Angiotensin II mediates the axonal trafficking of tyrosine hydroxylase and dopamine ß-hydroxylase mRNAs and enhances norepinephrine synthesis in primary sympathetic neurons.

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 ß-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.

Nuclear-Encoded Mitochondrial mRNAs: A Powerful Force in Axonal Growth and Development.

Axons, their growth cones, and synaptic nerve terminals are neuronal subcompartments that have high energetic needs. As such, they are enriched in mitochondria, which supply the ATP necessary to meet these demands. To date, a heterogeneous population of nuclear-encoded mitochondrial mRNAs has been identified in distal axons and growth cones. Accumulating evidence suggests that the local translation of these mRNAs is required for mitochondrial maintenance and axonal viability. Here, we review evidence that suggests a critical role for axonal translation of nuclear-encoded mitochondrial mRNAs in axonal growth and development. Additionally, we explore the role that site-specific translation at the mitochondria itself may play in this process. Finally, we briefly review the clinical implications of dysregulation of local translation of mitochondrial-related mRNAs in neurodevelopmental disorders.

Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons.

Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50-bp sequence element in the 3'untranslated region (3'UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.e., zip-code). In the present study, the hypothesis was tested that local translation of TH plays an important role in the biosynthesis of the catecholamine neurotransmitters in the axon and/or presynaptic nerve terminal. Toward this end, a targeted deletion of the axonal transport sequence element was developed, using the lentiviral delivery of the CRISPR/Cas9 system, and two guide RNA (gRNA) sequences flanking the 50-bp cis-acting regulatory element in rat superior cervical ganglion (SCG) neurons. Deletion of the axonal transport element reduced TH mRNA levels in the distal axons and reduced the axonal protein levels of TH and TH activity as measured by phosphorylation of SER40 in SCG neurons. Moreover, deletion of the zip-code diminished the axonal levels of dopamine (DA) and norepinephrine (NE). Conversely, the local translation of exogenous TH mRNA in the distal axon enhanced TH levels and activity, and elevated axonal NE levels. Taken together, these results provide direct evidence to support the hypothesis that TH mRNA trafficking and local synthesis of TH play an important role in the synthesis of catecholamines in the axon and presynaptic terminal.

MicroRNA-338 modulates cortical neuronal placement and polarity.

The precise spatial and temporal regulation of gene expression orchestrates the many intricate processes during brain development. In the present study we examined the role of the brain-enriched microRNA-338 (miR-338) during mouse cortical development. Reduction of miR-338 levels in the developing mouse cortex, using a sequence-specific miR-sponge, resulted in a loss of neuronal polarity in the cortical plate and significantly reduced the number of neurons within this cortical layer. Conversely, miR-338 overexpression in developing mouse cortex increased the number of neurons, which exhibited a multipolar morphology. All together, our results raise the possibility for a direct role for this non-coding RNA, which was recently associated with schizophrenia, in the regulation of cortical neuronal polarity and layer placement.

Altered expression of circadian rhythm and extracellular matrix genes in the medial prefrontal cortex of a valproic acid rat model of autism.

Autism spectrum disorders (ASD) are a highly heterogeneous group of neurodevelopmental disorders caused by complex interplay between various genes and environmental factors during embryonic development. Changes at the molecular, cellular and neuroanatomical levels are especially evident in the medial prefrontal cortex (mPFC) of ASD patients and are particularly contributing to social impairments. In the present study we tested the hypothesis that altered neuronal development and plasticity, as seen in the mPFC of ASD individuals, may result from aberrant expression of functionally connected genes. Towards this end, we combined transcriptome sequencing and computational gene ontology analysis to identify the molecular networks impaired in the mPFC of a valproic acid (VPA) rat model of autism. This investigation identified two subsets of genes differentially expressed in the mPFC of VPA rats: one group of genes being functionally involved in the regulation of the circadian rhythm, while the second group encompasses a set of differentially expressed collagen genes acting within the extracellular matrix. Ultimately, our integrated transcriptome analysis identified a distinct subset of altered gene networks in the mPFC of VPA rats, contributing to our understanding of autism at the molecular level, thus providing novel insight into the genetic alterations associated with this neurodevelopmental disorder.

Molecular determinants of cytochrome C oxidase IV mRNA axonal trafficking.

In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.

The schizophrenia risk gene MIR137 acts as a hippocampal gene network node orchestrating the expression of genes relevant to nervous system development and function.

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.

MicroRNA-338 Attenuates Cortical Neuronal Outgrowth by Modulating the Expression of Axon Guidance Genes.

