Conserved 3′-Untranslated Region Sequences Direct Subcellular Localization of Chaperone Protein mRNAs in Neurons^*

DNA Expression Constructs

All reporter constructs for analyses of RNA localization were based on myristoylated, green fluorescent protein (GFP^myr) plasmid originally provided by Erin Schuman (7). GFP expression constructs containing the β-actin and γ-actin 3′UTRs have been described (7). 3′UTRs of rat calreticulin and Grp78/BiP mRNAs (GenBank^TM accession numbers NM_022399 and M14050.1, respectively) were generated by RT-PCR and cloned into GFP^myr plasmid after sequence verification. The primer sequences and RT-PCR conditions are included in the supplemental material. The Kif5C⁵⁶⁰-dTomato, Kif1^CC-YFP, and pPrlSSmCherry constructs were kindly provided by Dr. Gary Banker.

Cell Culture and Transfections

Dissociated dorsal root ganglion (DRG) neurons were prepared as described previously (25). Neuron cultures were prepared from E18 embryonic rat cortexes using previously described dissociation and plating conditions (26). For expression of reporter constructs, both DRG and cortical neurons were transfected immediately after dissociation using the AMAXA Nucleofector^TM apparatus in rat neuron Nucleofector solution with 1–5 μg of plasmid DNA (program G-13; Lonza, Inc.). After electroporation, the cells were gently resuspended in culture media and plated immediately; medium was replaced after 4 h. For viral transduction, lentivirus (LV) was added to the DRG cultures immediately prior to plating. Generation of LV is fully described in the supplemental material.

In Situ Hybridization

The methods for fluorescence in situ hybridization (FISH) varied with the probe design and preparation; individual hybridization methods are described in detail in the supplemental material. Antisense oligonucleotide probes were designed to detect endogenous mRNAs in cultures and in tissue sections as described previously (7). These were synthesized with the 5′-amino modifier C6 at four thymidines per oligonucleotide, and then chemically labeled with digoxigenin succinimide ester (Roche Applied Science). Rat calreticulin probes spanned nucleotides 1360–1410 and 1637–1686 and rat Grp78/BiP probes spanned nucleotides 1422–1471 and 2164–2213; probes for rat β-actin and γ-actin mRNAs are as published previously (7). Scrambled oligonucleotide probes were used to test for specificity. cRNA probes were used to detect the GFP reporter mRNA in transfected neurons (27, 28). For this, linearized pcDNA3-eGFP (Addgene, Plasmid 13031) was in vitro transcribed with SP6 or T7 RNA polymerases using digoxigenin-labeled nucleotide mixture (Roche Applied Science) to generate antisense and sense riboprobes.

After hybridization, samples were processed for immunodetection. The following primary antibodies were used: chicken anti-neurofilament (NF) H (1:1,000; Chemicon), chicken anti-NF M (1:1,000; Aves Laboratories), chicken anti-MAP2 (1:10,000; Abcam), rabbit anti-Tau (1:5,000; Sigma), and rabbit anti-S100 (1:500; Dako). Probes were detected using Cy3-conjugated mouse anti-digoxigenin antibody, and proteins were detected using Cy5- and fluorescein isothiocyanate-conjugated anti-rabbit and anti-chicken antibodies (1:400 each; Jackson ImmunoResearch). To increase sensitivity for the tissue sections, an additional Cy3-conjugated anti-mouse IgG F′ab fragment antibody (1:400; Jackson ImmunoResearch) was included with secondary antibody incubation.

Epifluorescent microscopy was used to capture the pixel intensity of the FISH signals for cultured neurons. ImageJ was used to quantitate pixel intensities for the GFP mRNA in growth cone images (matched for exposure, gain, and offset). For this, the growth cone was visualized by differential interference contrast, and NF signal was used to determine the end of the axon shaft.

Leica DMRXA2 microscope fitted with Orca-ER CCD camera (Hamamatsu) was used for epifluorescent microscopy. Leica SP2 confocal microscope was used to image tissue sections and for live cell imaging (see below).

