The Journal of Neuroscience, April 24, 2013 • 33(17):7165–7174 • 7165

Cellular/Molecular Intra-axonal Synthesis of Eukaryotic Initiation Factors Regulates Local Synthesis and Axon Growth in Rat Sympathetic Neurons

Amar N. Kar, Margaret A. MacGibeny, Noreen M. Gervasi, Anthony E. Gioio, and Barry B. Kaplan Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892-1381

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 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 metaboliclabelingstudiesshowedthatdownregulationofaxonaleIF2B2andeIF4G2expressionalsoinhibitedlocalproteinsynthesisand 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.

Introduction thetic neurons of the rat superior cervical ganglia (SCG) (Natera- Early studies, conducted in both invertebrate and vertebrate an- Naranjo et al., 2010). One of the most abundant miRs identified imal species, have identified numerous axonally localized mR- in SCG axons was miR16. A bioinformatic search for en- Ј NAs encoding a functionally diverse set of , such as coding mRNAs that contain miR16-binding sites in the 3 un- Ј cytoskeletal elements, translation factors, ribosomal proteins, translated region (3 UTR) revealed eukaryotic translation molecular chaperones, signaling molecules, transcription factors, initiation factors (eIFs), eIF2B2 and eIF4G2, as potential candi- and nuclear-encoded mitochondrial proteins (Perrone-Capano dates. In this communication, we report that the mRNAs encod- et al., 1987; Moccia et al., 2003; Willis et al., 2007; Vogelaar et al., ing eIF2B2 and eIF4G2, components involved in eukaryotic 2009; Tcherkezian et al., 2010; Zivraj et al., 2010; Gumy et al., translation initiation pathway, are present in the axon and that 2011). It has also been shown that local translation plays a key role axonal translation of these factors is regulated by miRNA16. In in axonal functions, such as axon growth, regeneration, synaptic this communication, we also demonstrate that local translation plasticity, signal transduction, and long-term viability (Martin et of eIF2B2 and eIF4G2 plays an important role in the regulation of al., 1997; Campbell and Holt, 2001; Zhang and Poo, 2002; Hanz the axonal protein synthetic system and axon growth. et al., 2003; Si et al., 2003; Verma et al., 2005; Wu et al., 2005; Leung et al., 2006; Hillefors et al., 2007; Cox et al., 2008; Dubacq Materials and Methods et al., 2009; Yoon et al., 2012; Donnelly et al., 2013). Neuronal cell cultures. SCGs were obtained from 3-day-old Harlan In addition to these mRNAs, axons contain a diverse popula- Sprague Dawley rats of either sex. Neurons were dissociated using Milte- tion of small, noncoding RNAs, namely, microRNAs (miRs), nyi Biotec gentle MACS Dissociator and Neuronal Tissue Dissociation which play key roles in the regulation of local protein synthesis Kit according to the manufacturer’s protocol. Dissociated primary neu- (Schratt et al., 2006; Aschrafi et al., 2008; Muddashetty et al., rons were plated into the center compartment of a three-compartmented 2011; Olde Loohuis et al., 2012). Recently, it has been reported Campenot culture chamber (Hillefors et al., 2007). Cells were grown in serum-free Neurobasal medium (Invitrogen) containing nerve growth that ϳ130 different miRNAs are present in the axons of sympa- factor (NGF; 50 ng/ml), 20 mM KCl, and 20 U/ml penicillin and 20 mg/ml streptomycin (Hyclone) for 2–7 d before use. The culture medium was replaced every 3–4 d. Two days after plating, 5-fluoro-2Ј-deoxyuridine Received April 27, 2012; revised Jan. 29, 2013; accepted March 5, 2013. (50 ␮M) was added to the culture medium to inhibit the growth of non- Author contributions: A.N.K., A.E.G., and B.B.K. designed research; A.N.K., M.A.M., and N.M.G. performed re- neuronal cells and remained in the medium for the duration of the ex- search; A.N.K., M.A.M., and N.M.G. analyzed data; A.N.K., M.A.M., and B.B.K. wrote the paper. periments. Culture media also contained NGF at all times. The side This work was supported by the Division of Intramural Research Programs of the National Institute of Mental compartments, which contained the distal axons used in the experi- Health. We thank Ms. Sanah Vohra for invaluable technical assistance and critical reading of the manuscript. The authors declare no competing financial interests. ments, were devoid of neuronal soma and non-neuronal cells, as judged CorrespondenceshouldbeaddressedtoDr.BarryB.Kaplan,LaboratoryofMolecularBiology,NationalInstituteof by phase-contrast microscopy and 5-bromodeoxyuridine staining. Mental Health, Building 10, Room 4N-212, Bethesda, MD 20892. E-mail: [email protected]. Fluorescence in situ hybridization (FISH). In situ hybridization for DOI:10.1523/JNEUROSCI.2040-12.2013 miR16, eIF2B2, and eIF4G2 was performed according to the protocol Copyright © 2013 the authors 0270-6474/13/337165-10$15.00/0 described previously (Natera-Naranjo et al., 2012). Briefly, 7-day-old 7166 • J. Neurosci., April 24, 2013 • 33(17):7165–7174 Kar et al. • Translational Regulation of Axonal Protein Synthesis

