Novel mouse model of alternating hemiplegia of childhood exhibits prominent motor and seizure phenotypes

Figures

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  Fig. 1.. Generation and validation of the Atp1a3G947R mouse model. A. Genomic amplicon sequence electropherograms comparing WT and Atp1a3G947R (R/+) mice. B. Atp1a3 mRNA transcript expression in brain normalized to Gapdh was not different between WT and R/+ mice (p = 0.43). C. Atp1a3 protein expression in brain normalized to GRP75 was not different between WT and R/+ mice (p = 0.31). In panels B and C, symbols are data from individual mice, horizontal lines represent mean values and error bars indicate SEM, n = 8–11 mice per genotype.

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  Fig. 2.. Physical characteristics and survival of heterozygous Atp1a3G947R mice. A. R/+ mice weighed less than their sex-matched WT littermates. WT males and females had average weights of 28.4 ± 0.35 g and 21.9 ± 0.25 g, while R/+ males and females had average weights of 23.0 ± 0.4 g and 19.1 ± 0.2 g (****p < 0.0001, 43–51 mice of each sex per genotype). B. R/+ mice were smaller than their sex-matched WT littermates. WT males and females had average lengths of 17.3 ± 0.1 cm and 16.5 ± 0.1 cm, while R/+ males and females had average lengths of 16.1 ± 0.1 cm and 15.6 ± 0.1 cm (****p < 0.0001, 20–26 mice of each sex per genotype). C. R/+ mice exhibited premature lethality. Kaplan Meier plot comparing WT and R/+ survival. R/+ mice began to sporadically die after the first month of life with increasing mortality during 3 to 5 months of life (****p < 0.0001, 161–178 mice per genotype). In panels A and B, symbols represent individual mice, horizontal lines represent mean values and error bars indicate SEM.

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  Fig. 3.. Impaired motor function of heterozygous Atp1a3G947R mice. A. R/+ mice traveled farther in an open field arena compared to WT (R/+: 180.1 ± 10.8 m; WT: 130.3 ± 4.6 m; ****p < 0.0001, 15–18 mice per genotype). B. R/+ mice spent less time in the center of an open field arena compared to WT (R/+: 10.4 ± 0.95 %; WT: 13.4 ± 0.86 %; *p < 0.03, 15–18 mice per genotype). C. Motor behaviors during tail suspension. WT mice had an average tail suspension score of 3.45 ± 0.15, due to spending the most time in an immobile hang position or trying to right themselves. R/+ mice displayed uncoordinated and erratic trunk and limb movements, had trouble curling to right themselves and scored an average of 7.2 ± 0.28 (****p < 0.0001, 14–19 mice per genotype). D. Forelimb motor task. WT mice exhibited an average latency of 8.2 ± 1.1 s to remove an adhesive sticker from their head using forelimbs, while R/+ mice had a longer average latency of 36.7 ± 8.9 s (****p < 0.0001, 14–19 mice per genotype). E. Success in traversing an elevated beam. Most WT mice (11/19; 58.8 %) traversed the beam with a median time of 19 s. Only 15.4 % (2/13) of R/+ mice traversed the beam, with a median time of 34.5 s. The…

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  Fig. 4.. Heterozygous Atp1a3G947R mice exhibit dystonic-like behaviors. A. R/+ mice became dystonic during rapid cooling. Kaplan-Meier curves for occurrence of dystonic-like behaviors upon cooling from 42° to 30 °C core temperature (**p < 0.004, 20 mice per genotype). B. Comparison of movement disorder score (labeled as ‘Dystonia Score’) between WT and R/+ mice (****p < 0.0001, 16–20 mice per genotype). Symbols are data from individual mice, horizontal line represents mean values and error bars indicate SEM C. Comparison of latency to recovery from dystonic behaviors between WT and R/+ mice (****p < 0.0001, 16–20 mice per genotype). Symbols are data from individual mice, horizontal lines and error bars represent median values with 95 % CI. D. Representative image of a WT mouse, exhibiting normal appearance and mobility after cooling. E. Representative image of a R/+ mouse, exhibiting inability to right self, tail rigidity, hindlimb clasping, and core/body twisting after cooling. F. Representative image of a separate R/+ mouse, exhibiting hyperextension and splaying of hindlimbs with tail rigidity after cooling.

