Loss of CDKL5 disrupts kinome profile and event-related potentials leading to autistic-like phenotypes in mice

Abstract

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in neurodevelopmental disorders including atypical Rett syndrome (RTT), autism spectrum disorders (ASDs), and early infantile epileptic encephalopathy. The biological function of CDKL5 and its role in the etiology of these disorders, however, remain unclear. Here we report the development of a unique knockout mouse model of CDKL5-related disorders and demonstrate that mice lacking CDKL5 show autistic-like deficits in social interaction, as well as impairments in motor control and fear memory. Neurophysiological recordings reveal alterations in event-related potentials (ERPs) similar to those observed in RTT and ASDs. Moreover, kinome profiling uncovers disruption of multiple signal transduction pathways, including the AKT-mammalian target of rapamycin (mTOR) cascade, upon Cdkl5 loss-of-function. These data demonstrate that CDKL5 regulates signal transduction pathways and mediates autistic-like phenotypes and together establish a causal role for Cdkl5 loss-of-function in neurodevelopmental disorders.

Fig. 1.

Generation of Cdkl5 knockout mice. (A) Targeting strategy. Three loxP sites and a neomycin positive selection cassette (Neo) were inserted surrounding the genomic locus of Cdkl5 exon 6 via homologous recombination. Upon Cre-directed recombination, both the Neo cassette and exon 6 were excised. (B) Schematic of CDKL5 protein in WT and knockout. The excision of exon 6 causes a reading frame shift, resulting in a TAA stop codon in the 5′ end of exon 7, leading to truncation of CDKL5 in its kinase domain (red). (C) PCR of genomic DNA using primers flanking exon 6. A 300-bp PCR product in Cdkl5^–/y mice indicates the absence of Cdkl5 exon 6. (D) Sequencing of cDNA generated from Cdkl5 mRNA. Excision of exon 6 in Cdkl5^–/y mice causes the reading frame, highlighted in black, to be shifted in Cdkl5^–/y mice, resulting in a premature stop codon (TAA, circled in red) at the 5′ end of exon 7. (E) Western blot probed with an antibody directed against CDKL5. Full-length CDKL5 protein is absent in Cdkl5^–/y mice.

Fig. 2.

Behavioral phenotyping of Cdkl5^–/y mice. (A) Sixty-minute locomotor assay, measured by infrared beam breaks in a home cage-like environment. Cdkl5^–/y mice (n = 19) display increased activity relative to wild type (WT, Cdkl5^+/y) littermates (n = 15). Two-way repeated measures (RM) ANOVA, P < 0.0001 (interaction). (B) Rotarod assay, measuring latency to fall from an accelerating rotating rod. Testing was performed five trials per day for 5 consecutive days. Latency to fall is decreased in Cdkl5^–/y mice (n = 18) relative to WT littermates (n = 15), indicating impaired motor coordination in Cdkl5^–/y mice. Two-way ANOVA, P < 0.01 (main effect of genotype). (C) Cdkl5^–/y mice (n = 14) spend more time in the open arms and less time in the closed arms of a zeromaze assay relative to WT littermates (n = 12), showing decreased anxiety. *P < 0.05, unpaired two-tailed Student t test. (D) Three-chambered social approach assay. Cdkl5^–/y mice (n = 17) spend less time in a social chamber containing a stimulus mouse (S) and more time in a nonsocial chamber containing a novel object (NS) relative to WT mice (n = 15). C, center. Two-way ANOVA with Bonferroni correction, P < 0.0001 (interaction); **P < 0.01, ***P < 0.001. (E) Cdkl5^–/y mice (n = 17) spend significantly more time sniffing a novel object (NS) and trend toward less time sniffing a stimulus mouse (S) relative to WT mice (n = 15). Two-way ANOVA with Bonferroni correction, P < 0.01 (interaction); *P < 0.05. (F) Cdkl5^–/y mice (n = 17) spend less time directly interacting with a freely moving stimulus mouse compared with WT littermates (n = 15). ***P < 0.001, unpaired two-tailed Student t test. (G) Cdkl5^–/y mice (n = 12) show impaired nesting behavior relative to WT littermates (n = 11) at 4–5 postnatal weeks. ***P < 0.001, unpaired two-tailed Student t test. (H) Fear conditioning paradigm, measuring time spent immobile. While Cdkl5^–/y mice (n = 14) freeze in response to a mild footshock similarly to WT littermates (postshock), they show decreased freezing upon return to the testing chamber (context) and upon hearing the testing tone (cue) relative to WT littermates (n = 14), demonstrating impaired learning and memory in Cdkl5^–/y mice. Two-way ANOVA with Bonferroni correction, P < 0.01 (interaction); *P < 0.05. All data are presented as mean ± SEM.

