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In the sympatho-adrenal system, angiotensin II (Ang II) acts as a key neuromodulatory component. At sympathetic nerve terminals, Ang II influences sympathetic transmission by enhancing norepinephrine (NE) synthesis, facilitating NE release and inhibiting NE uptake. Previously, it was demonstrated that tyrosine hydroxylase (TH) mRNA is trafficked to the distal axons of primary superior cervical ganglia (SCG) neurons, directed by a cis-acting regulatory element (i.e. zipcode) located in the 3’UTR of the transcript. Results of metabolic labeling studies established that the mRNA is locally translated. It was further shown that the axonal trafficking of the mRNA encoding the enzyme plays an important role in mediating dopamine (DA) and NE synthesis and may facilitate the maintenance of axonal catecholamine levels. In the present study, the hypothesis was tested that Ang II induces NE synthesis in rat primary SCG neurons via the modulation of the trafficking of the mRNAs encoding the catecholamine synthesizing enzymes TH and Dopamine β-Hydroxylase (DBH). Treatment of SCG neurons with the Ang II receptor type 1 (AT1R) agonist, L-162,313, increases the axonal levels of TH and DBH mRNA and protein and results in elevated NE levels. Conversely, treatment of rat SCG neurons with the AT1R antagonist, Eprosartan, abolished the L-162,313-mediated increase in axonal levels of TH and DBH mRNA and protein. In a first attempt to identify the proteins involved in the Ang II-mediated axonal transport of TH mRNA, we used a biotinylated 50-nucleotide TH RNA zipcode as bait in the affinity purification of TH zipcode-associated proteins. Mass spectrometric analysis of the TH zipcode ribonucleoprotein (RNP) complex immune-purified from SCG neurons led to the identification of 163 somal and 127 axonal proteins functionally involved in binding nucleic acids, the translational machinery or acting as subunits of cytoskeletal and motor proteins. Surprisingly, immune-purification of the TH axonal trafficking complex, results in the acquisition of DBH mRNA, suggesting that these mRNAs maybe transported to the axon together, possibly in the same RNP complex. Taken together, our results point to a novel mechanism by which Ang II participates in the regulation of axonal synthesis of NE by modulating the local trafficking and expression of TH and DBH, two key enzymes involved in the catecholamine biosynthetic pathway.

Contains fulltext : 144740.pdf (Publisher’s version ) (Closed access) Autism spectrum disorders are severe neurodevelopmental disorders, marked by impairments in reciprocal social interaction, delays in early language and communication, and the presence of restrictive, repetitive and stereotyped behaviors. Accumulating evidence suggests that dysfunction of the amygdala may be partially responsible for the impairment of social behavior that is a hallmark feature of ASD. Our studies suggest that a valproic acid (VPA) rat model of ASD exhibits an enlargement of the amygdala as compared to controls rats, similar to that observed in adolescent ASD individuals. Since recent research suggests that altered neuronal development and morphology, as seen in ASD, may result from a common post-transcriptional process that is under tight regulation by microRNAs (miRs), we examined genome-wide transcriptomics expression in the amygdala of rats prenatally exposed to VPA, and detected elevated miR-181c and miR-30d expression levels as well as dysregulated expression of their cognate mRNA targets encoding proteins involved in neuronal system development. Furthermore, selective suppression of miR-181c function attenuates neurite outgrowth and branching, and results in reduced synaptic density in primary amygdalar neurons in vitro. Collectively, these results implicate the small non-coding miR-181c in neuronal morphology, and provide a framework of understanding how dysregulation of a neurodevelopmentally relevant miR in the amygdala may contribute to the pathophysiology of ASD. 12 p.

