Mechanisms Underlying The Regulation Of BDNF Expression And Its Receptor Signaling In Memory And Emotion-related Behaviors

BackgroundsThe brain plays vital roles in controlling the human perception,cognition,learning and memory and emotion.However,with the development of society and the increasing environmental pressures,more and more people are suffering from anxiety,major depressive disorde(MDD),post-traumatic stress syndrome(PTSD)and schizophrenia.These mental diseases not only threaten human health,but also have a tendency to light up the disease in adulthood,and bring a huge burden to the society and the family.Therefore,it is particularly important to study the pathological and physiological mechanisms associated with the development of these diseases and to find effective drug targets.Occurrence of mental illness is the result of genetic and environmental factors,such as acute or chronic environmental stress can induce mental illness,especially depression and post-traumatic stress syndrome,and these diseases are related to individual fear memory extinction disorder.Therefore,it is of great significance for understanding the mechanisms of gene regulation underlying these emotional disorders.The neurotrophic factor family is a protein widely distributed in the nervous system and plays an important neuromodulatory role.Brain-derived neurotrophic factor(BDNF)is a widely distributed cytokine in the central nervous system.The abnormal expression of BDNF is closely related to the occurrence and development of mental diseases.For example,BDNF knockout mice exhibit marked anxiety and depression-like behavior;likewise,BDNF expression is reduced in the prefrontal,hippocampal brain regions,and peripheral blood of human that have long-term stress and depression.In addition,antidepressant drugs(fluoxetine,citalopram,ketamine,etc.)can produce antidepressant effects by promoting the synthesis of BDNF.The above results indicate that decreased BDNF production is an important factor leading to depression.Therefore,elucidating the molecular mechanism regulating BDNF expression is of great significance in revealing the pathogenesis of psychotic diseases and the development of drugs.The synthesis of BDNF is regulated by transcription,mRNA trafficking and translation:(1)At transcriptional level,CREB transcription factor is the main molecule regulating the transcription of BDNF gene.After phosphorylation of CREB,it is transported from the cytoplasm to the nucleus and binded to the BDNF promoter region.The binding promotes the transcription of BDNF;(2)At the level of mRNA transport,the 3' UTR of BDNF mRNA has the function of promoting mRNA transport,and the long-form BDNF 3' UTR can help BDNF mRNA transport to dendrites and promote synapse formation;(3)At the translational level,binding of the HuD protein to the 3'UTR of BDNF promotes BDNF mRNA translation.However,the translation mechanism of BDNF mRNA by regulating its 5'UTR is unknown.Pdcd4 is a protein translation inhibitory factor that binds eIF4A competitively with the alpha helix domain MA3 and eIF4G,thereby inhibiting the formation of ribosome complexes and protein synthesis,and it has a 5' UTR selectively manner to regulate the gene expression.Our group has been engaged in the study of the regulation mechanism of Pdcd4 in disease.However,whether Pdcd4 regulates BDNF protein expression is currently unknown.BDNF binds to high-affinity tyrosine kinase-like receptor(TrkB)and promotes cell proliferation,differentiation,survival,growth of neuronal processes,and synaptic plasticity.The normal function of BDNF-TrkB is closely related to the advanced cognitive,learning and memory.It participates in various memory processes such as memory formation,consolidation,and regression.Studies have shown that BDNF binding to TrkB receptors which distributed at postsynaptic membranes can cause phosphorylation and dimerization of TrkB to produce kinase activity,activate of intracellular MAPK/ERK1/2,PLCy,and PI3K signaling transduction.Thereby,with the help of AMPA and NMDA receptors,it is involved in long-term potentiation(LTP)production and memory processes.However,whether the presynaptic membrane TrkB is involved in memory behavior has not been reported yet.ObjectiveOverall,BDNF and its receptor TrkB play a very important role in maintaining the normal physiological functions of the central nervous system.There are two ways to regulate the function of BDNF.On one hand,conditioned stimulus can regulate the expression and release of BDNF.On the other hand,the released BDNF could combine with the receptor distributed in the presynaptic or postsynaptic membrane and active the intracellular signal transduction.The aim of this study is to explore how the BDNF-TrkB system plays a regulatory role in the process of learning and memory and emotional disorders.It mainly includes two aspects:?.First,the mechanism of Pdcd4 molecules regulating BDNF expression and its role in depression;?.Second,the role and mechanism of presynaptic BDNF receptor TrkB in memory extinction.Part ?Pdcd4 regulates BDNF expression and involves in depressionlike behaviorMethods1.Pdcd4 participates in chronic restraint stress induced anxiety and depression like behavior(1)Using 6-8 weeks C57/BL male mice,a chronic restraint stress model was established to cause anxiety and depression like behaviors in mice.