Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and core autism-related deficits

Abstract

Although many genes predisposing to autism spectrum disorders (ASD) have been identified, the biological mechanism(s) remain unclear. Mouse models based on human disease-causing mutations provide the potential for understanding gene function and novel treatment development. Here, we characterize a mouse knockout of the Cntnap2 gene, which is strongly associated with ASD and allied neurodevelopmental disorders. Cntnap2(-/-) mice show deficits in the three core ASD behavioral domains, as well as hyperactivity and epileptic seizures, as have been reported in humans with CNTNAP2 mutations. Neuropathological and physiological analyses of these mice before the onset of seizures reveal neuronal migration abnormalities, reduced number of interneurons, and abnormal neuronal network activity. In addition, treatment with the FDA-approved drug risperidone ameliorates the targeted repetitive behaviors in the mutant mice. These data demonstrate a functional role for CNTNAP2 in brain development and provide a new tool for mechanistic and therapeutic research in ASD.

Figure 1. Cntnap2^−/− mice show epileptic seizures and abnormal EEG pattern

(A) Presence of reactive astrocytes in the hipocampal hilus (inset) of P180 but not P14 mutant mice without significant changes in neuronal density. GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei. Scale bar: 50 µm. GFAP quantification is shown as % of area occupied by reactive astrocytes. n=4 mice/genotype for each age. Data are presented as mean ± S.E.M. ***p<0.001 (B) EEG recording from mutant mice show abnormal spike discharges (arrows) after seizure onset. n=3 mice for each age. RF, right frontal; RP, right parietal. See also Table S1, Movie S1 and Figure S3C.

Figure 2. Cntnap2^−/− mice show neuronal migration abnormalities

(A) Presence of ectopic neurons in the corpus callosum of Cntnap2^−/− mice. NeuN, neuronal nuclei; CTX, cortex; STR, striatum. Scale bar: 20µm. (B) Expression of Cux1, a marker for upper layer projection neurons, in somatosensory cortex of WT and Cntnap2^−/− mice. Note the abnormal distribution of CUX1 positive cells in groups (arrowheads) and rows (arrow) in deep cortical layers of mutant mice. Scale bar: 50µm (C) Neuronal birth-dating analysis. BrdU injected at E16.5 was immunostained at P7. Note abnormal distribution of neurons in groups (arrowheads) and rows (arrow) in deep cortical layers of mutant animals. Scale bar: 50µm. Data are presented as mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001. n=3 mice/genotype for each age. See also Figure S2.

Figure 3. Reduced number of GABAergic interneurons in Cntnap2^−/− mice

(A) Network plot showing the top 300 connections within the Cntnap2 module (B) Expression of Gad1 at P14 shows a reduced number of GABAergic interneurons in somatosensory cortex of KO mice. Scale bar: 50µm (C) Expression of the interneuron markers PAVLB, CALB2, and NPY at P14. Scale bar: 50µm (D) Expression of the GABAergic interneurons PAVLB and NPY in striatum. Scale bar: 100µm. Interneuron quantification is shown as % of WT. *p<0.05, **p<0.01, ***p<0.001. For (B), (C), (D) and (E) n=4 mice/genotype. Data are presented as mean ± S.E.M. See also Table S2 and Figure S3.

Figure 4. Reduced neuronal synchronization in Cntnap2^−/− mice

(A) Representative images of the calcium (top) and astrocytic (bottom) signals of a 3 min movie stack. OGB-1, Oregon green-1; SR-101, sulforhodamine-101 (B) Correlation coefficient of neuronal firing for every pair of neurons over cell distance (C) Mean correlation coefficient over the distance range analyzed (240 µm) (D) Firing amplitude presented as the summed fluorescence change across all imaged cells in a 3 minute time window (E) Mean number of firing events per cell in a 3 minute time window. n=4 mice/genotype. Data are presented as mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001.

Figure 5. Cntnap2^−/− show motor stereotypic movements and behavioral inflexibility

(A-F) Morris water maze (MWM) test. (A-C) Learning, (D-F) Reversal learning (A) Learning curve as indicated by the latency to locate a hidden platform (up to 60s) during a 5 day training period. The average of 4 trials per day is presented (B) Probe test (the platform is removed) showing the percentage of time spent in each pool quadrant (in 60s). Note that both genotypes spend significantly more time in the target quadrant. TA, target; AR, adjacent right; AL, adjacent left; OP, opposite (C) Number of platform site crossings during the probe test (D) Learning curve for the reversal task of the MWM test showing the latency to locate the new platform (E) Probe test. Note that WT but not KO mice spend significantly more time in the target quadrant (F) Number of platform site crossings during the probe test (G) Number of no alternations in the T maze spontaneous alternation test (10 trials) (H) Time spent grooming over a 10 min period. *p<0.05, **p<0.01, ***p<0.001. n=10 mice/genotype. Data are presented as mean ± S.E.M. See also Table S3 and Figure S4.

Figure 6. Cntnap2^−/− mice show communication and social behavior abnormalities

(A) UsV. Number of calls from pups when separated from their mother at P3, P6, P9 and P12 (5 min) (B) Juvenile play. Time involved in social interaction, as well as repetitive behaviors (grooming and digging) in pairs of mice matched in genotype and sex at age P21 (10 min) (C) Three-chamber social interaction test. Time interacting with either an unfamiliar mouse or an inanimate object (empty cup) in 10 min (D) Nesting behavior. The nesting score represents the amount of nesting material used after a 16 hour period (1, poor; 5, good). n=10 mice/genotype. Data are presented as mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001. See also Figure S5.

Figure 7. Risperidone rescues hyperactivity and repetitive behavior/perseveration in Cntnap2^−/− mice

(A-B) Open field test. Distance travelled (A) and velocity (B) for vehicle (PBS) and drug treated WT and KO mice (20 min) (C) Nesting behavior. Risperidone improves the nesting score of Cntnap2^−/− mice (D) Hot plate. Risperidone did not have an effect in the hyper-reactivity to thermal stimuli (E) Grooming behavior is reduced in risperidone treated mutant mice (10 min) (F) Risperidone improved spontaneous alternation of mutant mice (G) Drug treatment did not have an effect in the three-chamber social interaction test. n=10 mice/genotype and treatment condition. Data are presented as mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001.