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. 2018 Apr 4;38(14):3394-3413.
doi: 10.1523/JNEUROSCI.1686-17.2018. Epub 2018 Feb 26.

Swedish Nerve Growth Factor Mutation (NGFR100W) Defines a Role for TrkA and p75NTR in Nociception

Kijung Sung  1 , Luiz F Ferrari  2 , Wanlin Yang  1   3 , ChiHye Chung  4 , Xiaobei Zhao  1 , Yingli Gu  1   5 , Suzhen Lin  1   3 , Kai Zhang  6   7 , Bianxiao Cui  6 , Matthew L Pearn  8   9 , Michael T Maloney  10 , William C Mobley  1 , Jon D Levine  2 , Chengbiao Wu  11   9
Affiliations

Swedish Nerve Growth Factor Mutation (NGFR100W) Defines a Role for TrkA and p75NTR in Nociception

Kijung Sung et al. J Neurosci. .

Erratum in

Abstract

Nerve growth factor (NGF) exerts multiple functions on target neurons throughout development. The recent discovery of a point mutation leading to a change from arginine to tryptophan at residue 100 in the mature NGFβ sequence (NGFR100W) in patients with hereditary sensory and autonomic neuropathy type V (HSAN V) made it possible to distinguish the signaling mechanisms that lead to two functionally different outcomes of NGF: trophic versus nociceptive. We performed extensive biochemical, cellular, and live-imaging experiments to examine the binding and signaling properties of NGFR100W Our results show that, similar to the wild-type NGF (wtNGF), the naturally occurring NGFR100W mutant was capable of binding to and activating the TrkA receptor and its downstream signaling pathways to support neuronal survival and differentiation. However, NGFR100W failed to bind and stimulate the 75 kDa neurotrophic factor receptor (p75NTR)-mediated signaling cascades (i.e., the RhoA-Cofilin pathway). Intraplantar injection of NGFR100W into adult rats induced neither TrkA-mediated thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia based on agonism for TrkA signaling. Together, our studies provide evidence that NGFR100W retains trophic support capability through TrkA and one aspect of its nociceptive signaling, but fails to engage p75NTR signaling pathways. Our findings suggest that wtNGF acts via TrkA to regulate the delayed priming of nociceptive responses. The integration of both TrkA and p75NTR signaling thus appears to regulate neuroplastic effects of NGF in peripheral nociception.SIGNIFICANCE STATEMENT In the present study, we characterized the naturally occurring nerve growth factor NGFR100W mutant that is associated with hereditary sensory and autonomic neuropathy type V. We have demonstrated for the first time that NGFR100W retains trophic support capability through TrkA, but fails to engage p75NTR signaling pathways. Furthermore, after intraplantar injection into adult rats, NGFR100W induced neither thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia. We have also provided evidence that the integration of both TrkA- and p75NTR-mediated signaling appears to regulate neuroplastic effects of NGF in peripheral nociception. Our study with NGFR100W suggests that it is possible to uncouple trophic effect from nociceptive function, both induced by wild-type NGF.