(2)Western blot was used to detect the expression of Pdcd4 in the hippocampus and prefrontal cortex after stress.(3)Immunohistochemical staining was used to detect the expression of Pdcd4 after stress.(4)After chronic restraint stress,open field and elevated plus maze test were used to detect anxiety like behavior in mice;tail suspension test,forced swimming test and sucrose preference test were used to detect depression like behavior in mice.(5)AAV virus overexpressing Pdcd4 was packaged and injected into the hippocampus of wild type mice by stereotactic injection.Behavioral tests were applied to detect the anxiety and depression like behaviors in mice.2.Pdcd4 regulates synaptic plasticity and microglia polarization in the hippocampus of mice(1)We used Golgi staining to observe the number of spines in dendrites of hippocampus in Pdcd4 knockout mice and wild type mice after stress.(2)The mice brain that affected by Pdcd4 contained AAV virus was stained with GFP antibody to observe the number of spines in dendrites of GFP positive neurons.(3)Immunohistochemical staining was performed using Iba1 antibody and the changes of microglia morphology in brain tissue were detected.3.Pdcd4 mediates mTORC1 regulated neuronal synaptic plasticity(1)Western blot was used to detect the activation of mTORC1 signal in the hippocampus of mice after stress.(2)Immunoprecipitation was used to detect the ubiquitination of Pdcd4 after stress.(3)During the chronic restraint stress,rapamycin,a mTORC1 inhibitor,was used to detect the phosphorylation of mTORC1 and Pdcd4.(4)ELISA was used to detect the expression of BDNF in hippocampus of the wild type and Pdcd4 knockout mice.(5)The spine density in dendrites of the hippocampal neurons in mice was examined by Golgi staining.4.Pdcd4 regulates the expression of BDNF at the post-transcriptional level(1)qRT-PCR was used to detect the mRNA levels of BDNF in Pdcd4 knockout and wild type mice after stress.(2)ELISA was used to detect the protein levels of BDNF in Pdcd4 knockout and wild type mice after stress.5.the molecular mechanisms underlying Pdcd4 regulated BDNF mRNA translation(1)The RNA-IP experiment showed that Pdcd4 combined with BDNF mRNA.(2)Construction of BDNF mRNA 5' UTR or 3' UTR contained luciferase reporter.(3)Luciferase detection system was used to exam the function of BDNF mRNA 5 'UTR or 3' UTR on Pdcd4 regulated BDNF expression.Results1.Chronic restraint stress leads to increased Pdcd4 expression in the hippocampusWe detected gene expression in the hippocampus and prefrontal cortex of mice after chronic restraint stress and found that mRNA and protein expression of Pdcd4 were significantly increased in the hippocampus but not in the prefrontal cortex.We further clarified the expression profiles of Pdcd4 in the brain and found that Pdcd4 was widely distributed in the brain,and was mainly concentrated in neurons rather than astrocytes.2.Knockout of Pdcd4 shows resistance to chronic restraint stress induced anxiety and depression like behaviors in miceWe randomly divided the Pdcd4 knockout and littermate wild type mice into unstressed and stressed group.Tail suspension test,forced swimming test and sucrose preference test were used to detect depression like behavior in each group.The anxiety like behavior of each group mice were detected by open field test and elevated plus maze test.It was found that chronic restraint stress induced anxiety and depression like behaviors in wild type mice but not in the Pdcd4 knockout mice.3.Mice with overexpression of Pdcd4 in the hippocampus display spontaneous anxiety and depression like behaviorsPdcd4 was packed into type 9 adeno-associated virus and injected into the hippocampus by stereotaxic injection.Behavioral tests showed that mice injected with Pdcd4 virus showed obvious anxiety and depression like behavior compared with the control virus injected group.4.Pdcd4 participates in neuronal synaptic plasticity and microglia polarizationChronic stress leads to synaptic plasticity impairment in the hippocampus of mice,we used Golgi staining to detect the neuronal morphology in brain.The spine density of neurons in the hippocampus of wild type mice was significantly decreased after chronic stress,while Pdcd4 knockout mice showed resistance to chronic stress induced loss of spine.Besides,overexpression of Pdcd4 in the hippocampus of mice significantly decreased the dendritic spine density.In addition,Pdcd4 knockout mice were resistant to CRS-induced activation and proliferation of microglia in the hippocampal brain region and upregulated the expression of the anti-inflammatory cytokine IL-10.Therefore,Pdcd4 is an important molecule that regulates neuronal synaptic plasticity and microglial activation.5.Pdcd4 participates in mTORC1 regulated BDNF expression and synaptic plasticity changeChronic stress inhibits the activation of mTORC1 signaling and