Nav16 in Injured Axons in EAE

As documented elsewhere in this volume, there is extensive evidence for axonal degeneration that is a substrate for nonremitting disability in MS. Stys et al. (1992) demonstrated that sodium channels can participate in the production of calcium (Ca)-mediated axonal degeneration of CNS axons by providing a route for sustained sodium influx that drives reverse (calcium-importing) activity of the Na/Ca exchanger. The available evidence indicates that a persistent (noninactivating) sodium conductance carries the sustained sodium influx (Stys et al., 1993). Consistent with this schema, Imaizumi et al. (1997) have provided evidence for involvement of sodium channels and the Na/Ca exchanger in axonal degeneration in the anoxic spinal cord studied in vitro, and sodium channel blockers are protective, preventing axonal degeneration in a number of models of white matter injury in vivo (see, for example, Agrawal and Fehlings, 1996; Rosenberg et al., 1999).

There is also more direct evidence that sodium channels contribute to axonal degeneration in vivo in neuroinflamma-tory models of MS (see Waxman, 2003). Lo et al. (2002, 2003) have demonstrated that treatment with the sodium channel blocker phenytoin has a neuroprotective effect, preventing the degeneration of axons within the spinal cord and optic nerve, maintaining axonal conduction, and improving clinical outcome in progressive EAE. Bechtold et al. (2004) have demonstrated a similar protective effect of flecainide in chronic-relapsing EAE.

Figure 3 Upregulation of Nav1.2 mRNA within retinal ganglion neurons, which gives rise to demyelinated axons, in EAE. Representative digital images of retinal sections demonstrate upregulation of Nav1.2 mRNA in retinal ganglion neurons in EAE (A) compared with control (B). From Craner et al. (2003a).

Figure 3 Upregulation of Nav1.2 mRNA within retinal ganglion neurons, which gives rise to demyelinated axons, in EAE. Representative digital images of retinal sections demonstrate upregulation of Nav1.2 mRNA in retinal ganglion neurons in EAE (A) compared with control (B). From Craner et al. (2003a).

As described previously, Nav1.6 and Nav1.2 sodium channels are both expressed diffusely along demyelinated axon regions that can extend for tens of microns in EAE. This raises the question of whether one, or both, of these sodium channel isoforms might be associated with axonal injury. Interestingly, Nav1.6 channels can produce a persistent current that becomes larger with depolarization (Herzog et al., 2003), which is less prominent in Nav1.1 or Nav1.2 channels (Smith et al., 1998; Raman and Bean, 1997; Tanaka et al., 1999). Reasoning that co-expression of Nav1.6 and the Na/Ca exchanger could predispose these axons to injury, Craner et al. (2004a) studied spinal cord white matter in mice with EAE to determine whether there is a correlation between the expression of Nav1.6 sodium channel immunoreactivity, expression of the Na/Ca exchanger, and immunoreactivity to P-amyloid precursor protein (P-APP), a marker of axonal injury (Cochran et al., 1991; Trapp et al., 1998; Bitsch et al., 2000; Kuhlmann et al., 2002).

P-APP immunostaining demonstrated robust axonal injury in this EAE model. To test the hypothesis that Nav1.6, Nav1.2, or both are associated with axonal injury, Craner et al. (2004a) asked whether P-APP-positive axons tend to express Nav1.6 or Nav1.2. 92% of P-APP-positive axons were observed to be Nav1.6 immunopositive (either expressing Nav1.6 alone, 56.0%; or co-expressing Nav1.6 and Nav1.2, 36.2%), compared to only 1.8% of P-APP positive axons that expressed Nav1.2. Because these findings suggest that Nav1.6 is preferentially associated with axonal injury in EAE, Craner et al. (2004a) next asked whether Nav1.6 and the Na/Ca exchanger are co-localized in P-APP-positive axons, using triple-labeling fluorescence immunohistochem-istry. Representative fields are shown in Fig. 4 (right panels), which demonstrates co-localization of Nav1.6 (Fig. 4B) and the Na/Ca exchanger (Fig. 4C) along extensive regions of P-APP positive axons. In all, 73.5% of P-APP-positive axons displayed extensive regions labeled for both Nav1.6 and Na/Ca exchanger, but only 4.4% of P-APP-negative axons displayed immunolabeling for Nav1.6 and the Na/Ca exchanger (Fig. 4, left panel). Thus, Nav1.6 and the Na/Ca exchanger tend to be co-localized within injured axons in this model of MS.

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