Research Articles, Neurobiology of Disease

Cnksr2 Loss in Mice Leads to Increased Neural Activity and Behavioral Phenotypes of Epilepsy-Aphasia Syndrome

Eda Erata, Yudong Gao, Alicia M. Purkey, Erik J. Soderblom, James O. McNamara and Scott H. Soderling
Journal of Neuroscience 17 November 2021, 41 (46) 9633-9649; https://doi.org/10.1523/JNEUROSCI.0650-21.2021

Article Figures & Data

Figures

  • Figure 1.
    Figure 1.

    Generation of Cnksr2-/Y mice. A, Schematic diagram of strategy to generate Cnksr2-/Y mice. A targeting vector was designed to disrupt the Cnksr2 gene via homologous recombination in mouse embryonic stem cells, where two loxP sites were inserted upstream and downstream of exon 2. The resulting floxed line was crossed to FLP deleter line to remove the Neo cassette and establish germline transmission. Next, the founder line was crossed to CMV-Cre generate Cnksr2-/Y mice. B, Left, Schematic of PCR strategy to detect WT and KO alleles. Arrows indicate primers used for detection. Right, Representative PCR genotyping results. C, Left, Representative image of mice of each genotype. Right, Graphs of body weight of adult (P60-P75) Cnksr2+/Y and Cnksr2-/Y mice. D, Schematic showing three different peptides targeted for PRM LC-MS assay to quantify Cnksr2 protein levels. ITHQELEDLGVSR is encoded by exon 2, INMLTAGYAER by exon 18, and CHLQDHYGPYPLAESER by exon 20. E, Depletion of Cnksr2 in Cnksr2-/Y mice. PRM LC-MS assay to quantify relative protein expression levels across WT (left) and KO (right) animals. PRM chromatograms of the example ITHQELEDLGVSR peptide (m/z 499.5950 3+ precursor) for the four most abundant product ions from each mouse are shown. F, Representative mass spectrum of ITHQELEDLGVSR following higher-energy collisional dissociation fragmentation. G, Protein abundance of Cnksr2 and control proteins Bsn (Bassoon), Myh10 (Myosin-10), Sptan1 (Spectrin α chain), Plec (Plectin), and Cltc (Clathrin heavy chain 1) in Cnksr2+/Y and Cnksr2-/Y synaptosomes. Protein abundance was calculated by averaging the abundance of all three peptides targeted for all proteins. Cnksr2 protein was significantly depleted in Cnksr2 KO compared with the WT control. No differences were observed in control proteins: ****p < 0.0001 (two-way ANOVA with Bonferroni's multiple comparisons). n = 3 animals per group. Data were normalized by transforming the WT mean value into 100% and represent mean ± SEM. See Extended Data Table 1-1. H, Left, Representative images of NeuN (magenta) staining in Cnksr2+/Y and Cnksr2-/Y CA1 coronal slices. Scale bar, 20 μm. Right, Quantification of NeuN-positive cell numbers in CA1 and ACC reveals no significant differences in total the number of cells between genotypes. p > 0.05 (Student's t test). Three WT and 3 KO animals were used.

  • Figure 2.
    Figure 2.

    Expression of Cnksr2. A, Top, RNAscope assay used to target and detect mouse Cnksr2 mRNA in the brain. Bottom, Control slices incubated with positive control probes (PPIB) and negative control (dapB). B, Cnksr2 mRNA detection in sagittal sections (10 μm in thickness) from WT mice at ages P0, P12, and P30. Cnksr2 mRNA expression can be seen in cortex, hippocampus, striatum, and cerebellum. Scale bar, 1000 μm.

  • Figure 3.
    Figure 3.

