Nav1.6 promotes inflammation and neuronal degeneration in a mouse model of multiple sclerosis

Abstract

Background: In multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) model of MS, the Nav1.6 voltage-gated sodium (Nav) channel isoform has been implicated as a primary contributor to axonal degeneration. Following demyelination Nav1.6, which is normally co-localized with the Na⁺/Ca²⁺ exchanger (NCX) at the nodes of Ranvier, associates with β-APP, a marker of neural injury. The persistent influx of sodium through Nav1.6 is believed to reverse the function of NCX, resulting in an increased influx of damaging Ca²⁺ ions. However, direct evidence for the role of Nav1.6 in axonal degeneration is lacking.

Methods: In mice floxed for Scn8a, the gene that encodes the α subunit of Nav1.6, subjected to EAE we examined the effect of eliminating Nav1.6 from retinal ganglion cells (RGC) in one eye using an AAV vector harboring Cre and GFP, while using the contralateral either injected with AAV vector harboring GFP alone or non-targeted eye as control.

Results: In retinas, the expression of Rbpms, a marker for retinal ganglion cells, was found to be inversely correlated to the expression of Scn8a. Furthermore, the gene expression of the pro-inflammatory cytokines Il6 (IL-6) and Ifng (IFN-γ), and of the reactive gliosis marker Gfap (GFAP) were found to be reduced in targeted retinas. Optic nerves from targeted eyes were shown to have reduced macrophage infiltration and improved axonal health.

Conclusion: Taken together, our results are consistent with Nav1.6 promoting inflammation and contributing to axonal degeneration following demyelination.

Keywords: Adeno-associated virus; Conditional knockout; Experimental autoimmune encephalitis; Inflammation; Multiple sclerosis; Nav1.6; Optic nerve; Optic neuritis; Retinal ganglion cells; Scn8a; Sodium channel.

Fig. 1

Experimental timeline and AAV transduction of inner retinal cells. a Experimental timeline with intravitreal injection of the AAV2-Cre-GFP or AAV2-GFP virus (+AAV), as well as the induction and clinical stages of EAE in Scn8a-flox mice. b Representative confocal scanning laser ophthalmoscopy (CSLO) images of GFP-labeled inner retinal cells in a single animal up to 78 days post-AAVCre injection. c Quantification of AAV transduction progression (n = 4)

Fig. 2

Chronic stage EAE mice have increased RGC survival in retinas with reduced Scn8a (Nav1.6). a A population of RGCs (RBPMS-positive) in a normal (−EAE/−AAVCre) retina is shown in comparison to b a representative image of an uninjected (−AAV) EAE mouse, and c a representative image of a EAE mouse retina from a control AAVGFP-treated eye (+EAE/+AAVGFP) showing RBPMS-positive degenerating RGCs (white arrowheads) with GFP occasionally co-localizing with cell remnants. d A representative image of an EAE mouse retina from an AAVCre-treated eye (+EAE/+AAVCreGFP) showing normal appearing GFP-positive RGCs. e RGC quantification in +EAE retinas treated with AAVGFP (n = 3) or AAVCreGFP (n = 3). Lines link data points for retinas from the same animal. f Percent of expression change for Scn8a and Rbpms in AAVCre-treated (+EAE/+AAVCreGFP; n = 4) eyes relative to their contralateral control uninjected (+EAE/−AAV; n = 4) or GFP (+EAE/+AAVGFP; n = 4) eye. Scale bar = 50 μm. Data are presented as the mean ± SEM. *P ≤ 0.05, paired t test

Fig. 3

Nav1.6 promotes inflammation in EAE mice. Expression in the retina of the markers of inflammation. a Il6 (gene that encodes IL-6) and b Ifng (IFN-γ) is compared between untreated (−EAE) or EAE-induced (+EAE) mice. The eyes of untreated (−EAE) mice are either left uninjected (−AAVCre, open triangles) or injected with AAVCreGFP (+AAVCre, closed triangles). In the EAE-induced mice, a comparison is made between AAVCreGFP-injected (+AAVCre, black dots) and the contralateral eye, which is either left uninjected (blue dots) or injected with a GFP-only control (AAVGFP, green dots). c Analysis of the marker of reactive gliosis Gfap (Glial Fibrillary Acidic Protein). Lines link data points for retinas from the same animal. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, paired t test

Fig. 4

Targeting of Nav1.6 results in reduced infiltration of myeloid cells in EAE optic nerves. a Hematoxylin and eosin-stained optic nerves from control non-EAE-induced/uninjected eye (−EAE/−AAVCre) and from EAE-induced (+EAE) mice with eyes left uninjected (−AAVCre, uninj), injected with AAVGFP (+AAVGFP), or injected with AAVCreGFP (+AAVCre). Arrowheads indicate cellular aggregates. b Quantification of optic nerve nuclei from non-EAE-induced/uninjected eyes (−EAE/−AAVcre, open triangles) and from EAE-induced mice where a comparison is made between AAVCreGFP-injected (+AAVCre, black circles; n = 7) and the contralateral eye, which is either left uninjected (blue circles; n = 2) or injected with AAVGFP (green circles; n = 5). c Representative dot plot flow cytometry analysis showing the gating strategy used to identify the macrophage population expressing marker CD11b⁺ F4–80⁺ from a population of CD45⁺ cells isolated from optic nerves. d Flow cytometry analysis for CD11b⁺ F4–80⁺ macrophages. Lines link data points for retinas from the same animal. Scale bar, 500 μm. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, paired t test

Fig. 5

The axonal pathology is improved in optic nerves with reduced Nav1.6 levels. Representative ultra-thin transversal sections of optic nerves were obtained from control non-EAE-treated/uninjected mice (a, −EAE/−AAVCre), EAE-treated/uninjected (b, +EAE/−AAVCre), and EAE-treated/AAV2Cre-injected (c, +EAE/+AAVCre). Images in b and c are of retinas from the same animal. Ax, axolytic; *, demyelinating. Scale bar, 5 μm

Fig. 6

Reduced Nav1.6 levels in optic nerve is associated with decreased demyelination and reduced axonal damage. Electron micrographs of optic nerves from control non-EAE-induced (−EAE) or EAE-induced (+EAE) mice were analyzed. The non-EAE-induced optic nerves are from eyes either left uninjected (−AAVCre, open triangles) or injected with AAVCreGFP (+AAVCre, closed triangles). In the EAE-induced mice, a comparison is made between AAVCreGFP-injected (+AAVCre, black circles) and the contralateral eye, which is either left uninjected (blue dots) or injected with a GFP-only control AAV (green circles). We examined the frequency of a axolytic axons and b demyelinated axons (axons completely devoid of myelin). Based on the g-ratio (see "Materials and methods" section, we also examined the frequency of c optimally myelinated and d demyelinating axons. Data are presented as the mean ± SEM. *P ≤ 0.05; paired t test