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Axonal mRNA transport is robust in cultured neurons but there has been limited evidence for this in vivo. We have used a genetic approach to test for in vivo axonal transport of reporter mRNAs. We show that β-actin’s 3’UTR can drive axonal localization of GFP mRNA in mature DRG neurons, but mice with γ-actin’s 3’UTR show no axonal GFP mRNA. Peripheral axotomy triggers transport of the β-actin 3’UTR containing transgene mRNA into axons. This GFP-3’β-actin mRNA accumulates in injured PNS axons before activation of the transgene promoter peaks in the DRG. Spinal cord injury also increases axonal GFP signals in mice carrying this transgene without any increase in transgene expression in the DRGs. These data show for the first time that the β-actin 3’UTR is sufficient for axonal localization in both PNS and CNS neurons in vivo.
In cultured neurons, axonally synthesized proteins contribute to growth cone turning, growth cone formation, and axon survival (Verma et al., 2005; Leung et al., 2006; Yao et al., 2006; Aschrafi et al., 2008; Cox et al., 2008; Andreassi et al., 2010). There has been limited evidence for axonal mRNA localization and translation in vivo. The 3’ untranslated region (UTR) of EphA2 mRNA has been shown to localize a reporter mRNA into embryonic spinal cord axons (Brittis et al., 2002). Localization of ribosomes and β-actin mRNA in mature myelinated PNS axons has also been reported (Koenig et al., 2000; Sotelo-Silveira et al., 2008). The Fainzilber laboratory provided evidence for synthesis of proteins in mature PNS axons in vivo (Hanz et al., 2003; Perlson et al., 2005; Yudin et al., 2008). Although these studies support the hypothesis that mature PNS axons contain mRNAs and translational machinery, a report on ribosome transfer from Schwann cells to axons raises the possibility that these molecules may derive from sources other than transport from the neuronal cell body (Court et al., 2008). Ultrastructural studies of rodent hippocampus also suggested that ribosomes are excluded from mature CNS axons in vivo (reviewed in, Steward and Schuman, 2003). To date there has been no direct evidence that neuronal derived mRNAs are transported into and translated within mature PNS or CNS axons in vivo. Here, we have used a transgenic approach to directly test for axonal mRNA transport in vivo. Our data indicate that the 3’UTR of β-actin is sufficient to target a reporter mRNA for transport into distal axons in vivo.
All animal procedures were approved by respective IACUCs. cDNA for destabilized GFP (dzGFP) with a myristoylation motif and 5' and 3' UTRs of CamKIIα (dzGFPmyrCamKIIα) was used for transgenic constructs. The 3'UTRs of rat β-actin and γ-actin mRNAs were amplified by reverse transcription (RT) coupled PCR to add Not1 and Xho1 restriction sites. Primer sequences for transgene preparation and PCR analyses (see below) are available upon request. PCR products were cloned into pTOPO vector (Invitrogen), sequence verified, and then subcloned into dzGFPmyrCamKIIα replacing the CamKIIα 3'UTR. Mouse Tα1 promoter was amplified adding 5’ and 3’ AflII sites, and cloned into the above constructs to generate transgenes designated Tα1-GFP-3'β-actin and Tα1-GFP-3'γ-actin, respectively. Transgenes were digested with Xho1, gel purified, and microinjected into zygotes of FVB/N inbred mice (Taconic Farms) (Nagy et al., 2003). 3 Tα1-GFP-3'β-actin and 1 Tα1-GFP-3'γ-actin founders were generated.
PCR and Southern blotting tested GFP transgene insertion. Genomic DNA from tail clippings was used for duplex PCR for GFP and MeCP2 to confirm transgene integration and determine relative copy number (i.e., through ratio of the GFP and X-linked MeCP2 products). Copy number was further verified by Southern blot and quantitative PCR (qPCR).
