Nogo in multiple sclerosis: Growing roles of a growth inhibitor

Journal of the Neurological Sciences 245 (2006) 201 – 210 www.elsevier.com/locate/jns

Nogo in multiple sclerosis: Growing roles of a growth inhibitor Paulo Fontoura a,b,*, Lawrence Steinman c a

Department of Immunology, Faculty of Medical Sciences, New University of Lisbon, 1169-056 Lisbon, Portugal b Inflammation Group, Gulbenkian Science Institute, Oeiras, Portugal c Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA Received 1 April 2005; received in revised form 28 June 2005; accepted 12 July 2005 Available online 6 May 2006

Abstract In recent years, knowledge about the physiological functions of the Nogo-A protein has grown considerably, and this molecule has evolved from being one of the most important axonal regrowth inhibitors present in central nervous system (CNS) myelin, to several other potentially important roles in different areas such as nervous system development, epilepsy, vascular physiology, muscle pathology and CNS tumors. Therapeutically, targeting the Nogo-A protein by means of the immune response has been tried in an attempt to block neurite growth inhibition and promote regeneration in spinal cord injury models; the immune response to Nogo-A, however, has not been extensively studied. We propose to review recent evidence that Nogo-A may also play an important role in autoimmune demyelinating diseases such as experimental autoimmune encephalomyelitis and multiple sclerosis, including that Nogo-66 derived epitopes are encephalitogenic antigens in susceptible mouse strains, and that the immune response to Nogo-66 antigens includes both strong T cell and B cell activation, with epitope spreading of the antibody response to other myelin molecules. In CNS immunotherapy, careful targeting of neural self-antigens is a prerequisite in order to avoid unexpected deleterious effects, and increasing knowledge about the immune response to Nogo-A may provide a safe basis for the development of relevant therapeutic alternatives for several neurological conditions. D 2006 Elsevier B.V. All rights reserved. Keywords: Nogo-A; Autoimmune demyelination; Experimental autoimmune encephalomyelitis; Antigen-specific therapy; Multiple sclerosis

1. Introduction The career of the axonal regrowth inhibitor Nogo-A has been a success story from the start, as much as the propensity of its name for easy double entendres and catchy review titles. Following its simultaneous description by three independent groups in early 2000 [1– 3], there has been a flurry of activity around this central nervous system myelin component. The presence of white matter-associated inhibitors of axonal regeneration was suspected almost since the inception of the neuron doctrine, but only after the identification of individual myelin components capable of mediating such effects, and the subsequent characterization * Corresponding author. Department of Immunology, Faculty of Medical Sciences, New University of Lisbon, Campo Martires da Pa´tria 130, 1169056 Lisbon, Portugal. Tel.: +351 21 880 3045. E-mail address: [email protected] (P. Fontoura). 0022-510X/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2005.07.020

of the Nogo protein did that hypothesis come to fruition (reviewed in [4]). The identity of axonal regrowth inhibitors, their physiological roles and mechanisms of action have become increasingly clear and are the focus of intense research efforts (reviewed in [5]). Among the several inhibitors that have been characterized, Nogo-A continues to have a prominent role, and several therapeutic strategies aimed at improving axonal regeneration in spinal cord injury have been directed towards blocking interactions in the Nogo –Nogo receptor system [6– 11]. It has also become increasingly apparent that Nogo-A probably has a number of different roles, not limited to neurite growth inhibition; the focus of this review is to briefly review these roles, to specifically address the role of Nogo as an antigen for the immune response and to provide evidence that Nogo-A contains immunogenic regions with relevance for experimental autoimmune encephalomyelitis, and possibly for Multiple Sclerosis.