SCG axons were fixed in 4% formalin for 15 min and then washed three was measured by monitoring the Click-iT AlexaFluor-594 signal. Fluo- times in TBS-Triton X-100 (1 ϫ TBS, 0.1% Triton X-100) for 5 min. rescence images were captured using a Nikon Eclipse TE 300 fluorescence Axons were subsequently permeabilized in 0.5% Triton X-100 in TBS for microscope, and images were deconvoluted using the Deconvolution 10 min and postfixed with 4% formalin. After washes with TBS-T for 5 Lab Plugin (Vonesch and Unser, 2008). min, acetylation was performed in 25% acetic anhydride, 0.1 M HEPES All fluorescence quantification used original unprocessed image data, for 10 min followed by equilibration in 4ϫ sodium–saline citrate (SSC), with no pixels at zero intensity or saturated. Fluorescence levels were 50% formamide for 20 min. FITC-conjugated locked nucleic acid eIF2B2 quantified using NIH software ImageJ. For quantification of fluores- antisense (5Ј GAAACAAACAUAGCCUAGUCAC 3Ј), eIF4G2 antisense cence, the thick axon bundles in the field were analyzed. The boundary of (5Ј ACAUUUCUGGUUCGGUUUUCAA 3Ј), and negative control the axon bundle was selected by the drawing/selection tool, and the mea- probes (5Ј GUGUAACACGUCUAUACGCCCA 3Ј) were hybridized at surement function of the analysis tool was used to measure the area, 55°C overnight (25 nM probes in 10% dextran sulfate, 4 ϫ SSC, 1 ϫ integrated density, and the mean gray value for each bundle. The inte- Denhardt’s solution, 40% formamide, 1 mM DTT, 0.1 mg/ml yeast tRNA, grated density value was normalized for the area by dividing with the area AND 0.1 mg/ml salmon sperm DNA). After incubation, axons were of the selected bundle. To determine the background, several regions washed once in 40% formamide, 1 ϫ SSC at 55°C for 20 min, and three near the bundle were selected and the mean fluorescence of background times in 1 ϫ SSC at 55°C, 5 min each. The signal intensity of the locked reading was obtained. The relative fluorescence intensity for each bundle nucleic acid probes was amplified with AlexaFluor-488 signal amplifica- was determined as the difference between normalized integrated density tion kit (Invitrogen). value and the product of mean background fluorescence intensity and Luciferase reporter assay. The pEZX-Reporter containing the eIF2B2 the area of the selected bundle. All axon bundles in each culture dish were and eIF4G2 3ЈUTR constructs and the eIF2B2 and eIF4G2 miR16 miR measured, and statistical comparisons were made by measuring at least targeting sequence (MTS) deletion vectors were obtained from GeneCo- 35–40 axon bundles per treatment. poeia. SCG neurons were cotransfected with pEZX-reporter as control, Transfection of neurons with miR precursor, anti-miR, or siRNAs. The or the dual luciferase reporter constructs containing either the full rat precursor miR16, nontargeting precursor control miR-NT (pre-NT), eIF2B2 or eIF4G2 3ЈUTR or eIF2B2 and eIF4G2 3ЈUTR with miR16 MTS inhibitor anti-miR16, and nontargeting control anti-NT were obtained deletion, together with either precursor miR16 or inhibitors for miR16 from Ambion. eIF2B2- and eIF4G2-specific and NT-siRNAs were pur- (anti-miR16) or a nontargeting control (NT), respectively. Twenty-four chased from Dharmacon. The small RNAs (miRs or siRNAs, each at final hours after transfection, cells were assayed for firefly luciferase and Re- concentration of 25 nM), were transfected into distal axons located in the nilla luciferase expression: Renilla luciferase levels were used to normal- lateral compartments of Campenot cultures using siPORT NeoFX (Am- ize differences in transfection efficiency. The Dual-Light luminescent bion). Axons were exposed to media containing the transfection reagents reporter assay kit (Promega) was used for the detection of firefly and for 16 h. The two lateral compartments that harbored the distal axons Renilla luciferase in the same sample, using the GloMax 96 Microplate were transfected independently, and the total RNA was processed sepa- Luminometer (Promega). rately. Two DIV cultures were used for axon growth experiments, and Metabolic labeling of newly synthesized proteins. To label newly synthe- 6–8 DIV were used for all other experiments. sized proteins, the Click-iT Protein Labeling Kit (Invitrogen) was used. Western analysis. Distal axons, were harvested and lysed in 50 mM SCG axons were incubated in methionine-free Neurobasal medium for Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and complete 1 h. The medium was then replaced with methionine-free Neurobasal protease inhibitor mixture (Sigma). Equal amounts of each lysate were medium containing 0.2 ␮M of the methionine analog, L- azidohomoala- fractionated by 4–12% gradient SDS-PAGE (Invitrogen) and electro- nine (AHA). The axons were incubated in AHA-containing medium for blotted onto Hybond-LFP PVDF membrane (GE Healthcare). Mem- 6 h, allowing incorporation of AHA into nascent proteins. Negative con- branes were blocked with ECL Advance Blocking Agent (GE Healthcare) trol cultures were grown in Neurobasal medium containing methionine for 1 h, followed by overnight incubation at 4°C with antibodies against ␤ instead of AHA. After incubation, cells were lysed in 50 mM Tris-HCl, pH either eIF2B2, eIF4G2 (Sigma) or -actin (Cell Signaling Technology). 