of the antiYNogo A antibody (11C7) (85). The moderate ROCK inhibitor (Ki, 330 nM) (90), but the modified rigorous behavioral improvements (i.e. hindlimb kinematic dimethyl-fasudil is the most potent ROCK inhibitor to date patterns) correlated with the promotion of axonal regrowth Y Y (91). Clinical trials using fasudil in patients after subarach- and neurite sprouting events in the anti Nogo-A antibody noid hemorrhage have not produced serious side effects (92). treated group (85). We await the outcome of the clinical Y ROCK has recently been implicated in compromising the phase trial for the human anti Nogo-A antibody that may blood-brain barrier by phosphorylating claudin-5 and occlu- consolidate these exciting results and may have implications din, molecules involved in the maintenance of endothelial Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021 for the neurodegenerative phase of MS. cell tight junctions (93). Transendothelial migration of immune cells is a key event in the pathophysiology of EAE Inhibition of NgR1-Ligand Interaction and MS, and fasudil treatment inhibited T-cell migration into Recombinant fusion proteins using the soluble form of the CNS (94). In addition to reducing CNS inflammation, the ectodomain of NgR1 and the Fc portion of human IgG1 it also reduced axonal loss in the proteolipid protein model have been used in cell culture and in vivo to promote axonal of EAE (95). The investigators attributed these findings to regrowth (49, 86). It was recently shown that administration downregulation of IL-17Yproducing T cells and TH1 cells as of a fusion peptide corresponding to the ligand-binding do- a result of fasudil treatment, but we hypothesize that the main of the NgR1(310) resulted in neurite outgrowth (axonal treatment might also preserve axons through inhibition of regrowth and sprouting) after transection of corticospinal ROCKII activity, thereby reducing axonal degeneration. tract and raphespinal fibers and improved functional recovery Intraperitoneal administration of a single dose of fasudil in a mouse model of SCI (49). This may be caused by the in clip compression SCI in the rat improved locomotor per- fusion peptide competing with endogenous NgR1 for binding formance up to 4 weeks after injury (96). By contrast, oral to Nogo-A, OMgp, and MAG, thereby reducing NgR1 down- administration of the ROCK I and II inhibitor, Y-27632 stream signaling and promoting axonal regrowth. Recently, (Ki, 140 for ROCK I and 300 for ROCK II), did not produce the the binding of Nogo-66 to NgR1 was found not to be sialic same positive effects in locomotor performance (97). Never- acid dependent, whereas the interaction of the first 3 Ig-like theless, direct administration of Y-27632 to the transected domains of MAG to NgR1 and NgR2 is sufficient to confer Y spinal cord promoted axonal growth beyond the lesion site, sialic acid dependent binding. These data, along with a defi- decreased , and enhanced locomotor per- nition of molecular specificities for Nogo-66 and MAG bind- formance in rodents up to 30 days postinjury (58). These data ing to NgR, allowed for the generation of a receptor variant suggested that Y-27632 may not be as effective in crossing that exhibited potentiated binding to the MAIFs (86). The the blood-spinal cord barrier as fasudil. The C3 transferaseY investigators constructed a chimeric soluble receptor protein, OMN1 derived synthetic protein Rho antagonist, Cethrin, is more NgR , which had an increased binding avidity than the effective at penetrating the