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

doi:10.1523/JNEUROSCI.3338-08.2008

J Neurosci. Author manuscript; available in PMC 2012 November 13.

Published in final edited form as:

J Neurosci. 2008 November 19; 28(47): 12581–12590.

doi: 10.1523/JNEUROSCI.3338-08.2008

PMCID: PMC3496265

NIHMSID: NIHMS160444

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

Armaz Aschrafi, Azik D. Schwechter, Marie G. Mameza, Orlangie Natera-Naranjo, Anthony E. Gioio, and Barry B. Kaplan^*

Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Building 10, Room 4N222, Bethesda, MD 20892-1381, USA

^*Corresponding Author: Dr. Barry B. Kaplan, Molecular Neurobiology Section, Laboratory of Molecular Biology, NIMH, Building 10, Room 4A15, 10 Center Drive, Bethesda, MD 20892-138, Telephone: (301) 451-4512, Facsimile: (301) 594-3189, Email: kaplanb/at/mail.nih.gov

The publisher's final edited version of this article is available free at J Neurosci

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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 (C_T).

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

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