Neurochemical Research, Vol. 29, No. 11, November 2004 (Ó 2004), pp. 1999–2006
Cryptic Axonal Antigens and Axonal Loss in Multiple Sclerosis* George H. DeVries1;2 (Accepted July 26, 2004)
Axonal loss is well correlated with functional deficits in Multiple Sclerosis (MS); however, the molecular mechanisms that underlie this axonal loss are not understood. In this review we summarize evidence that antibodies to axolemma-enriched fractions (AEF) isolated from CNS myelinated axons may play a role in axonal destruction. AEF contains potent antigens that elicit high-titer antisera, which destroy neurites in vitro, prevent neurite outgrowth, cause reactive changes in the neuronal cell bodies of origin and prevent myelination. We propose that these AEF antigens are cryptic because they are shielded from immune surveillance in vivo via the tightly sealed paranodal loops of myelin. Antibodies to AEF are found in cerebrospinal fluid (CSF) and sera of MS patients at higher levels compared with CSF or sera derived from patients with other neurological diseases. The potential identity of these cryptic antigens and their role in the axonal destruction characteristic of MS is discussed.
Further evidence that supports the axonal hypothesis is the fact that some MS patients showed severe demyelination at autopsy, while they are neurologically normal (6). This implies that possibly axons reorganize sodium channels to conduct in a continuous manner and allow continued functioning of the nervous system. In addition, in animal models of MS, there were no observable symptoms in animals induced for experimental allergic encephalomyelitis (EAE) unless damage to the axons also was evident (3). Further morphological examination of the lesions of MS has allowed Trapp’s laboratory (2) to identify three types of lesions: Type 1 lesions, which are contiguous with subcortical white matter lesions; Type 2 lesions, which are confined to the cortex and are often perivascular, and the most interesting and prevalent type of lesions, Type 3, which extend from the pial surface to cortical layers three and four (2). The morphological hallmark of these lesions is the presence of transected axons, which is related to the activity of the lesion (1). Since axonal loss correlates well with functional loss in MS, it becomes impera-
Although Multiple Sclerosis (MS) has long been considered an inflammatory demyelinating disease with a primary attack on myelin, a new understanding of this disease has emerged. Recent data from Trapp’s laboratory (1,2) indicates that axonal loss is correlated with loss of functional activity. This data has led to the formation of the ‘‘axonal hypothesis,’’ which states that, in MS, axonal loss reaches a threshold above which further axonal loss results in irreversible neurological disability. A number of studies utilizing animal models (3), in addition to magnetic resonance studies of MS patients (4), have provided support for this hypothesis. It is clear that the cumulative axonal loss best correlates with the degree of disability of long-term MS patients (5).
* Special issue dedicated to Lawrence F. Eng. 1 Address reprint requests to: Research 151, Hines VA Hospital, Hines, Illinois 60141. Tel: 708-202-2262; Fax: 708-202-5969; E-mail:
[email protected] 2 Department of Anatomy and Cell Biology, University of Illinois Chicago, Chicago, Illinois 60612.
1999 0364-3190/04/1100–1999/0 Ó 2004 Springer Science+Business Media, Inc.
2000 tive to understand molecular mechanisms that lead to the loss of axons in this disease. MS has been classified into four categories by Lassmann and colleagues (7). One classification is antibody-mediated MS characterized by complement-mediated lysis of an antibody-targeted cellular component such as the axon. However, the cellular source and identity of the antigen generating the antibody is not known. Except for the nodal regions, molecules on the axonal surface membrane of myelinated axons in the normal brain are well shielded from surveillance by the immune system. We propose that antigens on the surface of axons beneath the myelin sheath are cryptic. These cryptic antigens exposed after demyelination, generate antibodies that damage axons. In addition to antibody lysis, a number of other molecular mechanisms have been proposed as responsible for transecting axons. For example, it has been reported (8) that cytotoxic T-lymphocytes are able to transect neurites that have been induced to express the Class I major histocompatibility complex (MHC). An interesting observation is that in a number of diseases, the cell bodies of neurons are relatively protected from this neuroinflammatory insult, while neurites are more susceptible (9–12). MHC can be induced in neurons by exposure to interferon-gamma (IFN-c), an inflammatory cytokine known to be present in MS lesions. Since the pioneering work of Bornstein and Appel in 1965 (13), it has been clear that humoral antibodies may play a role in demyelination in EAE. It is also well known that the c-immunoglobulin class of antibody is elevated in cerebrospinal fluid (CSF) of MS patients (14). Clearly, there is an interaction between cellular and humoral immune components that contribute to the immune-mediated demyelination that is present in MS. The antigen responsible for eliciting this immune response, which is evident by oligoclonal banding in the CSF, is not known. However, Alcazar et al. (15) have recently shown that CSF from patients with relapsing remitting MS can induce axonal damage in vitro. A number of other lines of evidence have been presented to indicate that antibodies play a crucial role in the cellular damage characteristic of MS. For example, IgG deposition, along with the presence of activated complement, is characteristic of demyelinating lesions in MS (16). In addition, serum-demyelinating activity related to immunoglobulins has been found in a sub-group of MS patients (17). Antigens that activate the lytic complement complex also have