Fig. 3.

Cdkl5^–/y mice display impaired ERP waveform and decreased event-related power and phase locking. (A) Grand-average ERP waveform following presentation of 250 85-dB white noise stimuli with 4-s interstimulus intervals in adult WT (Cdkl5^+/y) (n = 9) and Cdkl5^–/y mice (n = 9). Traces represent mean amplitude ± SEM. Characteristic polarity peaks P1, N1, and P2 in WT are labeled. [Scale bar, 100 ms (horizontal) and 20 mV (vertical).] (B) Amplitude and (C) latency of ERP peaks. Bars represent mean ± SEM; **P < 0.01, *P < 0.05, unpaired two-tailed Student t test with Bonferroni correction. (D) Time–frequency plots showing changes in event-related power following an 85-dB auditory stimulus. Color represents mean power, where warmer colors correspond to increased power and cooler colors correspond to decreased power relative to prestimulus baseline. (E) Changes in event-related mean power averaged across δ (2–4 Hz), θ (4–8 Hz), α (8–12 Hz), β (12–30 Hz), γ_low (30–50 Hz), and γ_high (70–140 Hz) oscillation frequencies. (Scale bars, length of a single δ oscillation cycle.) Insets show power traces on an expanded timescale, denoted by the length of a single oscillation cycle. Traces represent mean amplitude ± SEM. (F) Time–frequency plots showing changes in event-related phase-locking factor (PLF) following an 85-dB auditory stimulus. Color represents PLF, where warmer colors correspond to a higher PLF or lower circular variance in EEG phase across trials. (G) Changes in event-related PLF averaged across frequencies described above. (Scale bars, length of a single oscillation cycle.) Insets show traces on an expanded timescale. Traces represent mean PLF ± SEM.

Fig. 4.

Altered kinome profile and disrupted AKT–mTOR signaling in Cdkl5^–/y mice. (A) Summary of changes in kinome profile in Cdkl5^–/y mice relative to WT littermates. Whole cell lysates from the cerebellum, striatum, somatosensory cortex, olfactory bulb, hippocampus, and brainstem of WT (n = 7) and Cdkl5^–/y mice (n = 8) were probed with antibodies raised against different phosphorylation motifs representing known S/T kinases. Western blots were quantified using the Odyssey Infrared Imaging system and fold change in phosphorylation level between Cdkl5^–/y and WT mice across the six brain regions are expressed as log₂ (phosphorylation level in WT over phosphorylation level in Cdkl5^–/y). Color scheme indicates relative degree of phosphorylation reduction in Cdkl5^–/y mice: purple, strong; blue, moderate; and green, mild. (B and C) Whole cell lysate probed with an antibody specific for an RXRXX(s/t) phosphorylation motif representing AKT kinase substrates (B) and an antibody specific for an LXRXX(s/t) phosphorylation motif representing AMPK kinase substrates (C) shows a marked decrease in phosphorylation profiles in Cdkl5^–/y mice (KO) relative to WT littermates in the striatum, somatosensory cortex (ss cortex), and hippocampus, but moderate decrease in the brainstem, consistent with regions of high and low CDKL5 expression, respectively. (D) AKT S473 and mTOR S2448 phosphorylation is reduced in whole brain lysate from Cdkl5^–/y mice (KO) related to WT mice, whereas total levels of AKT and mTOR are unchanged. (E and F) Quantification of reduced AKT S473 (E) and mTOR S2448 (F) phosphorylation in Cdkl5^–/y mice. Phosphorylated protein levels are normalized to GAPDH loading control and expressed relative to WT levels. *P < 0.05, unpaired two-tailed Student t test with Bonferroni correction. (G and H) Quantification of total AKT (G) and mTOR (H) in WT and Cdkl5^–/y mice. Protein levels are normalized to GAPDH loading control and expressed relative to WT levels. NS, not significant; unpaired two-tailed Student t test.