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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2012 November 13.
Published in final edited form as:
PMCID: PMC3496265
NIHMSID: NIHMS160444

MICRORNA-338 REGULATES LOCAL CYTOCHROME C OXIDASE IV mRNA LEVELS AND OXIDATIVE PHOSPHORYLATION IN THE AXONS OF SYMPATHETIC NEURONS

Abstract

MicroRNAs (miRs) are evolutionarily conserved, non-coding RNA molecules of approximately 21 nucleotides 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 cytochrome c oxidase complex IV.

Keywords: Mitochondria, ATP synthesis, RNA localization, inhibitory RNA, oxidative phosphorylation, local translation, norepinephrine uptake

Introduction

Over the past few years, it has become widely accepted that a distinct subset of neuronal mRNAs are selectively transported to the distal structural/functional domains of the neuron, including the axon and presynaptic nerve terminal. Local proteins synthesized from these mRNAs play a key role in the development of the neuron and the function of the axon and nerve terminal (Cox et al., 2008; Wu et al., 2005; Hillefors et al., 2007; Campbell et al., 2001; Poon et al., 2006). The importance of local protein synthesis for mitochondrial function and viability of distal axons was demonstrated in previous studies (Hillefors et al., 2007). Mitochondria are thought to be closely associated with synapses and tethered to vesicle release sites (Zenisek and Matthews, 2000). Synaptic transmission requires mitochondrial ATP generation and control of local [Ca2+]i for neurotransmitter exocytosis, vesicle recruitment, and potentiation of neurotransmitter release (Chang et al., 2006). Results derived from an invertebrate model system revealed that about 25% of the total protein synthesized locally in the nerve terminal were destined for the mitochondria (Gioio et al., 2004). Other studies demonstrated that either the inhibition of local protein synthesis or the blockade of local protein transport into the organelle significantly reduced mitochondrial membrane potential and inhibited the mitochondria’s ability to restore axonal levels of ATP after KCl-induced depolarization (Hillefors et al., 2007; Gioio et al., 2004; Gioio et al., 2001).

Novel molecular mechanisms involving non-coding RNAs have recently been shown to spatially regulate mRNA translation in axons and dendrites. Ashraf et al. (Ashraf et al., 2006) demonstrated that memory-specific patterns of synaptic protein synthesis occur with the induction of a long-term memory in Drosophila, and that these patterns appear to be controlled by the proteasome-mediated degradation of a RISC pathway component. Other studies identified a dendritically localized miR that regulates the expression of the synaptic Limk1 protein, thereby controlling dendritic spine size (Schratt et al., 2006). Importantly, Hengst et al. (Hengst et al., 2006) have shown that key proteins involved in the RNAi / miR pathway, i.e. RISC complexes can assemble and function in developing axons.

Previously, we reported that several nuclear-encoded mitochondrial mRNAs, such as the mRNA encoding COXIV, were present in the distal axons of rat sympathetic neurons (Hillefors et al., 2007; Gioio et al., 2001). COXIV has been demonstrated to have an essential role in the assembly of the cytochrome c oxidase complex, suggesting a tight coupling of the local synthesis of cytochrome c oxidase and oxidative phosphorylation (Li et al., 2006). To assess the potential involvement of miRs in the control of the local synthesis of nuclear-encoded mitochondrial proteins in neurons, we analyzed the interrelationship between COXIV and one of its cognate miRs, miR-338. We found that levels of miR-338 increased during axonal outgrowth and maturation, and further demonstrated that this miR can modulate local COXIV levels and oxidative phosphorylation in the distal axons. Taken together, these findings identify a novel mechanism for the local regulation of axonal protein synthesis and respiration by miR in sympathetic neurons.

Materials and Methods

Neuronal cell cultures

SCG were obtained from 3 days-old Harlan Sprague–Dawley rats, and dissociated neurons plated in the center compartment of Campenot compartmented culture dishes as previously described (Hillefors et al., 2007). Cells were cultured in serum-free medium containing NGF (50 ng/ml) for 14–21 days prior to use with media changes every 3–4 days. The complete culture media, including NGF was present in both the central and side compartments throughout the culture period and during all experimental procedures. The side compartments, which contained the distal axons used in these experiments, contained no neuronal soma or non-neuronal cells, as judged by phase-contrast microscopy, as well as ethidium bromide and acridine orange staining.

Bioinformatics and miR target prediction

The miRanda algorithm (John et al., 2004) was used to investigate the 3′UTR sequence of rat COXIV mRNA for putative binding sites of miRs. MiR-338 was selected for further analyses as judged by the low predicted free energy of hybridization with the COXIV mRNA target (−14.9 kcal/mol), and the secondary structure prediction analysis of the COXIV 3′UTR using Mfold (Zuker, 2003). Constructs and primers used in this report were designed using VectorNTI (Invitrogen).