Contains fulltext : 135142.pdf (Publisher’s version ) (Open Access) BACKGROUND: Schizophrenia is a highly heritable neurodevelopmental disorder. A genetic variant of microRNA-137 (miR-137) has yielded significant genome-wide association with schizophrenia, suggesting that this miRNA plays a key role in its etiology. Therefore, a molecular network of interacting miR-137 targets may provide insights into the biological processes underlying schizophrenia. METHODS: We first used bioinformatics tools to obtain and analyze predicted human and mouse miR-137 targets. We then determined miR-137 levels in rat barrel cortex after environmental enrichment (EE), a neuronal plasticity model that induces upregulation of several predicted miR-137 targets. Subsequently, expression changes of these predicted targets were examined through loss of miR-137 function experiments in rat cortical neurons. Finally, we conducted bioinformatics and literature analyses to examine the targets that were upregulated upon miR-137 downregulation. RESULTS: Predicted human and mouse miR-137 targets were enriched in neuronal processes, such as axon guidance, neuritogenesis and neurotransmission. The miR-137 levels were significantly downregulated after EE, and we identified 5 novel miR-137 targets through loss of miR-137 function experiments. These targets fit into a glucocorticoid receptor-dependent signalling network that also includes 3 known miR-137 targets with genome-wide significant association with schizophrenia. LIMITATIONS: The bioinformatics analyses involved predicted human and mouse miR-137 targets owing to lack of information on predicted rat miR-137 targets, whereas follow-up experiments were performed with rats. Furthermore, indirect effects in the loss of miR-137 function experiments cannot be excluded. CONCLUSION: We have identified a miR-137-regulated protein network that contributes to our understanding of the molecular basis of schizophrenia and provides clues for future research into psychopharmacological treatments for schizophrenia.

Contains fulltext : 169125.pdf (Publisher’s version ) (Closed access) MicroRNAs (miRs) are small regulatory molecules, which orchestrate neuronal development and plasticity through modulation of complex gene networks. MicroRNA-137 (miR-137) is a brain-enriched RNA with a critical role in regulating brain development and in mediating synaptic plasticity. Importantly, mutations in this miR are associated with the pathoetiology of schizophrenia (SZ), and there is a widespread assumption that disruptions in miR-137 expression lead to aberrant expression of gene regulatory networks associated with SZ. To systematically identify the mRNA targets for this miR, we performed miR-137 gain- and loss-of-function experiments in primary rat hippocampal neurons and profiled differentially expressed mRNAs through next-generation sequencing. We identified 500 genes that were bidirectionally activated or repressed in their expression by the modulation of miR-137 levels. Gene ontology analysis using two independent software resources suggested functions for these miR-137-regulated genes in neurodevelopmental processes, neuronal maturation processes and cell maintenance, all of which known to be critical for proper brain circuitry formation. Since many of the putative miR-137 targets identified here also have been previously shown to be associated with SZ, we propose that this miR acts as a critical gene network hub contributing to the pathophysiology of this neurodevelopmental disorder.

A steady accumulation of experimental data argues that protein synthesis in neurons is not merely restricted to the somatic compartment, but also occurs in several discrete cellular micro-domains. Local protein synthesis is critical for the establishment of synaptic plasticity in mature dendrites and in directing the growth cones of immature axons, and has been associated with cognitive impairment in mice and humans. Although in recent years a number of important mechanisms governing this process have been described, it remains technically challenging to precisely monitor local protein synthesis in individual neuronal cell parts independent from the soma. This report presents the utility of employing microfluidic chambers for the isolation and treatment of single neuronal cellular compartments. Furthermore, it is demonstrated that a protein synthesis assay, based on fluorescent non-canonical amino acid tagging (FUNCAT), can be combined with this cell culture system to label nascent proteins within a discrete structural and functional domain of the neuron. Together, these techniques could be employed for the detection of protein synthesis within developing and mature neurites, offering an effective approach to elucidate novel mechanisms controlling synaptic maintenance and plasticity.

MicroRNAs (miRNAs) constitute a novel class of small, non-coding RNAs that act as post-transcriptional regulators of gene expression. Remarkably, it has been shown that these small molecules can coordinately regulate multiple genes coding for proteins with related cellular functions. Previously, we reported that brain-specific miR-338 modulates the axonal expression of cytochrome c oxidase IV (COXIV), a nuclear-encoded mitochondrial protein that plays a key role in oxidative phosphorylation and axonal function. Here, we report that ATP synthase (ATP5G1), like COXIV mRNA, contains a putative miR-338 binding site, and that modulation of miR-338 levels in the axon results in alterations in both COXIV and ATP5G1 expression. Importantly, miR-338 modulation of local COXIV and ATP5G1 expression has a marked effect on axonal ROS levels, as well as axonal growth. These findings point to a mechanism by which miR-338 modulates local energy metabolism through the coordinate regulation of the expression of multiple nuclear-encoded mitochondrial mRNAs in the axon.

