Haploinsufficiency for Pten and Serotonin transporter cooperatively influences brain size and social behavior

Abstract

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders that share deficits in sociability, communication, and restrictive and repetitive interests. ASD is likely polygenic in origin in most cases, but we presently lack an understanding of the relationships between ASD susceptibility genes and the neurobiological and behavioral phenotypes of ASD. Two genes that have been implicated as conferring susceptibility to ASD are PTEN and Serotonin transporter (SLC6A4). The PI3K and serotonin pathways, in which these genes respectively act, are both potential biomarkers for ASD diagnosis and treatment. Biochemical evidence exists for an interaction between these pathways; however, the relevance of this for the pathogenesis of ASD is unclear. We find that Pten haploinsufficient (Pten(+/-)) mice are macrocephalic, and this phenotype is exacerbated in Pten(+/-); Slc6a4(+/-) mice. Furthermore, female Pten(+/-) mice are impaired in social approach behavior, a phenotype that is exacerbated in female Pten(+/-); Slc6a4(+/-) mice. While increased brain size correlates with decreased sociability across these genotypes in females, within each genotype increased brain size correlates with increased sociability, suggesting that epigenetic influences interact with genetic factors in influencing the phenotype. These findings provide insight into an interaction between two ASD candidate genes during brain development and point toward the use of compound mutant mice to validate biomarkers for ASD against biological and behavioral phenotypes.

Fig. 1.

Macrocephaly in Pten^+/−; Slc6a4^+/− mice. (A) Representative dorsal-view images of brains from male Pten^+/+; Slc6a4^+/+, Pten^+/−; Slc6a4^+/+, Pten^+/+; Slc6a4^+/−, and Pten^+/−; Slc6a4^+/− mice. Brains were collected at 12 weeks of age. (B and C) As compared to wild-type controls, Pten and Slc6a4 haploinsufficient mice show a significant increase in brain mass. (B) Brain mass in females is significantly increased in Pten^+/−; Slc6a4^+/− mice as compared to Pten^+/+; Slc6a4^+/+, Pten^+/−; Slc6a4^+/+, and Pten^+/+; Slc6a4^+/− mice (F_3,42 = 20.6; P < 0.001). n = 10 Pten^+/+; Slc6a4^+/+, 10 Pten^+/−; Slc6a4^+/+, 10 Pten^+/+; Slc6a4^+/−, and 13 Pten^+/−; Slc6a4^+/− mice. (C) Brain mass in males is significantly increased in Pten^+/−; Slc6a4^+/− mice compared to Pten^+/+; Slc6a4^+/+, Pten^+/−; Slc6a4^+/+, and Pten^+/+; Slc6a4^+/− mice (F_3,33 = 30.0; P < 0.001). n = 6 Pten^+/+; Slc6a4^+/+, 6 Pten^+/−; Slc6a4^+/+, 11 Pten^+/+; Slc6a4^+/−, and 11 Pten^+/−; Slc6a4^+/− mice.*, P < 0.05; **, P < 0.01 [Tukey's honestly significant difference (HSD) test]. Ages were 8–12 weeks. Data are normalized to body mass to account for differences in body mass between animals.

Fig. 2.

Behavioral characterization of Pten^+/− mice. (A) Still images of the apparatus used to assay social approach behavior. A social stimulus mouse resides in an acrylic cage in chamber 1 and the acrylic cage in chamber 3 remains empty as a control. The subject mouse begins the assay in chamber 2, the assay is video recorded for 10 min, and the percentage of time spent in each chamber is then quantified. (B and C) Data showing percentage of time spent in a chamber containing a cage that holds a stimulus mouse (chamber 1), an empty chamber (chamber 2), or a chamber containing a cage with no stimulus mouse (chamber 3). All mice tested were 12 weeks of age. (B) In females, Pten^+/+ mice show a significant preference for chamber 1 over chamber 3, whereas this preference is not seen in Pten^+/− mice. (C) In males, both Pten^+/+ and Pten^+/− mice show a preference for chamber 1 over chamber 3. *, P < 0.05, ANOVA within-group comparison between chamber 1 and chamber 3. n = 17 males, 12 females for each genotype. Error bars indicate SEM. (D) Prepulse inhibition of the acoustic startle response in Pten^+/− mice. All mice tested were 12 weeks of age. As compared to Pten^+/+ mice, Pten^+/− mice have significant deficits in startle inhibition at prepulses 12 or 16 db above background. *, P < 0.05, ANOVA comparison between genotypes for given prepulse intensity. n = 12 mice (6 females) for each genotype.