Luciferase reporter gene constructs and luciferase assay

The sense and antisense strands of oligonucleotides coding for the full rat COXIV 3′UTR (for sequence see Fig. 1B), or the 3′UTR minus the putative miR-338 targeting site were synthesized (Invitrogen). Oligonucleotides were annealed and ligated into the HindIII and SpeI sites of pmir-Report luciferase vector (Ambion). Cells were co-transfected with pmir-REPORT β-galactosidase (β-gal), and the luciferase reporter constructs containing either the full COXIV 3′UTR or 3′UTR lacking the miR-338-binding site (ΔMTS), together with inhibitors for miR-338 or a non-targeting control (NT), respectively. SCG neurons were also transfected with a control luciferase reporter vector to determine the level of activity that can be achieved for an unmodified pmir-REPORT luciferase. Twenty-four hours post-transfection, cells were assayed for firefly luciferase and β-gal expression, and β-gal was used to normalize for differences in transfection efficiency. The Dual-Light luminescent reporter gene assay (Applied Biosystems) was used for the detection of firefly luciferase and β-gal in the same sample.

Figure 1
MiR-338 targets rat COXIV

Immunocytochemical analyses

SCG neurons grown in Campenot chambers for 15 days were first fixed with 4% paraformaldehyde in PBS for 1hr at ambient temperature. After washing in PBS, neurons were subsequently permeabilized with 0.3% Triton X-100 in PBS for 30 min and blocked with 10% normal donkey serum in PBS for 1hr. Rabbit polyclonal antibodies against DICER and eIF2c were kindly provided by Dr. Neil Smalheiser (University of Illinois, Chicago). Incubation with anti-DICER or anti-eIF2c antibodies diluted 1:700 and 1:1000, respectively, in blocking solution (2% normal donkey serum in PBS), was carried out overnight at 4°C. Cells were then incubated with donkey cy3-labeled, affinity purified anti-rabbit IgG (Jackson ImmunoResearch) diluted 1:200 in blocking solution for 1hr and washed in PBS. Images were captured using a Nikon Eclipse TE 300 fluorescence microscope equipped with a Nikon Digital Sight DS-L1 camera (Nikon, Melville, NY).

Analyses of miR and COXIV mRNA

For in situ hybridization of miR-338 and the scramble non-targeting miR, digoxigenin labeled antisense-locked nucleic acid (LNA) oligonucleotides were obtained from Exiqon (Woburn, MA). Tailed LNA oligonucleotides were purified and used for overnight hybridization at 37 °C. The probe concentration in the hybridization mix was 0.3 μM. All other hybridization conditions and procedures were previously described (Hillefors et al., 2007).

For the analysis of miR-338 expression by quantitative RT-PCR, distal axons located in the side chambers and soma and proximal axons in the central chamber were harvested separately, and total RNA prepared using the Cells-to-Signal lysis buffer (Ambion), and used directly for reverse transcription. The TaqMan microRNA Assay (Applied Biosystems) was used to quantify the expression of the mature miR-338 and values expressed relative to β-actin mRNA. TaqMan MicroRNA Assays use stem-looped primers that enable a two-step quantification of miRs present in a sample. In the first step, stem-looped primers anneal to target mature miRs and extend the length of the molecule by reverse transcription PCR. In the second step, a real-time PCR reaction that involves a forward primer, a reverse primer, and a TaqMan probe quantifies the number of mature miR molecules present in a sample based on fluorescent emission of a reporter dye (Chen et al., 2005).

For COXIV mRNA analyses, quantitative RT-PCR was performed on total RNA prepared from SCG axons and cell somas using the Cells-to-Signal lysis buffer (Ambion). RT-PCR was done essentially as previously described (Hillefors et al., 2007). The relative levels of each transcript were normalized to the β-actin mRNA to provide an internal control for reverse transcription and axonal density. RNA values are expressed relative to control by the comparative threshold method (CT).

Preparation of siRNA for COXIV silencing

Two independent siRNAs targeting rat COXIV were tested for silencing in SCG neurons. The siRNA sense and antisense strands were purchased from Dharmacon with the following sequences: siRNA-1, sense 5′-GGAGUGUUGUGAAGAGUGAUU-3′, antisense 5′-UCACUCUUCACAACACUCCUU-3′; siRNA-2, sense 5′-CCUCAUACCU UUGAUCGUGUU-3′, antisense 5′-CACGAUCAAAGGUAUGAGGUU-3′; and scramble control, sense 5′-UAGCGACUAAACACAUCAA-3′, antisense 5′-UAAGGCUAUGAAGAGAUAC-3′. The capacity of each siRNA to reduce the expression of COXIV was determined by transient transfection into the distal axons, or soma and proximal axons as explained below. The expression level of COXIV was determined by qRT-PCR, and Western blotting using rabbit monoclonal antibodies against rat COXIV (Cell Signaling Technology).

Transfection of neurons with luciferase reporter plasmids, siRNAs, miR precursors and anti-miRs

The transfection of reporter gene plasmids into the neuronal soma located in the central compartment of the Campenot cultures was conducted using NeuroPorter (Genlantis) according to the manufacturer’s instructions. The double-stranded RNA that mimics endogenous rat precursor miR-338, and miR-NT, employed as a non-targeting precursor control, were obtained from Ambion. In addition, the miR inhibitor, anti-miR-338, as well as non-targeting control anti-miR-NT were obtained from Ambion. The introduction of small RNAs (miRs or siRNAs, each at 25 nM final concentration) into the distal axons located in the side chambers of the culture dishes, or the soma and proximal axons in the center compartment was accomplished by lipofection using siPORT NeoFX (Ambion). The Campenot compartmentized culture used in the present studies contained two lateral compartments that harbored the distal axons, both were transfected independently, and the total RNA were tested separately from each other to increase the sample number of the analyses.