Immunofluorescence

For immunostaining only, sciatic nerve sections were equilibrated in phosphate-buffered saline (PBS) and then incubated in 20 mm glycine three times for 10 min followed by 0.25 m NaBH₄ for 30 min to quench autofluorescence. Samples were rinsed in PBS, permeabilized in 0.2% Triton X-100 for 15 min, and then incubated in 5% donkey serum for 1 h to block nonspecific binding. Sections were then incubated overnight at 4 °C in the following primary antibodies diluted in blocking buffer: rabbit anti-calreticulin (1:100; StressGen), rabbit anti-Grp78/BiP (1:100; StressGen), and chicken anti-NF H (1:1,000; Chemicon). Tissues were rinsed several times in PBS and incubated for 1 h in the following secondary antibodies diluted in blocking buffer as follows: fluorescein isothiocyanate-conjugated anti-chicken (1:400; Jackson ImmunoResearch) and Alexa555-conjugated anti-rabbit (1:750; Invitrogen). Samples were rinsed in PBS and then mounted with Prolong Gold Antifade (Invitrogen).

Fluorescence Recovery after Photobleaching

GFP expression in transfected neurons was confirmed by epifluorescence, and then distal processes were subjected to fluorescence recovery after photobleaching (FRAP) sequence at 37 °C using a Leica TCS/SP2 confocal microscope fitted with an environmental chamber (29). 40× oil immersion objective (numerical aperture = 0.7) was used for imaging, with the confocal pinhole set to 4 airy units to ensure full thickness of the axon/growth cone was photobleached. Prior to bleaching, neurons were imaged every 30 s for 2 min with a 488-nm laser and 15% power to establish base-line intensity. For photobleaching, the region of interest (ROI) was exposed to a 488-nm laser and 75% power every 1.6 s over 40 frames. GFP emission was then monitored every 60 s using a 488-nm laser, 15% power, 498–530-nm collection filter, with photomultiplier tube energy, offset, and gain matched for all collection sets. Photobleaching for Kif5C⁵⁶⁰-dTomato was performed with a 543 nm laser, 75% power, and recovery was monitored every 60 s at 15% power (595–640-nm band filter). To test for translation-dependent recovery, cultures were pretreated with 50 μm anisomycin for 30 min prior to photobleaching (29).

“Double-bleach” FRAP was performed as described with minor modifications (29). Briefly, an ROI in the distal axon was photobleached with a 488-nm laser, 100% power over 60 s. The proximal half of the original ROI was then repeatedly bleached by 15 successive scans at 70% power, 2-s intervals (continuous bleaching), whereas the distal half of the original ROI was monitored for the recovery by collecting GFP emissions (488-nm laser, 7% power, 498–530-nm collection filter) at the end of the 15th scan (monitoring scan). Continuous bleaching and monitoring scans were repeated for 61 cycles (30 s between each monitoring scan) to give an overall 30-min time-lapse sequence.

To determine the rate of vesicular transport, a 20-μm length of mid-axon shaft of pPrlSSmCherry-transfected DRG neurons was subjected to photobleaching and then monitored for recovery through anterograde transport of fluorescent vesicles. The laser and filter parameters for photobleaching and recovery were the same as those for dTomato outlined above. Image sequences were equally thresholded to minimize background using ImageJ. Each bleached ROI was then divided into 1-μm bins, and the duration for each bin to reach 50% recovery threshold was calculated by measuring the pixel intensity of the bleached ROI at 0–30 min.

ImageJ was used to calculate average pixels/μm² in the ROIs of the raw confocal images. The residual fluorescence signal at t = 0 min (post-bleach) was subtracted from intensity values. The corrected values were then normalized to the pre-bleach (set to 100%). For converting time-lapse sequences to video, the original WMV file was converted into AVI format using the Power Video Converter, version 1.5.43 (Apus).

Analyses of Ligand-dependent mRNA Transport

Both local and bath applied neurotrophin 3 (NT3) and myelin-associated glycoprotein (MAG) were used to evaluate regulation of axonal mRNA transport. For local application, NT3 (100 ng; Alomone Labs, Ltd.) and MAG-Fc (2.5 μg; R & D Systems) were covalently coupled to 2.5 × 10⁷ Fluoresbrite YO Carboxylate Microspheres (Polysciences, Inc.). Equimolar amounts of BSA (Sigma) and human IgG Fc (Sigma) were immobilized to microparticles as controls for NT3 and MAG, respectively. Efficiency of coupling was determined by the Bradford assay as described previously (7). For bath application, 100 ng/ml NT3 or 2.5 μg/ml MAG was added directly to the culture medium.

