Brain, Vol. 124, No. 6, 1114-1124,
June 2001
© 2001 Oxford University Press
Distribution of a calcium channel subunit in dystrophic axons in multiple sclerosis and experimental autoimmune encephalomyelitis
1 Department of Neuroimmunology, Brain Research Institute and 2 Department of Neurology, University of Vienna, 3 Department of Neurology, Hospital Lainz, Vienna, 4 Department of Neurology, Karl-Franzens-University, Graz, Austria, 5 Neuroimmunology Unit, Center of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden and 6 Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Germany
Correspondence to:
Professor Dr Hans Lassmann, Division of Neuroimmunology, Brain Research Institute, University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria E-mail: hans.lassmann{at}univie.ac.at
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are immune-mediated diseases of the CNS. They are characterized by widespread inflammation, demyelination and a variable degree of axonal loss. Recent magnetic resonance spectroscopy studies have indicated that axonal damage and loss are a reliable correlate of permanent clinical disability. Accordingly, neuropathological studies have confirmed the presence and timing of axonal injury in multiple sclerosis lesions. The mechanisms of axonal degeneration, however, are unclear. Since calcium influx may mediate axonal damage, we have studied the distribution of the pore-forming subunit of neuronal (N)-type voltage-gated calcium channels in the lesions of multiple sclerosis and EAE. We found that
1B, the pore-forming subunit of N-type calcium channels, was accumulated within axons and axonal spheroids of actively demyelinating lesions. The axonal staining pattern of 1B was comparable with that of ß-amyloid precursor protein, which is an early and sensitive marker for disturbance of axonal transport. Importantly, within these injured axons, 1B was not only accumulated, but also integrated in the axoplasmic membrane, as shown by immune electron microscopy on the EAE material. This ectopic distribution of calcium channels in the axonal membrane may result in increased calcium influx, contributing to axonal degeneration, possibly via the activation of neutral proteases. Our data suggest that calcium influx through voltage-dependent calcium channels is one possible candidate mechanism for axonal degeneration in inflammatory demyelinating disorders. voltage-gated calcium channels; multiple sclerosis; experimental autoimmune encephalomyelitis; axon degeneration
APP = amyloid precursor protein; CNPase = cyclic nucleotide phosphodiesterase; CO = control cases; EA = early active multiple sclerosis lesions; EAE = experimental autoimmune encephalomyelitis; IA = inactive multiple sclerosis lesions; LA = late active multiple sclerosis lesions; MOG = myelin oligodendrocyte glycoprotein; NWM = normal white matter in multiple sclerosis; PLP = proteolipid protein; PP = periplaque white matter in multiple sclerosis; RM = remyelinated multiple sclerosis lesions; VGCC = voltage-gated calcium channel
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Multiple sclerosis is an immune-mediated disease of the CNS. It is characterized by widespread inflammation, focal demyelination and a variable degree of axonal loss (Charcot, 1868
; Cuzner and Wekerle, 1996; Lassmann, 1998a). The disease usually follows a chronic disease course, resulting in persistent neurological deficits in most patients (Weinshenker et al., 1989).
A number of recent studies have provided evidence that axonal damage and loss within and around multiple sclerosis plaques are the major correlates for permanent clinical disability (Davie et al., 1995; Losseff et al., 1996; De Stefano et al., 1998; Lee et al., 2000; Reddy et al., 2000). Moreover, it has been demonstrated convincingly that the highest incidence of acute axonal injury occurs during active demyelination (Ferguson et al., 1997; Trapp et al., 1998; Bitsch et al., 2000; Kornek et al., 2000). Repeated episodes of demyelination and persistent chronic inflammation as well as other unknown mechanisms contribute further to ongoing fibre degeneration, leading to 75% and more of axonal loss in inactive demyelinated lesions (Lassmann; 1998b; Mews et al., 1998; Ganter et al., 1999; Evangelou et al., 2000; Lovas et al., 2000).
Axonal damage and loss have been described further in different models of experimental autoimmune encephalomyelitis (EAE) (Raine et al., 1989; Storch et al., 1998; Kornek et al., 2000; McGavern et al., 2000). Among these models, myelin oligodendrocyte glycoprotein (MOG)-induced EAE appears to be most suitable, since it reflects both clinical and pathological features of multiple sclerosis and leads to—in comparison with other EAE models—large plaques of demyelination (Storch et al., 1998). Similarly to multiple sclerosis, in MOG-induced EAE the highest incidence of acute axonal injury occurs during active demyelination, leading to a pronounced reduction of axonal density in all stages of demyelination (Kornek et al., 2000).
Although the kinetics and the extent of axonal damage have been described in detail in multiple sclerosis, the mechanisms of axonal degeneration are as yet unclear. Understanding these mechanisms, however, is necessary for the development of axon-protective strategies aimed at reducing permanent clinical disability in multiple sclerosis.