DeVries been described at sites undergoing active demyelination (18). Clearly, there is increased antibody production and activation in MS, but the nature of the antigen specifically responsible for the production of these antibodies is not known. Cryptic antigens are known to play a role in a number of diseases, such as Chronic Lymphocytic Thydroiditis (19). In this disease, antibodies to the thyroglobulin are produced as a consequence of unmasking a cryptic iodine-containing epitope. Antibodies to this epitope have been shown to induce thydroiditis in experimental animals. In addition, a condition known as Chagas Heart Disease develops in individuals infected with the parasite Trypanosoma cruisi, which results in exposure of cryptic self-epitopes, leading to the clinical symptoms of this disease (20). It was initially believed that the antibody found in CSF of MS patients is generated as a response to myelin self-antigens (21). However, it is now understood that the immune system is exposed to these myelin antigens in the periphery and exposure is not limited to demyelinating events in the CNS (21). Although generation of myelin-specific antibodies is a consequence of the demyelinating event, it may not be the primary causative event. A number of non-myelin antigens have been detected in patients with MS, but the exact identity of antigens responsible for generating antibodies that damage axons is still not known. The role of autoantibodies in Acute Motor Axonal Neuropathy (AMAN) may be instructive for understanding how cryptic antigens may function in MS. AMAN is a form of Guillain–Barre Syndrome. A hallmark of this disease is axonal degeneration of motor fibers with relative sparing of sensory fibers (22). It was discovered that many patients with this disease had a previous Campylobacter jejuni infection leading to the development of antibodies to gangliosides specific for the axolemma of motor fibers (23). Subsequently, in a complement-mediated manner, the autoantibodies attacked and destroyed the motor fiber axolemma. Although this is an example of molecular mimicry rather than cryptic antigens, it emphasizes that antibodies to axolemmal molecules can lead to axonal destruction. One interesting aspect of this disease is that the antibodies react more strongly with the PNS axolemma than with the CNS axolemma, possibly due to the different composition of the axolemma in the PNS, which has more GD1a ganglioside than CNS axolemma (24).
Cryptic Axonal Antigens and Axonal Loss The methodology for isolating an axolemmaenriched fraction (AEF) from the CNS and characterizing its molecular properties has been developed in our laboratory (25). Surprisingly, the lipids found in this membrane fraction contain a number of sphingolipids (cerebroside and sulfatide), which are generally considered to be characteristic of myelin (26). However, subsequent analysis of the fatty acid composition of these axonal sphingolipids showed that the average chain length was shorter than the comparable sphingolipid in myelin (27), raising the possibility of a separate metabolic origin for these lipids. Axolemma fractions actively signal to both Schwann cells and oligodendrocytes, causing proliferation of oligodendrocytes (28) and Schwann cells (29) as well as differentiation of Schwann cells (30). When isolated from CNS, AEF originates from internodal, nodal, paranodal and juxtaparan-
2001 odal axolemma since markers for each of the specialized regions of the axolemma are present in AEF (Fitzgerald and DeVries, unpublished observations). Specialized axonal molecules such as Caspr1, Caspr2, and neurofascin allow the paranodal loops of myelin to adhere to the axonal surface. Other specialized molecules are specifically found in the paranodal region such as the Kv1.1 and Kv1.2 channels (31). Clearly, these adhesive molecules effectively seal off the axolemma from immune surveillance of the axonal surface, which is beneath the paranodal and compact myelin (Figure 1). In this sense, these antigens can be considered as cryptic and should elicit a strong immune response when made accessible to the immune system after demyelination. These antibodies can then lyse axons in addition to contributing to further neuronal degeneration as
Fig. 1. Cryptic axonal antigens in CNS myelinated axons. Panel A: Typical myelinated axon of PNS and CNS showing location of cryptic antigens beneath paranodal loops. Panel B: Enlargement of paranodal loops and attached axolemma showing potential cryptic antigens that would be exposed during demyelination.