Accumulation of mRNA directly at sites of ligand stimulation was tested by FISH/immunofluorescence of dissociated DRG cultures grown overnight in the presence of immobilized microparticles (4.5 μm diameter) as described previously (7). Cultures were fixed and processed for FISH/immunofluorescence as outlined above. For quantifying signals, the pixel intensity was measured in bins (5 μm each 7 times) of the axon proximal to and distal to the center of the microparticle. Background was subtracted from the intensity values, and intensity in each bin was normalized to the mean intensity over the entire 35-μm binned region. Mean normalized intensity of each bin was calculated for 30 axons over at least three separate culture experiments.

Live cell imaging of GFP^myr-transfected cultures was used to visualize ligand-dependent changes in mRNA localization with accumulation of GFP as a readout. For this, ligand-coupled microparticles were contacted to axons using a pulled glass micropipette mounted onto a micromanipulator (Sutter Instruments). Microparticles prepared as above were held at the tip of the mounted micropipette using gentle suction pressure. For analyses, GFP signal intensity (pixels/μm²) was quantified in a 10-μm axon segment spanning 5 μm proximal to and distal from the center of the microparticle in each frame of the time-lapse sequence.

To quantify overall changes in axonal mRNA levels in response to these ligands, dissociated DRGs were cultured on porous membranes (8 μm diameter pores) as described previously (30). After 20 h in culture, the axonal compartment was selectively exposed to microparticles (20 μm diameter; 4.7 × 10⁵) for 4 h. Based on efficiency of protein coupling to microparticles and microparticles/ml used, an equivalent of 84 ng/ml NT3 and 4.16 μg/ml MAG-Fc was used. The axons were then sheared from cell bodies, and this “axonal preparation” was used for preparation of RNA as outlined below.

In some experiments, cultures were treated with pharmacological inhibitors to evaluate downstream signaling events for ligand-dependent axonal RNA transport. The following agents were used: K252A (200 nm; Calbiochem), PD98059 (50 μm; Biomol), and SP600125 (10 μm; Biomol). Additionally, some cultures were treated with colchicine (1 μg/ml; Sigma) to evaluate microtubule-dependent events.

RT-PCR Analyses of Axonal mRNA Levels

Axonal RNA was extracted using the RNAqueous Micro kit (Ambion). All axonal RNA samples were normalized to protein content before RT-PCR analyses as described previously (30). Normalized axonal RNA samples were reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad). The purity of axonal preparations was assessed by RT-PCR for γ-actin, MAP2, ErbB3, and GFAP mRNAs; amplification of β-actin mRNA was used as a positive control (see supplemental material for primer sequences).

After verification of purity, reverse-transcribed axonal RNA samples were processed for quantitative PCR using Prism 7900HT (Applied Biosystems) with 2× SYBR Green Master Mix (Qiagen). Axonal mRNA signals were analyzed as described previously with 12 S mitochondrial RNA amplification product providing normalization between isolates (7).

Statistical Analyses

Multiple statistical measures were used to analyze live cell imaging data. FRAP data were analyzed by one-way and repeated measures ANOVA. One-way ANOVA with Bonferroni post-hoc comparisons was performed using GraphPad Prism 5 software package. Significance of each time point post-bleach was compared with t = 0 min post-bleach. For repeated measures ANOVA, the differences of means between groups and differences within group mean over time were tested. The significance of difference of overall treatment group means as well as the difference of treatment group means for each assessment point, including within group difference from base line, were examined. The sphericity assumption of the model was checked using Mouchly test and Greenhouse-Geissure corrected method in case of violation of this model assumption. All analyses were two-tailed at 5% level of significance. The analyses were performed using statistical package SPSS version 17.0 (SPSS Inc., Chicago). The more conservative of the two statistical measures for FRAP data is indicated in the figures, with less conservative measure noted in the figure legends. For axonal GFP response to stimuli, two-way ANOVA was used to compare GFP signal intensities at different time points. All live cell imaging sequences were performed on at least 10 axons over at least three separate transfection experiments. For quantifying axonal mRNA levels by FISH or RT-quantitative PCR, significance was determined by Student-Newman-Keul test over at least three replicate experiments.

