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Keywords:

  • axon;
  • DRG;
  • dynein;
  • endosome;
  • MAPK;
  • microtubule;
  • NGF;
  • Rap1;
  • retrograde transport;
  • TrkA

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.

A compelling body of evidence points to the importance of retrogradely transported neurotrophin (NT) signals for neuronal survival and function (1–12). The mechanism(s) underlying retrograde transport of NT signaling has been explored (1,6,8,10,12). Under the ‘signaling endosome’ hypothesis, nerve growth factor (NGF) binds to and activates its receptor tyrosine receptor kinase A (TrkA) at the axon (AX) terminal, and the NGF–TrkA complex is internalized through clathrin-mediated and -independent pathways (13–20). The signaling endosome thus formed is retrogradedly transported to the cell body (CB) (21). Despite much support for the hypothesis (21–23), important questions remain as to how retrograde signals are trafficked (11,19,24). Indeed, some studies suggested that retrograde axonal transport of neither the NGF–TrkA complex nor the phosphorylated form of TrkA, pTrkA, is required to convey a survival signal to the CB (25,26).

Recent studies point to an important role for dynein-based transport of NT signals. Trk receptor appears to be associated with members of the cytoplasmic dynein family during retrograde transport. Both the 14 and the 74 kD chains of dynein could be coprecipitated with Trk in brain lysates, and both subunits accumulated together with Trk distal to a ligature site in the rat sciatic nerve (27). Proximity of TrkA to dynein has been shown by both confocal imaging (28) and immunoelectron microscopy (29). In addition, dynein-based transport was shown to be required for retrograde trafficking of activated Trks and thus played a critical role in promoting survival of NT-dependent DRG neurons (30). A role for intact microtubules in transporting NGF signals in sciatic nerve was also recently shown (28). Taken together with other reports (31,32), a strong case can be made for the importance of dynein and intact microtubules for retrograde transport of NT signals. Beyond a role for moving the signal within AXs, we envision the possibility that trafficking events also serve to regulate the nature of the signals generated, an issue addressed herein.

NGF induces prolonged activation of the mitogen-activated protein kinases (MAPK1/2) signaling pathway. While Ras mediates the transient phase of MAPK1/2 activation, Rap1, a small guanosine triphosphatase, contributes to the sustained activation of the MAPK1/2 signaling pathway (33), which is necessary for NGF-induced differentiation in PC12 cells (34). Following NGF treatment, Rap1 was shown to be concentrated and activated in the perinuclear region (35), and activated Rap1 was found in a long-lasting complex with both activated TrkA and activated MAPK1/2 (pMAPK1/2) in an early-endosome-enriched fraction (36).

We used PC12 cells and DRG neurons to pursue further the mechanism of NGF trafficking and signaling. We found that following NGF treatment, both NGF–TrkA complexes and Rap1 moved to the perinuclear region of PC12 cells where they were present in early endosomes in which persistent activation of MAPK1/2 was also registered. For both TrkA and Rap1, trafficking to the perinuclear region was prevented by the disruption of microtubules with nocodazole. Notably, nocodazole suppressed the activation of Rap1 and the ensuing persistent activation of MAPK1/2 without interfering with TrkA or Ras activation. Disrupting the dynein–dynactin complex by overexpresssing p50, dynamitin (37), also blocked trafficking of Rap1 to the perinuclear region and inhibited activation of MAPK1/2. Using a lentiviral system, we showed that overexpressing p50 inhibited retrograde transport of NGF and activated TrkA (pTrkA) and NGF-induced activation of MAPK1/2 in DRG neurons. Even for neurons whose CBs were bathed in NGF, the consequence of p50 overexpression was the death of immature neurons and atrophy of mature neurons. Our findings suggest that dynein and microtubules play an essential role in mediating not just the retrograde trafficking of activated TrkA but also activation of Rap1 signaling to MAPK1/2 in support of neuronal differentiation and survival.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Microtubule depolymerization prevents NGF-induced translocation of Rap1 to the perinuclear region

Microtubules, comprising α- and β-tubulin, form an elaborate network that extends throughout the cytoplasm in virtually all eukaryotic cells. One of the most important functions of the microtubule is to regulate the location of membrane-bound organelles. We reasoned that disruption of the microtubule network might alter NGF-induced trafficking events. First, we established that NGF treatment resulted in accumulation of NGF, TrkA, Rap1 and activated MAPK1/2 in early endosomes in the perinuclear region in PC12 cells (Figures S1 and S2). We then examined the microtubule network in PC12 cells using confocal microscopy. Cells, grown on glass coverslips, were serum starved overnight prior to treatment. The cells were pretreated with either the nocodazole vehicle (−nocodazole) or 5 μg/mL nocodazole (+nocodazole) for 2 h at 37°C. Cells were then treated with either the NGF vehicle or 50 ng/mL NGF (+NGF) for 30 min. The samples were prepared for indirect immunofluorescence and analyzed using confocal microscopy. Rap1-positive structures (magenta) and the microtubule network (green) were visualized and analyzed. (Please note that red color in all two-color images is presented as magenta and colocalization with green signal will be therefore denoted as white color hereafter.) In cells pretreated with the vehicle only (−nocodazole), microtubules were distributed throughout the cell and the Rap1-positive puncta were seen dispersed throughout the cytoplasm (Figure 1A–C). Treatment with NGF for 30 min had little or no effect on the distribution of α-tubulin (Figure 1A versus 1D). However, NGF treatment resulted in trafficking of Rap1 to the perinuclear region (Figure 1E versus 1B and Figure 1F versus 1C). The increase of Rap1 signal in the perinuclear region in NGF-treated cells was quantified in arbitrary unit (a.u.) of fluorescence intensity, and the results are presented in Figure 1M.

image

Figure 1. Nocodazole pretreatment blocked NGF-induced trafficking of Rap1 to the perinuclear region. PC12 cells were prepared as described in Materials and Methods. Following pretreatment with either the nocodazole vehicle alone (−nocodazole) (A–F) or 5 μm nocodazole (+nocodazole) (G–L) for 2 h, cells were treated with either NGF (50 ng/mL) (+NGF) (D–F, J–L) or the NGF vehicle (−NGF) (A–C, G–I). Indirect immunofluorescence was carried out using confocal microscopy. Tubulin (green) and Rap1 (magenta) were stained with a mouse antibody to α-tubulin and a rabbit antibody to Rap1, respectively. An Alexa 488 goat anti-mouse IgG conjugate was used to visualize the microtubules, and an Alexa 568 goat anti-rabbit IgG conjugate was used to detect Rap1. Colocalization of antigens is denoted by the white signal. Twenty-five cells were examined in each condition, and a typical representative image was shown. The size of the collection box for all panels is 30 × 30 μm. In (M), the average line fluorescence intensity of Rap1 staining in 16 control cells and 12 NGF-treated cells was measured. Because cells were different in size, we chose to measure the line fluorescence intensity of 10 μm distance (2.5 μm from the nuclear edge toward the center of nucleus and 7.5 μm from the nuclear edge toward the cell periphery). The results show that NGF treatment resulted inapproximately 30% increase in average fluorescence intensity (a.u.) in the perinuclear region compared with that in the control groups. Standard errors are also presented.

Pretreatment with 5 μm nocodazole at 37°C for 2 h resulted in a very different outcome. There was complete disintegration of the microtubule network in approximately 95% of cells (Figure 1; +nocodazole). The staining pattern for α-tubulin changed from the complex network seen in normal cells (Figure 1A,D) to a pattern that was diffused and evenly distributed (Figure 1G,J). The NGF vehicle caused no obvious change in the pattern for Rap1 immunostaining in cells pretreated with nocodazole, that is the Rap1-positive structures remained dispersed (Figure 1H versus 1B). However, Rap1 was no longer present in the perinuclear region in response to NGF treatment (Figure 1K versus 1E). Indeed, the pattern of Rap1 immunostaining showed little, if any, difference from that seen in cells treated with the NGF vehicle alone (Figure 1K versus 1H and Figure 1L versus 1I). These studies suggested that an intact microtubule network is required for trafficking of Rap1-containing organelles to the perinuclear region.