8.0, 1% SDS, and complete protease inhibitor mix (Sigma). The newly Membranes were washed three times with 1 ϫ TBS-Tween 20 (1 ϫ TBS synthesized, AHA-incorporated protein was crosslinked to alkyne- and 0.1% Tween 20) and incubated with HRP-labeled secondary anti- derivatized biotin (Invitrogen) by a copper (I)–catalyzed cycloaddition body for1hatroom temperature. After washing, membranes were de- (Click-iT) according to the manufacturer’s recommendation using the veloped with ECL Advance Blotting Detection Kit reagents (GE Click-iT protein reaction buffer kit (Invitrogen). After crosslinking, the Healthcare). AHA-labeled, biotin-crosslinked proteins were isolated by affinity pull- Measurement of axonal growth. Images of axons were obtained using down with streptavidin coated Dynabeads M-280 (Invitrogen). Matrix- an inverted phase-contrast microscope (Nikon). Distal axon length was bound proteins were eluted into LDS sample buffer (Invitrogen) by measured using the neurite tracing and quantification tool, Neuron J, boiling for 5 min. Total protein inputs and affinity-purified fractions from ImageJ software (NIH). All axons in each culture dish were mea- were separated by SDS-PAGE and eIF2B2, eIF4G2, ␤-actin, COXIV, and sured, and statistical comparisons were made by measuring at least hypoxia-inducible factor 1␣ (HIF1␣) proteins were detected by Western 35–40 axons per treatment. analysis. Antibodies against eIF2B2 and eIF4G2 were obtained from Statistical analyses. All statistical analyses were performed in Excel. Sigma. HIF1␣ antibody was purchased from Novus Biological, and Results are expressed as mean Ϯ SEM. p values were calculated by Stu- COXIV antibody was obtained from Cell Signaling Technology. To rule dent’s two-tailed t test. out leakage of AHA to the parental cell bodies in the center compartment during incubation of the axons located in the side compartment of the Results Campenot chambers, lysates from cell bodies of AHA-treated axons were As mentioned in the Introduction, we previously reported the pres- also used for biotin crosslinking, streptavidin affinity purification, and ence of a heterogeneous population of miRs in SCG axons. One of Western analyses. the most abundant miRNAs found in these axons was miR16 For fluorescence studies, AHA-labeled axons and corresponding cell (Natera-Naranjo et al., 2010). A bioinformatic search for potential body compartments were fixed in 4% formalin, permeabilized with Tri- targets of miR16, using various miRNA target prediction software, ton X-100, and incubated with alkyne-derivatized AlexaFluor-594 and revealed eIF2B2 and eIF4G2 mRNAs as candidates (Fig 1A). Click-iT cell reaction buffer kit (Invitrogen) according to the manufac- turer’s instructions. For time course experiments, axons were labeled miR16 targets rat eIF2B2 and eIF4G2 with AHA for 1, 4, and 6 h. After incubation, axons were fixed and Ј permeabilized, and AHA incorporation was measured by labeling with To assess whether miR16 specifically targets the 3 UTRs of Click-iT AlexaFluor-594. To assess the effects of the protein synthesis eIF2B2 and eIF4G2, SCG neurons were transfected at 3 DIV with inhibitor, emetine, on axonal translation, 7-day-old SCG axons were a dual luciferase reporter plasmid carrying both the eIF2B2/ treated with AHA and 50 or 100 ␮M emetine for 6 h. AHA incorporation eIF4G2 3ЈUTR downstream of the firefly luciferase coding se- Kar et al. • Translational Regulation of Axonal Protein Synthesis J. Neurosci., April 24, 2013 • 33(17):7165–7174 • 7167

quence and the Renilla luciferase open alone, which served as a tracking gene (Fig. 1B). Firefly luciferase activity was quantified 24 h after cotrans- fection with the dual reporter plasmid and either pre-miR16 or anti-miR16. The ra- tio of firefly reporter gene to Renilla track- ing was normalized for each reporter and was displayed as relative luciferase intensity. Overexpression of miR16 by treatment with pre-miR16 re- duced luciferase activity by ϳ40–50% in the context of both eIF2B2 and eIF4G2 3ЈUTRs compared with the nontargeting control, pre-NT (Fig. 1C). Conversely, treatment with the miR16 inhibitor, anti- miR16, resulted in an approximately twofold increase in relative luciferase inten- sity compared with the nontargeting con- trol, anti-NT (Fig. 1C). Deletion of the putative miR16 MTS from the eIF2B2 and eIF4G2 3ЈUTR in the dual luciferase re- porter constructs eliminated the response of the reporter gene to increased miR16 levels (Fig 1D). Together, the results of these ex- Figure 1. miR16 targets rat eIF2B2 and eIF4G2 mRNAs. A, Putative MTS in the eIF2B2 and eIF4G2 3ЈUTRs. B, Schematic of the vector backbone of miRNA 3ЈUTR target clone (GeneCopoeia) carrying dual luciferase reporters. Constructs contained the firefly luciferase open periments confirmed that the putative MTS Ј readingframe(hlucORF)upstreamofeitherthecompleteeIF2B2oreIF4G23ЈUTR,ortheeIF2B2andeIF4G23ЈUTRinwhichtheMTSswere in the eIF2B2 and eIF4G2 3 UTRs are func- deleted(␦16).Thedual-reporterconstructalsocontainedaRenillaluciferaseORF(hRlucORF).Thehlucluciferaseexpressionwasdrivenby tional and targeted by miR16. theSimianvirus40(SV40)promoter,whereashRlucexpressionwasdrivenbythecytomegalovirus(CMV)promoter.C,D,Luciferaseactivity ofthechimericeIF2B2andeIF4G23ЈUTRreportergenes,aswellastheRenillatrackinggene,wasmeasuredaftertransfectionofpre-miR16 eIF2B2 and eIF4G2 are expressed in rat oranti-miR16.pre-NToranti-NTwasusedascontrol.Luciferasereporterconstructswerecotransfectedwiththepre-miR16oranti-miR16 SCG axons or nontargeting controls, and luciferase activity was assessed 24 h after transfection. The ratio of reporter gene to tracking gene was To visualize miR16 and the mRNAs en- normalized for each reporter and expressed as relative luciferase intensity. Error bars indicate the SEM (n ϭ 9). The experiment was coding eIF2B2 and eIF4G2 in rat SCG ax- Ͻ repeated three times with similar results. *p 0.01 (Student’s ttest). ons, we used FISH. Consistent with our earlier results (Natera-Naranjo et al., 2010), the miR16-specific probe showed punctate hybridization signals in the ax- ons, compared with a scrambled probe used as a negative control (Fig. 2A). In subsequent experiments, eIF2B2- and eIF4G2-specific probes showed punctate signals along the length of the axon with regions of high density labeling. No fluo- rescent signals were observed with the nonspecific hybridization probe (Fig. 2B). Consistent with the compartmentalized culture results, FISH performed on single primary dissociated sympathetic neurons with probes specific to miR16 and mR- NAs encoding eIF2B2 and eIF4G2 showed punctate fluorescent labeling (Fig. 2C and Fig. 2D, respectively). These results estab- lished the axonal localization of miR16 and eIF2B2 and eIF4G2 mRNAs in SCG neurons. Based upon the above findings, we evaluated the hypothesis that eIF2B2 and eIF4G2 proteins are locally synthesized in Figure 2. miR16 and mRNAs encoding eIF2B2 and eIF4G2 are present in distal axons of sympathetic neurons. FISH was per- formed in primary SCG neurons (7 DIV) using FITC-conjugated locked nucleic acid probes for miR16 (A), eIF2B2, eIF4G2 (B), and the axon. To test this postulate, we used scrambled probes as negative controls. Insets, Enlargement of regions of the axons in A and B. C, D, Phase-contrast images of the bio-orthogonal labeling with AHA cou- axon fibers are provided in the corresponding panels. miR16 and mRNAs encoding eIF2B2 and eIF4G2 were also visualized in pled with Click-iT technology. In this ex- primary dissociated sympathetic neurons by FISH. Bottom, Magnified images of distal regions of the axon showing specific periment, SCG axons in the lateral punctate labeling. Arrows indicate the locations of punctate FISH signals in axons. compartments were exposed to AHA in 7168 • J. Neurosci., April 24, 2013 • 33(17):7165–7174 Kar et al. • Translational Regulation of Axonal Protein Synthesis

Figure3. mRNAsencodingeIF2B2andeIF4G2aretranslatedinthedistalaxonsofSCGneurons.A,Aschematicdiagramshowingthemetaboliclabelingstrategyusedtodetectnewlysynthesized axonal proteins. AHA, an analog of L-methionine, was added to axonal compartments, biosynthetically incorporated into newly synthesized proteins, and detected by a copper (I)–catalyzed cycloaddition (Click-iT) reaction to either an alkyne-derivatized fluorophore or biotin-alkyne followed by streptavidin affinity absorption and Western analysis. Local synthesis of eIF2B2 and eIF4G2 proteinswasassessedbymetaboliclabelingofaxonallysynthesizedproteinswithAHAfor6h,followedbybiotinylationandaffinitypurificationbystreptavidinbeads.Ascontrol,somalysatesfrom center compartments whose axonal compartments were labeled with AHA were also subjected to biotinylation and affinity purification by streptavidin beads. B, A Western analysis was performed on the affinity-purified fraction to detect the presence of eIF2B2 and eIF4G2 using monoclonal antibodies. There is a lack of AHA-labeled (pulldown fraction) eIF2B2 and eIF4G2 in the lysates from the cell bodies. Ten percent of the total input and 50% of the pulldown were loaded in each lane of the SDS gel for the Western analysis.

