The focus of the current project is to determine the role of microRNAs (miRNAs) in neurodevelopmental deficits associated with Fetal Alcohol Spectrum Disorders (FASD). We have determined earlier that ethanol skews the lineage commitment of mouse embryonic stem (ES) cells towards mesoderm at the expense of neuronal formation, a process mediated by an imbalance of Oct4 (Pou5f1) and Sox2 expression. Recently we identified 48 ethanol-modulated miRNAs during neuronal formation, using a hybridization-based Nanostring microarray. Four miRNAs (miR-143, miR-145, miR-669a and miR-690) exhibited a reversed pattern of expression with ethanol, and a graded response to ethanol concentration (50, 100 mM) with exposure time (2-4 days). Based on these findings, we hypothesize that (i) manipulating these microRNAs will rescue ethanol-mediated diversion from neuronal lineage, and (ii) such a rescue will result in a restored balance in miRNA and targeted mRNA network of lineage specifiers, like Oct4 and Sox2, and potentially other transcription factors, signaling and regulatory factors. Our approach utilizes Locked-Nucleic Acid (LNA) miRNA inhibitors and mimics, Nanostring nCounter digital counting for miRNA expression, simultaneous multiplex miRNA-mRNA assays, and immunocytochemistry, common to two specific aims.
Aim 1. Test whether inhibiting mesodermal activator miRNAs counters ethanol action of diversion from neuronal lineage. We will determine whether inhibition of miR-143/miR-145 individually and in combination, restores the neuronal phenotype and the underlying balance in the miRNA-mRNA network.
Aim 2. Counter ethanol-mediated diversion from neuronal lineage by increasing mesodermal suppressor miRNAs. We will employ mimics to increase the levels of mir- 669a/miR-690 individually and in combination, and assess the recovery of neuronal phenotype, and the rebalance of the miRNA/mRNA network. We will determine from both Aims the extent to which miRNA inhibitors and mimics compensate for ethanol changes using Clustering and Sample Progression Discovery approaches. Completion of this project will provide insights into the role of miRNAs in ethanol-mediated diversion from neuronal lineage. They will also form the basis for studies focused on understanding the causal miRNA-mRNA interactions driving ethanol-driven abnormal neurogenesis. Moreover, new miRNA-mRNA targets will be identified for in vivo translational studies, enabling early diagnosis and treatment of FASD.
Our overall goal is to understand the miRNA networks mediating the effects of ethanol on the early differentiation of embryonic stem cells to neuronal lineage, by integrating changes in miRNAs with that of target mRNAs of lineage specifying regulators. Understanding the adverse effects of ethanol on the dynamics of neuronal lineage is essential for novel diagnosis markers and therapeutic targets of Fetal Alcohol Spectrum Disorders.
