InteractiveFly: GeneBrief

mir-124 stem loop: Biological Overview | References


Gene name - mir-124 stem loop

Synonyms -

Cytological map position - 36D1-36D1

Function - microRNA

Keywords - Post-transcriptional regulation, negative regulation of synaptic transmission; dendrite morphogenesis; neuroblast proliferation; gene silencing by miRNA

Symbol - mir-124

FlyBase ID: FBgn0262398

Genetic map position - chr2L:17566370-17566469

Classification - mir-124 precursor RNA

Cellular location - cytoplasmic



EntrezGene

Recent literature
Kong, Y., Wu, J., Zhang, D., Wan, C. and Yuan, L. (2015). The role of miR-124 in Drosophila Alzheimer's disease model by targeting Delta in Notch signaling pathway. Curr Mol Med 15: 980-989. PubMed ID: 26592243
Summary:
Alzheimer's disease (AD) is a neurodegenerative disorder which mainly affects elderly population. MicroRNAs (miRNA) are small RNA molecules that fine-tune gene expression at posttranscriptional level and exert important functions in AD. MicroRNA-124 (miR-124) is a kind of miRNA abundantly expressed in the central nervous system. It is highly conserved from Caenorhabditis elegans to humans. However, its function in AD is still elusive. This study found miR-124 is significantly down-regulated in AD flies. miR-124 mutant flies show impaired climbing ability and shortened lifespan. In contrast, miR-124 expression rescues locomotive defects of AD flies. Using microarray analysis to test gene expression profiles of miR-124 mutant flies, Notch signaling pathway was found to be potentially targeted by miR-124. Further experiments showed that miR-124 regulates Notch ligand Delta expression by acting on specific site of Delta 3'UTR. In addition, reduced Delta expression by RNA interference extends lifespan and ameliorates learning defects of AD Drosophila. Notch inhibitor DAPT can also alleviate AD phenotypes. In conclusion, this study indicates that miR-124 plays neuroprotective roles in AD Drosophila by targeting Delta in Notch signaling pathway, which helps further in the understanding of miRNAs in the molecular pathology of AD.

Zhang, Y., Lamba, P., Guo, P. and Emery, P. (2016). miR-124 regulates the phase of Drosophila circadian locomotor behavior. J Neurosci 36: 2007-2013. PubMed ID: 26865623
Summary:
Animals use circadian rhythms to anticipate daily environmental changes. Circadian clocks have a profound effect on behavior. In Drosophila, for example, brain pacemaker neurons dictate that flies are mostly active at dawn and dusk. miRNAs are small, regulatory RNAs (≈22 nt) that play important roles in posttranscriptional regulation. This study identifies miR-124 as an important regulator of Drosophila circadian locomotor rhythms. Under constant darkness, flies lacking miR-124 (miR-124(KO)) have a dramatically advanced circadian behavior phase. However, whereas a phase defect is usually caused by a change in the period of the circadian pacemaker, this is not the case in miR-124(KO) flies. Moreover, the phase of the circadian pacemaker in the clock neurons that control rhythmic locomotion is not altered either. Therefore, miR-124 modulates the output of circadian clock neurons rather than controlling their molecular pacemaker. Circadian phase is also advanced under temperature cycles, but a light/dark cycle partially corrects the defects in miR-124(KO) flies. Indeed, miR-124(KO) shows a normal evening phase under the latter conditions, but morning behavioral activity is suppressed. In summary, miR-124 controls diurnal activity and determines the phase of circadian locomotor behavior without affecting circadian pacemaker function. It thus provides a potent entry point to elucidate the mechanisms by which the phase of circadian behavior is determined.