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Cortical neuron of origin for demyelinated axon Anti-axolemmal antibodies
Reactive changes in Neuronal Parikaryon
Exposed cryptic antigens
Anti-axolemmal antibodies Displaced nucleus Blocked axon-OPC signals
Oligodendrocyte precursor cell
Fig. 2. Anti-axolemmal antibodies induce neurodegeneration and prevent remyelination. Cryptic antigens, exposed as a consequence of the loss of myelin, elicit an antibodies which react with cryptic antigens causing reactive changes in the cell body of origin and potentially block axonal oligodendrocyte precursor interactions required for remyelination.
shown in Figure 2. The antibody-mediated axonal destruction may then lead to destabilization of the neuron from which the axons originate. Ultimately the neurons would die as a result of the axonal transection. Since premyelinating oligodendrocytes are found in MS lesions (32), the axolemma antibodies may block crucial axon-oligodendrocyte precursor interactions, which are necessary for remyelination (Figure 2). There should be a number of correlates to the ‘‘cryptic antigen’’ theory, which would relate cryptic axonal antigens to axonal loss in MS. First of all, since the antigens have not been seen by the immune system, the membrane preparation should be relatively antigenic. Secondly, in the presence of complement, the antibody should destroy neurites. Furthermore, it is predicted that neurite-outgrowth would not proceed in the presence of antibodies since the neurites would be destroyed as rapidly as they grew. In addition, damage to the neurites may result in reactive changes in the neuronal cell body of the damaged axons. These axonal-specific antibodies may also prevent remyelination by blocking specific signaling molecules required for the reestablishment of the critical relationship between axons and oligodendrocytes prior to myelination. Finally, one would predict that these antibodies would be found in higher proportions, specifically in MS, when compared with other neurological diseases. We now present evidence, based on previously published work, that antibodies to the AEF meet all of these criteria.
AEFs isolated from rat, human and bovine sources were used to immunize rabbits and raise polyclonal antisera. The membrane fractions were extremely antigenic, ranging from end-point titers of 1:6400 for human CNS AEF and 1:1250 for a rat CNS AEF. A number of antigens were shown to be immunoreactive to the antisera ranging in molecular weight from 38.5 to 120 kDa. The antisera reacted with axolemma of PNS sciatic nerve as well as CNS axon preparations (33). Therefore, the AEF fulfilled the first criterion of producing a hightiter antisera after immunization. To evaluate biological activities such as inhibition of neurite outgrowth and destruction of mature neurites, spinal cord explant cultures were cultured in vitro in the presence or absence of control antibodies or antibodies raised to AEF (34). As shown in Figure 3, in the presence of antibody, there was virtually no outgrowth of neurites. Moreover, addition of the anti-axolemmal antisera in the presence of complement caused complete fragmentation and destruction of neurites. In addition to preventing neurite outgrowth, the neuronal cell bodies from which the damaged neurites originated were studied morphologically. As shown in Figure 4, there were clear morphological changes in the neuron cell bodies derived from dorsal root ganglion after exposure to the antiaxolemmal antisera. These reactive changes included displacement of the nuclei to the periphery, cell body shrinkage, densely clumped neurofibrils in the cell body, and eccentrically displaced
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Fig. 3. Polyclonal antisera to CNS AEF destroys neurites in culture and presents neurite outgrowth. Embryonic spinal cord cultures with attached dorsal root ganglia were fixed as whole-mount preparations, impregnated with silver and photographed with darkfield optics. A: normal spinal cord ( c ) and DRG (g) explants 18 days in vitro (DIV). The outgrowth zone (o) contains abundant axons. B: spinal cord ( c ) and DRG (g) explants (18 DIV) after 14 days of exposure to antiserum to AEF. There are few axons in the outgrowth zone (o). C: outgrowth zone of a control Schwann cell-DRG culture (18 DIV) demonstrating normal axons. D: outgrowth zone of a culture (18 DIV) after 4 days of exposure to antiserum to AEF. There are many axon fragments and no intact axons. Magnification: 37 (A, B); 58 (C, D) (Taken from Figure 1 reference 34).
