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Recent evidence suggests growth cone responses to guidance cues require local protein synthesis. Using chick neurons, we investigated whether protein synthesis is required for growth cones of several types to respond to guidance cues. First, we found that global inhibition of protein synthesis stops axonal elongation after two hr. When protein synthesis inhibitors were added 15 min before adding guidance cues, we found no changes in the typical responses of retinal, sensory and sympathetic growth cones. In the presence of cycloheximide or anisomycin, ephrin-A2, slit-3, and semaphorin3A still induced growth cone collapse and loss of actin filaments, NGF and NT-3 still induced growth cone protrusion and increased F-actin, and sensory growth cones turned toward an NGF source. In compartmented chambers that separated perikarya from axons, axons grew for 24-48 hr in the presence of cycloheximide and responded to negative and positive cues. Our results indicate that protein synthesis is not strictly required in the mechanisms for growth cone responses to many guidance cues. Differences between our results and other studies may exist because of different cellular metabolic levels in in vitro conditions, and a difference in when axonal functions become dependent on local protein synthesis.
Neurons are highly polarized with elaborate shapes and highly localized activities. Protein synthesis is not confined to the perikaryon; rather, proteins made in dendrites and axons have important functions (Steward and Schuman, 2003). Defective dendritic protein translation underlies some behavioral deficits and mental retardation (Bramham and Wells, 2007; Dahm and Macchi, 2007; Wang et al., 2007). Axonal protein synthesis is critical in functions of terminals, as well as in development and regeneration (Campenot and Eng, 2000; Hengtst and Jaffrey, 2007; Koenig and Giuditta, 1999; Piper and Holt, 2004; Twiss and van Minnen, 2006; Verma et al., 2005; Zhang and Poo, 2002).
Is local protein synthesis important in axonal growth and guidance? In vitro experiments with compartmented chambers indicate that axonal protein synthesis is not required for axonal growth (Blackmore and Letourneau, 2007; Eng et al., 1999). Yet, there are reports that responses to guidance cues require axonal protein synthesis (Brunet et al., 2005; Campbell and Holt, 2001; Farrar and Spencer, 2008; Guirland et al., 2003; Leung et al., 2006; Lin and Holt, 2007; Piper et al., 2006; Wu et al., 2005; Yao et al., 2006). When growth cones enter new environments, local synthesis of receptors could sensitize growth cones to new guidance cues (Brittis and Flanagan, 2002). In addition, growth cone adaptations to changed balances of cues may involve local synthesis of receptors or signaling components (Ming et al., 2002).
Thus, local synthesis of receptors or signaling components may be necessary for growth cone navigation. Is it also necessary to locally make proteins with general roles in growth cone motility? Recent papers report that RhoA GTPase and ß-actin must be rapidly and locally synthesized for growth cones to respond to several cues (Leung et al., 2006; Wu et al. 2005, Yao et al., 2006). In light of the diverse mechanisms that rapidly regulate RhoA and ß-actin activity, it is unexpected that these components must be locally synthesized to enable responses to guidance cues.
Because previous studies involved few neuronal types, we investigated whether protein synthesis is required for growth cones of other neurons to respond to guidance cues. First, we found that globally inhibiting protein synthesis leads to slowing and arrest of axonal growth over several hours. When protein synthesis inhibitors were added before several positive and negative cues, we found normal growth cone responses. Repulsive cues, ephrin-A2, slit-3, Semaphorin3A (Sema3A) and Semaphorin6A (Sema6A), induced growth cone collapse in the presence of protein synthesis inhibitors. F-actin polymer was lost after adding repulsive cues. Furthermore, attractive cues, nerve growth factor (NGF) and neurotrophin-3 (NT-3), induced growth cone protrusion and increased F-actin despite inhibiting protein synthesis. In addition, growth cones turned toward an NGF source in the presence of protein synthesis inhibitors. In compartmented cultures where axonal protein synthesis was inhibited for many hrs, growth cones still responded to guidance cues. Our data indicate that protein synthesis in distal axons of chick retinal, sympathetic, and DRG neurons and mouse DRG neurons is not required for responses to several guidance cues.
F12 medium, DMEM medium, B27 additives, laminin, poly-D-lysine, Alexa Fluor 488 phalloidin, Alexa Flour 568 secondary antibodies, Click-iT AHA (cat. no. C10102) and Click-iT Tetramethylrhodamine Protein Analysis Detection Kit (cat. no. C33370) reagents were purchased from Invitrogen (Carlsbad CA). NGF, NT-3, L1-Fc, slit-3, ephrin-A2-Fc, Sema3A-Fc and Sema6A-Fc were purchased from R & D Systems (Minneapolis MN). Anti-RhoA monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz CA), and anti-eIF-4EBP-P rabbit antibodies were purchased from Cell Signaling Technology (Danvers MA). Rabbit anti-ß-actin was a gift from Dr. James Ervasti (University of Minnesota; Prins et al., 2008). Collagen was purchased from Inamed (Fremont CA). Cycloheximide and anisomycin were purchased from Calbiochem (San Diego CA), and MG-132 was from Tocris Bioscience (Ellisville MD).
Culture dishes, glass coverslips, or 24- or 96-well dishes were coated overnight with 20 μg/ml laminin or 4 μg/ml recombinant L1-Fc mixed with 8 μg/ml Fc in PBS. Explants of E7 and E13 DRGs, E7 temporal retina, and E7 sympathetic ganglia were dissected from chick embryos, and DRGs were removed from E15 mouse embryos, according to procedures approved by the University of Minnesota Institutional Animal Care and Use Committee. Neural tissues were cultured on experimental substrates in F-12 with B27 additives and buffered to pH 7.4 with 10 mM HEPES. Neurotrophins, NGF or NT-3, were added to cultures, as noted. Neural tissues were cultured overnight in a humidified incubator at 37° C.
E7 chick DRGs were dissected and dissociated with 0.2% bactotrypsin in CMF-PBS for 10 minutes then rinsed with F12H containing 10% serum. Approximately 2×106 cells were transfected with 3μg of a plasmid encoding the fluorescent F-actin probe, GFP-UtrCH, kindly provided by Dr. W.M. Bement of University of Wisconsin-Madison (Burkel et al., 2007), using the G-13 program of the Amaxa Biosystems Nucleofector (Amaxa, Inc., Gaithersburg MD). Cells were then rinsed with F12H and cultured overnight in F12 with B27 additives on video dishes coated with PDL, nitrocellulose, and L1.
