miRNAs are small non-coding RNA molecules that regulate gene expression by targeting the 3'-untranlated regions (UTRs) of mRNAs leading to mRNA degradation and suppression of protein synthesis. By coordinating and tuning gene expression, miRNAs play important roles in nervous system development and function. Dysregulation of miRNAs may lead to neural developmental disorders and mental illness. Here we propose to study two brain-enriched miRNAs, miR-9 and miR-139-5p, which potentially regulate the FOXP1 and FOXP2 genes. FOXP1 and FOXP2 are paralog transcription factors that function as a dimer and regulate the expression of numerous downstream genes important in neural circuit development. Mutations in the FOXP2 gene cause speech and language impairments, and both FOXP1 and FOXP2 have also been implicated in the Autism Spectrum Disorders (ASDs). An emerging hypothesis suggests that an adequate amount of functional FOXP2 is required for the proper development of the distributed neural circuitry for procedure learning and motor sequencing required for speech and language. However, regulation of the expression of FOXP1 and FOXP2 is not clear. Both the FOXP1 and FOXP2 genes have long 3'-UTRs (~4 kb), twice as long as their respective protein coding regions, suggesting that they are regulated by miRNAs. miR-9 and miR-139-5p are evolutionarily conserved and their targeting sites in the 3'-UTRs of FOXP1 and FOXP2 are also conserved, allowing us to study them in the zebra finch in the context of neural circuit development and vocal learning behavior. In the zebra finch brain, FOXP1 and FOXP2 are expressed in Area X, a nucleus in a cortical-striatal circuit required for vocal learning in juvenile zebra finches. Knockdown of FoxP2 in Area-X impairs vocal learning and reduces dendritic spine density in juvenile finches. Area X is largely composed of spiny neurons and a considerable number of new spiny neurons are generated in the germinal lining of the lateral ventricle adjacent to Area X and being recruited into Area X in young and adult zebra finches. We have shown that both miR-9 and miR-139-5p are expressed in Area X and in the ventricular zone. miR-9 is also known to regulate neural progenitor proliferation and differentiation during embryonic brain development. These collective observations led us to hypothesize that miR-9 and miR-139-5p regulate FOXP1 and FOXP2, and play important roles in regulating postnatal neurogenesis, neuronal dendritic development, and vocal learning behavior. This hypothesis will be tested using a combination of molecular, cellular, and behavioral approaches. First, we will test whether miR-9 and miR-139-5p regulate FoxP1 and FoxP2 in vitro; we will also examine how the expression of miR-139-5p and FoxP1 is regulated in the zebra finch brain during vocal development and in adults (we already studied miR-9 and FoxP2). Then we will use lentiviruses to manipulate the expression of miR-9 and miR-139-5p in Area X and test the effects on vocal learning and performance. We will also study the roles of miR-9 and miR-139-5p in regulating neurogenesis in postnatal and adult Area X, and study the roles of miR-9 and miR-139-5p in regulating dendritic structures in spiny neurons in Area X. This research promises to establish novel post-transcriptional regulatory relationships between miR-9/miR- 139-5p and FOXP1/FOXP2, and to delineate the roles of miR-9 and miR-139-5p in regulating neurogenesis and dendritic development in a neural circuit important for vocal learning behavior. As the striatum is at the center of many mental illness and neurological disorders, these results may open new opportunities to study how genetic and environmental factors contribute to these conditions via miRNA-mediated gene regulatory networks. With further research, these miRNAs may be developed into diagnostic markers or therapeutic agents.
The proposed research will establish a new regulatory relationship between miRNAs miR-9 and miR-139-5p with the FOXP1 and FOXP2 genes, and establish novel functions of miR-9 and miR-139-5p in the development and functions of a striatal circuit important for vocal learning behavior. The insights gained here will further our understanding of how genetic and environmental factors may contribute to many neural developmental disorders and mental illness related to the striatum. With further research, these miRNAs may lead to the development of novel diagnostic markers or therapeutic targets.
|Shi, Zhimin; Piccus, Zoe; Zhang, Xiaofang et al. (2018) miR-9 regulates basal ganglia-dependent developmental vocal learning and adult vocal performance in songbirds. Elife 7:|