EFFECTS OF BEHAVIORAL THERAPY ON MOUSE MODEL OF ASD ASSOCIATED WITH TUBEROUS SCLEROSIS COMPLEX AND DNA METHYLATION ANALYSIS
Authors: Kotajima *Hiroko , Sato Atsushi , Kasai Shinya , Hagino Yoko , Tanaka Miho , Takamatsu Yukio , Nishito Yoshimasa , Uchino Shigeo , Ikeda Kazutaka 
Published: February 2025

Abstract

Background
Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impaired social interaction. Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder and approximately half of the patients with TSC are diagnosed with ASD. Behavioral therapy has been conducted to acquire social communication skills in children with ASD, and when started early, it facilitates them to adapt to social life. However, the mechanism of improvement in behavioral therapy is unclear. Peer intervention programs have been reported to improve social-communicative behaviors in patients with ASD. Social deficits in model mice for ASD were improved when living with wild-type mice in the same cage (1). We focused on epigenetics as one of the improvement mechanisms of behavioral therapy. In children with anxiety disorders, the DNA methylation rate increased in the group that responded to behavioral therapy treatment (2). These results suggest that behavioral therapy affects DNA methylation.

Aim and Objectives
We aim to investigate the effects of ASD-like behavior of peer rearing of Tsc1+/- (heterozygous defects in Tsc1) and wild-type mice and analyze DNA methylation in the mouse whole brain.

Methods
At 3 weeks of age, the juveniles were weaned and reared with age-matched cage mates, two mice per cage. Each group consisted of a) 2 wild-type mice (ww-w); b) 1 wild- type and 1 Tsc1+/- mice (wh-w, wh-h); c) 2 Tsc1+/- mice (hh-h). Peer rearing was conducted from 3 weeks of age to the end of all behavioral tests. After completion of all behavioral tests, whole mouse brains were collected and analyzed genome-wide DNA methylation using Mouse CpG Island Microarray. All of the animal experiments were performed under the Guidelines for the Care of Laboratory Animals of the Tokyo Metropolitan Institute of Medical Science.

Results
While hh-h showed a decrease in social interaction time compared with ww-w, wh-h showed a significant increase in social interaction time compared with hh-h. This result showed that social deficits in Tsc1+/- mice were improved when living with wild-type mice. We are also analyzing social interaction in the cage during the peer-rearing period to determine what behaviors lead to improvement of social deficit in Tsc1+/- mice. DNA methylation patterns were calculated, suggesting that methylation patterns may differ depending on genotype and peer rearing.
Furthermore, we analyzed the ratio of DNA hyper/hypo-methylation. 9651 genes were altered when comparing ww-w and hh-h. 6173 genes were hypermethylation and 3478 genes were hypomethylation. 5470 genes were altered when comparing wh-h and hh-h. 3395 genes were hypermethylation and 2075 genes were hypomethylation. These results suggest that DNA methylation contributes to the improvement of social deficits in ASD. We are currently analyzing methylated genes to distinguish between genetic factors (genes affected by the mouse genotype) and environmental factors (genes affected by the genotype of cohabiting mice).

Discussion and Conclusion
These results suggest that early peer rearing with wild-type mice improves social impairment in Tsc1+/- mice and that DNA methylation is one of the mechanisms involved.

References
1. Yang et al., 2011. Social peers rescue autism-relevant sociability deficits in adolescent mice. Autism Res. doi: 10.1002/aur.163.
2. Roberts et al., 2014. Serotonin transporter [corrected] methylation and response to cognitive behaviour therapy in children with anxiety disorders. Transl Psychiatry. doi: 10.1038/tp.2014.83.