The effect of protein synthesis inhibitors on axon elongation was determined by time-lapse microscopy of axon elongation by DRG or retinal explants cultured in F12 with B27 (and 10 ng/ml NGF for DRGs) on laminin- or L1-coated 96-well dishes. A dish was placed on the warmed stage of a Nikon inverted microscope, and a microscope field of axons was chosen. CHI or anisomycin (or PBS for control) was added at 20 μM, and time-lapse images were collected at 2-5 minute intervals for 2-5 hr. Axon elongation rates of sample populations were determined, using Metamorph software (Molecular Devices) to measure the distance a growth cone migrated in 5-10 minute intervals over a 3-5 hr period.
Collapse assays were conducted in 24- or 96-well dishes. Explants were cultured overnight. Ten ng/ml NGF was added to cultures of DRGs or sympathetic ganglia. CHI or anisomycin were added in PBS at 20 μM for 15 min. Control dishes received PBS. After 15 min guidance molecules, Sema3A (0.5 μg/ml), ephrin-A2 (1 μm/ml), or slit-3 (1 μg/ml) were added for 30 min in continued presence of protein synthesis inhibitors. Control dishes received PBS. After 30 min, explants were fixed with 0.5% glutaraldehyde in PBS for 20 min at 37° C. After rinsing in PBS, the wells were visualized with a 20X phase contrast objective, and growth cones were counted as collapsed if they had one or no filopodia.
DRG explants were cultured overnight in 96-well dishes. A dish was placed on a warmed stage of an inverted microscope, a field of axons was chosen and images were collected once per min for 15 min. Then, 40 ng/ml NGF or NT-3 was added, and images of the same field were collected every minute for 30 min. Growth cone spreading was measured using Metamorph software to outline the borders of growth cones and determine the enclosed areas.
DRG explants were cultured overnight in F12 with B27 on laminin-coated coverslips glued over a hole in the bottom of a culture dish. Micropipette tips were dipped in a 1% nitrocellulose solution and dried. After drying, a tip was dipped several times in a solution of one μg/ml NGF or BSA in PBS. The micropipette was mounted on a micromanipulator. A culture dish was placed on the warmed microscope stage, and the micropipette tip was positioned 50 μm from a growth cone at a 45° angle to the direction of axon elongation. Images were acquired at 30 sec intervals for 15 min before and 45 min after introducing the micropipette tip. CHI was added to some dishes at the beginning of image acquisition. Growth cone turning angles were determined as the change in direction of growth cone migration between the beginning and end of the image acquisition period (Ming et al., 1997).
Compartmented cultures were made by putting an E7 DRG explant with attached peripheral root on a substrate, and adhering a 5 mm glass cloning cylinder to the substrate with silicone grease, so the DRG was within the lumen and the peripheral root extended under the 1 mm cylinder wall with its terminus outside the wall. This created two compartments and allowed us to expose the DRG and the peripheral root to different media. For chemotaxis experiments, a 3 mg/ml collagen gel was created in the outside compartment, following the manufacturer's instructions (PurCol, Inamed). Affi-Gel BlueGel chromatography beads were soaked in one μg/ml NGF or BSA in PBS for one hour, rinsed in PBS, and then placed in the collagen gel near the peripheral nerve root with an NGF-bead on one side and a BSA-bead on the other side of the root.
For immunocytochemistry explants cultured on laminin- or L1-coated coverslips were fixed with warmed 4% paraformaldehyde in PBS with 5% sucrose at ph 7.4 for 30 min. After rinsing, explants were treated 15 min with 0.1 M glycine in PBS, and extracted with 0.1% TX-100 in PBS with 2% goat serum and 1% BSA for one hr. Coverslips were incubated with the primary antibodies diluted in PBS containing 1% BSA for one hr. Primary antibody dilutions were 1/100 for anti-RhoA and anti- eIF-4EBP-P, and anti-ß-actin was used, per instructions from Dr. Ervasti (Prins et al., 2008). For labeling F-actin, Alexa Fluor 488-phalloidin was applied at 2.5 μl/100 μl mixed with the primary antibodies. The coverslips were rinsed and then incubated in 0.1% TX-100 in PBS with 2% goat serum and 1% BSA for one hr. Then, Alexa Fluor 568 goat anti-rabbit or anti-mouse antibodies at 1/1000 dilution were applied in PBS with 1% BSA for one hr. After rinsing, the coverslips were incubated in 0.1% TX-100 in PBS with 2% goat serum and 1% BSA for 30 min, then rinsed, and mounted in anti-fading medium.
To measure effects of protein synthesis inhibitors CHI and anisomycin on cultured DRG neurons, we used Click-iT reagents from Invitrogen. E7 DRGs were dissociated with 0.2% bacto-trypsin in CMF-PBS for 15 min, then rinsed and plated on poly-D-lysine and laminin-coated glass coverslips in DMEM with B27 additives and 10 ng/ml NGF. After overnight culture, the dishes were rinsed twice with warmed PBS and incubated in methionine-free DMEM for 45 min. During the last 15 min of this period 20 μM CHI or anisomycin or control buffer were added to experimental dishes. Then, the medium was replaced with methionine-free DMEM to which 50 μM of the methionine analog L-azidohomoalanine (AHA; Invitrogen C10102) was added. AHA was omitted from some dishes as a negative control. The dishes were returned to the incubator for 60 min for incorporation of the AHA into nascent proteins with or without protein synthesis inhibitors. After 60 min the dishes were rinsed twice and incubated 30 min in methionine-free DMEM with or without protein synthesis inhibitors. The cultures were fixed with 2% paraformaldehyde and 5% sucrose in PBS for 30 min and then rinsed and incubated in PBS with 0.1 M glycine for 15 min. The coverslips were then drained and placed on parafilm in a humidified 150 mm Petri dish. Using the nomenclature of the Click-iT reagent kit (Invitrogen C3337, the carboxytetramethylrhodamine alkyne (TAMRA) reagent was prepared by rapidly mixing 150 μl of the Step 1 solution with 120 μl distilled water, then 15 μl of Component C was added with 5 sec vortexing, followed by 15 μl of Step III solution with 5 sec vortexing. Fifty μl of this solution was applied to each coverslip, followed by 5 μl of the Step 2 solution, which was rapidly mixed with the solution on each coverslip. The coverslips were then incubated 30 min in the dark. Then, the coverslips were drained, rinsed twice with distilled water, then twice with methanol for 15 min, before being returned to distilled water and finally mounted on glass slides in anti-fading medium.
