Axonal loss is responsible for the development of chronic neurological deficits in MS, but the pathomechanisms that initiate or exacerbate axonal injury in this inflammatory demyelinating disease remain obscure. Previous studies demonstrated that axonal injury is most pronounced in areas of inflammatory demyelination. This led to the concept that axonal loss was a nonspecific “bystander” response to inflammatory demyelination mediated by soluble factors generated by activated macrophages such as nitric oxide or T cells (10
). However, although inflammatory demyelination is a ubiquitous feature of MS, axonal loss does not necessarily correlate with lesion distribution and load in defined spinal cord tracts (30
) and continues despite resolution of CNS inflammation in patients with progressive disease (31
). These observations led to suggestions that, in addition to the effects of inflammation, other mechanisms are involved in the development of axonal pathology in MS.
Using an unbiased proteomic approach, we identify neurofascin as a novel target of autoantibodies in MS. Neurofascin is a member of the L1 family of cell adhesion molecules, and its two isoforms play essential roles in maintaining the structural and functional integrity of myelinated fibers (26
). These isoforms are derived from a single gene by alternative splicing, and their extracellular domains contain six identical Ig domains and a variable number of identical fibronectin-like repeats, differing only in that NF155 uses an alternative fibronectin type III repeat and lacks a mucin-like domain (33
). As a consequence, mAbs raised against these proteins are commonly cross-reactive. This is also the case for the autoantibody response to neurofascin in patients with MS, which recognizes the intact extracellular domains of both NF155 and NF186, as we demonstrate by flow cytometry.
In vivo in animals with EAE, we demonstrate that antibodies with this specificity bind selectively to NF186 at the node of Ranvier to initiate axonal injury in the CNS and exacerbate clinical disease. The inability of A12/18.1 to recognize NF155 in these animals is presumably related to its localization within the paranodal axo–glial junctional complex. The antibody may be unable to penetrate into the junctional complexes or, alternatively, access to the target epitope is blocked owing to local protein–protein interactions. How antibody binding to the node leads to loss of function in EAE remains to be clarified. Unlike autoantibody-mediated demyelination, the clinical deficit in this model is associated with neither enhanced inflammation nor demyelination. However, the codeposition of C9 with antibody suggests involvement of complement, as is the case in other autoantibody-mediated neurological diseases (34
). This supposition is supported by our demonstration that A12/18.1 only mediates a functional deficit in vitro in the presence of fresh serum, and that this effect is abolished if the serum is heated at 56°C before the experiment. However, irrespective of the effector mechanisms involved, antibody-mediated axonal injury and the resulting functional deficit are reversible. Not only did the majority of animals recover clinically, but β-APP-immunoreactivity also returns to background levels with recovery, and there was no evidence of axonal loss. This suggests that the node of Ranvier is relatively resistant to antibody-mediated damage. This may involve clearance of antibody–complement complexes by endocytosis or ectocytosis (36
), or up-regulation of complement inhibitors such as CD55 by neurons in EAE lesions (37
Our experiments demonstrate that neurofascin-specific antibodies mediate axonal injury after recognition of NF186, but we cannot exclude they do not have other pathological effects. The molecular architecture of the paranode is disrupted after both experimental demyelination (38
) and in MS plaques (39
), indicating that NF155 may become accessible to bind antibody in demyelinating lesions. Neurofascin-specific antibodies may also inhibit remyelination by binding to NF155 reexpressed on the surface of remyelinating oligodendrocytes/oligodendrocyte processes (42
). Moreover, NF186 is not lost from the axonal surface after demyelination but is simply more diffusely distributed (40
), suggesting that it will continue to provide a target for autoantibody-mediated attack in demyelinated lesions. Neurofascin is also expressed on myelinated fibers in the peripheral nervous system, suggesting that it could also be involved in the pathogenesis of autoimmune-mediated peripheral neuropathies. In the current study, we did not observe any enhanced β-APP immunoreactivity or C9 deposition in the peripheral nervous system, although some antibody was detected bound to nodes in root entry zones. The blood–nerve barrier is known to have a limited permeability to serum Ig's at these sites (43
), and in the absence of a local inflammatory response, diffusion of antibody into the nerve is unable to initiate a functional deficit (44
). In the absence of peripheral nerve damage, the sudden death of some animals during the recovery phase of the disease cannot be explained satisfactorily, although it might reflect central autonomic dysfunction.
We demonstrate that neurofascin-specific antibodies mediate axonal damage, impair neuronal conduction, and exacerbate clinical disease in an animal model of MS; however, are they relevant in human disease? For a circulating autoantibody response to mediate tissue damage within the CNS, two criteria must be met. First, the blood–brain barrier must be breached so that autoantibodies circulating in the blood can gain access to the CNS parenchyma (12
), and second, once within the CNS compartment, the antibody must be able to access and bind to its target antigens. In the case of the neurofascin-specific autoantibody response in MS, these criteria are both met. First, MS is associated with an increased permeability of the blood–brain barrier to serum Ig's (46
). In the initial inflammatory phase of MS, this is most prominent in areas of perivascular inflammation. However, there is also evidence that as the disease progresses patients develop chronic blood–brain barrier abnormalities that result in increased leakage of serum protein into the CNS (47
). This indicates that antineurofascin antibodies in the periphery will gain access to the CNS parenchyma. Second, the polyclonal neurofascin-specific autoantibody response recognizes the native extracellular domain of NF186. As we demonstrate using mAb A12/18.1, these antibodies will bind to NF186 in vivo to exacerbate axonal injury and associated functional deficits in patients with MS. These effects will be most pronounced in patients with high titer antibody responses to NF155/186 and may have a considerable impact on disease progression. This may be the case in the 20–30% of MS with high titer antibody responses, but it should be noted that neurofascin-specific antibodies were also detected in occasional control donors. This lack of absolute disease specificity is not unexpected and is similar to that reported for autoimmune responses to other CNS autoantigens in MS (46
). In summary, we identify neurofascin as a novel autoantigen in a subset of MS patients and demonstrate that neurofascin-specific antibodies can induce reversible axonal injury and conduction block in inflammatory demyelinating diseases of the CNS. These findings should open new perspectives for therapeutic inhibition of antibody-mediated axonal injury.