Neurobasal medium without methionine for 6 h. After incuba- transfection, eIF2B2 and eIF4G2 mRNA levels in the axon de- tion, axons were lysed, and the newly synthesized, AHA-labeled creased ϳ40–50% (Fig. 4C,E), whereas no effect on parental proteins were crosslinked to the alkyne-derivatized biotin using somal levels of eIF2B2 and eIF4G2 transcripts were observed (Fig. the Click-iT reaction (Fig. 3A; see Materials and Methods). The 4D). To evaluate the specificity of these effects, we compared the AHA-labeled, biotin-crosslinked proteins were then affinity pu- effects of miR16 levels on the relative abundance of eIF4E, eIF3G, rified with streptavidin beads, and Western analysis was per- and eIF2S1, three eukaryotic translation mRNAs formed to detect the presence of eIF2B2 and eIF4G2 proteins. that do not contain a miR16 binding site in their 3ЈUTRs. As The results of Western analyses showed that both eIF2B2 and shown in Figure 4C, D, the introduction of pre-miR16 into the eIF4G2 were present in the streptavidin-purified fraction of the distal axons had no effect on the levels of these mRNAs either in AHA-labeled axons, whereas no bands were observed in the non- the distal axon or parental cell bodies. Consistent with the de- labeled (ϪAHA) axons (Fig. 3B). Additionally, to exclude the crease in mRNA levels, introduction of pre-miR16 into axons possibility of leakage of AHA into the cell body compartment, lead to a 50–60% decrease in eIF2B2 and eIF4G2 protein levels soma lysates from central compartments of AHA-treated axons (Fig. 4F). Conversely, transfection of anti-miR16 directly into the were also subjected to biotin-crosslinking and streptavidin affin- distal axons resulted in a 50% increase in eIF2B2 and eIF4G2 ity purification followed by Western analyses. No specific AHA- mRNA and a 60% increase in eIF2B2 and eIF4G2 protein (Fig. labeled eIF2B2 or eIF4G2 bands were present in the pulldown 4G,H) compared with the nontargeting anti-NT control. These fraction obtained from the cell bodies, indicating that eIF2B2 and results demonstrate that miR16 can regulate the local expression eIF4G2 bands in the AHA-labeled axonal lysates were indeed of eIF2B2 and eIF4G2. derived from de novo protein synthesis in the axon (Fig. 3B). Modulation of axonal miR16 levels alters axon growth rate in miR16 regulates axonal eIF2B2 and eIF4G2 expression SCG neurons To evaluate the role of miR16 in the regulation of axonal eIF2B2 Next, we evaluated the functional consequences of the modula- and eIF4G2 expression, we assessed the effects of pre-miR16 tion of miR16 levels by assessing its effects on axon elongation. In transfection on axonal eIF2B2 and eIF4G2 mRNA and protein this experiment, we transfected distal axons of 2-day-old SCG levels. Axonal compartments were transfected with either pre- cultures with pre-miR16 or anti-miR16. Sixteen hours after miR16 or anti-miR16, and mRNA and protein levels were mea- transfection, axon length in the lateral compartments was mea- sured by quantitative PCR and Western analysis, respectively. sured by phase-contrast microscopy. The results of these experi- Transfection of the lateral compartment with pre-miR16 for 16 h ments showed that transfection of the axons with pre-miR16 lead to an ϳ40-fold increase in mature miR16 levels in the axons decreased axon length by ϳ40% compared with nontargeting (Fig. 4A), whereas no significant change in miR16 levels was ob- controls (Fig. 4I). Interestingly, the inhibition of endogenous served in the corresponding cell bodies (Fig. 4B). At 16 h after miR16 activity by anti-miR16 transfection resulted in an increase Kar et al. • Translational Regulation of Axonal Protein Synthesis J. Neurosci., April 24, 2013 • 33(17):7165–7174 • 7169

sured using Click-IT assay (Invitrogen). To determine the length of AHA labeling re- quired for the study, we first performed a time course experiment by labeling newly synthesized, axonal proteins with AHA for 1, 4, or 6 h time intervals. The newly synthe- sized, AHA-labeled proteins were visualized by covalent crosslinking of the incorporated AHA to an AlexaFluor-594 tag (Fig. 5A). The results of the time course experiment showed that incorporation of AHA into SCG axons was time-dependent, with higher AHA incorporation observed in 4–6 h time periods compared with1hAHA exposure. AHA labeling for longer than 6 h did not show a significant increase in AHA incorporation in the axon (data not shown). Importantly, covalent crosslinking of the AlexaFluor-594 tag in the parental cell bod- ies after AHA labeling of the axonal com- partment showed no AHA signal in the cell body compartments during the labeling pe- riods used in this study (Fig. 5A,B). These results suggest that, within the experimental paradigm used in this study, no significant leakage of AHA occurred into the central compartment of Campenot cultures. In the subsequent studies, we evalu- ated whether AHA incorporation into axonal protein was sensitive to perturba- tions in the protein synthetic machinery. In these experiments, we measured AHA incorporation in SCG axons labeled (6 h) in the presence of emetine dosages of ei- ther 50 or 100 ␮M. The reagent vehicle containing DMSO served as a negative control. The data indicated that increas- ing concentrations of emetine reduced AHA labeling to near background levels Figure4. miR16regulatesaxonalmRNAandproteinlevelsofeIF2B2andeIF4G2andinfluencesaxonoutgrowth.Quantification (Fig. 5B). Together, the results of these ex- of mature miR16 levels or transcripts encoding eukaryotic translation initiation factors eIF4E, eIF3S, eIF2S1, eIF4G2, and eIF2B2 in periments showed that fluorogenic label- axons (A and