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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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 12

IHC postsynaptic defects in clarin-1–deficient mice.

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IHC postsynaptic defects in clarin-1–deficient mice. (A) Representative ...
(A) Representative electrically evoked brainstem responses (EEBRs) in the cochleae of P20 control, Clrn1ex4fl/fl Myo15-Cre+/–, and Clrn1ex4–/– mice. The first large downward inflection is due to an electrical artifact. In these stimulation conditions, wave I is not visible, as signal processing by the IHC synapse is bypassed. EEBR wave II (EII) and later waves (EIII and EIV), corresponding to the responses of higher auditory centers, are clearly visible in the control mouse (black trace, representative of 10 mice), absent in Clrn1ex4–/– mice (blue trace, representative of 6 mice), and significantly delayed and reduced in Clrn1ex4fl/fl Myo15-Cre+/– mice (red trace, representative of 10 mice). (B) Cochleae immunostained with Neurofilament 200 (NF200; green) showing a significant loss of auditory nerve fibers in P25 Clrn1ex4–/– mice (arrows) relative to control and Clrn1ex4fl/fl Myo15-Cre+/– mice of the same age. (C) IHCs of P20 control and Clrn1ex4fl/fl Myo15-Cre+/– mice. The postsynaptic GluA2/3-immunoreactive domain (green) is abnormally expanded in the nerve terminals underneath the IHCs of Clrn1ex4fl/fl Myo15-Cre+/– mice (n = 7). (D) Representative micrographs of the IHC synaptic region highlighting the expansion of postsynaptic terminals in Clrn1ex4fl/fl Myo15-Cre+/– mice (red dotted line; representative of 3 mice and 10 IHCs) and the swelling (artificially colored in green) of the postsynaptic boutons in Clrn1ex4–/– mice (representative of 3 mice and 10 IHCs) as compared with control mice (representative of 3 mice and 13 IHCs). (E) Quantification of the loss of parvalbumin-positive neurons in Rosenthal’s canal in control (4 cochleae from 3 mice), Clrn1ex4–/– (8 cochleae from 4 mice), and Clrn1ex4fl/fl Myo15-Cre+/– mice (6 cochleae from 3 mice) (values are mean ± SD; ANOVA, post hoc Holm-Sidak test). Scale bars: 500 nm (D), 20 μm (B and E), 5 μm (C).