Chaperone Protein mRNAs Are Transported into Axons of Peripheral Nervous System Neurons

mRNAs encoding the endoplasmic reticulum chaperone proteins calreticulin and Grp78/BiP were previously detected in sensory axons by RT-PCR and array hybridization (7, 30). Because these analyses used lysed axons that could contain proximal axonal segments, we used FISH to directly visualize the mRNAs in dissociated cultures of DRG neurons. DRG neurons showed punctate FISH signals for β-actin, calreticulin, and Grp78/BiP mRNAs that extended into growth cones (Fig. 1A). This granular distribution is similar to other mRNAs localizing to dendrites and axons (21).

Sotelo-Silveira et al. (31) recently provided some in vivo evidence for intra-axonal localization of β-actin mRNA in myelinated peripheral nervous system axons. By FISH analyses of adult rat sciatic nerve, both calreticulin and Grp78/BiP mRNA signals overlap with NF signals in regions distinct from the S100 immunoreactivity that marks the Schwann cell cytoplasm (Fig. 1B). XZ and YZ views generated from multiple optical XY planes through the nerve sections confirmed that the FISH signals for calreticulin and Grp78/BiP mRNAs are indeed within the axoplasm (supplemental Fig. S1, A and B). We had previously shown that axons of cultured DRG neurons contain calreticulin and Grp78/BiP proteins (30). Sciatic nerve axons also showed immunoreactivity for calreticulin and Grp78/BiP proteins that again overlapped with NF immunoreactivity (Fig. 1C). Taken together, these data indicate that endogenous calreticulin and Grp78/BiP mRNAs are transported into peripheral nervous system axons both in culture and in vivo, where they are likely used to generate proteins.

3′UTRs of Calreticulin and Grp78/BiP mRNAs Are Sufficient for Axonal Localization

mRNA localization is frequently driven by sequences within the 3′UTR. Analyses of rat calreticulin and Grp78/BiP mRNA 3′UTRs showed no clear homology to the zipcode element or any other region of rat β-actin mRNA. Calreticulin and Grp78/BiP 3′UTRs also showed no homology to one another (data not shown). However, by BLAST analyses, calreticulin and Grp78/BiP 3′UTRs show considerable homology to 3′UTRs from their mammalian orthologs. 3′UTRs of available mammalian calreticulin mRNAs contain two spans of >150 nucleotides in length showing ≥85% sequence identity, with shorter spans showing ≥95% sequence identity (supplemental Fig. S2A). 3′UTRs of mammalian Grp78/BiP mRNA show even higher homology, with ≥95% identity over nearly their entire 3′UTR (supplemental Fig. S2B). In contrast, the 3′UTR of γ-actin mRNA, which does not localize into axons (Fig. 1A) (12, 14), shows <50% sequence homology between mammalian orthologs using the same BLAST parameters (data not shown).

To determine whether subcellular mRNA localization is driven by these conserved regions of rat calreticulin and Grp78/BiP mRNAs, the 3′UTRs were cloned downstream of the coding sequence of the diffusion-limited GFP^myr (7, 29, 33). We have previously used axonal fluorescence of GFP^myr as an estimation of localized protein synthesis (7, 29, 33). As positive and negative controls for axonal localization, we used GFP^myr constructs containing the 3′UTRs of rat β-actin and γ-actin mRNAs, respectively (GFP^myrβ-actin and GFP^myrγ-actin) (7). DRG cultures transfected with GFP^myrβ-actin and GFP^myrγ-actin plasmids showed the anticipated axonal localization of GFP, with the 3′UTR of β-actin mRNA giving cell body and axonal signals and the 3′UTR of γ-actin mRNA giving cell body restricted signals (Fig. 2, A and B). The GFP mRNA distribution mirrored the localization of GFP fluorescence, with cell body and axonal FISH signals in GFP^myrβ-actin expressing neurons and only cell body signals in GFP^myrγ-actin expressing neurons (Fig. 2, E and F). Neurons transfected with GFP^myr carrying the 3′UTRs of calreticulin and Grp78/BiP mRNAs (GFP^myr-Cal and GFP^myr-BiP) showed axonal GFP fluorescence and mRNA localization that was comparable with the β-actin 3′UTR construct (Fig. 2, C, D, G, and H). These data suggest that the 3′UTRs of rat calreticulin and Grp78/BiP mRNAs have axonal localization elements.

