microRNA and the Posttranscriptional Response to Oxidative Stress During Neuronal Differentiation

Oxidative stress is an important environmental exposure associated with psychiatric disorders, but the underlying molecular mechanisms remain elusive. We recently observed enrichment of neurodevelopmental processes and diseases among a large number of differentially expressed genes during or after exposure to oxidative stress in an in vitro model of neuronal differentiation. To further explore the regulatory mechanisms that might account for a coordinated response to this exposure, we investigated changes in the expression of small non-coding miRNA in this system and estimated their interaction with differentially expressed mRNA. These molecules are thought to play a crucial role in brain development and its response to stress. Here we observed more than a hundred differentially expressed miRNAs, including 72 previously reported to be dysregulated in psychiatric disorders. The 7 most influential miRNAs associated with pre-treatment exposure were miR-138-5p, miR-96-5p, miR-34c-5p, miR-1287-5p, miR-497-5p, miR-195-5p, and miR-16-5p. These were sup-ported by at least 10 negatively correlated mRNA connections, and formed hubs in interaction network with 134 genes enriched with neurobiological function. Whereas in the co-treatment con-dition, miRNA-mRNA interaction pairs were enriched not only in neuronal processes, but also in cardiovascular and immunity-related disease ontologies. Interestingly, 12 differentially expressed miRNAs originated from the same genomic location, DLK1-DIO3, which encodes a schizophre-nia-associated miRNA signature. Collectively, these findings suggest that early exposure to oxida-tive stress, before and during prenatal neuronal differentiation, might increase the risk of mental illnesses in adulthood by disturbing the expression of miRNAs that regulate neurodevelopmental-ly significant genes and networks. Hydrogen peroxide (H2O2) at a final concentration of 10 µM was applied through two different protocols to induce chronic oxidative stress in neuroblasts. In the co-treatment approach, the cell culture medium was simultaneously supplemented with all-trans retinoic acid (ATRA) and H2O2 for a 7-day period, while in the pre-treatment regimen, cells were first exposed to H2O2 for 72 hours, and upon its removal were treated with ATRA for 7 days. All experiments were performed in biological triplicates, such that for each approach, there existed 3 replicates for peroxide-treated and 3 replicates for untreated samples. One of the samples was known as an outlier and removed from analysis.