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2. Nogo is a member of the Reticulon family Nogo belongs to the Reticulon (RTN) gene family [12,13], which consists of proteins 200 –1200 amino acids in length sharing a common C-terminus domain (reticulon homology domain, RHD), composed of two hydrophobic regions flanking a circa 66 aa loop. In the Nogo protein, this loop (Nogo-66) has been the focus of intense interest, since it mediates growth cone collapse through interaction with a recently described receptor, NgR [14]. Nogo-66 has been conserved throughout evolution, even in animals who show regeneration after injury, which indicates that it must have other functions in these beings [13]. Oligodendrocytes, some neuronal populations, the heart and testis express RTN4-A/Nogo-A; Nogo-C, the shortest transcript, is particularly abundant in muscle cells, whereas Nogo-B is widespread in distribution [12]. Most of the nogo transcript is probably retained in the endoplasmic reticulum (ER), but a low percentage of Nogo-A is present in the surface membrane of oligodendrocytes [1,2] and together with OMgp is also expressed in neurons [15 –18]. Membrane topology studies have proposed that Nogo-A can assume at least two different configurations depending on its localization either in the ER or plasma membrane: in the more prevalent ER-shape, the 66-loop faces the lumen, whereas at the surface it faces the outside [12,14]. Furthermore, since both hydrophobic stretches can span membranes either once or twice, both Nogo-66 and the Nterminus might be able to face the same inward or outward direction [12]. This possibility is particularly interesting inasmuch as the growth inhibitory capacity of Nogo-A appears to be mediated by both Nogo-66 (human Nogo1024 –1090) and a stretch in the N-terminus, also called NiG and which specifically corresponds to mouse Nogo544 –725 (human Nogo567 –748) [2,3,14,19]. Therefore, a membrane conformation in which both inhibitory domains faced outward might mean that even in normal circumstances –without the need for myelin destruction to expose them –both domains could exercise their physiological functions. The Nogo-66 specific receptor was identified in 2001 and its structure clarified in 2003 [14,20,21]. This GPIlinked receptor, expressed mainly in the brain, mediates Nogo-66’s capacity for neurite growth cone collapse after activation of the RhoA GTPase [5,14,22]. More recently, two other homologous proteins to this receptor have been found [23], although their function is yet to be elucidated. The Nogo-66 receptor complex seems to consist of at least three proteins: NgR, which interacts directly with Nogo-66; the p75NTR co-receptor which is probably the signalling part of the complex [24,25] and the transmembrane brainspecific LRR protein LINGO-1 [26]. Two other myelin axonal regrowth inhibitors, myelin associated glycoprotein (MAG) and oligodendrocyte – myelin glycoprotein (OMgp), also interact with the NgR/p75 complex [27 – 29], making

this complex a final common signalling pathway for growth inhibition in CNS myelin. The secondary structure of Nogo-66 has only recently been partially characterized. Using an artificially synthesized Nogo1 –40 peptide, Li et al. [30] were able to show using NMR spectroscopy, that two helices exist over residues 7 –12 and 26– 37, and possibly another at residues 20– 24. The N-terminus of Nogo1 –40 is largely positive in charge, in contrast with the C-terminus, which is negatively charged. Given the recently described crystallographic structure of the NgR ectodomain, it is predicted that the positive N-terminus would bind to a negative cavity in the receptor [20,21,30]. Curiously, signaling through the NgR appears to be related to the C-terminus of this sequence, since Nogo1 –40 peptide although binding strongly and being an excellent antagonist of NgR, does not induce inhibitory signalling [19,30]. This portion of the molecule might bind to a yet unknown positive surface on NgR, or to another member of the NgR complex.

3. Nogo expression in the nervous system During development, Nogo-A is expressed by several neuronal populations and might actually have a role as a growth promoter and in fibre tract formation [18,31]. During the early stages of myelination, Nogo appears to also have an important role in the local distribution of potassium channels in the paranodal region, through an interaction with the Caspr-F3 axoglial complex mediated by the Nogo-66 region [32]. The Caspr-F3 complex is responsible for the architecture of the axolemmal– glial apparatus. The Nogo-A-Caspr complex directly interacts with Kv1.1 and Kv1.2 potassium channels and thereby influences their segregation to the juxtaparanodal region. To further emphasize the notion that Nogo is not involved in axonal growth at this stage of myelination, Nogo-A, but not NgR, localizes to the paranodes, and Nogo-A, Caspr and Kv1.1 channels have a similar spatial and temporal relationship during development. In diseases such as the MBP mutant shiverer mouse and rat EAE, there is a disorganization of the axoglial junctional complex, with reduction of Nogo-A expression and relocalization of K+ channels to the paranodal region [32]. This probably contributes to defects in axonal transmission and therefore neurological disability. Furthermore, in experimental models of periventricular leukomalacia, chronic ischemia was shown to reduce the levels of Nogo-A in the periventricular white matter, leading to aberrant axonal sprouting [33]. In the adult human nervous system, Nogo-A is expressed predominantly by oligodendrocyte cell bodies and myelin sheaths, and to some extent in other populations of brain and spinal cord neuronal populations, especially in most brain stem nuclei, dorsal root ganglion sensory cells, and spinal cord motor neurons and inter-