Garaulet, D.L., Sun, K., Li, W., Wen, J., Panzarino, A.M., O'Neil, J.L., Hiesinger, P.R., Young, M.W. and Lai, E.C. (2016). miR-124 regulates diverse aspects of rhythmic behavior in Drosophila. J Neurosci 36: 3414-3421. PubMed ID: 27013671
Summary:
Circadian clocks enable organisms to anticipate and adapt to fluctuating environmental conditions. Despite substantial knowledge of central clock machineries, there is less understanding of how the central clock's behavioral outputs are regulated. This study identifies Drosophila miR-124 as a critical regulator of diurnal activity. During normal light/dark cycles, mir-124 mutants exhibit profoundly abnormal locomotor activity profiles, including loss of anticipatory capacities at morning and evening transitions. Moreover, mir-124 mutants exhibit striking behavioral alterations in constant darkness (DD), including a temporal advance in peak activity. Nevertheless, anatomical and functional tests demonstrate a normal circadian pacemaker in mir-124 mutants, indicating this miRNA regulates clock output. Among the extensive miR-124 target network, heterozygosity for targets in the BMP pathway substantially correct the evening activity phase shift in DD. Thus, excess BMP signaling drives specific circadian behavioral output defects in mir-124 knock-outs.



BIOLOGICAL OVERVIEW

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, its single mir-124 locus was deleted, and it was found to be dispensable for gross aspects of neural specification and differentiation. In contrast, a variety of mutant phenotypes were detected that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124-expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology (Sun, 2012).

microRNAs (miRNAs) are ~22 nucleotide (nt) regulatory RNAs that function primarily as post-transcriptional repressors. In animals, miRNAs have propensity to target mRNAs via 6-7 nt motifs complementary to their 5' ends, termed 'seed' regions. This limited pairing requirement has allowed most miRNAs to capture large target networks. Analysis of multigenome alignments indicates that typical human miRNAs have hundreds of conserved targets, and that a majority of protein-coding genes are under miRNA control. The extraordinary breadth of animal miRNA:target networks has been extensively validated by transcriptome and proteome studies (Sun, 2012).

miR-124 is strictly conserved in both primary sequence and spatial expression pattern, being restricted to the nervous system of diverse metazoans, including flies (Aboobaker, 2005), nematodes (Clark, 2010) Aplysia (Rajasethupathy, 2009), and all vertebrates studied (LagosQuintana, 2002; Landgraf, 2007; Wienholds, 2005). Such conservation implies substantial functions of miR-124 in controlling neural gene expression. miR-124 has been a popular model for genomewide investigations of miRNA targeting principles. For example, studies of miR-124 yielded the first demonstration of the downregulation of hundreds of direct targets detected by transcriptome analysis, and that this activity was driven by the miRNA seed region (Lim, 2005). In addition, miR-124 provided one of the first illustrations of spatially anticorrelated expression of a miRNA and its targets and direct identification of Ago-bound target sites (Sun, 2012).

Functional studies have connected vertebrate miR-124 to various aspects of neural specification or differentiation. Studies in chick ascribed miR-124 as a proneural factor that inhibits the anti-neural phosphatase SCP1 (Visvanathan, 2007). However, no substantial effect of miR-124 on chick neurogenesis was found in a parallel study (Cao, 2007), although miR-124 was observed to repress neural progenitor genes such as laminin gamma1 and integrin beta1. In the embryonic mammalian brain, miR-124 was reported to direct neural differentiation by targeting polypyrimidine tract binding protein 1 (PTBP1), a global repressor of alternative splicing in non-neural cells (Makeyev, 2007). In the adult mammalian brain, miR-124 promoted neural differentiation of the immediate progenitors, the transit-amplifying cells (TAs). Here, miR-124 was shown to directly target the transcription factor Sox9, which maintains TAs and is downregulated during neural differentiation (Cheng, 2009). Other mammalian studies bolster the concept that miR-124 promotes neurogenesis (Maiorano, 2009) or neural differentiation (Yu, 2008). One mechanism involves direct repression by miR-124 of Baf53a, a neural progenitor-specific chromatin regulator that must be exchanged for a neural-specific homolog to consolidate neural fate (Yoo, 2009). However, complicating the picture is the recent report that Xenopus miR-124 represses neurogenesis by directly targeting the proneural bHLH factor NeuroD1 (Liu, 2011; Sun, 2012 and references therein).

