Despite the numerous investigative reports of NgR1 signalling in the CNS during disease and injury, there exists debate as to whether the limitation in axonal damage or enhanced regrowth potential is regulated through this mechanism. The current study supports the contention that limiting the NgR1-dependent phosphorylation of CRMP-2 (which normally functions to regulate axonal microtubule assembly), axonal degeneration and hence neurological impairment can be halted in EAE. We have demonstrated that phosphorylated CRMP-2 is localized only in degenerative neuronal somata and axons in chronic-active multiple sclerosis lesions, where myelin damage and inflammatory cells coincide. Our study advocates that targeting NgR1-signalling in EAE, and by extension multiple sclerosis, may be a feasible therapeutic approach to limit the neurodegenerative effects of these conditions.
Current therapeutics in multiple sclerosis target the inflammatory nature of the disease in an attempt to limit the ‘autoimmune attack’ on CNS myelin and, as a consequence, reduce the devastating neurological complications that characterize this disease. However, comprehensive neuropathological investigations from a vast array of multiple sclerosis brain samples suggest a degree of heterogeneity in the pathogenesis, with some lesions showing tangible neurobiological origins (Lucchinetti et al., 2000
). Both the ‘outside-in’ and ‘inside-out’ hypotheses of demyelination and axonal degeneration involve dysregulation of axo-glial signalling, driving axonal degeneration potentiating neurological decline and establishing disability.
Using the exon 2 nogo receptor
null mutant mice bred on a C57Bl/6 background (Kim et al., 2004
), we report that the signalling mechanism that is operative during the neurodegenerative phase of EAE relies on NgR1-dependent phosphorylation of CRMP-2. A significant increase in the phosphorylation of CRMP-2 at the Thr555 site, specific for Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) (Arimura et al., 2000
; Arimura and Kaibuchi, 2005
; Mimura et al., 2006
), was demonstrated to be operative in EAE. This mechanism has been previously identified to be important in neurite/axon retraction events in culture (Arimura et al., 2000
; Arimura and Kaibuchi, 2005
). Similarly, pThr555-CRMP-2 has been demonstrated to be responsible for axonal degeneration in an acute model of spinal cord injury (Mimura et al., 2006
). Our finding that levels of pThr555-CRMP-2 in the spinal cord and optic nerve of ngr1+/+
but not ngr1−/−
mice, incrementally increased during the clinical and pathological progression of EAE, is critical in defining a neurobiological basis for this signalling cascade during neuroinflammatory-mediated degeneration.
There is strong evidence that phosphorylation of CRMP-2, either by GSK-3β, Cdk-5 or ROCK2, inactivates the normal physiological functions of CRMP-2. Physiologically, CRMP-2 can associate with α- and β-tubulin heterodimers (Arimura and Kaibuchi, 2005
; Arimura et al., 2005
), kinesin-1 motor proteins (Kimura et al., 2005
), TrkB and the Sra/WAVE1 complex, to facilitate anterograde axonal transport, and thereby growth, repair, maintenance and communication (Kawano et al., 2005
; Arimura et al., 2009
). Disruption of transport of this molecular cargo is integral for the interruption in the maintenance of the axonal terminal and by extension, synaptic connectivity (Arimura et al., 2005
; Kawano et al., 2005
; Kimura et al., 2005
; Petratos et al., 2010
). The consequential loss of axo-dendritic connectivity, mediated through various negative signal transduction events, leads to actin and tubulin depolymerization and eventual axonal retraction (Schmidt and Rathjen, 2010
). However, the question remains as to whether these events can be initiated during neuroinflammatory diseases such as EAE and multiple sclerosis.