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neurons [34]. The presence of Nogo-A in adult neurons suggests that this protein might have other roles beyond axonal inhibition, even in the mature CNS. These roles might include attractive or repulsive signalling for other neurons, signal transduction for a yet to be identified ligand, or some other intracellular function [15]. Interestingly, Nogo-A expression in adult neurons does not appear to be influenced by the local presence of inflammatory cytokines such as TNFa or IL-1h, or neurotrophic factors such as BDNF [35]. More recently, it has been found in experimental animals that Nogo-A expression is apparently universal in neurons, if in different degrees; curiously, NgR expression is very limited, and absent in neuronal populations that maintain high regenerative capacity [36]. At a cellular level, Nogo-A and NgR are expressed in a pattern consistent with their role in axonal – glial interactions and limitation of axonal sprouting and plasticity in the adult CNS [37]. NgR is expressed in mature neurons, and Nogo-A in the adaxonal myelin sheath, as well as in the outermost myelin membranes [15]. After CNS injury the expression of Nogo-A is not significantly altered, unlike other myelin molecules. Nogo’s role in neurite growth inhibition, in physiological conditions, seems to be restricted to the developing nervous system, and after that to tonic inhibition of adult neuronal growth [15,17].

4. Knocking out Nogo Given the prominent roles theoretically attributed to Nogo-A in the developing and adult nervous system, it was expected that knocking out the gene for Nogo or the Nogo receptor might induce severe phenotypes and dramatically change the regenerative capacity of the injured CNS. Such was not the case, and in fact, three independent groups reported different and somewhat contradictory results [38 – 40]. All three different Nogo knockouts had normal behaviour and phenotype, but responded differently to spinal cord injury, from evident improvement in regeneration and functional recovery [39] to no significant enhancement of regeneration [40]. Functional recovery was also absent in an intermediate phenotype that exhibited some increase in axonal regrowth capacity [38]. The absence of a distinct neurological phenotype for disease animals seems to argue against a significant role for Nogo-A in normal CNS development. In any case, it is difficult to reconcile these results with the expected major role of Nogo in growth inhibition. With the discovery of the two other ligands for NgR (MAG and OMgp), it was thought that redundancy in the NgR signalling system could account for the lack of functional differences in Nogo KO animals. Deleting the ngr gene, therefore, was the logical next step to validate this hypothesis, and once again two independent groups generated different, but in this case not conflicting, results [41,42]. In one case [41], ngr / mice exhibited selective

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regeneration of brainstem tracts (raphespinal and rubrospinal) but not corticospinal axons, even across complete spinal cord transsections, and significant functional improvement. Knockout animals also showed altered behaviour patterns, such as hypoactivity in open field testing and some motor impairment in rotarod testing. In a different study, deleting the ngr gene did not significantly reduce the in vitro neuronal response to myelin inhibitors, and corticospinal tract axons did not show enhanced regeneration [42]. Functional recovery was also not apparent in open field testing. In the same study, deleting the p75NTR co-receptor had a significant influence on in vitro tests, but again showed no effect in anatomical regeneration. Taken together, the results of all these experiments seem to argue for the relevance of the NgR signalling pathway in the regeneration of at least some types of axonal fibres, consistent with the existence of several ligands for this receptor. They also point to the fact that the role of Nogo-A as a growth inhibitor is not as major as once thought, and that other functions of this protein might be physiologically more relevant.