C, respectively) and cell bodies (B and D, respectively) 16 h after transfection of distal axons with pre-miR16. ing of incorporated AHA can be used to QuantificationofeIF2B2andeIF4G2mRNA(E)andprotein(F)levelsinthedistalaxons,after16htransfectionwithpre-miR16,as document levels of local translation in determined by real-time quantitative PCR and Western analysis, respectively. The mRNA (G) and protein (H) levels for eIF2B2 and SCG axons by fluorescence microscopy. eIF4G2 in distal axons treated with anti-miR16 for 16 h. mRNA levels were plotted relative to ␤-actin mRNA, and ␤-actin protein ϭ To test the hypothesis that inhibition levels were used as a loading control. Error bars represent the SEM (n 6). To assess the effect of miR16 on axonal growth, SCG of the local expression of eIF2B2 or neurons were cultured for 2 d, and axons were subsequently transfected with pre-miR16 (I), anti-miR16 (J), or nontargeting (NT) control. The direction of axon elongation is from left (central compartment) to right (lateral compartment). Arrows indicate the eIF4G2 alters total axonal protein synthe- locationofaxonterminals.After16htreatment,axonlengthwasmeasuredinthelateralsidesofthecompartmentalizedcultures. sis, eIF2B2- or eIF4G2-specific siRNA was DataaremeanϮSEMfrom50to60axons.Theexperimentwasperformedthreetimeswithsimilarresults.**pϽ0.001,***pϽ introduced into distal axons (5–7 DIV). 0.0001 (Student’s t test). Sixteen hours after transfection, eIF2B2 and eIF4G2 mRNA and protein levels were measured by quantitative PCR and in axon length (Fig. 4J). Together, these results indicate that Western analyses, respectively. The results of these experiments modulation of the local levels of miR16 can regulate the growth of showed that eIF2B2 mRNA and protein levels decreased ϳ80%, the axon. whereas eIF4G2 mRNA and protein levels decreased ϳ50–60% (Fig. 6A). No change in eIF2B2 or eIF4G2 mRNA levels was ob- siRNA-mediated downregulation of axonal eIF2B2 and served in parental soma after the axonal compartment was trans- eIF4G2 leads to an inhibition of local protein synthesis fected with either eIF2B2- or eIF4G2-specific siRNA (Fig. 6A). To further evaluate the postulate that the effects of miR16 on Next, levels of local translation in SCG axons were assessed by axonal growth are mediated, at least in part, by the local synthesis measuring fluorescent labeling of incorporated AHA after 16 h of eIF2B2 and eIF4G2, we reasoned that inhibition of eIF2B2 and treatment with eIF2B2- or eIF4G2-specific siRNA. We observed a eIF4G2 expression would affect local, axonal translation. The marked decrease in fluorescence intensity of eIF2B2- and levels of local protein synthesis in the axons were directly mea- eIF4G2-siRNA-treated axons compared with NT-siRNA-treated 7170 • J. Neurosci., April 24, 2013 • 33(17):7165–7174 Kar et al. • Translational Regulation of Axonal Protein Synthesis axons (Fig. 6B,C). Quantification of the fluorescent signal in eIF2B2 or eIF4G2 siRNA-treated axons revealed that levels of local translation decreased by 70% or 50%, respectively (Fig. 6B,C). Together, these results demonstrate that local levels of eIF2B2 and eIF4G2 affect the activity of the axonal protein synthetic system.

Downregulation of eIF2B2 and eIF4G2 expression inhibits the translation of specific axonally localized mRNAs ␤-actin, COXIV, and HIF1␣ Because siRNA-mediated downregula- tion of eIF2B2 or eIF4G2 led to a reduc- tion in total axonal protein synthesis, we explored whether the knock-down of eIF2B2 or eIF4G2 inhibits the translation of specific mRNAs. Earlier studies have established that ␤-actin mRNA is local- ized and translated in axons cultured in various systems, such as squid giant axon, superior cervical ganglia, dorsal root gan- glia, cortical neurons, and retinal ganglion cells, and this local translation plays an important role in axon growth and devel- opment (Kaplan et al., 1992; Olink-Coux and Hollenbeck, 1996; Bassell et al., 1998; Zhang et al., 1999; Leung et al., 2006; Yao et al., 2006; Donnelly et al., 2013). Addi- tionally, we previously reported the pres- ence and functionality of COXIV mRNA in the axons of SCG neurons (Hillefors et al., 2007; Aschrafi et al., 2008). Another subset of mRNAs that have been shown to be present in axons is that encoding tran- scription factors (Willis et al., 2007; Gumy et al., 2011). Locally synthesized tran- scription factors have been reported to be involved in retrograde signaling in re- sponse to stimuli, such as exposure to Figure 5. Nascent protein synthesis can be visualized by fluorescent AHA labeling in rat sympathetic axons. A, Time course for NGF or peripheral nerve injury (Cox et the detection of newly synthesized proteins in axons. Axonal compartments of SCG cultures were incubated with culture medium al., 2008; Michaelevski et al., 2010; Ben- containing the methionine analog AHA or methionine, for the time intervals indicated. The AHA-containing, newly synthesized Yaakov et al., 2012). In light of these proteinswerevisualizedbycrosslinkingtoalkyne-derivatizedAlexaFluor-594usingtheClick-iTCellreactionkit(Invitrogen).Arrow findings, we assessed the effect of indicatesfluorescentlylabeled,newlysynthesizedproteininthetreatedaxonbundles.ThereisabsenceofAHAfluorescenceinthe siRNA-mediated downregulation of parental cell bodies of AHA-treated axons. Bottom, Quantitation of the fluorescence signals in the axon bundles at specified time points. B, SCG distal axons were incubated with growth medium containing either AHA or methionine (control) in the presence or eIF2B2 or eIF4G2 on axonal translation of ␮ ␤-actin, COXIV mRNA, and HIF1␣,a absence of either 50 or 100 M emetine for 6 