Immunoblot assay

Distal axons in the side chambers or soma and proximal axons in the central chamber were harvested separately and lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1mM EDTA, 1% NP-40, and Complete protease inhibitor cocktail (Roche). Equal amounts of each lysate were applied to Hybond ECL nitrocellulose membranes (GE Healthcare). Membranes were blocked using the reagent provided with the ECL plus Western blotting detection kit (GE Healthcare) for 1 hr and incubated for 1 hr with rabbit monoclonal antibodies against either rat COXIV, or rat β-actin (Sigma). Membranes were washed in 1X TBS-T and incubated with horseradish peroxidase-labeled secondary antibody for 1 hr at room temperature. After washing, membranes were developed with the ECL plus Western blotting detection reagents. Dot blots were quantified using the NIH software Image J. The specifity of the antibodies used was assessed by SDS-PAGE Western blot analysis. A single band was detected at the expected molecular weight corresponding to COXIV and β-actin polypeptides, respectively.

Mitochondrial functional assays

The Alamar Blue (AB) reduction assay was used to examine the metabolic activity of mitochondria in SCG neuronal cultures transfected with anti-miR-338, miR-338 precursor, or non-targeting short oligonucleotides in the axon and soma compartments. AB is an oxidation-reduction indicating dye that produces a colorometric change in response to metabolic activity. The AB assay evaluates the metabolic activity of cells based on the reduction of resazurin (blue and nonfluorescent) to resorufin (pink and highly fluorescent) in the presence of metabolically active cells (Nakayama et al., 1997). This indicator is set up to detect oxidation by the whole of the electron transport chain. With AB the cells remain fully functional and healthy, unaffected by the presence of the indicator. 10% AB (AbD Serotec) was added to either axonal side chambers or center chambers immediately after lipofection, and the cultures were incubated for 24 – 48 hrs as indicated. Absorbance was quantified by the measurements at 570 and 595 nm, using a microplate reader (UVSpectramax, Molecular Devices), and triplicate samples run from each side compartment sample. The relative levels of AB absorbance from anti-miR-338, or miR precursor lipofected axons were compared to non-targeted miR and sham-transfected controls, and expressed as percentage of control.

ATP levels were assessed using CellTiter-Glo luminescent cell viability assay from Promega using the manufacturer’s instruction. Briefly, SCG neurons were transfected with anti-miR-338 or the non-targeting control oligonucleotide (anti-miR-NT). After transfection (24 hrs), the distal axons and soma were independently harvested, and CellTiter-Glo reagent (100 μl) was added to lyse the soma and distal axons, and the luminescence was then recorded in a luminometer with an integration time of 1 second per well. The luminescent signals for the anti-miR-338- and anti-miR-NT-treated cells were normalized to the relative luminescent signal of mock-treated cells.

Statistical analysis

Quantitative data are presented as the mean ± SEM. Student’s t-test was used to determine significant differences between two groups. One-Way ANOVA was used to analyze significant differences among multiple groups; p ≤ 0.05 was considered significant.

Results

MiR-338 responsive element in rat COXIV mRNA

The relatively short rat COXIV 3′UTR contains a specific 8-mer which has the potential to function as a putative hybridization site for miR-338 (Fig. 1A). A RNA secondary structure prediction analysis using Mfold (Zuker, 2003) revealed that the apparent miR-338 target site (MTS) is positioned on a hairpin-loop structure, in an exposed position, that might facilitate miR accessibility (Fig. 1C). The predicted MTS in COXIV 3′UTR had a low predicted free energy of hybridization with the cognate miR (−14.9 kcal/mol), suggesting a stable miR:MTS duplex within the 8-nt seed region at the 5′ end of the miR. This seed sequence is an important determinant of miR-induced repression of gene expression (Doench and Sharp, 2004).

To assess whether miR-338 can specifically target COXIV mRNAs, dissociated superior cervical ganglia (SCG) neurons were isolated from 3 day-old rats and cultured in Campenot multi-compartment chambers for 14 – 21 days and subsequently transfected with either a luciferase reporter plasmid containing the COXIV 3′UTR, or a control luciferase plasmid (Fig. 1D). When compared to the control condition, the presence of the COXIV 3′UTR reduced luciferase activity ~25% – 50%. Luciferase levels did not change when the anti-miR-338 was co-transfected with this reporter plasmid, indicating that COXIV is specifically targeted by miR-338. Results of a deletion experiment confirmed that the putative MTS was specifically targeted by miR-338. For example, if the 8-mer seed sequence was removed from the 3′UTR construct, luciferase levels did not vary from those of the control reporter (Fig. 1D). Identical results were obtained using the rat neuroblastoma B35 cell line (data not shown).