Keywords: NGF; TrkA; nociception; p75; sensory neuron; trophic.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
NGFR100W does not induce acute sensitization in adult rats. A–C, Mechanical hyperalgesia. The Randall–Selitto method was used to measure mechanical hyperalgesia in adult rats. After intradermal injection of 200 ng of wtNGF (n = 6) or NGFR100W (n = 6) into the rat's hindpaws, mechanical threshold was measured at the indicated times. A significant decrease in mechanical nociceptive threshold in rats injected with wtNGF was seen within 15 min, reached a maximum by 1 h, and lasted at least 5 d. In contrast, NGFR100W did not produce a significant decrease in the threshold during the 5 d period. D–F, Thermal hyperalgesia. The Hargreaves test was used to measure thermal hyperalgesia. Baseline response was first measured for each test animals before NGF injection. Hindpaws were injected with 600 ng of either wtNGF or NGFR100W. Thermal threshold was measured at the indicated times. Animals that were injected with wtNGF showed a significant decrease at 30 min. The decrease was dissipated after 1 h. NGFR100W failed to reduce thermal nociceptive threshold during the entire 1 h test period. Six rats for each group (n = 6) were used in the test. Unpaired t tests were performed against the baselines within each NGF injection group to produce p-values. Comparions were done between the threshold before injection with either wtNGF or NGFR100W to produce p-values that were noted in the figure. *p < 0.05, **p < 0.01, ***p < 0.001. G–I, PGE2-hyperalgesic priming effect. On d 7 after NGF administration, PGE2 was injected intradermaly (100 ng/5 μl) to test hyperalgesic priming. Naive rats (nontreated with NGF) are shown as a control. Naive animals showed acute hyperalgesia within 30 min, but not at 4 h, after PGE2 injection. Rats pretreated with intradermal injection of wtNGF showed a decrease in the mechanical nociceptive threshold after PGE2 injection at 30 min, like the controls, but unlike controls, it lasted longer than 4 h. Rats pretreated with NGFR100W showed significant decrease in mechanical nociception after PGE2 injection, which lasted at least 4 h and was comparable to wtNGF. Data are presented as mean ± SEM. Unpaired t tests were performed versus values before PGE2 injection. ***p < 0.001 Six paws (n = 6) were used for wtNGF or NGFR100W.
Figure 2.
Figure 2.
Low H+-evoked response by single-cell patch clamping. Rat E15.5 DRG neurons were cultured as described in the Materials and Methods. At DIC5, DRG neurons were deprived of NGF for 2 h. A, Phase contrast image of DRG neurons (under 60× magnification) showing the experimental setup. The patch pipette approached from the left and another glass pipette that applied brief puffs of pH 5.5 solution to the nearby cell body was placed at a distance. This pipette did not induce mechanical responses. B, Whole-cell patch-clamp recording was performed at a holding potential of −60 mV in DRG. The proton-evoked response was measured after a brief application of moderate acidic solution of pH 5.5 (blue arrow) onto the cell body to induce inward current (designated as “before NGF response”). Then, 50 ng/ml of wtNGF or NGFR100W (green arrow) was applied to the bath solution for 10 min and three additional puffs were applied to record the after NGF response. C, The data were normalized by calculating the ratio of the after NGF response to the before NGF response. Bar graphs represent mean ± SEM. wtNGF sensitized the inward current by 1.45-fold, but not NGFR100W (p = 0.0096; wtNGF vs NGFR100W; unpaired t test). **p < 0.01.
Figure 3.
Figure 3.
DRG survival assays. Rat E15.5 DRG neurons were cultured as described in the Materials and Methods. Parallel cultures were supplied with either wtNGF or NGFR100W at a range from 0 to 100 ng/ml. A, Phase contrast images of DRG neurons at 8 d in vitro were captured and representative images are shown. Negative control (no NGF, 0 ng/ml) failed to maintain the survival of DRGs. NGFR100W maintained the survival as potently as wtNGF. B, The survival rate of DRG neurons (i.e., cell counts) by wtNGF or NGFR100W was not significantly different. E, Unpaired t test was performed on wtNGF versus NGFR100W (p > 0.9999 at 0, 10, and 50 ng/ml, p = 0.4000 at 100 ng/ml). Data are presented as mean ± SEM.
Figure 4.
Figure 4.
Classical hyperbolic saturation curves of wtNGF and NGFR100W bound to TrkA or p75NTR-expressing NIH3T3 cells. A, B, Either NIH3T3 TrkA or p75NTR cells were exposed to wtNGF-QD655/or NGFR100W-QD655 for 20–30 min at 20°C with a range of different NGF-QD655 concentrations. Nonlinear regression analysis using observed data were performed to examine the binding of NGF to the TrkA receptor (A) or p75NTR (B). Blue squares and red circles represent surface-bound wtNGF and NGFR100W, respectively. C, D, Biotinylated wtNGF (C) or NGFR100W (D) was incubated with recombinant extracellular domain of p75NTR at 4°C for 2 h. The complex that contained biotinylated NGF was pulled down using streptavidin–agarose (SA) beads at 4°C for 2 h. The beads were washed and boiled in SDS sample buffer. Both an aliquot of the supernatant (control) and the bead-bound samples were analyzed by Western blotting using a specific antibody against p75NTR (REX). Representative blot shows p75NTR bound to wtNGF-SA beads (C) and p75NTR bound to NGFR100W-SA beads (D). Relative REX signal from pulled-down beads was significantly increased with increased amount of wtNGF, as shown in C. E, ANOVA analysis was performed with different amounts of wtNGF, suggesting that pulled-down p75 ECDs were significantly increased according to the increased amount of wtNGF (F(4,5) = 44.41, p = 0.0004). D, In contrast, pulled-down p75 ECD was not detectable even with 10 ngf of NGFR100W and only reached the level of baseline Rex signal intensity. Data are presented as mean ± SEM.
