Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder

Abstract

CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female Cdkl5 knockout (Cdkl5 +/-) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous Cdkl5 +/- mice. We found that Cdkl5 +/- mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, Cdkl5 +/- mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female Cdkl5 +/- mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.

Figure 1

Intermediate Cdkl5 expression levels in the brain of Cdkl5 +/− female mice. (a, b) Western blot analysis of Cdkl5 levels normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase) levels in the cortex (Cx), hippocampus (Hp), and cerebellum (Cb) of wild-type (Cdkl5 +/+ n = 5), heterozygous (Cdkl5 +/− n = 6), and homozygous (Cdkl5 −/− n = 5) Cdkl5 KO female mice aged 3–4 months. Immunoblots in (a) are examples from one animal of each experimental group, and the graph shows the relative amounts of Cdkl5 expression in the different brain structures of Cdkl5 +/+ mice. Regardless of the relative amounts, Cdkl5 expression levels were reduced to about 35–46% in Cdkl5 +/− mice in comparison to wild-type mice and absent in Cdkl5 −/− mice in all analyzed brain regions (b). Data in (b) are expressed as a percentage of the values of Cdkl5 +/+ mice. Values are represented as means ± SE. ^∗ p < 0.05 and ^∗∗ p < 0.001 (Mann–Whitney test after unpaired t-test).

Figure 2

Reduced body weight, impaired olfaction, and autistic-like features in Cdkl5 +/− female mice. (a) Body weight at 8 and 14 weeks of age in Cdkl5 +/+ (n = 9), Cdkl5 +/− (n = 8 and n = 11, resp.), and Cdkl5 −/− (n = 11 and n = 8, resp.) mice. Only at 14 weeks of age Cdkl5 +/− and Cdkl5 −/− mice showed reduced body weight compared to Cdkl5 +/+ mice. (b) Olfactory ability evaluated using the buried food test. Cdkl5 +/− (n = 15) and Cdkl5 −/− (n = 18) mice showed an increased latency to find the buried cookie compared to Cdkl5 +/+ mice (n = 14), indicating olfactory impairment in Cdkl5 +/− and Cdkl5 −/− mice. (c) Nest building ability evaluated at 3 and 22 h. Cdkl5 +/− (n = 9) and Cdkl5 −/− (n = 10) mice showed impaired nesting behavior compared to Cdkl5 +/+ (n = 8) mice. (d) Marble burying test. Cdkl5 +/− (n = 19) and Cdkl5 −/− (n = 15) mice buried fewer marbles compared to Cdkl5 +/+ mice (n = 15). Values represent mean ± SEM. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 (datasets in (a), Fisher's LSD test after ANOVA; datasets in (b–d), Dunn's test after Kruskal-Wallis).

Figure 3

Impaired motor coordination, hyperactivity, and stereotypes in Cdkl5 +/− female mice. (a, b) Rotarod assay, measuring latency to fall (a) and frequency of passive rotations (b; rotations in which the mouse does not perform any coordinated movement but is passively transported from the rotating apparatus) on the accelerating rotating rod. Testing was performed in 4 trials with an intertrial interval of 1 h. Cdkl5 +/− (n = 21) and Cdkl5 −/− (n = 23) mice showed a decreased latency to fall (a) and an increased frequency of passive rotations compared to Cdkl5 +/+ (n = 17) mice, indicating impaired motor coordination in Cdkl5 +/− and Cdkl5 −/− mice. (c, d) Locomotor activity measured as total distance traveled (c) and average locomotion velocity (d) during a 20 min open-field test. Cdkl5 +/− (n = 19) and Cdkl5 −/− (n = 22) mice exhibited increased locomotor activity with a longer total distance traveled (c) at a greater average speed (d) compared to Cdkl5 +/+ (n = 16) mice. (e) Number of stereotypic jumps (repetitive beam breaks < 1 s) in the corners of the open-field arena during the 20 min trial. Cdkl5 +/− (n = 19) and Cdkl5 −/− (n = 21) mice showed an increased number of repetitive stereotyped jumps compared to Cdkl5 +/+ (n = 15) mice. (f) Time (cumulative duration) spent by the border, near the walls, and in the center of the open-field arena. Cdkl5 +/+, Cdkl5 +/−, and Cdkl5 −/− mice spent a comparable time at the border, near the walls, and in the center compared to controls, suggesting that hyperactivity was not due to increased anxiety. Values represent mean ± SEM. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 (datasets in (a–d, f), Fisher's LSD test after ANOVA; datasets in (e) Dunn's test after Kruskall-Wallis).

