| Background and ObjectiveAuditory sense possesses great significance for animals' survival and reproduction,as it is one of the most important ways that animals used to collect environmental information.How auditory signal occurs and conducts is strictly and intricately regulated,the whole system need to coordinate,to transfer and to deliver well organized to handle the intact information flow.Auditory can simply be divided into three parts,outer middle and inner ear,according to its anatomia structure.Outer ear charges sound collection from outside environment at the beginning of auditory signal transmission process,where middle ear deliver sound wave to next stop through ossicles and eardrum,then inner ear transforms mechanical signals to electrical signals and transmits electrical signals to auditory cortex through afferent nerves.In inner ear,hair cells located on basilar memberane are identified as a place where mechanical signals to electrical signals transduction finished.Hair cells are highly differenciated sensor cells,in mammalian auditory system,hair cells are thought to be non-renewable,any damage or death of hair cells will lead to irreversible severe auditory dysfunction.So,hair cells are decisive for auditory system's function.The ability of hair cells to participate in Mechano-electrical transduction(MET),due to its specific structured,regular,F-actin based stereocilia grow on the apical.In mammals,stereocilia of hair cells located on auditory epithelial rangs as three rows with decreased height,seems like a staircase.Sound waves spread to inner ear and lead to endolymph's flow accompanied with stereocilia swing,and then the deflection of stereocilia caused open of MET channels located on the tips of stereocilia.Cations flow into cell body through opened channels,and electrical signals produced,informations coded by mechanical signals finished transition here,next the electrical signals spread to auditory cortex through afferent nerves,and hearing occurs.So,well arrangement of stereocilia structure is crucial for hair cells to play their role in MET process.Ankle links are some transient,lateral links arise near root of stereocilia during early developmental stage.These links disappear around 9 days postnatal when stereocilia are mature.There are several research indicate that mutations of ankle link proteins encoding genes can induce auditory dysfunction in mice,however,the detailed mechanisms are not fully settled until now.The recent four identified proteins which participate in ankle link formation are ADGRV1,PDZD7,WHRN and USH2A.In this sduty,we did an in-depth investigation about the function of ADGRV1 and PDZD7 during stereocilia development.ADGRV1(Adhesion G Protein-coupled Receptor V1)has been known well as GPR98?MASS1?VLGR1,it is the longest identified G Protein-coupled Receptor.Mutation of Adgrv1 gives rise to type ? Usher syndrome,characterized by clinical retinitis pigmentosa and sensorineural hearing loss.Usher syndrome is a kind of single gene mutation caused hereditary dissease,classify to the autosomal recessive inheritance,total 10 pathogenic genes have been reported including Adgrv1.In auditory sensor-hair cells,ADGRV1 is a crucial component of ankle links,it forms protein complex with PDZD7,WHRN and USH2A at ankle region,so-called ankle link complex,other known ADGRV1 protein partners including MY07A and Harmonin.ADGRV1 knock out mice show typical type ? Usher syndrome characteristics,MET current of Adgrv1 knock out hair cells decreased,hearing threshold lifted significantly.ADGRV1 also expressed in rod photoreceptor and cone photoreceptor,except participate in ankle link formation,by the way,mutation of Adgrv1 is also related to audiogenic epilepcy.ADGRV1 as the largest identified G protein coupled receptor,we already found its mutation can generate USH ? syndrome and audiogenic epilepcy,but we did not ever figure out could it can couple G protein to participate in cell signaling for a long while,not to mention its ligand and target genes.One reason responds for this situation is because full-length Adgrv1 gene is encoded by more than 19000 base pairs,such long size fragment is hard to be cloned or to do some other genic manipulation.Reseacher have ever cascaded several important domains of ADGRV1 to express a mini-VLGR1,and they found that mini-VLGR1 can activate adenylate cyclase to elevate intracellular cAMP level via coupled G?s or G?q,then PKA or PKC sequentially be activated.Our previous study indicated ADGRV1 can auto-hydrolyze via GPS(GPCR proteolysis site)located at extracellular near the transmembrane part,then it is separated into two parts,named V? and V?,comprised with extracellular part and transmembrane part including intracellular domain respectively.We found V? can inhibit adenylate cyclase's activity to decrease intracellular cAMP level via coupled G?i,this induced phosphorylation of CREB decreased.We construct V?