Sodium Channel Expression along Axons in MS

Despite the utility of EAE as an animal model of MS, there is no perfect model that mimics all of the features of the human disorder. Thus the question "what sodium channels are expressed along demyelinated axons in MS?" requires examination of human tissue. Craner et al. (2004b) recently carried out such a study, examining postmortem

Figure 4 Degenerating spinal cord axons in EAE co-express Nav1.6 and sodium-calcium exchanger over extensive regions. Left: Histogram showing co-expression of NCX and Nav1.6 in P-APP-positive axons in EAE. Triple immunolabeling was used to determine the proportion of P-APP-positive axons and P-APP negative axons that co-express NCX and Nav1.6 overextensive regions. The proportion of axons that co-express Nav1.6 and NCX is significantly higher in P-APP positive axons (filled bar) compared with P-APP negative axons (light bar). *P < 0.001. (Right): Spinal cord axons in EAE spinal cord immunostained for P-APP (blue; A, E), sodium channel Nav1.6 (red; B) and Nav1.2 (red; F), and sodium-calcium exchanger (NCX) (green; C, G). Panels D and H correspond to merged images (white). Note the co-expression of Nav1.6, NCX and P-APP, a marker of axonal injury (A—D). In contrast, P-APP/NCX-positive profiles do not display Nav1.2 immunoreactivity. (E—H). (From Craner et al., 2004a.)

Figure 4 Degenerating spinal cord axons in EAE co-express Nav1.6 and sodium-calcium exchanger over extensive regions. Left: Histogram showing co-expression of NCX and Nav1.6 in P-APP-positive axons in EAE. Triple immunolabeling was used to determine the proportion of P-APP-positive axons and P-APP negative axons that co-express NCX and Nav1.6 overextensive regions. The proportion of axons that co-express Nav1.6 and NCX is significantly higher in P-APP positive axons (filled bar) compared with P-APP negative axons (light bar). *P < 0.001. (Right): Spinal cord axons in EAE spinal cord immunostained for P-APP (blue; A, E), sodium channel Nav1.6 (red; B) and Nav1.2 (red; F), and sodium-calcium exchanger (NCX) (green; C, G). Panels D and H correspond to merged images (white). Note the co-expression of Nav1.6, NCX and P-APP, a marker of axonal injury (A—D). In contrast, P-APP/NCX-positive profiles do not display Nav1.2 immunoreactivity. (E—H). (From Craner et al., 2004a.)

spinal cord and optic nerve tissue from controls without neurological disease and from patients with disabling secondary progressive MS, acquired via a rapid autopsy protocol (Newcombe and Cuzner, 1993). Analysis of acute lesions from this tissue has revealed a pattern of sodium channel expression that is similar to the pattern in EAE. Control white matter, from patients with no neurological disease, displayed abundant myelin basic protein (MBP) and the expected pattern of expression of Nav1.6, which was focally expressed at nodes of Ranvier. Nav1.2 was expressed along unmyelinated axons within control spinal cord tissue (but not within control optic nerves, as there are very few unmyelinated axons within that tract). Within acute MS plaques, which could be identified on the basis of attenuated MPB immunostaining and the presence of substantial numbers of ORO-positive macrophages containing neutral lipids resulting from myelin breakdown, Nav1.6 and Nav1.2 sodium channels were expressed along extensive regions, in many cases extending for tens of microns, along demyeli-nated axons (Fig. 5C-H). Fig. 5G-J illustrates staining of these profiles for neurofilaments, establishing their identity as axons. In some cases these extensive zones of Nav1.6 or Nav1.2 immunostaining were bounded by Caspr, a protein specifically expressed at the paranodal axoglial junction, further confirming the identity of these profiles as axons, but, as at normal nodes, there was no overlap between Nav1.6 and Caspr immunostaining (Fig. 5E,F).