Contains fulltext : 96723.pdf (Publisher’s version ) (Closed access) Background MicroRNAs (miRNAs) are non-coding gene transcripts involved in post-transcriptional regulation of genes. Recent studies identified miRNAs as important regulators of learning and memory in model organisms. So far, no mutations in specific miRNA genes have been associated with impaired cognitive functions. Methods and results In three sibs and two unrelated patients with intellectual disability (ID), overlapping 1p21.3 deletions were detected by genome-wide array analysis. The shortest region of overlap included dihydropyrimidine dehydrogenase (DPYD) and microRNA 137 (MIR137). DPYD is involved in autosomal recessive dihydropyrimidine dehydrogenase deficiency. Hemizygous DPYD deletions were previously suggested to contribute to a phenotype with autism spectrum disorder and speech delay. Interestingly, the mature microRNA transcript microRNA-137 (miR-137) was recently shown to be involved in modulating neurogenesis in adult murine neuronal stem cells. Therefore, this study investigated the possible involvement of MIR137 in the 1p21.3-deletion phenotype. The patients displayed a significantly decreased expression of both precursor and mature miR-137 levels, as well as significantly increased expression of the validated downstream targets microphthalmia-associated transcription factor (MITF) and Enhancer of Zeste, Drosophila, Homologue 2 (EZH2), and the newly identified target Kruppel-like factor 4 (KLF4). The study also demonstrated significant enrichment of miR-137 at the synapses of cortical and hippocampal neurons, suggesting a role of miR-137 in regulating local synaptic protein synthesis machinery. Conclusions This study showed that dosage effects of MIR137 are associated with 1p21.3 microdeletions and may therefore contribute to the ID phenotype in patients with deletions harbouring this miRNA. A local effect at the synapse might be responsible. 9 p.

Contains fulltext : 167946.pdf (Publisher’s version ) (Closed access) BACKGROUND: Altered levels of urocortin 1 (Ucn1) in the centrally projecting Edinger-Westphal nucleus (EWcp) of depressed suicide attempters or completers mediate the brain's response to stress, while the mechanism regulating Ucn1 expression is unknown. We tested the hypothesis that microRNAs (miRNAs), which are vital fine-tuners of gene expression during the brain's response to stress, have the capacity to modulate Ucn1 expression. METHODS: Computational analysis revealed that the Ucn1 3' untranslated region contained a conserved binding site for miR-326. We examined miR-326 and Ucn1 levels in the EWcp of depressed suicide completers. In addition, we evaluated miR-326 and Ucn1 levels in the serum and the EWcp of a chronic variable mild stress (CVMS) rat model of behavioural despair and after recovery from CVMS, respectively. Gain and loss of miR-326 function experiments examined the regulation of Ucn1 by this miRNA in cultured midbrain neurons. RESULTS: We found reduced miR-326 levels concomitant with elevated Ucn1 levels in the EWcp of depressed suicide completers as well as in the EWcp of CVMS rats. In CVMS rats fully recovered from stress, both serum and EWcp miR-326 levels rebounded to nonstressed levels. While downregulation of miR-326 levels in primary midbrain neurons enhanced Ucn1 expression levels, miR-326 overexpression selectively reduced the levels of this neuropeptide. LIMITATIONS: This study lacked experiments showing that in vivo alteration of miR-326 levels alleviate depression-like behaviours. We show only correlative data for miR-325 and cocaine- and amphetamine-regulated transcript levels in the EWcp. CONCLUSION: We identified miR-326 dysregulation in depressed suicide completers and characterized this miRNA as an upstream regulator of the Ucn1 neuropeptide expression in midbrain neurons. 12 p.