A number of recent studies have emphasized the involvement of calcium ions in the development of axonal disturbance in trauma (George et al., 1995; Büki et al., 1999) and ischaemia (Stys et al., 1991; Fern et al., 1995). These studies suggested that, under pathological conditions, calcium may enter the axon via ion-specific transport mechanisms such as voltage-gated calcium channels (VGCCs) and/or reverse operation of the Na+/Ca2+ exchanger (Stys et al., 1991, 1992; Stys and Lopachin, 1998; Fern et al., 1995; George et al., 1995).
Increased levels of intra-axonal calcium then lead to activation of neutral proteases, e.g. calpains, which are known to degrade major components of the cytoskeleton (Schlaepfer and Zimmermann, 1985; Banik et al., 1993; Kampfl et al., 1997). Furthermore, pharmacological blockade of the respective channels can reduce axonal damage in trauma (George et al., 1995) and ischaemia (Stys et al., 1992; Fern et al., 1995). In order to determine a possible role of calcium channels in axonal degeneration in inflammatory demyelinating disorders, we have studied the distribution of the pore-forming subunit of N-type VGCCs in the lesions of multiple sclerosis and EAE by immunohistochemistry.
|
Material and methods |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Multiple sclerosis patients and controls
The study was performed on autopsy tissue from 13 patients. In addition, two control brains were included in this series from patients without evidence of neurological disease or neuropathological alterations. Clinical background data are summarized in Table 1
. All material was fixed in 4% paraformaldehyde and embedded in paraffin wax.
|
EAE material
Lewis.1N rats were kindly provided by Professor H. Hedrich (Hedrich, 1990
). EAE was induced in 18 rats by active sensitization with 20–100 µg of recombinant MOG as described in detail previously (Storch et al., 1998). In addition, three healthy rats of the same strain served as controls. Animals were weighed and examined daily for clinical signs of EAE. At various time points after sensitization (days 11–61), animals were sacrificed and perfused via the aorta with 4% paraformaldehyde. Brains and spinal cords were dissected and routinely embedded in paraffin wax. All experimental procedures were approved by the Bavarian and Swedish governments and performed in compliance with international animal welfare standards.
Neuropathology
Sections 2–4 µm thick were cut on a microtome and stained with haematoxylin–eosin, Luxol fast blue and periodic acid–Schiff, and Bielschowsky silver impregnation to assess inflammation, demyelination and axonal loss, respectively.
Immunohistochemistry was performed in adjacent serial sections using an avidin–biotin or an alkaline phosphatase–anti-alkaline phosphatase technique. The following primary antibodies were used: MOG (anti-MOG; Department of Biochemistry, Cardiff, UK), proteolipid protein (anti-PLP; Serotec, Oxford, UK), 2',3'-cyclic nucleotide phospho- diesterase (anti-CNPase; Affiniti Research Products, Ilkeston, UK), myelin basic protein (anti-MBP; BioGenex, San Ramon, USA), macrophages/activated microglia in rats (anti-ED 1; Serotec), early activated human haematopoietic macrophages (anti-MRP 14; BMA Biomedicals, Augst, Switzerland), late activated macrophages (anti-27E10; BMA Biomedicals), ß-amyloid precursor protein (anti-APP; Boehringer, Mannheim, Germany), 1B, the pore-forming subunit of N-type VGCCs (anti-1B; Alamone Laboratories, Jerusalem, Israel), and SMI 33 (anti-non-phosphorylated neurofilament M,H; Affiniti Research Products Ltd, Exeter, UK). Control sections were incubated in the absence of primary antibody. Staining was omitted when the 1B antibody was incubated together with a specific anti-1B-peptide (data not shown).
In situ hybridization for PLP mRNA was performed according to Breitschopf and colleagues (Breitschopf et al., 1992).
Laser scanning confocal microscopy
For double staining of APP and 1B, essentially the same immunocytochemistry protocol was used as for normal light microscopy. Briefly, sections were incubated with both primary antibodies overnight, then incubated with secondary antibodies (Cy3 goat anti-rabbit; Jackson ImmunoResearch Laboratories, West Grove, Pa., USA) and biotinylated sheep anti-mouse (Amersham, UK) for 2 h at room temperature. In a third step, sections were incubated with streptavidin-conjugated Cy2 (Jackson) for 1 h at room temperature. After rinsing with PBS (phosphate-buffered saline), sections were embedded in PBS/glycerol (1 : 9) with 3% DABCO (Sigma) and placed on a coverslip. Fluorescent preparations were examined using a Zeiss laser scan microscope as described previously (Akassoglou et al., 1998) (Cy2 signal green, Cy3 signal red, overlay yellow).
Electron microscopy
Immune electron microscopy was performed according to Vass and colleagues (Vass et al., 1986). Briefly, spinal cord tissue of EAE animals was fixed by perfusion with 4% paraformaldehyde. Tissue sections 50–100 µm thick were incubated with the primary antibody against the 1B subunit of VGCCs for 18 h at 4°C. The blocks were then washed extensively in PBS, incubated in biotinylated anti-rabbit immunoglobulin and in avidin peroxidase subsequently. The peroxidase reaction product was developed with di-amino benzidine reagent. The material was then routinely osmicated and embedded in resin. For control, primary antibody was omitted.