Disruption of the microtubule network blocks translocation of TrkA to the perinuclear region

To examine whether TrkA trafficking was impaired when microtubules were disrupted, PC12 cells were pretreated with either 5 μm nocodazole (+nocodazole) or its vehicle (−nocodazole) for 2 h. Cells were then treated for 30 min with either 50 ng/mL NGF (+NGF) or NGF vehicle (−NGF) (Figure 2). The samples were prepared for confocal microscopy to analyze the distribution of TrkA (Figure 2). A corresponding transmitted light image was also captured and superimposed to show the outline of the cell (Figure 2). In the absence of nocodazole (−nocodazole), treatment with NGF resulted in the expected increase in TrkA immunostaining in the perinuclear region (Figure 2B versus 2A). However, in cells pretreated with nocodazole, TrkA-positive puncta remained dispersed following NGF treatment (Figure 2D versus 2C).

image

Figure 2. Nocodazole pretreatment disrupted NGF-induced intracellular trafficking of TrkA. Following pretreatment with either the nocodazole vehicle alone (−nocodazole) (A, B) or 5 μm nocodazole (+nocodazole) (C, D) for 2 h, PC12 cells were treated with either NGF (50 ng/mL) (+NGF) (B, D) or the NGF vehicle (−NGF) (A, C). Cells were then rinsed, fixed with 100% methanol and processed for indirect immunofluorescence using confocal microscopy as described in Materials and Methods. TrkA (red) was stained with a mouse antibody to Trk (B3) and with an Alexa 568 goat anti-mouse IgG conjugate. A corresponding image from the transmitted light detector was also captured and superimposed onto the fluorescence image of TrkA to illustrate the outline of the cell. Twenty-five cells were examined in each condition, and a typical representative image was shown. The size of the collection box for all panels is 30 × 30 μm.

To confirm that TrkA trafficking was altered in cells pretreated with nocodazole, we examined the distribution of TrkA using subcellular fractionation on OptiPrep step gradients of 5:10:15:20:25% (36). Serum-starved PC12 cells were pretreated with either 5 μm nocodazole (Figure 3B) or its vehicle (Figure 3A) prior to treatment with either NGF (+NGF) or the NGF vehicle (−NGF). Cells were then rinsed and prepared for subcellular fractionation. Membrane fractions were collected from the four interfaces, and proteins were precipitated, washed and air-dried. The samples were analyzed using SDS–PAGE/immunoblotting. As shown previously, plasma membrane proteins such as the receptor for epidermal growth factor and Ras were concentrated in the light fractions (fractions 1 and 2), while the early endosomal markers Rab5 and EEA1 were enriched in the heavy fraction (fraction 4) (36).

image

Figure 3. Nocodazole pretreatment disrupted the intracellular trafficking of TrkA as analyzed using a step gradient of OptiPrep. PC12 cells were starved and pretreated with either vehicle (A, control) or 5 μm nocodazole (B) for 2 h and were subsequently treated with NGF (+NGF, 50 ng/mL) or the NGF vehicle (−NGF) for 30 min. Cells were rinsed and homogenized. The homogenates were centrifuged to remove nuclei. The resulting supernatants were fractionated by centrifugation using the step-density gradient system as described in Materials and Methods. Four membrane fractions (1, 5:10%; 2, 10:15%; 3, 15:20% and 4, 20:25%) were collected from the indicated interface, and proteins from each fraction were precipitated in 7% trichloroacetic acid and washed in acetone. The pellets were then dried and boiled in SDS–PAGE loading buffer. The proteins were analyzed using SDS–PAGE and immunoblotting. The blots were independently probed with the indicated antibodies. In a separate experiment, following treatment with either the NGF vehicle (−) or 50 ng/mL BtNGF (+) for 30 min, cells were fractionated as in (A). Fractions 1 and 4 from the vehicle- and BtNGF-treated samples were lysed and precipitated with streptavidin–agarose beads as described in Materials and Methods. The precipitates were washed, separated on SDS–PAGE and blotted with a rabbit antibody to pTrkA (C). In (D), membrane fractions (fraction 4) were collected from both NGF-treated (+NGF) and vehicle-treated (−NGF) samples and were reconstituted into an in vitro Elk-1 phosphorylation assay system. Phospho-Elk-1 and Elk-1 were detected using specific antibodies. All blots were visualized using SuperSignal, and the results shown are representative of at least three independent experiments.

In cells that were pretreated with the nocodazole vehicle (Figure 3A), NGF treatment did not alter the fractionation of EEA1, Rab5B, Rap1 or MAPK1/2; all were enriched in fraction 4 (Figure 3A). Without NGF treatment, TrkA (*) was mostly detected in fraction 2, with a small amount in fraction 1 (Figure 3A; −NGF). Following NGF treatment, TrkA (*) was predominantly seen in fraction 4 (Figure 3A; +NGF). The phosphorylated form of TrkA (pY490), pTrkA (indicated by arrow), was detected in the same fraction, as was pMAPK1/2 (Figure 3A; +NGF). The pattern for the 74 kD dynein intermediate chain (DIC74) was also examined. It was present in fractions 1 and 4 in the control sample (Figure 3A; −NGF). NGF treatment caused the disappearance of DIC74 from fraction 1; instead, it was now more concentrated in fraction 4 (Figure 3A; +NGF). These findings are consistent with the earlier results showing that a significant amount of TrkA is present in a light buoyant-density fraction prior to NGF treatment (38) and that NGF treatment for 30 min resulted in the movement of activated TrkA together with pMAPK1/2 to the heavier, early-endosome-containing fraction (20,36).

The fractionation patterns for Rab5B, Rap1 and MAPK1/2 were not affected by pretreatment with nocodazole (Figure 3B versus 3A). This treatment did alter the fractionation pattern for DIC74 and TrkA following NGF treatment. In cells treated with nocodazole but not with NGF, DIC74 was present in fraction 3 and more concentrated in fraction 4 (Figure 3B; −NGF). Most TrkA (*) was detected in fraction 2, with a small amount in fraction 1 (Figure 3B; −NGF). However, when nocodazole-pretreated cells were treated with NGF, DIC74 was captured in fraction 1 as well as in fraction 3 (Figure 3B; +NGF). Most TrkA (*) as well as most pTrkA (indicated by arrow) were now present in fraction 3 but not in fraction 4 (Figure 3B; +NGF). It is noteworthy that activation of TrkA (arrows in Figure 3A,B) was not affected by nocodazole, suggesting that NGF-induced TrkA activation at the plasma membrane or initial internalization of activated TrkA was not hindered. Furthermore, nocodazole pretreatment markedly inhibited NGF-induced activation of MAPK1/2 (i.e. pMAPK1/2) in fraction 4 (Figure 3B versus 3A). Thus, depolymerization of microtubules alters the trafficking of activated TrkA and impairs its downstream signaling.

We also treated PC12 cells with biotinylated NGF (BtNGF) and isolated fractions 1 and 4. Lysates of these fractions were then incubated with streptavidin–agarose beads to capture BtNGF and its binding proteins. The protein complex was analyzed by SDS–PAGE and immunoblotting with pTrkA antibody (pY490). Lysates of fraction 4 from BtNGF-treated cells, but not from vehicle-treated cells, contained pTrkA bound to BtNGF (Figure 3C). As a control, pTrkA was not recovered from fraction 1 of either BtNGF- or vehicle-treated cells (Figure 3C). We conclude that NGF–TrkA complexes are present in early endosomes.

To show if pMAPK1/2 activated by NGF in fraction 4 were accessible to its downstream signaling molecules, we carried out an in vitro kinase phosphorylation assay using recombinant Elk-1, a substrate immediate downstream to pMAPK1/2 in NGF signaling cascade. Fraction 4 from cells treated with NGF containing pMAPK1/2 was capable of phosphorylating Elk-1 (Figure 3D; +), while fraction 4 from cells that were treated with NGF vehicle did not contain pMAPK1/2 and was incapable of phosphorylating Elk-1 (Figure 3D; −). Therefore, a population of early endosomes is capable of transmitting the NGF signal.

