JIP3 mediates TrkB axonal anterograde transport and enhances BDNF signaling by directly bridging TrkB with kinesin-1

Abstract

Brain-derived neurotrophic factor (BDNF), secreted from target tissues, binds and activates TrkB receptors, located on axonal terminals of the innervating neurons, and thereby initiates retrograde signaling. Long-range anterograde transport of TrkB in axons and dendrites requires kinesin-mediated transport. However, it remains unknown whether anterograde TrkB transport mechanisms are the same in axons versus in dendrites. Here, we show that c-Jun NH(2)-terminal kinase-interacting protein 3 (JIP3) binds directly to TrkB, via a minimal 12 aa domain in the TrkB juxtamembrane region, and links TrkB to kinesin-1. The JIP3/TrkB interaction selectively drives TrkB anterograde transport in axons but not in dendrites of rat hippocampal neurons. Moreover, we find that TrkB axonal transport mediated by JIP3 could regulate BDNF-induced Erk activation and axonal filopodia formation. Our findings demonstrate a role for JIP3-mediated TrkB anterograde axonal transport in recruiting more TrkB into distal axons and facilitating BDNF-induced retrograde signaling and synapse modulation, which provides a novel mechanism of how the TrkB anterograde transport can be coupled to BDNF signaling in distal axons.

Figure 1.

Both TrkB-FL and TrkB.T1 receptors undergo anterograde transport. A, Sciatic nerves were ligated unilaterally at the midpoint for 1 d and processed for immunoblotting. Ligated and contralateral unligated sciatic nerves were dissected, and the extracts were analyzed with the indicated antibodies. Tubulin is used as a loading control. B, Quantification of the proximal and distal pool fraction. Data were shown as the mean ± SEM from five independent experiments (*p < 0.05, the ligated pool vs the control unligated pool; one-way ANOVA). Values were normalized to the level of the unligated pool. C, Schematic representation of the structure of TrkB-FL and TrkB.T1 receptors. The shared identical intracellular region (454–465 aa) was marked with black color.

Figure 2.

Colocalization of JIP3 and TrkB in vitro and in vivo. A, Subcellular colocalization of endogenous TrkB and JIP3 in hippocampal neurons visualized by confocal microscopy. Staining was performed with rabbit anti-TrkB (green) and mouse anti-JIP3 (red) antibodies. Colocalization of both proteins was shown in yellow. Bottom, Enlarged images of the framed regions, with the white arrows indicating the colocalization of TrkB and JIP3. Scale bar, 20 μm. B, The sciatic nerve from a male rat was ligated for 24 h, and longitudinal sections of ligated (left sciatic nerve) and the control unligated sections (right sciatic nerve) were immunostained with anti-TrkB antibodies. The bilateral part around the ligation in the white rectangle was enlarged in C. Scale bar, 100 μm. C, Colocalization of TrkB and JIP3 in the ligated sciatic nerve. Longitudinal sections from a ligated sciatic nerve were double immunostained with anti-TrkB (green) and anti-JIP3 (red) antibodies; the proximal portion is at the top, whereas the distal end of the ligation is at the bottom. The proximal part near the ligation in the white rectangle was enlarged in D. Scale bar, 50 μm. Colocalization of TrkB and JIP3 is indicated in yellow.

Figure 3.

