A knock-in mouse model for KCNQ2-related epileptic encephalopathy displays spontaneous generalized seizures and cognitive impairment

Abstract

Objective: Early onset epileptic encephalopathy with suppression-burst is one of the most severe epilepsy phenotypes in human patients. A significant proportion of cases have a genetic origin, and the most frequently mutated gene is KCNQ2, encoding Kv7.2, a voltage-dependent potassium channel subunit, leading to so-called KCNQ2-related epileptic encephalopathy (KCNQ2-REE). To study the pathophysiology of KCNQ2-REE in detail and to provide a relevant preclinical model, we generated and described a knock-in mouse model carrying the recurrent p.(Thr274Met) variant.

Methods: We introduced the p.(Thr274Met) variant by homologous recombination in embryonic stem cells, injected into C57Bl/6N blastocysts and implanted in pseudopregnant mice. Mice were then bred with 129Sv Cre-deleter to generate heterozygous mice carrying the p.(Thr274Met), and animals were maintained on the 129Sv genetic background. We studied the development of this new model and performed in vivo electroencephalographic (EEG) recordings, neuroanatomical studies at different time points, and multiple behavioral tests.

Results: The Kcnq2^Thr274Met/+ mice are viable and display generalized spontaneous seizures first observed between postnatal day 20 (P20) and P30. In vivo EEG recordings show that the paroxysmal events observed macroscopically are epileptic seizures. The brain of the Kcnq2^Thr274Met/+ animals does not display major structural defects, similar to humans, and their body weight is normal. Kcnq2^Thr274Met/+ mice have a reduced life span, with a peak of unexpected death occurring for 25% of the animals by 3 months of age. Epileptic seizures were generally not observed when animals grew older. Behavioral characterization reveals important deficits in spatial learning and memory in adults but no gross abnormality during early neurosensory development.

Significance: Taken together, our results indicate that we have generated a relevant model to study the pathophysiology of KCNQ2-related epileptic encephalopathy and perform preclinical research for that devastating and currently intractable disease.

Keywords: KCNQ2; epileptic encephalopathy; mouse model.

FIGURE 1

Electroencephalographic (EEG) recordings of the female carrying the p.(Thr274Met) variant at day 9, day 12, day 15, and 3 months of life. The first three EEGs display a suppression‐burst pattern, with periods of high‐amplitude bursts of paroxysmal activity, alternating with periods of flattening of the traces. At 3 months of age, the EEG became continuous, with delta rhythms and no spike

FIGURE 2

A, Schematic representation (not to scale) of the Kcnq2 endogenous, recombined, and Cre‐excised loci. The binding sites of the genotyping primers 284‐Oht and 281‐Oht are shown. Asterisks show the position of Thr274 in exon 3d of the mouse Kcnq2 gene. B, Representative example of a genotyping polymerase chain reaction (PCR) using mouse genomic DNA as a template and primers 284‐Oht and 281‐Oht. PCR without DNA served as a negative control (C−). The results are shown for 10 different animals, including six heterozygous knock‐in mice. Multiplexing is performed with SRY genotyping primers to confirm gender. MWM, Molecular Weight Marker. C, Kernel density estimates computed using R, with the time of spontaneous death as a variable. The graph shows the time of death in 175 knock‐in animals. Ninety‐one percent of spontaneous death in our colony occurred in heterozygous knock‐in animals. D, Kaplan‐Meier survival plot for the two genotypes from postnatal day 0 (P0) to P300

FIGURE 3

A, Schematic representation of a section at lateral +0.60 mm. Colored regions indicate the presence of at least one significant parameter within the brain region at the 0.05 level. White coloring indicates P > .05, and gray indicates that there were not enough data to calculate a P value. The left panel is for males, and the right is for females. B, Histograms for males (left) and females (right) showing variation (decreased, minus scale; increased, positive scale) in areas and lengths expressed as percentage of wild type (WT) together with a color map indicating the significance level. Numbers on the x‐axis correspond to brain regions illustrated in A, and bars are listed in the order of parameters provided in Table S1. C, Illustrating example of Kcnq2 ^+/+ (WT) and Kcnq2 ^Thr274Met/+ (knock‐in [KI]) male brain sizes in parasagittal sections double‐stained for Nissl and Luxol. D, Representative image of the corpus callosum enlargement in Kcnq2 ^Thr274Met/+ male brains shown across a parasagittal section

FIGURE 4

Representative example of the different phases of a spontaneous generalized tonic‐clonic seizure observed in the Kcnq2 ^Thr274Met/+ mouse model. These images are extracted from a video where a heterozygous knock‐in mouse is housed with a wild‐type animal (Video S3)

FIGURE 5

Prefrontal cortex electrocorticogram of a P32 Kcnq2 ^Thr274Met/+ mouse during a seizure. Artifacts are caused by violent seizure movements

FIGURE 6

Cognitive differences between Kcnq2 ^Thr274Met/+ and Kcnq2 ^+/+ mice. A, Time to reach the platform in the Morris Water Maze task. The Kcnq2 ^+/+ wild‐type mice (WT) are represented by blue squares, and Kcnq2 ^Thr274Met/+ mice (KI) are represented by red diamonds. The time is expressed in seconds, and the figure indicates the median value of a group of 10 mice for each block. Plus signs beside the curve indicate the significance of the within‐group, between‐blocks variation (χ², ⁺⁺ P < .001). The asterisk beside the left bracket indicates the learning slopes difference (t, *P < .01), and the x beside the right bracket indicates the probe test difference (t, ^x P < .01). B, Time to enter into the shelter in the Barnes Maze task. Time is expressed in seconds, and the figure shows the median value of a group of 11 mice for each block. Plus signs beside the curve indicate the significance of the within‐group, between‐blocks variation (χ², ⁺⁺⁺ P < .001). The asterisk beside the bracket indicates the learning slopes difference (t, * P < .01). C, Number of errors before entering into the shelter in the Barnes Maze task. The median value of the blocks is reported. Plus signs beside the curve indicate the significance of the within‐group, between‐blocks variation (χ², ⁺⁺⁺ P < .001). Asterisks beside the bracket indicate the learning slopes difference (t, **P < .001). D, Number of head dips in the Hole Board test. Data are given as mean ± SEM for the KI mice and WT mice. t, *P < .01