All vertebrate miR-124 loss-of-function studies have relied on antisense strategies and have yet to be validated by bona fide mutant alleles. However, as the three vertebrate mir-124 loci are co-expressed in the nervous system, analysis of the null situation will require a triple knockout. So far, a mir-124 knockout has only been described in C. elegans, which harbors a single copy of this gene. Like most other miRNA mutants in this species, the loss of miR-124 did not cause obvious developmental, physiological or behavioral phenotypes. Nevertheless, comparison of gene expression in mir-124-expressing cells from wildtype and mir-124 mutant animals revealed strong enrichment in miR-124 target sites amongst upregulated transcripts, revealing the impact of miR-124 on neuronal gene expression (Clark, 2010). The broad, but phenotypically-tolerated, misregulation of miR-124 targets in this species is potentially consistent with the 'fine-tuning' model for miRNA regulation (Sun, 2012).

This study analyzed a knockout of the sole mir-124 gene in Drosophila. Although this mutant is viable and exhibits grossly normal patterning, numerous phenotypes were documented, including short lifespan, increased variation in the number of dendritic branches of sensory neurons, decreased locomotion and aberrant synaptic release at CNS motoneuron synapses. All of these phenotypes were rescued by a single copy of a 19 kilobase (kb) genomic transgene encompassing the mir-124 locus. A transcriptional reporter of mir-124 was generated that recapitulated the CNS expression of endogenous pri-mir-124, and this was used to purify mir-124-expressing cells from stage-matched wild-type and mir-124-mutant embryos. Transcriptome analysis revealed strong enrichment of direct miR-124 targets amongst genes upregulated in mir-124-mutant cells. The miR-124 target network included coordinate repression of multiple components in the retrograde BMP signaling pathway, whose activity controls synaptic release. Loss of miR-124 further correlated with increased activity of other neural miRNAs and the neural translational regulator Pumilio, and had the net effect of impairing transition from the neuroblast to neuronal gene expression signature. Altogether, it was demonstrated that endogenous miR-124 has substantial impact on CNS gene expression, which underlie its requirement for organismal behavior and physiology (Sun, 2012).

These studies of Drosophila mir-124 demonstrate that its loss is compatible with grossly normal neural development and differentiation, despite broad changes in gene expression and global upregulation of direct miR-124 targets. Nevertheless, many clear defects are detected in these mutants, including short lifespan of adult males, defective larval locomotion, and aberrant synaptic transmission. The latter phenotype is perhaps reminiscent of reports that inhibition of Aplysia miR-124 similarly results in an increase in evoked EPSP amplitude (Rajasethupathy, 2009). These phenotypes were confirmed phenotypes to be due to miR-124 loss, as shown by their rescue by a mir-124 genomic transgene. Importantly, these phenotypes were obvious even under optimal culture conditions, demonstrating palpable requirements for this miRNA in the intact animal. It remains to be seen if synaptic overactivity in the mir-124 mutant can be directly linked to the behavioral defects observed at the organismal level. The electrophysiological defects in mir-124 mutants phenocopy activation of BMP signaling at the synapse, and miR-124 directly targets multiple components of this pathway. Still, it remains possible that the many other gene expression changes in mir-124 mutant neurons contribute to its loss of function phenotype. The detailed in vivo transcriptome-wide analysis of endogenous miR-124 targets sets the stage for future studies of how individual targets might affect different settings of miR-124 function (Sun, 2012).

Only a handful of other miRNA mutants are lethal or exhibit overt morphological defects, suggesting that many miRNAs serve as robustness factors. For example, a Drosophila mir-7 mutant exhibits minor cell specification defects, but these are enhanced by heat shock. In addition, the introduction of many C. elegans 'benign' miRNA mutants into genetically sensitized backgrounds uncovers a high frequency of phenotypes. Interestingly, miR-124 is not required for normal dendrite formation per se, but its absence caused a broader distribution of dendrite numbers on ddaD and ddaE neurons, i.e. a 'robustness' defect. It is speculated that environmental or genetic stress may reveal additional requirements for miR-124 in development and differentiation of the nervous system (Sun, 2012).

