Prenatal BPA exposure is associated with a sex-stratified TF–RBP–Ccar1 splicing regulatory network, purkinje cell layer alterations, and transcriptomic concordance with PTEN I135L cerebellar organoid models

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental condition characterized by social communication deficits and restricted, repetitive behaviors, exhibiting a consistent male bias in prevalence. Emerging evidence suggests that prenatal exposure to bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical, may perturb neurodevelopmental processes relevant to ASD. The cerebellum is increasingly recognized as an important brain region in ASD pathophysiology and is among the regions in which postmortem neuropathological abnormalities have been repeatedly reported. However, the effects of prenatal BPA exposure on cerebellar alternative splicing and its candidate upstream regulatory mechanisms remain incompletely understood. To investigate candidate upstream regulatory mechanisms for previously identified differentially alternatively spliced (DAS) genes, we integrated cross-dataset overlap analyses using Harmonizome TF-target resources, RBPDB, and oRNAment with molecular docking and RT-qPCR assessment of BPA-responsive RNA-binding protein (RBP) expression. Transcription factor occupancy at selected RBP gene promoters was assessed by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Purkinje cell layer cellular dynamics at PND1 were assessed using late-gestation BrdU labeling together with NeuN and Calb1 immunofluorescent staining; cross-species transcriptomic concordance was explored using a published single-cell RNA-seq dataset of isogenic PTEN I135L and PTEN WT cerebellar organoids from the Chap and Apex genetic backgrounds (GSE310490). Cross-dataset overlap analysis prioritized candidate TF–RBP relationships involving ASD-associated transcription factors, including AR, YY1, ESR1, and CUX1, and RBPs predicted to target Ccar1 pre-mRNA. ChIP-qPCR showed BPA-associated, sex-stratified differences in ERα and YY1 occupancy at selected RBP promoters, including Sart3, Tia1, and Tardbp. In parallel, Sart3, Tia1, Tardbp, and Hnrnpc were upregulated in the male cerebellum and occurred alongside previously observed Ccar1 exon-skipping and motor/gait phenotypes in male offspring. Prenatal BPA exposure was associated with sex-stratified Purkinje cell layer alterations, including reduced total cell density in males and altered neuronal marker-defined cellular composition in females. Finally, exploratory cross-species comparison identified significant transcriptomic overlap between BPA-responsive rat cerebellar DEGs and PTEN I135L-associated DEGs within the Chap isogenic organoid background; global concordance was not observed in the Apex background. Male BPA-downregulated DEGs showed cell-type-specific concordance in mature neuronal and Purkinje cell populations, and SART3 was the only examined BPA-responsive RBP gene whose peak expression fell within the predefined progenitor-to-neuron pseudotime interval. Together, these findings support a candidate, sex-stratified regulatory model linking BPA-associated changes in TF promoter occupancy and RBP expression with Ccar1 splicing in the neonatal rat cerebellum. Cross-species analyses identified shared transcriptomic patterns in selected cell populations of PTEN I135L cerebellar organoid models, providing contextual evidence for convergent developmental vulnerability without establishing causal or ASD-specific equivalence between the models. ASD is a neurodevelopmental condition characterized by differences in social communication and restricted or repetitive behaviors, with motor difficulties frequently co-occurring. Prenatal BPA exposure has been investigated as an environmental factor that may influence neurodevelopmental processes relevant to ASD. While BPA is known to perturb brain development, this study explores how it disrupts alternative splicing, a critical post-transcriptional process that generates protein diversity from a single gene, within the developing cerebellum. We found that prenatal BPA exposure was associated with changes in the promoter occupancy of selected transcription factors and with altered expression of downstream RNA-binding proteins in a sex-stratified pattern. In male rats, this regulatory pattern occurred alongside increased exon skipping of Ccar1, a gene involved in transcriptional and chromatin-associated regulatory processes. This male-biased molecular pattern occurred alongside reduced Purkinje cell layer density and previously observed motor and gait abnormalities. Female rats showed a different pattern of Purkinje cell layer alterations and did not show the same Ccar1 splicing change observed in males. Finally, comparison with a published PTEN I135L cerebellar organoid dataset identified shared transcriptomic patterns in selected analyses, suggesting partially convergent developmental responses across the two experimental systems. Overall, these findings support a model in which prenatal BPA exposure may influence cerebellar RNA-splicing regulation and cellular development in sex-stratified ways that are relevant to neurodevelopmental phenotypes.