L-type voltage-gated calcium channel agonists mitigate hearing loss and modify ribbon synapse morphology in the zebrafish model of Usher syndrome type 1.

Alaa Koleilat,Stephen C Ekker,Joseph A Dugdale,Mark A Masino,Jeffrey L Bellah,Aaron M Lambert,Lisa A Schimmenti,Trace A Christenson

doi:10.1242/dmm.043885

Abstract

ABSTRACTThe mariner (myo7aa−/−) mutant is a zebrafish model for Usher syndrome type 1 (USH1). To further characterize hair cell synaptic elements in myo7aa−/− mutants, we focused on the ribbon synapse and evaluated ultrastructure, number and distribution of immunolabeled ribbons, and postsynaptic densities. By transmission electron microscopy, we determined that myo7aa−/− zebrafish have fewer glutamatergic vesicles tethered to ribbon synapses, yet maintain a comparable ribbon area. In myo7aa−/− hair cells, immunolocalization of Ctbp2 showed fewer ribbon-containing cells in total and an altered distribution of Ctbp2 puncta compared to wild-type hair cells. myo7aa−/− mutants have fewer postsynaptic densities – as assessed by MAGUK immunolabeling – compared to wild-type zebrafish. We quantified the circular swimming behavior of myo7aa−/− mutant fish and measured a greater turning angle (absolute smooth orientation). It has previously been shown that L-type voltage-gated calcium channels are necessary for ribbon localization and occurrence of postsynaptic density; thus, we hypothesized and observed that L-type voltage-gated calcium channel agonists change behavioral and synaptic phenotypes in myo7aa−/− mutants in a drug-specific manner. Our results indicate that treatment with L-type voltage-gated calcium channel agonists alter hair cell synaptic elements and improve behavioral phenotypes of myo7aa−/− mutants. Our data support that L-type voltage-gated calcium channel agonists induce morphological changes at the ribbon synapse – in both the number of tethered vesicles and regarding the distribution of Ctbp2 puncta – shift swimming behavior and improve acoustic startle response.

Highlights

Up to 11% of deaf/hard of hearing children and 1 in 6000 people in the United States have Usher syndrome (USH) (Kimberling et al, 2010)
The mean number of tethered vesicles for wild type is 19±1 vesicles (n=32 ribbons, six zebrafish), which is statistically different from the mean for the myo7aa−/− mutants 15±1 vesicles (n=34 ribbons, five zebrafish) (Fig. 2H, t-test, P
We investigated the distribution of Ctbp2 puncta and identified that most hair cells from the myo7aa−/− mutant neuromasts had two puncta per cell and most wild-type hair cells had three puncta per cell (Fig. 3E)

Summary

Introduction

Up to 11% of deaf/hard of hearing children and 1 in 6000 people in the United States have Usher syndrome (USH) (Kimberling et al, 2010). USH is characterized by congenital onset of sensorineural deafness/ hard of hearing and the later onset of retinitis pigmentosa, resulting in significant vision impairment (Usher, 1914). There are three types of USH, characterized by the degree of hearing loss, vestibular abnormalities and onset of vision loss (Davenport SLH, 1977). Usher syndrome type I (USH1) is the most severe form of Usher syndrome, with profound bilateral congenital hearing loss and onset of retinitis pigmentosa within the first decade of life. Pathogenic variants of CDH23, PCDH15, USH1C ( known as harmonin) and USH1G ( known as sans) are responsible for 19-35%, 11-19%, 6-7% and 7% of incidences, respectively (see the Hereditary Hearing Loss Homepage). Each gene encodes structural and motor proteins important for mechanotransduction in the inner ear hair cells (Beurg et al, 2009; Grati and Kachar, 2011; Grillet et al, 2009a; Kazmierczak et al, 2007; Marcotti, 2012; Pepermans and Petit, 2015; Siemens et al, 2004)

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Conclusion