In light of the broad roles ascribed to endogenous miR-124 in neurogenesis, neural differentiation, and neural physiology (Gao, 2010), all from antisense strategies, the extensive negative data from the current Drosophila mir-124 knockout are equally compelling. While the relevant neural subpopulation may not have been examined, these studies indicate that miR-124 is not required for gross aspects of neurogenesis and differentiation in the embryonic and larval nervous system. Similarly, C. elegans deleted for mir-124, which is expressed mostly in ciliated sensory neurons, do not reveal obvious defects in neural development (Clark, 2010). Given that these invertebrate orthologs of miR-124 are identical in sequence to their vertebrate counterparts, and are highly and specifically expressed in their respective nervous systems, there is not strong reason a priori to suspect that miR-124 should not have comparable requirements amongst different animals. The analysis of vertebrate mir-124 knockouts is therefore highly anticipated (Sun, 2012).

The Drosophila system has been critical for elucidating fundamental features of miRNA target recognition in animals, and for studying specific miRNA-target interactions that mediate phenotype. However, it has been little-used to analyze the effects of miRNA-mediated gene regulation in the animal at the transcriptome-wide level. Perhaps the clearest example is the broad upregulation of maternal transcripts in early embryos lacking the mir-309 cluster. However, most miRNAs are tissue or cell-specific, and while it is much simpler to profile transcripts from whole flies, the inclusion of irrelevant cells can mask the action of the miRNA. For example, only 4/200 transcripts upregulated in mir-8 mutant pupae appeared to be direct conserved targets (Sun, 2012).

By purifying cognate miRNA-expressing cells from wild-type and miRNA-mutant backgrounds, this study succeeded in assessing transcriptome-wide effects of genetic removal of miR-124 with precision. The data provide a new perspective on the utilization of 'anti-targeting' in Drosophila. Previously, miR-124 was selected as a particularly compelling case in which its Drosophila targets were depleted for in situ terms related to nervous system development, and enriched for terms related to epidermal development (Stark, 2005). Since these tissues derive from a common developmental progenitor, the neuroectoderm, this led to a model in which miR-124 may solidify the neural fate by widespread suppression of epidermal genes that should be absent from neurons. This bioinformatic correlation has not been confirmed using an independently-derived set of miRNA targets (Sun, 2012).

Nevertheless, two observations suggest that the feature of mutual exclusion in the Drosophila miR-124 network is of subtle consequence. First, derepressed target genes were not enriched for epidermally-expressed genes. This is consistent with the view that on the transcriptome-wide level, the exclusion of epidermal genes from miR-124-expressing cells is primarily enforced by transcriptional mechanisms. Second, miR-124 targets were preferentially amongst the higher-expressed transcripts in miR-124+ cells, even in wild-type. Moreover, as well-conserved targets were expressed at overall higher absolute levels than poorly-conserved targets in miR-124+ cells, it is concluded that a dominant feature of the miR-124 target network has selected for substantial co-expression of the miRNA and its targets, perhaps to fine-tune their levels. This viewpoint is consistent with analyses of miR-124 targets in human, indicating a unifying theme for this particular miRNA across animals (Sun, 2012).

Early manifestations of the miRNA world emerged from pervasive control of the C. elegans heterochronic pathway and the D. melanogaster Notch pathway by miRNAs, and a few similar situations have been documented, i.e. direct targeting throughout the branched amino acid catabolism pathway by miR-277 or repression of multiple components of fatty acid metabolism by miR-33 (Davalos, 2011). Nevertheless, it is rare for such dedicated target networks to be seen amongst the miRNA oeuvre. Amongst the broad network of miR-124 targets, coordinate targeting of multiple components of the retrograde BMP signaling pathway is striking, including all three receptors (Sax/Tkv/Wit), the downstream transcription factor (Mad) and its cofactor (Medea). It was recently shown that misexpression of activated Sax and Tkv receptors in motoneurons increases evoked excitatory junctional potentials without affecting spontaneous activity, very similar to that of mir-124 mutants (Ball, 2010). This study extends this finding by analysis of activated Tkv alone. Therefore, deregulation of BMP signaling may contribute to the electrophysiological defects observed in mir-124 mutants (Sun, 2012).

