| BackgroundA typical neuron is divided into three parts:the soma or cell body, dendrites, and axon. Neurons develop axons and dendrites to communicate with distant cells. The precise targeting and localization of proteins within these domains are critical to every aspect of neuronal function. Long-range anterograde transport of molecules in axons and dendrites is mainly mediated by microtubule-dependent motors, kinesins. In neurons, conventional kinesin (kinesin-1), which consists of two heavy chains (KHC) and two light chains (KLC). is a multifunctional transporter of both axonal cargo such as synapsin and GAP43 and dendritic cargo such as mRNA and the AMPA receptor. Several binding partners for kinesin-1 have been identified that are soluble adaptor proteins and could mediate the attachment of membrane-bound cargoes to kinesin-1. However, in neurons, the cargo proteins transported by kinesin-1 remain primarily unknown.Neurotrophins. including nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), NT-3, and NT-4/5,have been considered to play important roles in the differentiation, neurite outgrowth, and survival of a variety of neurons, as well as in modulating the function of neural circuits. In particular, brain-derived neurotrophic factor(BDNF) plays an essential role in activity-dependent changes in synaptic function. BDNF, released from target tissues, binds and activates TrkB receptors located on axonal terminals of the innervating neurons and thereby initiates retrograde signaling. Therefore. BDNF signaling in neurons depends on proper cellular processing, transport, and localization of its TrkB receptor. Delivery of TrkB in the anterograde direction along axons or dendrites (from soma to nerve terminals) requires kinesin-1-mediated transport. It is not known whether the mechanisms responsible for TrkB anterograde transport in neurons are different in axons versus dendrites. Recently, it was reported that TrkB transport can be mediated by an adaptor complex comprising Slpl, Rab27B, and CRMP-2 to kinesin-1 and can be anterogradely transported. However, knockdown of any of these adaptors only reduces axonal targeting by 30-50%, suggesting the existence of additional transport mechanisms.C-JunNH2-terminal kinase (JNK)-interacting protein 3 (JIP3) is exclusively expressed in the brain and distributed in dendrites, perikarya, and axons of neurons. JIP3 was originally identified as a JNK-binding protein and functions as a scaffold protein to trigger specific JNK signaling modules. Subsequent genetic studies revealed that JIP3 is an ortholog of both Drosophila Sunday driver (syd) and the Caenorhabditis elegans UNC-16, which are implicated as adaptor proteins in kinesin-dependent cargo transport to axons. It has been shown that JNK3 can bind to JIP3 and be trafficked in the anterograde axonal transport pathways. However, it remains to be determined whether JIP3 can directly bind to other cargoes and mediate their anterograde transport.Objective1. To find out how TrkB receptor be anterogradely transported in neuron. Is there an adaptor protein?2. If we find out the transport mechanism, does it also works in dendrites?3. To investigate whether the transport mechanism affect BDNF signaling Methods1. Sciatic nerve ligationSciatic nerve ligation analysis is a well established in vivo tool used to identify transported molecules in axons biochemically and immunohistochemically. Male rats were anesthetized with 5% chloralhydrate, and the sciatic nerve on one side was ligated using a 5-0 polypropylene monofilament. One day after ligation, the animals were killed, and the sciatic nerves were frozen in liquid nitrogen immediately. For immunoblotting analysis of sciatic nerve, ligated and contralateral unligated sciatic nerves (3mm apart from the ligature) were dissected, and extracts were analyzed with the indicated antibodies. For immunohistochemistry staining of sciatic nerve, fresh longitudinal frozen sections were prepared (20μm). The contralateral sciatic nerve sections were prepared in parallel as a control.2. Immunofluorescence analysisHippocampal neurons cultured for 5 days were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min. Cultures were washed with PBS for three times, and then, permeabilized with 0.4% Triton X-100 in PBS for 10 min. After washed in PBS again, the cells were incubated with blocking solution (PBS containing 10% normal goat serum) for 1 h at room temperature. Following incubation with the primary antibodies at 4℃overnight, neurons were washed with PBS and incubated with fluorescent secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 594 or Cy5 for 1 hr at room temperature. All the immunostained cells