Disruption of the microtubule network suppresses Rap1 and MAPK1/2 signaling

To pursue further the idea that aberrant trafficking of TrkA and Rap1 would compromise activation of the MAPK1/2 signaling pathway, we analyzed the effects of nocodazole pretreatment on the activation of Ras and Rap1. We first confirmed that TrkA was activated to a level similar to that in control samples when microtubules were disrupted. Serum-starved PC12 cells were pretreated with either vehicle (−nocodazole) or 5 μm nocodazole (+nocodazole) prior to treatment with NGF or the NGF vehicle (Figure 4). Cells were rinsed, lysed and centrifuged. The supernatant was immunoprecipitated with a specific antibody to Trk (MCTrk). The immunoprecipitate was analyzed using SDS–PAGE/immunoblotting. As shown in Figure 4A, nocodazole pretreatment did not significantly alter the magnitude or the duration of TrkA activation induced by NGF.

image

Figure 4. Nocodazole pretreatment selectively inhibited Rap1 activation and the sustained phase of MAPK1/2 activation induced by NGF. PC12 cells were starved and treated with either the vehicle (−nocodazole) or 5 μm nocodazole (+nocodazole) for 2 h prior to treatment with NGF (50 ng/mL) or NGF vehicle (0 min) for the indicated time intervals. In (A), TrkA was immunoprecipitated from all samples with a mouse antibody to Trk (MCTrk) and was analyzed using SDS–PAGE/immunoblotting. TrkA was detected using B3, and pTrkA was revealed using a rabbit antibody to pTrkA (pY490). In (B), the level of RasGTP and Rap1GTP was analyzed as described in Materials and Methods. In addition, cells were washed and then lysed in RadioImmunoPrecipitation Assay (RIPA) buffer. Ten micrograms of proteins from the resulting supernatant of each sample was separated on SDS–PAGE and analyzed by immunoblotting with a mouse antibody to the phosphorylated MAPK1/2. The blot was stripped and reprobed for the total amount of MAPK1/2 to show equal loading of protein samples. Samples from the lysates were also immunoblotted with a rabbit antibody to phosphorylated Akt (Ser473) and reprobed with a rabbit antibody to total Akt. The results shown are representative of at least three independent experiments.

Samples were also prepared to assay the active forms (i.e. GTP-bound form) of Ras and Rap1 using an established protocol (36). As shown in Figure 4B, nocodazole pretreatment had little effect on GTP loading of Ras. In contrast, nocodazole significantly suppressed the activation of Rap1 (Figure 4B). Consistent with this result, there was also suppression of NGF-induced sustained activation of MAPK1/2. While the early phase of MAPK1/2 activation was unaffected by nocodazole, the activation of MAPK1/2 seen at 30 min of NGF treatment was markedly reduced (Figure 4B). The levels for total MAPK1/2 were not affected by nocodazole. We also examined activation of the phosphatidylinositide 3-kinase (PI3K)/Akt pathway following NGF treatment. Nocodazole pretreatment caused partial suppression of this pathway, as evidenced by a decrease in the level of phosphorylated Akt (pAkt, Ser473) but not total Akt (Figure 4B). Thus, disrupting the microtubule network suppressed NGF-induced activation of Rap1 signaling, the sustained phase of MAPK1/2 activation and activation of PI3K/Akt.

The overexpression of p50 blocks NGF-induced retrograde transport of Rap1 and abolishes the sustained phase of MAPK1/2 activation

DIC74 was found to be associated with the Trk receptor in mouse brain lysates (27). We confirmed the association of DIC74 with TrkA in PC12 cells by immunoprecipitation (data not shown). Thus, cytoplasmic dyneins might play a role in intracellular trafficking of Trk (30). To examine what role dynein plays in retrograde trafficking of TrkA and Rap1 signaling, we inhibited the activity of the dynein–dynactin complex by overexpressing the 50 kD subunit of dynactin and dynamitin (p50) (37,39). The overexpression of p50 causes dissociation of the microtubule- and cargo-binding subunits of the dynein–dynactin complex, thus serving to disrupt dynein-based transport (39,40). In a transgenic mouse model, p50 overexpression led to marked inhibition of retrograde axonal transport (41).

We reasoned that p50 overexpression in PC12 cells might block NGF-induced retrograde trafficking of TrkA and Rap1 and, as a result, inhibit the sustained activation of MAPK1/2. To test this idea, we transiently transfected PC12 cells with a pcDNA3 construct that contained the full-length human p50 complementary DNA (cDNA) sequence. We used immunostaining to examine the effects of p50 overexpression. Forty-eight hours posttransfection, PC12 cells were serum starved for 16 h prior to receiving treatment with either the NGF vehicle (−NGF) or the NGF (+NGF, 50 ng/mL for 30 min) (Figure 5). Cells were then processed for immunostaining using a mouse monoclonal antibody to p50 and a rabbit polyclonal antibody to Rap1. To better illustrate the perinuclear region, we also co-stained with Hoechst 33258 to visualize the nucleus by epifluorescence (Figure 5A/b,d,f,h). In untransfected cells (Figure 5A/a,b,e,f), NGF induced accumulation of Rap1 signal in the perinuclear region. In cells that overexpressed p50 (Figure 5A/c,d,g,h), NGF treatment failed to cause concentration of Rap1 in the perinuclear region (Figure 5A/b–f versus 5A/d–h). The effect was specific for p50 because overexpression of enhanced green fluorescent protein (EGFP) did not affect NGF-induced trafficking of Rap1 (data not shown).

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Figure 5. The overexpression of p50 inhibited NGF-induced translocation of Rap1 and pMAPK1/2 signals to the perinuclear region. PC12 cells were transiently transfected with pcDNA3 plasmids that contained the full-length human dynamitin cDNA sequence for 48 h as described in Materials and Methods. Cells were then reseeded on glass coverslips and starved. Cells were treated with either the NGF vehicle (−NGF) or NGF (+NGF, 50 ng/mL for 30 min). In both (A) and (B), p50 (green) was immunostained with a specific mouse antibody (1/500 dilution) and detected with a secondary goat anti-mouse IgG–Alexa 488 conjugate. Rap1 (magenta) in (A) was stained with a specific rabbit antibody and visualized with a secondary goat anti-rabbit IgG–Alexa 568 conjugate. The sample in (A) (b, d, f and h) was also co-stained with Hoechst 33258 (blue) to mark the nucleus. The resulting samples were captured and analyzed using a Nikon E800 epifluorescence microscope. Similarly, the pMAPK1/2 signals (magenta) in (B) were stained with a specific rabbit antibody to pMAPK1/2 and visualized with a secondary goat anti-rabbit IgG–Alexa 568 conjugate. The p50 (green) was stained as in (A). The resulting samples were analyzed using a confocal microscope (B). Typically, 25 cells were examined in each condition, and a typical representative image is shown. The size of the collection box is 60 × 60 μm for (A) and 30 × 30 μm for (B).

We next investigated the sustained phase of MAPK1/2 activation in cells that transiently overexpressed p50. Without NGF treatment (−NGF; Figure 5B), the baseline level for pMAPK1/2 signal in transfected (Figure 5B/c) cells was similar to that in nontransfected (Figure 5B/a) cells (Figure 5B/d versus 5B/b). Treatment of nontransfected (Figure 5B/e) cells with NGF for 30 min showed a marked increase in pMAPK1/2 in the perinuclear region (Figure 5B/f versus 5B/b). Similarly, a marked increase in pMAPK1/2 was also seen in cells that were transfected with a plasmid vector expressing EGFP, pEGFP (data not shown). In contrast, NGF treatment failed to elicit this response when p50 was overexpressed (Figure 5B/g,h versus 5B/e,f). Thus, inhibition of cytoplasmic dynein blocked NGF-induced retrograde transport of Rap1 and abolished the sustained phase of MAPK1/2 activation.

The overexpression of p50 blocks retrograde transport of the NGF–pTrkA complex in mature DRG neurons in culture

We next examined the trafficking of NGF signals in cultured neurons. Rat embryonic DRGs were cultured on collagen-coated cover glasses for 21 days (DIV-21) as described in Materials and Methods. At this point, the cultures contain large, mature neurons with well-developed AXs. At least 94% of neurons were judged to be NGF responsive as evidenced by immunopositivity for TrkA (data not shown). Of note, these neurons no longer require NGF for survival (42). Following NGF deprivation for 24 h, cells were treated with either 100 ng/mL NGF (Figure 6B,D,F) or NGF vehicle (Figure 6A,C,E) and were prepared for immunostaining. NGF treatment resulted in redistribution of both Rap1 and TrkA to the perinuclear region, where they significantly colocalized (Figure 6A versus 6B). Consistent with our results for PC12 cells, NGF induced activation of MAPK1/2 and the signal for pMAPK1/2 overlapped with that for Rap1 in the perinuclear region (Figure 6D). Finally, we confirmed colocalization of Rap1 with EEA1 in the perinuclear region following NGF treatment (Figure 6F versus 6E). Based on these observations, we conclude that NGF treatment of DRGs resulted in an increase of these signaling proteins within the perinuclear region as expected.

