Targeting a Cap-dependent Translation Repressor to Distal Neuronal Processes
To assess the contribution of intra-axonal translation to synapse development we made a translational repressor that could be expressed at sites remote to the cell body. The translation of most cellular mRNAs is cap-dependent and relies on a translation initiation complex (eIF4F) that includes cap binding protein (eIF4E), an RNA helicase, and a scaffolding protein (eIF4G) that bridges the complex to ribosomes. eIF4E binding proteins (4E-BPs) competitively displace eIF4G, and repress translation. The 4E-BP binding to eIF4E is negatively regulated by phosphorylation and this regulatory step can be exploited by exogenous expression of a non-phosphorylatable 4E-BP mutant in which three threonines and one serine have been mutated to alanines (4E-BP1–4A) (Gingras et al., 1999; Rong et al., 2008). Because most translation requires only low levels of eIF4E, this and similar manipulations affect preferentially a set of highly regulated mRNAs, including many of those targeted to distal sites (Gingras et al., 2001; Klann and Dever, 2004; Gkogkas et al., 2013). However, because 4E-BP1–4A is rapidly turned over (Yanagiya et al., 2012) its expression in cell bodies is unlikely to repress distally targeted mRNAs. To circumvent this, we generated a 4E-BP1–4A mutant encoded by a mRNA transcript having a 3′UTR from ß-actin mRNA, which contains a sequence recognized and transported by zipcode-binding protein 1 (ZBP1) into axons and dendrites where it can be translated locally (Kislauskis et al., 1994; Tiruchinapalli et al., 2003); and we placed this fusion gene downstream of an IRES (internal ribosome entry site) in order to escape its own negative regulation (Jang and Wimmer, 1990) [Fig. 1(A)]. We called this mutant, remotely targeted 4E-BP1–4A, or simply “rem4A.”
Figure 1. Development and characterization of a targeted translation repressor. Schematic diagrams (A) illustrate the basic domain organization and features of rem4A and control vectors. PC12 cells (B) were transfected with tetOFF rem4A and the effect of 48 h doxycycline (Dox) withdrawal was tested using AHA (L-azidohomoalanine) incorporation followed by a click chemistry reaction using azide-reactive Seta-650-DBCO. Cells were solubilized and run on 12% SDS PAGE and visualized using 680 nm channel in Licor after transfer to PVDF membrane and blot against FLAG-tag to detect rem4A expression. Confocal images (C) show axons from cultured hippocampal neurons expressing dUTR4A or rem4A that were immunostained for GFP and p-eEF2. Processes were straightened using Image J. (D) Intensity measurements along the lengths of neuronal processes (excluding cell bodies) were binned. Neurons expressing rem4A (blue) show higher p-eEF2 intensity than those expressing dUTR4A (brown) and levels remain higher distally. (E) Confocal image pairs are of the same axon expressing the constructs indicated in headings and imaged before (0') and after application of Sema3A (15'). Collapse response seen in axons expressing vector only is prevented by 4EGI or rem4A, but not by dUTR4A (values provided in text). Consistent with these functional data, confocal images (F, G) show AHA-Met incorporation (green) is enhanced by Sema3A exposure in neurons expressing dUTR4A relative to vehicle (F), but not in growth cones expressing rem4A (G). GFP fluorescence from transfected neurons is shown in blue. Growth cone morphology and collapse response to Sema3A is shown with Texas red phalloidin labeling (red). (H) Bar graph shows relative increase in AHA labeling intensity following Sema3A exposure in growth cones expressing dUTR4A (*p < 0.05, one way ANOVA). Mag bars = 50 µm in E, F, G; 15.5 µm in C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Fluorescence-based metabolic labeling (FUNCAT) was used to evaluate total new protein synthesis in PC12 cells expressing rem4A. PC12 cells were transfected with an inducible version of rem4A (tetOFF4A) and maintained in or withdrawn from doxycycline for 48 h, metabolically labeled with AHA (L-azidohomoalanine), a Met-like amino acid, and AHA incorporation was evaluated in lysates separated by SDS-PAGE. As expected rem4A expression had little impact on overall levels of new protein synthesis (Graff et al., 2007; Gkogkas et al., 2013) [Fig. 1(B)]. Hypophosphorylation of 4E-BP is usually accompanied by phosphorylation of elongation factor 2 (eEF2) (Liu et al., 2006; Rose et al., 2009), a modification that also represses translation (Ryazanov et al., 1988). Thus, as a proxy for rem4A effects, localization of p-eEF2 was evaluated in hippocampal neurons expressing rem4A or dUTR4A [a non-targeting transcript lacking the 3'UTR; Fig. 1(A)] as a control. p-eEF2 immunolabeling was enriched at distal as well as in proximal processes in neurons expressing rem4A compared to neurons expressing dUTR4A, the non-targeting transcript [Fig. 1(C,D)].