Selection of demyelinated plaques and definition of lesional staging
In multiple sclerosis and EAE, areas of normal white matter, periplaque white matter, actively demyelinating plaques, inactive demyelinated plaques and remyelinated shadow plaques were selected for further analysis of axonal pathology. Normal white matter was defined as an area which showed no evidence of demyelination by macroscopic inspection and histology within the area and the surrounding tissue. In multiple sclerosis, periplaque white matter represented a strip of tissue of 5 mm adjacent to the border of active or inactive plaques. In EAE, periplaque and normal white matter were not analysed separately for the evaluation of axonal pathology.
The following categories for demyelinated plaques were defined (Brück et al., 1995)
Early active lesions (EA)
These lesions were heavily infiltrated by T cells and macrophages. Myelin sheaths were being disrupted and macrophages contained degradation products, which were stained by Luxol fast blue and immunoreactive for all myelin proteins, including MOG and CNPase.
Late active lesions (LA)
In these lesions, myelin was already destroyed and removed from axons. Macrophages contained degradation products reactive for major myelin proteins, such as PLP, but were negative for MOG and CNPase.
Inactive lesions with active border (IA + A)
The inactive centre of radially expanding lesions still showed pronounced inflammation and macrophage infiltration. The macrophages revealed empty vacuoles and showed no immunoreactivity for myelin proteins.
Inactive lesions (IA)
These lesions showed no evidence for ongoing myelin destruction at their borders. Although these lesions also contained some T cells and macrophages, their number was much lower compared with active lesions. However, microglia activation was prominent.
Remyelinated shadow plaques (RM)
These lesions were characterized by myelin pallor, due to abnormally thin myelin sheaths and a pronounced expression of PLP mRNA in oligodendrocytes. Similarly to inactive lesions, residual inflammation and microglia activation were present.
Quantitative determination of 1B reactivity
Camera lucida drawings of demyelinating lesions were made in order to define precisely the pattern of myelin destruction for each lesional area. In the selected area, the demyelinating activity was determined by the presence or absence of myelin degradation products within macrophages immunoreactive for MOG, PLP and CNPase as described above. On adjacent serial sections, the number of 1B-positive elements stained per square unit of tissue was counted as described previously (Kornek et al., 2000). A 0.01 mm2 wide field, defined by an ocular morphometric grid, was selected in the centre of each lesional area. In this field, 1B-positive fibres were counted under a x100 objective. In inactive demyelinated lesions, shadow plaques, periplaque and normal white matter for each lesional area, the average number of 1B-positive axons in 10 adjacent fields of 0.01 mm2 were taken for quantification because of the low density of injured axons.
EAE
Total number of lesional areas analysed: n = 53; among those, n = 11 early active (EA), n = 11 late active (LA), n = 11 inactive plus active border (IA + A), n = 4 remyelination with concomitant demyelinating activity (RM-A), n = 16 periplaque white matter (PP), n = 12 white matter of control animals (CO).
Multiple sclerosis
Total number of lesional areas analysed: n = 91; among those, n = 16 EA, n = 10 LA, n = 15 IA + A, n = 7 inactive (IA), n = 7 remyelination (RM), n = 20 PP, n = 16 normal white matter in multiple sclerosis brains (NWM), n = 8 white matter of controls (CO).
The Mann–Whitney U test and the 
2-test were used for statistic analysis.
|
Results |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Distribution of the N-type channel in normal CNS tissue
The pattern of the pore-forming subunit of N-type calcium channels (
1B) was studied in grey and white matter of the rat spinal cord and in selected areas of the human brain (Fig. 1).
|
Corresponding to previous observations (Westenbroek et al., 1992
, 1998a; Day et al., 1996), the 1B staining was prominent throughout the CNS. In the rat spinal cord, immunoreactivity was found along the surface of neurones in the ventral and dorsal horn (Fig. 1A and B
) and throughout the laminae of the dorsal horn (Fig. 1A). Immunoreactivity for 1B was most pronounced in regions of high synaptic density (Fig. 1A).
At the light microscopic level, we observed a strong punctate staining along neuronal cell bodies and dendritic shafts, suggesting the presence of 1B in nerve terminals forming synapses on the neurones (Fig. 1C). This was confirmed by immune electron microscopy which was performed on rat spinal cord tissue. We found smooth and punctate surface staining consistent with the presence of 1B in the cell surface of nerve terminals forming synapses (Fig. 3A and B). In addition, some immunoreactive vesicles were found within the neuronal perikarya and in synaptic boutons (Fig. 3A).
|
No staining for
1B was observed in axons of the normal white matter at the light microscopic level (Fig. 1D). Yet, at the ultrastructural level, a low density of immunoreactive vesicles, which were not inserted into the axonal plasma membrane (Fig. 3C), was found within the axolemma.