Still, a 'one size fits all' description of miR-124 activity is not appropriate, since a number of functional miR-124 targets were observed whose predominant activities are in epidermal or other non-neural derivatives. Thus, the large miR-124 network accommodates a range of target properties. Derepression of a sufficient number of such non-neural transcripts may contribute collectively to the incomplete capacity of mir-124 mutant cells to transition from a neuroblast to neuronal gene expression signature (Sun, 2012).

One may speculate that dysfunction of miRNAs, which have large networks of targets, may trigger global changes in other modes of gene regulation. For example, overexpression of individual miRNAs or siRNAs can de-repress endogenous regulation via non-cognate miRNAs, possibly reflecting a titration mechanism. In addition to a global effect on neuroblast-to-neural transition, it was observed that genes downregulated upon in vivo loss of miR-124 were enriched for seeds of K box miRNAs and miR-10-5p. This is potentially consistent with a model in which absence of this abundant miRNA frees up AGO1 complexes to accept other neural miRNAs, yielding their overactivity. Another plausible mechanism might be that miR-124 represses a transcriptional repressor of these other miRNAs (Sun, 2012).

Pumilio binding sites were strongly associated with downregulated transcripts in mir-124 mutants. Pumilio is well-characterized as a neural RNA binding protein and translational regulator, and affects synaptic function and dendrite morphogenesis, which was also observed to be miR-124-regulated settings. Predictions of conserved miRNA binding sites (e.g., TargetScan or mirSVR) did not identify miR-124 target sites in the annotated pumilio 3' UTR or CDS; however modENCODE data revealed that pumilio transcription extends ~2 kb downstream of its annotated 3' end. The regulatory potential of such long pumilio 3' UTR isoforms remains to be studied. Other possibilities are that miR-124 regulates a transcriptional regulator of pumilio, or that Pumilio activity is altered in mir-124 mutants. Future studies should address the cross-talk of post-transcriptional regulation in neurons mediated by miR-124, neuronal miRNAs and Pumilio (Sun, 2012).

Drosophila miR-124 regulates neuroblast proliferation through its target anachronism

MicroRNAs (miRNAs) have been implicated as regulators of central nervous system (CNS) development and function. miR-124 is an evolutionarily ancient, CNS-specific miRNA. On the basis of the evolutionary conservation of its expression in the CNS, miR-124 is expected to have an ancient conserved function. Intriguingly, investigation of miR-124 function using antisense-mediated miRNA depletion has produced divergent and in some cases contradictory findings in a variety of model systems. This study investigated miR-124 function using a targeted knockout mutant and evidence is presented for a role during central brain neurogenesis in Drosophila. miR-124 activity in the larval neuroblast lineage is required to support normal levels of neuronal progenitor proliferation. anachronism (ana), which encodes a secreted inhibitor of neuroblast proliferation, was functionally identified as an important target of miR-124 acting in the neuroblast lineage. ana has previously been thought to be glial specific in its expression and to act from the cortex glia to control the exit of neuroblasts from quiescence into the proliferative phase that generates the neurons of the adult CNS during larval development. Evidence is provided that ana is expressed in miR-124-expressing neuroblast lineages and that ana activity must be limited by the action of miR-124 during neuronal progenitor proliferation. The possibility is discussed that the apparent divergence of function of miR-124 in different model systems might reflect functional divergence through target site evolution (Weng, 2012).

The finding that miR-124 activity is required in the NB lineage to support proliferation contrasts with findings from vertebrate systems that have suggested a role for miR-124 in limiting neuronal progenitor proliferation and in promoting neuronal differentiation. Several independent studies have reported that miR-124 promotes neuronal differentiation in mouse neural progenitor cells in culture by downregulating inhibitors of differentiation including the RNA splicing regulator PTB1, the SCP1 phosphatase, the transcription factor SOX9 and the ephrin B1 receptor. Depletion of miR-124 using antisense oligonucleotides has been reported to promote proliferation and reduce differentiation of neuronal progenitors isolated from the subventricular zone (SVZ) of the mouse embryonic CNS. Comparable results were obtained by injection of a pump to deliver antisense oligonucleotides to deplete miR-124 in vivo in the SVZ of the mouse brain. Taken together, the analysis of vertebrate miR-124 function mainly lends support to the idea that its expression in differentiating neurons acts to turn off negative regulators of differentiation and that loss of the miRNA supports proliferation of neuronal precursors (Weng, 2012).