MiR-338 abundance during axonal outgrowth and maturation

Even though miR-338 is known to be specifically expressed in neuronal tissue (Kim et al., 2004; Wienholds et al., 2005), little is known about its abundance and function during neuronal maturation and axonal outgrowth. Using in situ hybridization (ISH), the subcellular localization of miR-338 RNA within SCG neurons cultured in Campenot multi-compartment chambers (Campenot, 1977) was examined. Unlike the scrambled miR probe, employed as a negative control, hybridization with the miR-338-specific probe revealed the presence of miR-338 in the distal axons (Fig. 2A), as well as the soma and proximal axons (Fig. 2B). In addition, the miR-338 expression during axonal outgrowth was assessed using a TaqMan microRNA assay. MiR-338 expression levels were normalized to β-actin mRNA, which is relatively abundant in the axons of these neurons (Eng et al., 1999). After three days in culture, miR-338 was expressed at low levels throughout the neuron, but after 21 days in culture, its relative abundance had increased approximately 3–4 fold in the soma, and 6-fold in distal axons (Fig. 2E, F), suggesting that this miR may play a role in neuronal maturation, particularly in the elaboration of the structure and function of SCG axons.

Figure 2
Mature miR-338 is expressed in SCG neurons and is present in proximal and distal axons

Regulation of axonal levels of COXIV mRNA and protein by miR-338

To investigate the possibility that mature miRs might function in the axons, the expression and localization of Dicer and the RNA-induced silencing complex (RISC) component eIF2c in the distal axons, as well as proximal axons and soma of sympathetic neurons was studied by immunocytochemistry. A recent report has shown that DRG axons in culture are capable of autonomously silencing a gene without the contribution of the cell body, thereby spatially regulating gene expression (Hengst et al., 2006). Antibodies against rat eIF2c and DICER were used to visualize RISC complexes in SCG neurons by fluorescence microscopy. DICER and eIF2c antibodies revealed the presence of granule-like structures in the cell bodies and along the entire length of the axon (Fig. 3A and B). These granular structures are believed to represent complexes of RNA and proteins (RNP particles). The demonstration that the proteins DICER and eIF2c are localized in the distal axons supports the hypothesis that microRNA plays a role in the regulation of mRNA levels in the axons.

Figure 3
Localization of Dicer and eIF2c in the soma and axons of sympathetic neurons

To assess pre-miR processing in the distal axons, SCG axons were transfected with the precursor miR-338. Subsequently, the steady-state levels of mature miR-338 was determined using specific stem-loop primers for reverse transcription (RT) of mature miR-338 followed by real-time TaqMan within a two-step RT-PCR assay. Transfection with precursor miR-338 resulted in an approximately 10- and 42-fold increase in mature miR-338 levels compared to the endogenous miR-338 levels in sham-transfected axons, one, and four hours post transfection, respectively. After 24 hours, we observed a 42,000-fold increase in mature miR-338 levels in the axons, as compared to miR-338 levels in sham-transfected control axons (data not shown). These results demonstrated that the distal axons of SCG neurons have the capability of processing microRNA precursors to the mature form of the molecule.

To explore whether mature miR-338 regulates COXIV mRNA levels in the distal axons of SCG neurons, we monitored COXIV mRNA levels after transfecting the soma or the distal axons with the miR-338 precursor (pre-miR-338). COXIV mRNA levels decreased ~80% in both neuronal compartments when compared to the non-targeting pre-miR-NT (Fig. 4A). Conversely, lipofection of anti-miR-338 into the distal axons resulted in a 2-fold increase in COXIV mRNA as early as 4 hrs after axonal transfection (Fig. 4C), and in a 3.5-fold increase within 24 hrs (Fig. 4B), when compared to the non-targeting anti-miR control. The rapid nature of the response argues against the possibility that increases in axonal COXIV mRNA levels derive from the transport of mitochondria located in the cell soma. To evaluate this postulate, the levels of COXII mRNA, which codes for one of the three subunits of the COX complex IV encoded by the mitochondrial genome was assessed by qRT-PCR. In contrast to the alterations observed in COXIV mRNA levels after miR-338 inhibition, no difference in COXII mRNA levels was observed after transfection (Fig. 4D). Taken together, these findings demonstrate that anti-miR-338-induced elevation of COXIV mRNA levels is not mediated by significant increases in the number of mitochondria present in the distal axons.

Figure 4
miRNA-338 reduces COXIV expression in SCG neurons

To assess whether miR-338-mediated modulation of mRNA also correlated with an alteration in axonal COXIV protein levels, miR-338 was over-expressed in SCG neurons by transfection of miR-338 precursor RNA, and COXIV protein levels were evaluated by immunoblot analysis. Overexpression of miR-338 lead to a ~40% decrease in COXIV protein levels in both the soma and distal axons of SCG neurons (Fig. 4E, G). Inhibition of endogenous miR-338 by its specific anti-miR in the soma and distal axons led to a 2 – 4 fold increase in the expression of COXIV protein, while having no effect on β-actin protein levels (Fig. 4F, H).