5. The growing roles of Nogo—I go, you go, we all go for Nogo RTN proteins have been postulated to have roles in ER pore or transport complexes and structure stabilization, transport of ER products to membrane organelles such as the Golgi or endossomes, and to the plasma membrane, and possibly in cell division [12]. Increasingly, Nogo has been implicated in functions other than neurite growth inhibition. Among the many clues that nogo transcripts might have other roles is the growing list of molecules with which they can interact besides the Nogo receptor (reviewed in [43]). These include several diverse targets, such as mitochondrial proteins NIMP, UQCRC1 and UQCRC2 [44], the anti-apoptotic Bcl-2 and Bcl-XL [45], the axoglial contact protein Caspr [32], the endoplasmic reticulum protein ASYIP [46], and myelin basic protein and microtubular a-tubulin [47]. Like other members of the RTN family, Nogo is an ER-enriched protein, and interactions with other ER, mitochondrial and cytoplasmic proteins could have relevance in diverse aspects of cellular physiology that are yet not apparent. The fact that Nogodeficient mice apparently exhibit a normal phenotype could be related to the compensatory role that other members of the RTN family might perform in normal physiology [43]. Gradually, the role of the Nogo family of molecules has spread far from the field of axonal guidance and regrowth, and its main connection to spinal cord injury, to several other areas of neurological disease unrelated to trauma (reviewed in [48]). We will, therefore, for the purposes of this paper, concentrate on these novel roles and abstain from reviewing the extensive literature on neurotrauma.

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5.1. Nogo in epilepsy

5.3. Nogo in muscle pathology

The expression of Nogo and NgR in the mammalian hippocampus is tightly connected to the development and maturation of hippocampal afferents [31]. Hippocampal E16 pyramidal neurons express increasing amounts of Nogo mRNA, with smaller amounts in the dentate, and in the entorhinal cortex. NgR is expressed later, mainly from P0 to P5, especially in the CA3 area, and in the upper portion of the granule cell layer. In the embryonic hippocampus, Nogo-66 and its receptor probably do not interact and Nogo-A might have other roles during development. After adult injury to the hippocampus, however, Nogo-66 may play a role in inhibiting axonal regrowth. Soon after hippocampal injury, Nogo-A is upregulated by reactive astrocytes in the molecular layer of the dentate gyrus, and may play a role in regulating the migration of reactive astroglia. NgR, on the other hand, is downregulated in granule and hilar cells 15 days after lesions. Nogo and NgR transcription may also be connected with neuronal activity. Hippocampal granule cells transiently downregulate expression of both genes after kainic acid administration. Both in experimental models of temporal lobe epilepsy (TLE) and in human TLE samples, an upregulation of Nogo-A has been demonstrated [49,50] both in mesial temporal sclerosis and nonsclerotic specimen. In TLE samples, most hippocampal neurons were found to upregulate Nogo-A; in this case, given that aberrant neurite sprouting is an integral part of TLE, Nogo-A might be performing other actions besides growth inhibition, or its capacity for inhibition might be overcome by other stimuli present locally at the lesion site.

Nogo isoform expression in muscle cells might also be a marker for amyotrophic lateral sclerosis (ALS) [55]. In a transgenic mutant SOD1 ALS mouse model and in human ALS patients, an increase in the Nogo-A transcript was detectable concomitant to the onset of disease, together with a downregulation of Nogo-C expression. These changes might reflect a denervation-related dedifferentiation of muscle cells, since Nogo-A mRNA is abundant in embryonic muscle cells. An increase in Nogo-A transcripts in muscle biopsy samples might therefore be a good diagnostic marker for ALS. Nogo-C mRNA downregulation is present not only in ALS but also in other denervating diseases, such as neuropathies, and presumably reflects the repression of the growth-inhibiting capacities of this molecule in an attempt to regain muscle function by promoting muscle regeneration. The severity of ALS progression is apparently also related to the expression of Nogo-A and B [56].

5.2. Nogo, vascular physiology and stroke Nogo-B has been recently implicated in vascular remodelling after injury [51]. Authors were able to show that Nogo-B was highly expressed in vascular smooth muscle and endothelial cells, and that it was downregulated after injury. Nogo-A/B KO mice exhibited marked neointimal regrowth after injury, leading to complete stenosis of the vessel. Contrary to the role Nogo assumes in the CNS, in vessel wall biology this molecule appears to be a chemoattractant for endothelial cells, promoting adhesion in endothelial and muscle cells, and inhibiting PDGF-mediated smooth muscle cell migration. Furthermore, different regions of Nogo-B appear to have diverse functions. The N-terminus enhances endothelial cell migration; Nogo-66 inhibits smooth muscle cell migration through a yet-to-be identified receptor, but which is not NgR. Blocking the inhibitory function of Nogo with specific antibodies, or signalling through NgR was shown to improve experimental stroke models [52 – 54], although how much of this effect is mediated by actions on axon regrowth and how much by vascular remodelling are unsure.