lesion when applied subdurally to wild-type soluble NgR1. The successful therapeutic admin- effect neurological improvement in patients than either com- istration of the NgR1(310)-Fc fusion protein in SCI models OMN1 pound (98). Cethrin is currently under a multicenter clinical- (49, 87) and the development of the NgR chimeric pro- phase trial for SCI and is awaiting development for clinical tein that provides a highly specific MAIF inhibitor for use in use. Despite the robust data pertaining to the use of these various in vivo models of CNS axonal degeneration point to ROCK inhibitors in axonal regrowth and locomotor recovery application in EAE and MS. after SCI, their use in animal models of MS has been minimal. Studies have mainly defined the role of ROCK Inhibition of LINGO-1 inhibitors in reducing inflammatory infiltrates (95, 99). Ap- Inhibition of the function of LINGO-1 by either genetic propriate experiments remain to be done in relation to the means or the use of an antibody antagonist can promote direct role of ROCK inhibitors in preventing axonal de- axonal integrity and remyelination in MOG-induced EAE Y generation or alternatively promoting axonal regrowth after (12). Moreover, the function-blocking LINGO-1 Fc fusion axonal transection has occurred as a consequence of neuro- protein potentiated oligodendrocyte differentiation in vitro, Y inflammatory CNS lesions. In a similar context, ROCK in- and an anti LINGO-1 enhanced re- hibitors may also play a significant role in remyelination myelination and reduced EAE severity in vivo (44). The Y because the molecular signaling mechanisms driving this remyelinating potential of the anti LINGO-1 antibody was process in the demyelinated adult CNS are very similar to also shown in 2 neurotoxicant models of demyelination those of axon outgrowth (99, 100). (lysophosphatidylcholine or cuprizone), indicating that effects are unrelated to the inflammatory nature of the demyelination (88). These data advocate for the use of LINGO-1 antagonistic CONCLUSIONS therapies (e.g. LINGO-1YFc or antiYLINGO-1 antibody) in The mechanisms of axonal injury and degeneration in human demyelinating conditions. MS and its animal models, at both cellular and molecular

Ó 2010 American Association of Neuropathologists, Inc. 331

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. Petratos et al J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010 levels, need to be investigated thoroughly to identify novel 16. McKerracher L, David S, Jackson DL, Kottis V, Dunn RJ, Braun PE. therapeutic targets and limit or prevent axonal injury and the Identification of myelin-associated glycoprotein as a major myelin- derived inhibitor of neurite growth. Neuron 1994;13:805Y11 resultant neurological manifestations. It is likely that multiple 17. Mukhopadhyay G, Doherty P, Walsh FS, Crocker PR, Filbin MT. A mechanisms are involved in axonal injury in these disorders. novel role for myelin-associated glycoprotein as an inhibitor of axonal We propose that the molecular machinery that mediates axon regeneration. Neuron 1994;13:757Y67 retraction, particularly the NgR1YLINGO-1/RhoA/ROCKII/ 18. Chen MS, Huber AB, van der Haar ME, et al. Nogo-A is a myelin- associated neurite outgrowth inhibitor and an antigen for monoclonal CRMP-2 that promotes disassembly of the tubulin network antibody IN-1. Nature 2000;403:434Y39 within the axon, should be a focus of intense investigation. 