-Y6236fsX1 expression vector to mimic pathogenic mutation Y6244fsX1 in human ADGRV1,tranfected cells with V?-Y6236fsX1 expression vector,we found Y6236fsX1 point mutation do not affect V? expression level or its subcellular location compared to wild type,but,what is noticeable,mutant V?'s ability to inhibit cAMP level is strengthened,intracellular cAMP decreased significantly compared to wild type Vp.Deeply investigation showed,PDZD7 can regulate V? coupling with G?i,however Y6236fsX1 point mutation abolished ADGRV1 C-terminus PBI(PDZ binding interface)motif,so PDZD7 can not bind to V?-Y6236fsX1,and V?-Y6236fsX1 resulting for intracellular cAMP decrease can not be rescued by PDZD7.This indicates PDZD7 can regulate ADGRV1's cell signaling activity.PDZD7 is classified to scaffold protein,its whole CDS coding 3 PDZ domains,a HNL domain and a PR region.Mutations of Pdzd7 can give rise to non-syndrome deafness,and it is supposed as an Usher syndrome modifier.What corresponding to this is PDZD7 can interact with all known USH? proteins(ADGRV 1,WHRN and USH2A),and forms hetero-tetra protein complex to participate in ankle links formation.It reminds us ankle link complex is very likely to play an important role in stereocilia developmental process,so,uncover ankle complex and its proteins'function can help us to well understand the detailed process of stereocilia development and maintainance.Among ankle link complex,WHRN and PDZD7 possess similar protein structure,it also has 3 PDZ domains,and WHRN encodes a short isoform in inner ear,which includes only former two PDZ domains.Full length WHRN participates in formation of ankle links,while short WHRN located at the tips of stereocilia.Deficiency of full length WHRN can result to height changes of sterecilia,and hearing loss,and vestibular function disorder.Same as WHRN,USH1C(Harmonin)also includes full legth and short isoforms,and they exhibit different functions in inner ear.PDZD7 as a homolog of WHRN and Harmonin,it also expresses the short isoform with only former two PDZ domains in inner ear,but it is unclear which isoform of PDZD7 contributes to ankle links formation.On the basis of these research and hypothesis,we tried to deeply investigate the mechanism of how ADGRV1 and PDZD7 regulate stereocilia development and hair cell's function.The specific problems are:1)What is the function of ADGRV1 C-terminus during stereocilia development?2)Does ADGRV1 have cell signaling abililty in hair cell via coupling G?i?3)What target genes regulated by ADGRV1 in hair cells are?4)Which isoform of PDZD7 contributes to ankle links formation?5)What the function of PDZD7 long isoform during stereocilia development?In this work,we combine auditory phenotypic analysis of Adgrv1/Y6236fsX1,Adgrvl/de17TM,Pdzd7?PDZ3/?PDZ3 mutant mice models and so on,and molecular or cellular methods to investigat ADGRV1's function during hair cell stereocilia development and tried to resolved the mechanism why ADGRV1's mutation caused profound deafness.Meanwhile,we indicated PDZD7 long isoform's function during hair cell stereocilia development.Even more,we screened PDZD7 binding proteins with yeast two hybrids system,and we analyzed several PDZD7 partners' function in inner ear try to uncover the detailed molecular mechanism that why PDZD7's mutation can induce deafness.All these explorations will help us understand the function of ADGRV1 and PDZD7 deeply,and explain the molecular mechanism of their mutation caused hearing loss,so we can provide more solid theoretical directions for clinical therapy of related deafness patients.Key scientific problems:1)Does ADGRV1 C-terminus is essential for stereocilia development?2)Does ADGRV1 have cell signaling abililty in hair cell via coupling Gai,and what target genes regulated by ADGRV1 in hair cells are?3)Which isoform of PDZD7 contributes to ankle links formation?4)How PDZD7 long isoform affects stereocilia development?5)What is the molecular mechanism of PDZD7 mutation caused deafness?Research programs and the resultsTo answer these questions,we first generated Adgrv1/Y6236fsX1 mutant mouse model by CRISPR/Cas9 technology,and we investigated how ADGRV1 regulate stereocilia development and hair cell function through not only comparison of auditory phenotype between Adgrv1/Y6236fsX1 mutant mouse and Adgrv1/del7TM knock out mouse,but we also combined with some molecular and cytology sdudy methods.ADGRV1 is an important component of ankle links,we