were observed with a Zeiss LSM710 confocal microscope (Carl Zeiss, Thornwood, NY), and quantification of labeling intensities was carried out using MetaMorph Software (Universal Imaging Corporation, West Chester, PA).3. Co-immunoprecipitation AssayForty eight hours after being electroporated (Amaxa Biosystems, Koln, Germany) with the indicated constructs, HEK293 cells were extracted by TNE buffer (10 mM Tris pH 8.0,150 mM NaCl,1 mM EDTA,1% NP-40,10% glycerol with protease inhibitors). Lysates were clarified by centrifugation at 14,000g for 15 min at 4℃. After centrifugation. the soluble supernatants were incubated with indicated antibodies for more than 2 hr at 4℃. The immunocomplex was then precipitated with protein A or G-Sepharose (Sigma) overnight at 4℃. The beads were then washed for 3 times with TNE buffer and eluted by boiling in sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and at last subjected to immunoblotting to analyze with the indicated antibodies.For endogenous interaction, rat brain was homogenized and lysed in TNE buffer (8ml TNE/g brain lysate) with protease inhibitors and then clarified by centrifugation at 14,000g for 15 min at 4℃. After centrifugation, the proteins were quantified using Bicinchoninic acid (BCA) protein assay. Approximately 10 mg samples of rat brain lysates were incubated with mouse anti-JIP3 antibodies, and the same amount of lysates were incubated with mouse IgG sepharose as a negative control. The immunocomplex was precipitated using protein G-Sepharose beads. The bound proteins were eluted and analyzed by immunoblotting with anti-JIP3, anti-TrkB or anti-KLC1 antibodies. To confirm the result, rat brain lysates were also incubated with goat anti-KLC1 antibodies, then precipitated using protein G-Sepharose beads. Immunoblotting were done with the above antibodies to detect the bound proteins.4. Preparation of glutathione S-transferase fusion proteins and in vitro binding assayThe various glutathione S-transferase (GST)-TrkB juxtamembrane mutant constructs pGEX-TrkB-JM1 (454-465); pGEX-TrkB-JM2 (454-485); pGEX-TrkB-JM3 (454-508); pGEX-TrkB-JM (454-537). These cDNA fragments were generated by PCR and subcloned into the pGEX-4T-1 vector. All the fusion proteins were expressed in BL21 Escherichia coli and immobilized on glutathione-Sepharose 4B beads. JIP3-CC1 (amino acids 451-520) fragment was subcloned into pET28a to yield His-CC1. Purified his-tagged CC1 were incubated with GST-fusion proteins bound beads in pull down buffer at 4℃for 1-2 hr. After extensive wash with PBS buffer, the bound proteins were analyzed by SDS-PAGE followed by staining with Coomassie blue or immunoblotting with anti-His antibodies. 5. Live cell imaging.For the hippocampal neurons used for live cell imaging, the transfection of constructs was performed by Nucleofector device before plating. Five days after being seeded in the glass-bottomed 35-mm dishes coated with 0.1 mg/ml poly-D-lysine, the transfected neurons were observed by a Nikon Eclipse TE 2000-U inverted fluorescence microscope equipped with a motorized Z drive using a 40X oil immersion objective lens (1.0 NA). The cells selected for imaging had healthy morphologies and images were acquired every 1 s continuously for 1 min with a HQ2 cool CCD camera. The positive vesicle was tracked and processed by MetaMorph Software. A kymograph was generated using NIH Image J. The relative frequency of immobile, directional and bidirectional movements was calculated and then presented as the average of some neurites. For RNAi experiments,80-120 vesicles of 20 axons or 40 dendrites were counted in each condition and showed the relative frequency. These results were carried out at least in triplicate, and the data were analyzed by one-way ANOVA by SPSS 13.0.6. Quantification of filopodia.Filopodia were defined as any protrusion under 10μm in length. The number of filopodia and the length of axonal or dendritic shaft were then computed in order to obtain the filopodia density (number of filopodia/10μm). For each neuron, an axon or dendrite length ranging between 30 and 80μm was analyzed. Each experimental condition was repeated at least three times. In each experiment, more than 35 cells were examined at random, the results of more than three independent experiments were compiled and the mean±SEM was calculated.Results1. Both TrkB-FL and TrkB.T1 receptors are anterogradely transported in sciatic nerveWestern immunoblotting analyses of anterogradely and retrogradely transported molecules in 1 d ligated rat sciatic nerve confirmed previous findings that TrkB-FL protein levels are increased in extracts from both proximal (anterograde) and distal (retrograde) sides of the