To examine the relationship of Nav1.6 compared to Nav1.2 expression and axonal injury within MS lesions, Craner et al. (2004b) examined the co-localization of these channel isoforms with P-APP. This analysis demonstrated a large number of P-APP-positive axons within MS plaques, suggesting the presence of approximately 7,500 injured axons per cubic millimeter of tissue within these acute lesions, similar to the value (11,000 per mm3) reported by Trapp et al. (1998). Of importance, P-APP-immunopositive axons within MS lesions tended to express Nav1.6 over extensive regions. A total of 82% of P-APP-immunopositive axons expressed diffuse Nav1.6 sodium channel immunostaining, whereas only 21% of P-APP-immunopos-itive axons expressed diffuse Nav1.2 sodium channel immunostaining.

This analysis also demonstrated, as shown in Fig. 6, that Nav1.6 and the Na/Ca exchanger tend to be co-localized within P-APP-positive axons within acute MS lesions (Craner et al., 2004b). A total of 60% of P-APP-positive axons (but only 19% of P-APP-negative axons) displayed co-expression of Nav1.6 and the Na/Ca exchanger (Fig. 6, left panel). Nav1.2 and the Na/Ca exchanger tended to be co-expressed within P-APP-negative axons. A total of 56% of P-APP-negative axons co-expressed Nav1.2 and the Na/Ca exchanger, but only 18% of P-APP-positive axons showed this co-expression. Thus, the majority of P-APP-positive axons in these MS lesions display extensive regions where both Nav1.6 and the Na/Ca

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Figure 5 Changes in Nav1.6 and Nav1.2 expression along axons in human spinal cord, from active lesions identified by ORO staining in tissue obtained via a rapid protocol from patients with disabling secondary progressive MS. Note expression of Nav1.6 (panel A, red), bounded by Caspr (A, green), while Nav1.2 (B, red) is not detectable at nodes from controls without neurological disease. Panels C and D show the edge of active lesions, where extensive regions of diffuse expression of Nav1.6 (C, red) and Nav1.2 (D, red) are present at regions where myelin basic protein (C, D, green) is absent or markedly attenuated. Co-localization of neurofilament immunostaining (I, J, blue) with Nav1.6 (G, red) and Nav1.2 (H, red) establishes the identity of these profiles as axons. In some cases extensive regions of Nav1.6 (E, red) or Nav1.2 (F, red) are bounded by Caspr (E, F, green), without overlap, consistent with expression of Nav1.6 and Nav1.2 within the axon membrane. (From Craner et al., 2004b.)

Figure 5 Changes in Nav1.6 and Nav1.2 expression along axons in human spinal cord, from active lesions identified by ORO staining in tissue obtained via a rapid protocol from patients with disabling secondary progressive MS. Note expression of Nav1.6 (panel A, red), bounded by Caspr (A, green), while Nav1.2 (B, red) is not detectable at nodes from controls without neurological disease. Panels C and D show the edge of active lesions, where extensive regions of diffuse expression of Nav1.6 (C, red) and Nav1.2 (D, red) are present at regions where myelin basic protein (C, D, green) is absent or markedly attenuated. Co-localization of neurofilament immunostaining (I, J, blue) with Nav1.6 (G, red) and Nav1.2 (H, red) establishes the identity of these profiles as axons. In some cases extensive regions of Nav1.6 (E, red) or Nav1.2 (F, red) are bounded by Caspr (E, F, green), without overlap, consistent with expression of Nav1.6 and Nav1.2 within the axon membrane. (From Craner et al., 2004b.)

exchanger are present, in contrast to P-APP-negative axons, which tend to co-express Nav1.2 and the Na/Ca exchanger. In summary, this recent study of human MS tissue reveals a pattern of sodium channel expression within acute MS lesions that is similar to EAE, with Nav1.2 and Nav1.6 diffusely deployed along extensive regions of axons lacking myelin, and with Nav1.6 (but not Nav1.2) co-expressed together with the Na/Ca exchanger in injured axons.

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