Our data suggest that pThr555CRMP-2 is involved as a neurodegenerative molecule of axons and neurons in active multiple sclerosis lesions and that this signal can be limited by ablation of the NgR1
gene or by therapeutically targeting Nogo-A, during acute neuroinflammation, without any immune-dependent mechanisms being directly responsible for this signalling. However, we did observe that the phosphorylation of CRMP-2 was reduced at the chronic stage of EAE, a stage where neurodegeneration continues to progress. We are currently investigating this finding to delineate whether there is a clearance of extracellular myelin-associated inhibitory factors or NgR1 expression changes at the chronic stage of EAE. However, one line of investigation may derive from data that show that CRMP-2 is cleaved by calpain during overt neurodegeneration, forming a 55
kDa truncated form (Taghian et al., 2012
). Such forms of CRMP-2 have been linked to neuronal cell death in various models of neurodegeneration (Taghian et al., 2012
). Despite the uncertain mechanism occurring during the chronic stage of EAE, we importantly showed that the downstream target of NgR1 signalling, the phosphorylation of CRMP-2 at the Thr555 site, can be abrogated in retinal ganglion cell axons by transduction using a rAAV2 Flag-T555ACRMP2-GFP. The limitation of this signalling cascade during EAE-induced optic neuritis maintains the fidelity of these optic nerve axons and their fast axonal transport mechanisms.
The CNS tissue milieu during injury or disease is rich in axonal outgrowth inhibitors such as Nogo-A, potentiating axonal retraction events and blocking regeneration (Profyris et al., 2004
). It has recently been shown that limiting NgR1 signalling in the MOG35–55
-induced or the B10.PL models of EAE, by using small interfering RNA knock-down of Nogo-A
, can ameliorate the disease severity and enhance axonal repair (through increase in GAP-43-positive axons within the spinal cord) without altering myelin-specific T cell proliferation and cytokine production (Yang et al., 2010
). Our study extends these findings by pinpointing the signalling mechanism responsible for axonal degeneration in EAE and has demonstrated that downstream pThr555CRMP-2 is present in degenerative neurons and axons of chronic-active multiple sclerosis lesions.
It was recently discovered that the co-receptor of NgR1, Lingo-1, can promote demyelination in the MOG35–55
EAE murine model (Mi et al., 2007
). These investigators showed that in MOG35–55
mice or wild-type EAE-induced mice treated with a function-blocking anti-Lingo-1 antibody, demyelination was limited thereby blunting the neurological decline. Although these findings implicate the NgR1 signalling complex in the promotion of demyelination during neuroinflammation, our data suggest that targeting the NgR1 high-affinity receptor for myelin associated inhibitory factor-binding limits axonal degeneration during the onset and peak stages of EAE. However, at the early pre-onset stage of EAE, an equivalent proportion of demyelination in the optic nerves of both the ngr1−/−
wild-type littermate mice was demonstrated. This initial thinning of myelin (increasing G
-ratio) does not persist with progression of disease in the ngr1−/−
mice, suggesting that possible remyelination may in fact be preserving axons at the early stage of neuroinflammation (Harrington et al., 2010
). Despite these data, we believe that by limiting the axonal phosphorylation of CRMP-2 during neuroinflammation, we can preserve the axons during the peak stage of EAE as we demonstrated in the optic nerve following the rAAV2 Flag-T555ACRMP2-GFP transduction of retinal ganglion cells. These in vivo
data for the first time demonstrate that inhibition of the phosphorylation of CRMP-2 can prevent neuroinflammatory-mediated axonal degeneration. We not only demonstrated the limitation of axonal degeneration through the introduction of a site-specific phosphorylation mutant of CRMP-2 but also showed that when the capacity of Nogo-A to bind to its cognate receptor(s) is blocked, the inhibition of axonal degeneration in EAE could limit the phosphorylation of CRMP-2. This suggests that abrogating Nogo-A/NgR1 binding prevents axonal degeneration by blocking CRMP-2 phosphorylation.
