Panou, Iliana

English

The tryptophan-rich basic protein (WRB) has recently been proposed to act as the receptor of tail-anchored (TA) proteins. Together with the cytosolic protein TRC40 it presumably builds the main components of the posttranslational membrane insertion pathway in mammalian cells. A mutation in the wrb homologue gene in zebrafish causes visual, hearing, and balance deficits, as observed in the pinball-wizard (pwi) zebrafish mutants. In this study, I investigated the role of WRB in hearing by studying the synaptic transmission at the first auditory synapse, the cochlear inner hair cell (IHC) ribbon synapse in the Pwi conditional knockout mice. In this mouse model, the Wrb gene was targeted for excision through Cre-mediated homologous recombination in tissues that express the vesicular glutamate transporter-3. The findings indicated a progressive hearing loss accompanied by epileptic attacks and vestibular deficits. Further, the knockout animals did not survive beyond two months after birth. Patch-clamp recordings revealed a normal exocytosis of the readily-releasable pool of vesicles in IHCs, but diminished sustained
component of exocytosis, suggesting a defect in vesicle replenishment. These findings were further supported by ultrastuctural analysis of the knockout IHC ribbon synapses, where unusually large vesicle clusters were observed together with significantly fewer ribbon-associated vesicles. Otoferlin, a putative calcium sensor for synaptic vesicle fusion and/or priming factor at the IHC ribbon synapse, has recently been predicted to be a TA protein. In support of the hypothesis that the disrupted pathway of membrane insertion could affect the molecular components of the IHC synapse, otoferlin protein levels in IHCs of the Pwi knockout mice were decreased. Moreover, the expression of TRC40 was also strongly reduced. In conclusion, the IHC synaptic dysfunction probably contributes to the progressive hearing loss in the Pwi knockout mice. Although not yet quite understood, this synaptic defect might be attributed to deficits in certain IHC synaptic proteins, like otofelin that are disrupted upon deletion of the WRB protein. Furthermore, as a parallel study, a viral silencing approach was established, utilizing adeno-associated viruses for the delivery of miRNA-like silencing molecules in the developing otocyst. The results of the effort to knock-down the protein levels of otoferlin were suggestive of the success of the method, which nevertheless, needs further optimization

Georg-August-University Göttingen

The role of tryptophan-rich basic protein (WRB) in inner hair cell synaptic transmission and hearing

https://ediss.uni-goettingen.de/bitstream/11858/00-1735-0000-0022-5ED9-7/1/IlianaPanou_thesisX.pdf

The role of tryptophan-rich basic protein (WRB) in inner hair cell synaptic transmission and hearing

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Georg-August-University Göttingen