372. DEVELOPMENTAL INHIBITION OF PURKINJE CELLS CAUSES MALE-SPECIFIC ASD-LIKE PHENOTYPES - VULNERABLE MALE CEREBELLUM HYPOTHESIS

Abstract Background Autism spectrum disorder (ASD) is a common neurodevelopmental disease whose prevalence is a few percent in developed countries. Although the etiology of ASD has been controversial, a recent epidemiological study showed that perinatal cerebellar injury raised the ASD risk by 36-fold (Limperopoulos et al., 2007). Moreover, the number of Purkinje cells consistently decreased in the postmortem brains of ASD patients (Fatemi et al., 2012). The pathogenic role of cerebellar dysfunction was verified in animal models: Purkinje-specific deletion of Tsc1 reproduced the main three traits of ASD (Tsai et al., 2012), and pharmacological excitation of juvenile Purkinje cells caused lobule-specific ASD-like phenotypes in adults (Badura et al., 2018). However, most of these animal studies exclusively used male subjects. What accounts for the male-biased ASD prevalence in humans and its relationship with cerebellar dysfunction remains to be elucidated. Aims & Objectives Method We crossed L7-Cre heterozygous mice with LSL-hM4Di-mCitrine homozygous mice to achieve Purkinje-specific inhibitory DREADD (Designer receptors exclusively activated by designer drugs) expression. We orally administered 5 mg/kg CNO (clozapine N-oxide) to the pups from a) postnatal day 11 to 15, b) 11 to 20, c) 21 to 30. Behavioral experiments were performed after 8 weeks of age. For RNA-sequencing, we collected the cerebellum and frontal cortex of postnatal day 15 pups after five-day CNO administration. Importantly, we used animals of both sexes for all experiments. Results Purkinje-inhibited male mice exhibited mildly decreased social preference in a three-chamber test and fewer strategy changes in a reversal Barnes maze, while female mice showed no phenotypes. Total grooming time was unchanged in both sexes. As for common accompanying symptoms of ASD, male mice with developmental Purkinje cell inhibition showed impaired fine motor coordination and increased sleep duration. These phenotypes were mainly observed in the mice that received earlier intervention. Prepulse inhibition was altered in both males and females. RNA-sequencing revealed that developmental perturbation of the cerebellar activity changed the gene expression pattern not only in the cerebellum but also in the cortex in a sex-specific manner. We found that the expression of the Gabra2 and Pop4 (involved in activity-dependent histone H3.3 incorporation) genes was increased in the cerebellum and cortex only in males. Discussion & Conclusions We showed that transient inhibition of developing Purkinje cells could cause some ASD-like phenotypes, and male mice with earlier intervention were more susceptible. RNA-sequencing revealed male-specific differential gene expression in GABA signaling and activity-dependent histone incorporation. Interestingly, the gene expression in the cortex changed as well. As the same intervention resulted in sex-specific phenotypes, we hypothesize that the developing male cerebellum is vulnerable to perturbation, which may account for the biased sex ratio of ASD.