19. Prinjha R, Moore SE, Vinson M, et al. Inhibitor of neurite outgrowth in We also postulate that many of the promising experimental humans. Nature 2000;403:383Y84 therapeutics that have been examined in animal models of 20. GrandPre T, Nakamura F, Vartanian T, Strittmatter SM. Identification MS, at least in part, exert their effects through modulation of of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 2000;403:439Y44 this molecular machinery. Because demyelination is a domi- 21. Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glyco- nant event in limiting regeneration within the mammalian protein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021 CNS, understanding the control of these complicated molec- 2002;417:941Y44 ular events will be of major significance for attenuating the 22. Moreau-Fauvarque C, Kumanogoh A, Camand E, et al. The trans- membrane semaphorin Sema4D/CD100, an inhibitor of axonal growth, severity of devastating neurological diseases that involve is expressed on oligodendrocytes and upregulated after CNS lesion. axonal abnormalities such as traumatic spinal cord and brain J Neurosci 2003;23:9229Y39 injury, stroke, and demyelinating diseases. 23. Benson MD, Romero MI, Lush ME, Lu QR, Henkemeyer M, Parada LF. Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proc Natl Acad Sci U S A 2005;102:10694Y99 24. Oertle T, Schwab ME. Nogo and its paRTNers. Trends Cell Biol 2003; REFERENCES 13:187Y94 1. Langer-Gould A, Popat RA, Huang SM, et al. Clinical and demo- 25. Kottis V, Thibault P, Mikol D, et al. Oligodendrocyte-myelin glyco- graphic predictors of long-term disability in patients with relapsing- protein (OMgp) is an inhibitor of neurite outgrowth. J Neurochem remitting multiple sclerosis: A systematic review. Arch Neurol 2006; 2002;82:1566Y69 63:1686Y91 26. McKerracher L, Winton MJ. Nogo on the go. Neuron 2002;36:345Y48 2. Fugger L, Friese MA, Bell JI. From to function: The next 27. Lauren J, Hu F, Chin J, Liao J, Airaksinen MS, Strittmatter SM. challenge to understanding multiple sclerosis. Nat Rev Immunol 2009; Characterization of myelin ligand complexes with the neuronal 9:408Y17 NOGO-66 receptor family. J Biol Chem 2006;282:5715Y25 3. Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M, 28. Fournier AE, GrandPre T, Strittmatter SM. Identification of a receptor Lassmann H. Heterogeneity of multiple sclerosis lesions: Implications mediating Nogo-66 inhibition of axonal regeneration. Nature 2001;409: for the pathogenesis of demyelination. Ann Neurol 2000;47:707Y17 341Y46 4. Bjartmar C, Trapp BD. Axonal and neuronal degeneration in multiple 29. Mi S, Lee X, Shao Z, et al. LINGO-1 is a component of the Nogo-66 sclerosis: Mechanisms and functional consequences. Curr Opin Neurol receptor/p75 signaling complex. Nat Neurosci 2004;7:221Y28 2001;14:271Y78 30. Niederost B, Oertle T, Fritsche J, McKinney RA, Bandtlow CE. 5. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L. Nogo-A and myelin-associated glycoprotein mediate neurite growth Axonal transection in the lesions of multiple sclerosis. N Engl J Med inhibition by antagonistic regulation of RhoA and Rac1. J Neurosci 1998;338:278Y85 2002;22:10368Y76 6. Steinman L, Zamvil SS. Virtues and pitfalls of EAE for the develop- 31. Jalink K, van Corven EJ, Hengeveld T, Morii N, Narumiya S, ment of therapies for multiple sclerosis. Trends Immunol 2005;26: Moolenaar WH. Inhibition of lysophosphatidate- and thrombin-induced 565Y71 neurite retraction and neuronal cell rounding by ADP ribosylation of 7. Johns TG, Kerlero de Rosbo N, Menon KK, Abo S, Gonzales MF, the small GTP-binding protein Rho. J Cell Biol 1994;126:801Y10 Bernard CC. Myelin oligodendrocyte glycoprotein induces a demyeli- 32. Hu F, Strittmatter SM. The N-terminal domain of Nogo-A inhibits nating encephalomyelitis resembling multiple sclerosis. J Immunol cell adhesion and axonal outgrowth by an integrin-specific mechanism. 