To measure the effects of NGF and Sema3A on protein synthesis in DRG neurons, we used the Click-iT reagents as described above. Dissociated DRG neurons were cultured overnight on poly-D-lysine and laminin-coated coverslips in DMEM and B27 additives with 0.1 ng/ml NGF. After rinsing and 45 minute incubation in methionine-free DMEM, the medium was replaced with methionine-free DMEM containing 50 μM AHA and either 40 ng/ml NGF, 1 μg/ml Sema3A or PBS as a control for addition of a guidance cue. Other samples were incubated in methionine-free DMEM without AHA, as a control for the TAMRA reagent procedure. After one hour incubation with AHA and the guidance cues, the dishes were rinsed twice and incubated in methionine-free DMEM for 30 min, followed by fixation and incubation with the TAMRA reagents, as described above.
A Spot digital camera mounted on an Olympus XC-70 inverted microscope was used to acquire all images for quantitative fluorescence measurements. In any one experiment, all coverslips with explants or cells were fixed and processed together. For collection of fluorescence images, all images for one experiment were acquired in a single session. Parameters of time interval and gain setting on the digital SPOT camera were adjusted so the brightest areas did not reach saturation, and the same gain and time interval was used to capture all images of any particular staining. All analysis of image intensities was conducted from a similar region at the center of the microscope field.
Metamorph software (Molecular Devices, Downington PA) was used for all image analysis. Software tools were used to determine fluorescence intensity along axons, using a linescan tool, within growth cones, using a line tool to outline the terminal 25 μm of each distal axon and growth cone, and a square-shaped tool placed within the center of neuronal somata. After acquisition of each fluorescent neuronal image, a measure of background fluorescence was acquired by moving the software tool slightly outside the boundary of the neuron. The background fluorescence intensity value was subtracted from the fluorescence intensity value of the accompanying neuronal measurement.
Effects of NGF and Sema3A on phospho-eIF-4EBP content of distal axons E7 DRG explants were cultured overnight on laminin-coated coverslips. Forty ng/ml NGF, one μg/ml Sema3A or PBS were added for 15 min, and then the cultures were fixed and processed for immunofluorescence staining, as described above. For measuring anti-eIF-4EBP-P staining, images of imunofluorescence staining of a sample population of distal axons and growth cones were acquired using a 60X oil immersion objective. The linescan tool was used to determine mean fluorescence intensity of a line 2 pixels wide extending proximally 15 μm from the C-domain of a growth cone into the axon.
Effects of NGF, Sema3A and protein synthesis inhibitors on nascent protein synthesis Cultures of dissociated E7 DRG cells were treated with NGF, Sema3A, cycloheximide or anisomycin, then fixed, and incubated with AHA and the Click-iT reagents, as described in detail above. Fluorescence images of of labeled neurons were acquired with a 20X objective, and mean intensity of axonal and perikaryon carboxytetramethylrhodamine fluorescence was quantified as described above.
Effects of NGF and Sema3A on ß-actin and RhoA content, respectively, of DRG growth cones E7 DRG explants were cultured overnight on laminin-coated coverslips. Twenty μM CHI or PBS was added to cultures for 15 min, and then forty ng/ml NGF, one μg/ml Sema3A or PBS were added to a culture dish for 15 min in the continued presence of CHI or PBS. The cultures were fixed and processed for immunofluorescence staining with anti-RhoA (Sema3A-treated and controls) or anti-ß-actin (NGF-treated and controls). Images of immunofluorescence staining of a sample population of growth cones were acquired using a 60X oil immersion objective. To measure growth cone content of RhoA or ß-actin, the boundary of the distal 25 μm of each growth cone and distal axon was outlined with an outline tool, and the mean integrated pixel intensity of anti-RhoA or anti-ß-actin staining was determined.
Effects of Sema3A and ephrin-A2 on F-actin distribution in DRG and retinal growth cones For measuring effects of ephrin-A2 and Sema3A on F-actin content of growth cones, E7 DRGs and temporal retina explants were cultured overnight on L1-coated coverslips. Twenty μM CHI or PBS was added \for 15 min, and then 40 ng/ml NGF (DRGs) or 2 μg/ml ephrin-A2 (temporal retina) was added for 15 min in the continued presence of CHI or PBS. The cultures were fixed with 4% paraformaldehyde and 5% sucrose for 30 min and then extracted with 0.1% TX-100 in PBS for 15min. F-actin was stained by incubation with 2.5 μl/0.1 ml Alexo Fluor 568 phalloidin in PBS for 60 min. After rinsing, the coverslips were mounted in anti-fading medium. Images of sample populations of growth cones were acquired with a 60X oil immersion objective. Using the linescan tool of Metamorph, phalloidin fluorescence was determined along a two pixel wide by 6 μm line backward from the center of the growth cone leading margin.
Effects of NGF on F-actin content of DRG growth cones E7 DRG explants were cultured overnight on L1-coated coverslips. Twenty μM CHI or PBS was added to cultures for 15 min, and then 40 ng/ml NGF was added for 15 min in the continued presence of CHI or PBS. The cultures were fixed with 4% paraformaldehyde and 5% sucrose for 30 min and then extracted with 0.1% TX-100 in PBS for 15min. F-actin was stained by incubation with 2.5 μl/0.1 ml Alexa Fluor 568 phalloidin in PBS for 60 min. After rinsing, the coverslips were mounted in anti-fading medium. Images of growth cones were acquired with a 60X oil immersion objective. To measure F-actin content, the boundary of each growth cone was outlined with the outline tool, and the mean pixel intensity and integrated pixel intensities of F-actin staining were determined for each growth cone.
