CADM1 Mutation Knock-in Mice As Mice Model of ASD Showing Abnormal Excitatory-Inhibitory Synaptic Balance

Background: Previously, we detected two missense mutations (Y251S, H246N) on Cell adhesion molecule1 (CADM1) in autism spectrum disorder (ASD) patients. CADM1 is expressed in the membrane of both presynaptic and postsynaptic neurons, and acts as synaptic adhesion protein. Although Cadm1-KO mice showed increased aggression and anxiety behavior, repetitive behavior was not observed. In primary mouse nerve cell culture induced each missense mutations of CADM1, inhibition of dendrites extension and aggregation of mutant proteins in the endoplasmic reticulum were observed. Endoplasmic reticulum (ER) stress is considered to be induced by the genetic mutation and caused neural dysfunction.

Objectives: We speculated the two mechanisms as Cadm1 pathogenicity; the loss of function and the gain of function caused by ER stress. To analyze these possibilities and contribution of CADM1 to ASD, we established CADM1 (Y251S) knock-in (KI) mice and analyzed.

Methods: For behavior analysis, social recognition test, foot print test and stereotyped behavior were performed. Western blotting for α-tubulin, PSD-95, vGlut1 and GABBR2, and fluorescence immunohistochemistry for synaptophysin, vGlut1, vGAT, GABBR2 and calbindin were performed focused in the cerebellum.

Results: A) Behavior analysis: Preference index of heterozygotes and homozygotes of Cadm1(Y251S)-KI mice were significantly decreased. Both heterozygotes and homozygotes showed abnormal social behavior.Foot print test did not detected significant difference between three groups.Cadm1 (Y251S) -KI mice showed repetitive jumping behavior considered to be stereotyped behavior. Frequency were, WT; 0% and 0% in male/female, Heterozygotes; 12.7% and 6.4%, Homozygotes: 4.3% and 0%. On randomly chosen ten mice observation for 2 hours, average numbers of jumping/min for each mouse were 0.08 in WT, 9.26 in heterozygotes and 0.98 in homozygotes.

B) Brain tissue analysis: In immunohistochemical staining of cerebellum, Synaptophysin, a marker for pre-synaptic vesicles, and vGlut1, an excitatory pre-synaptic marker, were not different between WT and homozygotes. vGAT, an inhibitory pre-synaptic marker and GABBR2, inhibitory post-synaptic marker were stained strongly in homozygotes. On Western blotting analysis, GABBR2 was also expressed strongly in homozygotes compared. Expression of vGlut1 and PSD95, a postsynaptic expression marker, were not different between WT and homozygotes.

Conclusions: In behavior analysis, social behavior was impaired in both heterozygotes and homozygotes of Cadm1(Y251S)-KI mice. Repetitive jumping in heterozygote male mice may reflect the higher frequency in male in ASD. Abnormal social behavior is common feature of Cadm1-KO and Cadm1(Y251S)-KI. However, characteristic stereotypic behavior of ASD patient was seen in Cadm1(Y251S) -KI mice but not in Cadm1 KO mice. CADM1 mutation was suggested to act as a Gain of function in ASD pathogenesis. Synaptic balance was shifted to the inhibitory in Cadm1 (Y251S)-KI mice. Addition to the report that inhibitory synapses were increased in KI mice that carries Nlgn3 (R451C) mutation identified in ASD patient. Synaptic balance shifted toward inhibitory is considered to be common mechanism of ASD. From these results, ASD was considered to be caused not only loss of function of mutant protein but also Gain of function induced by mutant protein, and candidate of the mechanisms were ER stress and imbalance of synaptic function.