Distribution of 1B in the lesions of multiple sclerosis and EAE
A prominent reactivity for 1B was found in axons of actively demyelinating lesions of multiple sclerosis (Fig. 2D, G and K) and EAE (Fig. 2J and L; Tables 3 and 4). Actively demyelinating lesions were frequently located around vessels, both in multiple sclerosis (Fig. 2A) and in EAE (Fig. 2H). Within the multiple sclerosis lesions, axonal density was reduced (Fig. 2B). A prominent axonal staining pattern for APP as a marker for acute axonal injury (Gentleman et al., 1993; Sherriff et al., 1994) was found in multiple sclerosis (Fig. 2C and F) and EAE lesions (Fig. 2I).
|
|
Similarly to APP, the immunoreactivity for
1B was present in large calibre axons remininscent of axonal spheroids (Fig. 2K), in focal axonal swellings of non-transected fibres (Fig. 2L) as well as in some demyelinated axons with normal calibre.
In addition, many swollen axons displayed immunoreactivity for APP, 1B and SMI 33, a marker for non-phosphorylated subunits of neurofilaments, in multiple sclerosis lesions, as seen on serial sections (Fig. 1E–G).
Some glial staining was also observed, at both the light (Fig. 2B and C) and electron microscopic level (Fig. 3D and E). However, there was no difference in glial reactivity for 1B in control tissue or lesional areas, as described previously in diverse forms of brain injury, but also hypomyelination (Westenbroek et al., 1998b). In addition, some endothelial cells displayed immunoreactivity for 1B (Fig. 2D and J). At the light microscopic level, the accumulation of 1B in axons was comparable with that of APP. APP is a well-recognized marker for disturbance of axonal transport (Gentleman et al., 1993; Sherriff et al., 1994). As described in our previous study (Kornek et al., 2000), the highest number of APP-positive axons was also found in actively demyelinating lesions of multiple sclerosis and EAE.
Confocal microscopy of actively demyelinating lesions of multiple sclerosis and EAE
When confocal microscopy was performed, the co- localization of APP and 1B in dilated axons in multiple sclerosis (Fig. 1K, L and M) and EAE lesions (Fig. 1H, I and J) could be demonstrated. Most interestingly, however, in some large, swollen axons, 1B staining was accentuated along the axonal plasma membrane, suggesting the integration of the channel subunits into the axolemma (Fig. 1J, L and M). In order to discern the distinct localization of 1B within the axon, immune electron microscopy for 1B was performed.
Electron microscopy of actively demyelinating lesions of EAE
The axonal accumulation of 1B in areas of active demyelination was confirmed by electron microscopy, which was performed on the EAE material. In some demyelinated axons and, in particular, in dystrophic axonal swellings, the number of 1B-positive vesicles was clearly increased (Fig. 3D). Moreover, as suggested by confocal laser scanning, immune electron microscopy revealed that 1B is not only accumulated, but also integrated in the axonal plasma membrane in areas of active demyelination (Fig. 3D and E).
Quantitative evaluation of 1B-positive axons in the lesions of multiple sclerosis and EAE
Actively demyelinating lesions of multiple sclerosis and EAE
While myelinated axons of the normal white matter showed no immunoreactivity for 1B at the light microscopic level (Fig. 1D), some apparently normal axons as well as many axonal swellings of actively demyelinating lesions (Fig. 2D, G and J) were intensely stained by this antibody, for the pore-forming subunit of N-type calcium channels (multiple sclerosis + EAE: CO/EA, P < 0.0001; CO/LA, P < 0.0001). Both in multiple sclerosis and in EAE, the number of 1B-positive axons was slightly higher in early compared with late active lesions; the difference, however, was not statistically significant (multiple sclerosis + EAE: EA/LA, n.s.) (Tables 2 and 3).
|
Inactive lesions with an actively demyelinating edge
In both multiple sclerosis and EAE, a significant number of
1B-positive axons was found in the inactive centre of lesions with ongoing demyelinating activity at the edge. Again, the staining pattern for 1B was comparable with that of APP (multiple sclerosis + EAE: CO/IA + A, P < 0.0001) (Tables 2 and 3).
Inactive lesions of multiple sclerosis
In inactive lesions of multiple sclerosis, the number of 1B-positive axons was low, but it was significantly increased compared with controls (multiple sclerosis: CO/IA, P < 0.05) (Table 2). The EAE sample did not contain entirely inactive lesions.
Remyelinated lesions of multiple sclerosis and EAE
Remyelinated shadow plaques in multiple sclerosis showed no evidence of recurrent demyelinating activity. The number of 1B-positive axons was not significantly increased compared with control white matter (multiple sclerosis: CO/RM, n.s.). In contrast to multiple sclerosis, remyelinated EAE plaques, which showed signs of recurrent demyelinating activity, also showed 1B-positive axons (EAE: CO/RM-A, P < 0.05) (Tables 2 and 3).
Periplaque and normal white matter of multiple sclerosis and EAE
There was a low, but significant increase of 1B in the periplaque white matter of multiple sclerosis lesions (multiple sclerosis: CO/PP, P < 0.05). In normal white matter, no significant increase of 1B-positive axons was observed (multiple sclerosis: CO/NWM, n.s.). In EAE, no statistically significant increase of 1B-positive axons was noted in the periplaque/normal white matter (Tables 2 and 3).