To test whether targeted repression of cap-dependent translation is functionally relevant we took advantage of a well-characterized effect of Sema3A on axonal growth cones. When applied at a low dose, Sema3A promotes a local protein synthesis dependent form of growth cone collapse (Campbell and Holt, 2001; Manns et al., 2012). Cortical neurons were transiently transfected at 2 DIV with vector alone, rem4A, or dUTR4A and at 3 DIV growth cones were imaged before and 15 min after exposure to Sema3A. In neurons expressing vector alone, Sema3A provoked collapse in 61% of growth cones similar to previous studies (Carcea et al., 2010). Consistent with a requirement for cap-dependent translation, addition of 4EGI, a small molecule inhibitor of eIF4G binding to eIF4E (Moerke et al., 2007), 20 min prior to the addition of Sema3A abrogated collapse (12% collapse). Axons expressing rem4A showed no collapse (11%) similar to 4EGI, while dUTR4A growth cones responded similar to vector expressing controls (52%) [Fig. 1(E)] (Chi Square, p < 0.001 for GFP vs. 4EGI or rem4A; 50 neurons per group).
That the effect of rem4A is due to repression of protein translation is further supported by FUNCAT localization. In dUTR4A expressing neurons, Sema3A increased AHA incorporation in growth cones with no change observed in rem4A neurons or vehicle-treated controls [Fig. 1(F–H)]. In contrast Sema3A modestly decreased AHA incorporation in cell bodies, but there were no differences between untransfected cell bodies and those expressing dUTR4A or rem4A (one way ANOVA, p = 0.22). Additionally, no differences were detected in FLAG-tagged protein levels generated by the two constructs (data not shown). In adult dorsal root ganglia, ZBP1 levels are very low and can be rate limiting for the transport of ß-actin transcripts (Donnelly et al., 2011). However, limited ZBP1 supply does not appear to be relevant for the transport of rem4A here as ZBP1 levels are high in developing hippocampal neurons (Gu et al., 2002; Tiruchinapalli et al., 2003) and F-actin levels are similar between dUTR4A and rem4A expressing axons (t test, mean rhodamine phalloidin intensity, p = 0.4378). The latter is also consistent with previous work showing that Sema3A-mediated collapse requires local translation of RhoA and NF-protocadherin (Wu et al., 2005; Leung et al., 2013), but occurs independent of local actin translation (Leung et al. 2006). Taken together, the data show that transient expression of a translational repressor interferes with intra-axonal translation machinery and growth cone function and that the strategy is effective only when transcripts encoding the repressor are targeted to remote sites.
Presynaptic Translation Regulates Recycling Vesicle Pool
To assess whether targeted repression of intra-axonal translation alters vesicle recycling, we generated mosaic cultures in which a small number of neurons expressing either rem4A or vector alone were surrounded by a much larger number of untransfected neurons. At 10–12 DIV (18 h after transfection), neurons were stimulated with a hyperkalemic solution in the presence of FM4–64, a lipophilic styryl dye used to fluorescently label recycling synaptic vesicles. When vesicles fuse, the compound is incorporated into the exposed internal leaflets, and after endocytosis, the recycled vesicles remain fluorescent (Betz et al., 1996). Area and intensity of recycling vesicle clusters in transfected axons contacting unlabeled dendrites (cis) or vice versa (trans) [Fig. 2(A,B)] were measured using a semi-automated approach (Schmitz et al., 2011) (Supporting Information Fig. S1). The data show that presynaptic expression of rem4A increased intensity and area of FM-dye labeled sites [Fig. 2(C)], while postsynaptic expression of rem4A exerted no trans-synaptic effect on either the intensity or area of FM-dye labeled sites [Fig. 2(D)]. By contrast, postsynaptic expression of rem4A reduced PSD95 intensity (Supporting Information Fig. S1) consistent with previous work showing PSD95 levels can be regulated by local, dendritic translation (Muddashetty et al., 2007).
