Fig. 5: Activation of the DLK/JNK/c-Jun pathway in chronically demyelinated Myrf^ΔiSox10 mice.

a Experimental schematic and images of laser microdissection of the GCL of the retina. b Heatmap of expression of RGC-specific transcripts in the micro-dissected GCL samples relative to whole retina. c Heatmap of select transcripts from the GCL known to be activated by c-Jun/DLK signaling compared between genotypes. In situ hybridization in the retina at 10 weeks post tamoxifen with probes against Rbpms and Hrk (d) or Ecel1 (e). Arrowheads indicate double-positive cells. f Retina at 10 weeks post tamoxifen stained with phosphorylated (Ser63) c-Jun. g Quantification of phosphorylated c-Jun cells in the retinae of each genotype. ****p < 0.0001, and Myrf^ΔiPlp1 relative to Myrf^fl/fl at 10 (**p = 0.0088) and 20 weeks post tamoxifen (**p = 0.0092). Week 10 Myrf^fl/fl n = 16, Myrf^ΔiPlp1 n = 4, Myrf^ΔiSox10 n = 8, week 12 Myrf^fl/fl n = 7, Myrf^ΔiPlp1 n = 3, Myrf^ΔiSox10 n = 3, and week 20 Myrf^fl/fl n = 5, Myrf^ΔiPlp1 n = 7 mice. h Phosphorylated c-Jun costained with RBPMS cells in Myrf^ΔiSox10 mice. Inlays are of boxed area. Arrowheads indicate colabeled cells. i Representative images of phosphorylated c-Jun stained with AP-2α/β in Myrf^ΔiSox10 retinae. Inlays are of boxed area. Arrows indicate phosphorylated c-Jun positive cells negative for AP-2α/β. j Quantification of RBPMS and AP-2α/β expression in phosphorylated c-Jun+ cells. k Image of retinal layers stained with phosphorylated c-Jun, RBPMS and DAPI. Inlays are of boxed area. l Schematic of the DLK-mediated MAPK cascade and c-Jun. m Western blot of optic nerves for DLK, pMKK4, MKK4, pJNK, JNK, MOG and β-actin loading control from optic nerves of Myrf^fl/fl, Myrf^ΔiPlp1 and Myrf^ΔiSox10 mice. n Quantification of western blots in Myrf^ΔiPlp1 mice relative to Myrf^fl/fl. ***p = 0.0007 and *p = 0.0309. n = 4 per group. o Quantification of western blots in Myrf^ΔiSox10 mice relative to Myrf^fl/fl. pMKK4 (*p = 0.0434), pJNK (**p = 0.0072), total JNK (**p = 0.0034) and MOG **p = 0.0038). Myrf^fl/fl n = 4 and Myrf^ΔiSox10 n = 3. Scale bars are 50 µm in (a, f, h, i, k), and 5 µm in (d, e), insets in (h, i, k). Two-way ANOVA with Tukey’s post hoc at 10 and 12 weeks post tamoxifen, and Student’s t test to compare groups at 20 weeks post tamoxifen in (g). Student’s t test in (n, o). All statistical tests are two-sided. Error bars are SEM. NA not applicable. Source data for this Figure are provided as a Source Data file. Schematic in a created in BioRender. Duncan (2023). BioRender.com/n39a548. l created in BioRender. Duncan (2023) BioRender.com/g94u157.