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Figure 6. NGF-induced trafficking of Rap1, TrkA and pMAPK1/2 to the perinuclear region in DRGs (DIV-21). DRGs (DIV-21) were cultured and starved as described in Materials and Methods. Cells were treated with either NGF vehicle (−NGF; A, C and E) or 100 ng/mL NGF (+NGF; B, D and F) for 60 min. Cells were rinsed, fixed and prepared for immunostaining with indicated antibodies. Alexa 568 goat anti-rabbit IgG conjugates were used to visualize Rap1 (magenta). Alexa 488 goat anti-mouse IgG conjugates were used to reveal Trk, pMAPK1/2 and EEA1 (all in green). Representatives of confocal microscopic images from at least three independent experiments were shown. The size of the collection box is 50 × 50 μm.

To show whether or not p50 overexpression blocks retrograde axonal transport of NGF, we used Campenot chamber cultures (CAMP10; Figure 7A), in which distal axons (DAXs) were separated from their CBs and proximal axons (PAXs) (9,25). DRGs (DIV-21) were transduced with either a control virus or lenti-p50 or were not transduced, as described in Materials and Methods. The lentiviral system is highly efficient and results in sustained transduction of nondividing cells, such as DRG neurons (43). Following NGF withdrawal together with serum starvation for 24 h, BtNGF (100 ng/mL) was added to the DAX chamber. We elected to carry out most observations at 60 min because at this time-point there was robust transport of BtNGF to CBs. Samples were rinsed, fixed and incubated with a Streptavidin–Alexa 488 conjugate to detect BtNGF in neuron CBs and PAXs. The fluorescence signal for BtNGF/Streptavidin–Alexa 488 was measured and quantified as described in Materials and Methods. The results showed that p50 overexpression markedly decreased the amount of BtNGF present in the CB in comparison with the no-virus control (35 ± 60 a.u. versus 210 ± 40 a.u.). This represents a decrease of approximately 83% (p < 0.01; t-test) in retrogradely transported BtNGF when p50 was overexpressed (Figure 7B). Transduction with a control virus had no significant effect on transport as judged in relation to nontransduced neurons (210 ± 40 a.u. versus 244 ± 57 a.u.; p > 0.05).

image

Figure 7. Ectopic expression of p50-blocked retrograde transport of NGF–pTrkA signaling in rat DRGs (DIV-21) in Campenot chambers. A) Diagrams of two types of Campenot nerve cell growth chamber (CAMP10 and CAMP15). In (B), 20 000 cells from dissociated rat E15-16 DRGs were plated in the inner chamber of CAMP10. Cells were maintained for 21 days and then transduced with the indicated lentivirus. Seven days posttransduction, both the inner and the outer chambers were washed extensively and starved. Samples were rinsed again, and 50 ng/mL BtNGF was added to the outer chamber, followed by incubation at 37°C for 1 h. BtNGF was detected using a Streptavidin–Alexa 488 conjugate, and the amount of BtNGF in a.u. that was transported to the CBs and part of the PAXs from the DAX was quantified as described in Materials and Methods. In (C), DRGs (DIV-21) were cultured in CAMP15 and transduced as in (B). Following NGF deprivation, 100 ng/mL BtNGF was added to the axonal chamber, and the samples were incubated at 37°C for 1 h. Samples were rinsed, and the CBs were lysed, collected and cleared by centrifugation. The resulting supernatant was incubated with streptavidin–agarose conjugate. The precipitates were analyzed using SDS–PAGE/immunoblotting to detect BtNGF and pTrkA as indicated. In (D), NGF (100 ng/mL) (+) or NGF vehicle (−) was added to the DAX chamber for 15 min, and the AXs were collected for SDS–PAGE/immunoblotting analysis with the pTrkA antibody. Representative blots from at least three independent experiments were shown. In (E), DRG neurons (DIV-21) were transduced with either a control virus or lenti-p50 overnight, and the cultures were continued for an additional 7 days. Cells were extensively washed and prepared for immunostaining with a mouse antibody to TGN38 and a rabbit antibody to flag for visualizing p50. Alexa 568 goat anti-mouse IgG conjugates were used to visualize TGN38 (magenta). Alexa 488 goat anti-rabbit IgG conjugates were used to reveal dynamitin (green). Representatives of confocal microscopic images from at least three independent experiments were shown. The size of the collection box is 50 × 50 μm.

To show that retrograde transport of BtNGF/pTrkA was also blocked by p50 overexpression, we cultured DRGs (DIV-21) in CAMP15 chambers (Figure 7A). Cells were transduced with either lenti-EGFP or lenti-p50 or were not transduced, as described previously. Following NGF deprivation and serum starvation, BtNGF (100 ng/mL) was added to the DAX chamber for 60 min. Samples from the CB were rinsed, collected and lysed. BtNGF in lysates was precipitated with a streptavidin–agarose conjugate. The precipitates were washed and analyzed by SDS–PAGE/Western blotting using a rabbit-specific antibody to mouse NGF. BtNGF was detected in the CB chamber in the control virus sample (Figure 7C). In addition, pTrkA (pY490) was present in the precipitate, as assessed by reprobing with a rabbit-specific antibody to pTrkA (pY490) (Figure 7C). Transduction with lenti-EGFP resulted in relatively small (approximately 20%) decrease in pTrkA under this condition (Figure 7C). However, in cells transduced with lenti-p50, the amount of BtNGF in the CB was markedly reduced and there was little pTrkA (Figure 7C). To ensure that NGF activated TrkA in the AXs of these cells, we treated the DAX chamber with NGF (100 ng/mL) for 15 min and collected the axonal lysate for Western blotting. The level of pTrkA induced by NGF in lenti-p50 DAXs was approximately 75% of controls (Figure 7D). These results show that p50 overexpression blocks retrograde transport of the NGF–pTrkA complex in DRG neurons.

Overexpression of p50 in dividing cells causes disintegration of the Golgi complex (39,44). Because DRG neurons are postmitotic, we expected that the Golgi would remain intact. To confirm this, we stained DRG neurons with a mouse antibody (1/200) that specifically recognizes the 38 kD trans Golgi network protein TGN38. The flag-tagged p50 was visualized using a rabbit antibody to the flag tag (1/100). Alexa 568 goat anti-mouse immunoglobulin G (IgG) and Alexa 488 goat anti-rabbit IgG conjugates were used to visualize the respective primary antibodies. We compared the pattern of TGN38 staining in cells transduced with lenti-p50 with that in cells not transduced (Figure 7E). There was no apparent change in the appearance of the overall structures as marked by TGN38 antibody following viral transduction. Moreover, there was no apparent loss of DRG neurons during these experiments.

Although these experiments showed that DRG neuron survival was not affected by p50 overexpression, we noticed an apparent decrease in the size of neuronal somas even when neurons were bathed in NGF. Cellular images were captured and quantified. In the continuing presence of NGF, p50-overproducing cells were smaller, with a mean profile of 685 ± 200 μm2 (n = 50) compared with 943 ± 193 μm2 (n = 50) for cells transduced with the control virus (i.e. a decrease to approximately 73% of the control, p < 0.01). We conclude that it is possible that p50 overexpression led to a change in cell function, resulting in cellular atrophy. An alternative explanation is that NGF signaling from the surface of neuron CBs was inadequate to sustain cellular differentiation in the absence of the ability to traffic endosomes.

