Altered neurological and neurobehavioral phenotypes in a mouse model of the recurrent KCNB1 -p.R306C voltage-sensor variant

Figures

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  Figure 1.. Characterization of Kcnb1-p.R306C variant allele. (A) Chromatogram confirmation of Kcnb1 R306C genomic PCR product. Genomic PCR products were TOPO cloned and Sanger sequenced. The top trace represents a WT allele with 100% identity to the mouse C57BL/6J reference genome (GRCm39). The bottom trace shows a mutated allele (Mut) that encodes p.R306C, as well as a silent mutation to destroy the PAM site (p.I304=). (B-C) Transcript (B) and protein (C) expression levels did not differ between genotypes genotypes (p=0.28 and 0.68 respectively, Kruskal-Wallis). Symbols represent samples from individual mice, line represents median and error bars represent 95% confidence interval. (D) A representative western blot of forebrain membrane proteins probed for Kv2.1 protein and GRP75 as a normalization control show no difference. Kcnb1fs/fs that results in truncated protein with absent epitope was used as a negative control.

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  Figure 2.. Subcellular localization Kv2.1 in cultured hippocampal neurons. (A) Cultured hippocampal neurons were immuno-labeled with Kv2.1 and neuronal processes (MAP2 for dendrites and AnkG for axon initial segment); 40x magnification for MAP2 and 60x for AnkG staining. (B) Length measurements of AIS were not different across different genotypes (p = 0.1671, one-way ANOVA; n = 57–62 per genotype).

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  Figure 3.. Kcnb1R306C mice have elevated exploratory locomotion in open field assay. (A) Total distance traveled in 30 minutes was affected by genotype (F(2,44) = 20.6, p < 0.0001; one-way ANOVA; ****p < 0.0001, Tukey’s). (B) Locomotion assessed in 10-minute bins across the 30-minute session. Kcnb1C/+ and Kcnb1C/C mice had elevated distance at all time points compared to WT (F(2,44) = 19.11, p < 0.0001; Two-way repeated measures ANOVA, Genotype). All groups showed similar habituation, with locomotion slowing over time and no significant genotype-by-time interaction (see Table 3). (C) Total number of zone transitions was affected by genotype (F(2,44) = 4.86, p < 0.02; one-way ANOVA; *p < 0.02, Tukey’s). (D) Time spent in the center of area was not affected by genotype (F(2,44) = 1.201, p > 0.31). (E) Representative examples of open field paths for WT, Kcnb1C/+ and Kcnb1C/C mice. For A, C and D, symbols represent individual mice and horizontal lines represent mean. Symbols in B represent group means. Error bars represent SEM (A-D).

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  Figure 4.. Altered susceptibility to seizures induced by chemoconvulsants in Kcnb1C/+ and Kcnb1C/C mice. (A) Latency to the first flurothyl-induced MJ was affected by Kcnb1 genotype (Kruskal-Wallis H(2) = 12.28, p < 0.003; *p < 0.04 and **p < 0.004, Dunn’s). (B) Latency to flurothyl-induced GTCS was affected by Kcnb1 genotype (Kruskal-Wallis H(2) = 35.83, p < 0.0001). Kcnb1C/+ mice exhibited the longest median latency to GTCS, while Kcnb1C/C mice had the shortest latency to GTCS (*p < 0.03, **p < 0.003, ****p < 0.0001, Dunn’s). For A-B, symbols represent individual mice, horizontal line represents median and error bars are 95% CI. (C) Progression from MJ to GTCS differed between genotypes (Kruskal-Wallis H(2) = 13.50, p < 0.002). Kcnb1C/C mice progressed the fastest between stages compared to WT (‡ p < 0.005) and Kcnb1C/+ (‡ p < 0.003). (D) Progression of seizure stages scored on a modified Racine scale post KA administration. Kcnb1C/C mice progressed faster through stages 3–6 compared to WT and to stage 6 compared to Kcnb1C/+ (WT v. Kcnb1C/C Red *p < 0.02 **p < 0.008, ****p < 0.0001; Kcnb1C/+ v. Kcnb1C/C Black *p < 0.03). Kcnb1C/+ mice progressed faster to stages 5 and 6 compared to WT (WT…

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  Figure 5.. EEG abnormalities in Kcnb1C/+ and Kcnb1C/C mice. (A) EEG trace of the single GTC seizure identified in a Kcnb1C/+ mouse with corresponding spectral density array. Corresponding with the electrographic seizure, the Kcnb1C/+ mouse displayed head bobbing and facial automatisms. All observable behaviors concluded when the baseline EEG activity returned to normal. One additional GTCS was detected in a Kcnb1C/C mouse with similar electrographic and behavioral manifestations. (B-D) Top trace represents a two-hour segment of continuous spike-wave discharges identified in a Kcnb1C/+ mouse with corresponding spectral density array (B). Red line represents a ~7-minute segment expanded in panel C and the purple line represents a ~40 second segment expanded in panel D. (E-G) Top trace represents a ~30-minute segment of continuous spike-wave discharges identified in a Kcnb1C/C mouse with corresponding spectral density array (E). Red line represents a ~7-minute trace expanded in panel F and purple line represents a ~ 40 second expanded in panel G.

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  Figure 6.. Abnormal EEG power in Kcnb1C/+ and Kcnb1C/C mice. (A) Spike train frequency and durations for Kcnb1R306C mice. During manual review of EEG records (45–91 hrs per mouse), four Kcnb1C/+ (66%) and 3 Kcnb1C/C (60%) mice exhibited recurrent spike trains of various durations, while never detected in the EEG files reviewed from WT mice. Bars represent median length of spike trains. Symbols represent durations of individual trains. Left side shows trains during wakeful behavior and right side shows trains during restful behavior. (B) Max power frequency for WT, Kcnb1C/+ and Kcnb1C/C mice. WT mice had a calculated average max power frequency of 3.6 ± 0.4 Hz, while Kcnb1C/+ averaged 2.3 ± 0.2 Hz (*p < 0.03) and Kcnb1C/C averaged 2.6 ± 0.4. Bars represent mean and error bars SEM. Symbols represent individual values. No difference in max frequency was detected between light and dark cycles, therefore they were combined, resulting in 22–24 values per genotype. P values calculated by one-way ANOVA with Tukey’s. (C) Power spectra in Kcnb1 mice. Power band comparisons for delta (0.4 – 4 Hz) theta (4 – 8 Hz), alpha (8 – 13 Hz) and beta (13 – 30 Hz) during light and dark phases. In the light…