Priliminary Study Of The Role Of CDK5 In The Mouse Auditory System

Background and objectiveThe auditory system is an important peripheral sensory systel and is an important source of information for animals to obtain outside information.Auditory production and conduction process is an extremely fine and complex process,it needs a variety of signal conversion and transmission and the process requires a large number of proteins and molecular coordination.In the auditory system,inner ear hair cells are sensory cells that transform the external sound stimuli into neural signals.In the cochlea,the inner ear hair cells can sense the external environment of the acoustic stimulation,and they are converted into auditory signals transmitted into the brain.In mammals,hair cells are considered to be non-regenerable,and any loss of hair cells can lead to permanent damage to the hearing function,the hair cells play an extremely important role in the auditory system.The most important structural feature of the inner hair cells is the hair bundles present in the top of the hair cells.In malnals,the hair bundles of the outer hair cells are mainly composed of three rows of staggered stereocilia.Lymphatic fluid flow.resulting in the hair cell stereocilia swing.and stereocilia detlection xvill cause the mechanical electrical conduction channel opening.leading to extracellular cation iiitlux.which will be outside the mechanical signal into electrical signals,and the electrical signals transmitted into the brain,so that we produce hearing.Therefore,the integrity of the stereocilia on the maintenance of hair cell function plays a vital role.Cell-cycle-dependent kinase 5(CDK5)is an important proline-dependent serine/threonine protein kinase,which is highly expressed in postlitotic cells,such as neuronal.In addition,CDK5 plays an important role in many cellular physiological processes,such as cytoskeleton regulation,signal transduction,gene expression and cell activity.At present,many studies have shown that CDK5 activity regulation is the main cause of many neurodegenerative diseases,such as Alzheimer's disease,amyotrophic lateral sclerosis,Parkinson's syndrome and so on.So far,CDK5 has been studied more deeply in the neural field.Although the auditory system 1s also important in the peripheral nervous system,CDK5 is also highly expressed in the inner ear,the function of CDK5 in inner ear development has not been studied yet.In previous studies,we found that CDK5 knockout mice have a serious defect in the cortical lamellar cell structure due to the extensive expression of CDK5 and its important biological functions in the neurological field,leading to perinatal death in mice,which greatly hindered the biological function of CDK5 in vivo.In recent years,in order to better study the biological function of CDK5 in vivo,many conditional knockout mice have been used,but for CDK5 in the mouse inner ear function remains to be improved.In this study,CDK5 was specifically knocked out in the inner ear using the inner ear-specific knockout tool,and the function of CDK5 in inner ear development was studied in detail by observing the hearing of the knockout mice and the observation of hair cell morphology.In addition,although CDK5 and other members of the traditional CDK in the structure of a high degree of homology,but studies have shown that it is not involved in the regulation of cell cycle.In addition,CDK and other members of the family is significantly different.CDK5 has its own specific activator,common and has been proved p35,p39 and CCNI.However,our previous study found that p35,p39 is mainly expressed in nerve tissue,the expression in the inner ear is very low,so we speculated that CDK5 may exist in the inner ear of the other activating factor?therefore,in order to find CDK5 interacting proteins in the inner ear.Cyclin1(CCNI)and cyclin 12(CCNI2),two CDK5-interacting proteins were obtained from the inner ear library by yeast two-hybrid system in a previous study.Co-immunoprecipitation and co-localization of the cells were used to verify the interaction between the two.Although CCNI2 is a homologous protein with CCNI,the function of CCNI2 has not been studied so far.Therefore,the function of CCNI2 and its influence on the activity of CDK5 kinase are to a great extent still unknown,and the function of CCNI has been proved to be an activator of CDK5.In this study,we examined the effect of CCNI2 on the kinase activity of CDK5 and examined its effect on cell cycle and cell proliferation,which will provide a better insight into the role of CDK5 in regulating the development of the inner ear.Scientific questions to be solvedIn this paper,we need to solve the scientific problems are the following:1)The role of CDK5 in hair cell development of mouse inner ear.2)CDK5 regulates the molecular mechanism of inner ear hair cell development3)Regulating of CDK5 kinase activity by CCNI2.4)CCNI2 effects on cell cycle and cell proliferationResearch programs and the resultsIn order to solve the above scientific problems,we first mate Cdk5LoxP/Loxp mice with the inner ear-specific Cre mice to obtain Atohl-Cren-mediated CDK5-specific knockout mouse lodel Atohl-CDK5 conditioned knockout mice,and the biological function of CDK5 protein in mouse auditory system was