| BackgroundNeurons are highly polarized and compartmentalized cells. The growth of neuronal processes into an extended axon and highly branched dendrites is crucial for the development of neuronal connectivity. Axon outgrowth is essential for the proper development of the nervous system as well as for axon regeneration after injuries. Although the regulation of axon outgrowth has been a subject of intense investigation, the signaling mechanisms that mediate axon outgrowth in the mammalian brain remain to be elucidated.JNK-interacting proteins (JIPs) comprise a family of proteins that were first identified for their role in organizing JNK signaling cascades. As a member of the JIPs, JIP3has been suggested to tether specific JNK signaling modules, thereby promoting signal transmission. The JNK pathway is involved in axon formation/polarization, extension, synaptic plasticity and dendrite development. Moreover, as a homology of UNC-16in Caenorhabditis elegans and Sunday Driver in Drosophila, JIP3is implicated as an adaptor protein in kinesin-dependent vesicular transport to axons and has recently been reported to be a mediator in TrkB anterograde axonal transport in hippocampal neurons. Interestingly, JIP3is selectively expressed in the embryonic and adult mouse brains. Furthermore, the expression of JIP3is predominantly localized in the cell bodies and axons of developing neurons and concentrates at axon tips in cultured hippocampal neurons. Together, these studies prompt the hypothesis that JIP3might regulate axon development. Recently, it was reported that JIP3could restrict axon branching via the GSK3β/DCX signaling pathway. However, whether and how JIP3plays a role in other aspects of axon development such as specification and elongation remain unknown.JIP1is another member of the JIPs, it also tethers specific JNK signaling modules and enhances signal transmission. Samely, it can mediate many cargos transporting as an adaptor protein. TrkB is a receptor of BDNF and it is very important for neurons to transport TrkB from cell body to axonal tips to accept BDNF signal. It is reported that TrkB can be anterograde transported mediating by JIP3or complex of Slp1, Rab27B and CRMP-2. But how TrkB axonal anterograde transporting is strictly regulated and if JIP1take part in this course, remain to be elucidated.Objective1. To explore the function of JIP3on axon development.2. To identify the molecular mechanism for JIP3influenced axon development.3. To study the function of JIP1on TrkB axonal anterograde transport.Methods1、Neuronal cultures and transfectionHippocampi were dissected from the embryos at embryonic day18(E18), dissociated with0.05%trypsin-EDTA, and gently agitated with a sterile, fire-polished glass Pasteur pipette. Neurons were cultured in Neurobasal medium (Invitrogen) supplemented with2%B27and0.5mM glutamine, and an incubator with saturated humidity,5%CO2, and invariant temperature at37℃was used for cell culture. For immunofluorescence staining, neurons were cultured on coverslips coated with0.1mg/ml poly-D-lysine (PDL)(Sigma-Aldrich) in six-well plates (Corning) in the same medium described above. Neurons were electroporated with various constructs in a Nucleofector device (Amaxa Biosystems), according to the manufacturer’s instructions, before plating.2、Immunofluorescence analysisHippocampal neurons cultured for5days were fixed with4%paraformaldehyde in PBS for10min and permeabilized with0.4%Triton X-100in PBS for10min. After three washes, the cells were incubated with blocking solution (PBS containing10%normal goat serum or donkey serum) for1h at room temperature. After incubation with the primary antibodies at4℃overnight, cells were washed three times and incubated with secondary antibodies for1h at room temperature.3、In utero electroporationThe constructs described above were transfected by in utero electroporation. Briefly, pregnant C57BL/6mice were used. Constructs that had been mixed with Fast Gree were injected at E15.5into the lateral ventricle of each embryonic brain using a pulled glass micropipette. A pair of electrodes with a diameter of7mm (CUY650-P7, NEPA Gene) that were attached to the electroporator (CUY21SC, NEPA Gene) transmitted five square electric pulses at30V for50ms at950ms intervals through the uterine wall.4、Neuronal culture in microfluidic chambersThe microfluidic chambers were