In both multiple sclerosis and EAE, there was a highly significant correlation between the frequency of injured axons immunoreactive for APP and 1B in the respective lesional areas (APP/1B: P < 0.0001).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Recent magnetic resonance spectroscopy studies have provided evidence that axonal damage and loss within and around multiple sclerosis lesions are the major correlates of permanent clinical disability (Davie et al., 1995
; Truyen et al., 1996; De Stefano et al., 1998; Lee et al., 2000; Reddy et al., 2000). Axonal injury as a common feature of multiple sclerosis pathology was already recognized at the turn of the century (Kornek and Lassmann, 1999). However, only recently have new techniques provided the tools for accurate quantification of acute axonal damage as well as definite axonal loss (Ferguson et al., 1997; Trapp et al., 1998; Mews et al., 1999; Bitsch et al., 2000; Evangelou et al., 2000; Kornek et al., 2000; Lovas et al., 2000). Finally, histopathological correlates of MRI-defined lesions have clarified further when and to what extent axonal injury occurs in multiple sclerosis (Brück et al., 1997; van Walderveen et al., 1998; Bitsch et al., 1999). The mechanisms of axonal damage, however, are not yet understood.
Axonal degeneration is a common mechanism in a variety of disorders of the CNS (Mohr et al., 1977; Fisher, 1979; McQuin and O'Leary, 1987; Büki et al., 1999). The morphology is very similar under all these circumstances, suggesting a common final pathway in which an increase in the intra-axonal calcium levels appears to be a key step (Ransom et al., 1990; George et al., 1995). External calcium is required for and is sufficient to produce axonal degeneration in ischaemia (Waxman et al., 1993) and in trauma (George et al., 1995), where increased permeability of the axonal membrane may persist for a surprisingly long period (Povlishock and Pettus, 1996).
Under pathological conditions, calcium influx may therefore be mediated through an intact axolemma via calcium-specific transport mechanisms such as voltage gated sodium channels (Agrawal and Fehlings, 1996; Stys and Lopachin, 1998), reverse operation of the Na+/Ca2+ exchanger (Stys et al., 1991, 1992) or via voltage-dependent calcium channels (Fern et al., 1995; George et al., 1995). Blockade of these channels can inhibit or at least delay axonal degeneration after injury.
While most authors have suggested a primary route of calcium entry through reverse operation of the Na+/Ca2+ exchanger in experimental white matter anoxic injury (Stys and Lopachin, 1998), Fern and colleagues observed a significantly increased protection when N-type calcium channels were blocked in addition to L-type calcium channels (Fern et al., 1995). They suggested a low density of N-type channels in white matter compared with L-type channels. They concluded that Ca2+ influx through N-type channels might only become important during anoxia when L-type channels were blocked.
We have investigated a possible involvement of the N-type calcium channel in axonal degeneration in inflammatory demyelination. N-type channels are broadly, if not ubiquitously, expressed in different CNS regions and may be blocked specifically by 
-conotoxin GVIA (Westenbroek et al., 1992, 1998a). They are present on nerve terminals forming synapses on many central neurones (Westenbroek et al., 1992) and play an important role in neurotransmitter release. Like most membrane-bound proteins, calcium channels pass from their sites of synthesis in the endoplasmic reticulum to the Golgi apparatus. From here, packed in membrane vesicles, they are carried by fast axonal transport along tracks formed by microtubules to the synaptic membrane (Alberts et al., 1989). Our study confirmed previous findings of the presence of the 1B subunit in the presynaptic terminals of neurones by electron microscopy. Furthermore, we could show the presence of 1B-immuno- reactive vesicles within the axoplasma, showing their way of transport along the axon by the axonal transporting system.
Most interestingly though, we found a prominent axonal accumulation of 1B-positive vesicles in demyelinated axons and axonal swellings in actively demyelinating lesions of multiple sclerosis and EAE. The pattern was comparable with the axonal accumulation of APP in acutely injured axons of multiple sclerosis and EAE (Ferguson et al., 1997; Kornek et al., 2000). The preferential localization of 1B and APP in acutely damaged axons has been shown by light and confocal microscopy. We therefore can assume that the axonal accumulation of 1B in areas of active demyelination is due to the breakdown of axonal transport. In addition, however, 1B was not only found to be accumulated, but also integrated in the axonal membrane of acutely injured axons, as shown by immune electron microscopy in actively demyelinating EAE lesions. In normally myelinated axons, VGCCs are absent in the internodal axonal plasma membrane (Waxman and Ritchie, 1993).
The abnormal localization of the N-type channel under pathological conditions may have profound consequences for the development of axonal degeneration: Since pore-forming subunits alone can function as intact calcium channels (Westenbroek et al., 1992; Walker and De Waard 1998), an influx of calcium through this particular channel may lead to abnormally high levels of intracellular calcium. Increased levels of intracellular calcium may then potentiate axonal perturbation by the activation of calpain (Banik et al., 1997; Kampfl et al., 1997; Büki et al., 1999) or other unknown mechanisms. These alternative modes of action may evolve over a relatively prolonged temporal framework, leading to delayed secondary axotomy (Büki et al., 1999).