Figure 2. rem4A alters presynaptic function in cis. Confocal images in (A) and (B) illustrate the preparations used to evaluate FM dye uptake (shown in green in color overlays or in white) in living axons transfected with vector only (A) or rem4A (B). GFP labeling from transfected neurons is shown in magenta together with DIC image of neurons. Boxed areas are shown at higher mag to the right of each image, first as a color overlay and then with FM dye in gray scale so that labeling can be seen clearly. Scatterplots (C, D) show changes in recycling site intensity and area at sites expressing the construct in cis (in axons; C) or in trans (in dendrites; D). Inverted confocal image pairs in E show neurons transfected with SypHluorin alone (con) or together with DsRed-rem4A (rem4A) as indicated, before (rest) and after stimulation (stim) in the presence of bafilomycin. Data are summarized in F. *p < 0.05; **p = 0.002, unpaired two-tailed t-tests. Numbers in µg beneath x axis labels indicate the amount of vector or rem4A construct transfected. Mag bars = 30 and 15 µm (enlargement) in A, B; 30 µm, E. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FM-dyes can modulate vesicle recycling on their own (Zhu and Stevens, 2008). Thus to confirm our findings, neurons were transfected with SypHluorin, a pH sensitive probe that is targeted to the lumenal surface of vesicles and shows increased fluorescence intensity at higher pH (following exocytosis) (Zhu and Stevens, 2008), alone or together with DsRed-rem4A. Neurons were imaged and depolarized in the presence of bafilomycin, to prevent vesicle re-acidification, and the change in fluorescence (ΔF) between resting and stimulated status was compared. The data show that ΔF in rem4A axons was significantly greater than in controls, consistent with an increase in the vesicle recycling pool [Fig. 2(E,F)].
To test whether size of the total pool of vesicles (which includes recycling and reserve pools) is altered, immunolabeling intensity for vesicular glutamate transporters (vGluts)1–3 was compared in neurons expressing rem4A or vector alone. There were no significant differences between the groups (mean intensity 547 ± 34 (GFP, n = 11) vs. 525 ± 42 (rem4A, n = 13), t test, p = 0.7). Taken together the data indicate that targeted repression of cap-dependent translation in axons increases the recycling pool of vesicles without changing the total pool. This could occur by recruiting vesicles from the reserve pool, but could also reflect changes in vesicle organization and packing.
Cdk5/p35 Signaling is Regulated by Cap-dependent Translation
The increased recycling pool size observed in axons expressing rem4A resembles effects of cyclin-dependent kinase 5 (Cdk5) inhibition on presynaptic terminals (Tomizawa et al., 2003; Kim and Ryan, 2010). Cdk5 activity is regulated by p35, a protein that can be regulated by degradation (Patrick et al., 1998; Kusakawa et al., 2000; Lee et al., 2000). We asked whether p35 levels are also controlled by cap-dependent translation. To test this, we co-expressed full-length p35 cDNA in HEK cells with or without rem4A. Western blots of whole cell lysates show that rem4A expression decreased p35 levels and that this effect required an intact 5′UTR; replacing the 5′UTR with an IRES sequence upstream of the p35 coding region rendered p35 protein levels resistant to rem4A mediated repression [Fig. 3(A,B)]. To confirm this effect in a more relevant environment, Neuro2A cells were transiently transfected with rem4A. While transfection efficiency is <100% (∼70%), it is higher and far more consistent than in primary neurons so that the impact of rem4A on endogenous proteins can be measured biochemically. Western blots show that rem4A expression decreased p35 levels compared to expression of vector alone [Fig. 3(C,D)]. Levels of CaMKIIα also appeared reduced by rem4A as expected, although the result failed to reach statistical significance (Burgin et al., 1990; Benson et al., 1992; Aakalu et al., 2001). Levels of a regulatory subunit of PKA, PKARIIß, were unchanged [Fig. 3(C,D)].
Figure 3. p35 is regulated by cap-dependent translation. (A) HEK cells were transfected with the constructs indicated and then immunoblotted for p35 and GAPDH (as a loading control). Data were visualized and documented using a LiCor (examples shown are taken from the same blot). Numbers at right and in C are molecular weights of size standards (in kDa) (B) Bar graph of p35 intensity normalized to loading control. One way ANOVA, p = 0.0001, Tukey's post test, ***p = 0.0001, compared to all other conditions. Examples of western blots of lysates from Neuro2A cells expressing the constructs indicated in the headings (C) and then blotted for the antibodies listed. (D) Bar graph shows quantification of western blot data normalized to loading control (*p = 0.04, t-test). Confocal images (E, F) show endogenous p35 immunolabeling (red in overlay) in the context of vGlut (green in overlay) and SMI31 labeling for axons (blue in overlay) at sites of axodendritic contact (E) or in free axons having no contacts (F). Arrows indicate colocalized clusters of vGlut and p35 labeling. Scatter plots (G) show significantly decreased p35 immunolabeling in masks defined by FM dye uptake in axons transfected with GFP vs. rem4A (strategy as in Supporting Information Fig. 1). Pairs of confocal images (H) show examples of axons transfected with GFP (left) and the indicated constructs (written below each pair) and then loaded with FM dye (right); processes were straightened using an Image J plugin. Scatter plots compare FM dye labeling intensity (I) and area (J). One way ANOVA p < 0.001; Tukey's post-test ***p < 0.001, **p < 0.01 all groups compared to vector. Numbers in µg beneath x-axis labels indicate the amount of cDNA construct transfected. Mag bar = 15 µm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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In neurons, immunolabeling for endogenous p35 extended throughout all processes with greater enrichment in cell bodies and dendrites than in axons, which were identified by labeling for an axonal marker [SMI31, Fig. 3(E,F)]. In both dendrites and axons, immunolabeling was partially punctate. Some p35 puncta colocalized with vGlut-labeled clusters along dendrites, which are presumably synapses [Fig. 3(E)], but also with vGlut clusters at nonsynaptic hot spots in axons having no nearby dendrites [Fig. 3(F)]. To test whether p35 levels at vesicle recycling sites are diminished by rem4A, neurons expressing rem4A or vector alone were stimulated in the presence of a fixable version of FM dye and then fixed and immunolabeled for p35. At recycling sites in axons expressing rem4A, labeling intensity for p35 was significantly diminished relative to control [Fig. 3(G)].
