Viral transfer of mini-otoferlins partially restores the fast component of exocytosis and uncovers ultrafast endocytosis in auditory hair cells of otoferlin knock-out mice

Abstract

Transmitter release at auditory inner hair cell (IHC) ribbon synapses involves exocytosis of glutamatergic vesicles during voltage activation of L-type Ca v 1.3 calcium channels. At these synapses, the fast and indefatigable release of synaptic vesicles by IHCs is controlled by otoferlin, a six-C2-domain (C2-ABCDEF) protein that functions as a high-affinity Ca 2ϩ sensor. The molecular events by which each otoferlin C2 domain contributes to the regulation of the synaptic vesicle cycle in IHCs are still incompletely understood. Here, we investigate their role using a cochlear viral cDNA transfer approach in vivo, where IHCs of mouse lacking otoferlin (Otof Ϫ/Ϫ mice of both sexes) were virally transduced with cDNAs of various mini-otoferlins. Using patch-clamp recordings and membrane capacitance measurements, we show that the viral transfer of mini-otoferlin containing C2-ACEF, C2-EF, or C2-DEF partially restores the fast exocytotic component in Otof Ϫ/Ϫ mouse IHCs. The restoration was much less efficient with C2-ACDF, underlining the importance of the C2-EF domain. None of the mini-otoferlins tested restored the sustained component of vesicle release, explaining the absence of hearing recovery. The restoration of the fast exocytotic component in the transduced Otof Ϫ/Ϫ IHCs was also associated with a recovery of Ca 2ϩ currents with normal amplitude and fast time inactivation, confirming that the C-terminal C2 domains of otoferlin are essential for normal gating of Ca v 1.3 channels. Finally, the reintroduction of the mini-otoferlins C2-EF, C2-DEF, or C2-ACEF allowed us to uncover and characterize for the first time a dynamin-dependent ultrafast endocytosis in IHCs.

Synaptic Release Potentiation at Aging Auditory Ribbon Synapses

“…When labeling the IHCs cytosol with an antibody against myo-7a, an unconventional myosin essential for the hair bundle functional integrity and found in the apical sterocilia as well as the cytoplasm of the hair cells (Hasson et al, 1995), we measured a drastic reduction in length of the infra-nuclear synaptic zone of Otof -/- IHCs lacking otoferlin (Fig.7). The reduction of the IHC synaptic zone was consistent with a large reduction of the resting cell membrane capacitance, measured under whole-cell patch clamp at -70 mV, in P21 Otof -/- IHCs as compared to P21 Otof +/+ IHCs (respectively, 8.7 ± 0.1 fF, n = 23, and 10.8 ± 0.1 fF, n = 44, p < 0.05), as previously described (Tertrais et al, 2019). Similar reduction in the infra-nuclear IHC synaptic zone, as well as a reduced resting membrane capacitance, was observed in old P360 Otof +/+ IHCs (Fig.…”

“…The lack of ABR waves arises from a severe defect of Ca 2+- evoked synaptic vesicle exocytosis at IHC ribbon synapses (Michalski et al, 2017). This hair cell synaptopathy is associated with an early loss of ribbons at P16 (Vincent et al, 2017; Tertrais et al, 2019). We confirmed here that Otof -/- mice have IHCs with an early low number of synaptic ribbons, with a 34% reduction in ribbon number at P15 as compared to P15 Otof +/+ C57BL/6J mice (10.7 ± 0.3 and 16.3 ± 0.3 synaptic ribbon/IHC, respectively, p < 0.05; Fig.…”

“…Cdh23 encodes cadherin 23, a protein necessary for inner ear development and maintenance of the sensory hair cell structures, in particular the tip and lateral links of the stereocilia at the hair bundle (Siemens et al, 2004; Söllner et al, 2004; Kazmierczak et al, 2007; Michel et al, 2005; Lagziel et al, 2005), but growing anatomical evidence also suggests that synaptic rearrangements on sensory hair cells also contribute to auditory functional decline in C57BL/6J mice (Stamataki et al, 2006; Zachary and Fuchs, 2015; Jiang et al, 2015). Remarkably, an even earlier degeneration of auditory ribbon synapses has been reported in C57BL/6J mice when the otoferlin gene is lacking ( Otof -/- mice) (Roux et al, 2006; Vincent et al, 2017; Tertrais et al, 2019). We recall that otoferlin is a crucial Ca 2+ sensor that controls synaptic vesicle exocytosis at auditory inner hair cell (IHC) ribbon synapses (Beurg et al, 2010; Michalski et al, 2017) and vestibular hair cell synapses (Dulon et al, 2009).…”

Auditory Neuropathy Spectrum Disorders: From Diagnosis to Treatment: Literature Review and Case Reports

“…OTOF mutations were found in 2.4% of individuals with hearing loss [62]. Virally mediated gene therapy has been put forward as a promising prospect in restoring hearing in knock out mice with impaired OTOF [63].…”

Auditory Neuropathy Spectrum Disorder due to Two Novel Compound Heterozygous OTOF Mutations in Two Chinese Families

“…The C2 domains are highly conserved, and the last four domains have five conserved aspartyl residues predicted to bind Ca 2+12 . This suggests that otoferlin is involved in docking of the synaptic vesicles to the plasma membrane and mediating their fusion and exocytosis in a Ca 2+ -dependent manner in rodents [13–16].…”