Our study therefore presents the first ultrastructural observation of an abnormal redistribution of N-type calcium channels in acutely injured axons. This may be due to a rearrangement of the axonal membrane after injury. Since sodium channels are known to redistribute along demyelinated axons (Black et al., 1991; Moll et al., 1991), a similar mechanism may also exist for VGCCs.
It is unlikely that Ca2+ influx through VGCCs is the first step in axonal degeneration in inflammatory demyelinating diseases. In fact, the accumulation of 1B in axons closely reflecting that of APP suggests that a primary injury, leading to disturbance of axonal transport, must precede the accumulation of VGCCs. Nevertheless, a further increase in calcium ions in partially injured axons through these channels may augment pathological calcium load and further potentiate axonal damage. This may explain why the blockade of VGCCs in trauma and ischaemia reduced but did not completely prevent axonal degeneration.
Since the pattern and timing of axonal injury as seen by the accumulation of APP (Kornek et al., 2000) and of 1B (this study) was comparable in multiple sclerosis and EAE, MOG-induced EAE appears to be an ideal model for testing axon-protective strategies in inflammatory demyelinating diseases.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The authors wish to thank Helene Breitschopf, Angela Kury, Marianne Leisser, Petra Tassotti and Jutta Wakley-Neuninger for expert technical assistance, and Dr Helmut Rauschka for reading the manuscript and for discussion. The project was supported by the Austrian Science Foundation Project P 12658-MED and the EC Biomed 2-Project BMH 4-97-2027.
|
References |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Agrawal SK, Fehlings MG. Mechanisms of secondary injury to spinal cord axons in vitro: role of Na+, Na+-K+-ATPase, the Na+–H+-exchanger, and the Na+–Ca2+ exchanger. J Neurosci 1996; 16: 545–52.
Akassoglou K, Bauer J, Kassiotis G, Pasparakis M, Lassmann H, Kollias G, et al. Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55TNF receptor signaling in the central nervous system of transgenic mice. Models for multiple sclerosis with primary oligodendrogliopathy. Am J Pathol 1998; 153: 801–13.
Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. The cells of the nervous system: an overview of their structure and function. In: Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD, editors. Molecular biology of the cell. 2nd edn. New York: Garland; 1989. p. 1059–63.
Banik NL, Hogan EL, Xu J, Fischer I, Lobo D. Calpain activity in spinal cord injury [abstract]. J Neurochem 1993; 61 Suppl: S112.
Banik NL, Matzelle DC, Gantt-Wilford G, Osborne A, Hogan EL. Increased calpain content and progressive degradation of neurofilament protein in spinal cord injury. Brain Res 1997; 752: 301–6.[ISI][Medline]
Bitsch A, Bruhn H, Vougioukas V, Stringaris A, Lassmann H, Frahm J, et al. Inflammatory CNS demyelination: histopathologic correlation with in vivo quantitative proton MR spectroscopy. AJNR Am J Neuroradiol. 1999; 20: 1619–27.
Bitsch A, Schuchardt J, Bunkowski S, Kuhlmann T, Brück W. Acute axonal injury in multiple sclerosis: correlation with demyelination and inflammation. Brain 2000; 123: 1174–83.
Black JA, Felts P, Smith KJ, Kocsis JD, Waxman SG. Distribution of sodium channels in chronically demyelinated spinal cord axons: immuno-ultrastructural localization and electrophysiological observations. Brain Res 1991; 544: 59–70.[ISI][Medline]
Breitschopf H, Suchanek G, Gould RM, Colman DR, Lassmann H. In situ hybridization with digoxigenin-labeled probes. Sensitive and reliable detection method applied to myelinating rat brain. Acta Neuropathol (Berl) 1992; 84: 581–7.[Medline]
Brück W, Porada P, Poser S, Rieckmann P, Hanefeld F, Kretzschmar HA, et al. Monocyte/macrophage differentiation in early multiple sclerosis lesions. Ann Neurol 1995; 38: 788–96.[ISI][Medline]
Brück W, Bitsch A, Kolenda H, Brück Y, Stiefel M, Lassmann H. Inflammatory central nervous system demyelination: correlation of magnetic resonance imaging findings with lesion pathology. Ann Neurol 1997; 42: 783–93.[ISI][Medline]
Büki A, Siman R, Trojanowski JQ, Povlishock JT. The role of calpain-mediated spectrin proteolysis in traumatically induced axonal injury. J Neuropathol Exp Neurol 1999; 58: 365–75.[ISI][Medline]
Charcot JM. Histologie de la sclérose en plaques. Gaz Hop Civ Milit 1868; 41: 554–5, 557–8, 566.
Cuzner ML, Wekerle H. Symposium: immunopathogenesis of demyelinating diseases. Brain Pathol 1996; 6: 229–30.
Davie CA, Barker GJ, Webb S, Tofts PS, Thompson AJ, Harding AE, et al. Persistent functional deficit in multiple sclerosis and autosomal dominant cerebellar ataxia is associated with axon loss. Brain 1995; 118: 1583–92.