h. Arrows indicate fluorescently labeled, newly synthesized protein in the treated axonbundles.Fluorescenceintensitywasmeasuredforeachtreatment.Bottom,Quantitativedata.DataaremeanϮSEMfrom30 transcription factor whose mRNA has axon bundles. **p Ͻ 0.001 (Student’s t test). been shown to be present in the axons (Ben-Yaakov et al., 2012). SCG axons observed an ϳ70% decrease in COXIV translation in eIF2B2- (5–7 DIV) were treated with siRNA targeted against eIF2B2 or siRNA treated axons, whereas eIF4G2-siRNA treatment showed eIF4G2 for 16 h and were subsequently labeled with AHA for 6 h. a 40–50% decrease in axonal COXIV synthesis. The knock-down After labeling, AHA-incorporated proteins were crosslinked to of eIF4G2 led to a 60–70% decrease in axonal translation of biotin and purified with streptavidin beads. Western analyses ␣ were performed to assess the levels of locally synthesized ␤-actin HIF1 , whereas eIF2B2 knock-down did not show a statistically ␣ or COXIV or HIF1␣. As shown in Figure 6D, eIF2B2 and eIF4G2 significant effect on HIF1 levels (Fig. 6D). Additionally, no sig- ␤ siRNA-treated axons exhibited a 50–60% decrease in axonal nificant effect was observed on axonal mRNA levels of -actin, ␣ ␤-actin translation compared with NT-siRNA-treated controls. COXIV, and HIF1 after treatment of axons with eIF2B2 or It is important to note that the decrease in ␤-actin protein levels eIF4G2-specific siRNA (Fig. 6E). These findings establish that, in observed after eIF2B2 or eIF4G2 knock-down only corresponded addition to inhibiting global axonal protein synthesis, siRNA- to the locally synthesized pool, and the total levels of ␤-actin mediated downregulation of eIF2B2 or eIF4G2 reduces the trans- protein in the axon did not change significantly. Interestingly, we lation of specific axonally localized mRNAs. Kar et al. • Translational Regulation of Axonal Protein Synthesis J. Neurosci., April 24, 2013 • 33(17):7165–7174 • 7171

Figure 6. siRNA-mediated knock-down of eIF2B2 and eIF4G2 expression inhibits nascent protein synthesis in rat sympathetic axons. siRNA oligonucleotides targeted against either eIF2B2 or eIF4G2ornontargetingNT-siRNAweretransfectedintodistalaxonsofSCGneurons.A,TheeIF2B2andeIF4G2mRNAlevelsfromaxonsandcellbodiesweremeasuredbyquantitativePCRandaxonal protein levels quantified by Western analysis. The mRNA levels are expressed relative to ␤-actin mRNA, and ␤-actin protein levels served a loading control in the Western analyses. The axonal compartments of 1-week-old SCG cultures were treated with siRNA targeted against either eIF2B2 (B) or eIF4G2 (C) or NT control for 16 h and were subsequently labeled with AHA for 6 h. After incubation, cells were either fixed, and the newly synthesized AHA-labeled proteins were visualized by fluorescence microscopy (B, C), or alternatively cells were lysed, and the newly synthesized AHA-labeled proteins were isolated by affinity purification with streptavidin beads and specific proteins detected by Western analyses (D). B, C, Arrows indicate fluorescently labeled, newly synthesizedproteininthetreatedaxonbundles.MeanfluorescenceintensitiesofeIF2B2oreIF4G2siRNA-treateddistalaxonbundlesegmentsrelativetonontargetedcontrolaxonsareshown.Data are mean Ϯ SEM of 30 axon bundles per treatment. D, Western analyses of affinity purified pulldown fractions show the levels of newly synthesized ␤-actin, COXIV, and HIF1␣ proteins. QuantificationofimmunoblotswithImageJshowedchangesin␤-actin,COXIV,andHIF1␣signalinpulldownfromAHA-labeledaxonstreatedwitheIF2B2oreIF4G2siRNAcomparedwithNT-siRNA treatment. E, Axonal ␤-actin, COXIV, and HIF1␣ mRNA levels were quantified after transfection of eIF2B2, eIF4G2, and NT-siRNA. Values represent mean Ϯ SEM from three independent experiments. *p Ͻ 0.05, **p Ͻ 0.001, ***p Ͻ 0.0001 (Student’s t test).

Downregulation of both eIF2B2 and eIF4G2 has an additive specific siRNA alone (Fig. 7A,B). Interestingly, simultaneous inhibitory effect on axonal protein synthesis knock-down of both eIF2B2 and eIF4G2 levels in SCG axons also To investigate whether the knock-down of axonal eIF2B2 and resulted in a marked change in axon morphology (Fig. 7A). eIF4G2 has an additive inhibitory effect on local protein synthe- sis, we transfected SCG axons (5–7 DIV) with siRNAs targeted Knock-down of local eIF2B2 and eIF4G2 expression reduces against eIF2B2, eIF4G2, or both eIF2B2 and eIF4G2. After trans- axonal growth fection, axons were treated with AHA for 6 h, and AHA incorpo- To evaluate the postulate that inhibition of local protein synthesis ration was assessed by fluorescence microscopy. AHA labeling in could have deleterious effects on the development and/or viabil- axons showed that knock-down of local levels of both eIF2B2 and ity of the axon, distal axons located in the lateral compartments of eIF4G2 together resulted in a significant decrease in axonal transla- 2-day-old SCG neuron cultures were transfected with either tion, compared with axons treated with either eIF2B2- or eIF4G2- eIF2B2- or eIF4G2-specific siRNA or a combination of both siR- 7172 • J. Neurosci., April 24, 2013 • 33(17):7165–7174 Kar et al. • Translational Regulation of Axonal Protein Synthesis