MicroRNAs (miRs) are small non-coding RNAs that confer robustness to gene networks through post-transcriptional gene regulation. Previously, we identified miR-338 as a modulator of axonal outgrowth in sympathetic neurons. In the current study, we examined the role of miR-338 in the development of cortical neurons and uncovered its downstream mRNA targets. Long-term inhibition of miR-338 during neuronal differentiation resulted in reduced dendritic complexity and altered dendritic spine morphology. Furthermore, monitoring axon outgrowth in cortical cells revealed that miR-338 overexpression decreased, whereas inhibition of miR-338 increased axonal length. To identify gene targets mediating the observed phenotype, we inhibited miR-338 in cortical neurons and performed whole-transcriptome analysis. Pathway analysis revealed that miR-338 modulates a subset of transcripts involved in the axonal guidance machinery by means of direct and indirect gene targeting. Collectively, our results implicate miR-338 as a novel regulator of cortical neuronal maturation by fine-tuning the expression of gene networks governing cortical outgrowth.

A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons.

Mitochondria are enriched in subcellular regions of high energy consumption, such as axons and pre-synaptic nerve endings. Accumulating evidence suggests that mitochondrial maintenance in these distal structural/functional domains of the neuron depends on the "in-situ" translation of nuclear-encoded mitochondrial mRNAs. In support of this notion, we recently provided evidence for the axonal targeting of several nuclear-encoded mRNAs, such as cytochrome c oxidase, subunit 4 (COXIV) and ATP synthase, H+ transporting and mitochondrial Fo complex, subunit C1 (ATP5G1). Furthermore, we showed that axonal trafficking and local translation of these mRNAs plays a critical role in the generation of axonal ATP. Using a global gene expression analysis, this study identified a highly diverse population of nuclear-encoded mRNAs that were enriched in the axon and presynaptic nerve terminals. Among this population of mRNAs, fifty seven were found to be at least two-fold more abundant in distal axons, as compared with the parental cell bodies. Gene ontology analysis of the nuclear-encoded mitochondrial mRNAs suggested functions for these gene products in molecular and biological processes, including but not limited to oxidoreductase and electron carrier activity and proton transport. Based on these results, we postulate that local translation of nuclear-encoded mitochondrial mRNAs present in the axons may play an essential role in local energy production and maintenance of mitochondrial function.

Axonal localization and mitochondrial association of precursor microRNA 338.

MicroRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons, and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can locally be processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatiotemporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs.

MicroRNA-326 acts as a molecular switch in the regulation of midbrain urocortin 1 expression.

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.

The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons.

Synthesis and regulation of catecholamine neurotransmitters in the central nervous system are implicated in the pathogenesis of a number of neuropsychiatric disorders. To identify factors that regulate the presynaptic synthesis of catecholamines, we tested the hypothesis that the rate-limiting enzyme of the catecholamine biosynthetic pathway, tyrosine hydroxylase (TH), is locally synthesized in axons and presynaptic nerve terminals of noradrenergic neurons. To isolate pure axonal mRNA and protein, rat superior cervical ganglion sympathetic neurons were cultured in compartmentalized Campenot chambers. qRT-PCR and RNA in situ hybridization analyses showed that TH mRNA is present in distal axons. Colocalization experiments with nerve terminal marker proteins suggested that both TH mRNA and protein localize in regions of the axon that resemble nerve terminals (i.e., synaptic boutons). Analysis of polysome-bound RNA showed that TH mRNA is present in polysomes isolated from distal axons. Metabolic labeling of axonally synthesized proteins labeled with the methionine analog, L-azidohomoalanine, showed that TH is locally synthesized in axons. Moreover, the local transfection and translation of exogenous TH mRNA into distal axons facilitated axonal dopamine synthesis. Finally, using chimeric td-Tomato-tagged constructs, we identified a sequence element within the TH 3'UTR that is required for the axonal localization of the reporter mRNA. Taken together, our results provide the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated. These findings raise the interesting possibility that the biosynthesis of the catecholamine neurotransmitters is locally regulated in the axon and/or presynaptic nerve terminal.

Monitoring mRNA Translation in Neuronal Processes Using Fluorescent Non-Canonical Amino Acid Tagging.

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.

MicroRNA-181 promotes synaptogenesis and attenuates axonal outgrowth in cortical neurons.

MicroRNAs (miRs) are non-coding gene transcripts abundantly expressed in both the developing and adult mammalian brain. They act as important modulators of complex gene regulatory networks during neuronal development and plasticity. miR-181c is highly abundant in cerebellar cortex and its expression is increased in autism patients as well as in an animal model of autism. To systematically identify putative targets of miR-181c, we repressed this miR in growing cortical neurons and found over 70 differentially expressed target genes using transcriptome profiling. Pathway analysis showed that the miR-181c-modulated genes converge on signaling cascades relevant to neurite and synapse developmental processes. To experimentally examine the significance of these data, we inhibited miR-181c during rat cortical neuronal maturation in vitro; this loss-of miR-181c function resulted in enhanced neurite sprouting and reduced synaptogenesis. Collectively, our findings suggest that miR-181c is a modulator of gene networks associated with cortical neuronal maturation.

Dysregulation of the axonal trafficking of nuclear-encoded mitochondrial mRNA alters neuronal mitochondrial activity and mouse behavior.