For Southern blotting, genomic DNA was digested with Xho1 and BamH1, electrophoresed on agarose gels, denatured, and transferred to Hybond-N (Amersham). Hybridization was carried out using 32P-labeled GFP probe generated by random priming (Stratagene). Blots were imaged using a Typhoon 9400 (GE Healthcare). For qPCR, a standard curve was generated with serial dilutions of the transgene template. DNA from 3 animals for each line was diluted to 30 ng/µl (~1 × 104 genome copies) and three 1:5 serial dilutions were made. qPCR was performed for GFP on each dilution using the Prism 7900HT (ABI) with 2x SybrGreen Master Mix (Qiagen).
Mice homozygous for transgenes were used for all studies, including the four copy Tα1-GFP-3'β-actin mice that were generated by crossing homozygous Tα1-GFP-3'β-actin2a and Tα1-GFP-3'β-actin2b mice.
Dorsal root ganglion (DRG) cultures were prepared as described (Zheng et al., 2001). For immunostaining, neurons were cultured at low density on polylysine/laminin coated glass coverslips. For isolation of axons, neurons were cultured on polyethylene tetrapthalate membranes, and axons were isolated and tested for purity by RT-PCR as described (Vuppalanchi et al., 2010).
Mice were subjected to sciatic nerve crush at mid-thigh as previously described (Zheng et al., 2001). The nerve grafting procedure was performed for the common fibular nerve (CF) as described (English et al., 2005). CF segment from wild type mice was grafted into transected CF of transgenic mice. For acellular grafts, wild type CF segment was repeatedly frozen in liquid nitrogen prior to grafting (English et al., 2007).
Infinite Horizon Spinal Cord Impactor (Precision Sys. & Instr.) was used for T9 contusive spinal cord injury at 65 kDynes force. Sham mice underwent laminectomy alone. Mice received injections of saline and Baytril via IP for 7 days after injury and were given vitamin C supplemented drinking water throughout the study. Bladder expression was performed twice daily for injured animals until voluntary control was resumed.
RNA was isolated from tissues using a motorized homogenizer (Omni International) and RNAqueous kit (Ambion). RNA was isolated from cultured cells using RNAqueous-micro kit (Ambion). 100 ng DNAse-treated RNA was used as template for iScript RT kit (BioRad). Reactions were diluted 5-fold for PCR. Standard PCR (≤ 30 cycles) was analyzed by ethidium bromide stained agarose gels. Prism 7900HT with 2X SybrGreen Master Mix was used for qPCR with quadruplicate reactions for all samples.
Tissue samples were fixed in 4% paraformaldehyde (PF), cryoprotected overnight in 30% sucrose at 4°C and then cryosectioned at 10 µm. Immediately before use, sections were warmed to room temperature, washed in phosphate buffered saline (PBS) and incubated in 20 mM glycine for 30 min followed by 0.25 M NaBH4 for 30 min. For cultured neurons, coverslips were fixed in 4%PF for 20 min and then rinsed in PBS. Samples were processed identically for subsequent steps as described (Vuppalanchi et al., 2010). The following primary antibodies were used: chicken anti-Neurofilament Heavy (NFH, 1:1000; Millipore), mouse anti-neurofilaments (NFH, NFM & NFL, 1:1500 each; Sigma), Alexa546 goat anti-chicken (1:1000; Invitrogen), and Alexa555 goat anti-rabbit (1:1000; Invitrogen).
Fluorescence in situ hybridization (FISH) on nerve tissues was performed as described (Muddashetty et al., 2007). Digoxigenin (DIG) labeled oligonucleotide probes were used to detect GFP mRNA with Cy5-labeled mouse anti-DIG (Jackson Immunores) for detection. Rabbit anti-S100 antibody (1:200; Abcam) was used with secondary antibodies as above. Scrambled probes and primary antibody omission served as negative controls. These controls did not reveal any fluorescent signals (data not shown).
