The Mechanisms For Limk1/Cofilin Signaling Pathway Regulating BDNF Induced Axonal Elongation And Aversive Memory Extinction

BackgroundActin is a cytoskeletal protein, which plays important roles in cell physiological functions such as cytokinesis, endocytosis, neurite extension and synaptic plasticity. Actin exerting its physiological function depends on actin remodeling, that is dynamic changes of globular actin (G-actin) and filamentous actin (F-actin). Actin remodeling is regulated by many proteins including ADF/cofilin, Arp2/3, Eps8, Profilin, myosin Ⅱ and myosin Ⅴ. Since ADF/cofilin is ubiquitously expressed in eukaryote, directly regulating actin remodeling and is essential for growth and development, it is gradually becoming research focus in the fields of tumor metastasis, diseases of reproductive system, diseases of circulatory system and disorder of nervous system.Neurons play physiological functions requiring properly growth and differentiation. The outgrowth of axons is earlier than dendrites. The terminal enlargement of axon is growth cone, composing filopodia and lamellipodia. Growth cone is rich of actin network especially beneath the membrane. Actin dynamic is the basis of neurite extension. Neurotrophins especially the most studied BDNF are important for neuronal growth, differentiation, survival and synaptic plasticity. However, the exact mechanism for BDNF promoting neurite outgrowth is pending further study.In the central nervous system (CNS) in adults, the vast majority of neurons form a complex network in the form of chemical synapse interconnection. Chemical synapses, which are specialized junctions between axonal terminals and dendritic spines, have synaptic plasticity. Synaptic plasticity is the ability of synapse to change in strength responding to either use or disuse of transmission over synaptic pathways. Synaptic plasticity results from the alteration of neurotransmitter release, the number of receptors located on synapse and the morphology of dendritic spines. Actin dynamics is not only the basis of synaptic morphology but also closely linked to postsynaptic AMPA receptor endocytosis and insertion. Actin dynamics, which directly modulates synaptic transmission efficiency including LTP and LID, is the foundation of learning and memory. Although cofilin is the key molecule in regulating actin reorganization, the specific mechanism for cofilin regulating learning and memory remains unclear.Objective1. To explore the relationship between LIMK1/cofilin signaling pathway and BDNF/TrkB signaling pathway, and identify the molecular mechanism for BDNF induced axonal elongation. So that it provides the theory basis for deeply researching the therapy of BDNF mutant induced abnormalities in the nervous system.2. To study the effect of LIMK1/cofilin signaling pathway on aversive memory extinction, and provide the theory basis for searching a potential treatment strategy for memory disorders.Methods 1. The mechanism for LIMK1/cofilin signaling pathway regulating BDNF induced axonal elongation1.1Detection of interaction between LIMK1and TrkB48h after electroporation with HA-LIMK1and Flag-TrkB, HEK293cells were lysed and collected for co-immunoprecipitation and immunoblotting. We performed immunostaining experiments to compare the subcellular distribution of LIMK1and TrkB.1.2The short term effect of BDNF on LIMK1After serum starvation, cultured neurons were stimulated with BDNF for30min. cell lysates were collected for SDS-PAGE to detect phosphorylation of LIMK1. Cell extracts were subjected to SDS-PAGE in the absence or presence of dithiothreitol, and then immunoblotted with anti-LIMK1to test homo-dimerization level of LIMK1. Additionally, neuron lysates were separated into membrane and cytosol fraction to examine the distribution of LIMK1before and after BDNF treatment.1.3Sequence analysis of TrkB/LIMK1interactionVarious sequences were gradually deleted from the full-length of TrkB (TrkB-FL), and co-immunoprecipitation was carried out between TrkB mutants and LIMK1. The responsible sequence for TrkB/LIMK1interaction was linked to T1protein which could not associate with LIMK1. Then we detected the interaction between chimeric receptor and LIMK1.1.4Design the specific inhibitor for TrkB/LIMK1interactionBased on our previous TrkB/LIMK1interaction domain mapping study, we used a synthesized peptide consisting of the9-amino acid sequence of JMBox4in TrkB fused to the membrane permeability peptide-Tat. We examined the membrane permeability by immunostaining and tested the interfering efficiency by co-immunoprecipitation endogenously and exogenously. 