A possible basis for the difference between these findings and those in vertebrate systems is that the expression of miR-124, although broadly CNS specific, differs in detail between insects and vertebrates. This study observed expression in neural progenitors, which has not been generally reported in the vertebrate, although the presence of the miR-124 primary transcript in the NB and GMC is consistent with the expression of the miR-124-GFP reporter. In situ hybridization does not reveal significant levels of mature miR-124 in vertebrate neural progenitors, but one report using an in vivo sensor for miRNA activity has indicated that miR-124 activity can be detected in mouse neural progenitor cells. Taken together, these findings prompt the question of whether there might be a corresponding function in the neural progenitors of the vertebrate CNS that might have been overlooked owing to low-level expression of the mature miRNA (Weng, 2012).

The possibililty has also been considered the possibility that the difference between the current findings and those reported using vertebrate models reflect methodological differences, i.e., the use of a genetic null mutant versus miRNA depletion using injected or transfected antisense oligonucleotides to reduce miRNA activity. Previous analysis of genetic mutants has not supported the conclusions of antisense injections to deplete miRNA function in Drosophila embryos. Antisense methods allow partial reduction of function and might introduce a degree of experimental variability. It will be of interest to learn whether mouse knockouts of miR-124 support the findings reported using antisense methods (Weng, 2012).

A third and perhaps more interesting possibility is that the different effects of miR-124 on neuronal progenitor proliferation reflect evolutionary divergence of miR-124 function. miR-124 has hundreds of potential targets as identified by computational prediction, by expression profiling of miRNA overexpression and depletion and by immunopurification of miRNA-containing ribonucleoproteins. There is little evidence of conservation of the identified or predicted targets between insects, nematodes and vertebrates. Furthermore, each of the reports on miR-124 function in vertebrates has attributed its role to the regulation of different targets. This might reflect subtly different roles for the miRNA in different neuronal progenitor cell models in vitro, in different regions of the developing CNS or at different stages of development. Evidence has been presented for different miR-124 functions at different stages in Xenopus eye development. It is also possible that miR-124 acts via several functionally significant targets that each serve as repressors of neuronal differentiation in vivo (Weng, 2012).

In Drosophila, this study has identified ana as a target of miR-124 in vivo and provided direct genetic evidence that downregulation of ana expression in neuronal progenitors is required to support a normal level of proliferation within the larval central brain. The ana gene is not conserved beyond the Drosophila family. This leads to the intriguing proposal that miR-124 might have acquired a novel target in Drosophila, which has led to an entirely distinct function in the control of CNS proliferation from that found in vertebrates. It might also suggest that an evolutionarily ancient and presumably conserved role of miR-124 awaits discovery (Weng, 2012).

miR-124 controls Drosophila behavior and is required for neural development

MicroRNA-124 (miR-124) is an evolutionarily conserved, small, noncoding RNA molecule that participates in the central nervous system (CNS) developmental control of gene expression. This study found that Drosophila embryos lacking the miR-124 gene did not exhibit detectable defects in axon growth or CNS development. In contrast, adult mutants showed severe problems in locomotion, flight, and female fertility. Furthermore, the deficits that were observed in the adult stage could be marginally rescued with elav-GAL4 driven expression of miR-124, making elav-GAL4 an excellently simulated driver to induce entopic over-expression of miR-124. Further developmental assessment in the third instar larval neuromuscular junction (NMJ) and dendritic arborization (DA) neurons was performed with miR-124 knock outs, flies over-expressing miR-124, and rescue models. Typically, the absence and over-abundance of a molecular signal exerts opposite effects on development or phenotype. However, it was determined that both miR-124 knock-outs and over-expressing flies displayed reduced NMJ 6/7 bouton number and branch length. Similarly, reduced ddaE branching numbers were observed between the two mutant lines. As to ddaF, branching number was not influenced by miR-124 knock out, but was statistically reduced by miR-124 over-expression. While it was not possible determine any causal relationship between behavioral defects and dysplasia of NMJs or DA neurons, there were some accompanying relationships among behavioral phenotypes, NMJ abnormalities, and ddaE/ddaF dendritic branching which were all controlled by miR-124 (Wang, 2014).