In cultured neurons, β-actin mRNA is transported into axons and dendrites, while γ-actin mRNA remains in the cell body (Bassell et al., 1998; Zheng et al., 2001; Tiruchinapalli et al., 2003). This axonal localization is driven by the 3'UTR of β-actin mRNA through a conserved 'zip code' element (Kislauskis et al., 1994). We have taken advantage of this zip code element to test for axonal localization in vivo. For this, we generated transgenic mice expressing a destabilized, myristoylated GFP with the 3'UTR of rat β-actin or γ-actin mRNAs (Fig. 1A). The 54 nt zip code region of rat β-actin shows 100% identity to the corresponding region of mouse β-actin mRNA 3'UTR; rat γ-actin 3'UTR shows 94% identity with the mouse γ-actin mRNA (data not shown). Myristoylation limits GFP diffusion in neuronal processes to provide a reporter for sites of translation (Aakalu et al., 2001; Willis et al., 2007; Yudin et al., 2008). The neuronal-specific Tα1 tubulin promoter, which is activated during periods of axon growth (Gloster et al., 1994), was used to drive transgene expression.
Genotyping by PCR and Southern blotting confirmed transgene integration (Fig. 1). Two β-actin lines and one γ-actin line were chosen for subsequent studies based on robust GFP expression in DRGs. Tα1-GFP-3'γ-actin line has four transgene copies and Tα1-GFP-3'β-actin2a and Tα1-GFP-3'β-actin2b lines have two transgene copies (Fig. 1B). The two Tα1-GFP-3'β-actin lines were crossed to generate a line carrying four transgene copies (Tα1-GFP-3'β-actin2ax2b; Fig. 1B). DRG cultures from the Tα1-GFP-3'β-actin lines showed GFP in cell bodies and neurites, but those from the Tα1-GFP-3'γ-actin line showed GFP only in the neuronal cell bodies (Fig. 1C). RT-PCR of axonal isolates from DRGs cultured from the transgenic lines also confirmed axonal localization of GFP-3’β-actin but not GFP-3’γ-actin mRNA (data not shown). RT-PCR and immunoblotting showed no evidence for GFP expression in Schwann cells in these mice (Fig. 1D).
To determine if the 3'UTR of β-actin can support in vivo localization of GFP mRNA in the PNS, sciatic nerve was crushed to activate the Tα1 promoter and L4–5 DRGs and sciatic nerve were analyzed. Both transgenes showed increase in GFP fluorescence in the DRG after injury (Fig. 2A). GFP signals were highest in the DRGs over 5–14 d after crush injury, consistent with previous reports for Tα1 activation (Gloster et al., 1994). Only the nerve sections from the Tα1-GFP-3’β-actin mice showed GFP signals (Fig. 2B). The Tα1-GFP-3'γ-actin nerves did not show signals above the autofluorescence of wild type nerve.
Since we saw no transgene expression in non-neuronal cells (Figs. 1D and and2A),2A), we used RT-qPCR to analyze GFP mRNA levels. DRGs from both transgenic lines showed increase in GFP mRNA peaking 5 d after crush (Fig. 3A). Relative transgene induction in the DRGs was overall higher in Tα1-GFP-3'β-actin than in Tα1-GFP-3'γ-actin lines, but the absolute levels of the GFP mRNA were not significantly different prior to injury (Fig. 3B). Curiously, the sciatic nerve GFP-3’β-actin mRNA levels showed peak accumulation at 1 d after injury, which precedes the 5 d peak in GFP-3’β-actin mRNA levels in the DRG (Fig. 3A). Relative nerve GFP mRNA levels in Tα1-GFP-3'γ-actin mice could not be calculated over 0–28 d samples since threshold values were not reached for this ΔΔCt analyses. Thus, we analyzed raw RT-qPCR amplification plots. DRG and sciatic nerve RT-qPCR plots for Tα1-GFP-3'β-actin mice rose from baseline within a few cycles of one another (Fig. 3C,D). Plots for the DRG RNA from Tα1-GFP-3'γ-actin mice were similar to the Tα1-GFP-3'β-actin mice. However, the sciatic nerve RTqPCR plots from Tα1-GFP-3'γ-actin mice were essentially the same as wild type (Fig. 3C,D). Thus, GFP-3'γ-actin mRNA does not appear to be transported into distal sciatic nerves.