then inhibits the ubiquitin-mediated degradation of Pdcd4.mTORC1 regulates neuronal plasticity.During stress,rapamycin inhibited mTORC1 signaling and the expression of BDNF can be further inhibited in wild type mice while in Pdcd4 knockout mice,the effect of rapamycin was blocked.The spine density in hippocampus of wild type mice was also decreased after chronic stress which could be blocked by Pdcd4 knockout.6.Pdcd4 regulates the post-transcriptional process of BDNF mRNATo explore the mechanisms underlying Pdcd4 regulated BDNF expression,we detected the changes of BDNF mRNA and protein in the hippocampus of mice after stress.It was found that Pdcd4 knockout could only block chronic stress induced decrease of BDNF protein,but not mRNA in the hippocampus.Furthermore,overexpression of Pdcd4 can directly reduced the protein level,but no mRNA level of BDNF.7.Pdcd4 regulating IIc isoform BDNF mRNA translationThe isoform of BDNF is determined by the 5 'UTR of BDNF mRNA.We constructed different isoforms of BDNF mRNA 5 'UTR contained luciferase reporter plasmid and detected the effect of Pdcd4 on the expression of luciferase.The results showed that Pdcd4 inhibited IIc isoform BDNF mRNA expression.We further cloned the IIc-5'UTR BDNF into GFP expressing plasmid to form a fusion protein.We then detected the expression of GFP to reflect the effect of Pdcd4 on IIc isoform BDNF expression and the result was consistent with the luciferase reporter experiment.Finally,we demonstrated that the role of Pdcd4 in inhibiting IIc isoform BDNF mRNA translation depending on the RBD-2 and MA3 domains.8.Pdcd4 inhibits eIF4A-dependent BDNF mRNA translationPdcd4 can bind to eIF4A through its MA3 domain,thereby inhibiting eIF4A-dependent protein translation processes.We found that siPdcd4 promotes the expression of reporter genes,and that simultaneous transfection with sieIF4A blocked the effect of siPdcd4 on the expression of reporter genes.Therefore,we conclude that the translational expression of IIc 5'UTR-BDNF mRNA also depends on the regulation of eIF4A.9.Silencing hippocampal Pdcd4 expression can prevent and correct CRS-induced depressive behaviorWe packaged the siPdcd4 lentivirus that interferes with Pdcd4 expression and injected the virus into the mouse hippocampal region before and after CRS.We observed that reducing the expression of Pdcd4 in hippocampal regions can prevent and treat stress-induced emotional disorder.Through suspension and forced swimming experiments,we found that regardless of the injection of interfering virus before or after CRS,compared with the siNC control group,the immobility time of mice was significantly reduced,showing the occurrence of depression-like behaviors that are resistant to CRS.Similarly,the sucrose preference experiment also demonstrated that the injection of interfering virus before or after CRS can correct the CRS-induced decrease in sugar preference.Finally,we conducted open-field experiments and an elevated plus-maze test to show that the siPdcd4 virus injection before CRS was able to resist the CRS-induced decrease the time spent in the central and open arms of the mice;after the CRS was injected with the virus,The anxiety-like behavior induced by CRS was partially rescued,but there was no statistical difference.The above results show that reducing the expression of Pdcd4 molecules can play a good role in prevention and treatment of depression-like behavior caused by CRS,and can prevent anxiety-like behavior to a certain extent.Conclusions1.It was found that Pdcd4 plays an important role in the regulation of depression and anxiety-like behaviors,that is,Pdcd4 overexpression promotes the occurrence of depression,knocking out or silencing Pdcd4 expression can effectively inhibit the occurrence of depression;2.It was found that BDNF is a novel target gene of Pdcd4 and that Pdcd4 selectively controls the translation of BDNF mRNA of subtype IIc through an eIF4A-dependent manner;3.The mTORC1-Pdcd4-BDNF axis was found to play an important role in depression.The mTORC1 signal was found to promote the ubiquitination of Pdcd4 and control the expression of Pdcd4,and Pdcd4 expression can inhibit the expression of BDNF and regulate the synaptic plasticity of neurons.The stress can inhibit the activation of mTORC1 signal and block the expression of Pdcd4.Ubiquitin degradation results in increased expression levels of Pdcd4,in turn,inhibits BDNF expression,decreases neural plasticity,and promotes the development of depression.Innovation and significance1.This study first proposed that Pdcd4 is a new molecule regulating BDNF post-transcriptional translation level.It has important theoretical significance for the study of the mechanism of promoting Pdcd4 and clarifying the molecular mechanism of BDNF expression regulation.2.To propose a new mechanism of mTORC1-Pdcd4-BDNF axis modulating synaptic plasticity of neurons,to deepen the understanding of the involvement of mTORC1 signaling pathway in anxiety and depression behavior and the mechanism of mTOR regulation of BDNF.3.We first discovered that Pdcd4 is a key regulatory molecule in stress-induced