To test whether the axonal GFP^myr-Cal and GFP^myr-BiP mRNAs are locally translated, we performed FRAP analyses of transfected DRG cultures. For this, the terminal axons of neurons were photobleached, and the bleached ROI was then monitored for recovery of GFP fluorescence by time-lapse imaging (29). GFP^myr-Cal expressing neurons showed significant recovery by 6 min after photobleaching (Fig. 3, A and C; supplemental Video 1), and GFP^myr-BiP expressing neurons showed significant recovery by 8 min after photobleaching (Fig. 3, B and D). Fluorescence for both UTR reporter constructs showed a steady increase in intensity over the 20-min observation period, and the recovery for both was protein synthesis-dependent (Fig. 3, A–D; supplemental Video 2).

Despite the above protein synthesis-dependent recovery from photobleaching in these axons, closer inspection of the GFP signals in the more proximal segments of the anisomycin-treated cultures showed a decline in intensity over time (supplemental Fig. S3A). This could reflect movement of existing reporter protein from proximal axonal segments into the bleached ROI. As outlined below, several independent experimental approaches were used to rule out the possibility of protein diffusion versus localized translation of reporter.

First, we co-transfected DRG neurons with GFP^myr-Cal and a constitutively active kinesin isoform (Kif5C⁵⁶⁰) that was shown to accumulate selectively in axonal growth cones (34). In contrast to cortical neurons expressing Kif5C⁵⁶⁰-dTomato, DRG neurons showed robust accumulation of the Kif5C⁵⁶⁰-dTomato in every growth cone (supplemental Fig. S4); this is consistent with the previously published axonal nature of DRG processes in culture (14, 35). Photobleaching growth cones with both GFP^myr-Cal and Kif5C⁵⁶⁰-dTomato signals showed rapid recovery of GFP fluorescence but no recovery of dTomato fluorescence over the 30-min observation (data not shown; also see Fig. 6 below). Although Kif5C⁵⁶⁰-dTomato would move by fast axonal transport, the post-bleach observation period might not adequately reflect transport if detection thresholds for the dTomato and GFP^myr were vastly different. To more directly measure fast axonal transport rates in these DRG cultures, we transfected DRG neurons with an expression construct consisting of the signal peptide of prolactin fused to mCherry (pPrlSSmCherry). The protein generated from pPrlSSmCherry is translated in the cell body with the signal peptide directing the nascent polypeptide into the default secretory pathway with some vesicular mCherry transported into axons. Monitoring rates of mCherry movement in the axon shaft gave a transport rate of 2.6 ± 0.3 μm/s, consistent with known rates of fast axonal transport (36). With an average axon length of 1,200 μm by 3 DIV for the cultured rat DRGs, GFP could reach from cell body to the growth cone in 7–9 min through microtubule-based transport. Thus, we could not exclude the possibility that transport of cell body-derived GFP^myr protein contributed to the recovery from photobleaching.

To distinguish locally synthesized GFP^myr from transported and diffusing GFP^myr protein, we used a double-bleach FRAP sequence to continuously photobleach signals in the proximal axon of the GFP^myr-Cal transfected DRGs, whereas recovery was monitored in distal axons. Although the GFP recovery is overall lower and more variable with the near continual laser exposure required for this double-bleach sequence (29), the distal axon still showed significant recovery from photobleaching (supplemental Fig. S3B).