1995;154:5536Y41 J Neurosci 2008;28:1262Y69 8. Gresle MM, Shaw G, Jarrott B, et al. Validation of a novel biomarker 33. Hu F, Liu BP, Budel S, et al. Nogo-A interacts with the Nogo-66 for acute axonal injury in experimental autoimmune encephalomyelitis. receptor through multiple sites to create an isoform-selective subnano- J Neurosci Res 2008;86:3548Y55 molar agonist. J Neurosci 2005;25:5298Y304 9. Wang D, Ayers MM, Catmull DV, Hazelwood LJ, Bernard CC, 34. Schweigreiter R, Walmsley AR, Niederost B, et al. Versican V2 and Orian JM. Astrocyte-associated axonal damage in pre-onset stages of the central inhibitory domain of Nogo-A inhibit neurite growth via experimental autoimmune encephalomyelitis. Glia 2005;51:235Y40 p75NTR/NgR-independent pathways that converge at RhoA. Mol Cell 10. Satoh J, Onoue H, Arima K, Yamamura T. Nogo-A and nogo receptor Neurosci 2004;27:163Y74 expression in demyelinating lesions of multiple sclerosis. J Neuropathol 35. Lauren J, Hu F, Chin J, Liao J, Airaksinen MS, Strittmatter SM. Exp Neurol 2005;64:129Y38 Characterization of myelin ligand complexes with neuronal Nogo-66 11. Karnezis T, Mandemakers W, McQualter JL, et al. The neurite out- receptor family members. J Biol Chem 2007;282:5715Y25 growth inhibitor Nogo A is involved in autoimmune-mediated demye- 36. Mimura F, Yamagishi S, Arimura N, et al. Myelin-associated glyco- lination. Nat Neurosci 2004;7:736Y44 protein inhibits microtubule assembly by a Rho-kinaseYdependent 12. Mi S, Hu B, Hahm K, et al. LINGO-1 antagonist promotes spinal cord mechanism. J Biol Chem 2006;281:15970Y79 remyelination and axonal integrity in MOG-induced experimental auto- 37. Mehta NR, Lopez PH, Vyas AA, Schnaar RL. Gangliosides and Nogo immune encephalomyelitis. Nat Med 2007;13:1228Y33 receptors independently mediate myelin-associated glycoprotein inhib- 13. Huber AB, Schwab ME. Nogo-A, a potent inhibitor of neurite out- ition of neurite outgrowth in different nerve cells. J Biol Chem 2007; growth and regeneration. Biol Chem 2000;381:407Y19 282:27875Y86 14. Schwab ME, Caroni P. Oligodendrocytes and CNS myelin are non- 38. Syken J, Grandpre T, Kanold PO, Shatz CJ. PirB restricts ocular- permissive substrates for neurite growth and fibroblast spreading in dominance plasticity in . Science 2006;313:1795Y800 vitro. J Neurosci 1988;8:2381Y93 39. Atwal JK, Pinkston-Gosse J, Syken J, et al. PirB is a functional receptor 15. Huang DW, McKerracher L, Braun PE, David S. A therapeutic vaccine for myelin inhibitors of axonal regeneration. Science 2008;322:967Y70 approach to stimulate axon regeneration in the adult mammalian spinal 40. Kim JE, Li S, GrandPre T, Qiu D, Strittmatter SM. Axon regeneration cord. Neuron 1999;24:639Y47 in young adult mice lacking Nogo-A/B. Neuron 2003;38:187Y99

332 Ó 2010 American Association of Neuropathologists, Inc.

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010 Mechanisms of Axonal Degeneration in MS