Fig. 3.

Social behavior and prepulse inhibition in Pten and Slc6a4 haploinsufficient mice. (A) Social approach data for 8-week-old female Pten^+/+; Slc6a4^+/+ (n = 13), Pten^+/−; Slc6a4^+/+ (n = 13), Pten^+/+; Slc6a4^+/− (n = 11), and Pten^+/−; Slc6a4^+/− (n = 13) mice. *, P < 0.05, ANOVA within-group comparison between chamber 1 and chamber 3. Error bars indicate SEM. (B) Social approach data from A presented as approach–avoidance score for analysis across genotypes. Time spent with a social stimulus mouse in chamber 1 is significantly decreased in Pten^+/−; Slc6a4^+/− mice as compared to Pten^+/+; Slc6a4^+/+, Pten^+/−; Slc6a4^+/+, and Pten^+/+; Slc6a4^+/− mice (F_3,49 = 25.3; P < 0.001). *, P < 0.05; **, P < 0.01 (Tukey's HSD test). (C) Social approach and recognition data for 8-week-old male mice. During trial 1, the stimulus (located in chamber 1) and the subject mouse interacted for 10 min. Mice were then separated for 30 min. Trial 2 then took place, during which the subject and the stimulus mouse interacted for 5 min. Pten^+/+; Slc6a4^+/+ (n = 12), Pten^+/−; Slc6a4^+/+ (n = 10), Pten^+/+; Slc6a4^+/− (n = 8), and Pten^+/−; Slc6a4^+/− (n = 8) mice are shown. *, P < 0.05, ANOVA within-group comparison between chamber 1 and chamber 3. Error bars indicate SEM. (D) Prepulse inhibition of the acoustic startle response in 8-week-old Pten^+/−; Slc6a4^+/− mice. Pten^+/−; Slc6a4^+/+ and Pten^+/−; Slc6a4^+/− mice have significant deficits in startle inhibition at a prepulse 16 db above background (F_3,46 = 3.6; P < 0.05). *, P < 0.05 (Tukey's HSD test). n = 11 Pten^+/+; Slc6a4^+/+ (8 female), 10 Pten^+/−; Slc6a4^+/+ (7 female), 13 Pten^+/+; Slc6a4^+/− (7 female), and 13 Pten^+/−; Slc6a4^+/− (9 female) mice.

Fig. 4.

Correlation between brain mass and sociability across and within genotypes. (A) Plot of population means for brain mass (normalized to body mass) (X-axis) and social approach–avoidance scores (Y-axis) for 8-week-old female Pten^+/+; Slc6a4^+/+ (n = 7), Pten^+/−; Slc6a4^+/+ (n = 8), Pten^+/+; Slc6a4^+/− (n = 10), and Pten^+/−; Slc6a4^+/− (n = 11) mice. r = −0.98. (B–E) Individual subjects from A, arranged by genotype, plotted for brain mass (normalized to body mass) (X-axis) and social approach–avoidance scores (Y-axis). (B) Pten^+/+; Slc6a4^+/+: r = 0.77; P < 0.05 (r to P conversion). (C) Pten^+/−; Slc6a4^+/+: r = 0.70; P < 0.05. (D) Pten^+/+; Slc6a4^+/−: r = 0.60; P = 0.07. (E) Pten^+/−; Slc6a4^+/−: r = 0.65; P < 0.05. Group sizes are different from those reported in Fig. 3 A and B because not all animals assayed for social approach were measured for brain mass.