ligation. Interestingly, we found that the truncated TrkB isoform, TrkB.T1 receptor, which lacks the tyrosine kinase domain, is also anterogradely transported. A previous study has indicated that TrkB receptors undergo anterograde transport via the interaction between the TrkB tyrosine kinase domain and the Slp1/Rab27B/CRMP-2/kinesin-1 complex. However, the anterograde transport of truncated TrkB.T1 receptor suggests that TrkB tyrosine kinase domain-independent anterograde transport mechanisms exist. It is known that TrkB.T1 and TrkB-FL receptors share a small part of JM domain containing 12 amino acids (K454-G465), which might be a key domain involved in TrkB anterograde transport. We screened a human brain cDNA library using the 12 aa as bait by yeast two-hybrid assay. Among the 35 positive clones, three clones, which encoded JIP3, were isolated. JIP3 has been implicated as an adaptor protein in kinesin-1-dependent anterograde transport. Therefore, we investigated whether JIP3 could mediate TrkB anterograde transport.2. JIP3 colocalizes and interacts with TrkB in vitro and in vivoWe first compared the subcellular distribution of JIP3 and TrkB in cultured hippocampal neurons (5 DIV) by immunocytochemistry staining. We used TrkB and JIP3 antibodies to examine the localization of endogenous TrkB and JIP3. The results indicated that TrkB containing vesicles were present in the perinuclear region, axons, and dendrites, partially colocalized with JIP3. To test the hypothesis that anterograde transport of TrkB is associated with JIP3 in vivo,we performed sciatic nerve ligation to follow the localization of TrkB and JIP3. A remarkable accumulation of TrkB receptor was observed in the proximal portion of the sciatic nerve after ligation and JIP3 and TrkB showed higher colocalization in the proximal sides of the ligation, which is consistent with their in vivo interaction.Next, we examined whether JIP3 could form a complex with TrkB. First, we transfected FlagTrkB-FL or FlagTrkB.T1 with N-terminal HAJIP3 in HEK293 cells. After immunoprecipitation of JIP3 with HAantibodies, an association of TrkB-FL or TrkB.T1 with JIP3 was observed as assessed by immunoblotting analysis using Flag antibodies, although TrkB.T1 showed weaker association.The complex between the FlagTrkB-FL/FlagTrkB.T1 and HAJIP3 was also detected after immunoprecipitation of Flag and immunoblotting with anti-HA antibodies. To examine whether this interaction occurs under physiological conditions, we performed endogenous JIP3/TrkB coimmunoprecipitation assays from brain lysates. Lysates from rat brain were immuno precipitated with anti-JIP3 antibodies, and we found TrkB coimmunoprecipitated with JIP3 under the endogenous level. As expected, KLC1 also associated with the TrkB/JIP3 complex, which was further confirmed by the immunoprecipitation with anti-KLC1 antibodies from brain lysate. Thus, we determined that TrkB/JIP3/KLCl could form a complex both in vitro and in vivo.3. JIP3 is an intermediate interacting protein that links TrkB-FL binding with KLC1To address which molecule is the intermediate bridge between TrkB and KLC1, we examined whether JIP3 could contribute to the TrkB and KLC1 interaction. We found JIP3 is an intermediate interacting protein that links TrkB-FL binding with KLC1. Interestingly, we found the elimination of TrkB-FL binding with KLC1 by knockdown of JIP3 was not complete. Double knockdown of JIP3 and Rab27B could further decrease the association between TrkB-FL and KLC1, compared with knockdown of JIP3 or Rab27B alone. This result suggests that TrkB-FL could synchronously form a complex with KLC1 through JIP3 and Slpl/Rab27/CRMP-2, respectively, and these two systems appear to function in a subadditive manner.4. The JM1 domain in TrkB is not only necessary but also sufficient for TrkB/JIP3 interactionTo address whether the JM1 12 amino acids shared by TrkB-FL and TrkB.T1 play an important role in this interaction, various mutants of TrkB receptor were constructed:TrkB-JM1 retaining only the 12aa in the TrkB intracellular domain, TrkB-JMO with the entire TrkB intracellular domain deletion, and TrkB JM1 with the 12 aa deletion in the TrkB intracellular domain. The coimmunoprecipitation assay indicated that the JM1 domain in TrkB is not only necessary but also sufficient for TrkB/JIP3 interaction. The TrkB/JIP3 association could be enhanced by the amino acids between JM2 and JM3, which explained the weaker association of JIP3 with TrkB.T1 compared with the TrkB-FL.It is well known that there are three most common types of Trk receptors:TrkA. TrkB. and TrkC. We want to know whether JIP3 could form complex with all three Trk receptors. Interestingly, we found