Day NC, Shaw PJ, McCormack AL, Craig PJ, Smith W, Beattie R, et al. Distribution of 1A, 1B, and 1E voltage-dependent calcium channel sububnits in the human hippocampus and parahippocampal gyrus. Neuroscience 1996; 71: 1013–24.[ISI][Medline]
De Stefano N, Matthews PM, Fu L, Narayanan S, Stanley J, Francis GS, et al. Axonal damage correlates with disability in patients with relapsing–remitting multiple sclerosis. Results of a longitudinal magnetic resonance spectroscopy study. Brain 1998; 121: 1469–77.
Evangelou N, Esiri MM, Smith S, Palace J, Matthews PM. Quantitative pathological evidence for axonal loss in normal appearing white matter in multiple sclerosis. Ann Neurol 2000; 47: 391–5.[ISI][Medline]
Ferguson B, Matyszak MK, Esiri MM, Perry VH. Axonal damage in acute multiple sclerosis lesions. Brain 1997; 120: 393–9.
Fern R, Ransom BR, Waxman SG. Voltage-gated calcium channels in CNS white matter: role in anoxic injury. J Neurophysiol 1995; 74: 369–77.
Fisher CM. Capsular infarcts: the underlying vascular lesions. Arch Neurol 1979; 36: 65–73.[Abstract]
Ganter P, Prince C, Esiri MM. Spinal cord axonal loss in multiple sclerosis: a post-mortem study. Neuropathol Appl Neurobiol 1999; 25: 459–67.[ISI][Medline]
Gentleman SM, Nash MJ, Sweeting CJ, Graham DI, Roberts GW. Beta-amyloid precursor protein (beta APP) as a marker for axonal injury after head injury. Neurosci Lett 1993; 160: 139–44.[ISI][Medline]
George EB, Glass JD, Griffin JW. Axotomy-induced axonal degeneration is mediated by calcium influx through ion-specific channels. J Neurosci 1995; 15: 6445–52.
Hedrich HJ, editor. Genetic monitoring of inbred strains of rats. Stuttgart: Gustav Fischer; 1990.
Kampfl A, Postmantur RM, Zhao X, Schmutzhard E, Clifton GL, Hayes RL. Mechanisms of calpain proteolysis following traumatic brain injury. Implications for pathology and therapy: a review and update [Review]. J Neurotrauma 1997; 14; 121–34.[ISI][Medline]
Kornek B, Lassmann H. Axonal pathology in multiple sclerosis: a historical note. Brain Pathol 1999; 9: 651–6.[ISI][Medline]
Kornek B, Storch MK, Weissert R, Wallstroem E, Stefferl A, Olsson T, et al. Multiple sclerosis and chronic autoimmune encephalo-myelitis: a comparative quantitative study of axonal injury in active, inactive and remyelinated lesions. Am J Pathol 2000; 157: 267–76.
Lassmann H. Neuropathology in multiple sclerosis. New concepts [Review]. Mult Scler 1998a; 4: 93–8.
Lassmann H. The pathology of multiple slerosis. In: Compston A, Ebers G, Lassmann H, McDonald I, Matthews B, Wekerle H, editors. McAlpine's multiple sclerosis. 3rd edn. London: Churchill Livingstone; 1998b. p. 323–58.
Lee MA, Blamire AM, Pendlebury S, Ho KH, Mills KR, Styles P, et al. Axonal injury or loss in the internal capsule and motor impairment in multiple sclerosis. Arch Neurol 2000; 57: 65–70.
Losseff NA, Webb SL, O'Riordan JI, Page R, Wang L, Barker GJ, et al. Spinal cord atrophy and disability in multiple sclerosis: a new reproducible and sensitive MRI method with potential to monitor disease progression. Brain 1996; 119: 701–8.
Lovas G, Szilagyi N, Majtenyi K, Palkovits M, Komoly S. Axonal changes in chronic demyelinated cervical spinal cord plaques. Brain 2000; 123: 308–17.
Lublin FD, Reingold SC. Defining the clinical course of multiple sclerosis: results of an international survey. Neurology 1996; 46: 907–11.
McGavern DB, Murray PD, Rivera-Quinones C, Schmelzer JD, Low PA, Rodriguez M. Axonal loss results in spinal cord atrophy, electrophysiological abnormalities and neurological deficits following demyelination in a chronic inflammatory model of multiple sclerosis. Brain 2000; 123: 519–31.
McQuinn BA, O'Leary DH. White matter lucencies on computed tomography, subacute arteriosclerotic encephalopathy (Binswanger's disease), and blood pressure. Stroke 1987; 18: 900–5.
Mews I, Bergmann M, Bunkowski S, Gullotta F, Brück W. Oligodendrocyte and axon pathology in clinically silent multiple sclerosis lesions. Mult Scler 1998; 4: 55–62.
