Functional Study Of Phospholipid Flippase Beta Subunit Tmem30a In Auditory System

BackgroundDeafness is the second largest disability disease after physical disability.About 466million people worldwide suffer from disabling hearing loss.Among them,sensorineural hearing loss caused by damage or defects of cochlear hair cells accounts for 90%.The mammalian auditory sensory organ is a Corti device,which consists of a special set of hair cells and supporting cells.The inner hair cells are essential for the production of hearing,and most of the hearing impairments are caused by hair cells and spiral ganglion cells.Apoptosis is caused by damage to hair cells that can cause permanent hearing damage.Tmem30a is the?subunit of most P4-ATPase which plays an important role in maintaining the asymmetric distribution of plasma membrane phospholipids,cell membrane structure stability,vesicle protein transport,cell polarity establishment and cell differentiation.Previous studies have shown that P4-ATPase plays an important role in the formation of vision and the maintenance of nervous system function.It has been reported that Atp8a2 and Atp8b1 knockout lead to increased hearing threshold in mice,but their damage and damage mechanisms to the auditory system are still lack of research,and the function of the auditory system in the?subunit Tmem30a,which interacts with it and plays a role in P4-ATPase function,is still unclear.PurposeThis study hopes to specifically knock out Tmem30a through the cochlea to explore its impact on the auditory system and the damage mechanism to the auditory system.MethodAfter hybridization of Gfi^Cre/-mice with Tmem30a^loxp/loxp mice,the progeny obtained by hybridization were bred to obtain Temm30a^loxp/loxpGfi^Cre/-inner ear-specific Tmem30a knockout mice.The knockout mice were screened by Genotyping and qPCR.The hearing of mice was detected by Auditory Brainstem Response.The localization of Tmem30a in the inner ear measured by immunohistochemistry.The morphology of hair cells was observed by cochlear patching method.The morphology and arrangement of stereociliary on hair cells were observed by scanning electron microscopy.Finally,the mechanism of apoptosis was discussed by immunohistochemistry.ResultThis study found that Tmem30a is distributed on inner ear hair cells.Auditory Brainstem Responseresults indicate that the specific knockout of Tmem30a gene in the cochlea leads to loss of hearing in mice.The results of hair cell plating showed that the hair cells of normal mice were arranged neatly,and the hair cells of the knockout mice were disorderly arranged and a large area of deficiency appeared.Scanning electron microscopy showed that the stereociliary on the hair cells of the knockout mice were disorderedandalargenumberofflocculentconnectionsappeared.Immunohistochemistry results showed that Chop staining of hair cells and spiral ganglion cells of knockout mice was positive,while no positive staining was found in normal mice.ConclisionKnockout of the Tmem30a gene results in loss of hearing,disordered distribution of hair cells and stereociliary,and even deficiency,which occurs through the endoplasmic reticulum stress pathway.This indicates that Tmem30a plays an important role in the production of hearing,the development of the auditory system,and the development and morphology of hair cells and cilia.