systematically studied by this mouse model.In the study.ABR results showed that the hearing threshold appears to increase of Atol1-CDK5 knockout mice at the age of one month,and progressive hearing loss occurs with age.Because DPOAE is mainly to detect the a1plillcation of outer hair cells.the threshold of DPOAE in knockout mice was significantly higher than in wild-type mice,so we hypothesized that knockout of CDK5 may affect hair cell function.We tested the MET function of mouse hair cell stereocillia bv FM1-43fx dye uptake assay.The results showed that knockout of CDK5 in mouse hair cells of p8 appeared on the FM I-43fx dye intake anomalies,and with the increase of age has increased the trend.In order to more clearly observe the morphology of the stereocillia,we observed by phalloidin staining and SEM on the hair cells,the results show that knockout hair cells from the stereocillia p8 began to appear disorder,p14 appeared on the hair cell loss,with time,the knockout hair cell depletion gradually increased,and the hair cells in the basal membrane of the knockout mice were almost completely lost at 12month.In view of the time difference between the hair cell dysplasia and the hair cell loss,we think that CDK5 gene knockout may cause the hair cells to be lost due to the disorder of the stereocillia,leading to the abnormal phenotype of the louse hearing.The main actin connexins are ERM,fimbrin,villin,FASCIN2 and so on,which have a significant effect on the structure maintenance and stability of the mouse hair cells stereocillia.Their abnormality may lead to the abnormal assembly of the mouse hair cells with stereocillia,which may cause the hearing loss of mice.ERK is the major phosphorylated substrate of CDK5.In our study,ERM protein phosphorylation was significantly decreased in knockout mice,suggesting that ERM may be an important reason for the failure of assembly of stereocillia actin in knockout mouse.Extracellular signal-regulated kinase(ERK)is a member of the mitogen-activated protein kinase(MAPK)family and is one of the major phosphorylated substrates of CDK5.A variety of extracellular stimuli can cause ERK protein phosphorylation,the activation of ERK protein activity,and ERK activity of abnormal cells will affect the normal physiological processes,such as cell survival,protein synthesis,cell proliferation,growth,migration and apoptosis.Our results suggest that ERK protein phosphorylation in the inner ear of CDK5 knockout mice is significantly reduced,suggesting that this may be the main reason for loss of hair cells in mice.In order to find the pathways of hair cells loss,we used Caspase3 antibody to detect the apoptosis of hair cells in the inner ear of mice by the immunohistochemistry of the inner ear basement membrane.The results showed that there was no detection ot any apoptotic signal,suggesting that the absence of hair cells may not be achieved through the apoptotic pathway.Subsequently,we used Atol1-CDK5 mice to mate with GFP-LC3 transgenic mice to express GFP-LC3 transgenic Atoh1-CDK5 homozygous nice.In the mouse inner ear basement membrane,we observed GFP-LC3 aggregation,so we initially speculated that the cell autophagy pathway may play an important role in knockout mouse hair cells loss in the inner ear.In order to further clarify the regulation mechanism of CDK5 in the inner ear,we found a new role of CDK5-CCNI2 by yeast two-hybrid method,In order to better prove the activation of CDK5 by CCNI2,In addition,the phosphorylation level of CDK5-specific phosphorylated substrate PAK1 was detected by IP kinase assay.The results showed that CCN12 could activate the kinase activity of CDK5.It is a new activator of CDK5.And the activation of CDK5 by CCNI2 is stronger than CCNI on CDK5 activation,but weaker than that of p35 on CDK5.BrdU intake and MTT results showed that interfering RNA using CCNI2 in the cells slowed down cells proliferation level.We examined the change of cell cycle after CCNI2 knockdown by flow cytometry.The results showed that the number of cells in G1 phase after CCNI2 knockdown was significantly increased,the number of cells in S phase was significantly decreased,and the effect of CCNI2 on cell cycle was depending on the presence of the CDK5 protein.In summary,CCNI2 can activate CDK5 kinase activity,participate in cell proliferation and cell cycle regulation,and this regulation depends on the presence of CDK5 protein.Innovation and significanceIn this paper,Inner ear specific knockout CDK5 mice as the object of study,we have explored the regulation of CDK5 protein in the development of louse inner ear.We have the innovative research results and significance of the following two points:1.Inner ear specific knockout CDK5 mice as the study object.found that knockout mice hair cell stereocillia disorder or even loss,hair cell death,hearing abnormalities.This fully shows that CDK5 plays an important role in the development and maintenance of hair cells during ihe development of mouse inner ear and the survival of hair cells.2.It is proved that CCNI2 is a new activator of CDK5.which can regulate cell proliferation and cell cycle,and this regulation process depends on the role of CDK5 protein.This finding provides important information for our in-depth understanding of the CDK5 regulatory mechanism.