To test whether p35 levels regulate vesicle recycling, FM dye uptake was stimulated and recycling pools were compared in neurons expressing vector alone, p35 shRNA, or exposed to 4EGI. The data show increased FM dye intensity and area in p35 knockdown axons as well as following bath application of 4EGI [Fig. 3(H–J)]. These data support that the increased recycling pool size observed in rem4A expressing axons is due at least in part to decreased presynaptic p35 levels.
Local mRNA Translation Contributes to Axonal p35 Levels
Our data suggest that p35 is translated locally in axons. A recent study reported that p35 mRNA is transported into axons (Zivraj et al., 2010); and this was confirmed using single-molecule (sm)-FISH, a fluorescence in situ hybridization method utilizing multiple fluorescent oligonucleotide probes (Raj et al., 2008; Akins et al., 2012; Cajigas et al., 2012). The greatest density of p35 mRNA labeling was in cell bodies, dendrites showed a decreasing gradient, and axons displayed an irregular distribution pattern with regions of greater and lesser intensity [Fig. 4(A–C)].
Figure 4. Intra-axonal de novo synthesis of p35. (A) Inverted confocal image of sm-FISH labeling for p35 mRNA in a cultured neuron. Fluorescent clusters, probably indicative of mRNA granules, are observed in dendrites (B, enlargement of region in A) and axons (C, enlargement of region in A). (D) Cartoons show p35 translation reporters driven by pCAGG (chicken beta-actin promoter with CMV enhancer) promoter and flanked by p35 5'UTR and either full length or truncated p35 3'UTR. Photoswitchable mEOS2 is preceded by the first 16 aa of p35, which contains a myristoylation sequence (Myr). p35 translation reporters were introduced at 9DIV and images were taken ∼16 h later with green filter (E, F) and red filter (F and not shown). At t = 0, green fluorescence in the entire neuron was photoswitched to red and accumulation of green was then monitored in axons at the indicated times (E and G). Cell bodies were photoswitched every 2 min throughout the duration of the experiment to exclude contributions from this source (as in F). Two-way ANOVA of construct vs. time (G), p <0.0001; Mag bars = 14 µm (A) and 7 µm (B, C, E), and 65 µm (F). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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To test whether p35 can be translated locally in axons and dendrites as well as in cell bodies, we generated a p35 translation reporter in which mEOS2, an irreversible photoswitchable tag (McKinney et al., 2009), was flanked by the full length 5′ and 3′ UTRs of p35 mRNA (p35mEOS2-fL) and a control reporter having a truncated 3′UTR (p35mEOS2-dUTR) (Aakalu et al., 2001; Leung et al., 2006). In both constructs mEOS2 was preceded by the first 16aa of p35, which encodes a myristoylation site (Hisanaga and Saito, 2003), so that the reporter will be anchored to the membrane near its site of synthesis (Aakalu et al., 2001) [Fig. 4(D)]. Neurons were transfected with the reporters at 9 DIV and identified by green fluorescence ∼16 h later. New protein synthesis in axons was assessed by measuring accumulation of green in axons that had been photoswitched to red and isolated “optically” (Aakalu et al., 2001) to eliminate contributions of newly synthesized green proteins transported from cell bodies. At t = 0, the entire neuron was photoswitched from green (excitation λ = 488 nm, emission λ = 516 nm) to red (561 nm; 581 nm) using 405 nm laser light; cell bodies were photoswitched repeatedly every 2 min throughout the duration of the experiment [Fig. 4(F)]; and appearance of green was monitored in axons every 10–20 min. Recovery of green p35mEOS2-fL fluorescence was detected by 10 min and continued to increase over 60 min. Truncating the 3′UTR (p35mEOS2-dUTR) eliminated this effect [Fig. 3(E,G), 2-way ANOVA, p <0.0001]. These data indicate that p35 can be generated locally within axons at sites distant from growth cones.