MiR-338-mediated COXIV control of axonal mitochondrial function and neurotransmitter uptake

To determine whether miR-338-dependent COXIV down-regulation affects mitochondrial function in the distal axons, axons in the side compartments of the Campenot cultures were transfected with either pre-miR-338, or non-targeting pre-miR-NT. Alamar Blue (AB), a redox dye employed to assess metabolic activity of mitochondria was added to the culture media and the samples were incubated for 24 hrs before the measurement of the relative levels of the reduced and oxidized forms of the dye. The overexpression of miR-338 significantly reduced mitochondrial oxygen consumption in the axons as determined by the reduced form of AB (Fig. 5A). Consistent with the decrease in the COXIV levels and a subsequent decrease in mitochondrial metabolic activity upon overexpression of miR-338, axons transfected with anti-miR-338 displayed a ~50% increase in metabolic oxygen consumption as compared with the non-targeting miR (Fig. 5B). In addition, an anti-miR-338-mediated increase of mitochondrial activity could also be observed in the soma and proximal axons, suggesting a cellular role of miR-338 in the regulation of COXIV levels. Further assessment of the change in mitochondrial activity mediated by miR-338 was observed by the measurement of axonal ATP levels 48 hrs after transfection of distal axons with anti-miR-338. Using the luminometric ATP assay, a marked increase of axonal ATP levels upon local inhibition of endogenous miR-338 was observed, further underlining the importance of the miR-338 in the regulation of axonal respiration (Fig. 5C).

Figure 5
MiR-338 mediated alteration in COXIV levels modulates metabolic activity of mitochondria in the soma and distal axons of sympathetic neurons

To investigate the impact of miR-338-mediated changes in mitochondrial ATP synthesis and respiration on axonal function, distal axons cultured in compartmented culture dishes were transfected with the precursor miR-338 or anti-miR-338, incubated in medium containing [3H] NE (2 μCi/ml), and NE uptake was than measured by liquid scintillation spectrometry. Under these experimental conditions, preincubation of control axons with desipramine (1μM), a NE uptake inhibitor, reduced the content of [3H] NE into distal axons by approximately 85% (mean CPM in control axons: 4465.8 ± 580 SEM; mean CPM in desipramine-treated axons: 708 ± 266.48 SEM, independent t-test, two-tailed p value = 0.0002, n = 12 measurements). As shown in Fig. 6A and B, transfection of distal axons with anti-miR-338 or precursor miR-338 resulted in a 50% increase, or 50% decrease in norepinephrine uptake, respectively. Taken together, these results establish that modulation of miR-38 levels alters ATP synthesis and metabolic rates, as well as NE uptake in the distal SCG axons.

Figure 6
MiR-338 modulates norepinephrine uptake in the axons of sympathetic neurons

To further evaluate the postulate that the axonal effects of miR-338 are mediated, at least in part, by the local synthesis of COXIV, siRNAs targeted against COXIV mRNA were lipofected into the distal axons. After 24 hrs, COXIV mRNA and protein levels were reduced by 75% and 50%, respectively (Fig. 7A, B). In contrast to these findings, the introduction of siRNA into the axons had no effect on somal levels of COXIV mRNA and protein 24 hrs post-transfection. Similar to the findings obtained with miR-338, reduction in COXIV expression by RNA interference resulted in a significant decrease in basal oxygen consumption (Fig. 7C), and axonal ATP levels (Fig. 7D) compared to sham- controls. Transfection of distal axons with non-targeted siRNA (NT) had no effect on these experimental variables. Additionally, knock-down of the COXIV levels resulted in a 25 – 30% reduction in the uptake of [3H] NE in the distal axons (Fig. 7E). These data provide evidence that the local translation of COXIV mRNA plays a key role in regulating the oxidative capacity of the axon.

Figure 7
SiRNA-mediated knock-down of axonal COXIV levels decreases axonal respiration and ATP levels, and diminishes NE uptake in distal axons

Discussion

The regulation of gene expression by miRs represents a remarkable mechanism of posttranscriptional regulation, widely used in plants and animals. MiRs are single-stranded RNA molecules of about 21–23 nucleotides in length, first discovered in Caenorhabditis elegans as regulators of genes involved in developmental timing, and are now believed to modulate the expression of a myriad of genes in animals and plants (Ambros, 2004; Stark et al., 2005; Foshay and Gallicano, 2007; Ambros and Chen, 2007; Kosik, 2006; Kosik and Krichevsky, 2005; Krichevsky et al., 2003). They are highly conserved and involved in the regulation of a subset of biological processes such as cell proliferation, apoptosis, metabolism, cell differentiation, and morphogenesis (Varez-Garcia and Miska, 2005; Ambros and Chen, 2007; Scalbert and Bril, 2008; Nelson et al., 2008; Fiore et al., 2008; Ivanovska et al., 2008). The present studies identify an axonally localized miR that regulates the expression of COXIV, a key protein within the electron transfer chain in mitochondria, thereby controlling local levels of ATP production in the axons of sympathetic neurons. We hypothesized that the non-coding RNA miR-338 acts as a local regulator of COXIV by binding to the 3′UTR of its mRNA, thereby modulating mitochondrial oxidative phosphorylation. To investigate the mechanism of miR-338 repression, luciferase gene reporter constructs that contained the COXIV 3′UTR were utilized and their expression studied in transfected SCG cells. We found that the presence of the 3′UTR of COXIV significantly repressed luciferase activity, while co-transfection of the specific inhibitor anti-miR-338, but not the not-target anti-miR, increased the activity of luciferase in SCG neurons. In addition, we found that overexpression or inhibition of miR-338 in the soma or axons altered COXIV mRNA and protein levels, as determined by qRT-PCR and dot-blot analysis.