Diffusion of abundant mRNAs was previously suggested to account for axonal localization in hypophyseal and olfactory nerve axons (37–40). To determine whether GFP^myr-Cal and GFP^myr-BiP transcripts nonspecifically diffuse into the axons, we used LV-mediated transduction to express GFP^myr-Cal. With serial dilution of the LV, we obtained near homogeneous expression in the DRG neurons at much lower overall GFP fluorescence levels than by the electroporation method used above. Despite low GFP fluorescence in the cell body, axonal GFP signals were seen as long as GFP fluorescence was visible in the cell body (supplemental Fig. S3C). This observation plus the restriction of the GFP^myrγ-actin mRNA to the neuronal cell body (see Fig. 2B) argues that the axonally localized GFP mRNA and protein are the result of specific mRNA transport driven by the conserved 3′UTR of calreticulin mRNA.

Finally, we performed FRAP analyses on the GFP^myr-Cal- or GFP^myr-BiP-transfected DRGs after microtubule depolymerization to determine the contribution of microtubule-based transport to the recovery of GFP fluorescence above. Even in the presence of colchicine, both GFP^myr-Cal- and GFP^myr-BiP-expressing neurons showed significant fluorescence recovery after photobleaching (Fig. 3, C and D). Although diffusion rather than transport of GFP protein could contribute to this recovery, the double photobleach experiments shown in supplemental Fig. S3B effectively rule out this possibility. Moreover, the myristoylation tag greatly reduces diffusion of the GFP (33); FRAP performed on axons of DRGs expressing soluble GFP shows recovery in seconds compared with the minutes required for recovery of the GFP^myr with the axonally localizing 3′UTRs shown here (data not shown). Finally, GFP^myr constructs with γ-actin 3′UTR and deletions of calreticulin 3′UTR (see below) show no axonal fluorescence despite expressing exactly the same protein. Taken together, these data point to a microtubule-dependent transport of GFP^myr mRNA into axons that is specifically driven by the 3′UTRs of calreticulin and Grp78/BiP mRNAs.

Conserved RNA Elements Drive Axonal Localization of Calreticulin mRNA

Because axonal localization of a heterologous mRNA could be driven by the 3′UTR of calreticulin or Grp78/BiP mRNAs and these show high sequence identity between mammalian orthologs, we hypothesized that the conserved regions of these UTRs contain RNA structures needed for axonal localization. We generated 3′UTR deletion mutants of the GFP^myr-Cal reporter to test this possibility (Fig. 4A). Because the 3′UTR of calreticulin showed less conservation than Grp78/BiP, we reasoned that this 3′UTR should provide a more stringent test for functionality of conserved regions. GFP^myr constructs that included nucleotides 1315–1412 or 1682–1780 of rat calreticulin mRNA showed axonal fluorescence with protein synthesis-dependent recovery from photobleaching (Fig. 4, B, D, and E). GFP^myr with nucleotides 1413–1681 of calreticulin mRNA showed only weak axonal fluorescence with no recovery from photobleaching (Fig. 4C). To determine whether the reporter mRNA with nucleotides 1413–1681 localized into the axons but was not efficiently translated, we performed FISH for GFP mRNA on the transfected DRGs. No axonal GFP mRNA was seen in the axons of cultures expressing GFP^myr plus nucleotides 1413–1681, but the neuronal cell bodies showed strong FISH signals for the reporter mRNA (Fig. 4C). In contrast, each construct that showed axonal GFP fluorescence recovery also showed axonally localized reporter mRNA (Fig. 4, B, D, and E). These data suggest that nucleotides 1315–1412 or 1682–1780 of rat calreticulin mRNA (Fig. 4A) are necessary for subcellular localization.

To further define the cis-elements needed for calreticulin mRNA localization, we generated GFP reporter mutants that included just nucleotides 1315–1412 and 1675–1780 in the 3′UTR. Both reporters showed axonal localization of GFP mRNA- and protein synthesis-dependent recovery from photobleaching in distal axons (Fig. 4, F and G). Thus, either of the conserved 3′UTR segments of nucleotides 1315–1412 or 1681–1780 from rat calreticulin mRNA are sufficient for axonal mRNA localization.

Calreticulin mRNA 3′UTR Is Sufficient for Localization in Central Nervous System Neurons

To determine whether calreticulin and Grp78/BiP mRNAs also localize into processes of more polarized neurons, we performed FISH on cortical neuron cultures prepared from rat embryos. Co-labeling for Tau and MAP2 was used to distinguish axons and dendrites, respectively. After 9 DIV, both the axonal and dendritic processes showed granular FISH signals for calreticulin and Grp78/BiP mRNAs (Fig. 5, A–C). Thus, these endogenous chaperone protein mRNAs can also localize into axons and dendrites of cultured central nervous system neurons.

