Study On The Role Of Junction-mediating And Regulatory Protein In Learning And Memory And The Regulation Mechanism Of Brain Excitability In Mice

Backgroud:Junction-mediating and regulatory protein（JMY）is one of the newly discovered key transcriptional cofactors of p53.The important role of p53in the nervous system has been gradually discovered,involving brain development,epilepsy,brain tumor,Alzheimer’s disease and so on.Thus,we propose that JMY may have a potential role in the brain.It was found that JMY could regulate the growth of neurites through regulating actin nucleation in vitro.Up to now,studies about JMY has been focused on cells and embryos,but there are few studies on the expression and biological function of JMY in intact adult brain.Objective:To understand the distribution of JMY in the body and brain of mice,and to explore the effects of JMY on high-level brain functions such as learning and memory and the related regulation mechanism of neuronal excitability.We have carried out the following research work.Methods:In this study,we combined genetics,histomorphology,animal behavior,molecular biology and proteomics to carry out the research.Firstly,the expression of JMY in distinct nerve cell types,organs and different brain regions were observed by immunohistochemistry,RT-PCR and Westernblot,then Jmy conditional gene knockout mice,prepared by Cre-loxp system,examined by a series of behavioral experiments to analyze the biological function of JMY in the brain.Furthermore,we used pilocarpine-induced epilepsy model and electrical stimulation induced-kindling model to observe the phenotypic changes of Jmy conditional gene knockout mice and analyze the regulatory role of JMY in the brain hyperexcitability.finally,we explored the related molecular mechanism of JMY function in the brain through mass spectrum analysis and molecular biology experiments.Results:Firstly,we observed the expression of JMY in distinct types of cells in the brain.The results showed that JMY was expressed in neurons.We further detected the expression of JMY in adult mice.It was observed that JMY was expressed in brain,spinal cord and other organs and tissues in different degrees.We then examined the tissues of various brain regions and found that JMY was widely expressed in the whole brain,including olfactory bulb,cortex,hippocampus,thalamus,and cerebellum.These results imply a potential biological function of JMY in the brain.In order to further clarify the biological function of JMY in the brain,we generated Jmy conditional knockout mice,Nestin-Cre;Jmy^-/-by Cre-loxp system and then studied the role of JMY in brain function through a series of behavioral experiments,such as Morris water maze,Y maze,novel object recognition,three chamber test and so on.The results showed that after knocking out Jmy in the brain,the abilities of spatial learning and memory,spatial recognition memory and object recognition were impaired,and the social memory may also be impaired.In order to further study the brain region where JMY plays a role in learning and memory,we prepared Emx1-Cre;Jmy^-/-conditional knockout mice to knock out Jmy in the limbic lobe system,which is the key brain region of learning and memory.Using the behavioral experiments,we observed that the learning and memory ability of the mice with Jmy knockout in the limbic lobe system was impaired,which was similar to the whole brain knockout Jmy mice.Excitability is an important characteristic of the brain,and its balance is necessary for learning and memory.Brain hyperexcitability is always the cause of a variety of diseases.We used epilepsy model as an extreme paradigm of brain hyperexcitability to study the regulatory effect of JMY on brain hyperexcitability.We firstly used pilocarpine-induced model to cause sustained hyperexcitation of neurons in the brain,and found that the expression of JMY was temporarily up-regulated.We also observed a similar phenomenon in the electrical stimulation kindling model.Therefore,the expression of JMY changes during overactivation in the brain,suggesting that JMY may have a potential regulatory effect on brain excitability.Next,we used Nestin-Cre;Jmy^-/-mice to make electrical stimulation kindling model to quantitatively observe the role of JMY in the process of gradual increasing brain excitability.The results showed that after Jmy was knocked out in the brain,the seizure process induced by electrical stimulation was accelerated and the afterdischarge duration was prolonged.We further used Timm staining to detect mossy fiber sprouting in the electrical stimulation model.The results showed that the degree of mossy fiber sprouting in the brain increased after Jmy was knocked out.We further used Emx-Cre;Jmy^-/-mice to make electrical stimulation kindling model to observe the effect of JMY on the epileptiform activity and the corresponding pathological changes in the limbic lobe system.The results showed that the duration of afterdischarge induced by electrical stimulation was prolonged and the degree of mossy fibers sprouting in the brain was aggravated after Jmy knockout in the limbic lobe.In order to further explore the specific mechanism of the physiological function of JMY in the brain.Through mass spectrometry analysis,we screened33 proteins that were differentially expressed after Jmy knockout in the brain,of which 21 proteins were up-regulated and 12 proteins were down-regulated.The highest up-regulation multiple was TP53RK（2.5）and the highest down-regulation multiple of KCNS2（0.65）.We further analyzed the function of domains,GO functions and KEGG pathways,suggesting that the differential protein may be related to neuronal excitability.The genes encoding differential proteins were screened by comparing the changes of m RNA in the brain of Nestin-Cre;Jmy^-/-mice and the control group at basal level and 12 hours after Kindling stimulation.Conclusions:In the physiological state,JMY plays an important role in maintaining the learning and memory functions of adult mice,such as spatial learning ability,spatial reference memory,spatial working memory and object recognition memory,while JMY plays a negative role in the brain hyperexcitability induced by seizures.The regulation of neuronal excitability is the potential mechanism for the function of JMY in the brain of mice.Our research reveals the new biological function of JMY in the brain.This study expands the knowledge about the regulatory mechanisms of learning and memory.It also helps to improve the understanding of the internal molecular mechanism of brain hyperexcitability regulation,and promote the mechanism research and diagnosis and treatment strategy development of related diseases.