Gestational immune activation and Tsc2 haploinsufficiency cooperate to disrupt fetal survival and may perturb social behavior in adult mice

Abstract

Approximately 40-50% of individuals affected by tuberous sclerosis (TSC) develop autism spectrum disorders (ASDs). One possible explanation for this partial penetrance is an interaction between TSC gene mutations and other risk factors such as gestational immune activation. In this study, we report the interactive effects of these two ASD risk factors in a mouse model of TSC. Combined, but not single, exposure had adverse effects on intrauterine survival. Additionally, provisional results suggest that these factors synergize to disrupt social approach behavior in adult mice. Moreover, studies in human populations are consistent with an interaction between high seasonal flu activity in late gestation and TSC mutations in ASD. Taken together, our studies raise the possibility of a gene × environment interaction between heterozygous TSC gene mutations and gestational immune activation in the pathogenesis of TSC-related ASD.

Fig. 1

Percentage of pregnancies lost in Poly I:C-injected and saline-treated animals. The vast majority of saline-injected females carried pregnancy to term; in contrast, a considerable portion of pregnancies was aborted as a consequence of Poly I:C injection. The table shows the proportion of Poly I:C-related lost pregnancies either associated with fetal resorption (abdominal collapse without abortion of dead fetuses) or frank abortion (premature discharge of dead fetuses).

Fig. 2

Interactive effects of Tsc2 haploinsufficiency and gestational Poly I:C on intrauterine survival and adult social approach behavior. (a) Graph shows the number of pups born in surviving litters, plotted by genotype (Tsc2^+/− and WT) and treatment (Poly I:C and saline). Fewer Tsc2^+/− pups were born to Poly I:C-injected females compared to saline-injected controls. (b) Graph shows the time (s) spent actively exploring the social cage and the empty cage (sniffing, rearing), as determined by an experienced observer. WT/saline, Tsc2^+/−/saline and WT/Poly I:C mice spent significantly more time exploring the social cage than the empty cage, demonstrating normal social approach behavior. Tsc2^+/−/Poly I:C mice, in contrast, did not spent more time exploring the social cage than the empty cage. (c,d,e) These graphs show the time spent exploring (c), the number of exploratory episodes (d) and the average duration of exploratory episodes (e) during the social approach behavioral task as quantified with an automated system. While WT/saline, Tsc2^+/−/saline and WT/Poly I:C mice tended to show higher values for the social cage than the empty cage, this was not the case for Tsc2^+/−/Poly I:C mice. ** P < 0.01, * P < 0.05, n.s. P > 0.05. Data represent means +/− S.E.M.

Fig. 3

(a) Behavioral testing, using the open field, revealed no significant effect of Tsc2 haploinsufficiency or gestational Poly I:C on ambulatory behavior, demonstrating normal locomotion in Tsc2^+/−/Poly I:C mice. (b) Ambulatory distance in the center zone did also not differ between groups, suggesting normal levels of anxiety-related behaviors in Tsc2^+/−/Poly I:C mice. (c) The graph shows the latency (min) to retrieve a buried food item in an olfactory sensitivity test. There was no significant difference with respect to latencies across the experimental groups, suggesting that olfactory dysfunction did not account for the behavioral impairments in Tsc2^+/−/Poly I:C mice. Data represent means +/− S.E.M.

Fig. 4

Advanced paternal age confers autism susceptibility and also is a risk factor for other neurodevelopmental disorders. To combine Tsc2 haploinsufficiency with advanced paternal age in mice, we bred old Tsc2^+/− males with WT females and generated controls by crossing young Tsc2^+/− males with WT females and assessed social behavior in the adult offspring. Tsc2^+/− and WT offspring of both young and old fathers showed normal social approach behavior. Plotted is the time (s) test subjects spent exploring either a cage containing a conspecific (social cage) or an empty cage. * P < 0.05, ** P > 0.01. Data represent means +/− S.E.M.