The local regulation of gene expression by non-coding RNA is facilitated at different levels in the distal compartments of neurons (Kosik, 2006). In axons, the RNA-induced silencing complex (RISC) may modulate the expression of genes by one of two mechanisms: direct cleavage (in the case of perfect complementarities between the miR guide strand and the mRNA target) or translational attenuation (in cases of partial complementarities between the miR guide strand and the 3′ UTR of target genes)(Anderson et al., 2008; Doench and Sharp, 2004; Doench et al., 2003). The first mechanism is likely to apply to miR-338-mediated modulation of COXIV levels, even though miRBase tool data suggested an imperfect hybridization between miR-338 and the COXIV 3′UTR. This does not preclude the possibility that translation attenuation is an early event in lowering of COXIV protein levels in the distal axons followed by mRNA degradation (Mathonnet et al., 2007).

Our studies demonstrate that regulation of local translation of nuclear-encoded mitochondrial genes is an important contributor for the maintenance of mitochondrial function in the axon. In previous studies, we have shown that several nuclear-encoded mitochondrial mRNAs were present in distal axons and that the local translation of mRNAs is crucial for mitochondrial function. For example, the inhibition of local protein synthesis for 4 hours resulted in a lowering of mitochondrial membrane potential, oxygen consumption and ATP synthetic capacity (Hillefors et al., 2007). These findings suggest that key proteins regulating mitochondrial activity are rapidly turned over in the distal axons. Interestingly, Li et. al. (Li et al., 2006) showed that COXIV plays a rate-limiting role in the assembly of enzyme complex IV and that dysfunctional cytochrome c oxidase resulted in a comprised mitochondrial membrane potential, as well as decreased respiration and ATP levels. These deficits could ultimately have marked effects on axonal growth and function.

To assess the potential regulatory impact of miR-338, we searched the miRBase for nuclear-encoded mitochondrial mRNAs that contained putative miR-338 target sites. This search revealed eighteen nuclear-encoded mRNAs (Table 1), approximately 1% of the total mRNAs which encode mitochondrial proteins. Subsequently, we selected a subset of these mRNAs (n = 8), representing a diversity of mitochondrial functions and structure, and tested for their presence in the distal axons of SCG neurons using qRT-PCR. This experiment identified 4 mRNAs that were present in the axon (data not shown). This finding suggests that miR-338 might regulate multiple, diverse mitochondrial functions by modulating the expression of specific nuclear-encoded mitochondrial genes in the soma or distal parts of SCG neurons.

Table 1
List of nuclear-encoded mitochondrial target genes of miR-338

Our studies further provide evidence that the “overexpression” of miR-338 in the axons diminished COXIV levels and subsequently axonal oxidative phosphorylation. This reduction in respiration had minimal effects on neuronal morphology or viability. The lack of morphological abnormalities in the neuron expressing reduced oxidative phosphorylation actively has been demonstrated by other investigations into axonal transport of mitochondria (Stowers et al., 2002b; Verstreken et al., 2005). One possibility for the stability of the morphology is that intracellular transport of ATP alleviates the global effects caused by an increase in miR-338. It is also possible that only minimal respiration and ATP production are required to maintain neuronal morphology and viability under basal conditions. However, a reduction in ATP production and reduced oxidative phosphorylation may comprise additional axonal functions under prolonged activity or stress that are not observed through morphological alteration. Previous studies demonstrated that ATP and Ca2+ are needed locally at the synapses and mitochondria are highly abundant in axon terminals and are essential for vesicle cycling and neurotransmitter release and uptake (Shepherd and Harris, 1998; Rowland et al., 2000). In the Drosophila mutant, Milton, a defect in synaptic transmission is associated with the loss of mitochondria from the axon terminal (Stowers et al., 2002a). Mitochondria are important regulators of cell survival and death and a dysfunction of mitochondrial energy metabolism leads to reduced ATP production, impaired calcium buffering, and increased generation of reactive oxygen species (ROS) (Beal, 2007; Petrozzi et al., 2007) that are implicated in a subset of neurodegenerative diseases, such as Alzheimer’s disease (Stokin et al., 2005; Stokin and Goldstein, 2006), and Parkinson’s disease (Murdock et al., 2000). In addition, the generation of ROS contributes to aging since overexpression of mitochondrially localized antioxidant enzymes lengthens life span in Drosophila (Ruan et al., 2002; Calingasan et al., 2008).

In conclusion, we have provided evidence that the non-coding miR-338 is a novel regulator of mitochondrial oxidative phosphorylation and axonal function (e.g. NE uptake) in the distal axons through local modulation of COXIV levels. These findings point to a novel mechanism for a soma-independent regulation of respiration in distal axons through neuronal microRNA. In future studies, we will utilize the elegant Campenot culture system to further characterize the subset of miRs that regulates distinct set of target genes involved in axonal maintenance and function.

Acknowledgments

The authors wish to thank Dr. Neil Smalheiser (University of Illinois) for the kind gift of the DICER and eIF2c antibodies. We thank Elena Perry for the assistance with the miR maturation studies. The authors express their gratitude to Dr. Howard Nash (National Institute of Mental Health) for a critical evaluation of the manuscript. This work was supported by the Division of Intramural Research Programs of the National Institute of Mental Health.