Effects of NGF gradient on GFP-UtrCH distribution in DRG growth cones Immediately after placing a video dish with GFP-UtrCH infected neurons on a warmed microscope stage, 20 μM cycloheximide or the same volume of control media was added for 15 min. A transfected growth cone was identified and a fluorescent image was taken. An NGF or BSA-coated micropipette was brought to one side of the growth cone, and after 2 min another image was acquired using the same exposure and gain settings. Using Metamorph software, a 15 pixel-thick line was drawn across the growth cone (perpendicular to its neurite axis) at the two time points before and after applying the NGF gradient. The “linescan” function was used to obtain fluorescence intensity measurements for each point along the line across the growth cone. Ten growth cones for the control and cycloheximide conditions were used in the analysis.
Statistical analysis Parameters of population values are reported as mean ± S.E.M. All statistical analysis was by unpaired Student's t-test or Mann-Whitney U test.
It is reported that guidance cues trigger inactivation of the translation repressor elF-4EBP1 in distal axons (Campbell and Holt, 2001; Cox et al., 2008; Li et al., 2004). Inactivation of e1F-4EBP1 by phosphorylation releases elongation factor elF-4E to initiate mRNA translation. We used phospho-specific antibodies to ask whether the guidance cues NGF and Sema3A induce elF-4EBP1 phosphorylation (elF-BP1-P) in axons extended from E7 chick dorsal root ganglia (DRG). DRG explants were cultured overnight and exposed to NGF or Sema3A for 15 min before fixation and staining with anti- elF-4EBP1-P (Figure 1a-c). Panel 1d shows analysis of mean anti-elF-4EBP-P staining of a line scanned from the center of each growth cone proximally for 15 μm in the axon. The mean staining intensity for elF-4EBP1-P was significantly greater in distal axons of both NGF- and Sema3A-treated DRG cultures than in axons of untreated DRGs. Thus, like previous reports, signaling triggered by NGF and Sema3A inhibits a repressor of mRNA translation in distal axons of DRG neurons.
The inactivation of elF-4EBP1 and activation of elongation initiation factor 4E are associated with increased protein synthesis in response to growth factors and guidance cues (Campbell and Holt, 2001; Li et al., 2004; Takei et al., 2001). We investigated whether protein synthesis in DRG neurons is altered by NGF and Sema3A. To detect nascent proteins we used a non-radioactive reagent, L-azidohomoalanine (AHA), a methionine analog, which is incorporated into proteins instead of methionine and is detected by reaction of the azido-modified protein with a fluorescent alkyne (see Materials and Methods for details). Overnight cultures of dissociated E7 DRGs were exposed to NGF or Sema3A in AHA-containing media for 60 min before fixation, and reaction with the fluorescent alkyne. To control for the labeling procedures, some samples were not given AHA, and to control for the addition of guidance cues, other samples were given AHA but neither NGF nor Sema3A. As shown in Figure 2a-e, the mean staining intensity for nascent proteins in both cell bodies and distal axons was increased 60% by NGF addition, compared to cultures not treated NGF. After addition of Sema3A the nascent protein amounts were also elevated from the levels in untreated control neurons. Because this experiment involved one time point, we make no conclusion about effects of NGF or Sema3A on rates of nascent protein synthesis or degradation. Because of the global presentation of ligands and the duration of treatment, we infer nothing about the location of nascent protein synthesis. However, our results indicate that NGF and Sema3A stimulate increased amounts of nascent proteins in cell bodies and axons of DRG neurons. Thus, like previous reports we found that guidance cues increase nascent proteins in neurons (Campbell and Holt, 2001; Zhang et al., 1999; 2001).
Before assessing the effects of inhibiting protein synthesis on responses to guidance cues, we showed that cycloheximide (CHI), which inhibits peptide chain elongation, and anisomycin, which inhibits peptide bond formation, are effective inhibitors of chick neuronal protein synthesis. These drugs were previously used to inhibit protein synthesis in chick neurons (Blackmore and Letourneau, 2007; Luduena, 1973; Oppenheim et al., 1990). To measure the effects of CHI and anisomycin, we used the same Click-iT reagents that we used to determine the effects of NGF and Sema3A on nascent protein synthesis. After washing out methionine-containing DMEM medium, DRG cells were incubated one hr with 50 mM AHA with or without 20 μM CHI or anisomycin. As shown in Figure 2 f-i and quantitated in panel j, CHI- and anisomycin-treated neurons were as weakly labeled as control neurons that were not incubated with the methionine analog, AHA, but only with the fluorescent alkyne reagents. Thus, we could not detect protein synthesis in DRG neurons treated with CHI or anisomycin.
Because it was previously reported that global application of protein synthesis inhibitors did not inhibit axonal extension during a one hr period (Campbell and Holt, 2001), we investigated axonal elongation by DRG and retinal neurons in the global presence of protein synthesis inhibitors. Explants of E7 DRGs and temporal retina were grown in dishes coated with laminin or the cell adhesion molecule L1. CHI, anisomycin or the drug vehicle was added, and rates of axonal elongation were calculated after time-lapse imaging. The mean elongation rate for untreated DRG or retinal axons did not change over four hr of recording, though the elongation rate for DRG axons was significantly faster on laminin than on L1 (130±16 vs 41±6 μm/hr, respectively). During the first hr of treatment with 20 μM CHI or anisomycin, the axonal elongation rate of treated DRG or retinal neurons was nearly equal to that of control, untreated neurons (Table 1; Figure 3a). During the second hr, axonal growth rates began to slow in the presence of protein synthesis inhibitors (Table 1; Figure 3a). Some growth cones became smaller and less motile, and on laminin some axons retracted. On L1 axonal retraction during the second hr of treatment was less frequent than on laminin, but the rate of axon elongation still slowed (Table 1). Continuing after 2 hr of protein synthesis inhibition, nearly all axons retracted or became quiescent (Figure 3b; Supplemental Figure S1).
We asked whether concurrent inhibition of proteolysis might extend the time that axons elongate in the presence of protein synthesis inhibitors. We added MG-132, an inhibitor of proteosome activity previously used with neurons (Lee and Goldberg 1996; Tursun et al., 2005), to some wells that received CHI. MG-132 alone did not reduce axon elongation over 4 hr (Figure 3b). When CHI was combined with MG-132, fewer axons retracted during hr 2-5 in CHI + MG-132 than in wells treated with CHI alone, but axonal growth had still stopped in CHI + MG-132 by 4-5 hr (Figure 3b). Thus, global inhibition of protein synthesis does not significantly inhibit axonal elongation during the first hr of treatment, but during the second hr axonal growth slowed, and axons began to retract or lose growth cone activity. Concurrent inhibition of protein degradation prolonged the period during which axons could elongate in the absence of protein synthesis.
