Activity-dependent regulation of miRNAs in different subcellular compartments of neurons and its implications for neuronal morphogenesis and plasticity

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

Functional interdependence of the actin regulators CAP1 and cofilin1 in control of dendritic spine morphology

The vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Actin filaments (F-actin) are the major structural component of dendritic spines, and therefore, actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Studies of the past decade identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission, and behavior, and they emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. Here, we report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperates with cofilin1 in spines and that its helical folded domain is relevant for this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that is essential for synaptic cofilin1 activity

Mef2-mediated transcription of the miR379–410 cluster regulates activity-dependent dendritogenesis by fine-tuning Pumilio2 protein levels

Neuronal activity orchestrates the proper development of the neuronal circuitry by regulating both transcriptional and post-transcriptional gene expression programmes. How these programmes are coordinated, however, is largely unknown. We found that the transcription of miR379–410, a large cluster of brain-specific microRNAs (miRNAs), is induced by increasing neuronal activity in primary rat neurons. Results from chromatin immunoprecipitation and luciferase reporter assays suggest that binding of the transcription factor myocyte enhancing factor 2 (Mef2) upstream of miR379–410 is necessary and sufficient for activity-dependent transcription of the cluster. Mef2-induced expression of at least three individual miRNAs of the miR379–410 cluster is required for activity-dependent dendritic outgrowth of hippocampal neurons. One of these miRNAs, the dendritic miR-134, promotes outgrowth by inhibiting translation of the mRNA encoding for the translational repressor Pumilio2. In summary, we have described a novel regulatory pathway that couples activity-dependent transcription to miRNA-dependent translational control of gene expression during neuronal development

Activity-dependent regulation of miRNAs in different subcellular compartments of neurons and its implications for neuronal morphogenesis and plasticity

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

A comprehensive characterization of the nuclear microRNA repertoire of post-mitotic neurons

MicroRNAs (miRNAs) are small non-coding RNAs with important functions in the development and plasticity of post-mitotic neurons. In addition to the well-described cytoplasmic function of miRNAs in post-transcriptional gene regulation, recent studies suggested that miRNAs could also be involved in transcriptional and post-transcriptional regulatory processes in the nuclei of proliferating cells. However, whether miRNAs localize to and function within the nucleus of post-mitotic neurons is unknown. Using a combination of microarray hybridization and small RNA deep sequencing, we identified a specific subset of miRNAs which are enriched in the nuclei of neurons. Nuclear enrichment of specific candidate miRNAs (miR-25 and miR-92a) could be independently validated by Northern blot, quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH). By cross-comparison to published reports, we found that nuclear accumulation of miRNAs might be linked to a down-regulation of miRNA expression during in vitro development of cortical neurons. Importantly, by generating a comprehensive isomiR profile of the nuclear and cytoplasmic compartment, we found a significant overrepresentation of guanine nucleotides at the 3’ terminus of nuclear-enriched isomiRs, suggesting the presence of neuron-specific mechanisms involved in miRNA nuclear localization. In conclusion, our 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 and possibly other polarized cell types

Functional Redundancy of Cyclase-Associated Proteins CAP1 and CAP2 in Differentiating Neurons

Cyclase-associated proteins (CAPs) are evolutionary-conserved actin-binding proteins with crucial functions in regulating actin dynamics, the spatiotemporally controlled assembly and disassembly of actin filaments (F-actin). Mammals possess two family members (CAP1 and CAP2) with different expression patterns. Unlike most other tissues, both CAPs are expressed in the brain and present in hippocampal neurons. We recently reported crucial roles for CAP1 in growth cone function, neuron differentiation, and neuron connectivity in the mouse brain. Instead, CAP2 controls dendritic spine morphology and synaptic plasticity, and its dysregulation contributes to Alzheimer’s disease pathology. These findings are in line with a model in which CAP1 controls important aspects during neuron differentiation, while CAP2 is relevant in differentiated neurons. We here report CAP2 expression during neuron differentiation and its enrichment in growth cones. We therefore hypothesized that CAP2 is relevant not only in excitatory synapses, but also in differentiating neurons. However, CAP2 inactivation neither impaired growth cone morphology and motility nor neuron differentiation. Moreover, CAP2 mutant mice did not display any obvious changes in brain anatomy. Hence, differently from CAP1, CAP2 was dispensable for neuron differentiation and brain development. Interestingly, overexpression of CAP2 rescued not only growth cone size in CAP1-deficient neurons, but also their morphology and differentiation. Our data provide evidence for functional redundancy of CAP1 and CAP2 in differentiating neurons, and they suggest compensatory mechanisms in single mutant neurons

The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development

MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA‐generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain‐derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca2+‐dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre‐miR16, was impaired. Decreased production of miR‐16‐5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane‐targeted TRBP. Moreover, miR‐16‐5p or membrane‐targeted TRBP expression blocked BDNF‐induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity‐dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.ISSN:1469-221XISSN:1469-317

A microRNA-129-5p/Rbfox crosstalk coordinates homeostatic downscaling of excitatory synapses

Synaptic downscaling is a homeostatic mechanism that allows neurons to reduce firing rates during chronically elevated network activity. Although synaptic downscaling is important in neural circuit development and epilepsy, the underlying mechanisms are poorly described. We performed small RNA profiling in picrotoxin (PTX)-treated hippocampal neurons, a model of synaptic downscaling. Thereby, we identified eight microRNAs (miRNAs) that were increased in response to PTX, including miR-129-5p, whose inhibition blocked synaptic downscaling in vitro and reduced epileptic seizure severity in vivo. Using transcriptome, proteome, and bioinformatic analysis, we identified the calcium pump Atp2b4 and doublecortin (Dcx) as miR-129-5p targets. Restoring Atp2b4 and Dcx expression was sufficient to prevent synaptic downscaling in PTX-treated neurons. Furthermore, we characterized a functional crosstalk between miR-129-5p and the RNA-binding protein (RBP) Rbfox1. In the absence of PTX, Rbfox1 promoted the expression of Atp2b4 and Dcx. Upon PTX treatment, Rbfox1 expression was downregulated by miR-129-5p, thereby allowing the repression of Atp2b4 and Dcx. We therefore identified a novel activitydependent miRNA/RBP crosstalk during synaptic scaling, with potential implications for neural network homeostasis and epileptogenesis

A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (Galpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha(13) both suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Galpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis