Study On The Mechanism Of Hearing Impairment In Connexin 30 Gene Knockout Mice

Hearing impairment(HI)is one of clinically common disabling diseases.According to the latest statistics from the World Health Organization,360 million people in the world are disabled due to hearing impairment,which accounts for 5.3%of the total population.The genetic causes of hereditary hearing impairment(HI)are very heterogeneous.Among of the deafness-related genes,mutations of GJB genes which encoding the connexins(Cxs)are one of the main reasons of non-syndromic hereditary hearing impairment.Among of these genes,the GJB2 and GJB6 gene mutations which encoding Cx26(Connexin 26,Cx26)and Cx30(Connexin 30,Cx30)respectibely are the most common in human non-syndromic hereditary hearing impairment.Hearing impairment affects children's learning,behavior and cognitive development,these will bring bad quality of life.hearing impairment of adults not only cause difficulties in their social interaction but also has ill life.So far,we do not have a complete and sophisticated system to prevent and cure hearing impairment.Hence,preventing the incidence of hearing impairment,especially reducing the incidence of hereditary hearing impairment,and further improve hearing impairment in deaf patients is the key to clinical treatment of hereditary sensory hearing impairment.Today,as we know,cochlear implants are the only treatment for hearing impairment.However,it is well-known that surgery is invasive and expensive treatment spending,and the difference in the language training performance of post-period of surgical patients and it still not the most ideal way to obstruct the incidence of hereditary hearing impairment.Therefore,the search for new ways of prevention and treatment to hearing impairment has become a problem that we urgently need to solve.How to do with that critical question,we need to study in more detail of the pathogenesis about the hereditary hearing impairment.From a basic view,exploring the best way to blocking the hearing impairment,especially the hereditary hearing impairment.Many scholars reported that Cxs mutations were responsible for the non-syndromic hereditary hearing impairment,but the details of the mechanism still unclearly.Therefore,in this study,we will use Cx30 knockout mice as subjects and perform protein grouping on their cochlear tissues.Researches in studies to make efforts to reveal its pathogenesis and explore the prevention strategies.And then,we will perform the results of proteomics for therapeutic efficacy,disease classification,individualized treatment,and disease pattern analysis.About 200 GJB2 mutations have been reported to cause hearing impairment.They can be classified into many different types: large deletions that remove the whole GJB2 gene,large deletions that remove regulatory sequences that are needed for the expression of GJB2 but keep the gene intact,and a plethora of small-scale alterations,including nonsense,missense and splice-site point mutations,as well as frameshifting small insertions and deletions.The frequencies of these mutations are diverse,with different mutant alleles overrepresented in different populations.Since the genetic epidemiology of Cx26 gene mutation has been widely studied,we will focus on the mechanism of non-syndromic hereditary hearing impairment caused by the abnormal Cx30 protein expression after Cx30 gene deletion.The inner ear is a very complex sensory organ which the development and function depend on the stable balanced interactions among diverse cell types.The support cells of inner ear play the essential roles in the physical and physiological support to the sensory hair cells and in maintaining cochlear homeostasis.The support cells in the cochlea communicate with each other through a series of gap junctions(GJs)which formed by Cxs.GJs are also involved in many biological activities in this organ.GJs can perform an important role to maintain the internal environment which the tissue cells living and the ion exchange between the neighbor cells rapidly.Therefore,the gap junction network,which consisted by Cxs,between the adult cochlear cells is thought to contribute to homeostasis in the cochlea environment.It has been reported that 30%-50% of the non-syndromic inherited deafness in all races are caused by the mutations in the genes GJB2 and GJB6 which encode Cx26 and Cx30.In Europe and the United States,the mutant genes of Cx26 and CX30 have been screened as one of the routine items for deafness gene screening in many medical institutions.As the reports,Cx30 