Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants

Abstract

The multi-C 2 domain protein otoferlin is required for hearing and mutated in human deafness. Some OTOF mutations cause a mild elevation of auditory thresholds but strong impairment of speech perception. At elevated body temperature, hearing is lost. Mice homozygous for one of these mutations, Otof I515T/I515T , exhibit a moderate hearing impairment involving enhanced adaptation to continuous or repetitive sound stimulation. In Otof I515T/I515T inner hair cells (IHCs), otoferlin levels are diminished by 65%, and synaptic vesicles are enlarged. Exocytosis during prolonged stimulation is strongly reduced. This indicates that otoferlin is critical for the reformation of properly sized and fusion-competent synaptic vesicles. Moreover, we found sustained exocytosis and sound encoding to scale with the amount of otoferlin at the plasma membrane. We identified a 20 amino acid motif including an RXR motif, presumably present in human but not in mouse otoferlin, which reduces the plasma membrane abundance of Ile515Thr-otoferlin. Together, this likely explains the auditory synaptopathy at normal temperature and the temperature-sensitive deafness in humans carrying the Ile515Thr mutation.

A dual‐AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock‐out mice

“…For gene replacement therapy in Otof −/− mice, we used mouse otoferlin transcript variant 4 cDNA (NM_001313767), coding for the 1977 amino acid‐long protein and previously confirmed to be expressed endogenously in wild‐type cochleae (Strenzke et al , ). We designed otoferlin dual‐AAV‐trans‐splicing (dual‐AAV‐TS) and dual‐AAV‐hybrid (dual‐AAV‐Hyb) half‐vectors, both containing the N‐terminal otoferlin CDS in the 5′‐AAV half‐vector and the C‐terminal CDS in the 3′‐AAV vector (Fig EV2).…”

“…Maximum intensity projections of optical confocal sections. Scale bars: 5 μm. DSynapse numbers quantified from IHCs in apical cochlear turns (C) of B6 wild‐type (P6: n = 53 IHCs; P14: n = 73 IHCs) and B6‐ Otof −/− (P6: n = 62 IHCs; P14: n = 65 IHCs) mice at two different developmental stages (P6 and P14). ECa 2+ ‐current–voltage relationship of control CD1B6F1 wild‐type ( n = 6 IHCs), dual‐AAV‐TS‐transduced ( n = 8 IHCs), and non‐transduced CD1B6F1‐ Otof −/− ( n = 10 IHCs) IHCs (P14–18). FRepresentative Ca 2+ ‐currents (I ca ) and IHC plasma membrane capacitance increments (ΔC m ) of a wild‐type control, transduced, and non‐transduced Otof −/− IHC in response to a 20 ms depolarization pulse at maximum Ca 2+ ‐current potentials (typically −14 mV). G, HAverage exocytosis level measured as ΔC m (G) and corresponding Ca 2+ ‐current integrals (Q Ca2+ ) (H) in wild‐type [CD1B6F1: n = 6 IHCs; B6: n = 11 IHCs (B6 data replotted from Strenzke et al , )], dual‐AAV‐TS‐transduced Otof −/− ( n = 8 IHCs), and non‐transduced ( n = 11 IHCs) Otof −/− IHCs. Individual dual‐AAV‐TS transduced Otof −/− IHCs expressing eGFP, that had exocytosis (thinner red lines) and almost no exocytosis (broken red lines; not included into the average), are depicted. Data information: In (B, D), individual animals are depicted with open symbols.…”

“…Apparently, a click ABR threshold of ~50 dB can be established by very few dual‐AAV‐transduced IHCs. Improving dual‐AAV vectors to gain higher otoferlin protein levels might result in even lower ABR thresholds, because synapses contacting low threshold SGNs might require a high vesicle replenishment rate, which correlates with the amount of otoferlin in IHCs (Strenzke et al , ).…”

“…The ABR wave I amplitude, representing the summed activity of the auditory nerve, was very small and slightly delayed (Fig EV5D and I). This might be explained by the presence of fewer fully functional IHCs, fewer synapses in these cells, and lower otoferlin protein levels in treated Otof −/− compared to wild‐type mice, the latter most likely reducing spike synchrony (Buran et al , ; Bourien et al , ; Strenzke et al , ). The normal ABR wave II‐IV latencies (Fig EV5I) and the increase in ABR wave amplitudes along the auditory pathway (Fig EV5D–H) suggest some degree of central auditory compensation of the peripheral sound encoding.…”

Hair Cell Transduction, Tuning, and Synaptic Transmission in the Mammalian Cochlea

“…A missense mutation of the C2F domain in the pachanga mutant showed reduced exocytosis and slowed priming of vesicles in the readily releasable pool, implying otoferlin may underlie rapid replenishment of vesicles as well as fusion (225) (293). A mutation in the C2C domain, I515T, diminished otoferlin levels in the plasma membrane, and exocytosis during prolonged stimulation was strongly reduced, again indicating that the protein is critical for the reformation of fusion-competent synaptic vesicles (281). Because of the requirement for rapid and sustained neurotransmitter release at the IHC ribbon synapse, replenishment of the pool of readily-releasable vesicles is probably a rate-limiting step.…”