Given the recent implication that NgR1 is involved in the efflux of activated macrophages from injured peripheral nerves (Fry et al., 2007
), we wanted to investigate the possibility of a putative immune role for NgR1 during CNS inflammation. We found no discernible difference in the proliferation response of T cells isolated from ngr1−/−
mice at pre-onset, peak and chronic stages of EAE (7, 18 and 30 days post-injection) in response to the MOG35–55
peptide (Supplementary Fig. 4
). Furthermore, we were unable to demonstrate differences in cytokine profiles from EAE-induced ngr1−/−
(12 and 18 days post-injection) compared with ngr1+/+
mice. However, when we analysed the populations of CD3+/CD8+, CD3+/CD4+ and B220 lymphocytes, Gr-1+, F4-80+ and CD-11c+ granulocytes in the spleen and CNS of ngr1−/−
, we could not demonstrate differences in these populations of immune-lineage cells from ngr1+/+
mice 18 days post-EAE immunization (data not shown). The inference would therefore be that the potential for immune-mediated induction of disease is not altered in the ngr1−/−
mice. Thus, it would appear that the limitation of the neurological decline in the ngr1−/−
mice is dependent on a neurobiological mechanism. Despite this, recent data show that monocytes, T and B cells isolated from patients with multiple sclerosis express NgR1 (Pool et al., 2009
). Given that stimulation of immune cells with Nogo led to an alteration of their adhesion properties, it is possible that in immune cells, NgR1 signalling regulates their cytoskeletal dynamics through RhoA activation (Pool et al., 2009
). Therefore, further investigation is required to implicate or exclude a role of NgR1 in immune cell activation and migration in multiple sclerosis.
Current evidence defines pharmacological blockade of myelin associated inhibitory factors as a means of enhancing regeneration in the CNS (McGee and Strittmatter, 2003
; Zorner and Schwab, 2010
). Blocking the signalling of Nogo-A in CNS disease and injury has been well studied and includes EAE (Karnezis et al., 2004
; Mi et al., 2007
). In this study, we used the anti-Nogo(623–640) antibody, previously demonstrated to promote axon outgrowth in the presence of the Nogo peptide, as well as the prevention of neurological decline when injected intravenously over the course of EAE (Karnezis et al., 2004
). We showed that after four injections of anti-Nogo(623–640) antibodies, the decreased severity of EAE correlated with a decreased amount of axonal degeneration. Importantly, reduced levels of pThr555-CRMP-2 in these animals corresponded with replenished tubulin association.
Function-blocking antibodies of Nogo-A have been successfully used in preclinical experiments enhancing locomotor performance in neurotrauma paradigms in rodents and adult monkeys (Schnell and Schwab, 1990
; Freund et al., 2006
), improving manual dexterity after a cervical spinal cord injury in the latter (Freund et al., 2006
). Over the space of two decades, these significant studies advocating therapeutic targeting of Nogo-A in neurotrauma have encouraged the Phase I clinical trials of anti-human Nogo-A antibody (ATI 355) by Novartis (Kwon et al., 2010
). With 45 patients enrolled in this study, intrathecal administration of the antibody in these patients has shown remarkable tolerance with no side-effects manifest in this group (Kwon et al., 2010
). With a Phase II trial currently underway, we now propose that one of the major mechanisms by which anti-Nogo-A antibody treatment is clinically effective may well be through the eventual decrease of the phosphorylated levels of CRMP-2 limiting NgR1-dependent axonal degeneration. Interestingly, other pharmacological studies using the ectodomain of NgR1 and co-receptors such as Lingo-1 have recently shown enhanced neurological improvement in injury and disease models of the CNS by blocking the binding of the myelin associated inhibitory factors (Lee et al., 2004
; Li et al., 2004
; Wang et al., 2006
). It is tantalizing to suggest that similar therapeutic effects could be met in the treatment of multiple sclerosis.
This study has provided new insights into the molecular mechanisms that govern axonal degeneration during inflammatory demyelination of the CNS (as occurs in multiple sclerosis) as well as offering novel therapeutic strategies for this disease. Since myelin damage is a dominant event in neurodegeneration, the work presented here may provide novel therapeutic avenues to attenuate axonal abnormalities, which accompany demyelination in a variety of disorders such as spinal cord and brain injury, stroke and multiple sclerosis. We suggest that blockade of NgR1 signalling and reduction of CRMP-2 phosphorylation limits axonal degeneration in EAE, allowing for the normal physiological function of CRMP-2 to ensue, and by virtue, limiting the axonal degeneration and alleviating clinical symptoms of EAE.