The RT-qPCR amplification plots for the naïve Tα1-GFP-3'β-actin sciatic nerves show higher Ct values, indicative of less GFP mRNA, than the naïve DRG and 5 d crushed nerve RNA samples, but clearly indicate presence of GFP-3'β-actin mRNA in the mature uninjured nerve (Fig. 3C). Thus, the neuronally expressed transgene mRNA with the β-actin 3'UTR also localizes into uninjured PNS axons.
As a more rigorous test for axonal localization of the GFP-3'β-actin mRNA, we transected the CF nerve of transgenic mice and provided a wild type nerve graft as a regeneration substrate (English et al., 2005). FISH for GFP mRNA showed signals in 14 d grafts from Tα1-GFP-3'β-actin mice that were clearly distinct from Schwann cells (Fig. 3E). The Tα1-GFP-3'γ-actin lines only showed background GFP mRNA signals in grafts (Fig. 3E). Axons from transected nerves similarly regenerated into acellular allografts (data not shown); RT-qPCR for acellular grafts also showed Ct values for Tα1-GFP-3'β-actin nerves indicative of GFP mRNA localization while grafts of Tα1-GFP-3'γ-actin mice showed no evidence for GFP mRNA (Fig. 3F).
Axonal mRNA localization has been detected in the developing spinal cord (Brittis et al., 2002). Thus, we asked if Tα1-GFP-3' β-actin mice showed axonal GFP fluorescence in their spinal cord axons. Since PNS nerve injury increased axonal GFP signals in the Tα1-GFP-3'β-actin mice, we reasoned that spinal cord injury might also lead to localization of the GFP-3’ β-actin mRNA into ascending DRG axons. Scattered GFP positive axons of the sham Tα1-GFP-3'β-actin mice (Fig. 4A–D). Strong GFP fluorescence was seen in axons of the Tα1-GFP-3'β-actin mice at 10 d after contusion injury (Fig. 4E–H). By confocal imaging, these GFP signals overlapped with axonal markers but were distinct from glial markers (Fig. 4F,G). No GFP fluorescence was seen in the Tα1-GFP-3'γ-actin spinal cord (Fig. 4I–L). Lower thoracic DRGs in these animals showed no increase in GFP signals with spinal cord injury (Fig. 4B,F, and G inset panels), suggesting that this injury did not activate the Tα1 promoter.
The data presented here indicate that β-actin’s 3’UTR but not γ-actin’s 3’UTR can drive axonal mRNA localization in vivo, both in the peripheral and central processes of sensory neurons. The identical coding sequences of these transgenes make it highly unlikely that protein diffusion could account for the appearance of GFP selectively in the axons of the Tα1-GFP-3'β-actin mice. The lack of any transgene expression in Schwann cells points to axonal transport of the GFP-3’ β-actin mRNA as the only plausible explanation for its presence in these axons. This is particularly true for the nerve grafts, where axons from the transgenic mice are regenerating into a wild type substrate that contains no cells with the transgene. These data provide unequivocal proof for transport of mRNAs in adult mammalian sensory axons in vivo.
The vast majority of studies of axonal mRNA transport and local translation have focused on developing and regenerating axons (reviewed in, Willis and Twiss, 2010). The presence of GFP-3’ β-actin mRNA in the naïve nerve combined with absence of any expression in Schwann cells indicates that this transgene mRNA is transported into adult axons in vivo, irrespective of their growth status. Thus, the transgenic approach used here specifically overcomes the unavoidable limitations of in vitro models where neurons cannot be cultured without axotomy. This is consistent with works showing that both injury and neuropathic pain-invoking stimuli can activate translation in PNS axons, presumably by utilizing mRNAs that reside in the mature axon prior to injury or stimulation (Hanz et al., 2003; Perlson et al., 2005; Jimenez-Diaz et al., 2008; Yudin et al., 2008; Melemedjian et al., 2010).