detected whether C-terminus defective ADGRV1 can still mediate ankle links formation by immunostaining with antibody which recognizes ADGRV1 N-terminus.We do not find any positive fluorescence signals in 5 days postnatal inner ear whole mount staining,which reminds us Adgrv1/Y6236fsX1 mutation most probably affected ankle links formation or the stability of ankle link complex,so that mutant ADGRV1 can not located at ankle region.We examined earlier mouse,staining images show ADGRV1 accumulates at apical surface of basal hair cells from 1 day postnatal mouse's inner ear.This phenomenon indicates mutant ADGRV1 might still be transported to cell membrane,but it can not be anchored to ankle region.Section staining results also agree with the lack of ADGRV1 in stereocilia,but what noticeable is,ADGRV1 seems accumulate at synapse in del7TM knock out and Y6236fsX1 mutant mouse compared to wild type.Meanwhile,we examined other USH ? proteins' distribution in ADGRV1 defective mouse inner ear with specific antibodies,we found that PDZD7 diffuses to whole stereocilium,where WHRN and USH2A disappears from ankle region in ADGRV1 defective mouse.Auditory Brainstem Response(ABR)shows hearing shreshold is already lifted obviously in two weeks old Adgrv1/Y6236fsX1 mice,it is similar to Adgrvl/del7TM mice,which indicates Adgrv1/Y623 6fsX1 mice exhibit severe hearing loss from early stage.Outer hair cells can amplify sound signals actively,which can be detected by Distortion Product Otoacoustic Emission(DPOAE).We find,compared to control mice,DPOAE threshold of Adgrv1/Y6236fsX1 mice is significantly elevated,which means Adgrvl/Y6236fsX1 outer hair cells' function are impaired.Because integrity of stereocilia has crucial function for hearing maintaining,so we analyzed hair cell stereocilia morphology of Adgrv1 mutant mice by staining with phalloidin.The results show Adgrv1/Y6236fsX1 mouse's outer hair cells stereocilia have already obviousely disorganized in 4 days postnatal,the regular "V" shape disappeared,and the orientation of "W" shape changed.When mutant mice grew older,hair cell stereocilia disorganized more severe,and accompanied with stereocilia total degeneration of partial hair cells.Outer hair cells have already begins to loss in 1 month old ADGRV1 mutant mice,especially for basal turn of basilar membrane,according to DAPI staining counting.High resolution of SEM analysis indicates stereocilia became unsymmetrical in P8 mouse,and stereocilia arrange extremely irregularly.All these results told us ADGRV1 plays a key role in stereocilia development and maturity.C-terminus deficiency of ADGRV1 is likely to affect hair cell's function,so we tested 6236 point mutation mouse's hair cells' electrical physiological function.As expected,Adgrv1/Y6236fsX1 hair cells can only generate about 170 pA current at the point of maximal mechanical stimulus,while wild type hair cells can generate about 700 pA current,which illustrates 6236 point mutation caused decrease of MET channel's efficiency.As we demonstrated before that ADGRV 1 participate in cell signaling,so we analyzed gene expression level in Adgrv1/del7TM mouse inner ear using Microarray.We found some genes' expression levels are upregulated in Adgrv1/del7TM mouse.We verified several genes' expression levels in Adgrvl/Y6236fsXl mutant mouse's inner ear by quantitative real time PCR,the results showed target genes' expression level decrease in Adgrv1/Y6236fsX1 mouse's inner ear.In another way,we built the Hek293-VLGR1 a stable express cell line,and we examined expression level changes of those corresponding target genes in stable express cell line.Consistant with our finding,all examined target genes in over-expression cell line became downregulated.In addition,we cloned promoter regions of two target genes' to pGL3-Basic express vector,by detection of luciferase activity,we found ? subunit of ADGRV1 can indeed inhibit target genes' transcriptional level.To figure out which isoform of PDZD7 is more important for auditory system's function,we generate the mouse model PDZD7?PDZ3,which disturbs the third PDZ domain expression of PDZD7 but not affect short isoforms expression.We examined PDZD7 expression in PDZD7?PDZ3 mouse's inner ear by whole mount staining of basilar membrane with antibodies reconganized PDZD7 N-terminus or C-teminus respectively.Compared to control,none of these two antibodies detect positive signals in PDZD7?PDZ3/?PDZ3 mouse,which means only PDZD7 long isoform locate at ankle region.Meanwhile,we over expressed long or short isoform of PDZD7 by electrotransfection in cultured basilar membrane explants,like the result of antibody staining,only PDZD7 long isoform is distributed in stereocilia.ABR