Cadm1-Expressing Synapses on Purkinje Cell Dendrites Are Involved in Mouse Ultrasonic Vocalization Activity

Foxp2(R552H) knock-in (KI) mouse pups with a mutation related to human speech–language disorders exhibit poor development of cerebellar Purkinje cells and impaired ultrasonic vocalization (USV), a communication tool for mother-offspring interactions. Thus, human speech and mouse USV appear to have a Foxp2-mediated common molecular basis in the cerebellum. Mutations in the gene encoding the synaptic adhesion molecule CADM1 (RA175/Necl2/SynCAM1/Cadm1) have been identified in people with autism spectrum disorder (ASD) who have impaired speech and language. In the present study, we show that both Cadm1-deficient knockout (KO) pups and Foxp2(R552H) KI pups exhibit impaired USV and smaller cerebellums. Cadm1 was preferentially localized to the apical–distal portion of the dendritic arbor of Purkinje cells in the molecular layer of wild-type pups, and VGluT1 level decreased in the cerebellum of Cadm1 KO mice. In addition, we detected reduced immunoreactivity of Cadm1 and VGluT1 on the poorly developed dendritic arbor of Purkinje cells in the Foxp2(R552H) KI pups. However, Cadm1 mRNA expression was not altered in the Foxp2(R552H) KI pups. These results suggest that although the Foxp2 transcription factor does not target Cadm1, Cadm1 at the synapses of Purkinje cells and parallel fibers is necessary for USV function. The loss of Cadm1-expressing synapses on the dendrites of Purkinje cells may be associated with the USV impairment that Cadm1 KO and Foxp2(R552H) KI mice exhibit

A complex of synaptic adhesion molecule CADM1, a molecule related to autism spectrum disorder, with MUPP1 in the cerebellum

Mutations in the synaptic adhesion protein CADM1 (RA175/SynCAM1) are associated with autism spectrum disorder (ASD), a neurodevelopmental disorder of uncertain molecular origin. Cadm1-knock out (KO) mice exhibit smaller cerebella with decreased number of synapse of Purkinje cells and some ASD-like symptoms, including impaired ultrasonic vocalization. In this study, we examined the alteration of the Cadm1 synaptic complex in the mouse cerebellum at post-natal stages. The C-terminal peptide of Cadm1 associated with Mupp1 at PSD-95/Dlg/ZO-1 (PDZ)(1-5), a scaffold protein containing 13 PDZ domains, which interacted with gamma-aminobutyric acid type B receptor (GABBR)2 at PDZ13, but not with PSD-95. The GABBR2 was detected in a set of proteins interacting with Cadm1 C-terminal. Cadm1 colocalized with Mupp1 and GABBR2 on the dendrites of Purkinje cells in the molecular layers of the developing cerebellum and on the dendrites of hippocampal neurons cultured in vitro. These observations suggest that the Cadm1 synaptic receptor complex, including Mupp1-GABBR2, is located on the dendrites of Purkinje cells. The amount of GABBR2 protein, but not mRNA, was increased in the cerebella of Cadm1 KO mice, suggesting that lack of Cadm1 does not affect transcription of GABBR2, but may stabilize the Mupp1-GABBR2 complex; the Mupp1-GABBR2 interaction may be stabilized by conformational change in Mupp1 or association with other adhesion molecules and by anchorage to the post-synaptic membrane. Up-regulation of GABBR2 in the cerebellum in the absence of CADM1 may be associated with ASD pathogenesis

Abnormal cerebellum development of <i>Cadm1</i> KO.

<p>(A) Wild-type, heterozygote, and homozygous <i>Cadm1</i> KO mice. (P50) (B) The difference in mean weight between homozygous <i>Cadm1</i> KO mice and their wild-type littermates (five each) was significant at P10 and increased over the next 20 days (A, B); at P30, the mean weight of the homozygous <i>Cadm1</i> KO mice was 20–25% less than that of the wild-type mice. In addition, the brains of homozygous <i>Cadm1</i> KO mice were smaller (C, n = 22), and the cerebellums of homozygous <i>Cadm1</i> KO mice had an approximately 20% reduction in size and weight (D, n = 10). Bars in the graph indicate mean±standard error (SEM). Student's <i>t</i>-test (*<i>p</i><0.05). Bars in the pictures indicate 1 cm (A), 5 mm (C), and 0.75 mm (D), respectively.</p

Distribution of Cadm1 in the cerebellum (P11).

<p>The Cadm1 intensity preferentially distributed in an apical–distal dendritic portion. Wild-type (A, B, upper panel) and <i>Cadm1</i> KO mice (B, lower panel). Green, Cadm1. Red, Calbindin. Blue, Hoechst. Bars, 30 µm.</p

The influence of Cadm1 deficiency on the expression of synaptic proteins and Foxp2 in the cerebellum.

<p>(A) Immunoblot analysis of the influence of the deficiency in the cerebellums of <i>Cadm1</i> KO and wild-type pups (P10). An example of the typical immunoblotting results is shown. Values are mean±standard error (SEM). Student's <i>t</i>-test (*<i>p</i><0.05, **<i>p</i><0.01). Pups: n = 5. All experiments were performed three times. (B) RT-PCR analysis of the influence of the Cadm1 deficiency on the expression of <i>Foxp2</i> in the cerebellum of wild-type and <i>Cadm1</i> KO pups (P10). Values are mean±standard error (SEM). Pups: n = 5. All experiments were performed three times. A comparison showed no significant difference (Student's <i>t</i>-test; <i>p</i><0.05).</p

Analysis of ultrasonic vocalizations (USVs) of <i>Cadm1</i> KO mice (P8).

<p>(A) Real-time spectrography of the USVs by pups after separation from the dam. (B) Major vocalization patterns of <i>Cadm1</i> KO and wild-type pups. Wild-type vocalization was mainly whistle-type USVs, but <i>Cadm1</i> KO mice exhibited only a small number of click-type vocalizations. (C) The number of whistle-type USVs per min by pups. Vocalizations were recorded for 3 min. Experiments were done three times for 5 pups in each group, and an example of typical results is shown. Values are mean±standard error (SEM). Student's <i>t</i>-test (**<i>p</i><0.01).</p

Altered distribution of Cadm1 in the molecular layer of wild-type and <i>Foxp2</i>(R552H) KI mice (P11).

<p>Cadm1 preferentially distributed in the apical–distal dendritic portion in the molecular layer. The immunoreactivity of Cadm1 as well as that of Synaptophysin and VGluT1, pre-synaptic markers, was decreased in the molecular layer of the <i>Foxp2</i>(R552H) KI mice. Green, Calbindin. Red, Cadm1, VGluT1, Synaptophysin. Blue, Hoechst. Bar, 30 µm.</p