Aplysia miR-124 functions in constraining synaptic plasticity through CREB

Memory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic plasticity, massive parallel sequencing was carried out to profile the small RNAs of Aplysia californica. 170 distinct miRNAs were identified, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain enriched, and several of these were rapidly downregulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Further characterization of the brain-enriched miRNAs revealed that miR-124, the most abundant and well-conserved brain-specific miRNA, was exclusively present presynaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB. Direct evidence is presented that a modulatory neurotransmitter important for learning can regulate the levels of small RNAs, and a role is presented for miR-124 in long-term plasticity of synapses in the mature nervous system (Rajasethupathy, 2009).

miR-124 serves as a negative constraint on serotonin-induced long-term facilitation, since increased or decreased miR-124 levels in sensory neurons leads to a significant inhibition or enhancement, respectively, of synaptic facilitation. In particular, the inhibition of miR-124 confers to sensory-motor synapses a greater sensitivity for serotonin, since just one pulse of serotonin is sufficient to cause long-term facilitation. These physiology data also suggest that miR-124 inhibition is just one of many 5HT-mediated events that activate CREB to induce long-term facilitation, since the inhibition of miR-124 alone, in the absence of 5HT, does not lead to long-term facilitation. Therefore, while the observed effects of the miR-124 manipulations on LTF are of a significant magnitude, it is likely that these effects would be even greater if there were a coordinated manipulation of several miRNAs that act together in parallel pathways during synaptic plasticity. The observation that miR-124 levels affect facilitation both at 24 and 48 hr after exposure to spaced pulses of serotonin suggests that miR-124 regulation is required not only for the induction phase but that it is also critical for the maintenance phase of synaptic facilitation. Since miR-124 levels return back to baseline within 12 hr after exposure to serotonin, the initial drop in miR-124 during this time window appears to be sufficient enough to upregulate the relevant transcripts to allow for facilitation for up to 48 hr after exposure to serotonin. Indeed, the upregulation of many plasticity-related transcripts are transient and fall into this initial time window. The data also suggest that miR-124 does not significantly affect or contribute to serotonin-independent processes such as basal and constitutive synaptic activity. However, since all of the experiments were conducted on several-day-old cultures, at which point the cells and synapses are fully mature and stable, these studies leave open the possibility that miR-124 contributes to serotonin-independent processes in immature neurons such as neurite out-growth and synapse formation (Rajasethupathy, 2009).

The negative constraint that miR-124 imposes on synaptic facilitation is mediated, at least in part, by its direct regulation of CREB. The fact that miR-124 inhibition significantly and specifically increases CREB1 levels, along with immediate downstream genes such as UCH, C/EBP, and KHC, that miR-124 serotonin kinetics parallels the CREB1 serotonin kinetics, and that miR-124 inhibition can provide the switch necessary to convert short-term facilitation into long-term facilitation all strongly support the conclusion that miR-124 can tightly control CREB and CREB-mediated signaling during plasticity. CREB has been extensively studied over the years for its regulation by kinase-dependent posttranslational modifications, such as phosphorylation by PKA and MAPK. The present study, however, is one of the first to address posttranscriptional regulation of CREB. While this additional level of regulation might appear redundant, for example by paralleling the function of CREB2, it is likely that miR-124 inhibition allows for more rapid and transient control over CREB expression, as well as the opportunity for CREB to be drawn into various distinct downstream pathways once activated. It was also noticed that CREB, in turn, may be able to regulate miR-124 expression levels since there are several putative CREB binding sites in the presumed promoter region upstream of the Aplysia mir-124 gene. Although Aplysia and mammalian systems have clear differences in the complexities of their CNS, and also even in the types of neurotransmitters used during long-term memory processes, the underlying calcium-induced signaling pathways (including cAMP, PKA, MAPK, and CREB) and their functions are very much shared. It is therefore very likely that miR-124 is activity-regulated in the mammalian hippocampus and regulates CREB in much the same way as observed in this study, especially in light of the fact that the mammalian CREB1 UTR bears a conserved miR-124 target site as predicted by targetscan, which was recently confirmed as a site directly bound by Argonaute in mouse brain (Rajasethupathy, 2009).