test shows severe hearing loss at different frequencies when PDZDG7?PDZ3 mice are only two weeks old,ABR threshold is elevated to 60dB,and mutant mice show progressive hearing loss,ABR threshold become to 80dB in elder PDZD7?PDZ3 mice,which means,PDZD7 is essential for normal hearing ability maintaining.Same as ADGRV1 deficient mouse,lack of PDZD7 long isoform in hair cells interrupts the normal distribution of other USH? proteins too.Immunofluorescence of PDZD7APDZ3/?PDZ3 inner ear shows ADGRV1 occurs at the tips of stereocilia,and WHRN and USH2A's expression level at ankle region are reduced visibly.We analyzed hair cell stereocilia morphology with both phalloidin staining and SEM,Which indicate PDZD7?PDZ3 mouse's stereocilia begin to be disorganized as early as 4.5 days postnatal,the stereocilia have lose their symmetrical "V" shape compared to 0.5 days postnatal.Irregular of stereocilia become more severe when they are mature,the orientation of "V" shape changes,even some hair cells disappear because of stereocilia degeneration,consistant with progressive hearing loss we observed in PDZD7?PDZ3/?PDZ3 mice,we found its outer hair cells lost become more severe as mouse grow older by staining of DAPI.Electrophysiological test shows the current amplitudes are obviously reduced in both inner and outer hair cells of PDZD7?PDZ3/?PDZ3 mouse,FM1-43fx staining results fit with this quite well,and it indicates that mature hair cells completely lose their MET function.The 4 identified proteins related to ankle links formation are ADGRV1,PDZD7,WHRN and USH2A,all mutations in their coding sequence can lead to different degree hearing loss,and affect ankle links formation.However,what other ankle proteins exist,except those famous 4?Then we started from PDZD7,and screened its binding proteins by yeast two hybrids,all PDZD7 partners we got include classic PBI motif at their C-termini.We did advanced research about three interested genes(Cadm1,Frmpd4,Pip5k1c).Protein coded by Cadm1,a homolog of deafness gene Nectin1,can interact with PDZD7 Nterminal,we examined Cadm1-/-mouse's hearing ability with ABR test,and we found neither in normal physiological state nor after noise stimulation,Cadm1-/mice kept their hearing shreshold at normal level.Detection with RT-PCR shows,all homologous genes of Cadm1 expressed in inner ear,and they might compensate its function when Cadm1is deleted.FRMPD4 can both interact with PDZD7 Cterminal and LGN,while LGN regulates hair cell planar polarity.Unfortunately,we found hair cells' planar polarity of FRMPD4 deficiency mouse is not affected,hair cell stereocilia develop normally,knock out mouse's hearing ability is normal.Another PDZD7 partner PIP5K1c can even result for high frequency hearing loss by heterozygous mutation of its coding sequence.We verified the interaction between PIP5K1c and PDZD7 by co-immunoprecipitation and cell colocalization test,further more,we found PIP5K1c specifically bind to PDZD7 long isoform.By over express PIP5K1c in hair cell through electrical transfection,we observed PIP5K1c distribute in stereocilia.This suggest that lack of PDZD7 long isoform affects stereocilia development and hearing maintaining might because disruption protein interaction between PDZD7 and PIP5K1c.Innovation and SignificanceWe used several gene deficient mouse models to deeply investigate how ADGRV1 and PDZD7 long isoform regulate stereocilia development and hair cell's function,and we screened PDZD7 binding proteins by yeast two hybrids system,we check out the partner PIP5K1c,which one might contribute to the reason of deafness caused by Pdzd7 gene mutation,through analysis of several PDZD7 binding proteins knock out mice's auditory phenotypes.We have the innovative research results and significance of the following four points:1)We generate the Adgrv1/Y6236fsX1 mutant mouse model with CRISPR/Cas9 technology to mimic human pathogenic mutation,and we demonstrate ADGRV1 Cterminal plays key role in ankle links formation and stability,lack of ADGRV1 Cterminal has serious impacts on stereocilia development.2)We first time confirmed the concept that ADGRV1 has cell signaling activity through coupling with G?i in inner ear by study on Adgrv1/Y6236fsX1 mutant mouse model,and we identified several candidate target genes.3)We built PDZD7 long isoform specific knock out mouse model,and we proved PDZD7 long isoform is essential for stereocilia development and MET function maintaining.4)Through yeast two hybrids screen,we get several new PDZD7 binding proteins,and we found PIP5K1c expressed on stereocilia,which provide a candidate constitute protein for ankle links. |
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