5.4. Nogo in CNS tumours Human glioma cell lines endogenously express NgR and their in vitro migration and invasive properties are hampered by both Nogo-66 and MAG, consistent with signalling through the NgR complex [57]. If these findings can be confirmed in tumour biopsy samples, they open up several therapeutic opportunities, such as locally inducing the expression of myelin-associated inhibitors, or using alternative means of NgR stimulation to prevent progression of glial tumours. The role of Nogo-B in cancer cell apoptosis is the subject of conflicting findings. Initially, an apoptosis-inducing gene called ASY with no homology to other known apoptotic genes was identified and found to be identical with Nogo-B. The product of this gene was capable of inducing apoptosis in several cancer cell lines, presumably by association with endoplasmic reticulum proteins. Interestingly, small cell lung cancer cells were found to lack ASY expression, leading to the assumption that mutations in ASY or ASYassociated proteins may underlie cancer pathogenesis [58]. These findings were later contradicted by the finding that several cancers do in fact express Nogo-B consistently, and that these cells do not have increased sensitivity to apoptosis as might be expected. Also, other members of the RTN family, such as RTN1, are overexpressed in cancer cells, and therefore, the underexpression of Nogo-B might turn out to be a compensatory mechanism [59]. In another study, NogoB was found to interact with Bcl-2 and Bcl-xL and may decrease the effects of these anti-apoptotic molecules, even though it did not increase sensitivity to apoptotic stimuli [45]. The kinetics of expression of this molecule, and the induction of endoplasmic reticulum stress, might also be determining factors in the apoptotic role of Nogo-B, since different studies have used several methods of inducing Nogo-B expression. In conjunction, these studies point to

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several different areas where Nogo-related physiology might be relevant to the progression of CNS tumors (reviewed in [60]).

6. Nogo and the immune system: from therapeutic strategies to autoimmune demyelination Multiple Sclerosis (MS) is an immune-mediated disease caused at least in part by an autoimmune attack against central nervous system myelin (reviewed in [61]). Throughout the years, several myelin-associated targets have been identified, based on data gathered from multiple sclerosis animal models and patients, and pathophysiological mechanisms centered on the role of myelin reactive T CD4+ cells, antibodies, and several inflammatory molecules have been devised (reviewed in [62,63]). There is considerable heterogeneity in clinical and MRI findings in MS, and based on pathological examination of tissue samples from patients, a classification of MS has been proposed in which the T cell/macrophage, antibody/ complement-mediated myelin damage or oligodendrocytic pathology predominates [64]. Moreover, the recent rediscovery of abundant axonal pathology in acute and chronic MS lesions [65], and its relation to disability progression later on in the disease course, has led to the concept that MS evolves in two different stages, namely, an initial inflammatory phase, and a later neurodegenerative phase, and accordingly that therapies should be aimed at different targets in each phase (reviewed in [66]). In that perspective, Nogo-A assumes an added importance, since it could obviously be an important target for the neurodegenerative phase; might it also be a target for the inflammatory phase? The notion of using the immune response to self CNSrelated antigens, to improve the outcome after traumatic injury has been the focus of several papers, with mostly positive results, leading to the concept of neuroprotective autoimmunity (reviewed in [67]). In a different strategy, immunization with whole myelin was shown to induce the production of polyclonal antibodies that presumably blocked the actions of myelin-associated growth inhibitors and resulted in functional improvement ([68]. After the characterization of the Nogo inhibitory domains, Hauben et al. [69] vaccinated rats with a synthetic peptide derived from the Nogo N-terminus region (p472, Nogo623 –640—SYDSIKLEPENPPPYEEA) emulsified in CFA and were able to induce a strong T cell response, but not antibodies to p472. The adoptive transfer of Nogo-specific T cell lines improved outcome after experimental spinal cord contusion and apparently did not induce demyelination. More recently, intrasplenic immunization with a fusion protein of the Nogo-A specific region (NiG, amino acids 174 –979, which includes p472) and the C-fragment of tetanus toxin (TTC) was used to induce an antibody response in rats subjected to spinal cord injury [70]. The vaccination strategy used