1.5The long term effect of BDNF on LIMK1We detected the LIMK1level after5-20h BDNF treatment. To determine whether BDNF has an influence on LIMK1stability, we analyzed the degradation rate of the LIMK1protein in primary cultured hippocampal neurons pretreated with cycloheximide (CHX).1.6Effect of Tat-JMBox4on BDNF induced axonal elongationTo identify the importance of LIMK1in BDNF induced axonal elongation, we tested the effect of BDNF on axonal length of the cultured neurons that were transfected with HA-LIMK1or LIMK1siRNA, respectively. Then we examined the effect of BDNF on axonal length of neurons when TrkB/LIMK1interplay was blocked by Tat-JMBox4. In addition, we detected the level of p-LIMK1, p-cofilin and F-actin in growth cone before and after BDNF treatment.2. The mechanism for LIMK1/cofilin signaling pathway regulating aversive memory extinction2.1Establish the experimental model and test cofilin activityWe established the CTA model and recorded the aversive index (AI). We collected different brain regions at vary times, and detected cofilin activity by Western Blot analysis. We tested phosphorylation of LIMK1and SSH1as well.2.2The behavior change after Tat-peptide treatmentWe used synthetic peptides to alter cofilin activity or inhibit GluA2synaptic insertion. Specifically Tat-Ser3can enhance cofilin activity, Tat-pSer3can weaken cofilin activity and Tat-pepR845A can block GluA2synaptic insertion. We examined the behavior change after microinjection of drugs.2.3Expression level of AMPARs in synapse and postsynaptic membraneWe isolated crude particulate fraction of synapse termed synaptoneurosome (SNS) from the IrL, part of SNS was surface biotinylated and subsequently precipitated with neutravidin-agarose conjugate. All subunits of AMPA receptors (AMPARs) and NR1subunit of NMDA receptor were detected by immunoblotting.2.4Morphological changes of synapseWe carried out Golgi staining to observe the structure changes including ratios of spine’s head to neck and spine density.Results1. The mechanism for LIMK1/cofilin signaling pathway regulating BDNF induced axonal elongation1.1LIMK1interacts with TrkB receptorLIMK1associates with TrkB receptor endogenously and exogenously. In addition, LIMK1co-localizes with the TrkB receptor in cultured hippocampal neurons.1.2Short term treatment of BDNF induces LIMK1phosphorylation, homo-dimerization and redistribution30min after BDNF treatment, phosphorylation (Thr-508) of LIMK1was increased in a TrkB tyrosine kinase activity-dependent manner. BDNF induced LIMK1homo-dimerization independent of TrkB receptor tyrosine kinase activity. Additionally, BDNF stimuli increased membrane-associated LIMK1and decreased cytosolic LIMK1.1.3JMBox4region in TrkB is responsible for TrkB/LIMK1interactionWe found that JMBox4region which is the9-amino acid sequence between JM3and JM4is necessary and sufficient for TrkB/LIMK1interaction.1.4The short term effect of BDNF on LIMK1is dependent on TrkB/LIMK1interaction The synthesized peptide (Tat-JMBox4) which could efficiently block TrkB/LIMK1interaction disrupted BDNF induced LIMK1phosphorylation, homo-dimerization and redistribution.1.5Long term treatment of BDNF stabilizes LIMK1proteinWe found that endogenous LIMK1was gradually degraded with a half-life of～20h when CHX was present. However, when treated with BDNF, the half-life of LIMK1protein was prolonged to>20h. The effect of BDNF on LIMK1degradation was also blocked by Tat-JMBox4.1.7TrkB/LIMK1interaction is required for BDNF induced axonal growthWe found that up-regulation of LIMK1levels increase axonal length whereas siRNA knocking down LIMK1levels decreased axonal length. Neurons over-expressing LIMK1displayed significantly longer axon in the presence of BDNF, whereas neurons transfected with LIMK1siRNA lost their responsiveness to BDNF. Moreover, we found that the BDNF induced axonal extension was blocked by Tat-JMBox4treatment.2. The mechanism for LIMK1/cofilin signaling pathway regulating aversive memory extinction2.1CTA