Overexpression of p50 blocks NGF-induced activation of MAPK1/2 and results in atrophy in mature DRG neurons in culture

To examine if p50 overexpression suppressed NGF signaling in mature neurons, DRG neurons (DIV-21) were cultured on coated cover glasses and transduced with either a control virus or lenti-p50, as described previously. Cells were then starved and treated with either NGF or the NGF vehicle and processed for immunostaining for pMAPK1/2. As expected, NGF treatment of neurons transduced with a control virus induced a marked increase in pMAPK1/2 in both CBs and AXs (Figure 8B versus 8A). In cultures transduced with lenti-p50, approximately 90% of the neurons overexpressed p50 in both the CB and the AXs. In these cells, NGF failed to elicit activation of MAPK1/2 in either the CB or the AX (Figure 8B versus 8A).

image

Figure 8. Overexpression of p50 in mature rat DRG neurons inhibited NGF-induced activation of MAPK1/2. DRGs (DIV-21) were prepared and transduced as described in Figure 7. Prior to processing for immunostaining and immunoblotting, cells were starved, washed and treated with either the NGF vehicle (−NGF) or NGF (+NGF, 100 ng/mL for 60 min). In both (A) and (B), p50 (green) was immunostained with a specific mouse antibody and detected with a secondary goat anti-mouse IgG–Alexa 488 conjugate. pMAPK1/2 (magenta) was stained. The samples were captured and analyzed using a Nikon E800 epifluorescence microscope (A and B). Twenty-five cells were examined in each condition, and a typical representative image is shown. The scale bar in the CB and AX is 20 μm. In (C), lysates from cells that were treated with either the NGF vehicle (−NGF) or NGF (+NGF, 100 ng/mL for 60 min) were analyzed using SDS–PAGE/immunoblotting. The blots were probed with a rabbit antibody to either pMAPK1/2 or the phosphorylated form of TrkA (pY490) or to pAkt. Blots that fell within the linear range of exposures were scanned and measured using Scion Image Beta 4.0.2 (Scion Corporation). The relative activity in a.u. was obtained by subtracting the background value in the vehicle-treated sample from that of the NGF-treated sample. The results shown are representative of at least three independent experiments. In (D), images of 100 cells from each sample were analyzed, and the cell size was measured using Scion Image Beta 4.0.2 as described in Materials and Methods. The size distribution was shown.

To examine the effect of p50 overexpression on NGF signaling, we harvested cell lysates from cultures treated with either the vehicle or the NGF (100 ng/mL) for 60 min. Samples were analyzed using SDS–PAGE, followed by immunoblotting with an antibody to pTrkA (pY490). As shown in Figure 8C, there was a small (approximately 17%) and statistically insignificant reduction (p > 0.5) in the level of pTrkA induced by NGF in p50-overproducing cells compared with cells transduced with a control virus. Thus, consistent with previous results (Figure 7D), p50 overexpression did not appear to significantly affect TrkA activation. Next, we measured the level of pMAPK1/2. While a robust response was evident in cells that were transduced with the control virus, activation of MAPK1/2 by NGF was virtually completely inhibited in neurons that overexpressed p50 (Figure 8C). NGF-mediated activation of the PI3K/Akt was also suppressed, albeit to a lesser extent (Figure 8C). Therefore, as for PC12 cells, overexpression of p50 in DRG neurons blocked or severely inhibited the sustained phase of MAPK1/2 activation induced by NGF while having little effect on the activation of TrkA.

To address more thoroughly what role dynein-based transport has on NGF signaling, we examined the neuronal soma size, a well-recognized phenotype regulated by NGF signaling in mature DRG neurons (45) and in NGF-dependent basal forebrain cholinergic neurons (46,47). Rat DRG neurons cultured for DIV-21 on cover glasses were transduced with either a control virus or lenti-p50 in the continuous presence of NGF. Seven days later, cells were imaged using confocal microscopy. In control samples, cell size varied from 200 to 1800 μm2, with a mean value of 1010 ± 28 μm2. In cells that overproduced p50, the mean value was significantly decreased to 651 ± 28 μm2 (p < 0.01) (Figure 8D). Overexpression of p50 thus resulted in severe atrophy (to approximately 65% of control value), despite the fact that NGF was continuously present in the medium bathing the surface of CBs and their AXs. If compromised NGF signaling and not some other consequence of p50 overexpression was responsible, the same effect should be shown with simple removal of NGF from the bathing medium. This is what was observed. The NGF withdrawal from mature DRG neurons (DIV-21) for 5 days resulted in a decrease in somal size to approximately 70% of the NGF-treated control. We conclude that interrupting dynein function through p50 overexpression markedly inhibited the NGF signaling events needed to maintain the somal size of mature DRG neurons.

Overexpression of p50 in developing DRG neurons caused cell death even in the presence of NGF

Because the survival of developing DRGs is critically dependent on the availability of NGF (42), we asked whether or not overexpression of p50 in developing DRG neurons would result in cell death in the presence of NGF. Embryonic rat DRGs (E15-16) were dissected, dissociated and cultured for 7 days (DIV-7). Cultures were then transduced with either a control virus or lenti-p50 and maintained for additional 7 days in the continuous presence of NGF (100 ng/mL). Under these conditions, we routinely achieved >95% transduction efficiency. Cultures were rinsed and apoptotic neurons detected using the In Situ Cell Death Detection Kit, TMR red (Roche). This assay specifically labels DNA strand breaks (‘nicks’) in genomic DNA produced during apoptosis. Apoptotic cells, whose nuclei were fluorescently labeled with TMR red, were visualized and counted. Transmitted light images were simultaneously collected and superimposed onto the corresponding confocal images to allow for visualization of cells. Representative images (Figure 9) show cells transduced with no virus (Figure 9A), a control virus (Figure 9B) or lenti-p50 (Figure 9C). The DRG neurons transduced with lenti-p50 showed massive cell death (Figure 9C), whereas relatively few apoptotic cells were observed in either of the control cultures (Figure 9A,B). Scoring the percentage of apoptotic cells showed a marked and highly significant (p < 0.01) increase when neurons were transduced with lenti-p50 (Figure 9D). Thus, even though NGF (100 ng/mL) was continuously present throughout the culture period, transduction with lenti-p50 resulted in the death of approximately 85% of neurons. Consistent with other recent findings (30), we conclude that dynein-based transport of NGF signaling appear to play a critical role in promoting survival of developing DRG neurons.

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Figure 9. Ectopic expression of p50 in early developing cultured rat DRG neurons (DIV-7) caused apoptosis. DRG neurons were isolated and cultured for 7 days as described. Cells were transduced with no virus (A), a control virus (B) or lenti-p50 (C) overnight, and the cultures were continued for an additional 7 days in the presence of 100 ng/mL NGF. Seven days posttransduction, samples were processed, and apoptotic cells were detected using the In Situ Cell Death Detection Kit, TMR red. The samples were analyzed and images captured using a confocal microscope, and the nuclei of apoptotic cells were specifically labeled and revealed with magenta fluorescence. Images from transmitted light detector were simultaneously collected and superimposed onto their corresponding fluorescence images. The size of the collection box is 500 × 500 μm. In (D), we scored the number of apoptotic DRG neurons in each condition. The percentile of apoptotic cells was shown.

Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Herein, we show that intact microtubules and functional dynein are critical for the ability of NGF signaling to induce sustained activation of MAPK1/2 and to support the survival and maintenance of DRG neurons. The underlying trafficking events induced by NGF treatment included translocation of TrkA and Rap1 to the perinuclear region where they were present in early endosomes. Importantly, the trafficking events supported by microtubules and dynein were necessary not just for NGF signals created and transported retrogradely within AXs but also when signals arose at the level of CBs. The data are evidence that endosomal trafficking plays an important role in both the creation of NGF signals and their intracellular transport.

Our working hypothesis posits that several distinct steps are required for generating the TrkA/Rap1 signaling endosome. First, NGF binds to TrkA at the cell surface to activate the receptor. Second, the NGF–pTrkA complex is internalized through either clathrin-mediated or non-clathrin-mediated pathways (18,19,48). Third, the newly formed NGF–pTrkA signaling endosome is likely docked onto the microtubule network through the binding of both the intermediate and the light chains of the dynein complex (30), thus linking it to the retrograde transport system.

The studies reported herein provide evidence for another step in trafficking of the NGF–pTrkA complex, leading to ‘maturation’ of the NGF–pTrkA signaling complex into one that is now competent to persistently activate MAPK1/2. Rap1 plays a key role in transducing and sustaining the NGF signal from TrkA to MAPK1/2. Disruption of the microtubule network by nocodazole or the dynein–dynactin complex through ectopic expression of p50, dynamitin, prevents the genesis of a fully functional NGF–TrkA–Rap1 signaling endosome.

There is compelling evidence that the NGF–pTrkA–Rap1 signaling endosome contributes critically to sustained activation of MAPK1/2, a pathway important for PC12 cell differentiation (33,36). The current study provides some key evidence that following NGF treatment, TrkA and Rap1 are trafficked to signaling endosomes in the perinuclear region. This process likely represents an important step in delivering the NGF signal to the nucleus (9,48,49). There are significant potential advantages to the compartmentalization of TrkA signaling in endosomes. First, the signal would be specific for NGF. Second, by concentrating the signaling machinery used to transmit TrkA signals, the probability of conducting signals that are robust and of high fidelity may be increased. Third, providing the signal near the nucleus a local source of downstream signals, such as those propagated by activated MAPK1/2, would facilitate effective delivery of signals through diffusion.