Previously, compartmented chambers were used to show that protein synthesis in distal axons is not required for axonal elongation (Blackmore and Letourneau, 2007; Eng et al., 1999). We devised a compartmented dish to investigate whether distal protein synthesis is required for DRG axon elongation. An E7 DRG with the attached peripheral root was placed in the lumen of a 5 mm diameter glass cloning cylinder, which was stuck to the substrate with silicon grease. The distal portion of the peripheral root was extended under the cylinder wall, so the end of the root was outside the cylinder. We determined that the inside of the cloning cylinder and the dish outside the cylinder were separate compartments by showing that blue dye placed in one compartment did not diffuse into the other compartment over 24 hr. In addition, different heights of the liquid medium could be maintained in the two compartments. When control medium was in the distal compartment, Schwann cells migrated with axons that extended from the end of the peripheral nerve (Figure 3c). After 24 hr axons and Schwann cells had extended hundreds of μm from the nerve root. If 20 μM CHI was in the distal axon compartment, axons still elongated for many hr (Figure 3d), but Schwann cells did not leave the nerve roots (compare Figure 3c and d). Because of the separation of the compartments, axons and Schwann cells migrated from the explants in the perikaryon compartment of dishes with CHI in the distal axon compartment (Figure 3e and f are similar, despite CHI in the axon compartment). Axonal growth rates were comparable in untreated (144±13 μm/hr) or CHI-treated (140±14 μm/hr) distal axonal compartments. Thus, although axonal growth from DRG explants stops after one hr of global inhibition of protein synthesis, protein synthesis is not required in distal axons for DRG axonal growth for >24 hr.
Growth cone collapse assays were conducted with E7 and E13 DRG explants that were exposed to Sema3A, E8 sympathetic chain ganglia exposed to Sema6A, and E7 temporal retinal explants exposed to negative cues, ephrin-A2 and slit-3 (Table 2; Figure 4). Explants were cultured 24 hr in laminin-coated wells, and then 20 μM CHI or anisomycin or control vehicle were added for 15 min before adding Sema3A to DRG cultures, or ephrin-A2 or slit-3 to temporal retinal cultures for 30 min. As illustrated in Figure 4 and presented in Table 2, the low collapse rates of untreated growth cones and the high growth cone collapse induced by these repulsive guidance cues were not different in the presence of CHI than results from explants treated with the drug vehicle (Mann-Whitney U test). Without repulsive cues, the growth cone collapse frequency did not exceed 16%, despite the presence of protein synthesis inhibitors for 45 min. After adding repulsive cues, the collapse frequency was >80% for Sema3A, Sema6A and ephrin-A2 and >60% for slit-3 with or without protein synthesis inhibitors. In addition to studies with chick DRGs, we investigated whether protein synthesis was required for growth cones of embryonic mouse DRG neurons to respond to Sema3A. DRGs were explanted from E15 mice and cultured 24 hr on laminin. Cultures were treated 15 min with CHI or the vehicle for 15 min, before adding Sema3A. As shown in Figure 4m-p and Table 2, CHI treatment did not inhibit the collapse response of mouse DRG growth cones to Sema3A. Some E7 DRG explants were cultured 8 days before treatment. Many growing axons with active growth cones were present with a mean growth rate of 64 ±5 μm/hr (n=22), and the growth rate did not change, when 20 μM CHI was added for 30 min (69±4 μm/hr, n=22). When 1 μg/ml Sema3A was added to the CHI-treated dishes, 95% growth cones rapidly collapsed. Thus, protein synthesis is not required for growth cone collapse by several neuronal types in response to several negative cues.
We tested the effects of inhibiting protein synthesis on retinal growth cone responses to surface-bound ephrin-A2. Contact with 6 μm ephrin-A2-coated beads induced 90% of temporal retinal growth cones to collapse or turn away from the beads (n=20; Weinl et al. 2004), while contacts with BSA-coated beads induced no turning or collapse (n=8). In the presence of CHI added simultaneously with beads to E7 temporal retinal explants, growth cones did not collapse after contact with BSA-coated beads (n=9), while 95% of CHI-treated growth cones (n=23) collapsed or turned away after contacting ephrin-A2 beads (Figure 4q-v). These results, consistent with responses to soluble repulsive cues, indicate that protein synthesis is not required for responses to repulsive guidance cues.
Downstream signaling from guidance cue receptors regulates actin filament dynamics via Rho GTPases (Guan and Rao, 2003; Hu et al., 2001; Wahl et al, 2000). Because it was reported that local RhoA synthesis is required for Sema3A-induced growth cone collapse (Wu et al., 2005), we investigated the effects of ephrin-A2 and Sema3A on actin filament organization in control and CHI-treated retinal and DRG growth cones. E7 retinal and DRG explants were cultured on L1, because axonal retraction induced by repulsive cues is diminished on L1, while growth cone collapse still occurs. After 15 min pretreatment with CHI or the control vehicle, explants were exposed to repulsive cues for 15 min, fixed and stained with rhodamine-phalloidin to label F-actin. Figure 5 shows images and line scans of the distribution of F-actin at the leading margins of control, ephrin-A2 or Sema3A-treated retinal and DRG growth cones, respectively, without and with CHI. Control growth cones had a peak of actin filament density at the leading margin, followed by a flat level of filament density further back in the growth cone (Figure 5a, e, f, j). Actin filament distribution and density in growth cones treated 30 min with CHI was identical to controls (Figure 5b, g). In growth cones treated 15 min with ephrin-A2 (temporal retina) or Sema3A (DRG), the peak in actin filament density at the leading edge was absent, and as previously seen (Fan et al., 1993), actin filament density was significantly lower throughout the leading margin (Figure 5 c, e, h, j). Growth cones of neurons treated 15 min with CHI, and then 15 min with ephrin-A2 or Sema3A and CHI were not different from growth cones treated with repulsive cues alone (Figure 5 d, i). Thus, protein synthesis is not required for signaling downstream from repulsive cues that reduces actin filament content.