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primarily resulted in a strong B cell response with isotype class switching from IgM to IgG mediated by TTC-specific T cells. No T cell response to NiG was reported, and vaccination did not induce EAE. Nogo-66, given its probable extracellular location and interaction with the NgR complex, could possibly be a more logical target for immune interventions. In fact, immunization with Nogo-66 combined with MAG, in both incomplete Freund’s adjuvant (IFA) and aluminum hydroxide, was able to improve longdistance corticospinal axon regeneration and sprouting [71]. Even though this study was conducted in EAE-susceptible SJL/J mice, no signs of EAE were observed. Retrospectively, this is perhaps not surprising, since both these adjuvants are not good at inducing demyelinating T cell responses. Looking at the role of Nogo-A in multiple sclerosis, an increase in the expression of Nogo by oligodendrocytes was found in chronic active demyelinating plaques, where this molecule presumably interacted with NgR expressed on reactive astrocytes and microglia [72]. Two groups working independently addressed the role of Nogo in EAE focusing on different regions of this molecule, NiG and Nogo-66 [73 –75]. Both groups conducted EAE-induction experiments on Nogo A/B knockout mice in a C57BL/6 (H-2b) background with similar results, in that the deletion of nogo resulted in EAE improvement [74,75]. In some experiments, the absence of Nogo in the KO experiments also resulted in a poor proliferative response to myelin oligodendrocyte glycoprotein (MOG), as well as a reduction in IFNg production by MOG-reactive T cells [74]. Our own results did not show consistent results regarding an appreciable reduction of proliferation on two different mouse KO strains (P. Fontoura, unpublished observation). Karnezis et al. [74] chose to use Nogo623 – 640 for their experiments. This peptide was found not to be encephalitogenic in EAE-susceptible C57BL/6 mice, and furthermore, vaccination with Nogo623 – 640 in IFA was able to partially protect animals from EAE induced with MOG35 – 55, as well as reducing the number of demyelinating lesions and axonal loss. MOG-specific T cells from Nogo-vaccinated animals showed normal proliferative capacity but were phenotypically shifted towards a Th2 response. AntiNogo623 –640 antibodies, but not T cells, were detectable at peak of disease suppression and were found to block the inhibitory action of myelin on neurite outgrowth. Finally, passive immunization with polyclonal affinity-purified antibodies to Nogo was able to protect from MOG induced EAE. In essence, these results showed that the absence of Nogo or vaccination with a NiG peptide was able to beneficially influence the phenotype of MOG-reactive T cells, and these, together with anti-Nogo blocking antibodies elicited by vaccination, were able to reduce inflammation and possibly provide a better microenvironment for axonal survival and regeneration. Our own work [73,75] focused on the role of T and B cell responses to Nogo-66 (Nogo1024 –1090) in EAE,