Similar observations were made for the c-Jun NH(2)-terminal kinase (JNK) signaling pathway in neurons (50). The JNK-interacting protein-1, JIP1, that serves as a scaffold for the stress-activated JNK pathway was found to be highly concentrated in the neurite tips of primary hippocampal neurons. On anoxic stress (oxygen and glucose deprivation), JIP1 as well as activated JNK were translocated to the perinuclear region (50,51). Ablation of JIP1 prevented JNK activation and rendered neurons less susceptible to stress-induced apoptosis. Trafficking of signaling proteins from the cell periphery to the perinuclear region may contribute critically to the ability to transmit a variety of signals that modulate neuronal survival and function.

The mechanistic basis for trafficking events involving TrkA is the topic of recent studies (18–20). Our findings suggest that the cytoplasmic motor dynein plays an important role in trafficking of the NGF–pTrkA complex and in the activation of the MAPK1/2 and PI3K signaling pathways. It remains to be determined how the dynein–dynactin complex interacts with TrkA and, perhaps, with elements of the NGF–pTrkA–Rap1 signaling complex. Whether or not the activity of dynein itself is regulated by NGF signaling is another important question. In addition, the receptor for transforming growth factor-β (52,53) and the activated receptor for epidermal growth factor (54) were recently shown to undergo translocation to the perinuclear region through early endosomes and that this process was driven by dynein. It will be important to define whether or not dynein contributes routinely to the trafficking of the signals of other NTs and cytokines.

The signaling endosome hypothesis focuses on the retrograde transport of neurotrophic signals within AXs. We expected to show that overexpressing p50 would severely inhibit retrograde transport of NGF signaling from AX terminals of DRG neurons. Unexpectedly, we discovered that ectopic expression of p50 also inhibited NGF signaling from the somal surface of DRG neurons, leading to the death of immature neurons and the atrophy of mature ones. These findings make the case that NGF signaling events, including at least those that involve MAPK1/2 and PI3K, need to engage the endosomal system. Indeed, our findings are evidence that endosomal traffic not only delivers signals but is also used to create them. It is important to point out that the present study does not rule out the existence of other paradigms by which NGF signal can be conveyed to the CB. We speculate that it is advantageous to have multiple pathways coexit to support neuronal function.

Disruption of trafficking of NGF signal can readily be envisioned as adversely affecting neuronal function. Indeed, an emerging body of data suggests that such events are impaired or altered in the central nervous system of patients suffering from neurodegenerative disorders such as Alzheimer’s disease (6,55–62). It is imperative to pursue further the mechanisms of retrograde trafficking of NGF and other NT signaling and their possible involvement in neurodegenerative disorders.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Antibodies and reagents

Mouse antibodies to EEA1, dynamitin (p50), Ras, Rap1 and TGN38 were purchased from BD Transduction Laboratories. Rabbit antibodies to Rap1 (Krev-1) and EGFP and mouse antibodies to Trk (B3 and MCTrks), DIC74 and pMAPK1/2 were purchased from Santa Cruz Biotechnology, Inc. Rabbit antibodies to Elk-1, phospho-Elk-1, pTrkA (pY490), Akt, phosphorylated Akt (Ser473), MAPK1/2 (Erk1/2) and the phosphorylated (i.e. activated) MAPK1/2 were purchased from Cell Signaling, Inc. Rabbit antibody to flag was purchased from Upstate Biotechnology, Inc. A rabbit antibody to mouse NGF was purchased from Alomone labs. Control mouse IgGs were obtained from Chemicon. Nocodazole and n-Octyl-β-D-glucopyranoside were purchased from Calbiochem-Novabiochem Corp. Nocodazole was dissolved at a concentration of 5 mg/mL (1000 × stock) in dimethyl sulfoxide (DMSO). Mouse NGF was purified as previously described (63). The NGF was stored in 0.2% acetic acid (the NGF vehicle). The BtNGF was produced according to a published method (64). The efficiency of biotin labeling (>95%) was verified by retarded mobility of BtNGF versus NGF on SDS–PAGE. The BtNGF was fully potent in activating TrkA and in inducing neurite outgrowth as confirmed using PC12 cells (data not shown). Horseradish peroxidase (HRP) conjugated to goat anti-rabbit or anti-mouse IgGs was obtained from Jackson Immunoresearch Laboratories, Inc. Alexa 568 or Alexa 488 goat IgG conjugates (anti-mouse or anti-rabbit) and Hoechst 33258 (10 mg/mL), Streptavidin–Alexa 488 were purchased from Molecular Probes. Protein G–agarose conjugates and SuperSignal reagents were obtained from Pierce. OptiPrep was obtained from Life Technologies. Mouse IgGs against α-tubulin (Clone DM1A) and agarose–glutathione conjugates and all other chemicals were purchased from Sigma. In Situ Cell Death Detection Kit, TMR red (Cat# 2 156 792) was purchased from Roche Applied Science. Campenot nerve cell growth chambers (CAMP10 and CAMP15) were purchased from Tyler Research Corporation.

Cell culture and transient transfection

PC12 cells were maintained in DMEM (4.5 mg/L glucose) supplemented with 10% horse serum and 5% FBS. Cells were incubated at 37°C with 5% CO2. Cells that were 50–60% confluent and serum starved for 24 h prior to treatments were used in all experiments. Unless indicated otherwise, all pretreatments and treatments were carried out by adding growth factors and other reagents to the media at 37°C. The final concentrations for the nocodazole vehicle (DMSO) and the NGF vehicle (0.2% acetic acid) were 0.1 and 0.0001%, respectively. For transient transfection, the pcDNA3 plasmids containing the full-length, flag-tagged human dynamitin cDNA sequence (a generous gift from Dr E. Holzbaur, University of Pennsylvania) were introduced into 70–80% confluent PC12 cells using Lipofectamine 2000 (Invitrogen). For mock transfection, pEGFP-N1 (BD Clontech) was used as a control. In general, a transfection efficiency of 5–10% was obtained.

Rat DRG culture and lentiviral transduction

The DRG neurons were isolated aseptically from E15-16 Sprague Dawley rat following previously established methods (42,65). Dissociated neurons were plated on either collagen-coated 12-well plates (25 000) or 18-mm glass coverslips in 12-well plates (10 000/plate) in NGF (100 ng/mL) in ‘maintenance medium’ [(MM), containing sodium phosphate-free MEM with Earle’s salts, l-glutamine, 10% heat-inactivated fetal bovine serum and 4.5 g/L d-glucose]. Selection medium (MM with antimitotic factors) was added to the culture next day. The selection was continued for 2 days. Cultures were then maintained in MM containing NGF (100 ng/mL) for 18 additional days (a total of 21 days), feeding every 3 days. The 21-day-old cells in vitro are referred as DIV-21. At this time, they contain large, mature neurons, with an average diameter of 48 μm, at least 94% of which are immunopositive for TrkA and extend well-developed neurites that also stain positively for TrkA (data not shown).

For lentiviral transduction, a cDNA encoding either EGFP or dynamitin was cloned into the pLenti6/V5 expression plasmid using the ViraPower Lentivirus Expression System (Invitrogen) following the manufacture’s instruction. The plenti6-EGFP, pLenti6-dynamitin plasmid or the empty vector was cotransfected together with pLP1, pLP2 and pLP/VSVG into 293FT cells. The lentivirus (lenti-EGFP or lenti-dynamitin or control virus) was harvested from the supernatant (5000× g for 15 min). The DRG neurons of DIV-7 or DIV-21 were transduced with the resulting lentivirus at a multiplicity of infection of approximately 10. A control virus was produced using the empty pLenti6/V5 vector and was also introduced into DRGs in parallel experiments. Virus was removed from DRG culture following an overnight incubation. The DRG neurons were switched back to MM, and the culture was continued for an additional 7 days. Prior to experiments, DRG neurons were extensively washed in serum-free, NGF-free MM and were deprived of NGF for 24 h in serum-free, NGF-free MM containing a goat antibody against mouse NGF (1:500). After washing in MM, NGF (100 ng/mL) or equal volume of the vehicle control (0.05% acetic acid in PBS, pH 7.0) was added for 60 min. Cells were washed and processed for either immunostaining for confocal analysis or lysed in RadioImmunoPrecipitation Assay (RIPA) buffer for SDS–PAGE/immunoblotting as described subsequently.