    Cnksr2 loss leads to increased spontaneous firing and bursting rates of cortical neurons in vitro. A, Experimental design to record spontaneous local neural activity using a MEA. Cultured cortical neurons were prepared from Cnksr2+/Y and Cnksr2-/Y neonatal pups at P0, and spontaneous neuronal activity was recorded at DIV6, DIV8, DIV10, and DIV12. Three Cnksr2+/Y and 3 Cnksr2-/Y mice were used for three MEA plates. Figure created with www.BioRender.com. B, Representative raster plots showing spontaneous activity recorded from a Cnksr2+/Y (right) or Cnksr2-/Y (left) well on DIV8. C, Graph of spontaneous mean firing rates. Cnksr2-/Y neurons displayed a significantly increased mean firing rate as early as DIV6 and up to DIV12 (DIV6: *p = 0.02; DIV8: **p = 0.001; DIV8: ****p < 0.0001; DIV12: ***p = 0.0009; Mann–Whitney test). n = 3 per genotype. D, Graph of burst frequencies. Burst frequency in Cnksr2-/Y neurons compared with the WT control was also increased (DIV6: **p = 0.0027; DIV8: **p = 0.0010; DIV10: ****p < 0.0001; DIV12: *** p = 0.0008; Mann–Whitney test). n = 3 per genotype. Data are mean ± SEM.

  • Figure 4.
    Figure 4.

    Cnksr2-/Y mice exhibit abnormal EEGs with epileptiform discharges. For handling-induced tonic-clonic seizure example, see also Movie 1. A, Schematic of 2 EEG/1 EMG recording area for freely moving mice. B, Graph of EMG full-spectrum power. No significant difference in average EMG power between genotypes was observed (p > 0.05; unpaired t test). Data were normalized by transforming the WT mean value into 100% and represent mean ± SEM. C, Number of electrographic seizure events detected per day plotted for Cnksr2+/Y and Cnksr2-/Y mice. Five WT and 5 KO mice were used for the recordings, and each mouse was recorded for 1 or 2 sessions. A total of 35 events were detected in 5 KO (7 of 9 sessions) mice, and 0 events were detected in 5 WT mice (0 of 9 sessions). **p = 0.0011 (Fisher's exact test, one-tailed). D, Number of spike-wave discharges per hour quantified for Cnksr2+/Y and Cnksr2-/Y mice. Spike-wave complexes lasting >1 s and separated by <0.5 s were quantified and reported. Number of spike-wave complexes seen in EEGs were significantly increased in Cnksr2-/Y mice. ****p < 0.0001 (Mann–Whitney test). E, Representative EEG1 traces. Top, WT baseline EEGs. Bottom, Traces of electrographic seizures detected in KO mice. F, Baseline EEG-1 and EEG-2 electrode traces for ko-1 before the detected event. G, Electrographic seizures detected in EEG-1 for ko-1. H, Electrographic seizures detected in EEG-1 for ko-3. I, Baseline EEG-1 and EEG-2 electrode traces for ko-3 after the detected event. J, Identification of vigilance states in which electrographic seizure events occur in Cnksr2-/Y mouse. Nineteen events during NREMS, 12 events during awake/REMS, and 4 events during transition state between awake/REMS and NREMS were identified.

  • Figure 5.
    Figure 5.

    Loss of Cnksr2 causes increased anxiety and impaired reversal learning. A, Open field testing data for time in center, vertical activity, and total distance moved. Cnksr2-/Y mice spent significantly less time (*p = 0.013; unpaired t test) in the center area but did not show any difference in total distance traveled (p > 0.05; unpaired t test). Cnksr2-/Y mice also had decreased vertical activity (**p = 0.001; unpaired t test). Representative heat map shows locomotion traces for both Cnksr2+/Y and Cnksr2-/Y during 1 h exploration session. B, Elevated zero maze data. The percentage of time spent in the open areas of the elevated zero maze and the total distance traveled is shown for each genotype. Cnksr2-/Y mice spent significantly less time in the maze's open areas than Cnksr2+/Y mice (**p = 0.004; unpaired t test). Cnks2+/Y and Cnksr2-/Y mice did not show any differences in the total distance traveled (p > 0.05; unpaired t test). Data are mean ± SEM. Representative locomotion traces during testing session for each genotype are shown. C, Morris Water Maze testing data. Top, Schematic for the experimental plan and a representative image of quadrants within the pool. Bottom, No difference in swim velocities was found between genotypes (p > 0.05; unpaired t test). Data are mean ± SEM. Mice were tested in two daily sessions of four trials each for both acquisition and reversal phases. D, Acquisition phase of the water maze test. Top, Representative heat map traces of Cnksr2+/Y and Cnksr2-/Y mice during a single trial on day 3 of acquisition. Bottom, Graph of session averages ± SEM is represented. On the first day of the acquisition phase, Cnksr2-/Y mice spent significantly more time to reach the platform: ***p = 0.0006 (two-way ANOVA with Bonferroni's multiple comparisons). Cnksr2-/Y mice performed similarly to Cnksr2+/Y mice for days 2-8. E, Reversal phase. Top, Representative single-trial heat map traces of Cnksr2+/Y and Cnksr2-/Y mice during a single trial on day 3 of reversal. Bottom, Graph of session averages ± SEM is represented. During reversal testing, Cnksr2-/Y mice spent significantly more time to reach the platform on the first 3 d of testing: **p < 0.01; ***p < 0.001 (two-way ANOVA with Bonferroni's multiple comparisons). F, Reversal probe trials. Graphs of swim times for Cnksr2+/Y and Cnksr2-/Y within each quadrant on day 2 (top) and day 8 (bottom) are shown. On day 8, Cnksr2+/Y mice spent significantly more time in the target quadrant than the nontarget quadrants: p < 0.05 (two-way ANOVA with Sidak's multiple comparisons). For Cnksr2-/Y, the difference was not significant.