In summary, this study has identified a comprehensive set of brain-enriched miRNAs in Aplysia, many of which can be regulated by the neuromodulator serotonin, signifying potential roles in learning-related synaptic plasticity. Specifically, it was demonstrated that brain-specific miR-124 responds to serotonin by derepressing CREB and enhancing serotonin-dependent long-term facilitation. This initial study compels the exploration of how neuromodulators act through small RNAs during various forms of plasticity and whether some act locally at synapses. This study also provides evidence that some 5HT-regulated Aplysia miRNAs regulate plasticity-related genes involved in local protein synthesis at the synapse. The likelihood of a coordinated set of miRNAs combinatorially regulating events at the synapse makes possible a new and rich layer of computational complexity that could be responsible for the emergence of discrete and long-lasting states of activity at the synapse (Rajasethupathy, 2009).


REFERENCES

Search PubMed for articles about Drosophila mir-124

Aboobaker, A. A., Tomancak, P., Patel, N., Rubin, G. M. and Lai, E. C. (2005). Drosophila microRNAs exhibit diverse spatial expression patterns during embryonic development. Proc Natl Acad Sci U S A 102: 18017-18022. PubMed ID:16330759

Ball, R. W., Warren-Paquin, M., Tsurudome, K., Liao, E. H., Elazzouzi, F., Cavanagh, C., An, B. S., Wang, T. T., White, J. H. and Haghighi, A. P. (2010). Retrograde BMP signaling controls synaptic growth at the NMJ by regulating trio expression in motor neurons. Neuron 66: 536-549. PubMed ID:20510858

Cao, X., Pfaff, S. L. and Gage, F. H. (2007). A functional study of miR-124 in the developing neural tube. Genes Dev 21: 531-536. PubMed ID:17344415

Cheng, L. C., Pastrana, E., Tavazoie, M. and Doetsch, F. (2009). miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat Neurosci 12: 399-408. PubMed ID:19287386

Clark, A. M., Goldstein, L. D., Tevlin, M., Tavare, S., Shaham, S. and Miska, E. A. (2010). The microRNA miR-124 controls gene expression in the sensory nervous system of Caenorhabditis elegans. Nucleic Acids Res 38: 3780-3793. PubMed ID:20176573

Davalos, A., Goedeke, L., Smibert, P., Ramirez, C. M., Warrier, N. P., Andreo, U., Cirera-Salinas, D., Rayner, K., Suresh, U., Pastor-Pareja, J. C., Esplugues, E., Fisher, E. A., Penalva, L. O., Moore, K. J., Suarez, Y., Lai, E. C. and Fernandez-Hernando, C. (2011). miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling. Proc Natl Acad Sci U S A 108: 9232-9237. PubMed ID:21576456

Gao, F. B. (2010). Context-dependent functions of specific microRNAs in neuronal development. Neural Dev 5: 25. PubMed ID:20920300

Lagos-Quintana, M., Rauhut, R., Yalcin, A., Meyer, J., Lendeckel, W. and Tuschl, T. (2002). Identification of tissue-specific microRNAs from mouse. Curr Biol 12: 735-739. PubMed ID:12007417

Landgraf, P., et al. (2007). A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129: 1401-1414. PubMed ID:17604727

Lim, L. P., Lau, N. C., Garrett-Engele, P., Grimson, A., Schelter, J. M., Castle, J., Bartel, D. P., Linsley, P. S. and Johnson, J. M. (2005). Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433: 769-773. PubMed ID:15685193

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Biological Overview

date revised: 5 December 2014

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