is important to the function of the cochlea and the maintenance of hearing,but its exact role and detailed mechanism in the physiology and pathology of the mammalian cochlea is still unclear.At present,there are no prevention and treatment methods based on the cariogenic mechanism.In this study,we used Cx30 knockout mice as research subjects to study the pathophysiological role of Cx30 in the process of hearing development to find targets for the treatment and the prevention of deafness.After the mice born with Cx30 KO were observed,the cochlea's tissue cells began to show damage.Which is the first cells in the hair cells and supporting cells to be damaged? What is the order of damaged to outer hair cells and inner hair cells? Does the endolymphatic potential decrease or never develop? What is the reason for the ABR threshold to rising? Is the hair cell apoptosis or the EP decrease.Are there other unknown factors? To explore the molecular mechanism of hereditary deafness caused by mutations in the gap junctional Cx30 gene in the cochlea,we used the Cx30 KO mouse as the research subject.In part one,to investigate the effects of Cx30 gene mutations,we observed cochlear hair cell loss and OC morphological changes by whole-mount cochlear preparation and phalloidin-propidium iodide double staining at 10,18,30 and 60 days after birth.And to find the characteristics of CX30 gene mutation in affecting the development of mouse cochlea epithelial cells(including sensory epithelium cells and non-sensory epithelial cells).In part two,we examined the auditory brainstem response(ABR)and endocochlear potential(EP)of Cx30 KO mice and WT mice to observe the development of ABR and EP in the mice of both groups,and then talk about the mechanism.In part three,by observing the changes in proteomics of the cochlea of Cx30 KO mice compare with WT mice,we will investigate the characteristics of the related proteins in the cochlea of Cx30 knockout mice and further reveal the deafness mechanism of Cx30 KO mice.The successful implementation of this study will provide a strong scientific basis for the prognosis,disease classification,individualized treatment,and disease type of deafness,and provide a basis for the exploration of new effective methods for deafness prevention and treatment.Part I Pathological Changes of connexin30 knockout mice in the cochlea at different postnatal time pointsObjective:The connexin connexin 30 knockout mice were used as research model to nvestigate the effects of Cx30 gene mutations at different postnatal time points.Methods:Cochlear hair cell loss and support cells loss were observed by whole-mount cochlear preparation and phalloidin-propidium iodide double staining at 10,18,30 and 60 days after birth.And to find the characteristics of CX30 gene mutation how to affect the development of mouse cochlea epithelial cells(including sensory epithelium cells and non-sensory epithelial cells)(n=8).The expression of Cx30 in the mouse cochlea tissue was detected by Western Blot,and the cochlear basilar membranes,phalloidin(Pha)and propidium iodide(Propidium Iodide)staining were used.And the observation of growth and development of cochlear sensory epithelial cells and non-sensory epithelial cells were applied by laser confocal microscopy.Results:1.The hair cells of Cx30 KO mice began to die from the P10.Following the increase of age,the number of dead cells increased significantly.2.The earliest appearance of apoptosis in outer hair cells of Cx30 KO mice(at P10).3.With the increase of age,the apoptosis of outer hair cells was gradually aggravated,and the inner hair cells apoptosis occurred at the latest.4.The order of appearance of apoptosis in outer hair cells is from the inside to the outside,from the first layer to the second layer and the third layer.As the age increases,the hair cells turn from the basal to the top,and apoptosis gradually occurs.5.In addition to hair cells in inner ear cells,non-sensory epithelial cells also appeared necrotic and detached,but the apoptosis time was later than that of hair cells,that is,gradual apoptosis appeared from sensory cells to non-sensory epithelial cells.Conclusion:Damage to the cochlear cells of Cx30 KO mice began at P10 and the hair cell death gradually increased with time.The order of cell death is : from outer hair cells to inner hair cells;from the base to the top;from sensory cells to non-sensory epithelial cells.Outer hair cells are from the first row to the second and third rows.PART II Changes of ABR and EP in Connexin30 Knockout MiceObjective: To detect