Investigation On Auditory Function And Hearing Loss Mechanism In Arhgef6 Knockout Mice

Hearing loss is the most common genetic disease in clinic.Genetic factor plays important role in the development of hearing loss.Many gene mutations have been found to associate with hearing loss.The research on gene knockout mouse models provides a useful tool for the research of hearing function and deafness mechanism.Arhgef6 is the X-linked mental retardation gene(MRX46).Clinical feature of patients carrying Arhgef6 mutation include mental retardation and sensorineural hearing loss in some cases.ARHGEF6 is a guanine nucleotide exchange factor(GEF)for Rho GTPases,which specifically activates Rho GTPases Racl and Cdc42.Rho GTPases act as molecular switches in many cellular processes.Their activities are regulated by binding or hydrolysis of GTP,which are facilitated by GEFs and GTPase-activating proteins(GAPs),respectively.Rho GTPases Racl and Cdc42 have been shown to play important roles in hair cell(HC)stereocilia development and hearing function.However,the role of ARHGEF6 in the inner ear development and hearing function have not been investigated yet.Here,we found ARHGEF6 is detected in mouse cochlear HCs,including the HC stereocilia,which indicate that ARHGEF6 may take part in the development and function of hair bundles.Then we established Arhgef6 knockout mice using the clustered regularly interspaced short palindromic repeat(CRISPR)-associated Cas9 nuclease(CRISPR/Cas9)genome editing technique.We showed that ARHGEF6 was indispensable for the maintenance of hair cell stereocilia.ARHGEF6 disruption in mice causes HC stereocilia deficits,which eventually leads to progressive HC loss and hearing loss.However,loss of ARHGEF6 did not affect the synapse density and spiral ganglion cell(SGN)numbers.We speculate that ARHGEF6 mainly influence hearing function through hair bundle morphogenesis and function and is dispensable for cochlear synapse and SGN development.At the molecular level,active Racl and Cdc42 were dramatically decreased in the Arhgef6 knockout mice,as well as some cytoskeleton protein in the downstream of Rac1/Cdc42 such as Pakl and Radixin,suggesting that ARHGEF6 disruption may reduce the level of active Racl and Cdc42 thus influence the development of actin cytoskeleton in stereocilia and ultimately leads of hair cell degeneration and hearing loss.