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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.