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since this extracellular domain should be a more likely target for interactions with the immune system, and we were able to demonstrate that Nogo-66 contains at least three antigenically important epitopes. Peptides Nogo1-22 (RIYKGVIQAIQKSDEGHPFRAY), Nogo23 – 44 (LESEVAISEELVQKYSNSALGH) and Nogo45 – 66 (VNSTIKELRRLFLVDDLVDSLK), were synthesized to encompass the whole Nogo-66 region and used to induce EAE in SJL/J and C57BL/6 mouse strains. We found that all immunized animals exhibited clinical signs of meningitis (i.e., photophobia, irritability and drowsiness) after 10 days, and in a few SJL/J mice immunized with Nogo45 –66 there were evident signs of motor paralysis, such as loss of tail tone or tail paralysis, and hindlimb weakness (grade 2 on the EAE clinical scale). Histological evaluation of all these animals confirmed the existence of characteristic inflammatory infiltrates in the meninges and parenchyma [73,75]. These results confirmed that Nogo66 peptides could act as weak encephalitogens in EAEsusceptible mouse strains. Strong T cell responses were found against Nogo1-22 and Nogo45– 66 in SJL/J mice, and against Nogo1 – 22 and 23– 44 in C57BL/6 mice. Antigenic domains appeared to be strain specific: SJL/J (H-2s) animals had T cell reactions only to Nogo1 – 22 and Nogo45– 66, whereas C57BL/6 (H-2b) mice reacted only to Nogo1– 22 and Nogo23 –44. Adoptive transfer of T cell lines reactive to Nogo1 –22 and 45 – 66 peptides was performed in SJL/J animals and did not result in disease; moreover, when we performed the transfer experiments in animals suffering from PLP139 – 151 induced chronic EAE, we observed a statistically significant improvement in clinical scores [75], as well as in histological assessment of disease burden (P. Fontoura, unpublished observation). SJL/J derived Nogo45 – 66 T cell lines maintained in vitro were shown to spontaneously adopt Th2 phenotypes and secrete large amounts of IL-4 and IL-10, whereas Nogo1 –22 cell lines predominantly secreted IFNg [73,75]; in C57BL/6 cell lines, there was a usually a Th1 biased response (P. Fontoura, unpublished observation). We could find no evidence of epitope spreading of the T cell response from Nogo, or to Nogo from PLP139 – 151 or MBP85-99, either by standard proliferative reactions or ELISPOT assays (P. Fontoura, unpublished observation). In order to eliminate cross-reactivity to other encephalitogenic myelin antigens, anti-Nogo T cell lines were challenged with other known encephalitogens for the SJL and C57 strain (PLP139– 151, MBP85– 99, MOG35 – 55) and were shown to be non-reactive; conversely, anti-PLP T cells did not react to any of the Nogo peptides. Primary structure analysis of the Nogo sequences using measurements of hydropathicity (Kyte – Doolittle plots including hydrophilicity and hydrophobicity), bulkiness, recognition factors or polarity (Zimmerman plots) enabled us to identify probable antigenic domains in all three arbitrarily selected peptides [75]. As discussed above, the predicted secondary

conformation of Nogo1– 40 includes at least two helical structures over residues 7– 12 and 26 – 37 [30]. Unfortunately, no results were reported on the more antigenically important Nogo45 –66 region. It is, at present, unknown how such helices might interfere with binding in the H – 2s and H – 2b MHC cleft. Studying the B cell response in EAE has traditionally been limited to detection and quantification of antibodies against a limited set of myelin proteins and peptides, usually by ELISA or Western blot. Recently, our laboratory has pioneered the use of large-scale spotted arrays for multiplex detection of antibody responses in autoimmune and infectious diseases [76,77]. Using this technology, we were able to show that in EAE there is a progressive spreading of the antibody response from the primary immunizing antigens, e.g., proteolipidic protein (PLP)139 – 151, myelin basic protein (MBP)85 – 99 to other myelin proteins and peptides, and furthermore, that the extent of this spread was correlated with relapse rate in chronic EAE [78]. Applying this method to Nogoimmunized animals resulted in the detection of a similar phenomenon: an initial response to the immunizing antigen, especially strong for Nogo45 –66 in both strains, and to Nogo1-22 in SJL/J, followed by spread to several other myelin antigens, such as MAG, PLP, MBP, MOG, oligodendrocyte-specific glycoprotein (OSP), a-B-Crystallin and 2V,3V-cyclic nucleotide 3V-phosphodiesterase (CNPase) [75]. The extent of epitope spreading was particularly strong from Nogo45 – 66, and targeted mainly MAG (p193 – 208, p313 – 328), MBP (p85 –99, p121 –139) and MOG (p35 – 55) epitopes. Curiously, the reported T cell epitope mapping for MAG in the SJL/J mouse includes strong responses to both MAG193– 208 and 313– 328 [79] and MBP85 –99 is a well-known T cell epitope in this strain [80]. Spread might be due either to local release of myelin debris and local priming of the B cell response or might represent cross-reactivity to other myelin antigens. In either case, the end-result could be deleterious, especially if in conjunction with strong T cell responses. Alternatively, such a response might, if the antibodies block the action of myelin growth inhibitors, or neutralize the antigenic properties of myelin debris, result in amelioration of disease. In fact, antibodies to Nogo-A have been recently found in the blood and CSF of patients with inflammatory (including MS) and non-inflammatory acute CNS diseases [81]. The role these antibodies have in the pathophysiology of such diseases is uncertain, and they might represent an epiphenomenon of injury, contribute to immune-mediated injury or to a protective/regenerative response. Taken together, both reported studies were able to demonstrate that the immune response to Nogo can modulate experimental models of autoimmune demyelination and lead to clinical improvement. Comparisons between these studies are limited due to the different antigenic targets chosen by both groups, and naturally