Abbreviations

COXIV
cytochrome c oxidase, subunit IV
AB
Alamar Blue
miR
microRNA
UTR
untranslated region
MTS
miR target site
SCG
superior cervical ganglia
ISH
in situ hybridization
NE
norepinephrine
siRNA
small interfering RNA

References

  • Ambros V. The functions of animal microRNAs. Nature. 2004;431:350–355. [PubMed]
  • Ambros V, Chen X. The regulation of genes and genomes by small RNAs. Development. 2007;134:1635–1641. [PubMed]
  • Anderson EM, Birmingham A, Baskerville S, Reynolds A, Maksimova E, Leake D, Fedorov Y, Karpilow J, Khvorova A. Experimental validation of the importance of seed complement frequency to siRNA specificity. 2. RNA. 2008;14:853–861. [PubMed]
  • Ashraf SI, McLoon AL, Sclarsic SM, Kunes S. Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila. Cell. 2006;124:191–205. [PubMed]
  • Beal MF. Mitochondria and neurodegeneration. 8. Novartis Found Symp. 2007;287:183–192. [PubMed]
  • Calingasan NY, Ho DJ, Wille EJ, Campagna MV, Ruan J, Dumont M, Yang L, Shi Q, Gibson GE, Beal MF. Influence of mitochondrial enzyme deficiency on adult neurogenesis in mouse models of neurodegenerative diseases. Neuroscience. 2008;153(4):986–96. [PMC free article] [PubMed]
  • Campbell DS, Regan AG, Lopez JS, Tannahill D, Harris WA, Holt CE. Semaphorin 3A elicits stage-dependent collapse, turning, and branching in Xenopus retinal growth cones. J Neurosci. 2001;21:8538–8547. [PubMed]
  • Campenot RB. Local control of neurite development by nerve growth factor. Proc Natl Acad Sci U S A. 1977;74:4516–4519. [PubMed]
  • Chang DT, Honick AS, Reynolds IJ. Mitochondrial trafficking to synapses in cultured primary cortical neurons. J Neurosci. 2006;26:7035–7045. [PubMed]
  • Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005;33:e179. [PMC free article] [PubMed]
  • Cox LJ, Hengst U, Gurskaya NG, Lukyanov KA, Jaffrey SR. Intra-axonal translation and retrograde trafficking of CREB promotes neuronal survival. Nat Cell Biol. 2008;10:149–159. [PMC free article] [PubMed]
  • Doench JG, Petersen CP, Sharp PA. siRNAs can function as miRNAs. Genes Dev. 2003;17:438–442. [PubMed]
  • Doench JG, Sharp PA. Specificity of microRNA target selection in translational repression. Genes Dev. 2004;18:504–511. [PubMed]
  • Eng H, Lund K, Campenot RB. Synthesis of beta-tubulin, actin, and other proteins in axons of sympathetic neurons in compartmented cultures. J Neurosci. 1999;19:1–9. [PubMed]
  • Fiore R, Siegel G, Schratt G. MicroRNA function in neuronal development, plasticity and disease. Biochim Biophys Acta. 2008;1779(8):471–8. [PubMed]
  • Foshay KM, Gallicano GI. Small RNAs, Big Potential: The Role of MicroRNAs in Stem Cell Function. Curr Stem Cell Res Ther. 2007;2:264–271. [PubMed]
  • Gioio AE, Eyman M, Zhang H, Lavina ZS, Giuditta A, Kaplan BB. Local synthesis of nuclear-encoded mitochondrial proteins in the presynaptic nerve terminal. J Neurosci Res. 2001;64:447–453. [PubMed]
  • Gioio AE, Lavina ZS, Jurkovicova D, Zhang H, Eyman M, Giuditta A, Kaplan BB. Nerve terminals of squid photoreceptor neurons contain a heterogeneous population of mRNAs and translate a transfected reporter mRNA. Eur J Neurosci. 2004;20:865–872. [PubMed]
  • Hengst U, Cox LJ, Macosko EZ, Jaffrey SR. Functional and selective RNA interference in developing axons and growth cones. J Neurosci. 2006;26:5727–5732. [PubMed]
  • Hillefors M, Gioio AE, Mameza MG, Kaplan BB. Axon viability and mitochondrial function are dependent on local protein synthesis in sympathetic neurons. Cell Mol Neurobiol. 2007;27:701–716. [PubMed]
  • Ivanovska I, Ball AS, Diaz RL, Magnus JF, Kibukawa M, Schelter JM, Kobayashi SV, Lim L, Burchard J, Jackson AL, Linsley PS, Cleary MA. MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression. Mol Cell Biol. 2008;28(7):2167–74. [PMC free article] [PubMed]
  • John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human MicroRNA targets. PLoS Biol. 2004;2:e363. [PMC free article] [PubMed]
  • Kim J, Krichevsky A, Grad Y, Hayes GD, Kosik KS, Church GM, Ruvkun G. Identification of many microRNAs that copurify with polyribosomes in mammalian neurons. Proc Natl Acad Sci U S A. 2004;101:360–365. [PubMed]
  • Kosik KS. The neuronal microRNA system. Nat Rev Neurosci. 2006;7:911–920. [PubMed]