Ternary complex formation of JIP3, TrkB, and KLC1 in vitro and in vivo. A, Coimmunoprecipitation of JIP3 with TrkB-FL/TrkB.T1. Lysates from HEK293 cells transfected with HAJIP3 and Flag-tagged TrkB-FL/TrkB.T1 constructs were immunoprecipitated with anti-HA antibodies or anti-Flag antibodies. Then, immunoblotting analysis was performed to detect immunoprecipitated proteins. B, Endogenous JIP3 associates with TrkB and KLC1. Rat brain lysates were subjected to immunoprecipitation with mouse anti-JIP3 or goat anti-KLC1 antibodies, and the protein complex was eluted; the TrkB, KLC1, and JIP3 were detected by immunoblotting. C, JIP3-dependent interaction between TrkB and KLC1. Lysates from HEK293 cells cotransfected with MycKLC1 and FlagTrkB-FL/FlagTrkB.T1 constructs were immunoprecipitated with anti-Myc antibodies. Then, immunoblotting analysis was performed to detect immunoprecipitated proteins. For FlagTrkB-FL: TrkB-FL wild-type group, cotransfected with FlagTrkB-FL and MycKLC1, with endogenous JIP3 expression; JIP3 overexpressed group, cotransfected with FlagTrkB-FL, MycKLC1, and HAJIP3; siJIP3 group, cotransfected with FlagTrkB-FL, MycKLC1, and siJIP3. For FlagTrkB.T1: TrkB.T1 wild-type group, cotransfected with FlagTrkB.T1 and MycKLC1, with endogenous JIP3 expression; siJIP3 group, cotransfected with FlagTrkB.T1, MycKLC1, and siJIP3. Quantitation of immunoblotting in the top panel of Western blots in C is shown as a percentage of TrkB coimmunoprecipitation by KLC1 in different groups compared with TrkB-FL wild-type group. Data are shown as the mean ± SEM from three independent experiments [n = 3; *p < 0.05, vs FlagTrkB-FL wild-type (WT) group; ^##p < 0.01, vs FlagTrkB.T1 WT group, one-way ANOVA]. D, The KLC1-independent interaction between TrkB-FL and JIP3. Lysates from HEK293 cells transfected with HAJIP3 and FlagTrkB-FL constructs were immunoprecipitated with anti-HA antibodies. Then, immunoblotting analysis was performed to detect immunoprecipitated proteins. Wild-type group, Cotransfected with FlagTrkB-FL and HAJIP3, with endogenous KLC1 expression; KLC1 overexpressed group, cotransfected with FlagTrkB-FL, MycKLC1, and HAJIP3; siKLC1 group, cotransfected with FlagTrkB-FL, HAJIP3, and siKLC1. E, Simultaneous knockdown of JIP3 and Rab27B further decreases the association between TrkB-FL and KLC1. Lysates from HEK293 cells cotransfected with MycKLC1 and TrkB-FL constructs were immunoprecipitated with anti-Myc antibodies. Wild-type group, Cotransfected with FlagTrkB-FL and MycKLC1, with endogenous JIP3 and Rab27B expression; siJIP3 group, cotransfected with FlagTrkB-FL, MycKLC1, and siJIP3; siRab27B group, cotransfected with FlagTrkB-FL, MycKLC1, and siRab27B; siJIP3+siRab27B group, cotransfected with FlagTrkB-FL, MycKLC, siJIP3, and siRab27B. Quantitation of immunoblotting in the top panel of Western blots in E is shown as a percentage of TrkB coimmunoprecipitation by KLC1 in different groups compared with wild type. Data are shown as the mean ± SEM from three independent experiments (n = 3; *p < 0.05; **p < 0.01, vs the FlagTrkB-FL WT group; ^#p < 0.05, vs siJIP3+siRab27B group; one-way ANOVA). IP, Immunoprecipitation; CO-IP, coimmunoprecipitation.

Figure 4.

The JM1 domain of TrkB interacts directly with JIP3. A, Schematic representation of the TrkB mutants generated. HEK293 cells were transiently cotransfected with Flag-tagged TrkB-FL or TrkB mutants and HA-tagged JIP3. Immunoprecipitation was performed using rabbit anti-HA antibodies, and immunoblotting was performed with mouse anti-HA or mouse anti-Flag antibodies. B, Schematic representation of the TrkB juxtamembrane domain, IL2R, and chimeric IL2R–BJM mutants. HEK293 cells were transiently transfected with the indicated constructs. Immunoprecipitation was performed using mouse anti-Flag antibodies, and immunoblotting was performed with rabbit anti-HA or rabbit anti-Flag antibodies. C, Coimmunoprecipitation of JIP3 with TrkB-FL, TrkA-FL, and TrkC-FL. HEK293 cells were cotransfected with Flag-tagged Trks and HAJIP3; immunoprecipitation was performed using mouse anti-Flag antibodies, followed by immunoblotting with rabbit anti-Flag or rabbit anti-HA antibodies. Multiple sequence alignment on the JM1 domain of three Trk receptors was performed by ClustalW2. D, TrkB-JM1 confers TrkA the binding ability with JIP3. A schematic representation of TrkA and chimeric TrkA-BJM1 mutant is shown. Immunoprecipitation was performed using mouse anti-Flag antibodies, and immunoblotting was performed with rabbit anti-HA or rabbit anti-Flag antibodies.

Figure 5.

