microRNA as a Maternal Marker for Prenatal Stress-Associated ASD, Evidence from a Murine Model

Abstract

Autism Spectrum Disorder (ASD) has been associated with a complex interplay between genetic and environmental factors. Prenatal stress exposure has been identified as a possible risk factor, although most stress-exposed pregnancies do not result in ASD. The serotonin transporter (SERT) gene has been linked to stress reactivity, and the presence of the SERT short (S)-allele has been shown to mediate the association between maternal stress exposure and ASD. In a mouse model, we investigated the effects of prenatal stress exposure and maternal SERT genotype on offspring behavior and explored its association with maternal microRNA (miRNA) expression during pregnancy. Pregnant female mice were divided into four groups based on genotype (wildtype or SERT heterozygous knockout (Sert-het)) and the presence or absence of chronic variable stress (CVS) during pregnancy. Offspring behavior was assessed at 60 days old (PD60) using the three-chamber test, open field test, elevated plus-maze test, and marble-burying test. We found that the social preference index (SPI) of SERT-het/stress offspring was significantly lower than that of wildtype control offspring, indicating a reduced preference for social interaction on social approach, specifically for males. SERT-het/stress offspring also showed significantly more frequent grooming behavior compared to wildtype controls, specifically for males, suggesting elevated repetitive behavior. We profiled miRNA expression in maternal blood samples collected at embryonic day 21 (E21) and identified three miRNAs (mmu-miR-7684-3p, mmu-miR-5622-3p, mmu-miR-6900-3p) that were differentially expressed in the SERT-het/stress group compared to all other groups. These findings suggest that maternal SERT genotype and prenatal stress exposure interact to influence offspring behavior, and that maternal miRNA expression late in pregnancy may serve as a potential marker of a particular subtype of ASD pathogenesis.

Keywords: SERT; autism spectrum disorder; microRNA; serotonin; stress.

Figure 1

Social behavioral data obtained during the novel stranger versus novel object of the social approach task (A). A priori t-testing revealed that males born from HS dams spend less time interacting with the novel stranger than do wildtype controls as well as WS offspring. The HS males also spent significantly more time in the center chamber and interacting with the novel object than did the WN control. WS mice also spent significantly less time in the center chamber than the HS mice. (B). A priori t-testing showed HS females spent significantly less time interacting with the novel stranger than only the WN stressed mice. (C). Proportion of time spent with the novel stranger when compared to the novel object is quantified as the SPI in which male HS mice showed a lower preference for interacting with the novel stranger than did WN controls (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 for individual pairwise comparisons).

Figure 1

Social behavioral data obtained during the novel stranger versus novel object of the social approach task (A). A priori t-testing revealed that males born from HS dams spend less time interacting with the novel stranger than do wildtype controls as well as WS offspring. The HS males also spent significantly more time in the center chamber and interacting with the novel object than did the WN control. WS mice also spent significantly less time in the center chamber than the HS mice. (B). A priori t-testing showed HS females spent significantly less time interacting with the novel stranger than only the WN stressed mice. (C). Proportion of time spent with the novel stranger when compared to the novel object is quantified as the SPI in which male HS mice showed a lower preference for interacting with the novel stranger than did WN controls (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 for individual pairwise comparisons).

Figure 2

Social behavioral data obtained during the novel stranger versus familiar stranger portion of the social approach task. (A). There were no differences found amongst the male groups when comparing the amount of time spent interacting with the novel stranger, the familiar stranger, or time spent in the center chamber for the HS vs WN comparison. (B). A priori t-testing showed that prenatally stressed female offspring of HS dams spent less time interacting with the familiar stranger than did WN controls. (C). Females showed a main effect of genotype (p < 0.05), with SERT offspring revealing a higher in SNI than wildtypes. No differences were seen when measuring the proportional ratio of time spent with the novel stranger versus total time spent with either stranger in the male group. (* p < 0.05 for individual pairwise comparisons).

Figure 2

Social behavioral data obtained during the novel stranger versus familiar stranger portion of the social approach task. (A). There were no differences found amongst the male groups when comparing the amount of time spent interacting with the novel stranger, the familiar stranger, or time spent in the center chamber for the HS vs WN comparison. (B). A priori t-testing showed that prenatally stressed female offspring of HS dams spent less time interacting with the familiar stranger than did WN controls. (C). Females showed a main effect of genotype (p < 0.05), with SERT offspring revealing a higher in SNI than wildtypes. No differences were seen when measuring the proportional ratio of time spent with the novel stranger versus total time spent with either stranger in the male group. (* p < 0.05 for individual pairwise comparisons).

Figure 3

Measures used for anxiety-like behavior include the ratio of time spent in the open arm in comparison to time spent in the closed arm. There was a significant interaction between genotype and stress across the female OA ratios, driven primarily by a difference in stress in the WT offspring. (* p < 0.05, for individual pairwise comparisons).

Figure 4

(A). No differences were seen across all groups in movement measured by distance traveled during open-field testing. (B). HS males also spent significantly more time in the inner region of the open field apparatus than did the HN males. (C). No differences were seen in immobility across all conditions and sexes. (* p < 0.05, for individual pairwise comparisons).

Figure 4

(A). No differences were seen across all groups in movement measured by distance traveled during open-field testing. (B). HS males also spent significantly more time in the inner region of the open field apparatus than did the HN males. (C). No differences were seen in immobility across all conditions and sexes. (* p < 0.05, for individual pairwise comparisons).

Figure 5

Repetitive behaviors were measured in mice models using frequency of marbles buried, frequency of grooming behavior, and grooming duration. (A). Male WS controls buried significantly fewer marbles than did WN and significantly less than did HS mice. (B). HS males depicted a significantly higher frequency of self-grooming behavior than WN controls. (C). No significant differences were seen across the total grooming duration comparisons. (* p < 0.05, ** p < 0.01, *** p < 0.001, for individual pairwise comparisons).

Figure 5

Repetitive behaviors were measured in mice models using frequency of marbles buried, frequency of grooming behavior, and grooming duration. (A). Male WS controls buried significantly fewer marbles than did WN and significantly less than did HS mice. (B). HS males depicted a significantly higher frequency of self-grooming behavior than WN controls. (C). No significant differences were seen across the total grooming duration comparisons. (* p < 0.05, ** p < 0.01, *** p < 0.001, for individual pairwise comparisons).

Figure 6

Biological processes GO analysis. The top enriched functional categories based on Gene Ontology (GO) biological processes are depicted based on the 921 unique predicted projections of miRNAs identified at embryonic day 21. Dot size corresponds to the number of identified genes within a given class, and lower enrichment is shown in bluer shades, while increasing enrichment is more red.