Figure 4

Impaired hippocampus-dependent learning and memory in Cdkl5 +/− female mice. (a) Spatial learning assessed using the Morris water maze. Cdkl5 +/− (n = 20) and Cdkl5 −/− (n = 19) mice showed an increased latency to find the platform over the 5-day learning period compared to the Cdkl5 +/+ (n = 16) mice. (b, c) Maximum (b) and average (c) swim speed during the learning phase in mice as in (a). (d) Percentage of floating, defined as the percentage of time swimming at less than 4 cm/s during the learning phase in mice as in (a). (e–h) On day 6 (probe test), the platform was removed and spatial memory was assessed by evaluating different parameters. Cdkl5 +/− and Cdkl5 −/− mice showed an increased latency to enter the former platform zone (e), a reduced frequency to enter (f), and time spent in the quadrant (g) in which the platform had been located during the learning period. In addition, Cdkl5 +/− and Cdkl5 −/− mice swam a larger distance from the former platform (h) compared to the Cdkl5 +/+ mice, indicating defects in spatial memory. Values represent mean ± SEM. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 as compared to the Cdkl5 +/+ female mice; ^# p < 0.05 as compared to the Cdkl5 +/− female mice (dataset in a); ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 as compared to the Cdkl5 +/+ or Cdkl5 +/− female mice (dataset in b–h), Fisher's LSD test after ANOVA; datasets in (d), Dunn's test after Kruskall-Wallis).

Figure 5

Impaired memory in Cdkl5 +/− female mice. (a, b) Passive avoidance task to measure memory that involves contributions from both the hippocampus and amygdala. On the first day (a), no difference was observed between genotypes regarding the latency to enter the dark compartment. On the second day, Cdkl5 +/− (n = 12) and Cdkl5 −/− (n = 14) mice showed a decreased latency to enter the dark compartment compared to Cdkl5 +/+ (n = 13) mice, indicating defects in memory. Values represent mean ± SEM. ^∗ p < 0.05 and ^∗∗ p < 0.01 (Dunn's test after Kruskall-Wallis).

Figure 6

Sleep apnea occurrence rate in Cdkl5 +/− female mice. (a, c) Apnea occurrence rate during NREM sleep (a), REM sleep (b), and total sleep period (c). Cdkl5 +/− (n = 11) and Cdkl5 −/− (n = 8) mice showed an increased number of apneas during NREM sleep (a) and, consequently, an increased number of total sleep apneas (c) compared to Cdkl5 +/+ (n = 9) mice. Values represent mean ± SEM. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 (Fisher's LSD test after ANOVA).

Figure 7

Impaired hippocampal development in Cdkl5 +/− female mice. (a, b) Mean total dendritic length (a) and mean number of dendritic branches (b) of Golgi-stained mature granule neurons of Cdkl5 +/+ (n = 4), Cdkl5 +/− (n = 3), and Cdkl5 −/− (n = 4) mice. (c) Examples of the reconstructed dendritic tree of Golgi-stained mature granule neurons of one animal from each experimental group. (d, e) Dendritic spine density (number of spines per μm; d) and percentage of immature and mature spines (e) in relation to the total number of protrusions of granule neurons of mice as in (a). (f) Examples of Golgi-stained dendritic branches of mature granule neurons of one animal from each experimental group. The black triangles indicate mature spines (mushroom- and cup-shaped spines). Scale bar: 1 μm. (g) Number of fluorescent puncta per μm² exhibiting PSD-95 immunoreactivity in the molecular layer of the dentate gyrus of Cdkl5 +/+ (n = 4), Cdkl5 +/− (n = 4), and Cdkl5 −/− (n = 3) mice. (h) Confocal images of sections processed for PSD-95 immunohistochemistry of one animal from each experimental group. Scale bar: 2.5 μm. Values represent mean ± SEM. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 (datasets in (a–c, e–h), Fisher's LSD test after ANOVA; datasets in (d), Dunn's test after Kruskall-Wallis).

Figure 8

Altered ERK signaling pathway in Cdkl5 +/− female mice. (a) Western blot analysis of P-AKT-Ser 473 levels normalized to total AKT levels (left histograms) and P-ERK-Ser 42–44 levels normalized to total ERK levels (right histograms) in the hippocampus (a) and cerebellum (b) of Cdkl5 +/+ (n = 6 and n = 5, resp.), Cdkl5 +/− (n = 6 and n = 8, resp.), and Cdkl5 −/− (n = 6 and n = 5, resp.) mice. Immunoblots are examples from one animal of each experimental group. Data are expressed as a percentage of the values of Cdkl5 +/+ mice. Values are represented as means ± SE. ^∗ p < 0.05 and ^∗∗ p < 0.01 (Fisher's LSD after ANOVA).