Clarin-1 gene transfer rescues auditory synaptopathy in model of Usher syndrome
Didier Dulon, … , Christine Petit, Aziz El-Amraoui
Didier Dulon, … , Christine Petit, Aziz El-Amraoui
Published July 9, 2018
Citation Information: J Clin Invest. 2018;128(8):3382-3401. https://doi.org/10.1172/JCI94351.
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Research Article Neuroscience

Clarin-1 gene transfer rescues auditory synaptopathy in model of Usher syndrome

Abstract

Clarin-1, a tetraspan-like membrane protein defective in Usher syndrome type IIIA (USH3A), is essential for hair bundle morphogenesis in auditory hair cells. We report a new synaptic role for clarin-1 in mouse auditory hair cells elucidated by characterization of Clrn1 total (Clrn1ex4–/–) and postnatal hair cell–specific conditional (Clrn1ex4fl/fl Myo15-Cre+/–) knockout mice. Clrn1ex4–/– mice were profoundly deaf, whereas Clrn1ex4fl/fl Myo15-Cre+/– mice displayed progressive increases in hearing thresholds, with, initially, normal otoacoustic emissions and hair bundle morphology. Inner hair cell (IHC) patch-clamp recordings for the 2 mutant mice revealed defective exocytosis and a disorganization of synaptic F-actin and CaV1.3 Ca2+ channels, indicative of a synaptopathy. Postsynaptic defects were also observed, with an abnormally broad distribution of AMPA receptors associated with a loss of afferent dendrites and defective electrically evoked auditory brainstem responses. Protein-protein interaction assays revealed interactions between clarin-1 and the synaptic CaV1.3 Ca2+ channel complex via the Cavβ2 auxiliary subunit and the PDZ domain–containing protein harmonin (defective in Usher syndrome type IC). Cochlear gene therapy in vivo, through adeno-associated virus–mediated Clrn1 transfer into hair cells, prevented the synaptic defects and durably improved hearing in Clrn1ex4fl/fl Myo15-Cre+/– mice. Our results identify clarin-1 as a key organizer of IHC ribbon synapses, and suggest new treatment possibilities for USH3A patients.

Authors

Didier Dulon, Samantha Papal, Pranav Patni, Matteo Cortese, Philippe F.Y. Vincent, Margot Tertrais, Alice Emptoz, Abdelaziz Tlili, Yohan Bouleau, Vincent Michel, Sedigheh Delmaghani, Alain Aghaie, Elise Pepermans, Olinda Alegria-Prevot, Omar Akil, Lawrence Lustig, Paul Avan, Saaid Safieddine, Christine Petit, Aziz El-Amraoui

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Figure 4

Abnormal Ca2+ currents in mutant IHCs lacking clarin-1.

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Abnormal Ca2+ currents in mutant IHCs lacking clarin-1. (A–D) Ca2+ curre...
(A–D) Ca2+ currents in IHCs were activated with a depolarizing voltage-ramp protocol (1 mV/ms) from –90 to +30 mV. The amplitude of Ca2+ currents is normal on P9, but larger in Clrn1ex4–/– (blue curves, A and B) and Clrn1ex4fl/fl Myo15-Cre+/– (red curves, C and D) mice than in controls (black curves, A–D) on P13. (E) Comparative change in peak Ca2+ current density (peak ICa2+ at –10 mV normalized with respect to cell size) with age, at pre- and post-hearing stages (from P6 to P18), in IHCs from Clrn1ex4fl/fl Myo15-Cre+/– mice and control mice. (F) Boltzmann fit of the I-V curve for IHC Ca2+ currents (100-ms voltage steps) in Clrn1ex4fl/fl Myo15-Cre+/– and control mice on P18. (G) Comparative change in the half-maximal activation voltage of ICa2+ (V1/2) before and after hearing onset (from P6 to P18) in IHCs from Clrn1ex4fl/fl Myo15-Cre+/– and control mice. (H and I) ICa2+ activation time constant measured for various voltage steps from a holding potential at –80 mV in IHCs of P13 Clrn1ex4–/– mice (H), P18 Clrn1ex4fl/fl Myo15-Cre+/– mice (I), and the corresponding control mice. (J) IHCs from P18 Clrn1ex4fl/fl Myo15-Cre+/– (red) and control (black) mice were subjected to voltage steps from –80 mV to various membrane potentials for 100 milliseconds (inset: current traces for a Clrn1ex4fl/fl Myo15-Cre+/– mouse and a control mouse). The current reduction at the end of the 100-millisecond step (Ca2+-dependent inactivation, CDI) is expressed as a percentage of ICa2+ peak (%): CDI = [ICa2+ peak – ICa2+ 100 ms]/ICa2+p eak. The values shown are means ± SEM. *P < 0.05 (E–J, Student’s t test with Welch’s correction; and A–D, 2-way ANOVA); n, number of cells.