anxiety and depression behavior.This provides theoretical and experimental evidence for the use of Pdcd4 as a drug target and a more specific design antidepressant.Limitations1.There is a lack of rescue experiments in vivo to clarify that Pdcd4 participates in emotional expression by regulating BDNF expression;2.Pdcd4 specifically regulates the 5'UTR of BDNF which remains to be further explored;3.The lack of experiments to identify the mechanism of Pdcd4 regulation of microglia polarization.Part ?Pre-synaptic TrkB mediates BDNF signaling transmission in memory extinctionMethods1.The fusion protein of CC1 domain and EGFP was constructed.In vitro immunoprecipitation was used to detect the binding of CC1-EGFP to TrkB and JIP3.2.In vitro cultured neurons were used to quantitatively analyze the movement and distribution of TrkB in axons through live cell experiment and immunofluorescence.3.The method of labeling active presynaptic vesicles with FM1-43 living dyes was used to analyze whether CC1-EGFP affects BDNF induced release of presynaptic vesicles.4.Using the microfluidic chamber,we determined whether CC1-EGFP inhibits BDNF induced retrograde signal transduction.5.CC1-EGFP was packaged into type 5 adeno-associated virus,and was injected into the basolateral amygdala.The conditioned fear memory model was used to detect the memory extinction in rats.6.Western Blot was used to detect the neural circuit of BDNF signal transduction during the memory extinction process.Results1.CC1-EGFP blocked the interaction of JIP3 with TrkB JIP3 binds to TrkB with its CC1 domain.We fused the CC1 domain with EGFP to form a fusion protein.Through the immunoprecipitation experiment,we proved that CC1-EGFP can bind to TrkB and inhibite the interaction of JIP3 with TrkB.2.Effect of CC1-EGFP on the distribution of TrkB receptor at the tip of axonsWe transfected CC1-EGFP into hippocampal neurons and observed TrkB protein transport in axons using live cell imaging.We found that CC1-EGFP can specifically inhibited anterograde transport of TrkB in axons,but not in dendrites.In addition,immunofluorescent staining showed that CC1-EGFP reduced the distribution of TrkB at the end of the axons.3.Effect of CC1-EGFP on presynaptic TrkB mediated BDNF functionPresynaptic TrkB mediated BDNF signaling induces presynaptic vesicle release.Therefore,we used FM1-43 dye to label the active vesicles and observe the effect of CC1-EGFP on BDNF mediated release of presynaptic vesicles.The results showed that CC1-EGFP blocked BDNF induced release of presynaptic vesicles.We also used the microfluidic chamber to culture neurons in which the axonal terminal could be separated from the cell body.Our results showed that CC1-EGFP inhibited BDNF treatment in the axonal terminal induced Erk5 activation in the cell body.4.Effect of CC1-EGFP expression in the basolateral amygdala on memory extinctionIn order to study the effect of presynaptic TrkB on memory extinction,CC1-EGFP was packaged into type 5 AAV virus with a Camkll alpha promoter to enable the selectively expression of CC1-EGFP in long distance projective neurons.We injected the virus into the basolateral amygdala.After conditioned fear memory acquisition and extinction training,we found that overexpression of CC1-EGFP in the basolateral amydala could inhibit memory extinction.The results showed that presynaptic TrkB of basolateral amygdala neurons was involved in the extinction process of memory.5.CC1-EGFP inhibited the transmission of BDNF signaling from basolateral nucleus to medial prefrontal cortexIn order to investigate the neural circuit of presynaptic TrkB mediated BDNF signaling in memory extinction,we examined the levels of p-TrkB in basolateral amygdala and medial prefrontal cortex.We simultaneously injected CC1-EGFP and BDNF into the basolateral amygdala and detected the activation of TrkB both in basolateral amygdala and medial prefrontal cortex.It is found that CC1-EGFP can inhibit the transmission of BDNF signaling from basolateral amygdala to medial prefrontal cortex.Conclusions1.We developed a protein tool CC1-EGFP to study the role of presynaptic TrKB mediated BDNF function.2.CC1-EGFP could be used to study the role of presynaptic TrkB in basolateral amygdala in memory extinction.3.We found that presynaptic TrkB of basolateral amygdala mediated BDNF signaling transmission from basolateral amygdala to medial prefrontal cortex during memory extinction.Innovation and significanceWe demonstrated for the first time that the presynaptic TrkB of the basolateral amygdala neurons participates in the process of memory regression,which makes people more aware of the mechanism of dissipating memory formation and provides the possibility of manipulating memory later.Limitations1.This work does not investigate whether presynaptic TrkB regulates memory resolution is a sufficient condition;2.We have only demonstrated that presynaptic TrkB in the basolateral amygdala neurons participates in memory extinction,and it has not been demonstrated whether other presynaptic TrkBs in the brain regions associated with extinction are involved in memory regulation.