  • Figure 6.
    Figure 6.

    Behavioral characterization of Cnksr2 KO mice. A, Y-Maze spontaneous alternation test to evaluate working memory. No significant difference in % alternation and number of entries between genotypes: p > 0.05 (unpaired t test). B, Novel object recognition to evaluate episodic-like memory. No significant difference in short-term memory (STM) or long-term memory (LTM) preference index: p > 0.05 (unpaired t test). C, Object memory load testing to evaluate complex episodic-like memory. Top, Views of the testing arena in Trials 1 and 7. Bottom, No significant difference between genotypes in preference index across Trials 2-7: p > 0.05 (unpaired t test). D, Rotarod testing. No significant difference in latency to fall in accelerating rotarod and steady speed rotarod between genotypes: p > 0.05 (two-way ANOVA, multiple comparisons with Bonferroni correction).

  • Figure 7.
    Figure 7.

    Cnksr2-/Y mice show USV deficits but not impairment of social interaction behavior. A, Schematic of experimental design. Pup vocalizations were recorded during maternal separation on P5. Adult male vocalizations were recorded during courtship behavior. B, Graphs of percent of vocal animals per genotype for pup and adult assays. No significant difference was observed in the percent of vocal pups between genotypes. In contrast, a significant decrease in percent vocal adults was observed in Cnksr2-/Y mice compared with WT controls: ****p < 0.0001 (unpaired t test). C, Top, Example pup spectrogram patterns of USVs. Bottom, Graphs of calls per min as well as average duration and frequency for each genotype. No significant difference was observed between genotypes in the number of calls per min (p > 0.05, unpaired t test). Cnksr2-/Y pups showed decreased average call duration (*p = 0.038; unpaired t test) and increased call frequency (*p = 0.016; unpaired t test). D, Top, Example spectrogram pattern of USVs in adult male mice during courtship. Bottom, Graphs of calls per min, average call duration, and social preference in the three-chamber sociability test for each genotype. Both numbers of calls per min (***p = 0.0006; Mann–Whitney test) and average call duration (***p = 0.0002; Mann–Whitney test) were significantly decreased in Cnksr2-/Y mice; however, there was no significant difference in sociability index demonstrated by three-chamber sociability test between Cnksr2+/Y and Cnksr2-/Y mice (p > 0.05; unpaired t test). Data are mean ± SEM.

  • Figure 8.
    Figure 8.