It is reported that Sema3A stimulates RhoA mRNA translation, and Sema3A increased RhoA density in rat DRG growth cones (Wu et al., 2005). We conducted similar determination of RhoA staining intensity in growth cones of DRG neurons treated with Sema3A with or without inhibiting protein synthesis. As shown in Figure 5k-o, we found no significant changes in mean RhoA staining intensity in DRG growth cones after Sema3A treatment in control or CHI-treated dishes. Thus, treatment of chick DRG neurons with Sema3A did not significantly change the RhoA content of growth cones.
We examined the effects of CHI on responses of E7 DRG growth cones to attractive cues NGF and NT-3. E7 DRG explants were cultured overnight in L1-coated wells in F12 and B27 without neurotrophins. Growth cone behaviors were recorded before and after adding 40 ng/ml NGF (Figure 6a; Supplemental videos S1 and S2) or NT-3 (Figure 6b). Growth cones rapidly expanded in response to either neurotrophin. As measured in Figure 6c and d (also supplemental videos S3 and S4), this expansion response was not inhibited by 15 min pretreatment with CHI, which remained in the media with the neurotrophins. In another experiment we extended the CHI pretreatment period to two hr before adding NGF. We observed that after two hr in CHI the spreading response of growth cones to NGF addition was absent (Supplemental Figure S2). Thus, chick embryo growth cones normally contain sufficient components to spread in response to neurotrophins without need for protein synthesis.
We next determined the turning response of E7 DRG growth cones toward a soluble NGF gradient. When an NGF-coated micropipette was placed near growth cones, growth cones turned toward the micropipette with a mean turning angle of 12±2.5 ° (n=17; Figure 6e). Using criteria of Ming et al., (1997; >5° is a positive turn, -5° to +5° is no turn, <-5° is a negative turn), 65% of turns were positive, 35% did not turn, and 0% were negative turns. Turning response to a BSA-coated pipette was -0.6±2.4°(n=9), significantly different from the NGF response (p < 0.005; 11% of turns were positive, 11% negative, and 88% did not turn). We then conducted assays in the presence of CHI, which was added 15 min before placing the micropipette. As illustrated in Figure 6f, growth cone turning toward the pipette occurred with a mean turning angle of 5.4±2.1° (n=19; 42% positive turns, 53% no turns, 5% negative turns). This was statistically different from the turning angle of non-CHI treated growth cones toward NGF (p < 0.05, t test). However, the turning response of CHI-treated growth cones toward a BSA-coated pipette was -1.7±2.2° (n=11; 0% positive turns, 91% no turns, 9% negative turns), which was statistically different from the 5.4±2.1° turning response to an NGF micropipette (p < 0.05). Thus, global inhibition of protein synthesis did reduce growth cone turning toward NGF over a 45 min assay, although the attractive turning response was still present.
Asymmetric distribution of actin filaments is an early phase in growth cone turning toward NGF (Gallo and Letourneau, 2000). We assessed the effects of an NGF gradient on F-actin distribution in live control and CHI-treated growth cones, transfected to express a fluorescent F-actin probe, GFP-UtrCH. This probe contains the F-actin binding domain of utrophin and binds F-actin without altering the dynamics of actin-dependent events (Burkel et al., 2007). Figure 6 shows the average GFP-UtrCH intensity values along 15 pixel wide line scans taken across the width of growth cones before and after being exposed to BSA, NGF, or NGF gradients in the presence of CHI. A shift in F-actin distribution toward the NGF source was seen in both control and CHI-treated growth cones, but not toward a BSA source. A quantification of the F-actin distribution across the growth cones was estimated by summing the average GFP-UtrCH pixel intensities along the line scans in the growth cone halves toward the NGF source and in the halves away from the NGF source and then computing a proximal/distal ratio of the summed F-actin intensities. As a measure of change in F-actin distribution, the proximal/distal intensity ratios of images acquired 2 min after introducing NGF were divided by the ratios determined from images of the same growth cones acquired just before introducing NGF. For control growth cones this ratio was 1.31 ±0.15 SE (n=10) and for CHI-treated growth cones the ratio was 1.15 ±.09 SE (n=10). These values were significantly different from ratios computed for growth cones exposed to a local BSA source (0.94 ±0.06 SE), but were not significantly different from each other by either t-test or Mann-Whitney U test. Thus, introduction of a local NGF source induced a shift in growth cone F-actin distribution toward the NGF source, and this was not affected by global inhibition of protein synthesis.
Growth cone spreading is correlated with increased F-actin content in NGF-treated growth cones. To examine changes in actin filament content in DRG growth cones after adding NGF, E7 DRG explants were exposed to global NGF for 15 min, then fixed and stained with rhodamine-phalloidin to quantify F-actin. As shown in Figure 7, the total integrated staining for F-actin in DRG growth cones was increased 250% at 15 min after adding NGF (Figure 7 a, b, f). This increase in F-actin was unaffected by 15 min pretreatment with CHI before and during NGF treatment (Figure 7 c, d, f). Thus, protein synthesis is not required for the rapid increase in F-actin and growth cone spreading that is induced by NGF.
It is reported that 5 min treatment with netrin or BDNF increased the ß-actin content of Xenopus growth cones (Leung et al., 2006; Yao et al, 2006). We treated DRG explants with NGF for 15 min with or without 15 minute CHI pretreatment, and then fixed and stained the cultures with ß actin antibodies (Figure 7g-j). By quantitative immunofluorescence, we found that NGF treatment did not increase the total integrated staining for ß-actin in DRG growth cones and distal axons (Figure 7k). As noted above, NGF treatment did result in increased F-actin content (Figure 7f), and ß-actin distribution was altered in spread NGF-treated growth cones, being concentrated at the leading edge. CHI treatment did not alter total integrated anti-ß-actin staining of growth cones, whether exposed or not exposed to NGF (Figure 7 I, j, k). Thus, NGF treatment did not change the ß-actin content of DRG growth cones and distal axons.
We used compartmented cultures to ask whether 24 hr inhibition of distal protein synthesis affects growth cone collapse in response to Sema3A. When 1 μg/ml Sema3A was added to axonal compartments treated 24 hr with CHI, 73% of growth cones of E7 DRG neurons collapsed within 10 min (n=24; Figure 8a, b).