Contains fulltext : 107737.pdf (Publisher’s version ) (Open Access) MicroRNAs (miRNAs) are important gene regulators that are abundantly expressed in both the developing and adult mammalian brain. These non-coding gene transcripts are involved in post-transcriptional regulatory processes by binding to specific target mRNAs. Approximately one third of known miRNA genes are located within intronic regions of protein coding and non-coding regions, and previous studies have suggested a role for intronic miRNAs as negative feedback regulators of their host genes. In the present study, we monitored the dynamic gene expression changes of the intronic miR-338-3p and miR-338-5p and their host gene Apoptosis-associated Tyrosine Kinase (AATK) during the maturation of rat hippocampal neurons. This revealed an uncorrelated expression pattern of mature miR-338 strands with their host gene. Sequence analysis of the 3' untranslated region (UTR) of rat AATK mRNA revealed the presence of two putative binding sites for miR-338-3p. Thus, miR-338-3p may have the capacity to modulate AATK mRNA levels in neurons. Transfection of miR-338-3p mimics into rat B35 neuroblastoma cells resulted in a significant decrease of AATK mRNA levels, while the transfection of synthetic miR-338-5p mimics did not alter AATK levels. Our results point to a possible molecular mechanism by which miR-338-3p participates in the regulation of its host gene by modulating the levels of AATK mRNA, a kinase which plays a role during differentiation, apoptosis and possibly in neuronal degeneration.

Contains fulltext : 88234.pdf (Publisher’s version ) (Open Access) microRNAs (miRNAs) constitute a novel class of small, noncoding RNAs that act as negative post-transcriptional regulators of gene expression. Although the nervous system is a prominent site of miRNA expression, little is known about the spatial expression profiles of miRNAs in neurons. Here, we employed compartmentalized Campenot cell culture chambers to obtain a pure axonal RNA fraction of superior cervical ganglia (SCG) neurons, and determined the miRNA expression levels in these subcellular structural domains by microarray analysis and by real-time reverse-transcription polymerase chain reaction. The data revealed stable expression of a number of mature miRNAs that were enriched in the axons and presynaptic nerve terminals. Among the 130 miRNAs identified in the axon, miR-15b, miR-16, miR-204, and miR-221 were found to be highly abundant in distal axons as compared with the cell bodies of primary sympathetic neurons. Moreover, a number of miRNAs encoded by a common primary transcript (pri-miRNA) were differentially expressed in the distal axons, suggesting that there is a differential subcellular transport of miRNAs derived from the same coding region of the genome. Taken together, the data provide an important resource for future studies on the regulation of axonal protein synthesis and the role played by miRNAs in the maintenance of axonal structure and function as well as neuronal growth and development. 01 augustus 2010

MicroRNAs (miRs) are evolutionarily conserved, noncoding RNA molecules of ∼21 nt that regulate the expression of genes that are involved in various biological processes, such as cell proliferation and differentiation. Previously, we reported the presence of a heterogeneous population of mRNAs present in the axons and nerve terminals of primary sympathetic neurons to include the nuclear-encoded mitochondrial mRNA coding for COXIV. Sequence analysis of the 3′UTR of this mRNA revealed the presence of a putative binding site for miR-338, a brain-specific microRNA. Transfection of precursor miR-338 into the axons of primary sympathetic neurons decreases COXIV mRNA and protein levels and results in a decrease in mitochondrial activity, as measured by the reduction of ATP levels. Conversely, the transfection of synthetic anti-miR oligonucleotides that inhibit miR-338 increases COXIV levels, and results in a significant increase in oxidative phosphorylation and also norepinephrine uptake in the axons. Our results point to a molecular mechanism by which this microRNA participates in the regulation of axonal respiration and function by modulating the levels of COXIV, a protein which plays a key role in the assembly of the mitochondrial cytochromecoxidase complex IV.

Contains fulltext : 108666.pdf (Publisher’s version ) (Open Access) MicroRNAs (miRNAs) constitute a class of small, non-coding RNAs that act as post-transcriptional regulators of gene expression. In neurons, the functions of individual miRNAs are just beginning to emerge, and recent studies have elucidated roles for neural miRNAs at various stages of neuronal development and maturation, including neurite outgrowth, dendritogenesis, and spine formation. Notably, miRNAs regulate mRNA translation locally in the axosomal and synaptodendritic compartments, and thereby contribute to the dynamic spatial organization of axonal and dendritic structures and their function. Given the critical role for miRNAs in regulating early brain development and in mediating synaptic plasticity later in life, it is tempting to speculate that the pathology of neurological disorders is affected by altered expression or functioning of miRNAs. Here we provide an overview of recently identified mechanisms of neuronal development and plasticity involving miRNAs, and the consequences of miRNA dysregulation.