We next asked if the 3′UTR of rat calreticulin mRNA is sufficient for axonal or dendritic localization in central nervous system neurons. For this, we co-transfected dissociated cortical neurons with GFP^myr-Cal and Kif5C⁵⁶⁰dTomato plasmids. The Kif5C⁵⁶⁰ mutant selectively concentrates in axonal growth cones of hippocampal neurons at stage 3 and above (∼4 DIV) (34). After 5 DIV, the cortical neurons showed a single process with prominent Kif5C⁵⁶⁰-dTomato signal in its growth cone. These Kif5C⁵⁶⁰-positive processes were considered as axons, and the other neurites were presumed to be dendritic. Both the axonal and dendritic processes of the cortical neurons showed prominent GFP^myr-Cal fluorescence. As in the DRG axons, the Kif5C⁵⁶⁰-dTomato-positive axonal processes showed significant recovery of GFP fluorescence within 6 min after photobleaching. This recovery was also protein synthesis-dependent and preceded recovery of the dTomato fluorescence (Fig. 6, A and C).

The dendritic processes also showed significant recovery of fluorescence within 6 min that was similarly attenuated by protein synthesis inhibition (Fig. 6, B and C). Although not reaching significance, there was an initial increase in GFP fluorescence in the bleached ROI of the dendrites despite the anisomycin pretreatment (Fig. 6C). This raised the possibility that diffusion of GFP from cell body or more proximal regions of the dendrites could explain the recovery in dendrites. However, GFP mRNA was present in both axonal and dendritic processes of GFP^myr-Cal-transfected cortical cultures by FISH analysis (Fig. 6D). Furthermore, transfection of these cortical cultures with the GFP^myr reporter 3′UTR deletion mutants containing nucleotides 1315–1587 and 1586–1863 showed similar axonal and dendritic GFP mRNA localization, but the UTR mutant containing nucleotides 1413–1681 only showed cell body GFP mRNA similar to DRG neurons (Fig. 6D). Thus, like the DRG neurons, either of the conserved regions of the 3′UTR of calreticulin is sufficient for localization in the cortical neurons, and at least one of these elements is needed for localization. These data suggest that localization elements for calreticulin mRNA are shared between the peripheral and central nervous system neurons as well as between axons and dendrites.

Calreticulin mRNA Levels Are Altered at the Sites of Tropic Stimulation

We showed previously that axonal levels of calreticulin mRNA are specifically increased by exposure to NT3 and MAG by RT-quantitative PCR assays (7). By the quantitative in situ hybridization approach, calreticulin mRNA also accumulates focally in axons at sites of NT3 and MAG stimulation, with 4–5 times more abundant FISH signal intensity in the axon directly adjacent to the stimuli (supplemental Fig. S1, C and D). In DRG neurons transfected with the GFP^myr-Cal construct, axonal GFP fluorescence accumulated adjacent to NT3 microparticles (Fig. 7A). The GFP signals showed a significant increase within 15 min immediately adjacent to the ligand source (Fig. 7D; supplemental Video 3). This NT3-dependent increase in GFP fluorescence was attenuated by inhibition of protein synthesis (Fig. 7A), and there was no response in axons exposed to microparticles with equimolar quantity of BSA (Fig. 7B; supplemental Video 4). Using a micromanipulator to apply the microparticles allowed us to directly correlate responses with neuronal subtypes in these DRG cultures. Interestingly, only the large diameter neurons responded to NT3. The small diameter DRG neurons, which are typically NGF-responsive, showed no NT3-dependent increase in GFP fluorescence (Fig. 7D). Thus, this NT3 response is restricted to a neuronal subpopulation in the DRG neurons, consistent with the restricted expression of TrkC, the high affinity receptor for NT3, in the large diameter sensory neurons.

