Elucidating mechanisms associated with endoplasmic reticulum stress at the placental and fetal brain axis in response to oxycodone exposure

Prenatal oxycodone (Oxy) exposure has been associated with several neurodevelopmental outcomes in newborns. However, the molecular mechanisms involved in Oxy-induced placental and fetal brain damage during gestation remain unknown. Endoplasmic reticulum (ER) stress is a cellular stress response triggered by the accumulation of unfolded proteins, which activates the unfolded protein response. Here, we hypothesized that Oxy exposure induces ER stress in the fetal brain-placental axis. To test this hypothesis, we used three model systems: in vitro primary neurons, primary mixed neuroglial cells, and an in vivo model of maternal Oxy exposure. Quantitative Reverse Transcription PCR (qRT-PCR) was employed to detect mRNA expression of ER stress mediators, such as Binding Immunoglobulin Protein (Bip), activating transcription factor 4 (ATF4), spliced X-box binding protein 1 (sXBP1), and C/EBP homologous protein (CHOP). Immunoblot analysis was also used to detect protein expression of ER stress mediators, such as Bip; phosphorylated and total forms of inositol-requiring transmembrane kinase/endoribonuclease 1α (p-IRE1α and t-IRE1α) and protein kinase R-like endoplasmic reticulum kinase α (p-PERKα and t-PERKα); phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α); and sXBP1. Further, we detected pro-apoptotic markers such as Bcl-2 interacting mediator of cell death (Bim), p53 upregulated modulator of apoptosis (PUMA), and cleaved poly (ADP-ribose) polymerase (PARP). Exposure of Oxy to rat primary fetal neuronal cells increased mRNA expression of Bip and ATF4. sXBP1 and CHOP mRNA levels were upregulated in both rat primary fetal neuronal cells and mixed neuroglial cells. Prenatal Oxy exposure resulted in increased mRNA expression of all ER stress markers tested in both the placenta and fetal brain. We also observed that Oxy exposure increased protein levels of ER stress markers (Bip, p-IRE1α, t-IRE1α, p-PERKα, t-PERKα, p-eIF2α, and sXBP1), as well as pro-apoptotic and apoptotic markers (Bim, PUMA, and cleaved PARP). In conclusion, Oxy exposure increases ER stress mediators in both the placental and fetal brain compartments, which could further lead to apoptosis.