The activity-dependent spatiotemporal regulation of gene expression in neurons is essential for the formation and function of neuronal circuits within the brain. Recently microRNAs, a new class of post-transcriptional regulators of gene expression were implicated in the regulation of neuronal differentiation and development. Furthermore, in mature fully developed neurons, miRNAs (e.g. miR-134) were shown to be involved in the control of local protein synthesis in the vicinity of dendritic spines (Schratt et al., 2006). Activity-dependent local protein synthesis is required for synaptic plasticity, which is believed to be one of the molecular substrates of learning and memory. Nonetheless, the molecular mechanisms underlying the function and regulation of miRNAs during synaptic plasticity are poorly understood.
In a previous publication from our lab, it was shown that the activity of the brain-enriched miRNA - miR-134 is regulated by brain-derived neurotrophic factor, which is released upon synaptic stimulation in neurons (Schratt et al, 2006). Interestingly, in the mouse genome this miRNA is encoded in a large miRNA cluster (miR379-410 cluster) consisting of 39 miRNAs. The expression of the miR379-410 cluster is induced upon neuronal activity by virtue of myocyte-enhancing factor 2, a transcription factor that binds to a regulatory region upstream of this cluster (Fiore et al., 2009). The transcriptional upregulation of a subset of miRNAs from the miR379-410 cluster (miR-134, -381 and -329) is necessary for activity-dependent dendritic development of rat hippocampal neurons. Furthermore, we found that the post-transcriptional regulation of the RNA-binding protein Pumilio 2 by miR-134 is essential for activity-dependent dendritogenesis. Taken together, we defined a novel MEF2-miRNA-PUM2 pathway involved in the activity-dependent regulation of dendritogenesis in primary neurons.
MiR-134 localizes within dendrites of hippocampal neurons, where it can regulate the local translation of proteins important for spine structure and plasticity. However, at the beginning of this project, it was unknown how this miRNA is targeted to dendrites. I was involved in a project that aimed at identifying and characterizing the transport mechanism of miR-134 to dendrites. We found that the dendritic localization of miR-134 is mediated by the DEAH-box helicase DHX36 protein, which binds to a cis-acting element located within the loop region of the miR-134 precursor (pre-miR-134; Bicker et al., 2013). Furthermore, we showed that depletion of DHX36 increased protein levels of LIM kinase 1, a dendritically localized target of miR-134 (Schratt et al, 2006). Moreover, the depletion of DHX36 led to an increase in dendritic spine size, a similar phenotype as observed upon inhibition of miR-134 activity. In summary, we described a novel mechanism for dendritic targeting of pre-miR-134 relevant for the function of miR-134 in spine morphogenesis.
Activity-dependent regulation of gene expression in the nucleus is important for the development and function of the nervous system, including synaptic plasticity and memory formation. Interestingly, several recent reports suggested that miRNAs (and/or siRNAs) might be involved in the regulation of epigenetic modifications and alternative mRNA splicing events in the nucleus of non-neuronal cells. However, whether miRNAs employ this mechanism to regulate gene expression in the neuronal nucleus was not known. A prerequisite for the study of miRNA function in the nucleus of post-mitotic neurons is the a priori knowledge of the nuclear miRNA repertoire. Therefore, using microarray and deep sequencing technologies, I identified miRNAs which are enriched in the nuclei of rat primary cortical neurons (Khudayberdiev et al. 2013; Frontiers in Mol. Neurosci, accepted for publication). Subsequently, I validated differential expression of specific nuclear-enriched miRNAs by Northern blot, quantitative real-time PCR and fluorescence in situ hybridization. By cross-comparison to published reports, I found that nuclear accumulation of miRNAs might be linked to a down-regulation of their expression during in vitro development of cortical neurons. Importantly, I found a significant overrepresentation of guanine nucleotides at the 3’ terminus of nuclear-enriched miRNA isoforms (isomiRs), suggesting the presence of neuron-specific mechanisms involved in miRNA nuclear localization. In conclusion, these results provide a starting point for future studies addressing the nuclear function of specific miRNAs and the detailed mechanisms underlying subcellular localization of miRNAs in neurons.
Taken together, the results presented in my cumulative PhD thesis demonstrate that activity-dependent regulation of specific miRNAs in different subcellular neuronal compartments (dendrites, nucleus, and soma) plays an important role in neuronal morphogenesis (dendrite and spine development) and plasticity.
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