Ethanol selectively disrupts neuronal microexon regulation and chromatin marks in PC12 cells

Exposure to alcohol (ethanol) is the most common toxic insult during human development and is a teratogenic agent associated with abnormalities ranging from behavioral disorders to fetal alcohol syndrome. Ethanol disrupts gene regulatory programs crucial for neuronal identity, including alternative splicing. A distinctive feature of developing neuronal transcriptomes is the high prevalence of microexon inclusion, which consists of short exonic sequences ranging from 3 to 51 nucleotides. However, the impact of ethanol on microexon inclusion remains poorly understood.

In this study, we investigated whether ethanol exposure disrupts neuron-specific alternative splicing programs during neuronal differentiation. To this end, PC12 cells were exposed to 50 mM ethanol throughout five days of NGF-induced differentiation, alongside undifferentiated control cells, to analyze mRNA and protein expression dynamics of key splicing regulators and neurodevelopmental targets.

Our data show that in undifferentiated cells, ethanol exposure resulted in a significant reduction in Srrm4 expression, while concomitantly increasing Srrm3 mRNA levels, consistent with reduced neuron-specific microexon inclusion of the intersectin 1 (Itsn1) mRNA. While undifferentiated PC12 cells primarily express the canonical variants of lysine-specific demethylase 1 (LSD1) and PHD finger protein 21A (PHF21A), differentiation triggers the inclusion of microexons 8a and 14, generating the neuron-specific isoforms neuronal LSD1 and PHF21A, respectively. Under differentiated conditions, ethanol exposure increased neurite length but did not affect the mRNA levels of the neuronal variants of Lsd1, Phf21a, or Itsn1. Notably, ethanol exposure significantly increased the levels of LSD1 protein and its associated histone substrate, H3K4me2, in differentiated PC12 cells.

Taken together, these findings suggest that ethanol exposure modulates post-transcriptional and chromatin-based mechanisms during neuronal commitment and differentiation. This selective disruption highlights distinct molecular pathways that may contribute to persistent neurodevelopmental alterations relevant to alcohol use disorder, providing new insights into the teratogenic mechanisms of ethanol on the developing nervous system.