that JIP3 could also bind with TrkC but not with TrkA. This result indicates that JIP3 could specifically mediate TrkB/TrkC but not TrkA binding to kinesin-1, and this binding depends on their JM1 domain.5. JIP3 associates with TrkB-FL and KLC1 via its CC1 domain and LZ domain, respectively AND JIP3-CC1 could directly bind with the TrkB JM1 domainTo verify which domain in JIP3 is involved in the JIP3/TrkB interaction, we constructed a series of deletion mutants of JIP3 and tested their ability to bind to TrkB-FL by coimmunoprecipitation studies. The results indicate that the 424-625 amino acid region of JIP3 is critical for TrkB-FL and JIP3 interaction. The 424-625 amino acid region in JIP3 contains three conserved domains named leucine Zipper-like domain (LZ), coiledcoil 1 (CC1), and coiled-coil 2 (CC2). To define more precisely the region required for its binding with TrkB, three JIP3 mutants with respective LZ, CC1, or CC2 domain deletions were constructed, and their ability to bind to TrkB-FL was analyzed by coimmunoprecipitation studies. The results indicate that the CC1 deletion could abolish the JIP3 and TrkB-FL interaction. We also examined the ability of these JIP3 mutants to bind to KLC1 and found that the LZ deletion mutant lost its ability to bind to KLC1. Thus, our data suggests that JIP3 associates with TrkB-FL and KLC1 via its CC1 domain and LZ domain, respectively. We next examined whether the CC1 deletion mutant of JIP3 (JIP3CC1) could compete with wild-type JIP3 to bind with KLC1 and thus reduce the TrkB-FL and KLC1 association. As shown in Figure 5D, JIP3CC1 overexpression significantly decreased the TrkB-FL and KLC1 interaction, which suggests that JIP3△CC1 could be used as a dominant-negative (DN) construct to evaluate the role of JIP3 in regulating TrkB-FL transport and function. All the results above indicated that JIP3 may play an important role in the interaction between TrkB and KLC1.To confirm that JIP3 does interact directly with TrkB, we performed a GST pull-down in vitro interaction study between JIP3 and TrkB. The in vitro binding assay reveals that JIP3-CC1 could directly bind with the TrkB JM1 domain and the amino acids between the JM2 and JM3 could enhance this interaction, which is consistent with our previous coimmunoprecipitation experiments.6. JIP3 regulates axonal but not dendritic TrkB anterograde transportSince we showed that TrkB/JIP3/KLC1 could form a complex, it is conceivable that JIP3 may also mediate TrkB transport by kinesin-1. To address this question, we first examined the effect of overexpression/knockdown of JIP3 on the subcellular localization of TrkB-FLGFP in differentiated PC 12 cells. In a control group (transfected with scrambled siRNA), TrkB-FL-GFP accumulated at the tips of neurites. Depletion of JIP3 by siRNA or transfection of JIP3 DN construct JIP3CC1 could significantly decrease this distal localization of TrkB-FL-GFP. On the contrary, overexpression of JIP3 led to a significant increase in the amount of TrkB-FL-GFP at the tips of neurites. These results suggest that JIP3 facilitates anterograde transport of TrkB-FL-GFP in PC 12 cells.We repeated the experiments in cultured hippocampal neurons to investigate whether JIP3 could enhance TrkB anterograde transport under endogenous conditions in neurons. TrkB staining- staining levels in the distal 30μm of the axons was significantly enhanced by JIP3 overexpression. In contrast, either overexpression of a dominant-negative form of JIP3 (JIP3△CC1), which could form a complex with KLC1 but cannot interact with TrkB, or JIP3 knockdown via siJIP3 transfection decreased TrkB localization at the distal part of axon. Interestingly, the involvement of JIP3 in TrkB anterograde transport is specific for axons, as overexpression/knockdown of JIP3 had no effect on distal TrkB dendritic distribution. Simultaneously knocking down JIP3 and Rab27B could further decrease the endogenous TrkB distal axonal localization compared with their respective knockdown, which supports the idea that JIP3/TrkB and Slpl/Rab27B/CRMP-2/TrkB complex functions in an additive fashion to facilitate TrkB anterograde transport.7. Live cell imagng results:JIP3 has important functions on anterograde TrkB transport preferentially in axons. To further examine the function of JIP3 in TrkB transport under live cell conditions, we transfected neurons with TrkBFL-mRFP and monitored TrkB-FL-mRFP-containing vesicle movement in neurons at 5 DIV by using time-lapse fluorescence microscopy. TrkB-FL-mRFP-containing vesicles were found to be highly mobile, in axons, visibly moving anterogradely, retrogradely, or bidirectionally. Compared with axons, a weaker dynamic movement of