Mohr JP, Kase CS, Meckler MD, Fisher CM. Sensorimotor stroke due to thalamocapsular ischemia. Arch Neurol 1977; 34: 739–41.[Abstract]
Moll C, Mourre C, Lazdunski M, Ulrich J. Increase of sodium channels in demyelinated lesions of multiple sclerosis. Brain Res 1991; 556: 311–16.[ISI][Medline]
Povlishock JT, Pettus EH. Traumatically induced axonal damage: evidence for enduring changes in axolemmal permeability with associated cytoskeletal change. Acta Neurochir Suppl (Wien) 1996; 66: 81–6.[Medline]
Raine CS, Cross AH. Axonal dystrophy as a consequence of long-term demyelination. Lab Invest 1989; 60: 714–25.[ISI][Medline]
Ransom BR, Stys PK, Waxman SG. The pathophysiology of anoxic injury in central nervous system white matter. Stroke 1990; 21 (II Suppl): III52–7.
Reddy H, Narayanan S, Matthews PM, Hoge RD, Pike GB, Duquette P, et al. Relating axonal injury to functional recovery in MS. Neurology 2000; 54: 236–9.
Schlaepfer WW, Zimmerman UJ. Mechanisms underlying the neuronal response to ischemic injury. Calcium-activated proteolysis of neurofilaments. [Review]. Prog Brain Res 1985; 63: 185–96.[ISI][Medline]
Sherriff FE, Bridges LR, Sivaloganathan S. Early detection of axonal injury after human head trauma using immunocytochemistry for ß-amyloid precursor protein. Acta Neuropathol (Berl) 1994; 87: 55–62.[Medline]
Storch MK, Stefferl A, Brehm U, Weissert R, Wallstroem E, Kerschensteiner M, et al. Autoimmunity to myelin oligodendrocyte glycoprotein in rats mimics the spectrum of multiple sclerosis pathology. Brain Pathol 1998; 8: 681–94.[ISI][Medline]
Stys PK, Lopachin RM. Mechanisms of calcium and sodium fluxes in anoxic myelinated central nervous system axons. Neuroscience 1998; 82: 21–32.[ISI][Medline]
Stys PK, Waxman SG, Ransom BR. Na+–Ca2+ exchanger mediates Ca2+ influx during anoxia in mammalian central nervous system white matter. Ann Neurol 1991; 30: 375–80.[ISI][Medline]
Stys PK, Waxman SG, Ransom BR. Ionic mechanisms of anoxic injury in mammalian CNS white matter: role of Na+ channels and Na+–Ca2+ exchanger. J Neurosci 1992; 12: 430–9.[Abstract]
Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mörk S, Bö L. Axonal transection in the lesions of multiple sclerosis. N Engl J Med 1998; 338: 278–85.
Truyen L, van Waesberghe JH, van Walderveen MA, van Oosten BW, Polman CH, Hommes OR, et al. Accumulation of hypointense lesions (`black holes') on T1 spin-echo MRI correlates with disease progression in multiple sclerosis. Neurology 1996; 47: 1469–76.
van Walderveen MA, Kamphorst W, Scheltens P, van Waesberghe JH, Ravid R, Valk J, et al. Histopathologic correlate of hypointense lesions on T1-weighted spin-echo MRI in multiple sclerosis. Neurology 1998; 50: 1282–8.
Vass K, Lassmann H, Wekerle H, Wisniewski HM. The distribution of Ia antigen in the lesions of rat acute experimental allergic encephalomyelitis. Acta Neuropathol (Berl) 1986; 70: 149–60.[Medline]
Walker D, De Waard M. Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function. [Review]. Trends Neurosci 1998; 21: 148–54.[ISI][Medline]
Waxman SG, Ritchie JM. Molecular dissection of the myelinated axon. Ann Neurol 1993; 33:121–36.[ISI][Medline]
Waxman SG, Black JA, Ransom BR, Stys PK. Protection of the axonal cytoskeleton in anoxic optic nerve by decreased extracellular calcium. Brain Res 1993; 614: 137–45.[ISI][Medline]
Weinshenker BG, Bass B, Rice GP, Noseworthy J, Carriere W, Baskerville J, et al. The natural history of multiple sclerosis: a geographically based study. I. Clinical course and disability. Brain 1989; 112: 133–46.
Westenbroek RE, Hell JW, Warner C, Dubel SJ, Snutch TP, Catterall WA. Biochemical properties and subcellular distribution of an N-type calcium channel 1 subunit. Neuron 1992; 9: 1099–115.[ISI][Medline]
Westenbroek RE, Hoskins L, Catterall WA. Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals. J Neurosci 1998a; 18: 6319–30.
Westenbroek RE, Bausch SB, Lin RC, Franck JE, Noebels JL, Catterall WA. Upregulation of L-type Ca2+ channels in reactive astrocytes after brain injury, hypomyelination, and ischemia. J Neurosci 1998b; 18: 2321–34.
Received July 12, 2000. Revised November 11, 2000. Accepted February 1, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Dutta and B. D. Trapp Pathogenesis of axonal and neuronal damage in multiple sclerosis Neurology, May 29, 2007; 68(22_suppl_3): S22 - S31. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Chitnis, J. Imitola, Y. Wang, W. Elyaman, P. Chawla, M. Sharuk, K. Raddassi, R. T. Bronson, and S. J. Khoury Elevated Neuronal Expression of CD200 Protects Wlds Mice from Inflammation-Mediated Neurodegeneration Am. J. Pathol., May 1, 2007; 170(5): 1695 - 1712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|