41. Simonen M, Pedersen V, Weinmann O, et al. Systemic deletion of the 66. Jaffe AB, Hall A. Rho GTPases: Biochemistry and biology. Annu Rev myelin-associated outgrowth inhibitor Nogo-A improves regenerative Cell Dev Biol 2005;21:247Y69 and plastic responses after . Neuron 2003;38:201Y11 67. Poliakov A, Cotrina M, Wilkinson DG. Diverse roles of eph receptors 42. Zheng B, Ho C, Li S, Keirstead H, Steward O, Tessier-Lavigne M. and ephrins in the regulation of cell migration and tissue assembly. Dev Lack of enhanced spinal regeneration in Nogo-deficient mice. Neuron Cell 2004;7:465Y80 2003;38:213Y24 68. Arimura N, Menager C, Kawano Y, et al. Phosphorylation by Rho 43. Dimou L, Schnell L, Montani L, et al. Nogo-AYdeficient mice reveal kinase regulates CRMP-2 activity in growth cones. Mol Cell Biol 2005; strain-dependent differences in axonal regeneration. J Neurosci 2006; 25:9973Y84 26:5591Y603 69. Hsieh SH, Ferraro GB, Fournier AE. Myelin-associated inhibitors 44. Mi S, Miller RH, Lee X, et al. LINGO-1 negatively regulates regulate cofilin phosphorylation and neuronal inhibition through LIM myelination by oligodendrocytes. Nat Neurosci 2005;8:745Y51 kinase and Slingshot phosphatase. J Neurosci 2006;26:1006Y15 45. Lee X, Yang Z, Shao Z, et al. NGF regulates the expression of axonal 70. Amano M, Ito M, Kimura K, et al. Phosphorylation and activation of LINGO-1 to inhibit oligodendrocyte differentiation and myelination. myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 1996;271: J Neurosci 2007;27:220Y25 20246Y49 46. Kim JE, Liu BP, Park JH, Strittmatter SM. Nogo-66 receptor prevents 71. Goshima Y, Nakamura F, Strittmatter P, Strittmatter SM. Collapsin- raphespinal and rubrospinal axon regeneration and limits functional induced growth cone collapse mediated by an intracellular protein Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021 recovery from spinal cord injury. Neuron 2004;44:439Y51 related to UNC-33. Nature 1995;376:509Y14 47. Zheng B, Atwal J, Ho C, et al. Genetic deletion of the Nogo receptor 72. Minturn JE, Fryer HJ, Geschwind DH, Hockfield S. TOAD-64, a gene does not reduce neurite inhibition in vitro or promote corticospinal tract expressed early in neuronal differentiation in the rat, is related to regeneration in vivo. Proc Natl Acad Sci U S A 2005;102:1205Y10 unc-33, a C. elegans gene involved in axon outgrowth. J Neurosci 48. Cafferty WB, Strittmatter SM. The Nogo-Nogo receptor pathway limits 1995;15:6757Y66 a spectrum of adult CNS axonal growth. J Neurosci 2006;26:12242Y50 73. Hedgecock EM, Culotti JG, Thomson JN, Perkins LA. Axonal guid- 49. Wang X, Baughman KW, Basso DM, Strittmatter SM. Delayed Nogo ance mutants of Caenorhabditis elegans identified by filling sensory receptor therapy improves recovery from spinal cord contusion. Ann neurons with fluorescein dyes. Dev Biol 1985;111:158Y70 Neurol 2006;60:540Y49 74. Hamajima N, Matsuda K, Sakata S, Tamaki N, Sasaki M, Nonaka M. 50. Fontoura P, Ho PP, DeVoss J, et al. Immunity to the extracellular A novel gene family defined by human dihydropyrimidinase and domain of Nogo-A modulates experimental autoimmune encephalo- three related proteins with differential tissue distribution. Gene 1996; myelitis. J Immunol 2004;173:6981Y92 180:157Y63 51. Reindl M, Khantane S, Ehling R, et al. Serum and cerebrospinal fluid 75. Fukata Y, Itoh TJ, Kimura T, et al. CRMP-2 binds to tubulin heterodimers antibodies to Nogo-A in patients with multiple sclerosis and acute to promote microtubule assembly. Nat Cell Biol 2002;4:583Y91 neurological disorders. J Neuroimmunol 2003;145:139Y47 76. Cole AR, Causeret F, Yadirgi G, et al. Distinct priming kinases 52. Spillmann AA, Bandtlow CE, Lottspeich F, Keller F, Schwab ME. contribute to differential regulation of collapsin response mediator Identification and characterization of a bovine neurite growth inhibitor proteins by glycogen synthase kinase-3 in vivo. J Biol Chem 2006;281: Y (bNI-220). J Biol Chem 1998;273:19283Y93. 