The response to MAG was similarly translation-dependent but was overall slower than that seen with NT3, not reaching significance until 20 min after exposure (Fig. 7, C and D). However, in contrast to NT3, there was no difference in the responses of the large and small diameter DRG neurons to MAG stimulation (data not shown). GFP^myr-Cal transfected neurons exposed to human IgG Fc microparticles showed no change in axonal GFP fluorescence adjacent to microparticles over the 40-min analysis period (Fig. 7D). Taken together, these data indicate that the 3′UTR of calreticulin mRNA is sufficient for ligand-dependent localization of calreticulin mRNA in DRG axons.

Because calreticulin mRNA has two distinct localization elements, we asked if either of these UTR subregions confers ligand-dependent localization in sensory neurons. Only the GFP^myr containing the proximal element of calreticulin 3′UTR (nucleotides 1315–1587) showed accumulation of axonal GFP fluorescence in response to NT3 and MAG (Fig. 8). Neurons expressing GFP^myr with the distal element from calreticulin (nucleotides 1586–1863) showed no change in signal intensity with NT3 and MAG treatment (Fig. 8, C and D). However, GFP^myr containing just nucleotides 1315–1412 of the 3′UTR of calreticulin was responsive to both NT3 and MAG (Fig. 8, C and D).

Because RNA localization elements can also confer translational control (41, 42), we quantified axonal levels of the GFP reporter mRNAs in cultures treated with NT3 and MAG. FISH signal intensity in the growth cones of neurons expressing GFP^myr with the full 3′UTR of calreticulin (nucleotides 1315–1863) or just nucleotides 1315–1412 increased with both NT3 and MAG treatment (Fig. 8E and supplemental Fig. S5). This increase in mRNA levels is comparable with the fold increase in GFP fluorescence for GFP^myr-Cal observed in response to NT3 and MAG observed in Fig. 7. In contrast, neurons expressing GFP^myrCal^1413–1780 and GFP^myr-Cal^1586–1863 showed no change in growth cone GFP mRNA compared with controls after NT3 or MAG treatment (Fig. 8E and supplemental Fig. S5).

Stimulus-dependent Axonal Transport of Calreticulin mRNA Requires Activation of JNK

Previous work showed that NGF modulates transport of mRNAs into axons through activation of MEK1, at least for most mRNAs tested (7). To determine whether NT3 and MAG use similar signaling pathways to modulate calreticulin transport, we quantified endogenous axonal calreticulin mRNA levels after exposing axons to immobilized sources of ligand as described previously (7). After 4 h of exposure to ligand ± kinase inhibitors, axons were isolated from the cell body and used for RNA extraction. Purity of the axonal RNA isolates was verified by the absence of MAP2, γ-actin, ErbB3, and GFAP mRNAs (Fig. 9A). In DRG neurons, MAP2 and γ-actin mRNAs are restricted to the neuronal cell body, and ErbB3 and GFAP mRNAs provide a test for glial contents in these axonal preparations. As anticipated, the NT3-dependent increase in axonal calreticulin mRNA was attenuated by inhibition of the Trk receptor with K252A, but the response to MAG was not affected by K252A (Fig. 9B). This is consistent with the selective effect of NT3 on large diameter DRG neurons shown in Fig. 7. In contrast to the effects of NGF on axonal mRNA transport (7), inhibition of MEK1 with PD98059 had no effect on the NT3 or MAG response. However, treatment with the JNK pathway inhibitor SP600125 attenuated both the NT3- and MAG-dependent increases in axonal calreticulin mRNAs (Fig. 9B).

In our previous work, several mRNAs showed a similar transport profile to calreticulin, with regulation by MAG and NT3 in parallel but no response to brain-derived neurotrophic factor or NGF (7). Thus, we asked whether axonal localization of these other mRNAs might also require activation of JNK (Table 1). Shifts in axonal levels of HSP60 mRNA, whose transport is increased by NT3 and MAG similar to calreticulin mRNA, required activation of JNK. Inhibition of the JNK pathway also prevented NT3- and MAG-dependent shifts in axonal levels of HSP90 mRNA. Interestingly, axonal HSP90 mRNA are increased by MAG but decreased by NT3. Axonal levels of αB-crystallin and Kv3.1a mRNAs, which are selectively regulated by MAG or NT3, respectively, were not affected by the JNK inhibitor.