Mapping the JIP3 binding domain with TrkB. A, Coimmunoprecipitation of TrkB-FL with JIP3 mutants. HEK293 cells were cotransfected with FlagTrkB-FL and HAJIP3 mutants, and immunoprecipitation was performed using rabbit anti-HA antibodies, immunoblotted with mouse anti-Flag or mouse anti-HA antibodies. B, Coimmunoprecipitation of KLC1 with JIP3 mutants. HEK293 cells were cotransfected with MycKLC1 and HAJIP3 mutants, and immunoprecipitation was performed using rabbit anti-HA antibodies, immunoblotted with mouse anti-Myc or mouse anti-HA antibodies. C, Schematic representation of the HAJIP3 mutants. Amino acid numbers correspond to appropriate domains within the JIP3 deletion constructs. A summary of TrkB-FL/KLC1 interactions with the JIP3 mutants is provided. D, Interaction of TrkB with KLC1 was reduced by HAJIP3ΔCC1. HEK293 cells were cotransfected with MycKLC1, FlagTrkB-FL, and HAJIP3/JIP3ΔCC1 constructs, and immunoprecipitation was performed using rabbit anti-Myc antibodies, followed by immunoblotting with mouse anti-Myc, anti-Flag, or anti-HA antibodies. E, Direct interaction of the TrkB-JM1 domain with the JIP3-CC1 domain in vitro. GST-JM1, GST-JM2, GST-JM3, and GST-JM fusion proteins were incubated with the purified His-CC1. The precipitate was subjected to Coomassie Brilliant Blue staining and immunoblotting analysis using anti-His antibodies. A summary of JM mutants' interactions with CC1 is provided.

Figure 6.

JIP3 mediates anterograde TrkB transport. A, JIP3 influences the localization of TrkB-FL-GFP in PC12 cells. Distribution of TrkB-FL-GFP in the tips of differentiated PC12 cells, which were cotransfected with TrkB-FL-GFP and distinctive constructs (scramble siRNA, siJIP3, HAJIP3, or HAJIP3ΔCC1) and induced to differentiation for 2 d with 50 ng/ml NGF. The cells were fixed, and the relative TrkB-FL-GFP distribution in the tips of cells was measured. Arrows indicate the tips of PC12. The transfected siRNA is shown in red. The expression of JIP3 or JIP3ΔCC1 was detected by immunostaining with anti-HA antibodies (red); the localization of TrkB-FL-GFP is shown in green. B, Quantitative analysis of the localization of TrkB-FL-GFP in the tips of PC12 cells in A. The values expressed as mean ± SEM from three independent experiments (n = 3; *p < 0.05, vs the scramble siRNA group; one-way ANOVA). C, JIP3 influences the localization of endogenous TrkB at the distal axon. Cultured hippocampal neurons transfected with HAJIP3 or the dominant-negative form of JIP3 (HAJIP3ΔCC1), siJIP3, siRab27B, or siJIP3 + siRab27B at 3 DIV were stained for the endogenous TrkB at 5 DIV. The transfected siRNA is shown in red. The expression of HAJIP3 or HAJIP3ΔCC1 was detected by immunostaining with anti-HA antibodies (red), and the endogenous TrkB was detected by immunostaining with anti-TrkB antibodies (green). Scale bar, 10 μm. D, Quantitative analysis of the localization of endogenous TrkB at a distal axon or dendrites in C. The values shown are the mean ± SEM from three independent experiments (n = 3; *p < 0.05; **p < 0.01, vs scramble siRNA group; ^#p < 0.05, vs siJIP3 + siRab27B group; one-way ANOVA). E, Quantitative analysis for the localization of endogenous TrkB at a distal axon in the presence of MDC. Data shown are the mean ± SEM of three independent experiments (n = 3; *p < 0.05, vs the scramble siRNA + MDC group; one-way ANOVA).

Figure 7.

Depletion of JIP3 results in the inhibition of anterograde TrkB vesicle transport. A, A 3 DIV hippocampal neuron cotransfected with EGFP and TrkB-FL-mRFP; the white box indicates the area shown in B. Scale bar, 20 μm. B, Movement of TrkB-FL-mRFP. TrkB-FL-mRFP vesicles can be transported retrogradely (down arrows), anterogradely (arrowheads), bidirectionally (up arrows). Immobile TrkB-FL-mRFP- vesicles (bold arrows) are also observed. The numbers indicate seconds. C, Kymograph of some neurites indicated in A. The kymograph of the axon and three of the dendrites for the neuron in A is shown. Images were captured every 1 s for 60 s in hippocampal neurons. Scale bar, 20 μm. D, Quantitative analysis of relative frequency of anterograde (Antero), retrograde (Retro), bidirectional (Bidir), or immobile vesicles of TrkB-FL-mRFP in siJIP3-transfected neurons The numbers of cells analyzed for each group are >60. Data shown are the mean ± SEM of three independent experiments (n = 3; *p < 0.05, vs the scramble siRNA group; one-way ANOVA).