consecutive extinction training enhances cofilin activity in the IrLWe observe that the rats exhibited a transient increase in cofilin phosphorylation at30min and120min after E1, peaking at30min and returning to baseline at240min. Hence, we selected the time point30min after extinction training for measuring the p-cofilin during CTA extinction. Immunoblotting analysis showed that the p-cofilin levels were markedly reduced after E3when compared with E1.2.2LIMK1and SSH1coordinate to regulate cofilin activity during extinction Since LIMK1is activated by its Thr508phosphorylation and SSH1is inactivated by its Ser978phosphorylation, we used specific antibodies to determine the activity changes of LIMK1and SSH1. We observed that extinction training decreased LIMK1activity whereas increased SSH1activity.2.3Cofilin activity is required for CTA extinctionCompared with Tat control, Tat-S3microinfusion immediately after extinction training significantly facilitated memory extinction, while Tat-pS3had the opposite effect. However, we observed that administration of Tat-Ser3or Tat-pSer3peptide into the IrL4h after E1-E3had no effect on memory extinction.2.4Memory extinction facilitates GluAl and GluA2synaptic trafficking in the IrLThe levels of GluAl and GluA2in the SNS fraction were notably increased, whereas the level of GluA3, GluA4and NR1were not significantly changed during memory extinction. In a crude homogenized IrL fraction, the expression levels of GluAl and GluA2were unchanged. We also observed an increase in the surface levels of GluA1and GluA2subunits but no other subunits extinction.2.5cofilin activity regulates GluAl and GluA2synaptic trafficking during memory extinction We found that Tat-Ser3infusion immediately after exposure to E1-E3significantly increased the surface levels of GluA1and GluA2. On the contrary, Tat-pSer3injection decreased not only synaptic but also postsynaptic surface levels of GluAl and GluA2during memory extinction.2.6Morphology of synapse is unchanged during memory extinctionNeither the three consecutive extinction training nor the additional regulation of cofilin activity by Tat-Ser3or Tat-pSer3could change the ratios of spine’s head to neck and spine density during memory extinction.2.7Inhibition of GluA2membrane insertion leads to memory extinction impairmentWe observed that microinjection of Tat-pepR845A which could inhibit GluA2-containing AMPA receptors synaptic membrane recruitment impaired memory extinction, even when we combined the microinfusion of Tat-pepR845A and Tat-Ser3.Conclusion1. Our study revealed that TrkB interacted with LIMK1via its JMBox4region. BDNF induced LIMK1dimerization and phosphorylation depended on TrkB/LIMKl interaction but not TrkB tyrosine kinase activity. Furthermore, we found that BDNF induced LIMK1dimerization and transactivation played an essential role in BDNF enhanced actin polymerization and axonal elongation. These findings provide insights into the mechanistic link between LIMK1regulated actin dynamic and BDNF induced axonal elongation.2. In this study, we found memory extinction induced a temporal activation of cofilin, which stimulated GluA1and GluA2translocation to synapse and recruitment to postsynaptic membrane. Manipulating cofilin activity could alter extinction process, which was mediated by AMPARs synaptic trafficking. Finally we showed extinction triggered modifications of synaptic physiology and spine morphology are independent processes. Understanding the actin dynamics regulated relationship between synaptic physiological function (number of synaptic receptors) and spine structure (spine size and density) is crucial to our comprehension of mechanism of memory process. Innovation1. The mechanism for LIMK1/cofilin signaling pathway regulating BDNF induced axonal elongation1.1We found LIMK1could associate with TrkB receptor for the first time.1.2Our research showed that BDNF has short term effect and long term effect on LIMK1differently.1.3This study revealed new mechanism for BDNF induced axonal elongation.2. The mechanism for LIMK1/cofilin signaling pathway regulating aversive memory extinction2.1Deeply explore the molecular mechanism for LIMK1/cofilin signaling pathway regulating memory extinction.2.2We found that synaptic physiology and spine morphology are independent processes.