TrkB-FLmRFP vesicles was observed in dendrites. To quantify the TrkB-FL-mRFP-containing vesicle movements, we subdivided the vesicles into four categories:(1) the anterograde group (vesicles that moved only in the anterograde direction); (2) the retrograde group (vesicles that moved only in the retrograde directions); (3) the bidirectional group (vesicles that moved in both directions); and (4) the immobile group (vesicles that were immobile). Axonal and dendritic TrkB-FL-mRFP-containing vesicles had similar proportions of the four groups. Compared with the control group, neurons cotransfected with TrkB-FL-mRFP and siJIP3 had a significant decreased percentage of the anterogradely transported vesicles and the corresponding increased percentage of the immobile vesicles in axons. JIP3 knockdown had no effect on the percentage of TrkB-containing vesicle categories in dendrites. These results indicate that JIP3 has important functions on anterograde TrkB transport preferentially in axons.8. JIP3-dependent TrkB anterograde transport is required for BDNF-induced signalingTo assess the functional consequences of JIP3-dependent TrkB anterograde transport, next we investigated whether JIP3 could affect BDNF-induced signaling. The addition of BDNF (50 ng/ml) to hippocampal neurons for 15 min induced activation of Erk1/2, which is a well established downstream target of TrkB activation. Because of the low transfection efficiency in neurons, the phosphorylation of Erk1/2 induced by BDNF was analyzed by immunocytochemical staining. Phosphorylated Erk1/2 (pErk1/2) was attenuated by35% in JIP3 knockdown neurons despite identical levels of total Erkl/2, whereas pErk1/2 was increased by about 40% in JIP3-overexpressed neurons. The effect of JIP3 on BDNF induced Erk1/2 phosphorylation was more obvious in the axonal shaft and terminus.9. JIP3 might not play a significant role in TrkB plasmamembrane insertionAccording to our previous report (Zhao et al.,2009), we used ratiometric fluorescence assay to measure the TrkB cellsurface level. We found that overexpression or knockdown of JIP3 could increase or decrease total and cell-surface TrkB-FL levels in distal axons, respectively. However, the ratio of surface TrkB-FL versus total TrkB-FL remained unchanged.These results suggest that anterogradely transported TrkB-FL is successively inserted into plasma membrane and JIP3 might not play a significant role in TrkB plasmamembrane insertion.10. JIP3 regulates axonal filopodia formation in response to BDNFBDNF/TrkB signaling could drive activitydependent synaptic morphogenesis. To investigate the role of JIP3-dependent TrkB transport in BDNF-triggered synaptogenesis, we examined whether JIP3 could regulate BDNF induced filopodia formation, a process involved in synaptogenesis and neuronal development. Hippocampal neurons were transfected, respectively, with scramble siRNA, siJIP3, JIP3ACC1 (JIP3 DN construct), or JIP3, and the formation of filopodia was examined after a 20 min administration of BDNF. Consistent with the previous report. treatment of hippocampal neurons with BDNF induced the filopodia formation in both axons and dendrites. Compared with the control group, siJIP3 and JIP3 DN transfection abolished the BDNF-induced axonal filopodia formation, and overexpression of JIP3 could augment the axonal filopodia formation elicited by BDNF stimulation. On the contrary, JIP3 had no effect on the augmentation of dendritic filopodia density in response to BDNF treatment. Together, these results suggest that JIP3 could selectively influence BDNF-induced axonal filopodia formation, which might be mediated by the role of JIP3 in BDNF signaling.Conclusion1. JIP3 is an intermediate interacting protein that links TrkB-FL binding with KLC1 to form a complex TrkB/JIP3/KLC1. 2. A consensus 12 amino acids in the intracellular JM domain plays an important role in TrkB/JIP3 interaction, this domain in TrkB is not only necessary but also sufficient for this interaction.3. JIP3 associates with TrkB-FL and KLC1 via its CC1 domain and LZ domain, respectively; JIP3-CC1 could directly bind with the TrkB JM1 domain.4. JIP3 regulates axonal but not dendritic TrkB anterograde transport.5. JIP3-dependent TrkB anterograde transport is required for BDNF-induced signaling.Innovations:First, to our knowledge, this is the first evidence that JIP3, a KLC1 adaptor protein, directly binds to TrkB and links TrkB to the motor protein kinesin-1.Second, we found that JIP3 selectively mediates TrkB anterograde transport in axons but not in dendrites.Third, we showed that JIP3-mediated TrkB anterograde axonal transport regulates BDNF actions.
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