Figure 8.

JIP3 is involved in BDNF-induced Erk1/2 phosphorylation in neurons. A, BDNF-induced Erk1/2 phosphorylation in siJIP3- or HAJIP3-transfected neurons. Immunostaining of Erk1/2 and pErk1/2 was performed in cultured neurons. The hippocampal neurons were treated for 15 min with BDNF (50 ng/ml), and levels of total Erk1/2 and pErk1/2 were examined using anti-Erk1/2 and anti-pErk1/2 antibodies. GFP indicates the transfected neurons. Arrowheads indicate the untransfected neurons, and arrows indicate the transfected neurons. Scale bar, 50 μm. Bottom panels show enlarged images of the framed regions indicating immunostaining in axon terminals. B, Quantitative analysis of the mean fluorescence intensity of phosphorylation of Erk1/2 in HAJIP3- or siJIP3-transfected neurons compared with the control condition (scramble siRNA group). Data shown are the mean ± SEM from three independent experiments (n = 3; *p < 0.05; **p < 0.01, vs the scramble siRNA group; one-way ANOVA). C, Immunoblotting using anti-Erk1/2 and anti-pErk1/2 antibodies to assess activation of Erk1/2 in PC12 cells stably expressed TrkB (differentiated or undifferentiated) with JIP3 overexpression or knockdown when treated with BDNF. PhosphoTrkB was also analyzed by immunoprecipitation with rabbit anti-Flag antibodies and immunoblotting with anti-phosphoTyr antibodies. D, Histogram shows pErk1/2 and pTrkB levels in each group in C. Data were shown as the mean ± SEM from three independent experiments (n = 3; *p < 0.05, vs the vector control group; one-way ANOVA). E, Immunoblotting using anti-Erk1/2 and anti-pErk1/2 antibodies to assess activation of Erk1/2 in differentiated PC12 cells (with endogenous expression of TrkA) with JIP3 overexpression or knockdown when treated with NGF (50 ng/ml). PhosphoTrkA was also analyzed by immunoblotting. F, Histogram shows pErk1/2 and pTrkA levels in each group in E. Data were shown as the mean ± SEM from three independent experiments.

Figure 9.

JIP3 does not facilitate TrkB plasma membrane insertion. A, Hippocampal neurons expressing FlagTrkB-FL-GFP were stained with M2 anti-Flag antibody under nonpermeabilized conditions to label surface TrkB-FL. Total TrkB-FL was represented by GFP fluorescence. The neurons were transfected with HAJIP3-RFP, siJIP3, or siRab27B, respectively. Immunofluorescence images of the tips of axons are from a representative cell for each condition. B, Quantification of surface TrkB-FL-GFP levels in the tips of axons by ratiometric fluorescence assay of immunofluorescence images shown in A. C, Quantification of total TrkB-FL-GFP distribution in the tips of axons by ratiometric fluorescence assay of immunofluorescence images shown in A. D, Quantification of TrkB surface/total ratio in the tips of axons by ratiometric fluorescence assay of immunofluorescence images shown in A. The data were normalized to the scramble siRNA group. Data were shown as the mean ± SEM from three independent experiments (n = 3; *p < 0.05; **p < 0.01, vs the scramble siRNA group; one-way ANOVA).

Figure 10.

JIP3 regulates axonal filopodia formation in response to BDNF in hippocampal neurons. After staining with Alexa 488-phalloidin, filopodia were defined as any protrusion under 10 μm in length. The number of filopodia and the length of axonal or dendritic shaft were then computed to obtain the filopodia density (number of filopodia per 10 μm). A–D, Hippocampal neurons (5 DIV) transfected with scramble siRNA (A), siJIP3 (B), HAJIP3ΔCC1(C), or HAJIP3 (D) were treated with BDNF (100 ng/ml) for 20 min, with the unstimulated group as vehicle control. Existence of transfected siRNA could be detected by RFP fluorescence; the expression of HAJIP3 or HAJIP3ΔCC1 was detected by immunoblotting with HA antibodies (red). Bottom panels in A showed enlarged images of the framed regions with arrows highlighting the filopodia. E, F, The number of filopodia in axons (E) or dendrites (F) per 10 μm were calculated. For each neuron, the length of axon or dendrite length ranging between 30 and 80 μm was analyzed. The results of more than three independent experiments were compiled. Results are presented as the mean ± SEM (*p < 0.05, vs the vehicle control; ^#p < 0.05, vs scramble siRNA group stimulated with BDNF; one-way ANOVA). Scale bars: A, D, 10 μm.