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  Fig. 5.. Heterozygous Atp1a3G947R mice have lower induced seizure threshold. A. Comparison of latency to GTCS following flurothyl exposure between WT and R/+ mice (***p = 0.0001, 19–20 mice per genotype). Symbols are data from individual mice, horizontal lines represent mean values and error bars indicate SEM. B. Latency to myotonic jerk (MJ) and GTCS after flurothyl exposure. There was no difference in latency to onset of MJ between WT and R/+ mice, but mutant mice transitioned more quickly to GTCS (****p < 0.0001, 15–19 per genotype). C. Susceptibility to MES-induced seizures. A greater proportion of R/+ mice exhibited hindlimb extension (HLE) in response to a subthreshold corneal MES stimulation (****p < 0.0001, 9–10 mice per genotype). D. Comparison of latency to KA induced seizures between WT and R/+ mice. Mutant mice progressed faster to Racine stages 3 and 4 (**p < 0.009, ****p < 0.0001, 16–22 mice per genotype). E. Comparison of survival time after KA administration between WT and R/+ mice (****p < 0.0001, 16–22 mice per genotype). F. Heat maps depicting individual level seizure severity after KA administration organized by genotype. Individual mice are presented on the y-axis,…

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  Fig. 6.. Heterozygous Atp1a3G947R mice have spontaneous epileptiform EEG activity. A. Representative spontaneous seizure recorded by EEG from a R/+ mouse. Corresponding to the electrographic activity, this R/+ mouse exhibited behavioral arrest and returned to normal activity after seizure termination (Supplemental Video 9). B. Segment (~1 h) of a continuous spike train identified in a R/+ mouse. Yellow line represents a ~ 3.5 min segment expanded into middle trace. Green line represents a ~ 40 s segment expanded into bottom trace. C. Representative trace of a R/+ mouse with frequent high amplitude, high frequency bursts (blue lines) and spike train (green line). Blue lines with asterisks are expanded into bottom trace to highlight burst activity. D. Comparison of root square mean (RMS) between WT and R/+ mice (p > 0.76, 5–11 mice per genotype). E. Comparison of burst frequency between WT and R/+ mice (*p < 0.02, 5–11 mice per genotype). F. Duration of spike trains in WT and R/+ mice (5–11 mice per genotype). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 7.. Power spectral density (PSD) analysis of EEG1. A. R/+ mice exhibit lower activity across the power spectra during apparent sleep (3.1–9.4 Hz frequency range: *p < 0.03–0.0001). Data points represent mean values from 5 mice per genotype and shading indicate SEM. B. Comparison of total power between WT and R/+ mice during apparent sleep (**p < 0.007, 5 mice per genotype). C. R/+ mice exhibit lower activity across the power spectra during wakefulness (4.7–9.4 Hz frequency range: *p < 0.002–0.0001). Data points represent mean values from 5 mice per genotype and shading indicate SEM. D. Comparison of total power between WT and R/+ mice during wakefulness (*p < 0.02, 5 mice per genotype). PSDs and total power were averaged from artifact, spike, and seizure free 5-min epochs. In panels B and D, symbols are data from individual mice, horizontal lines represent mean values and error bars indicate SEM.

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  Fig. 8.. Heterozygous Atp1a3G947R mice exhibit shorter REM and longer nREM sleep periods by video-EEG. A. Rest and wake cycles for WT and R/+ mice over a 24-h period. Mice were housed in a 14:10 light cycle. Gray shaded area represents dark cycle. Blue represents time spent awake and black is time at rest. Y-axis represents individual mice (5 mice per genotype). B. Total number of transitions identified in 24 h between awake and rest states. No difference was determined. (p > 0.30, 5 mice per genotype). Symbols are data from individual mice, horizontal lines represent mean values and error bars indicate SEM. C. Total duration spent in awake and rest states in both light and dark cycles over 24 h. No differences were determined. (p > 0.99, 5 mice per genotype). Symbols are data from individual mice, horizontal lines represent mean values and error bars indicate SEM. D. Comparison of nREM and REM in a 25-min segment of sleep. 1 min epochs were scored on % of signal in nREM (1–4 Hz) and REM (6–9 Hz). R/+ mice spent less time in REM and more time in nREM compared to WT (***p = 0.0001, **p < 0.008, 5 mice per genotype). Symbols represent 1 min epochs, horizontal lines represent mean values and…