Prenatal BPA exposure perturbs RNA-binding protein-mediated splicing regulation and synaptogenesis in the developing...

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental condition characterized by social communication deficits, exhibiting a male bias in prevalence. Emerging evidence suggests that prenatal exposure to bisphenol A (BPA) may perturb neurodevelopmental trajectories relevant to ASD. While the cerebellum is increasingly recognized as a brain region implicated in ASD pathophysiology, the impact of gestational BPA exposure on its post-transcriptional alternative splicing machinery remains fundamentally undefined.

Here, we investigated sex-dependent effects of prenatal BPA exposure on the alternative splicing landscape of the neonatal rat cerebellum. We utilized RNA-seq to profile differential alternative splicing (DAS) events. Ingenuity Pathway Analysis (IPA) was used to predict biological functions and canonical pathways, and to construct the interactome network of DAS genes. To explore candidate upstream regulatory mechanisms, we performed in silico molecular docking and used high-resolution melting (HRM) qRT-PCR to validate selected splicing events. Furthermore, we assessed in vitro cellular phenotypes in primary cerebellar neurons by measuring MTS-based viability and Syn1/Psd95 puncta colocalization.

Prenatal BPA exposure was associated with widespread DAS in genes enriched for ASD-relevant pathways in the neonatal rat cerebellum. To our knowledge, this study is the first to report molecular docking analyses predicting favorable interactions between BPA and several candidate RNA-binding proteins (RBPs), including CPEB1, RALYL, HNRNPDL, and ACO1. Our findings support a model in which BPA may perturb RBP-associated splicing regulation, including altered splicing of chromatin regulators such as Ccar1 in males. These molecular and cellular findings were accompanied by sex-stratified differences in neuronal viability and synaptic puncta measurements. BPA exposure was associated with an increased MTS viability signal in male primary cerebellar neurons, together with significant reductions in Psd95 and Syn1 puncta density, whereas female neurons showed significantly increased synaptic puncta colocalization together with reduced viability.

In this study, we propose that prenatal BPA may be relevant to ASD-related neurodevelopmental pathways through sex-dependent changes in RBP-associated alternative splicing, including altered splicing of Ccar1 in males, together with distinct cellular outcomes. Together, these findings identify the developing cerebellum as a sensitive target of prenatal BPA exposure and highlight alternative splicing as a candidate pathway relevant to ASD biology.