Ras and Rap1 activation assay

Established methods were used to detect endogenous GTP-bound Ras and Rap1 proteins as described previously (36). The fusion constructs between glutathione S-transferase (GST) and either the Rap-binding domain of RalGDS (RalGDSRBD) or the Ras-binding domain of C-Raf (C-RafRBD) (gifts from Dr J. L. Bos, Utrecht University, The Netherlands) were overexpressed in E scherichia coli DH5α cells. The fusion proteins were purified and used to assay Rap1GTP and RasGTP, respectively. Briefly, an equal number of treated or untreated PC12 cells were lysed in ice for 30 min in fishing buffer (FB) (10% glycerol; 1% Nonidet P-40; 50 mm Tris–HCl, pH 7.5; 200 mm NaCl; 2.0 mm MgCl2; 250 μm phenylmethylsulfonyl fluoride; 2 μg/mL aprotinin; 1 μg/mL leupeptin; 10 μg/mL soybean trypsin inhibitor; 10 mm NaF and 1 mm Na3VO4). The samples were centrifuged at 16 000× g, for 30 min at 4°C. Either 5 μg RalGDSRBD/GST or 5 μg C-RafRBD/GST proteins that were prebound to agarose–glutathione conjugates was added to the resulting supernatants and incubated at 4°C for 60–120 min with gentle rotation. The beads were washed four times in cold FB and boiled in SDS–PAGE sample buffer. The amounts of RasGTP and Rap1GTP were analyzed using SDS–PAGE and immunoblotting.

Cell fractionation

Fractionation of PC12 cells was carried out using a published protocol (36). Briefly, an equal number of treated or untreated PC12 cells were rinsed and harvested by centrifugation (800× g for 5 min). Cells were resuspended and homogenized by douncing 20 times in a Teflon-coated homogenizer in 1 mL cold homogenization buffer (HB) (250 mm sucrose; 20 mm Tricine–NaOH, pH7.8; 1 mm ethylenediaminetetraacetic acid and 2 mm MgCl2). The samples were centrifuged (800× g for 10 min), and the supernatant was adjusted to 25% OptiPrep with 50% OptiPrep in HB. The resulting mixture (2 mL in 25% OptiPrep) was placed at the bottom of an Ultra-Clear™ Tube (14 × 89 mm; Beckman Instruments, Inc.) and was overlaid successively with 2 mL each of 20, 15, 10 and 5% OptiPrep in cold HB. The samples were centrifuged for 16–18 h at 27 000 r.p.m. at 4°C in a SW41 rotor (Beckman Instruments, Inc.). Membrane fractions were collected from each of the four interphases of the OptiPrep gradients. For all studies, proteins were precipitated from the membrane fractions using 7% trichloroacetic acid and washed with acetone. The pellets were air-dried, boiled in SDS–PAGE loading buffer and analyzed using SDS–PAGE and immunoblotting.

Elk-1 in vitro phosphorylation assay, immunoprecipitation, SDS–PAGE and immunoblotting

Briefly, protein samples were separated on 7.5–12.5% gels and proteins electrotransferred onto polyvinylidene fluoride (PVDF) membranes (NEN Life Science Products, Inc.). The PVDF membranes were preblotted with 5% nonfat milk (carnation) and probed with primary antibodies, as indicated, at concentrations suggested by the suppliers. The blots were washed in Tris Buffered Saline Tween 20 (TBST) (20 mm Tris–HCl, pH 7.4; 150 mm NaCl and 0.1% Tween-20), followed by incubation with either goat anti-mouse or anti-rabbit IgG–HRP conjugates at a dilution of 1/10 000 to 1/40 000. The blots were washed and developed with SuperSignal (Pierce). For quantification, exposures within the linear ranges were scanned and determined using the Scion Image program (Scion Corp.). For in vitro Elk-1 phosphorylation assay, a previously published method was followed (28).

Indirect immunofluorescence and confocal microscopy

The PC12 cells were grown for 24–48 h on glass coverslips coated with matrigel (Becton Dickinson). Cells were starved, washed twice briefly with cold PBS and fixed with ice-cold 100% methanol for 5 min at −20°C. The samples were rinsed thrice with PBS at room temperature. The fixed cells were preblocked with 1% BSA and 0.2% Triton-X-100 in PBS for 20 min at room temperature. Primary antibodies were diluted at 1/300 to 1/500 in PBS containing 1% BSA and were incubated with the fixed cells for 1 h at room temperature. The samples were washed with PBS containing 0.8% BSA thrice and then incubated with goat anti-rabbit– or anti-mouse–IgG Alexa conjugates (1/600) for 1 h at room temperature. The coverslips were washed with PBS containing 0.2% BSA thrice, followed by a rinse with PBS and dH2O. In experiments where nuclear staining was required (Figure S2C,D and 5A), samples were further incubated with 2 μg/mL Hoechst 33258 (final concentration) in PBS for 1 min at room temperature, followed by a rinse with PBS and dH2O. The coverslips were air-dried and mounted in antifade medium for observation. Images shown in Figure S2C,D and 5A were analyzed using a Nikon E800 epifluorescence microscope and were captured using a SPOT charge-coupled device (CCD) camera (Diagnostic Instruments). The images were processed using SPOT 3.4.3 and Adobe Photoshop 5.0 (Adobe Systems). For confocal microscopy, the images were captured using a Nikon Eclipse E800 microscope and a Bio-Rad Laser Scanning System Radiance2000. Using a 60× Plan-Apo immersion objective (NA1.4), images at the half height of the cell were collected in a 512 × 512 collection box. The images were processed using Confocal Assistant 4.02 (Bio-Rad) and Adobe Photoshop 7.0 (Adobe Systems). In experiments in which the periphery of cells was visualized, a bright field image was captured and superimposed onto the fluorescence image.

Transport of BtNGF in rat DRGs using campenot chamber

E15-16 rat DRGs were dissected and dissociated, and 20 000 cells were placed in the middle chamber of a CAMP10 Campenot chamber (Figure 7). After seven DIV, AXs crossed the barrier and extended into the outer chamber. Cultures were maintained for 3 weeks in NGF-containing medium as described previously. The DRGs were then transduced with lentivirus with different constructs and maintained for an additional 7 days. Cells were extensively rinsed and starved as described previously. We routinely checked and discarded those chambers with leakage problem. For transport assay, BtNGF (100 ng/mL) was added to the DAXs in the outer chamber and incubated for 60 min at 37°C. Cells were rinsed, fixed, permeabilized and incubated with Streptavidin–Alexa 488 (1/1000 dilution) for 1 h at room temperature. Samples were rinsed and mounted. The BtNGF transported to the CB was measured as follows: images of CBs in the middle chamber were acquired using an inverted Nikon fluorescence microscope (TE2000U) equipped with Nikon 10× Plan objective. The fluorescence signal was detected and captured using a CCD camera (CoolSNAP HQ; Roper Scientific, Inc.), with a detection area of 1392 × 1040 pixels (pixel size: 6.45 × 6.45 μm2). This cooled (−30°C) camera has a 12-bit dynamic range, which allows imaging very dim and very bright objects at the same time without saturation. Binary data were collected and saved using a home-built software package with 2 × 2 hardware binning and 0.5 seconds exposure time (Stanford University). Subsequent data analysis was performed using Data Interactive Language (idl) software with scripts written in the Chu laboratory. For each fluorescence image, the average signal intensity within a selected region of interest (ROI) encompassing 100 cells was measured. At least four ROIs were selected to obtain the mean and the standard deviation. A background value was obtained by incubating a sample with Streptavidin–Alexa 488 conjugate only. This value was subtracted from those obtained from the samples that were treated with BtNGF. All statistical analyses were carried out using Student’s t-test.

For detection of BtNGF that was transported retrogradely to the CB/PAXs by Western blot, 200 000 DRGs were placed in the small chamber of a CAMP15 apparatus (Figure 7) and maintained for 3 weeks. Following lentiviral transduction and NGF deprivation, BtNGF (50 ng/mL) was added into the larger chamber that harbored only the AXs and was incubated for 12 h. The CBs/PAXs were rinsed, lysed and cleared by centrifuge. Forty microliters of streptavidin–agarose conjugate slurries was added to the supernatant to precipitate BtNGF. The precipitates were analyzed by SDS–PAGE/Western blotting.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

We thank the members of the Mobley laboratory for their assistance. We also thank Drs Francisca Bronfman and Mike Fainzilber for their invaluable advice on the production of BtNGF. These studies are supported through the grants from National Institutes of Health (NS24054, NS38869, AG16999 and NS055371), the John Douglas French Alzheimer’s Foundation, the McGowan Charitable Trust, the Larry L Hillblom Foundation, the Adler Foundation and the Deane Johnson Alzheimer’s Disease Fund.

