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

• Published in: The Journal of neuroscience Vol. 39; no. 18; pp. 3394 - 3411
• Main Authors: Tertrais, Margot, Bouleau, Yohan, Emptoz, Alice, Belleudy, Séverin, Sutton, R Bryan, Petit, Christine, Safieddine, Saaid, Dulon, Didier
• Format: Journal Article
• Language: English
• Published: United States Society for Neuroscience 01-05-2019
• Subjects: Calcium; Calcium channels; Calcium channels (L-type); Calcium channels (voltage-gated); Calcium ions; Channel gating; Channels; Cochlea; Deactivation; Dynamin; Endocytosis; Exocytosis; Glutamatergic transmission; Hair; Hair cells; Human health and pathology; In vivo methods and tests; Inactivation; Life Sciences; Membrane capacitance; Neurobiology; Neurons and Cognition; Neurotransmitter release; Recovery; Reintroduction; Restoration; Sensory Organs; Synapses; Synaptic ribbons; Synaptic vesicles; Time dependence; Vesicles; hair cells; ribbon synapses; exocytosis; otoferlin; deafness; calcium sensor
• ISSN: 0270-6474; 1529-2401
• DOI: 10.1523/JNEUROSCI.1550-18.2018

Transmitter release at auditory inner hair cell (IHC) ribbon synapses involves exocytosis of glutamatergic vesicles during voltage activation of L-type Ca
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
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
, where IHCs of mouse lacking otoferlin (
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
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
IHCs was also associated with a recovery of Ca
currents with normal amplitude and fast time inactivation, confirming that the C-terminal C2 domains of otoferlin are essential for normal gating of Ca
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.
Otoferlin, a large six-C2-domain protein, is essential for synaptic vesicle exocytosis at auditory hair cell ribbon synapses. Here, we show that the viral expression of truncated forms of otoferlin (C2-EF, C2-DEF, and C2-ACEF) can partially rescue the fast and transient release component of exocytosis in mouse hair cells lacking otoferlin, yet cannot sustain exocytosis after long repeated stimulation. Remarkably, these hair cells also display a dynamin-dependent ultrafast endocytosis. Overall, our study uncovers the pleiotropic role of otoferlin in the hair cell synaptic vesicle cycle, notably in triggering both ultrafast exocytosis and endocytosis and recruiting synaptic vesicles to the active zone.