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different mechanisms were put forward in order to explain the beneficial results. Both antibodies against Nogo623 – 640 and T cells reactive to Nogo45– 66 were strongly implicated in ameliorating two different mouse models of chronic disease, C57BL/6 non-relapsing and SJL/J relapsing EAE. Notably, in both studies there was no apparent effect on axonal regrowth, and immunomodulatory effects were held responsible for the improvement in disease course. Also, in our case we show that ‘‘protective’’ immunization with neural antigens might have deleterious effects, depending on the type of response induced. Under some circumstances, immunization with Nogo peptides was able to induce a meningitis and encephalomyelitis with clinical and histological signs resembling EAE. This was related to the induction of strong T cell responses and marked epitope spreading of the B cell response to other myelin targets; only when the T cell response was Th2 shifted did we observe beneficial effects. Related to the work of Karnezis et al. [74], we found that Nogo623 – 640 immunization in SJL/J did not result in T cell responses, and that the B cell response did not show any appreciable spread to other myelin antigens (P. Fontoura, unpublished observation), which might account for the lack of observed demyelinating lesions. Caution must be exercised when immunizing against self-antigens; depending on the type of immune response induced, therapeutic strategies based on immunization with neural self-antigens may provoke unanticipated detrimental effects such as encephalomyelitis, as was recently demonstrated in human trials of amyloid beta peptide vaccination (reviewed in [82,83]).

7. Conclusions Based on our results, Nogo-66 might join the list of myelin components implicated in the immunopathogenesis of EAE and potentially MS, such as MBP, MAG, MOG, PLP or several other minor antigens (reviewed in [63]). Also, immune-targeting Nogo might prove to be an interesting therapeutic strategy in two different settings (Fig. 1). First, several types of antigen specific therapies such as altered peptide ligands or DNA vaccination against Nogo might prove to be relevant in human demyelinating diseases such as MS. A recent report of DNA vaccination against myelin growth inhibiting epitopes, such as Nogo-A and MAG, proved that although stimulating the immune system, such vaccines were not encephalitogenic [84]. Second, given the evidence for epitope spreading of the B cell response to other myelin antigens after Nogo-66 immunization, this phenomenon might provide a blocking response to other myelin-associated growth inhibitors with therapeutically relevant consequences in CNS injury. Careful targeting of neural antigens is a prerequisite for successful antigen-specific therapies in CNS disease; increasing knowledge of the immune response to Nogo-A, as is provided in recent work [69 – 71,74,75], provides the

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IMMUNIZATION WITH NOGO-A PEPTIDES

PERIPHERAL LYMPHOID ORGAN Naive T cell

APC

B cell

Th1 CELL ANTIBODIES Th2 CELL BBB CNS

Th1 RESPONSE IFNγ TNFα

TGFβ

Th2 RESPONSE

IL-10

Fig. 1. Mechanisms of Nogo-A immunization in experimental autoimmune encephalomyelitis. Immunization with Nogo peptides such as Nogo623640 or Nogo-66 leads to the generation of a T (green and red cells) and/or B (purple) cell response, depending on the peptide, adjuvant and animal species and strain. T cell responses may be either Th1 (red) or Th2 (green), and B cell responses may either be limited to the immunizing peptide (Nogo623-640) or exhibit epitope spreading to other myelin antigens. In the CNS, depending on the type of immune response generated previously the effects might be beneficial or deleterious. The combination of a strong Th1 response with secretion of proinflammatory cytokines (red hexagons) and antibody targeting of other myelin antigens can lead to inflammation and demyelination (top row). If a predominantly Th2 response is generated, anti-inflammatory cytokines (green hexagons) may be generated; furthermore, the antibody response to Nogo and spread to other myelin-associated) growth inhibitors (gray stars) may block their action and facilitate axonal regeneration at sites of injury (bottom row).

basis for the development of relevant therapeutic alternatives for several neurological diseases, including CNS trauma, stroke, tumors, epilepsy, neurodegenerative diseases and multiple sclerosis.

Acknowledgments PF gratefully acknowledges the support given by the Gulbenkian Foundation, Luso-American Foundation, Christopher Reeve Paralysis Foundation and Roman Reed Spinal Cord Injury Research Fund of California, Research for Cure.

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