Figure 5.
Figure 5.
Imaging analysis of binding and internalization into NIH3T3-TrkA- or -p75NTR-expressing cells. A, NIH3T3-TrkA cells were cultured on coverslips that were precoated with poly-L-lysine. Cells were rinsed and serum-starved for 2 h. Cells were then incubated with 0.2 nm of wtNGF, or NGFR100W, KKE, or delta9/13-QD655 conjugates or with QD655 alone for 30 min at 37°C. After extensive rinses, QD655 signals were captured by live cell imaging. Representative images are shown. wtNGF, NGFR100W, and the KKE mutant that were used as positive controls for the TrkA receptor showed bright QD655 signals inside the cells. To ensure that internalization of NGFR100W was receptor mediated, we premixed NGFR100W with 100-fold unlabeled NGF before live imaging. The results show little internalization of NGFR100W, indicating the internalization of NGFR100W into NIH3T3-TrkA cells was TrkA specific. B, Similarly, NIH3T3-p75NTR cells were used to investigate internalization of the different forms of NGF proteins and representative images are shown after incubating with NGF-QD655 for 2 h at 37°C. The results show that both wtNGF and Δ9/13, which are known to bind to the p75NTR receptor, were internalized into NIH3T3-p75NTR cells and the QD655 signals were mostly concentrated around the peripheries of the cell. No signals were observed in the NIH3T3-p75NTR cells when treated with either the KKE mutant or NGFR100W. C, Internalized QD655 within the cells were quantitated. Data are presented as mean ± SEM.
Figure 6.
Figure 6.
Analysis of TrkA- and p75NTR-mediated signaling pathways. A–C, PC12 cells were serum-starved and treated with 50 ng/ml of either wtNGF or NGFR100W for the indicated time intervals. Cell lysates were analyzed by SDS-PAGE and immunoblotting with specific antibodies as indicated. Treatment with either wtNGF or NGFR100W induced phosphorylation; i.e., activation of TrkA, Erk 1/2, and Akt. The blots were reprobed for total Akt or Erk1/2 as loading controls. We also measured the signals for pTrkA activated by wtNGF or NGFR100W. The signals for pTrkA were normalized against GAPDH (Fig. 6-1). D, E, Analysis of downstream signaling of p75NTR in PC12 nnr5 by immunoblotting. The cells were prepared, treated, and cell lysates analyzed by SDS-PAGE/immunoblotting with specific antibodies as in A. There was a significant reduction in phosphorylation of Cofilin by NGFR100W compared with wtNGF. F, Immunostaining of RhoA-GTP in PC12 nnr5. PC12 nnr5 were plated on the coverslip coated by poly-L-lys, starved for 2 h, treated with either wtNGF or NGFR100W, and the preparations were fixed and permeabilized, followed by the protocol. Differential interference contrast imaged cells show single staining for active form of RhoA (RhoA-GTP) or double staining for active RhoA and nucleus. RhoA-GTP staining revealed that RhoA activation was stronger in the cell treated with wtNGF than in the cell treated with NGFR100W. G, H, Analysis of PLC-γ signaling in PC12 cells by immunoblotting. G shows that PLC-γ stimulated by NGFR100W differs significantly from the one by wtNGF. In contrast, the same lysate showed similar amount of activation of Erk1/2 and Akt. PC12 cells were pretreated with the p75NTR inhibitor Pep15, followed by treatment with 50 ng/ml NGF. In parallel samples, cells were treated with vehicle, 50 ng/ml NGF, or 50 ng/ml NGFR100W. Cell lysates were analyzed by SDS-PAGE/immunoblotting with specific antibodies as indicated. The data show thatactivation of PLC-γ was markedly suppressed by NGFR100W compared with wtNGF. wtNGF failed to fully activate PLC-γ when p75NTR was functionally inhibited, similar to partially activated PLC-γ when treated by NGFR100W. Data are presented as mean ± SEM. p values were calculated using student unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7.
Figure 7.
Analysis of retrograde axonal transport by live imaging. A, B, Rat E15.5 DRG neurons were cultured in microfluidic chamber. Kymographs of axonal movement of wtNGF and NGFR100W based on real-time imaging series of axonal transport assays are shown. The graphs represent spatial position of QD605 signals (in micrometers) over time (seconds). The results for both wtNGF and NGFR100W showed similar slopes, suggesting that NGFR100W moves at a speed within the axon similar to that of wtNGF. C, Overlayed kymographs of displacement of axonal QD605 signals for wtNGF and NGFR100W. The 100–150 QD605 signals for either wtNGF (blue) or NGFR100W (red) were analyzed and superimposed. The results demonstrate that axonal movement of wtNGF and NGFR100W behaves in a strikingly similar fashion. D, Total average transport speeds including pausing for wtNGF and NGFR100W were calculated to be 1.5 and 1.4 mm/s, respectively. The moving velocity without pausing, that is, during the “go” motion period, was 1.7 mm/s for both wtNGF and NGFR100W.
Figure 8.
Figure 8.
Role of TrkA and p75NTR in nociceptive response and hyperalgesic priming. A, Five microliters (1 μg) of K252a, GW4869, or vehicle (10% DMSO) was administered intradermally on the dorsum of the hindpaw of adult rats at the same site where NGF was going to be injected. After NGF injection (200 ng of wtNGF), the Randall–Selitto method was used to measure mechanical hyperalgesia as in A. n = 4 for vehicle, K252a, GW4869; n = 6 for K252a + GW4869. Data are presented as mean ± SEM. p-values were calculated using unpaired t test. ***p < 0.001. B, Higher dosages of GW4869, 10 μg (n = 4) and 1000 μg (n = 4), were injected intradermally on the dorsum of the hindpaw of adult rats following by NGF injection as in A. The same method was applied to measure mechanical hyperalgesia used as in A. Data are presented as mean ± SEM. p-values were calculated using unpaired t test. *p < 0.05, **p < 0.01.
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