JIP3 Regulates Hippocampus-dependent Learning And Memory Of Adult Mice

BackgroundMitogen-activated protein kinase(MAPK)-c-Jun N-terminal kinase(JNK)signal pathway,plays a pivotal role in cell growth,cell differentiation,cell apoptosis and so on.JNK-interacting protein family(JIPs)have four family,including JIP1,JIP2,JIP3 and JIP4.All the JIPs contain the same highly conservative JNK binding domain,which promoting the activation of JNK signaling pathway.JIP3 was first discovered as JNK3's interacting protein.Further studies have illustrated that JIP3 interacts with JNK1 or JNK2,however,the interaction of JIP3 and JNK3 was stronger.The relation of JIP3 and JNK3 was very tightness,because JIP3 as JNK signal pathway scaffold protein to promoting JNK activation and JIP3 as JNK substrate was phosphorylated by JNK.In the other hand,previous studies have found that either of the JIP3's ortholog UNC-16 in Caenorhabditis elegans or Sunday driver in Drosophila can regulate cargoes axon transport as an adaptor protein.In mammal,JIP3 can regulates cargoes axonal transport alone or cooperate with JIP1.Above all have revealed that JIP3's two mainly functions in cell level,which is JIP3 regulates JNK signal pathway activation and cargo trafficking.Then what function will JIP3 plays in animal tissue level?First of all,recent studies have illustrated that JIP3 has important roles in individual development.For example,JIP3 can be detected in embryo 9th day and until has significant increase expression in embryo 13 days.This phenomenon reveals that JIP3 may be participate in neuronal development.In order to injuring the expression of JIP3,Kelkar etc.knock out the NO.1 exon of JIP3 by homologous recombination method in 2003.The results found that JIP3-/-mice was died in the early postnatal days with lacking of the telencephalic commissure and the random distribution of neurons.This is another evidence for the important roles of JIP3 in the development of mice again.In order to reduce the damage of knock out JIP3,Tronche ect.knock out JIP3 in central nervous system.They found that knock out JIP3 in central nervous system caused mice postal death shortly and lack telencephalic commissure by crossing Nestin-cre and JIP3 flox/flox mice in 2007.Above studies all illustrated that JIP3 plays important roles in mice development,especially for brain development.More interesting,studies have proved that the expression of JIP3 in brain is more than other organs in adult mice,which prompt that JIP3 may play important roles in the brain of adult mice.However,the above attempt about studying JIP3 knockout mice are failure,leading to there is no progress in studying the function of JIP3 in adult mice.For revealing the function of JIP3 in adult mice,we will have more comprehensive understanding the roles of JIP3.Brain is a sophisticated tissue,which is compose of several functional brain regions.These brain regions have different functional roles,but they restrict mutually influence each other and regulate brain function collectively.Hippocampus,a key brain region,plays important roles in cognition,learning and memory and emotion.In anatomy levels,hippocampus could divide into CA1,CA3 and DG,which could form a neural circuit.In our study,we found JIP3 mainly expressed in hippocampus and specifically focused on the part of synapse formation of CA1 and DG.In more detail,JIP3 widely expressed in both excitatory neurons and inhibitory neuron.These results suggest that JIP3 has relation with the function of hippocampus.Furthermore,we detected the expression of JIP3 after CFC and MWM,which all showed that the expression of JIP3 was increased in dorsal hippocampus,but no change in ventral hippocampus.The phenomena declared that JIP3 participates in hippocampus-dependent learning and memory.In order to illustrate JIP3 is necessary for hippocampus-dependent learning and memory,we knock down the expression of JIP3 by intracerebral microinjecting lenti-siJIP3 into dorsal hippocampus.We found that the CFC and spatial memory of mice are both deficit,which suggest that JIP3 is necessary for hippocampus-dependent learning and memory.Due to the trauma and poor precision of lentivirus injection,we hybridize the JIP3 conditional knockdown mice with Cre mice to confirm that JIP3 plays important roles in hippocampus-dependent learning and memory.Object:To investigate whether JIP3 participates in hippocampus-dependent learning and memory of adult mice.Methods:1.Protein extraction and Western blotDifferent brain tissue samples were isolated and the samples were homogenized.The protein samples were detected by western blot.The immunoreactive bands were analyzed by Quantity One.2.ImmunohistochemistryIn order to exam which region is JIP3 expressed,the adult mice were anesthetized deeply by 5%chloral hydrate.The mice were washed by and normal saline and then fixed by 4%paraformaldehyde.After the brains of mice were sliced by freezing microtome,the brain sections were used for immunohistochemistry.The concentration of JIP3 antibody was 1:500.In the end,the photographs were obtained by fluorescence microscope.3.Immunocytochemical stainingIn order to study JIP3 in which kind of neurons,we cultured hippocampus neurons in vitro.After cultured 7 days,the neurons were stained JIP3(1:200),CaMK?