Neuroprotective Role of DING protein in Fetal Alcohol Spectrum Disorders and Depression.

Introduction: An estimated 20% of women consume alcohol during pregnancy, and 10% of women receive antidepressants during their pregnancy. Women with depression are more likely to use alcohol (EtOH) in early pregnancy and are more likely to have a child with one of the fetal alcohol spectrum disorders (FASD). Previously, we provided evidence in rat neurons in vitro that DING phosphatase (p38SJ, a member of the DINGGG family of proteins that has neuroprotective effects under conditions of cellular stress) was neuroprotective against EtOH-mediated toxicity. Now, we examine the effects of DING, alone or in combination with EtOH and serotonergic (5-HT) pathway molecules and drugs, on EtOH-mediated neurotoxicity in human fetal tissues in vitro. Methods: Human fetal brain tissue was collected between 9 and 23 weeks' gestation. Primary neurons and neurospheres were prepared from a 16-week fetal brain. Exposures to EtOH and SSRI were assessed by detailed questionnaires. Neurotoxicity/apoptosis was assessed in synaptosome extracts with the Caspase-Glo 3/7 assay and a neurotoxicity microarray. Developmental expressions of DING, serotonin transporter (SERT), and 5-HT1A receptor (5HT1A) protein levels were quantified in the brain, placenta, synaptosomes, serum, and "fetal brain-derived exosomes" (FB-Es) by quantitative western blotting and digital droplet PCR. Results: Increasing the levels of activated DING, either by addition to culture media or by intracellular overexpression, was associated with increased cell survival. Developmental expressions of DING, in the presence of EtOH- or SSRI-exposure, were investigated in brain and placenta tissues. While 5HT1A, 5-hydroxytryptamine serotonin receptor 1A (5-HT1A) was strongly inhibited under EtOH stress conditions (>10-fold), DING was not only able to reverse the negative effects of stress, as measured by the synaptic plasticity array, but also upregulated the 5-HT receptor (5-fold compared to untreated control) upon treatment of neurons. Conclusions: Maternal EtOH use is associated with decreased 5-HT receptor and DING protein expression in the fetal brain and with increased neuronal apoptosis. Combined exposure to EtOH and SSRIs may have greater toxic effects than either one alone. DING reversed the toxic effects of EtOH on serotonin receptors in neurons and neurospheres. These findings provide potential mechanisms for the neuroprotective effects of DING, which might suggest neuroprotective approaches to preventing fetal alcohol spectrum disorders (FASD).