We found that the spreading response of E7 DRG growth cones to global NGF addition also occurred after inhibiting axonal protein synthesis for 24 hr (Figure 8c-f). To investigate whether protein synthesis in axons is required for a long term turning response to NGF, we used the compartmented culture and placed agarose beads soaked in either NGF or BSA into a collagen matrix that was formed in the axonal compartment (Figure 8g and h). The collagen matrix included either 20 μM CHI or the control vehicle. After 24 hr, we analyzed axon orientation with respect to the NGF or BSA beads. We drew a line halfway between the beads and measured the orientation angle of the terminal 200 μm of each axon with respect to the closer bead (Figure 8g and h). In gels with control vehicle the orientation angle to a BSA bead was 14±7°, while orientation to the NGF bead was 46±4°. These values were statistically different (p < 0.0005). In gels containing 20 μM CHI, axon orientation toward a BSA bead was 11±8°, while orientation angle toward an NGF bead was 65±7°(p < 0.0001). Thus, DRG axons elongating from a nerve root into collagen could orient toward an NGF-bead despite continuous presence of CHI. These results show that axonal protein synthesis is not required for DRG growth cones to be guided by an NGF gradient.
Axonal protein synthesis supports multiple functions in adult terminals, including neurotransmission, plasticity and regeneration (Jimenez-Diaz et al., 2008; Klann and Dever, 2004; Piper and Holt, 2004; Twiss and van Minnen, 2006; Verma et al., 2005; Zheng et al., 2001). Protein synthesis in developing axons totals approximately 5% of neuronal protein (Eng et al., 1999; Lee and Hollenbeck, 2003). However, studies with compartmented cultures indicate that axonal growth does not require axonal protein synthesis (Blackmore and Letourneau, 2007; Eng et al., 1999). Recent papers reported that immediate local protein synthesis is required for responses to guidance cues (Lin and Holt, 2007). We investigated this requirement for chick retinal, DRG and sympathetic neurons and mouse DRG neurons. Unlike other reports, we found that protein synthesis is not required for chick and mouse DRG and chick retinal and sympathetic growth cones to respond to ephrin-A2, slit-3, semaphorins, NGF and NT-3. Our assays included cues presented globally and cues locally presented on beads or in a gradient. Furthermore, in novel studies in a compartmented dish where distal axons were in the continuous presence of CHI, but neuronal perikarya were not, axons elongated for hours and growth cones responded to guidance cues in the absence of local protein synthesis. Our results are consistent with two recent publications, reporting that protein synthesis is not required for chick retinal growth cone responses to ephrin-A5 (Lang et al, 2008) or for recruitment of DCC receptors to growth cone surfaces (Bouchard et al, 2008).
Like previous reports, NGF and Sema3A stimulated the phosphorylation of the translation repressor, elF-4EBP1. Likewise, nascent protein synthesis in DRG neurons was increased by global addition of NGF or Sema3A. Thus, our findings taken with previous findings (Campbell and Holt, 2001; Cox et al., 2008; Takei et al., 2001), suggest that common mechanisms regulate protein synthesis in developing axons in response to extrinsic factors. However, the relative roles of distally synthesized proteins in axonal functions may depend on metabolic, developmental or physiologic circumstances of different neurons.
When protein synthesis inhibitors were globally applied to DRG or retinal explants, the axonal growth rate was normal for one hour, but eventually elongation slowed, growth cone motility diminished, and axons retracted. Axonal growth may stop because axonal assembly eventually uses all available components. It may also be that proteins with critical roles in growth cone motility are degraded. We noted that growth cones began to detach and retract during prolonged protein synthesis inhibition. Adhesion receptors that mediate growth cone traction are reutilized via endocytotic/exocytotic recycling (Caswell and Norman, 2006; Dequidt et al., 2007; Tojima et al., 2006). Perhaps, when protein synthesis is blocked, degradative events during recycling exhaust adhesive receptors. In support of this, growth cone detachment and axonal retraction that occurred after prolonged CHI treatment was delayed, when we also inhibited protein degradation.
When protein synthesis inhibitors were in the axonal compartment of a compartmented chamber, axonal elongation was undiminished for 24 hr or more. This indicates that all proteins necessary for axonal growth are made in perikarya and transported distally. As in prior studies (Blackmore and Letourneau, 2007; Eng et al., 1999), axons in our cultures grew several millimeters without distal protein synthesis. Axons in developing embryos reach their targets after similarly growing several millimeters or less. However, adult projection axons can be 10-100 times longer than when they connected to their targets. Even at these lengths fast axonal transport can deliver vesicle-bound proteins to terminals in a few hr, but proteins that travel via slow transport, especially cytoskeletal components, take days or weeks to reach ends of long axons. Thus, protein synthesis in adult axons is critical to replace degraded proteins, maintain cytoskeletal integrity, mediate plasticity, and support regeneration (Campenot and Eng, 2000; Hengtst and Jaffrey, 2007; Koenig and Giuditta, 1999; Piper and Holt, 2004; Twiss and van Minnen, 2006; Verma et al., 2005; Zhang and Poo, 2002).
Why are our results different from reports that local protein synthesis is required for growth cone responses to guidance cues? One possibility involves the neurons and animal species involved. We used DRG neurons, sympathetic neurons and retinal neurons from embryonic chicks and mice. Other labs used embryonic Xenopus retinal (Campbell and Holt, 2001; Leung et al., 2006; Piper et al., 2006) or spinal cord neurons (Guirland et al., 2003; Yao et al., 2006), and one study used fetal rat DRGs (Wu et al., 2005). Retinal and DRG neurons are common between these studies, so it is unclear that these differences are due to different neuronal phenotypes. The differentiation state of Xenopus spinal cord neurons is uncertain, as the spinal cords were from young embryos, and the neurons were uncharacterized.