fabricated in poly(dimethylsiloxane)(PDMS) using rapid prototyping and soft lithography as previously published. The microfluidic features consist of two parallel compartments, which have access ports (or wells) at both ends. A PDMS barrier contains400small microgrooves (450μm long,10μm wide,3μm high), connecting the two compartments. The dimensions of the microgrooves are designed to guarantee that the axons are specifically isolated, allowing selective stimulation of distal axons. The clean, sterilized, and dry microfluidic chambers were reversibly affixed to PDL-coated60-mm dishes. Suspensions of electroporated neurons were plated into the cell body compartment, where they would attach to the dish after10-15min. After4days in culture, axons grew through the microgrooves and extended into the axon compartment.Results1. JIP3regulates neuronal axon elongation in a kinesin-and JNK-dependent manner1.1Expression of JIP3increases rapidly in hippocampal neurons during developmentBy western blot, we found that the expression of JIP3gradually increased during development, with a robust elevation from DIV2to DIV4, a period suggested to be critical for axon elongation. Next, JIP3immunofluorescence staining was performed on hippocampal neurons at stages2and3of development. We found that upon reaching stage3, when cultured hippocampal neurons had developed a longer axon and several minor dendrites, JIP3was mainly distributed in the cell bodies and the distal segments of the axons. Our results extend this finding by examining the stages of hippocampal process outgrowth. The spatiotemporal expression characteristics of JIP3imply that JIP3has a potential role in axon development.1.2JIP3enhances axon elongation in hippocampal neurons in vitroBy immunofluorescence staining, gain-of-function and loss-of-function studies, we found that JIP3is able to enhance axon elongation but has no effect on axon specification or dendrite elongation in cultured hippocampal neurons.1.3JIP3enhances cortical neuronal axon elongation in vivoIn utero electroporation was performed to examine the effect of JIP3on axon elongation in vivo. Firstly, we proved our in utero electroporation system is reliable by comparing our results with previous reports. Then, we found that, in the IZ and SVZ regions of the E18-P0mouse cortex, there was no significant difference in the percentages of multipolar and unipolar/bipolar neurons between scramble, siJIP3and JIP3-IRES-EGFP groups. Next, compared with the scramble group, JIP3knockdown decreased and JIP3overexpression increased the length of callosal axons that invaded the contralateral cortex at P5. We thereby confirmed that JIP3plays a role in cortical neuronal axon elongation without affecting axon specification in vivo.1.4Identification of the key domains in JIP3that are critical for axon elongationTo verify which domain of JIP3is involved in the effect on axon elongation, various JIP3deletion mutants were constructed and successfully expressed, and their abilities to enhance axon elongation were examined. Then with the help of immunofluorescence staining, we found that deletion of KBD&LZ (JIP3AA) or JBD (JIP3AJBD) could abolish the enhancement of axon elongation. These data suggest that the KBD&LZ and JBD domains are critical for JIP3to enhance axon elongation.1.5JIP3must be transported to the axon tips to exert its effect on axon elongationIt is reported that JIP3ΔΔ lost its capacity to enhance axon elongation through its inability to be transported to the axon tips. So, siRNA oligonucleotides against each rat kinesin1heavy chain (KIF5) subtype were simultaneously introduced into hippocampal neurons, and knocking down KIF5expression significantly decreased the localization of JIP3at axon tips. More interestingly, in the absence of KIF5, JIP3lost its ability to enhance axon elongation, suggesting that kinesin-based transportation to axon tips is necessary for JIP3to enhance axon elongation.1.6JIP3enhances axon elongation by locally activating JNK at axon tipsPrevious studies indicated that the JBD domain of JIP3could mediate the interaction between JIP3and JNK, so we sought to determine the effect of exogenously expressed JIP3or JIP3AJBD on JNK activation. Immunoblots of lysates from hippocampal neurons transfected with different constructs showed no detectable difference in the levels of total JNK and phosphorylated JNK (pJNK). Whereas, immunocytochemistry results showed that pJNK/JNK ratio at axon tips was significantly increased by overexpressing JIP3-EGFP but decreased by overexpressing JIP3AJBD-EGFP. These results suggest that JIP3is able to selectively enhance JNK activation at the axon tips.We then surmised that JIP3would stimulate axon elongation by specifically increasing JNK phosphorylation at axon tips. To test this hypothesis, we employed a microfluidic chamber containing two parallel compartments connected by microgrooves. The JNK inhibitor SP600125was added to the cell body compartment;24h later, the levels of pJNK in the cell bodies were significantly decreased in the SP600125-treated group compared with the control group. However, administration of SP600125to the cell body compartment did not affect pJNK levels at the axon tips. And samely, the exposure of distal axons to SP600125had negligible effects on the levels of pJNK in the cell bodies. After24h of SP600125treatment in the cell body compartment, JIP3overexpression still led to a robust increase in axon elongation. In contrast, SP600125applied to the axon compartment significantly blocked axon elongation, whether JIP3was overexpressed or not. Taken together, these findings support a model in which JIP3promotes axon elongation by specifically stimulating JNK phosphorylation at axon tips.1.7JIP3regulates actin dynamics at axon tips through the JNK-cofilin pathwayHow could JIP3-enhanced JNK activation at axon tips lead to axon elongation? We found that JIP3overexpression could increase the number and length of filopodia at axon tips. This result suggested that the dynamics of the actin-based filopodia might be responsible for the enhancement of axon elongation by JIP3. JNK has been reported to promote PDGF-BB-induced dephosphorylation of cofilin. The dephosphorylated form of cofilin is active and can influence filopodial dynamics and neurite outgrowth by regulating F-actin dynamics. To investigate a more direct mechanism for the effect of JIP3on axon elongation, we performed immunofluorescence analysis of the levels of phosphorylated cofilin (p-cofilin) and F-actin in hippocampal neurons. We found that JIP3could enhance cofilin activity and F-actin formation, whereas JIP3ΔJBD had no effect on them. More interestingly, overexpression of the less active, pseudo-phosphorylated cofilin mutant cofilin S3E abolished the ability of JIP3to enhance axon elongation. Together, these data indicate that JIP3may regulate actin dynamics at axon tips through the JNK-cofilin pathway and thereby control axon elongation.2. JIP1mediates TrkB axonal anterograde transport2.1JIP1interacts with TrkB in vitro and in vivoWe confirmed that JIP1and TrkB can be co-immunoprecipitated when two moleculars were overexpressed or by endogenous JIP1/TrkB coimmunoprecipitation assay from brain lysates. These results showed that JIP1and TrkB can interact with each other in vitro and in vivo. At the same time, we found that when the brain lysates was immunoprecipitated by KLC1, JIP1and TrkB can be coimmunoprecipitated, which prompted that JIP1might influence TrkB anterograde transport.2.2JIP1influences the expression of TrkB at axonal tips of cultured hippocampal neuronsWe transfected JIP1-HA or siJIP1construct into cultured hippocampal neurons and tested the expression of TrkB at axonal or dendritic tips by immunofluorescence analysis. The results showed that in cultured hippocampal neurons, JIP1could influence the expression of TrkB at axonal tips, but not that at dendritic tips.2.3JIP1mediates TrkB axonal anterograde transport by the same way as JIP3By the same method as shown in section2.2, we found that when JIP1or JIP3was knocked down separately, or both of them were knocked down at the same time, the depressed expression levels of TrkB at axonal tips were all the same, which showed that JIP1could mediate TrkB axonal anterograde transport by the same way as JIP3.Conclusion 1、JIP3is able to enhance axon elongation but has no effect on axon specification or dendrite elongation.2、JIP3is able to selectively enhance JNK activation at the axon tips but not in the cell bodies.3、JIP3mediate axon elongation by regulating actin dynamics at axon tips through the JNK-cofilin pathway.4、JIP1mediates TrkB axonal anterograde transport by the same way as JIP3... |
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