Effect Of Inner Ear Conditional Knockout Fgf13 On Auditory Function And Mechanisms In Mice

Deafness is known to occur in more than 400 syndromes,designated as syndromic hearing loss(SHL),which accounts for almost 30% of hereditary hearing loss.But the molecular mechanisms that underlie the pathogenesis of most SHL have not yet been determined.Previous studies have shown that deafness has been a common feature in patients with three main syndromes,the B(?)rjeson-Forssman-Lehmann syndrome(BFLS),Wildervanck syndrome(WS),and Congenital Generalized Hirsutism(CGH).The gene mapping analysis shows that all of them are characterized with loss-of-function mutations in the Fgf13 gene.Whether the pathogenesis of syndromic deafness in these syndromes is associated with the loss-of-function mutations of Fgf13 and what roles of the gene play in auditory function are unclear.We hypothesized that Fgf13 may play important roles in the auditory system,and its abnormalities in expression or function may be a novel molecular mechanism for deafness.To elucidate this hypothesis,we examined the expression and localization of Fgf13 in the murine cochlear tissue.Furthermore,a transgenic mouse line with a conditional knockout of the Fgf13 gene in the inner ear area(Fgf13 cKO)was generated.Using this knockout mouse line,we first investigated the effect of Fgf13 knockout on auditory function in mice at the whole animal level,and then the effect of Fgf13 knockout on the gross morphological structure changes of cochlea was detected.Finally,we further explored the mechanisms of deafness caused by knockout Fgf13 in mice.This study reveals a novel role for Fgf13 in auditory function,and indicates that the gene could be a candidate for understanding deafness.These findings may provide new perspectives on the molecular mechanisms and novel therapeutic targets for treatment deafness.Objective: To investigate the effect of inner ear conditional knockout of Fgf13 on auditory function and its mechanisms in mice.Methods:1.Immunofluorescence staining and real-time quantitative PCR(qRT-PCR)were used to study the expression and localization of FGF13 in mice cochlea.2.A mouse line with conditional knockout of Fgf13 in the inner ear was generated by loxP/Cre system,and the knockout efficiency was verified.3.Auditory brainstem response(ABR)and distortion product otoacoustic emission(DPOAE)techniques were used to study the effect of Fgf13 knockout on auditory function in mice.4.Immunohistochemistry was applied to study the effect of Fgf13 knockout on the morphological changes of mice cochlea.5.Immunofluorescence staining and qRT-PCR were used to further explore the mechanisms of the loss of spiral ganglion neurons in cKO mice.Results:1.FGF13 was located primarily in the organ of Corti,spiral ganglion neurons,stria vascularis and supporting cells.At the cellular level,FGF13 was expressed in the cytoplasm,membrane,and neurites of the SGNs.In hair cells,staining was also observed in the cytoplasm and membrane,with more prominent expression in the inner rather than the outer regions.We did not observe FGF13 expression in the nucleus of either the SGNs or HCs.The expression levels of FGF13 showed no significant changes between P0-P60 in the mice cochlea.2.We crossed Fgf13-loxP mice with Atoh1-cre mice to generated a mouse line with conditional knockout of Fgf13 in the inner ear by loxP/Cre recombinant technique,and realized the knockout of gene in the cochlear spiral ganglion neurons,hair cells and some supporting cells.The qRT-PCR results showed that the expression level of FGF13 in cochlea decreased?66.2%.The fluorescence intensity of the immunostaining was significantly reduced,indicating the efficient deletion of Fgf13 in mice.3.The homozygous knockout Fgf13 mice displayed significantly higher auditory thresholds in response to click stimulus and tone test across the 4-32 k Hz auditory spectrum in comparison with WT and Atoh1-cre control mice,with significantly lower amplitude and increased latency of wave I in the auditory brainstem response.There were no significant changes in the interpeak latency between wave II-IV.However,the heterozygous knockout Fgf13 mice showed a normal hearing threshold.In addition,Fgf13 knockout did not significantly affect the auditory threshold of distortion product otoacoustic emission in mice,suggesting a normal function of the outer hair cells in Fgf13 cKO mice.4.Fgf13 knockout significantly reduced the cell densities of type I spiral ganglion neurons marked by Tuj1 from the apex to the base of the cochlea,with more significant loss in the base area.Deletion of Fgf13 had no significant changes for the gross morphological structure of cochlea,including the organ of corti,stria vascularis,spiral limbus and tectorial membrane,but caused significant reduction of cell densities of whole spiral ganglion neurons.The whole-mount staining results showed that Fgf13 deficiency had no significant effects on the morphology and densities of both the inner and outer hair cells from the apex to base of the mice cochlea.5.Fgf13 knockout induced significant increase of the TUNEL-and cleaved caspase-3 positive SGNs from the apex to the base of cochlea,indicating caspase-dependent apoptosis mediated the loss of spiral ganglion neurons.Further detection of the expression levels of apoptotic related factors showed significant higher expression levels of pro-apoptotic factors,such as caspase-9,caspase-3,caspase-12,P53,cytochrome C and Bak,and decreased expression of anti-apoptotic factors Bcl-2 and Bcl-xl in Fgf13 cKO mice.There were no significant alterations in caspase-8,AIF,Bim and Bax among the three groups.The results analysis of expression and localization of cytochrome C in SGNs showed that in both WT and Atoh1-cre groups,cytochrome C was distributed uniformly as puncta in SGNs.In contrast,in Fgf13 cKO mice,there was an uneven distribution as well as plaque aggregation accompanied by an increased expression in the cytoplasm of the neurons.These data indicate that knockout of Fgf13 induced release of cytochrome C into the cytoplasm and hence,activated the mitochondrial apoptotic pathway in SGNs.Conclusion: FGF13 was expressed predominantly in the organ of Corti,SGNs,stria vascularis and supporting cells of cochlea tissue.Fgf13 conditional knockout in the inner ear caused sensorineural hearing loss.Fgf13 knockout significantly reduced the cell densities of spiral ganglion neurons,which was mediated by mitochondrial apoptotic pathway.