Another source of differences may lie in different levels of metabolism in these in vitro studies. Perhaps, our neurons had high metabolic rates that generated sufficient proteins in the perikarya to supply all growth cone functions. In support of this, after 24 hr culture E7 DRG axons had extended >2000 μm, and E7 retinal axons had extended >1000 μm. E7 DRG explants maintained axonal growth rates of >1 mm/day for at least 8 days. These lengths far exceed axon lengths in published figures of Xenopus retinal and spinal neurons or rat DRG neurons. Xenopus spinal cord neuronal axons were 100-200 μm long and were cultured on poly-lysine (Guirland et al., 2003; Yao et al., 2006), unlike the natural substrates we used. Our liquid medium was F12 with B27 additives, while Xenopus neurons were cultured in diluted Leibovitz medium with 1% serum and Ringer's solution. The importance of specific medium components to neuronal metabolism has been appreciated for many years (Bottenstein and Sato, 1979; Chen et al, 2008). Axonal growth by Xenopus spinal neurons is most vigorous in the first 12 hours, most growth occurs in the first 24 hr, and few neurons survive 4 days (Tabi and Poo, 1991). Another indication of high activity in our neurons was the rapidity of growth cone responses to guidance cues. Upon adding NGF, growth cone spreading and increased F-actin was evident by 2 min, while retinal and DRG growth cones began to collapse within 2 min of adding ephrin-A2 or Sema3A. This rapid response is more consistent with mechanisms that activate signaling pathways rather than protein synthesis. Xenopus retinal growth cone turning toward netrin was not seen until ten min after netrin-induced ß-actin synthesis was detected (Leung et al., 2006).
Perhaps, growth cones in our cultures were rich in proteins that function in growth cone motility, while levels of these proteins were low in other studies. We found that 45 min CHI treatment produced no significant decreases in growth cone content of RhoA or of ß-actin, as expected if protein levels are high. Neither did 15-30 min treatment of DRG explants with Sema3A or NGF induce detectable increases in RhoA or ß-actin content of growth cones, respectively. Leung et al. (2006) reported that in Xenopus retinal growth cones ß-actin staining intensity was 30% higher after five min netrin treatment. Assuming a retinal growth cone contains 500-1000 ribosomes (Campbell and Holt, 2001), and it takes one min to synthesize an actin molecule (Trachsel, 1991), these growth cones would have to initially contain about 17,000 actin monomers for the addition of 5,000 monomers (1,000 molecules/min for 5 min) to constitute a 30% increase. Actin and tubulin are the most abundant proteins in chick brain growth cones (Cypher and Letourneau, 1991), and based on cytoplasmic G-actin pools in chick brain neurons (30-37 μM; Devineni et al., 1999), we estimate a 10 μm × 20 μm × 1 μm DRG growth cone contains about 4 × 106 ß-actin monomers, 100X more than Xenopus retinal growth cones. In the studies of Wu et al. (2005) rat DRG explants were cultured three days, and the rat DRG axons had reached 400-500 μm long, which is 1/4 the length of our E7 chick DRG axons after 20 hr. As seen in Figure 3a of Wu et al. (2005), RhoA was barely detectable in embryonic rat DRG growth cones. On the other hand, our DRG growth cones stained robustly for RhoA with or without Sema3A treatment, and we found that migrating growth cones in 8 day in vitro DRG cultures collapsed rapidly in response to Sema3A, even with CHI treatment. Our results are consistent with the hypothesis that growth cones of actively growing axons contain sufficient RhoA and ß-actin to respond to guidance cues without protein synthesis. Though our data show that guidance cues stimulate protein synthesis in axons of E7 DRG neurons, our data also show that these events do not detectably increase growth cone levels of RhoA or ß-actin and are not required for responding to guidance cues.
Global treatment with CHI did affect DRG growth cones. Although globally inhibiting protein synthesis for up to 45 min did not significantly affect growth cone responses, discrete changes from lacking key proteins may occur. After two hr CHI treatment, DRG growth cones did not spread in response to NGF. Globally applied CHI reduced the turning of DRG growth cones to toward NGF. Turning is more complex than growth cone spreading, and may involve exocytosis of vesicle-associated proteins (Tojima et al., 2006) that are transported at fast transport rates of 50-400 mm/day (Squire et al., 2008). Such proteins could be moved from the perikaryon to the tip of a 1000 μm axon in 4-30 min (or 30 sec-3 min in a 100 μm Xenopus spinal neuron axon), which is within the duration of a turning response. Thus, because vesicle transport is rapid, globally applied protein synthesis inhibitors cannot distinguish a growth cone location from a cell body location for protein synthesis that is necessary for growth cone turning. In an attempt to eliminate contributions of perikaryon-derived proteins to growth cone responses, some studies isolated growth cones from cell bodies by severing axons (Campbell and Holt, 2001; Ming et al., 2002; Wu et al, 2005). However, the isolated axons were damaged, as most did not recover growth cones (Ming et al., 2002). In another study of severed axons (Zheng et al., 2001), cycloheximide treatment induced retraction of isolated axons, but axons of intact neurons did not retract after cycloheximide treatment.
Therefore, unlike previous reports, we used compartmented dishes to examine whether distal axonal protein synthesis is required for responses to guidance cues. Because DRG growth cones responded to globally applied Sema3A or NGF and could turn towards an NGF source when distal protein synthesis was inhibited for 24 hr, our results clearly showed that proteins made in the perikaryon and transported to growth cones are sufficient for axonal elongation and growth cone guidance.
In summary, we confirmed that guidance cues stimulate protein synthesis in distal axons. Protein synthesis may occur in axons from the earliest neuronal polarization and axonal differentiation, via targeted mRNA transport into axons and signaling by extrinsic cues to regulate mRNA translation (Kiebler and Bassell, 2006). Extrinsic cues like Sema3A and NGF may regulate axonal mRNA translation throughout this period, during navigation to targets, later as axons branch and are remodeled into terminal fields, and in mature organisms to maintain terminals and to respond to stimuli or injury. Our in vitro results indicate that proteins made in distal axons of chick neurons are not necessary for axonal elongation and growth cone responses to guidance cues, while other in vitro studies indicate that distally synthesized proteins are required for responses to guidance cues. As noted above, higher metabolic activity in our neurons may explain these differences in in vitro studies. Perhaps, in embryos the relative contributions of locally translated proteins like RhoA and ß-actin to total growth cone pools is initially low as immature axons rapidly elongate to their targets. Later, the significance of distal axon protein synthesis may increase, as axons reach their targets, elongate by intercalary growth to become the greatest part of neuronal mass (Pfister et al., 2004), and form mature endings in a complex environment. Local protein synthesis may become highly important as axon terminals become locally distinct in form and function. In vivo studies across the time span of development are needed to more clearly understand the roles of distal protein synthesis in axonal morphogenesis.
This research was supported by N.I.H. grants HD19950 (P.C.L.) and NEI Training Grant EY07133 (B.M.M.) and by a grant from the Minnesota Medical Foundation. We thank Dr. Gianluca Gallo and Dr. Lorene Lanier for helpful comments.