  • Kosik KS, Krichevsky AM. The Elegance of the MicroRNAs: A Neuronal Perspective. Neuron. 2005;47:779–782. [PubMed]
  • Krichevsky AM, King KS, Donahue CP, Khrapko K, Kosik KS. A microRNA array reveals extensive regulation of microRNAs during brain development. RNA. 2003;9:1274–1281. [PubMed]
  • Li Y, Park JS, Deng JH, Bai Y. Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex. J Bioenerg Biomembr. 2006;38:283–291. [PMC free article] [PubMed]
  • Mathonnet G, Fabian MR, Svitkin YV, Parsyan A, Huck L, Murata T, Biffo S, Merrick WC, Darzynkiewicz E, Pillai RS, Filipowicz W, Duchaine TF, Sonenberg N. MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F. Science. 2007;317:1764–1767. [PubMed]
  • Murdock DG, Christacos NC, Wallace DC. The age-related accumulation of a mitochondrial DNA control region mutation in muscle, but not brain, detected by a sensitive PNA-directed PCR clamping based method. Nucleic Acids Res. 2000;28:4350–4355. [PMC free article] [PubMed]
  • Nakayama GR, Caton MC, Nova MP, Parandoosh Z. Assessment of the Alamar Blue assay for cellular growth and viability in vitro. J Immunol Methods. 1997;204:205–208. [PubMed]
  • Nelson PT, Wang WX, Rajeev BW. MicroRNAs (miRNAs) in Neurodegenerative Diseases. Brain Pathol. 2008;18:130–138. [PMC free article] [PubMed]
  • Petrozzi L, Ricci G, Giglioli NJ, Siciliano G, Mancuso M. Mitochondria and neurodegeneration. Biosci Rep. 2007;27:87–104. [PubMed]
  • Poon MM, Choi SH, Jamieson CA, Geschwind DH, Martin KC. Identification of process-localized mRNAs from cultured rodent hippocampal neurons. J Neurosci. 2006;26:13390–13399. [PubMed]
  • Rowland KC, Irby NK, Spirou GA. Specialized synapse-associated structures within the calyx of Held. J Neurosci. 2000;20:9135–9144. [PubMed]
  • Ruan H, Tang XD, Chen ML, Joiner ML, Sun G, Brot N, Weissbach H, Heinemann SH, Iverson L, Wu CF, Hoshi T. High-quality life extension by the enzyme peptide methionine sulfoxide reductase. Proc Natl Acad Sci U S A. 2002;99:2748–2753. [PubMed]
  • Scalbert E, Bril A. Implication of microRNAs in the cardiovascular system. Curr Opin Pharmacol. 2008;8(2):181–8. [PubMed]
  • Schratt GM, Tuebing F, Nigh EA, Kane CG, Sabatini ME, Kiebler M, Greenberg ME. A brain-specific microRNA regulates dendritic spine development. Nature. 2006;439:283–289. [PubMed]
  • Shepherd GM, Harris KM. Three-dimensional structure and composition of CA3-->CA1 axons in rat hippocampal slices: implications for presynaptic connectivity and compartmentalization. J Neurosci. 1998;18:8300–8310. [PubMed]
  • Stark A, Brennecke J, Bushati N, Russell RB, Cohen SM. Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3′UTR evolution. Cell. 2005;123:1133–1146. [PubMed]
  • Stokin GB, Goldstein LS. Axonal Transport and Alzheimer’s Disease. Annu Rev Biochem. 2006;75:607–27. [PubMed]
  • Stokin GB, Lillo C, Falzone TL, Brusch RG, Rockenstein E, Mount SL, Raman R, Davies P, Masliah E, Williams DS, Goldstein LS. Axonopathy and transport deficits early in the pathogenesis of Alzheimer’s disease 5. Science. 2005;307:1282–1288. [PubMed]
  • Stowers RS, Megeath LJ, Gorska-Andrzejak J, Meinertzhagen IA, Schwarz TL. Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein. Neuron. 2002b;36:1063–1077. [PubMed]
  • Stowers RS, Megeath LJ, Gorska-Andrzejak J, Meinertzhagen IA, Schwarz TL. Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein. Neuron. 2002a;36:1063–1077. [PubMed]
  • Varez-Garcia I, Miska EA. MicroRNA functions in animal development and human disease. Development. 2005;132:4653–4662. [PubMed]
  • Verstreken P, Ly CV, Venken KJ, Koh TW, Zhou Y, Bellen HJ. Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron. 2005;47:365–378. [PubMed]
  • Wienholds E, Kloosterman WP, Miska E, varez-Saavedra E, Berezikov E, de BE, Horvitz HR, Kauppinen S, Plasterk RH. MicroRNA expression in zebrafish embryonic development. Science. 2005;309:310–311. [PubMed]
  • Wu KY, Hengst U, Cox LJ, Macosko EZ, Jeromin A, Urquhart ER, Jaffrey SR. Local translation of RhoA regulates growth cone collapse. Nature. 2005;436:1020–1024. [PMC free article] [PubMed]
  • Zenisek D, Matthews G. The role of mitochondria in presynaptic calcium handling at a ribbon synapse. Neuron. 2000;25:229–237. [PubMed]
  • Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31:3406–3415. [PMC free article] [PubMed]