16591 98 53. Fry EJ, Ho C, David S. A role for Nogo receptor in macrophage 77. Uchida Y, Ohshima T, Sasaki Y, et al. Semaphorin3A signalling clearance from injured peripheral nerve. Neuron 2007;53:649Y62 is mediated via sequential Cdk5 and GSK3beta phosphorylation of 54. Tran EH, Hoekstra K, van Rooijen N, Dijkstra CD, Owens T. Immune CRMP2: Implication of common phosphorylating mechanism under- lying axon guidance and Alzheimer’s disease. Genes Cells 2005;10: invasion of the parenchyma and experimental Y allergic encephalomyelitis, but not leukocyte extravasation from blood, 165 79 78. Cole AR, Knebel A, Morrice NA, et al. GSK-3 phosphorylation of are prevented in macrophage-depleted mice. J Immunol 1998;161: the Alzheimer epitope within collapsin response mediator proteins 3767Y75 regulates axon elongation in primary neurons. J Biol Chem 2004;279: 55. Bourquin C, van der Haar ME, Anz D, et al. DNA vaccination efficiently 50176Y80 induces antibodies to Nogo-A and does not exacerbate experimental 79. Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A, autoimmune encephalomyelitis. Eur J Pharmacol 2008;588:99Y105 Kaibuchi K. GSK-3beta regulates phosphorylation of CRMP-2 and 56. Pool M, Niino M, Rambaldi I, Robson K, Bar-Or A, Fournier AE. neuronal polarity. Cell 2005;120:137Y49 Myelin regulates immune cell adhesion and motility. Exp Neurol 2009; Y 80. Arimura N, Inagaki N, Chihara K, et al. Phosphorylation of collapsin 217:371 77 response mediator protein-2 by Rho-kinase. Evidence for two separate 57. Zhang L, Zheng S, Wu H, et al. Identification of BLyS (B lymphocyte signaling pathways for growth cone collapse. J Biol Chem 2000;275: stimulator), a non-myelinYassociated protein, as a functional ligand for Y Y 23973 80 Nogo-66 receptor. J Neurosci 2009;29:6348 52 81. Kawano Y, Yoshimura T, Tsuboi D, et al. CRMP-2 is involved in 58. Fournier AE, Takizawa BT, Strittmatter SM. Rho kinase inhibition kinesin-1Ydependent transport of the Sra-1/WAVE1 complex and axon enhances axonal regeneration in the injured CNS. J Neurosci 2003;23: formation. Mol Cell Biol 2005;25:9920Y35 Y 1416 23 82. Rizvi S. Multiple sclerosis: Current and future treatment options. Endocr 59. Profyris C, Cheema SS, Zang D, Azari MF, Boyle K, Petratos S. Metab Immune Disord Drug Targets 2007;7:292Y99 Degenerative and regenerative mechanisms governing spinal cord injury. 83. Baptiste DC, Fehlings MG. Pharmacological approaches to repair the Neurobiol Dis 2004;15:415Y36 injured spinal cord. J Neurotrauma 2006;23:318Y34 60. Dent EW, Gertler FB. Cytoskeletal dynamics and transport in growth 84. Schwabb JM, Brechtel K, Mueller C-A, et al. Experimental strategies cone motility and axon guidance. Neuron 2003;40:209Y27 to promote spinal cord regenerationVan integrative perspective. Prog 61. Canty AJ, Murphy M. Molecular mechanisms of axon guidance in the Neurobiol 2006;78:91Y116 developing corticospinal tract. Prog Neurobiol 2008;85:214Y35 85. Maier IC, Ichiyama RM, Courtine G, et al. Differential effects of antiY 62. Buettner HM, Pittman RN, Ivins JK. A model of neurite extension Nogo-A antibody treatment and treadmill training in rats with incom- across regions of nonpermissive substrat: Simulations based on experi- plete spinal cord injury. Brain 2009;132:1426Y40 mental measurement of growth cone motility and filopodial dynamics. 