NAs. Axons in the contralateral compart- ment of the culture dish were treated with NT-siRNA and served as a negative con- trol. Axon length in both side compart- ments of the Campenot chambers was measured 16 h transfection by phase- contrast microscopy. Consistent with the findings obtained from pre-miR16 treatment, siRNA-mediated downregula- tion of the axonal expression of eIF2B2 or eIF4G2 resulted in a significant decrease in axonal elongation compared with the distal axons transfected with nontargeting siRNA (Fig. 8A,B). These data provide ev- idence that the local translation of eIF2B2 and eIF4G2 mRNAs plays a key role in regulating axonal growth. Because knock-down of both eIF2B2 and eIF4G2 together showed an additive inhibitory effect on local protein synthe- sis, we also evaluated the effect of the eIF2B2 and eIF4G2 double knock-down on axon growth. Consistent with the find- ings of the metabolic labeling experi- ments, the results of the axon growth experiment showed that simultaneous knock-down of both eIF2B2 and eIF4G2 Figure 7. Downregulation of both eIF2B2 and eIF4G2 in SCG axons shows additive inhibitory effects on local protein synthesis. led to a greater decrease in axon length, Axonal compartments of 1-week-old SCG cultures were transfected with siRNA targeted against eIF2B2, eIF4G2, a combination of compared with axons treated with eIF2B2 eIF2B2andeIF4G2,ornontargetingNTcontrolfor16h.Aftertransfection,axonswerelabeledwithAHAfor6h.A,Axonswerefixed, or eIF4G2-specific siRNAs individually and newly synthesized, AHA-containing proteins were labeled with AlexaFluor-594. Arrows indicate fluorescently labeled, newly (Fig. 8A,B). Together, these findings sug- synthesized protein in the treated axon bundles. B, Mean fluorescence intensities of treated distal axon bundle segments relative to nontargeted control neurons are shown. Data are mean Ϯ SEM from 30 axons. **p Ͻ 0.001 (Student’s t test). gest that these two translation initiation factors may regulate local protein synthe- sis and axonal growth. In addition, these findings suggest that the effects of miR16 on axonal growth are mediated, at least in part, by the local translation of eIF2B2 and eIF4G2. Discussion Studies using both invertebrate and vertebrate model systems have reported the presence of a number of axonally localized mRNAs encoding ribosomal proteins and factors involved in the regulation of protein synthesis (Perrone-Capano et al., 1987; Moccia et al., 2003; Willis et al., 2007; Taylor et al., 2009; Vogelaar et al., 2009; Zivraj et al., 2010). In this study, we investigated the role of locally synthesized translation factors in the regulation of axonal protein synthesis. To monitor axonal translation activity, we used metabolic labeling studies with AHA, an amino acid analog incorporated into proteins in place of methionine. The incorporation of AHA into newly synthesized proteins can be visualized after cell fixation by addition of a tagged fluorescent alkyne or can be affinity purified by tagging AHA-labeled pro- teins with biotin after cell lysis. Consistent with earlier experi- mental approaches, we observed a time-dependent accumulation of the AHA label in the axons (Dieterich et al., 2006, 2010; Tcherkezian et al., 2010). This labeling of axons with AHA was sensitive to treatment with the translation inhibitor, emetine. Also, within our experimental paradigm, in which only the ax- Figure8. Knock-downoflocaleIF2B2andeIF4G2expressionattenuatesaxonelongationinsym- onal compartments were labeled with AHA, no axonally applied pathetic neurons. A, SCGs were cultured for 2 d, and axons located in the lateral compartments were AHA label was observed in the cell bodies within the short- transfected with siRNA targeted against eIF2B2, eIF4G2, both eIF2B2 and eIF4G2-siRNA together, or labeling periods (6 h) used in these studies. This finding suggests nontargeted NT control siRNA. Axon elongation is from left to right; arrows indicate the location of that metabolic labeling with AHA in the axon is a valid technique axonal terminals. B, Axon length was measured 16 h after transfection. Data represent the mean Ϯ to assess local translation and that the newly synthesized, AHA- SEM of 50–60 axons. The experiments were repeated three times with similar results. ***p Ͻ labeled proteins we observed in the distal axons were not derived 0.0001 (Student’s ttest). Kar et al. • Translational Regulation of Axonal Protein Synthesis J. Neurosci., April 24, 2013 • 33(17):7165–7174 • 7173 from the cell body during the labeling time periods used in this grown in compartmentalized cultures (Hillefors et al., 2007; study. Manns et al., 2012). As previously discussed, differences between Interestingly, the AHA labeling experiments showed that the results reported in these studies could be attributed to differ- downregulation of axonal eIF2B2 and eIF4G2 expression led to a ences in the neurons used as well as differences in cell culture marked decrease in local mRNA translation. Consistent with the conditions, such as NGF concentrations, culture substratum, and decrease in global axonal protein synthesis after eIF2B2 or the use of serum-free culture medium. eIF4G2 knock-down, local translation of the axonally localized In conclusion, the findings reported here indicate that local mRNAs, ␤-actin, COXIV, and HIF1␣ was also inhibited. The synthesis of proteins involved in the translation machinery play local translation of ␤-actin mRNA, in response to chemotactic an important role in the regulation of local protein synthesis in cues, has been shown to be important for axon guidance (Bassell the axon and axonal function. Finally, we show that a noncoding et al., 1998; Leung et al., 2006; Yao et al., 2006) and elongation mRNA regulates axonal protein synthesis by modulating the local (Zhang et al., 1999). It bears mention here that studies from the levels of proteins involved in the control of mRNA translation. Letourneau and Ervasti laboratories have argued against the role of ␤-actin in axon pathfinding and motor neuron development respectively (Roche et al., 2009; Cheever et al., 2011). 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