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Supplemental Information.

NGF trafficks together with TrkA to early endosomes in PC12 cells The signaling endosome hypothesis predicts that NGF/TrkA complexes are trafficked within endosomes (1–3). We reexamined the issue, focusing on a specific endosomal compartment, the early endosome; it is within early endosomes in the perinuclear region that NGF treatment results in persistent pMAPK1/2 in PC12 cells (4). We used BtNGF, which possesses full biological activity, in combination with a strepavidin-Alexa 488 conjugate that allowed for detection by immunofluoresence. Serum-starved PC12 cells were chilled and were incubated with 50 ng/ml BtNGF for 45 min on ice to allow surface binding but not internalization of BtNGF. This was confirmed by showing under these conditions that >95% of the BtNGF-streptavidin Alexa 488 signals were susceptible to subsequent acid-stripping in 0.2 N Acetic Acid, 0.4 M NaCl at 4°C for 5 min (data not shown). Binding of BtNGF to the PC12 cell surface was receptor-mediated as there was little signal for BtNGF-strepavidin Alexa 488 when excess unmodified NGF (1.0 μM) was included in the incubation (data not shown). We then prepared two sets of samples. One was rinsed and fixed immediately following the binding step (Fig.1S A). The second set was rinsed and shifted to 37°C for 30 min to allow for endocytosis of BtNGF (Fig.1S B). Both sets were then processed for immunostaining with MCTrk, a mouse antibody to Trk, and analyzed by confocal microscopy. The signal for Trk overlapped extensively with that for BtNGF with or without warming (Fig.1S A). However, BtNGF/Trk concentrated in the perinuclear region following warming (Fig.1S B). To ask whether or not NGF was present in early endosomes, we compared the pattern for BtNGF with that for an early endosomal marker, EEA1 (Fig. 1S C and D). Whereas surface-bound BtNGF showed little, if any, co-localization with EEA1 (Fig.1S C), following warming, BtNGF was markedly co-localized with EEA1 (Fig.1S D). Similarly, without warming, Trk showed little co-localization with Rab5B (Fig.1S E), another early endosomal marker protein. Following warming Trk was co-localized with Rab5B (Fig.1S F). As expected, pMAPK1/2 that was induced by NGF following warming (Fig.1S H vs G) was also present in the Rab5B-positive compartment (Fig.1S H). Our results thus place NGF and TrkA in early endosomes in the perinuclear region.

NGF treatment induces trafficking of Rap1 to and activates MAPK1/2 in early endosomes.

To investigate the trafficking events induced by NGF treatment, we examined the distribution of TrkA, Rap1 and pMAPK1/2. PC12 cells were grown on glass coverslips and were serum-starved overnight. The cells were treated with either the NGF vehicle (-NGF) or 50 ng/ml NGF for 30 min (+NGF). This time point was chosen to maximize trafficking of TrkA to early endosomes and activation of Rap1 (4, 5). Samples were then prepared for indirect immunofluorescence and analyzed using confocal microscopy. To outline the cell perimeter, in some cases (Fig.2S A, B, E, F) images were also captured using transmitted light and were superimposed onto the fluorescent image. In control cells (-NGF), Rap1 (Fig.2S A) and TrkA (Fig.2S E) were found dispersed throughout the cytoplasm (also see Fig. 4). NGF treatment resulted in marked concentration of both Rap1 (Fig.2S B vs A) and TrkA (Fig.2S F vs E) in the perinuclear region. The perinuclear localization of Rap1 in NGF-treated cells was also illustrated in studies in which the nucleus was stained using Hoechst 33258 (Fig.2S D vs C).

Next, we examined the subcellular localization of Rap1 with respect to TrkA, pMAPK1/2 and EEA1. Again, in cells treated with vehicle only, both Rap1 and TrkA were seen dispersed throughout the cytoplasm and showed little colocalization (Fig.2S G). As expected, no signal for pMAPK1/2 was seen in these samples (Fig.2S I). Treatment with NGF resulted in the concentration of Rap1 and TrkA in the perinuclear region (Fig.2S H). NGF treatment also resulted in the appearance of pMAPK1/2 in the perinuclear region, where pMAPK1/2 partially co-localized with Rap1 (Fig.2S J). Rap1 was partially co-localized with EEA1 before and after NGF treatment (Fig.2S K, L). We conclude that NGF treatment induces trafficking events that lead to the concentration of TrkA, Rap1 and pMAPK1/2 within early endosomes in the perinuclear region.

Supplemental Figure 1: BtNGF was internalized into EEA1-positive endosomes in PC12 cells. PC12 cells were cultured for 24–48 hrs on cover glasses coated with matri-gel and were serum-starved overnight. Cells were chilled and incubated with BtNGF (50 ng/ml) for 45 min on ice. Cells were rinsed and were immediately processed for immunostaining (A, C, E, G). A parallel sample was shifted to 37°C for 30 min to allow endocytosis of BtNGF prior to immunstaining (B, D, F, H). BtNGF was detected with a streptavidin-Alexa 488 conjugate (green, A-D). The following specific primary antibodies were used: a mouse antibody to Trk, B3 (1/200) for TrkA (A, B, E, F); a mouse antibody to EEA1 (1/50) for EEA1 (1/50) (C, D); a rabbit antibody to Rab5B (1/200) for Rab5B (E, F, G, H); a mouse antibody to pMAPK1/2 (1/100) for pMAPK1/2 (G, H). Secondary antibody conjugated to either Alexa 568 or Alexa 488 was used to visualize the primary antibody. Co-localization of proteins is denoted by the white signal. The size of the collection box is 30 μm × 30 μm.

Supplemental Figure 2: NGF induced trafficking of Rap1, TrkA and pMAPK1/2 to the perinuclear region. PC12 cells were cultured as in Figure 1. Following pretreatment with either the NGF vehicle alone (-NGF) or with NGF (50 ng/ml) (+NGF), indirect immunofluorescence was carried out using confocal microscopy as described in Materials and Methods. Rap1 (A-D, G-L) was immunostained using a specific rabbit antibody (1/250) and detected with an Alexa 568 goat anti-rabbit IgG conjugate. TrkA (E-F, G-H) was detected using B3 (1/200) and visualized with either an Alexa-568 goat anti-mouse IgG conjugate (E-F) or an Alexa-488 goat anti-mouse IgG conjugate (G-H). The signals for pMAPK1/2 and EEA1 were each detected using a mouse monoclonal antibody specific for these proteins as in Fig. 1. The dilutions of primary antibodies were as follows: 1/100 for pMAPK1/2 and 1/50 for EEA1. An Alexa 488 goat anti-mouse IgG conjugate was used to visualize these antibodies. Co-localization of antigens is denoted by the white signal. In A, B, E and F, a corresponding image from the transmitted light detector was simultaneously captured and superimposed onto the fluorescence image of Rap1 or TrkA to illustrate the outline of the cell. In C and D, Rap1 (magenta) was co-stained with Hoechst 33258 to reveal the nucleus (blue). The resulting samples were captured and analyzed using a Nikon E800 epifluorescence microscope. 25 cells were examined in each condition (A-L) and a typical representative image was shown. The size of the collection box is 30μm × 30 μm for A-B, E-L, 60 μm × 60 μm for C, D.

References

Howe CL, Mobley WC. Signaling endosome hypothesis: A cellular mechanism for long distance communication. J Neurobiol 2004;58:207–16.

Ye H, Kuruvilla R, Zweifel LS, Ginty DD. Evidence in support of signaling endosome-based retrograde survival of sympathetic neurons. Neuron 2003;39:57–68.

MacCormick M, Moderscheim T, van der Salm LW, Moore A, Pryor SC, McCaffrey G, Grimes ML. Distinct signalling particles containing ERK/MEK and B-Raf in PC12 cells. Biochem J 2005;387:155–64.

Wu C, Lai CF, Mobley WC. Nerve growth factor activates persistent Rap1 signaling in endosomes. J Neurosci 2001;21:5406–16.

Mochizuki N, Yamashita S, Kurokawa K, Ohba Y, Nagai T, Miyawaki A, Matsuda M. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 2001;411:1065–8.

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tra_718_sm_8_11gfig1.jpg238KSupporting info item
tra_718_sm_8_11gfig2.jpg260KSupporting info item

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