Prenatal Alcohol Exposure Inhibits Transient Expression of Autophagy and Synaptic Proteins in Developing Brain.

Introduction: Neuronal apoptosis and consequent inhibition of autophagy, with loss of synaptic connections are central events in the genesis of fetal alcohol spectrum disorders (FASD). However, studies of molecular mechanisms of autophagy in human fetal brain are limited. Recently, prenatal exposure to EtOH was associated with reduced miRNA-9 levels in fetal brain-derived exosomes (FB- Es) isolated from maternal plasma, which correlated with small eyes, an anatomical hallmark of fetal alcohol syndrome (FAS). Since miR-9 targets several genes that regulate synaptic plasticity, EtOH-induced inhibition of miR-9 could potentially result in dysregulation of genes involved in synaptogenesis/plasticity. Methods: Rats were fed a 6.7% EtOH liquid diet from E16 to birth. Human studies: Fetal brain tissues from elective pregnancy terminations were collected at 9-23 weeks gestational age (GA; n=20 EtOH-exposed and 20 GA- and fetal sex-matched unexposed controls). EtOH consumption was assessed by questionnaire (adapted from NIAAA PASS). Expressions of 84 genes in a synaptic plasticity microarray were assessed in human fetal brain samples, verified by qRT-PCR, and for some mRNAs, copy number was determined in FB-Es by droplet digital PCR. Protein expression was measured in brain by qWestern blot assays or with a MAP kinase microarray. Exosomal protein levels were measured by ELISA. Results: Levels of pro-apoptotic caspase-3 and Bax were significantly increased in the brains of EtOH-exposed rat pups, while early expressions of anti-apoptotic Bcl2 and biphasic Bag3 were inhibited. Phosphorylation of GSK3β was increased, and during Bag3 inhibition, the GSK3β target β-catenin also was increased. EtOH-exposed P8 and P15 rats showed motor abnormalities during low Bag3 expression. EtOH exposure reduced expression of synaptophysin and synapsin. In most synaptic plasticity pathways, levels of mRNAs were reduced. Several immediate-early genes were upregulated, but SYNPO, which is involved late in synaptic plasticity was downregulated 78%. Genes involved in Long Term Potentiation (LTP) and long-term depression (LTD) were downregulated, but the neurotoxic cytokine TNF⍺ was upregulated. Conclusions: Prenatal exposure to EtOH was associated with reduced expression of autophagy genes in the fetal brains of rats and humans. Inhibition of Bag3 correlated with upregulation of GSK3β and its downstream targets, suggesting dysregulation of β-catenin signaling. Synaptic proteins, including those implicated in LTP and LTD also were inhibited by EtOH. The results in FB-E mirrored those in brain tissue. Reduced expression of miR-9 target synaptic genes in FB-Es might serve as novel biomarkers to predict FASD.