Fig 2: KCNQ3 dysregulation in cortical and retinal MS tissues.(A) Uniform manifold approximation and projection (UMAP) plot visualizes clustering of human excitatory (EN) and inhibitory (IN) cortical neurons based on a published snRNA-Seq data set (12). (B) Normalized KCNQ2/-3/-5 expression in control (Ctrl) and MS human cortical neurons. (C) Spatial KCNQ3 expression (ISH) in the human cortex. (D) Violin plots visualize average KCNQ2/-3/-5 expression levels (snRNA-Seq, A) in control neurons (n = 5; green dashed line) and representative MS samples from patients with various disease durations. (E) KCNQ3 ISH in human CGM and MS NAGM and DMGM lesion areas based on MOG IR. DL, deep layers. (F) Correlation of nucleus- and cytoplasm-associated KCNQ3 transcript counts within the same cell in human cortical tissues (ISH) from CGM (n = 35 areas, n = 5 patients), NAGM (n = 27 areas, n = 8 patients) and DMGM (n = 34 areas, n = 8 patients). (G) KCNQ3 upregulation in DMGM (ISH) independent of neuronal density. (H) Gradual loss of mean KCNQ3 expression in MS GM tissues (ISH) from patients with a prolonged MS disease duration approached CGM expression levels (n = 5, green dashed line). (I) Unsupervised trajectory inference of upper L2/-3 neuron branch and nuclei distribution along the trajectory (compare with Supplemental Figure 3, E and F) based on MS disease duration, demyelination (DM) extent, and lesion stage. (J and K) Pseudotime-dependent KCNQ2/-3/-5 expression in relation to disease duration and demyelination based on MOG IR. (L) Neuronal KCNQ2/-3/-5 expression grouped by lesion stage. (M) Sorting of retinal nuclei based on NeuN IR. (N) Normalized KCNQ2/-3/-5 expression by qPCR in human RGC nuclei (controls, n = 6; MS, n = 7). Scale bars: 500 µm (C); 100 µm (E). *P < 0.05, **P < 0.01, ***P < 0.001, by Wilcoxon rank-sum test with Bonferroni’s correction (B); generalized linear model by Wald test with Benjamini and Hochberg correction (D and L); simple linear regression (F and H); Kruskal-Wallis test (G); and mixed-effects model with Geisser-Greenhouse correction and Šidák’s multiple-comparison test (N). ACA, acute chronic active; CI, chronic inactive.

Fig 3: Dysregulation of Kv7 subunits in cortical and retinal EAE tissues.(A) Spatial Kcnq3 expression (ISH) in the mouse cortex. (B) Overview plot visualizes different EAE groups including endpoints at 15, 30, and 60 dpi; note chronic EAE groups (endpoint at 60 dpi) were divided into 2 groups separating animals with or without clinical worsening (rebound). (C) Kcnq3 expression (ISH) in Syt1+ Rorb+ L4 mouse neurons at 14, 30, and 60 dpi in EAE (each, n = 4) and control (n = 3) mice; Kcnq3-KO mouse tissue showed a strong reduction in Kcnq3 expression (n = 4). (D) Density of Kv7.2+ nodes (framed by Caspr+ IR) based on IR in L4 cortical areas at 14, 30, and 60 dpi in EAE (each, n = 4) and control (n = 3) tissues. (E) Kcnq2 expression based on ISH in L4 mouse neurons at 14, 30, and 60 dpi in EAE, control, and Kcnq3-KO (each, n = 4) mice. (F) Kcnq3 expression (ISH) of mouse retinal specimens comprising the inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), and outer nuclear layer (ONL) revealed specific Kcnq3 expression in RGCs (yellow arrows). Plots show normalized Kcnq2/-3/-5 expression (qPCR) in sorted mouse NeuNhi RGC nuclei (control, n = 9; 15 dpi, n = 6; 30 dpi, n = 3; 60 dpi chronic, n = 3; 60 dpi rebound, n = 4). (G) Cartoon illustrates dysregulated neuron-OL K+ shuttling during inflammatory demyelination resulting in neuronal hyperexcitability, axonal swelling, and impaired neuronal function in addition to OL-Kir4.1 loss (colorless channels with dashed borders) and transient upregulation of nodal Kv7 channels. Scale bars: 100 µm (A and F); 20 µm (C–E). Original magnification, ×63 (enlarged insets in D). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA (C–F).