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 1

Auditory thresholds in Clrn1ex4–/– and Clrn1ex4fl/fl Myo15-Cre+/– mice.

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Auditory thresholds in Clrn1ex4–/– and Clrn1ex4fl/fl Myo15-Cre+/– mice. ...
(A) The Clrn1-floxed mice (Clrn1ex4fl/fl) were engineered by addition of loxP sites on either side of exon 4. Clrn1ex4fl/fl mice were crossed with PGK-Cre+/– mice to achieve the early and ubiquitous elimination of clarin-1 (Clrn1ex4–/– mice), and with Myo15-Cre+/– mice to target clarin-1 elimination specifically to hair cells at postnatal stages (Clrn1ex4fl/fl Myo15-Cre+/– mice). A topological representation of the murine clarin-1, a protein containing 4 transmembrane domains with a C-terminal type II PDZ-binding motif (PBM, boxed), is shown. (B) RT-PCR shows mRNA expression of Clrn1 isoforms 1, 2, and 3 in the organ of Corti (P30) and only isoform 2 and 3 in P30 WT IHCs. Note that the expression of all isoforms is absent in P30 IHCs from Clrn1ex4fl/fl Myo15-Cre+/– (see uncut gels in supplemental material). (C) ABR thresholds in Clrn1ex4fl/fl Myo15-Cre+/– mice (light orange to dark red curves; mean ± SD) show a progressive increase between P15 and P18. A gray curve (control mice) and a blue curve (Clrn1ex4–/– mice) for P15–P20 are also shown. (D) In Clrn1ex4fl/fl Myo15-Cre+/– mice (red), ABR thresholds (mean ± SD) progressively increase to reach severe deafness at P21–P28 as compared with those in control mice (gray-black). (E) DPOAEs are absent in Clrn1ex4–/– mice (blue) as early as P15. By contrast, Clrn1ex4fl/fl Myo15-Cre+/– mice (red) display unaffected DPOAE thresholds up to P20 (green area). From P20 onward, DPOAE thresholds slowly increase by about 20–30 dB SPL. (F) Compound action potential (CAP) responses on P15–P19. CAPs were produced by 10-kHz, 95-dB-SPL, tone bursts. Bar charts represent CAP wave I latency and amplitude, which are significantly delayed and reduced, respectively, in Clrn1ex4fl/fl Myo15-Cre+/– mice. *P < 0.05 (2-tailed unpaired t test with Welch’s correction, n in the figure).