(1:200),GAD67(1:200).In the end,the photographs were obtained by fluorescence microscope.4.Stereotaxic location and lentivirus microinjectionAfter the mice were anesthetized deeply by 5%chloral hydrate,fixed in the stereotaxic apparatus.Lentivirus were injected in the dorsal hippocampus and the injective location was as follows:anteroposterior,-1.82 mm;lateral,±1.5 mm;dorsoventral,-2.5 mm.5.Behavioral tests5.1 Open field testOpen field test was the classical paradigm to exam the spontaneous activity and anxiety activity.The mice were putted in the center of square and recorded by Smart for 10 minutes.We based on the total movement distance to evaluate the spontaneous activity of mice and based on the ratio of spending time in center square to evaluate the anxiety activity.5.2 Contextual fear conditioning(CFC)The CFC was used to exam whether lentivirus of siJIP3 affect the capacity of fear memory.After the mice were received footshock 24 hours,then were placed back to same environment but no shock and recorded the freezing time,which is used for examining the capacity of fear memory.5.3 Morris water maze testThe mice were placed in the circular tank,which contains a platform and given 4 trials per days.After 5 days training,we removed the platform to exam the spatial learning and memory of the mice.We recorded the time spent in the target quadrant and the number of crossing the target quadrant by a video tracking system.6.Producing and identification of JIP3 conditional knockdown miceWe made the sequence of siJIP3 into loxp system according to the literature method and the molecular cloning technique.Then the conditional knockdown JIP3 mice were made by animal center of Tongji University in shanghai.After the mice birth,we first examined whether the exogenetic sequence was inserted into the genome by PCR.In the next step,the conditional mice crossed with Emxl-cre mice and the brain tissues of positive filial generation were examined the expression of JIP3 by western blot.In addition,we will analyze the change of morphology of conditional mice by Nissl's staining and brain volume.Results:1?The expression of JIP3 was abundant in the excitatory and inhibitory neurons in CA3 and DG of hippocampus.In order to investigate the function of JIP3 in the brain of adult mice,first of all,we extracted protein samples of some important brain regions,which include cerebral cortex,hippocampus,thalamus,cerebellum and hypothalamus.Then the samples were examined the expression of JIP3 by western blot.The results illustrated that JIP3 was expressed in these regions,but the expression in hippocampus was more than other regions.In the next step,we exam the expression of JIP3 in different subregions by immunohistochemistry.The results shown that JIP3 mainly focused on synapse formation of CA3 and DG.In the end,we examined JIP3 expressed in CaMKII positive excitatory neurons or GAD67 positive inhibitory neurons by immunocychemical staining.The results had illustrated that JIP3 expressed in the excitatory and inhibitory neurons of CA3 and DG regions in hippocampus,which suggested that JIP3 has relations with hippocampus-dependent learning and memory.2.JIP3 may participate in fear memory and spatial memory in dorsal hippocampus.In order to exam whether JIP3 participates in hippocampus-dependent activity programs,we first examined the expression of JIP3 after the mice were trained CFC.The results showed that the expression of JIP3 in DH was increased,but JIP3 in VH was no change,which suggested that JIP3 may participate in hippocampus-dependent fear memory.On the basis,we verified these phenomenons by another hippocampus-dependent learning and memory---MWM.The result found that JIP3 in DH has significantly increasement after training MWM 5 days,which suggested that JIP3 participates in hippocampus-dependent learning and memory.3?JIP3 is necessary for hippocampus-dependent learning and memoryIn order to exam JIP3 is necessary for hippocampus-dependent learning and memory,we injected the siJIP3 lentivirus into the DH of mice to exam what did JIP3 knockdown affect to learning and memory.After producing the lentivirus of siJIP3,we injected the lentivirus into CA3 and DG of dorsal hippocampus,which is JIP3 expression enrich region.After two weeks,we examined the activity of these mice.First of all,we examined whether siJIP3 affect the spontaneous activity and anxiety activity by Open Field Test.The results illustrated that the lentivirus of siJIP3 did not affect the spontaneous activity and anxiety activity of mice.On the basis,we examined whether siJIP3 affects learning and memory of mice by CFC and MWM.The results indicated that knockdown JIP3 in CA3 and DG could damage the fear and spatial learning and memory,which suggested that JIP3 is necessary for hippocampus-dependent learning and memory.4?Production and identification of the conditional knockdown JIP3loxp/-miceIt is hinder to study the function of JIP3 in adult mice,due to JIP3 knockout mice,which include knockout in whole and in local region,were all death after birth.Therefore,we took the measure that knockdown JIP3 in specific brain regions to get the adult mice of knockdown JIP3,which will provide a hope to study the function of JIP3 in adult period.Conclusion:1.JIP3 expresses in excitatory and inhibitory neurons of CA3 and DG widely.2.JIP3 participates in hippocampus-dependent learning and memory.3.JIP3 is necessary for hippocampus-dependent learning and memory.Innovation:1.We firstly illustrated that JIP3 participates in hippocampus-dependent learning and memory of adult mice2.We firstly got the normal development adult mice of conditional knockdown JIP3,which will provide a hope to study the function of JIP3 in adult period.