Genetic deletion of c-Jun amino-terminal kinase 3 (JNK3) modestly increases disease severity in a mouse model of multiple sclerosis

Abstract

The c-Jun amino terminal kinases (JNKs) regulate transcription, and studies suggest they contribute to neuropathology in the EAE model of MS. To examine the role of the JNK3 isoform, we compared EAE in JNK3 null mice to wild type (WT) littermates. Although disease severity was similar in female mice, in male JNK3 null mice the day of onset and time to reach 100% incidence occurred sooner, and disease severity was increased. While glial activation in spinal cord was similar, white matter lesions were increased in JNK3 null mice. These results suggest JNK3 normally limits EAE disease in a sex-dependent manner.

Keywords: Astrocytes; EAE; JNK3; Microglia; Multiple sclerosis; Spinal cord.

Figure 1.. EAE disease is modestly worsened in JNK3 null male mice.

>Male WT (n=10, ●) and JNK3 null (n=11, ○) mice were immunized with MOG _35-55 peptide to develop EAE. (A) The daily incidence of disease was significantly greater in the JNK3 null mice (p=0.0004, Wilcoxon matched-pairs signed rank test). (B) The average day of disease onset was shorter in the JNK3 null mice (p<0.05, unpaired T-test). (C) Compared to WT mice, average daily disease severity was slightly higher in JNK3 null mice, but this difference did not reach statistical significance. (D) Cumulative disease scores were significantly higher in the JNK3 null mice (mixed-effects model).

Figure 2.. EAE disease is not altered in JNK3 null female mice.

Female WT (n=7, ●) and JNK3 null (n=5, ○) mice were immunized with MOG _35-55 peptide to develop EAE. There were no significant differences observed between the 2 groups for either (A) Daily incidence of disease; (B) Average day of disease onset; (C) Average daily disease severity; or (D) Cumulative disease scores.

Figure 3:. Effect of JNK3 deletion on EAE-induced astrocyte activation.

Representative images of spinal cord sections (lumbar level) from sham and EAE WT male mice double stained with the nuclear marker DAPI (blue) and with antibody recognizing the astrocytic marker GFAP. Total spinal cord areas used for quantitation are delineated by a white line. (A) Compared to sham mice, EAE mice showed an increase in the percentage of total spinal cord area displaying GFAP immunoreactivity. This increase was particularly prominent in gray matter (yellow dashed line). (A’) Quantitative data confirms a significant increase in the percentage of GFAP-immunoreactive spinal cord area in EAE WT mice, compared to sham WT mice. (B, B’) Quantitative analysis shows similar GFAP-immunoreactive areas in EAE WT, compared to EAE JNK3 null mice. For panel A, data is mean ± sem % area stained; for panel B, data is relative staining of JNK3 null EAE samples, compared to WT EAE samples. n=3-4 mice per genotype; 6-9 sections imaged per mouse. **, p<0.01; ***, p<0.001 (unpaired T-tests)

Figure 4:. Effect of JNK3 deletion on EAE-induced microglia activation.

Representative images of spinal cord sections (lumbar level) from sham and EAE WT male mice double stained with the nuclear marker DAPI (blue) and with antibody recognizing microglial marker Iba1. (A) Compared to sham-treated mice, EAE WT mice showed an increase in the percentage of total spinal cord area displaying Iba1 immunoreactivity. (A’) The percentage of Iba1-immunoreactive area for the entire spinal cord was significantly increased in EAE WT mice, compared to sham WT mice. As observed for GFAP, no differences in Iba1-stained areas were observed between EAE WT JNK3 null mice (B, B’).

Figure 5:. Effect of JNK3 deletion on EAE-induced white matter spinal cord lesion size.

Representative images of spinal cord sections (lumbar level) from sham and EAE WT male mice (A) and EAE WT and JNK3 null mice (B). Sections were double stained with the nuclear marker DAPI (blue) and with an antibody recognizing myelin basic protein to help delineate white matter (WM, dashed yellow line). (A, A’) Compared to sham WT mice, EAE WT mice showed an increase in total white matter lesion areas. (B, B’) Compared to EAE WT mice, the area covered by DAPI-positive lesions in white matter was significantly higher in EAE JNK3 null mice. For panel A’, data is mean ± sem lesion volume. For panel B’, data is relative lesion area of samples from EAE JNK3 null mice compared to EAE WT mice. n=3-4 mice per genotype; 6-9 sections imaged per mouse. *, p<0.05; ****, p<0.0001 (unpaired T-tests).