nuclei (34). Similar changes were noted in CNS ventral root neurons (34). These changes were most noticeable in cultures in which neurite outgrowth had been inhibited by prolonged exposure to antisera to AEF. The anti-axolemmal antisera also were evaluated for their ability to inhibit myelination in embryonic spinal cord explants (Figure 5). In three out of the four sera, there was complete inhibition of myelination. The only anti-
axolemma antisera in which myelination occurred was one that did not contain immunoreactivity to cerebroside (35). This data, collectively, implies that exposure of antigens on the axolemma would give rise to a high-titer, potent antisera, which could destroy neurites, prevent neurite outgrowth, cause reactive changes in neurons, and prevent myelination. It is interesting to note that the antiaxolemmal antisera did not initiate demyelination
Fig. 4. Antisera to AEF alters neuronal morphology in vitro . Panel A: DRG neurons in normal culture (18 DIV) have centrally placed nuclei and a delicate cytoplasmic neurofibrillary pattern; (950 x ). Panel B: DRG neurons in an explant (18 DIV) after 14 days of exposure to antiserum to AEF are smaller than normal, contain clumped neurofibrils and have eccentrically placed nuclei; 1100 . (Taken from Figure 2 reference 34).
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Fig. 5. Antisera to AEF inhibits myelination in vitro. Antisera were tested for their ability to prevent myelination for 15–18 days in medium containing test sera. (See reference 35 for further details.) The number of cultures evaluated for each serum sample is indicated in each bar, which represents a single pre-immune or post-immune (anti-AEF) serum.
implying the immunological target was shielded by myelination (35). These in vitro studies provide convincing evidence that anti-axolemma antibodies possess biological activities consistent with the axonal destruction observed in MS. Therefore, we investigated the degree to which these antibodies were present in MS CSF and sera when compared with CSF and sera from other neurological diseases (OND) (36). Note that there is a significant increase in the reactivity of AEF antibodies in both CSF and serum of MS patients when compared with the reactivity of OND (Figure 6). This data is consistent with the view that
anti-axolemma antibodies are present in the CSF and serum of MS patients, where they may contribute to the axonal destruction that is characteristic of MS. Although we currently do not have evidence that the antigens to the AEF antisera are cryptic, experiments are in progress to test this possibility. In this regard, we have raised monoclonal antibodies to axolemma and will implant the monoclonal antibodysecreting hybridomas into the CNS of an animal that has been demyelinated by either a lysolecithin injection (37) or by inducing demyelination via cuprizone ingestion (38). Transplantation of hybridoma into the CNS has been used by Zhou et al. (39) to induce
Fig. 6. Immunoreactivity to AEF in CSF and serum of MS and other neurological control patients relative to a control sample. Cerebrospinal fluid samples from MS (n ¼ 24) or OND (n ¼ 17) patients as well as a standard control CSF sample were diluted 1:3 and analyzed by ELISA. (See reference 36 for further details.) The ratio of the MS or OND sample relative to the standard control were plotted as shown. The difference between the MS and OND ratios was significant at *P < 0.08. Serum samples from MS (n ¼ 21) and OND (n ¼ 13) patients as well as standard control samples were analyzed by ELISA. (See reference 36 for further details.) The ratios of the MS or OND samples relative to the standard control were plotted as shown. The difference between the MS and OND ratios was significant at **P < 0.001.
Cryptic Axonal Antigens and Axonal Loss demyelination in the CNS utilizing a hybridoma secreting a monoclonal antibody specific for oligodendrocytes. The prediction was that in the intact CNS containing the hybridoma-secreting antiaxolemmal antibody, no axonal damage was evident. However, in the demyelinated CNS, where cryptic antigens are exposed, there would be extensive and early axonal loss. In addition the physiological or functional deficits due to axonal loss should be evident earlier and to a greater degree. Rammohan (40) recently has proposed that molecular changes in antigens expressed at the nodal regions of myelinated axons could be a primary cause for axonal and functional loss in MS. The fact that axonal injury begins very early in MS, perhaps even before demyelination, supports this view (4, 41,42). The cryptic axonal antigens could be related to the axonal adhesive molecules found beneath the paranodal loops of myelin. In this regard, we previously reported that there is increased specific antigenic immunoreactivity to a 58 kDa cerebellar antigen specifically in the cerebellum of MS patients found when rabbits were immunized with cerebella of MS patients (43). A potential candidate for this cryptic antigen, which has a MW of 56.7 kDa, is the Kv1.2 alpha subunit of the potassium channel found in the juxtaparanodal region of myelinated axons (31). Exposure of this cryptic antigen after demyelination could lead to this specific immune response in MS brain. Further investigation of the role to identify the cryptic antigens that may be exposed as a result of demyelination and are contributory to axonal loss in demyelinating disease is under way in our laboratory.
ACKNOWLEDGMENTS The author is grateful for the expert editorial assistance of Jean Egerman. The author also acknowledges the advice and encouragement of Dr. Vincent Calabrese (McGuire VA Medical Center, Richmond, Virginia).
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