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.

Margot Tertrais, Yohan Bouleau, Alice Emptoz, Séverin Belleudy, R Bryan Sutton, Christine Petit, Saaid Safieddine, Didier Dulon
J. Neurosci.. 2019-03-04; : 1550-18
DOI: 10.1523/jneurosci.1550-18.2018

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1. J Neurosci. 2019 May 1;39(18):3394-3411. doi: 10.1523/JNEUROSCI.1550-18.2018. Epub 2019 Mar 4.

Tertrais M(1), Bouleau Y(1), Emptoz A(2), Belleudy S(1), Sutton RB(3), Petit C(2)(4), Safieddine S(2)(5), Dulon D(6).

Author information:
(1)Neurophysiologie de la Synapse Auditive, Institut National de la Santé et de la Recherche Médicale, UMR 1120, Université de Bordeaux, 33076 Bordeaux, France.
(2)Génétique et Physiologie de l’Audition, Institut National de la Santé et de la Recherche Médicale, UMR 1120, Institut Pasteur, 75015 Paris, France.
(3)Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, Texas 99430.
(4)Collège de France, 75005 Paris, France, and.
(5)Centre National de la Recherche Scientifique, 75016 Paris, France.
(6)Neurophysiologie de la Synapse Auditive, Institut National de la Santé et de la Recherche Médicale, UMR 1120, Université de Bordeaux, 33076 Bordeaux, France, .

Transmitter release at auditory inner hair cell (IHC) ribbon synapses involves exocytosis of glutamatergic vesicles during voltage activation of L-type Cav1.3 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 Ca2+ 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 -/- 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 -/- 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 -/- IHCs was also associated with a recovery of Ca2+ currents with normal amplitude and fast time inactivation, confirming that the C-terminal C2 domains of otoferlin are essential for normal gating of Cav1.3 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.

Copyright © 2019 the authors.

DOI: 10.1523/JNEUROSCI.1550-18.2018
PMCID: PMC6495124 [Available on 2019-11-01]
PMID: 30833506

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