86. Robak LA, Venkatesh K, Lee H, et al. Molecular basis of the interactions Dev Biol 1994;163:407Y22 of the Nogo-66 receptor and its homolog NgR2 with myelin-associated 63. Wang F, Liang Z, Hou Q, et al. Nogo-A is involved in secondary glycoprotein: Development of NgROMNI-Fc, a novel antagonist of CNS axonal degeneration of in hypertensive rats with focal cortical myelin inhibition. J Neurosci 2009;29:5768Y83 infarction. Neurosci Lett 2007;417:255Y60 87. Fournier AE, Gould GC, Liu BP, Strittmatter SM. Truncated soluble 64. Azari MF, Ozturk E, Profyris C, et al. LIF treatment reduces Nogo-A Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by deposits in spinal cord injury modulating Rho GTPase activity and myelin. J Neurosci 2002;22:8876Y83 CRMP-2 phosphorylation. J Neurodegen Regen 2008;1:23Y30 88. Mi S, Miller RH, Tang W, et al. Promotion of central nervous system 65. Buss A, Schwab ME. Sequential loss of myelin proteins during remyelination by induced differentiation of oligodendrocyte precursor Wallerian degeneration in the rat spinal cord. Glia 2003;42:424Y32 cells. Ann Neurol 2009;65:304Y15

Ó 2010 American Association of Neuropathologists, Inc. 333

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. Petratos et al J Neuropathol Exp Neurol  Volume 69, Number 4, April 2010

89. Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target 95. Sun X, Minohara M, Kikuchi H, et al. The selective Rho-kinase for neurological disorders. Nat Rev Drug Discov 2005;4:387Y98 inhibitor Fasudil is protective and therapeutic in experimental auto- 90. Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth immune encephalomyelitis. J Neuroimmunol 2006;180:126Y34 muscle mediated by a Rho-associated protein kinase in hypertension. 96. Hara M, Takayasu M, Watanabe K, et al. Protein kinase inhibition by Nature 1997;389:990Y94 fasudil hydrochloride promotes neurological recovery after spinal cord 91. Sasaki Y, Suzuki M, Hidaka H. The novel and specific Rho-kinase injury in rats. J Neurosurg 2000;93:94Y101 inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]- 97. Sung JK, Miao L, Calvert JW, Huang L, Louis Harkey H, Zhang JH. A homopiperazine as a probing molecule for Rho-kinaseYinvolved path- possible role of RhoA/Rho-kinase in experimental spinal cord injury in way. Pharmacol Ther 2002;93:225Y32 rat. Brain Res 2003;959:29Y38 92. Hirooka Y, Shimokawa H. Therapeutic potential of rho-kinase 98. Baptiste DC, Fehlings MG. Update on the treatment of spinal cord inhibitors in cardiovascular diseases. Am J Cardiovasc Drugs 2005;5: injury. Prog Brain Res 2007;161:217Y33 31Y39 99. Paintlia AS, Paintlia MK, Singh AK, Singh I. Inhibition of rho family 93. Yamamoto M, Ramirez SH, Sato S, et al. Phosphorylation of claudin-5 functions by lovastatin promotes myelin repair in ameliorating ex- and occludin by rho kinase in brain endothelial cells. Am J Pathol perimental autoimmune encephalomyelitis. Mol Pharmacol 2008;73: 2008;172:521Y33 1381Y93 94. Walters CE, Pryce G, Hankey DJ, et al. Inhibition of Rho GTPases 100. Baer AS, Syed YA, Kang SU, et al. Myelin-mediated inhibition of Downloaded from https://academic.oup.com/jnen/article/69/4/323/2917137 by guest on 30 September 2021 with protein prenyltransferase inhibitors prevents leukocyte recruitment oligodendrocyte precursor differentiation can be overcome by pharma- to the central nervous system and attenuates clinical signs of disease in cological modulation of Fyn-RhoA and protein kinase C signalling. an animal model of multiple sclerosis. J Immunol 2002;168:4087Y94 Brain 2009;132:465Y81

334 Ó 2010 American Association of Neuropathologists, Inc.

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.