the ABR and EP of Cx30 KO mice and WT mice with development process in defferent age respectively.To compare the changes of ABR and EP in Cx30 KO mice and WT mice.The effect of Cx30 on the development of ABR and EP and its mechanism were further discussed.Methods: The ABR and EP of 4KHz,8KHz,16 KHz and 32 KHz were detected in mice of Cx30 ko Group and WT Group(n=10)after birth l0 days(P10),18 days(P18),30 days(P30)and 60 days(P60).To analyze the effect of Cx30 on ABR and EP in mice.Results: 1.ABR results showed that the Hearing impairment of Cx30 ko mice was characterized by sensorineural hearing and almost no development(severe deafness).2.The hearing impairment of Cx30 ko mice is also gradually increased with the trend of time,until all deaf.3.The EP of Cx30 KO mice's inner ear was only 0-5mv at birth,obviously decreased compared with the normal control group,almost did not develop.The EP of WT mice's inner ear had reached the level of normal adult mice in P10 day.Conclusion: The EP of Cx30 KO mice were not produced,and the ABR increased significantly,which showed severe sensorineural deafness.PART 3 Proteomic study of cochlear tissue of Connexin30 gene knockout miceObjective: The changes of Cx30 in cochlear tissue in mice with different developmental stages were studied by using gap junction protein Cx30 gene knockout mice,and the mechanism of deafness in Cx30 ko mice was further discussed.Methods: To acquire the cochlear tissue of WT mice and Cx30 ko mice on 18 days after birth(P18).The differential expression of protein in cochlear tissue of mice was detected by Itraq quantitative mass spectrometry.To acquire the cochlear tissue of mice(n=4)on 10 days after birth(P10),18 days(P18),30 days(P30)and 60 days(P60),To detect the ROS levels in cochlear tissue after birth using H2 DCFDA fluorescence staining method(n=4).The expression of proteins related to redox reaction,ATP synthesis,K + Cycle Channel and EP formation in cochlear tissue was observed by Western blot(n=6).Results: 1.Quantitative mass spectrometry analysis of Itraq protein: Compared with WT mice,there were 1851 differentially expressed proteins in the cochlear tissue of Cx30 ko mice,There were 444 protein expression levels exceeding 1.5 times times,of which 204 species(45.9%)expressed increased;240 species(54.1%)protein expression decreased.It involves phosphate transport,oxidative stress,amino acid metabolism,hormone metabolism,cell cycle stagnation,actin system and other signal transduction pathways.2.Itraq quantitative mass spectrometry revealed high protein expression associated with oxidative stress in cochlear tissue of cx30 ko mice,H2 DCFDA fluorescence staining test showed that the overall level of ROS in cochlear tissue of Cx30 KO mice was no difference between WT group on P10,and higher than WT group mice on P18.Western Blot showed the expression of protein GPx1 and Prx2 in cochlear tissue: There was no obvious difference in 2 groups of mice on P10 days,and significantly enhanced in Cx30 ko group and WT mice Group on P18 and P60 days.The expression of mice in Cx30 ko group was slightly higher than that in WT group on P30 days.3.Itraq quantitative mass spectrometry revealed that the expression of key proteins(NDUFS3,NDUFV2,NDUFC2)in the mitochondria complexes of Cx30 ko mouse cochlea was reduced.Western Blot shows the expression of protein NDUFS3 in cochlear tissue: P10 Day,Cx30 ko Group is higher than WT group,there were no obvious difference in 2 group expression on P18 Day.Cx30 ko Group was significantly lower than WT group on P30 and P60.4.Itraq quantitative mass spectrometry showed that the expression of protein ATP1B2,ATP1A3,ATP4 A,ATP12A and SLC12A2 which were associated with transport of potassium ions in Cx30 ko mouse cochlea was reduced.Western Blot shows the expression of proteins necessary for EP formation in the cochlear tissue of Cx30 ko mice: The expression of KCNQ1,ATP1A1,SLC12A2,KCNJ10,SLC26A4 were significantly lower than those of WT group with corresponding time points(P10,P18,P30,P60).Conclusion: The absence of Cx30 affects the normal function of GJs which in the supporting cells of mice cochlear basaliar membrane.It block the recycle of ATP in the cochlea from support cells to hair cells which resulting in the loss of hair cell energy and hair cell damage.ATP decreasing can harm mitochondria,ROS accumulate,and the hair cell can be damaged by oxidative damage.ATP decreasing can aslo block the K + recycle and impaire EP formation.As the accumulation of K + in endolymphatic fluid and potassium poisoning in hair cells.