Regulation of microRNA function in neuronal development by the transcription co-activator Ncoa3

Tightly orchestrated gene expression programs enable proper neuronal development as well as the synaptic adaptations that are responsible for learning and memory processes. MicroRNAs (miRNAs) are a class of short regulatory RNA molecules which negatively affect the translation of target mRNAs, thereby contributing to the regulation of gene expression during brain development and cognitive functions. In the present cumulative thesis, I summarize my contributions to three research articles which describe the impact of specific miRNAs and an upstream regulator, the nuclear receptor co-activator 3 (Ncoa3), on neuronal growth and synaptic function. In the first publication, we identified miR-181a to be enriched at synaptic sites of the nucleus accumbens, a brain region of the dopaminergic mesolimbic system which is involved in the development of addiction. Using primary neurons, we demonstrated that miR-181a directly regulates the expression of the AMPA-receptor (AMPA-R) subunit GluA2. Neuromorphological analysis and electrophysiological measurements showed that miR-181a affects transmission at excitatory synapses. Dopamine signaling stimulated the expression of miR-181a which further influenced the dopamine-dependent control of GluA2 expression. Treatment of mice with several drugs of abuse specifically upregulated miR-181a levels in different brain regions. Taken together, this publication established miR-181a as novel regulator of synaptic efficacy and in the context of the present literature as a potential modulator of addiction behavior. Based on previous findings that showed the synaptic localization of miR-137 and that identified mutations in the MIR137 gene associated with schizophrenia and cognitive disabilities, we investigated the postsynaptic functions of miR-137 in the second publication. Manipulations of miR-137 expression provided evidence that the AMPA-R subunit GluA1 mRNA is a direct target of miR-137. Intriguingly, morphological and electrophysiological measurements revealed that miR-137 regulates the number but not the strength of excitatory synapses. MiR-137 further promoted the formation of silent synapses, since miR-137 manipulations affected AMPA-R-, but not NMDA-receptor (NMDA-R)-dependent currents. Furthermore, induction of miR-137 expression was required for mGluR-dependent long term depression (LTD). Therefore, this research article provides experimental support for a postsynaptic function of miR-137 in the regulation of synapse formation and plasticity, with possible implications for schizophrenia and cognitive disabilities. In the third publication, which includes the main part of my PhD project, 10 novel regulators of miRNA-dependent gene silencing in neurons were identified by performing an RNAi-based screen. One of the newly ascertained proteins was Ncoa3, a transcription co-activator whose function in hippocampal neurons was not studied. Reporter gene assays showed that Ncoa3 was required for miRNA-mediated repression of a specific set of miRNA target genes, including Limk1. In addition, Ncoa3-knockdown increased endogenous Limk1 protein levels and interfered with miR-134-induced spine shrinkage. At the same time, Ncoa3 deficiency by itself reduced the size of dendritic spines and the amplitude of miniature excitatory postsynaptic currents (mEPSCs) while it stimulated dendrite growth. The latter phenotype was dependent on proper miRNA-expression. Ago2 is a central effector of miRNA repression and we established it further as a direct transcriptional target gene of Ncoa3. Epistasis experiments confirmed that both impaired dendritogenesis and miRNA function upon Ncoa3 knockdown were a result of reduced Ago2 expression. Thus, this publication uncovered a novel transcriptional mechanism for the control of miRNA-dependent repression during neuronal development. In summary, these findings decipher neuronal gene expression programs which control synaptic adaptations and thus are potentially involved in learning and memory processes.