Local translation of nuclear-encoded mitochondrial mRNAs is essential for mitochondrial activity, yet there is little insight into the role that axonal trafficking of these transcripts play in neuronal function and behavior. Previously, we identified a 38 nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the Cytochrome C oxidase IV (COXIV) mRNA that directs the transport of a reporter mRNA to the axon of superior cervical ganglion neurons (SCG). Overexpression of a chimeric reporter mRNA with the COXIV zipcode competed with the axonal trafficking of endogenous COXIV mRNA, and led to attenuated axon growth in SCG neurons. Here, we show that exogenous expression of the COXIV zipcode in cultured SCG neurons also results in the reduction of local ATP levels and increases levels of reactive oxygen species (ROS) in the axon. We took advantage of this "competition" phenotype to investigate the in vivo significance of axonal transport of COXIV mRNA. Toward this end, we generated transgenic mice expressing a fluorescent reporter fused to COXIV zipcode under a forebrain-specific promoter. Immunohistological analyses and RT-PCR analyses of RNA from the transgenic mouse brain showed expression of the reporter in the deep layer neurons in the pre-frontal and frontal cortex. Consistent with the in vitro studies, we observed increased ROS levels in neurons of these transgenic animals. A battery of behavioral tests on transgenic mice expressing the COXIV zipcode revealed an "anxiety-like" behavioral phenotype, suggesting an important role for axonal trafficking of nuclear-encoded mitochondrial mRNAs in neuronal physiology and animal behavior.

MicroRNAs in the axon and presynaptic nerve terminal.

The distal structural/functional domains of the neuron, to include the axon and presynaptic nerve terminal, contain a large, heterogeneous population of mRNAs and an active protein synthetic system. These local components of the genetic expression machinery play a critical role in the development, function, and long-term viability of the neuron. In addition to the local mRNA populations these presynaptic domains contain a significant number of non-coding RNAs that regulate gene expression post-transcriptionally. Here, we review a small, but rapidly evolving literature on the composition and function of microRNAs that regulate gene expression locally in the axon and nerve terminal. In this capacity, these small regulatory RNAs have a profound effect on axonal protein synthesis, local energy metabolism, and the modulation of axonal outgrowth and branching.

Intra-axonal synthesis of eukaryotic translation initiation factors regulates local protein synthesis and axon growth in rat sympathetic neurons.

Axonal protein synthesis is a complex process involving selective mRNA localization and translational regulation. In this study, using in situ hybridization and metabolic labeling, we show that the mRNAs encoding eukaryotic translation initiation factors eIF2B2 and eIF4G2 are present in the axons of rat sympathetic neurons and are locally translated. We also report that a noncoding microRNA, miR16, modulates the axonal expression of eIF2B2 and eIF4G2. Transfection of axons with precursor miR16 and anti-miR16 showed that local miR16 levels modulated axonal eIF2B2 and eIF4G2 mRNA and protein levels, as well as axon outgrowth. siRNA-mediated knock-down of axonal eIF2B2 and eIF4G2 mRNA also resulted in a significant decrease in axonal eIF2B2 and eIF4G2 protein. Moreover, results of metabolic labeling studies showed that downregulation of axonal eIF2B2 and eIF4G2 expression also inhibited local protein synthesis and axon growth. Together, these data provide evidence that miR16 mediates axonal growth, at least in part, by regulating the local protein synthesis of eukaryotic translation initiation factors eIF2B2 and eIF4G2 in the axon.

MicroRNA-338 regulates the axonal expression of multiple nuclear-encoded mitochondrial mRNAs encoding subunits of the oxidative phosphorylation machinery.

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.

Local translation of ATP synthase subunit 9 mRNA alters ATP levels and the production of ROS in the axon.

To date, it has been demonstrated that axonal mRNA populations contain a large number of nuclear-encoded mRNAs for mitochondrial proteins. Here, we report that the mRNA encoding ATP synthase subunit 9 (ATP5G1), a key component of Complex V of the oxidative phosphorylation chain, is present in the axons of rat primary sympathetic neurons, as judged by in situ hybridization and qRT-PCR methodology. Results of metabolic labeling studies establish that this nuclear-encoded mRNA is translated in the axon. The siRNA-mediated knock-down of axonal ATP5G1 mRNA resulted in a significant reduction of axonal ATP5G1 protein and ATP levels. Silencing of local ATP5G1 expression enhanced the production of local reactive oxygen species (ROS). Importantly, reduction in the levels of ATP5G1 expression resulted in a marked attenuation in the rate of elongation of the axon. Exposure of the distal axons to nordihydroguaiaretic acid (NDGA), a ROS scavenger, mitigated the reduction in the rate of axon elongation observed after knock-down of ATP5G1. Taken together, these data call attention to the key regulatory role that local translation of nuclear-encoded mitochondrial mRNAs plays in energy metabolism and growth of the axon.

Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons.

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.