    Cnksr2 is localized to the postsynapse. A, Schematic for HiUGE labeling of endogenous Cnksr2. B, Left, Genomic PCR detection of smFP-V5 integration at the Cnksr2 locus. Right, Western blot detection of the Cnksr2-smFP-V5 fusion protein at the expected molecular mass (∼160 kDa). IP, Immunoprecipitation enrichment with V5-trap beads. C, Top, Knock-in expression of Cnksr2-smFP-V5 in cultured neurons (green) and DAPI (blue) is shown. Scale bars, 40 μm. Bottom left, Higher-magnification view of the boxed region shows colocalization (arrowheads) of Cnksr2-smFP-V5 immunoreactivity with Homer1 immunosignal (red). Scale bar, 2 μm. Bottom right, Higher-magnification view of the boxed region shows colocalization (arrowheads) of Cnksr2-smFP-V5 immunoreactivity with Vglut1 immunosignal (red). Scale bar, 2 μm. D, Top, Knock-in expression of Cnksr2-smFP-V5 in cultured neurons (green) and DAPI (blue). Scale bars, 40 μm. Bottom left, Higher-magnification view of the boxed region shows colocalization (arrowheads) of Cnksr2-smFP-V5 immunoreactivity with Gephyrin immunosignal (red). Scale bar, 2 μm. Bottom right, Higher-magnification view of the boxed region shows colocalization (arrowheads) of Cnksr2-smFP-V5 immunoreactivity with VGAT immunosignal (red). Scale bar, 2 μm. E, Graph represents colocalization ratios for Cnksr2 puncta with Homer1 (mean value 0.45) and Gephyrin (mean value 0.13). Colocalization ratios were calculated as the number of Cnksr2 puncta colocalized with Homer1 or Gephryin divided by total number of Cnksr2 puncta. Two or three dendrites were imaged from eight or nine neurons from multiple coverslips.

  • Figure 9.
    Figure 9.

    Cnksr2 as a scaffold protein and TMT proteomics workflow. A, Domain structure of Cnksr2. B, Cnksr2 interactor proteins. Curated from BioGRID and STRING databases. C, Workflow for differential protein expression analysis using TMT11plex multiplexed tagging with fractionated LC-MS/MS. Figure created with www.BioRender.com.

  • Figure 10.
    Figure 10.

    The loss of Cnksr2 results in changes to the synaptic proteome. A, Network of dysregulated proteins in Cnksr2-/Y mice; 109 proteins were differentially expressed in synaptosomes between Cnksr2-/Y and Cnksr2+/Y mice (two-tailed heteroscedastic t test on log2-transformed data p < 0.05, n = 4 for Cnksr2+/Y, n total = 4 for Cnksr2-/Y mice). See (Extended Data Table 1-1). B, Clustergram topology of proteins (red) in selected functional categories. C, Dysregulated proteins implicated in patient phenotypes of seizures, intellectual disability, and language delay/loss.

Tables

  • Table 1.

    Summary of clinical reports on CNKSR2 patients

    Houge et al., 2012Vaags et al., 2014Vaags et al., 2014Aypar et al., 2015Damiano et al., 2017Sun et al., 2018Bonardi et al., 2020Daoqi et al., 2020
    Number of patients15312141
    Age(s)58/12/13/6/856/58/62712/1885/21/12/95.5
    CNKSR2 variant(s)Deletion at Xp22.12Deletion at Xp22.12Frameshift mutation p.D152RfsX8Deletion at Xp22.12Nonsense mutation p.Arg712*Nonsense mutation p.Arg729*p.Glu675Glyfs*41
    p.T83Kfs*30
    p.Tyr153Serfs*5
    Deletion at Xp22.12    		Deletion of exon 14-17
    Intellectual disabilityMild to moderate 1/15/53/31/13/31/1Mild/moderate
    2/4    		1/1
    Epilepsy/seizures1/14/53/31/12/21/14/41/1
    CSWSNR5/5NRNR2/21/14/41/1
    ADHD1/15/53/3NR2/21/12/41/1
    Language delay/loss1/15/53/30/12/21/14/41/1

    • Clinical studies identifying CNKSR2 variants in patients. NR, Not reported.

Movies

  • Movie 1.

    Example video showing tonic-clonic seizure in Cnksr2 KO mouse during cage change.

Extended Data

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Cnksr2 Loss in Mice Leads to Increased Neural Activity and Behavioral Phenotypes of Epilepsy-Aphasia Syndrome
Eda Erata, Yudong Gao, Alicia M. Purkey, Erik J. Soderblom, James O. McNamara, Scott H. Soderling
Journal of Neuroscience 17 November 2021, 41 (46) 9633-9649; DOI: 10.1523/JNEUROSCI.0650-21.2021
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