Up-regulation Of C-Jun NH2-terminal Kinase-interacting Protein 3 (JIP3) Contributes To BDNF-enhanced Neurotransmitter Release

BackgroundIn addition to neuronal survival and differentiation, the brain-derived neurotrophic factor (BDNF) plays an important role in regulating synaptic plasticity, which enables the brain to build neuronal circuits during development and controls high order activities such as learning, memory and behaviors in the adult. BDNF exerts its presynaptic and postsynaptic actions on synaptic plasticity both in the short and long term. Acute application of BDNF was shown to modulate synaptic transmission by enhancing depolarization-evoked glutamate release in hippocampus at presynaptic sites. Meanwhile, at postsynaptic sites, BDNF rapidly activates ion channels including Na+, Ca2+ and K+channels and enhances the open probability of NMDA receptors and AMPA receptors. These rapid pre-and post-synaptic events induced by BDNF were mainly mediated by phosphorylation of existing proteins, which actions are likely to be transient. After long-term treatment of BDNF, these initial effects are followed by more sustained synaptic modulation including enhanced presynaptic neurotransmitter release and increased postsynaptic spine density and spine maturation, which involve the new proteins synthesis. Nevertheless, which BDNF-triggered newly synthesized proteins are involved in these processes, especially at the presynaptic sites, remains not fully understood.The c-Jun NH2-terminal kinase (JNK)-interacting protein 3 (JIP3) is expressed in the developing and adult brains, and originally identified for its role in organizing JNK signaling cascades as a scaffold protein. In addition, JIP3, also named Sunday Driver (SYD) in Drosophila and UNC-16 in Caenorhabditis elegans, is known as a scaffold protein mediating synaptic vesicles trafficking to axons terminal and has recently been reported to be an adaptor molecule for TrkB anterograde axonal transport.Interestingly, previous studies have reported that another neurotrophin member nerve growth factor (NGF) could up regulate the expression of JIP3 in differentiated PC12 cells. Combined with the previous finding that JIP3 is mainly expressed in cerebral cortex and hippocampus where BDNF is highly expressed, we tried to determine whether BDNF could regulate JIP3 expression. In this study, we found that BDNF/TrkB signaling could increase JIP3 protein synthesis via activation of CREB, which resulting in enhancement of neurotransmitter release.ObjectiveFinding out the mechanism of BDNF enhancing long-term postsynaptic plasticity.Results1. BDNF increases JIP3 protein levels in hippocampal neurons both in vitro and in vivo.Cultured hippocampal neurons (DIV7) were treated with 50 ng/mL BDNF for various times and the JIP3 protein levels were detected by western blot. We found that upon BDNF treatment, the JIP3 levels were gradually increased over time, with a significant elevation after BDNF treatment for 4 hours. Next, JIP3 immunofluorescence staining was performed on hippocampal neurons at DIV7. Upon BDNF treatment for 4 hours, the immunostaining intensity of JIP3 increased to 1.61±0.21 fold comparing with the control group. Hippocampal JIP3 protein levels in BDNF knock-out (BDNF+/-) mouse were detected by western blot, and we found that the JIP3 protein levels were significantly decreased (to～39.2%) in BDNF+/- mice compared with that in wild type mice.2. BDNF increases JIP3 levels in a TrkB dependent manner and via biosynthetic pathway.Inhibitors were used to respectively block TrkB or p75NTR mediated signaling. K252a is a selective inhibitor of the tyrosine protein kinase activity of the Trk family, and pep5 is the p75 receptor inhibitor. We found K252a pre-incubation could inhibit BDNF-induced TrkB phosphorylation and block BDNF-induced increase of JIP3 protein levels, whereas pep5 had no effects. Protein synthesis inhibitor cycloheximide (CHX) or degradation inhibitors (Leupeptin, Pepstatin A and MG132) were added 1 h before BDNF treatment. Interestingly, the increase of JIP3 protein levels upon BDNF treatment was abolished only in the presence of the CHX, but not degradation inhibitors. Next we carried out real-time PCR experiments to examine the mRNA levels of JIP3 in primary cultured neurons stimulated with BDNF for the indicated times. We found a significant increase in JIP3 transcripts after BDNF treatment for 2 hours.3. BDNF regulates JIP3 transcription via CREB.CREB inhibitor was used to inhibit CREB function by blocking the interaction of CREB and CREB-binding protein (CBP) in cultured neurons. We found that CREB inhibitor treatment decreased JIP3 protein levels under the basal condition. We further infected neurons with lent virus harboring a dominant negative mutant of CREB (CREB S/A); in which serinei 33 is mutated to alanine to inhibit endogenous CREB function. We found CREB S/A infection abolished BDNF-triggered increase in JIP3 expression levels. As a control, the JIP3 levels in neurons with wild-type CREB (CREB WT) infection could still respond to BDNF treatment.4. Mapping the CREB binding sites on JIP3 promoter.We constructed their JIP3 promoters into pGI3-basic reporter containing the 3311bp JIP3 promoter, a-2000 bp region or a-1000 bp region upstream of the ATG. From the result of dual luciferase assays, we found that all the three 5’ JIP3-luc constructs led to a significant increase in Iuciferase activity compared with the negative control. Further, we constructed the CRE sites deletion mutants into pGI3-basic and found fluorescence intensity significantly decreased in the -3265 to -3258 deletion mutant. Then we found that CREB can no longer interact this DNA fragment.5. JIP3 contributes to BDNF enhanced neurotransmitter release.DIV7 neurons were infected with lent virus harboring siJIP3 or scramble siRNA for four days. Then we carried FM1-43 staining to image synaptic vesicle endocytosis and exocytosis in the presynaptic terminal. we observed that under the basal condition, in the neurons infected with siJIP3, the releasable FM 1-43 fluorescence intensity at individual active button was decreased compared to the scramble siRNA group, indicating that JIP3 exerts an effect upon depolarization-evoked neurotransmitter release. Exposure to BDNF for 4 hours increased the releasable neurotransmitter both in the JIP3 knocked-down group and the control group. However JIP3 knockdown reduced the extent of BDNF-increased neurotransmitter release compared to the control group.Conclusions1. BDNF increases JIP3 protein levels in hippocampal neurons both in vitro and in vivo.2. BDNF increases JIP3 levels in a TrkB dependent manner and via biosynthetic pathway.3. BDNF regulates JIP3 transcription via CREB.4. JIP3 contributes to BDNF enhanced neurotransmitter release.Novelties1. Find the phenomenon and the mechanism of BDNF enhance JIP3 protein level.2. JIP3 contributes to BDNF enhanced neurotransmitter release in long term.