A multitude of mRNAs localize into axons of cultured CNS neurons (Taylor et al., 2009) and our data show that the 3'UTR of β-actin mRNA can drive transport of the GFP mRNA into the CNS processes of DRG neurons. Although transgene expression was limited to the DRG neurons in these mouse lines, our data justify future genetic approaches to test for functionality of axonal RNA localization motifs in the mature CNS. A parallel genetic approach for visualizing RNA localization in mice was also recently taken by the Singer lab (Lionnet et al., 2011). In this study, MS2 recognition stem loops were placed into the endogenous β-actin gene, effectively tagging the mRNA with a non-translated epitope. Our data complement this model, but also extend the findings to show in vivo axonal mRNA localization in adult mammals.
Immunolabeling studies have suggested that CNS axons have a much lower content of translational machinery than PNS axons, and Verma et al. (2005) hypothesized that this relates to the differing regenerative capacities of CNS vs. PNS neurons. Future studies will be needed to determine the extent to which RNA transport differs between PNS and CNS projections of the DRG neurons studied here. mRNAs have been detected in hypothalamic and olfactory axons in vivo but there was no ultrastructural evidence for ribosomes in these processes (Mohr et al., 1991; Mohr and Richter, 1992; Wensley et al., 1995; Denis-Donini et al., 1998; Mohr et al., 2001). Nonetheless, translationally active mRNAs were recently detected in olfactory axons suggesting that ultrastructural absence of ribosomes cannot be used to exclude the possibility for translation in vivo (Dubacq et al., 2009). This emphasizes the need for more sensitive tools and reagents to assess mRNA localization and translation in vivo as we report here.
The Tα1 promoter used for transgenic mice here generates an artificial situation since transgene expression is activated during periods of axonal growth (Gloster et al., 1994). Despite limitations introduced by this, our data suggest that injury can activate transport of mRNAs from the cell body rather than the axonal mRNA levels simply paralleling an increase in transcription. Consistent with this, tropic stimulation can mobilize transport of existing cellular mRNAs from the perikaryon into axons (Willis et al., 2007). CGRP mRNA also appears to be targeted for increased axonal delivery in injury-conditioned neurons despite a clear decline in expression of CGRP mRNA in the cell body after injury (Toth et al., 2009). It is intriguing to speculate that the injury-induced transport of GFP-3’β-actin mRNA could be independent of transcriptional activation of the transgene. The peak in sciatic nerve GFP-3’β-actin mRNA levels prior to the peak in transcriptional activation of the Tα1 promoter (Fig. 3A) and increased axonal GFP signals in ascending sensory axons without corresponding increase in DRG GFP expression after spinal cord injury (Fig. 4) are consistent with this notion.
Vogelaar et al. (2009) have recently shown that axons of cultured neurons have decreased regenerative capacity when β-actin mRNA is depleted (Vogelaar et al., 2009). However, it is not clear if this is the case in vivo. As more is learned of the mRNAs transported into axons, the mechanisms regulating their transport and translation, and the roles of localized protein synthesis in axons in culture settings, it will be of critical importance to move from culture to in vivo models so that axonal protein synthesis can be assessed in the neuron’s natural environment.
Erin Schuman provided the destabilized GFPmyr reporter used here. Freda Miller provided the Tα1 tubulin promoter. This work was supported by funds from NIH (R21-NS045880 and R01-NS041596 to JLT; K99-NR010797 to DEW; R21-NS060098 to GJB; and P20-RR15588 to CBKS) and the Christopher and Dana Reeve Foundation (TB2-0602 to JLT and SOK). Transgenic Core Facility of the University of Delaware was used to generate mice. The Nemours Core facilities provided technical assistance (Nemours Foundation and P20-RR020173 COBRE funding).
The authors have no conflict of interest with work reported in this manuscript.