Sex differences in ferroptosis-related vulnerability to autism-like deficits in the adolescent medial prefrontal cortex following embryonic valproic acid exposure

Abstract

Prenatal valproic acid (VPA) exposure is an important environmental risk factor for autism spectrum disorder (ASD), but the mechanisms underlying VPA-induced ASD-like phenotypes remain unclear. Emerging evidence implicates neuronal ferroptosis and neurodevelopmental disturbances, yet whether these alterations vary across developmental stages and between sexes is unknown. Adolescent male and female C57BL/6 mice exposed to VPA at embryonic day 12.5 (E12.5) or postnatal day 14 (PND14) were used to examine behavioral and neurobiological effects of VPA exposure across developmental stages and between sexes. Sociability and anxiety-like behaviors were assessed using the three-chamber social interaction test (3-CT) and open field test (OFT), respectively. Histological alterations in the medial prefrontal cortex (mPFC), hippocampus, and striatum were examined using hematoxylin-eosin (HE) and Nissl staining. Ferroptosis-related changes were evaluated using biochemical assays, immunofluorescence, transmission electron microscopy and Western blotting. In male mice, the effects of prenatal and early postnatal VPA exposure were compared to assess whether similar behavioral and ferroptosis-related alterations were observed across developmental windows. Both male and female adolescent offspring exposed to VPA exhibited social deficits and anxiety-like behaviors, with a more pronounced behavioral deficits in males, particularly in social preference and sociability. Histological analysis revealed variable degrees of neuronal abnormalities in the mPFC and hippocampus of VPA-exposed offspring, with the most prominent alterations observed in the mPFC. These histological changes were observed in both male and female offspring. Ferroptosis-related molecular changes were most evident in the mPFC and were accompanied by mitochondrial abnormalities and vacuolization. These alterations exhibited a distinct pattern of sex differences, with more pronounced changes observed in males than in females. Additionally, in male offspring, prenatal and early postnatal VPA exposure were associated with similar patterns of behavioral abnormalities and ferroptosis-related changes in the mPFC. This study suggests that prenatal VPA exposure induces ferroptosis-related abnormalities which could be linked to ASD-like behavioral and functional impairments, with more pronounced changes observed in male offspring. Among the brain regions examined, the mPFC showed the most prominent neurohistological and ferroptosis-related alterations. In male mice, prenatal and early postnatal VPA exposure were associated with similar patterns of ferroptosis-related molecular alterations, suggesting that ferroptosis-assiociated alterations may represent a shared molecular feature of VPA-induced neurodevelopmental abnormalities across different developmental windows. Autism is diagnosed more often in boys than in girls, but the biological reasons for this sex difference remain unclear. Valproic acid (VPA) is used to treat epilepsy and certain mood disorders, although prenatal exposure has been associated with an increased risk of autism-related developmental impairments. In our study, we explored whether VPA affects the developing brain differently in male and female mice. We used a well-established mouse model in which animals were exposed to VPA before birth. When the mice reached adolescence, we examined their social behavior, activity, and anxiety-like behavior. We also looked at several brain areas, including the medial prefrontal cortex (mPFC), which has been implicated in social behavior, emotional processing, and decision-making. We then compared male mice exposed to VPA before birth with male mice exposed during early life. Our findings showed that VPA exposure reduced social behavior and increased anxiety-like behavior in both male and female mice, with sex-related differences in the magnitude of these behavioral alterations. The clearest brain changes were found in the mPFC, where neuronal alterations were observed in both male and female mice. We also found signs of iron-related cell stress, known as ferroptosis, particularly in the mPFC. Similar patterns of behavioral and ferroptosis-related changes were observed in male mice following exposure before birth or during early life. Overall, our findings suggest that ferroptosis-related changes in the mPFC are associated with the more pronounced autism-like behavioral changes observed in male mice following VPA exposure. Prenatal VPA exposure induced social deficits and anxiety-like behaviors in adolescent mice, with significant differences observed between sexes. Prenatal VPA exposure was associated with region-specific neuronal alterations, with the most prominent changes observed in the mPFC. Prenatal VPA exposure was associated with ferroptosis-related alterations across the mPFC, hippocampus, and striatum, with sex-related differences most evident in the mPFC. Prenatal and early postnatal VPA exposure were associated with similar patterns of behavioral deficits and mPFC ferroptosis-related alterations in male mice. These findings suggest that ferroptosis-related alterations in the mPFC may represent a shared molecular feature associated with VPA-induced neurodevelopmental abnormalities across the developmental exposure windows. Prenatal VPA exposure induced social deficits and anxiety-like behaviors in adolescent mice, with significant differences observed between sexes. Prenatal VPA exposure was associated with region-specific neuronal alterations, with the most prominent changes observed in the mPFC. Prenatal VPA exposure was associated with ferroptosis-related alterations across the mPFC, hippocampus, and striatum, with sex-related differences most evident in the mPFC. Prenatal and early postnatal VPA exposure were associated with similar patterns of behavioral deficits and mPFC ferroptosis-related alterations in male mice. These findings suggest that ferroptosis-related alterations in the mPFC may represent a shared molecular feature associated with VPA-induced neurodevelopmental abnormalities across the developmental exposure windows.