RESUMO
Fetal alcohol spectrum disorders (FASD) are leading causes of neurodevelopmental disability. The mechanisms by which alcohol (EtOH) disrupts fetal brain development are incompletely understood, as are the genetic factors that modify individual vulnerability. Because the phenotype abnormalities of FASD are so varied and widespread, we investigated whether fetal exposure to EtOH disrupts ribosome biogenesis and the processing of pre-ribosomal RNAs and ribosome assembly, by determining the effect of exposure to EtOH on the developmental expression of 18S rRNA and its cleaved forms, members of a novel class of short non-coding RNAs (srRNAs). In vitro neuronal cultures and fetal brains (11-22 weeks) were collected according to an IRB-approved protocol. Twenty EtOH-exposed brains from the first and second trimester were compared with ten unexposed controls matched for gestational age and fetal gender. Twenty fetal-brain-derived exosomes (FB-Es) were isolated from matching maternal blood. RNA was isolated using Qiagen RNA isolation kits. Fetal brain srRNA expression was quantified by ddPCR. srRNAs were expressed in the human brain and FB-Es during fetal development. EtOH exposure slightly decreased srRNA expression (1.1-fold; p = 0.03). Addition of srRNAs to in vitro neuronal cultures inhibited EtOH-induced caspase-3 activation (1.6-fold, p = 0.002) and increased cell survival (4.7%, p = 0.034). The addition of exogenous srRNAs reversed the EtOH-mediated downregulation of srRNAs (2-fold, p = 0.002). EtOH exposure suppressed expression of srRNAs in the developing brain, increased activity of caspase-3, and inhibited neuronal survival. Exogenous srRNAs reversed this effect, possibly by stabilizing endogenous srRNAs, or by increasing the association of cellular proteins with srRNAs, modifying gene transcription. Finally, the reduction in 18S rRNA levels correlated closely with the reduction in fetal eye diameter, an anatomical hallmark of FASD. The findings suggest a potential mechanism for EtOH-mediated neurotoxicity via alterations in 18S rRNA processing and the use of FB-Es for early diagnosis of FASD. Ribosome biogenesis may be a novel target to ameliorate FASD in utero or after birth. These findings are consistent with observations that gene-environment interactions contribute to FASD vulnerability.
RESUMO
Introduction: Up to 9.9% of children have fetal alcohol spectrum disorders (FASD), the most frequent cause of intellectual disability in the US. FASD may involve abnormal brain development, including dysmyelination, suggesting abnormal development of oligodendrocytes (OLs), which make myelin and are rich in lipids. Indeed, low serum levels of omega-3 fatty acids (ω-3) have been reported in FASD. Free fatty acids bind to specific receptors (FFARs). We have isolated cell type-specific fetal brain-derived exosomes (FB-E) from maternal blood and sampled their contents to search for lipid-related biomarkers that predict FASD. Methods: Blood samples were collected from two groups of pregnant women: 1) those who consumed EtOH during pregnancy, and 2) non-EtOH using controls, under an IRB-approved protocol. Serum and OL-derived exosomes (OL-Es) were used to assay myelin basic protein (MBP) and FFAR by ELISA and droplet digital PCR (ddPCR), respectively. Results: FFAR and MBP proteins were downregulated in the EtOH group compared to controls, and this difference was greatest in OL-Es from maternal blood compared maternal serum. Conclusion: MBP and FFAR levels were reduced in OL-Es from EtOH-consuming pregnant women. The data suggest potential therapeutic targets to predict which children are at risk for developing FASD and reduce dysmyelination in developing.